An Epidemiological Surveillance Study to Evaluate the Incidence of Dengue in Endemic Regions of Brazil
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:
116606 (EPI-DENGUE-006 BOD BR)
Date of protocol:
Final: 07 June 2012
Date of Protocol Amendment
Protocol Amendment 1 Final: 14 June 2013
Protocol Amendment 2 Final: 24 July 2014
Protocol Amendment 3 Final: 09 January 2015
Protocol Amendment 4 Final: 16 October 2017
Title:
An epidemiological surveillance study to evaluate the 
incidence of dengue in endemic regions of Brazil.
Detailed Title:
A prospective, multicenter, multiyear, cohort study to 
determine dengue incidence in children and adults in 
communities from endemic regions in Brazil.
Co-ordinating author:
, Scientific Writer / 
, 
Scientific Writer, XPE working for GSK Biologicals 
GSK Contributing authors for previous versions:
Epidemiologist 
Epidemiologist, Senior Manager
Epidemiologist, Brazil
Project Statistician
Senior Manager Lead statistician
Study Manager
Study Manager
Study Delivery Lead
Local Delivery Lead
Global Study Manager (advisory role)
Clinical Laboratory Sciences Manager 
Project Statistician
Clinical Laboratory Sciences Manager
Global Vaccine Clinical Project Manager
Clinical Laboratory Project Manager
Senior Manager, Global Study Management 
Clinical Operations Manager
Clinical Laboratory Project Manager
Global Vaccine Clinical Laboratories Manager 
Clinical Safety representative 
Study Data Manager
Study Data Manager
Study Data Manager
Medical Affairs Director, Brazil
 
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eTrack study number and 
Abbreviated Title:
116606 (EPI-DENGUE-006 BOD BR)
Detailed Title:
A prospective, multicenter, multiyear, cohort 
study to determine dengue incidence in children 
and adults in communities from endemic regions 
in Brazil.
GSK Contributing authors for previous versions:
Medical Affairs Director, Brazil
Non-GSK Contributing authors:
Bio-Manguinhos, Fiocruz
Bio-Manguinhos, Fiocruz
Bio-Manguinhos, Fiocruz/ad hoc
Bio-Manguinhos, Fiocruz
Principal Investigator, Manaus, Fundação de 
Medicina Tropical Dr. Heitor Vieira Dourado
Principal Investigator, Salvador de Bahia, 
Fundação Oswaldo Cruz, Ministério da Saúde
Principal Investigator, Rio de Janeiro, Fundação 
Oswaldo Cruz, Ministério da Saúde
GSK Contributing authors for current version:
Epidemiologist, Brazil
Epidemiologist, Director
Local Delivery Lead
Local Delivery Lead
Manager, Clinical Laboratory Study 
Management
Lead Statistician
Study Delivery Lead
Oversight Data Manager
Study Data Manager
Study Data Manager
Project Lead, Chikungunya
Clinical Read-out Team Leader
Senior Manager Lead statistician
Study statistician
Medical Affairs Director, Brazil
GSK Biologicals’ protocol template for observational studies and interventional studies 
without administration of medicinal products as described in a research protocol based on 
the Protocol Document Standard version 13.2
© [2017] GSK group of companies or its licensor.
 
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Protocol Amendment 4 Sponsor Signatory Approval
eTrack study number and 
Abbreviated Title:
116606 (EPI-DENGUE-006 BOD BR)
Date of Protocol 
Amendment
Protocol Amendment 4 Final: 16 October 2017
Detailed Title:
A prospective, multicenter, multiyear, cohort study to 
determine dengue incidence in children and adults in 
communities from endemic regions in Brazil.
Sponsor signatory
Robert Paris,
CEPL, Early Development, 
Vaccines R&D,
GSK Biologicals US
Signature
Date
 
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Protocol Amendment 4 Rationale
Rationale/background for changes:
Additional exploratory objectives have been added in this protocol amendment to explore 
the seroprevalence of antibodies to Zika and chinkungunya viruses in the study 
population, as well as the potential aetiological role of these viruses in episodes of febrile 
illness detected during follow-up .
Zika is a flavivirus and chikungunya virus is a member of the alphavirus genus, and 
Togaviridae family. These cause emerging mosquito-borne viral diseases in Latin 
America especially in Brazil. Zika virus infections have been laboratory confirmed in 19 
states in Brazil between April and December 2015 [Boletim Epidemiológico n5, 2016]. 
In 2016, until epidemiological week 47, 196.976 cases were reported and 101,851 were 
confirmed [Boletim epidemiológico n 33, 2016]. 
In 2015, 20,661 autochthonous suspected chikungunya cases were reported in Brazil 
[Boletim Epidemiológico n5, 2016]. In 2016, 216,102 reported cases and 102,638 were 
confirmed up to epidemiological week 47 [Boletim epidemiológico n33, 2016]. Zika and 
chikungunya viruses cause symptoms often similar to dengue during the acute phase of 
illness.
The interpretation of the serological tests used to determine the seroprevalence of dengue 
antibodies or for diagnostic of acute dengue infection should take into account the 
possibility of cross-reaction with antibodies elicited by exposure to the Zika virus. The 
public health importance of Zika and chikungunya virus emergence, as well as the 
potential impact of these viruses on the interpretation of the laboratory assays, supports 
the inclusion of the exploratory objectives related to Zika and chikungunya infection.
Fever is the triggering event for a subject to be evaluated as a suspect dengue case in the 
study. While fever is common in suspected chikungunya cases, fever does not always 
present in suspected Zika cases.  For this reason, rash is being added as a triggering 
event, in addition to fever, for Zika virus disease evaluation. A prospective surveillance 
will be maintained for febrile cases and added for rash cases. Also, considering the 
epidemiological situation in Brazil for both Zika and chikungunya infection, a 
retrospective testing for these infections in subjects considered as suspect dengue case 
since the beginning of the study will also be done.
The current case definition for suspected dengue does not include a criteria specifying the 
maximum interval between fever onset and the initial medical visit. This is specified in 
section 5.7.1.1.
Currently, haematology testing (HCT, CBC) is planned to be performed for all suspected 
dengue cases at the initial visit, regardless of the time between fever onset and the visit. 
However, for patients presenting more than 14 days after fever and/or rash onset, the 
relevance of these tests is low. Therefore, a maximum interval will be defined in this 
amendment.
 
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The geographical distribution of the participants may impact the risk of infection. 
Therefore, updating information on the household of the study participants and their 
location at yearly visits is important during the follow-up period (which may extend to 4 
years). Hence, socio-demographic and household information will be collected at yearly 
visits.
Some sites may have more than 500 active participants enrolled in the penultimate year 
of the study. However, their site may have experience with high drop-out rate and can 
expect to drop below 500 participants during the last year of the study. To address this 
challenge, criteria to allow for replacement cohort recruitment was clarified.
Also, the NS1 test has been removed for the late presenters since this test does not have 
clinical relevance in the case of late presenters.
Collection of dengue vaccination history has been added since a competitor dengue 
vaccine is currently available in the Brazilian market.
 
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Protocol Amendment 4 Investigator Agreement
I agree:

To conduct the study in compliance with this protocol, any mutually agreed future 
protocol amendments or protocol administrative changes, and with any other study 
conduct procedures and/or study conduct documents provided by GlaxoSmithKline 
Biologicals (GSK Biologicals)

To assume responsibility for the proper conduct of the study at this site

That I am aware of, and will comply with, ‘Good Clinical Practice’ (GCP) 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 authorized 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:
116606 (EPI-DENGUE-006 BOD BR)
Date of Protocol Amendment
Protocol Amendment 4: 16 October 2017
Detailed Title
A prospective, multicenter, multiyear, cohort study to 
determine dengue incidence in children and adults in 
communities from endemic regions in Brazil.
Investigator name
Signature
Date
 
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Sponsor Information 
1.
Sponsor
GlaxoSmithKline Biologicals, 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.
Sponsor Study Contact for Reporting of a Serious Adverse Event
GSK Biologicals Central Back-up Study Contact for Reporting SAEs: refer to
protocol Section 6.2.3
GSK Biologicals Central Safety Physician and Back-up Phone contact: refer to
protocol Section 6.2.3
 
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SYNOPSIS
Detailed 
Title:
A prospective, multicenter, multiyear, cohort study to determine 
dengue incidence in children and adults in communities from 
endemic regions in Brazil.
Rationale for 
the study:
The estimation of dengue incidence is critical when designing an 
efficacy trial for a dengue vaccine. In a given city or region, 
dengue incidence is highly variable from one year to another 
(variations in geography, season, year-to-year variations in 
mosquito vector populations in different areas, etc.) and cannot 
yet be predicted with sufficient accuracy in many locations being 
considered for clinical trials. In a dengue vaccine efficacy trial, 
volunteers are recruited from geographically distinct sites. Some 
of these sites may experience high dengue transmission during the 
trial duration while others may have limited transmission. 
Therefore, making a valid assumption regarding the average 
incidence over time and across sites in order to assess the efficacy 
of an investigational vaccine is important.
In addition, a critical factor for the success of an efficacy trial is 
training staff at the various sites to perform study procedures such 
as febrile illness detection, dengue laboratory diagnosis and data 
collection in a cohort of subjects.
The proposed study will estimate the incidence of dengue 
infection and disease across geographically distinct locations over 
time in order to validate attack rate assumptions in future efficacy 
trials. We will enrol individuals from a random sample of 
households from selected communities of geographically different 
sites in Brazil and assess incidence of dengue infection and 
disease, by conducting scheduled serosurveys and an enhanced 
surveillance of febrile disease. The study will be conducted in at 
least three cities.
This study will also prepare potential sites for future clinical trials 
by setting up the logistics and training staff on site to enrol a 
cohort of subjects, perform dengue surveillance and other study 
procedures.
Finally, in line with the overarching strategy of building a strong 
partnership with the Ministry of Health (MoH), this study will 
serve as a useful comparison for evaluation of dengue 
surveillance systems.
 
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Objectives
Primary

To estimate the incidence of laboratory-confirmed 
symptomatic dengue infection in the study population by 
year/season.
Secondary

To estimate the serotype-specific incidence of virologically-
confirmed symptomatic dengue infection in the study 
population overall and by season.

To estimate the incidence of symptomatic dengue infection 
(including laboratory-confirmed and probable cases) in the 
study population by study site, gender, age-group, previous 
dengue exposure (primary or secondary), overall and by 
season.

To estimate the prevalence of previous dengue infection 
(dengue seroprevalence) in the study population overall and 
by study site, gender and age-group at enrolment.

To estimate the incidence of primary inapparent, dengue 
infection, in the study population overall and by study site, 
gender age-group and by season. 

To describe symptoms and spectrum of dengue disease in the 
study population.
Tertiary
(optional) 
objectives
(Amended 16 
October 
2017)

To investigate potential risk factors for symptomatic dengue 
infection.

To investigate the role of dengue neutralizing antibodies 
elicited through natural infection in protection against 
subsequent infection.

To estimate the degree of underreporting of dengue cases 
using the passive national notification surveillance.

To estimate the incidence of symptomatic Zika virus 
infection (including laboratory-confirmed and probable 
cases) in the study population by study site, gender, age-
group, previous flavivirus exposure, overall and by season.

To estimate the incidence of symptomatic chikungunya 
virus infection (including laboratory-confirmed and 
probable cases) in the study population by study site, 
gender, age-group, overall, and by season.

To describe previous Yellow Fever (YF) antibody profile in 
selected dengue cases to investigate the role of previous YF 
immunity on the dengue neutralizing antibody profile 
following dengue infection.
 
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
To describe the spatial and temporal distribution of dengue 
cases among cohort participants in the study areas.

To describe the spatial and temporal distribution of Zika
and chikungunya cases among cohort participants in the 
study areas.

To describe other infectious aetiologies related to 
differential diagnosis of dengue (chikungunya and Zika) in 
subjects with episodes of febrile illness referred to as 
“suspected dengue case”.

To estimate the seroprevalence of antibodies against Zika
and chikungunya virus at selected timepoints.
Study design

Type of design: Prospective, multicenter, community-based,
household-sampling, cohort study in Brazil.

Study population: Subjects six months of age and older at 
the time of enrolment who live in the selected study sites in 
Brazil, covering different regions of the country.

There will be 2 waves of enrolment for recruiting 3600 
subjects:

Initial Cohort = about 1800 subjects – up to 6 visits 
(Amendment 2) – INITIAL sites

Expansion Cohort = about 1800 subjects – up to 4 visits  
– NEW sites 

The study period, initially planned to be one year, has been
extended by additional three years (overall 4 years) to cover 
three additional dengue seasons (Amendment 2). 

Initial cohort subjects were invited to extend their 
participation. 
(See glossary of terms for the definition of initial and 
expansion cohort subjects)
In each cohort: replacement subjects may be enrolled in order 
to compensate for subjects who did not wish to extend their 
participation for three additional years (for subjects from the 
initial cohort – Amendment 2), subjects who prematurely 
terminate participation or subjects who are lost to follow-up.
This will be done to maintain a cohort size of at least 500 
subjects per site, in the Initial and Expansion cohorts, at the 
beginning of each additional study year/season.
At the end of the penultimate year of the study, the cohort 
size will be reviewed to address the potential need for 
 
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replacement cohort recruitment.
(See glossary of terms for the definition of replacement 
subjects, subjects who prematurely terminate participation 
and subjects lost to follow-up )

Informed consent (and assent, if applicable) will be obtained:

From the subject (and parents/LAR of the subjects,  if 
applicable) at the study start for the initial cohort.

From the subject (and parents/LAR of the subjects,  if 
applicable) of the initial cohort prior to participation in 
the additional three years of the study.

From the subject (and parents/LAR of the subjects,  if 
applicable) at the study start for the expansion cohort.

From the subject (and parents/LAR of the subjects, if 
applicable) prior to enrolment in the study for the 
replacement subjects.

Study visits: The visits will be as follows and shown in 
Synopsis Figure 1:

Initial cohort subjects consenting to continue 
participation for the three additional years will have:
o
three scheduled visits in the first year (Visit 1, 
Visit 2 and Visit 3) at six months intervals (+/- 28 
days), 
o
and thereafter, one scheduled visit per year during 
a period of low dengue transmission for the three 
additional years; i.e; one visit at study Year 2, Year 
3 and Year 4 (Visit 4, Visit 5 and Visit 6 
respectively).

Initial cohort subjects who do not consent to extend 
participation for three additional years will:
o
have three scheduled visits (Visit 1, Visit 2 and 
Visit 3) at six months interval (+/- 28 days). 
o
conclude their participation in the study at Visit 3
or the last follow up visit if the subject is suspected 
of having a dengue case on-going at Visit 3.

Subjects from the expansion cohort will have only three 
years of follow-up, with at most 4 scheduled visits.

For replacement subjects, the number of scheduled visits 
will depend on the study year in which they are enrolled. 
Replacement subjects will not be enrolled until after the 
third visit of the first study year is concluded for the 
initial cohort. The number of visits for the replacement 
subjects will range between two and four.
The yearly scheduled visits for the initial and expansion 
cohorts and replacement subjects will preferably occur 
 
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during the period of usually low dengue transmission.
Synopsis Figure 1
Schematic representation of the 
scheduled visits
V= Visit
The scheduled visits for initial cohort subjects are shown in black.
New enrolments will be done yearly (as needed) till the end of Study Year 3.
Enrollment of expansion subjects will start preferably during study Year 2.
For replacement subjects, the number of visits will depend on the year enrolled.

Suspected dengue, chikungunya and/or Zika case detection
(Amended 16 October 2017): Suspected dengue, Zika
and/or chikungunya cases will be detected using three 
sources:
1.
referred by study personnel during scheduled home 
visits; 
2.
through enhanced passive surveillance; and 
3.
as a result of active surveillance between scheduled 
visits; (see Section 5.5.3 for details). 

If dengue, Zika and/or chikungunya is/are suspected, a
medical appointment with a physician at a designated study 
hospital/clinic will be arranged (see Section 5.6 for 
management of suspected dengue, Zika and chikungunya
cases). 

If the suspicion is confirmed during the visit:
i.
A blood sample for dengue, Zika and/or chikungunya
diagnosis and for haematology (CBC and HCT) (mandated 
procedure for subjects presenting within the first 14 days 
following onset of fever and/or rash) will be collected at the 
first visit for suspected dengue, Zika and/or chikungunya
(acute blood sample) and 
ii.
a second blood sample (convalescent blood sample) will be 
collected approximately 21 (maximum 28 days) days later. 
iii. In between, a return visit may be scheduled as needed.

Data collection: eCRF

Type of study: self-contained
 
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
Duration of the study: Four years for the overall study. For 
an individual subject, it will range between one and four 
years.

Epoch 001: prospective data collection starting at Visit 1 
Day 0 and ending at the last subject last visit.
Synopsis Table 1
Study group and epoch foreseen in the study
Study Group
Number of subjects*
Age (Min/Max)
Epoch
Prospective study cohort
Initial Cohort:
1800 at enrolment (first year)
At least 1500 in each 
subsequent year 
6 months of age and older
Epoch 001
Expansion Cohort: 
1800 at enrolment (first year).
At least 1500 in each 
subsequent year
* The final number of participants will take into account the active subjects, subjects who prematurely terminate 
participation and subjects lost to follow-up.
Discussion 
of study 
design
Selection of study sites
At least six sites in Brazil will be selected for this study. Selection 
criteria include support from the Family Health Physician Program 
(FHP), the Larval Index Rapid Assay (LIRA) or with field research 
experience in the community. The FHP is a Brazilian government public 
health strategy that consists of multidisciplinary teams of health care 
workers that cover geographically defined areas to provide 
basic/preventive care to community members. The teams include a 
doctor, a nurse, a nurse auxiliary, other health professionals and 
community health care workers who pay regular visits to the household 
under its responsibility to deliver health promotion activities and 
facilitate triage and referral into the health unit, among other activities. 
Community health care workers are local residents who are trained for 
basic tasks.
LIRA is an alternative vector surveillance strategy that consists of 
random sampling of a number of dwellings in which surveillance of 
aedes-positive breeding places is carried out, and has been widely 
implemented in Brazil (Pontes, 2000). 
At least one community of households within each site will be selected.
 
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Number of 
subjects
Approximately 3600 subjects will be recruited in the study in 2 waves of 
enrolment. Each wave, initial and expansion cohorts, will enrol about 
1800 subjects. Replacement subjects may be recruited afterwards to 
compensate for subjects not willing to extend participation for the three 
additional years, subjects who prematurely terminate participation and 
subjects lost to follow-up. The recruitment of replacement subject aims at 
maintaining the number of subjects at a minimum of 500 subjects/site at 
the beginning of study Year 2, 3 and 4. The total number of subjects 
enrolled in the study may exceed 3600.
In order to reach the Brazilian population distribution, subjects enrolled 
in the expansion cohort should be composed of at least 30% of subjects 
younger than 18 years old and should not exceed 20 % of adults 50 years 
or older. Subjects enrolled as replacement subjects for the initial and 
expansion cohort will be recruited to approach as much as possible a 
minimal representation of 30% of children and a maximal representation 
of 20% for adults 50 years or older.
Endpoints
Primary

Laboratory-confirmed symptomatic dengue infection (all DENV 
types)
Symptomatic dengue infection is defined in Section 5.7.2
Secondary

DENV-type specific primary laboratory-confirmed symptomatic 
dengue infection

DENV-type specific secondary laboratory-confirmed symptomatic 
dengue infection

Primary symptomatic dengue infection (including laboratory-
confirmed and probable cases)

Secondary symptomatic dengue infection (including laboratory-
confirmed and probable cases)

Previous dengue infection(s) (dengue seroprevalence) at baseline. 
(defined in Section 5.7.5)

Primary inapparent dengue infection

Severity of symptoms of symptomatic dengue (using the 2009 WHO 
guidelines)
 
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Tertiary (optional) (Amended 16 October 2017)
Exploratory objectives may be assessed based on, but not limited to the 
following endpoints:

Symptomatic laboratory-confirmed Zika virus infection

Symptomatic Zika virus infection (including laboratory-confirmed 
and probable cases).

Symptomatic laboratory-confirmed chikungunya infection

Symptomatic chikungunya infection (including laboratory-
confirmed and probable cases).

Risk factors for dengue infection and disease

Neutralizing antibodies titers against DENV 1-4

Neutralizing antibody titers against YF virus

Spatial and temporal distribution of dengue cases (confirmed and 
probable symptomatic cases and inapparent infections)

Spatial and temporal distribution of laboratory confirmed and 
probable Zika virus cases

Spatial and temporal distribution of patients with laboratory 
confirmed and probable chikungunya virus infection

Occurrence of laboratory confirmed or probable Zika and/or 
chikungunya infection in suspected dengue cases (differential 
diagnosis of dengue), retrospectively

Antibody titre against Zika and chikungunya virus at the 
scheduled visits
 
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TABLE OF CONTENTS
PAGE
SYNOPSIS......................................................................................................................9
LIST OF ABBREVIATIONS...........................................................................................24
GLOSSARY OF TERMS ...............................................................................................25
1.
INTRODUCTION....................................................................................................29
1.1.
Background (Amended 16 October 2017)...................................................29
1.2.
Rationale for the study................................................................................30
2.
OBJECTIVES.........................................................................................................31
2.1.
Primary objective ........................................................................................31
2.2.
Secondary objectives..................................................................................31
2.3.
Tertiary objectives (optional) (Amended 16 October 2017) .........................31
3.
STUDY DESIGN OVERVIEW (AMENDED 16 OCTOBER 2017) ...........................32
3.1.
Discussion of study design .........................................................................35
3.1.1.
Selection of study sites ................................................................35
3.1.2.
Rationale for study design ...........................................................35
4.
STUDY POPULATION...........................................................................................36
4.1.
Selection of communities............................................................................36
4.2.
Selection of households..............................................................................36
4.3.
Overview of the recruitment plan ................................................................37
4.4.
Inclusion criteria for enrolment (Amended 16 October 2017) ......................38
4.5.
Exclusion criteria for enrolment...................................................................38
5.
STUDY CONDUCT ................................................................................................39
5.1.
Regulatory and ethical considerations, including the informed 
consent process..........................................................................................39
5.2.
Subject identification...................................................................................40
5.3.
General study aspects ................................................................................40
5.4.
Outline of study procedures ........................................................................41
5.5.
Detailed description of study procedures ....................................................49
5.5.1.
Procedures prior to study participation.........................................49
5.5.1.1.
Informed consent........................................................49
5.5.1.2.
Check inclusion and exclusion criteria ........................49
5.5.2.
Procedures at scheduled Visit 1 (enrolment visit) 
(Amended 16 October 2017)........................................................49
5.5.2.1.
Collect socio-demographic data..................................49
5.5.2.2.
Collect specific medical history data (Amended 
16 October 2017) .......................................................50
5.5.2.3.
Collect  blood sample .................................................50
5.5.2.4.
Instruction on enhanced passive dengue, 
chikungunya and Zika surveillance by the 
subject or subject’s parent(s)/LAR(s) 
(Amended 16 October 2017) ......................................50
 
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5.5.2.5.
Diary log distribution (Amended 16 October 
2017)..........................................................................50
5.5.2.6.
Recording of serious adverse events related to 
a study procedure.......................................................51
5.5.3.
Procedures at subsequent scheduled visits (2-6, as 
applicable) (Amended 16 October 2017) .....................................51
5.5.4.
Study conclusion (Amended 16 October 2017)............................52
5.5.5.
Dengue, chikungunya and Zika case detection (Amended 
16 October 2017).........................................................................52
5.5.5.1.
Case detection during scheduled home visits 
(Amended 16 October 2017) ......................................52
5.5.5.2.
Case detection through enhanced passive 
surveillance (Amended 16 October 2017)...................53
5.5.5.3.
Case detection through active surveillance 
(Amended 16 October 2017) ......................................54
5.6.
Management of suspected dengue, chikungunya and Zika cases 
(Amended 16 October 2017).......................................................................54
5.6.1.
Diary log instructions for suspected dengue, chikungunya 
and/or Zika cases (Amended 16 October 2017)...........................55
5.7.
Case definitions ..........................................................................................56
5.7.1.
Dengue........................................................................................56
5.7.1.1.
Suspected symptomatic dengue case 
(Amended 16 October 2017) ......................................56
5.7.1.2.
Laboratory-confirmed symptomatic dengue 
case (Amended 16 October 2017)..............................57
5.7.1.3.
Virologically confirmed symptomatic dengue 
infection......................................................................57
5.7.1.4.
Moderate to severe dengue........................................57
5.7.1.5.
Previous dengue infection (Amended 16 
October 2017) ............................................................58
5.7.1.6.
Primary symptomatic dengue case (Amended 
16 October 2017) .......................................................59
5.7.1.7.
Secondary symptomatic dengue case 
(Amended 16 October 2017) ......................................59
5.7.1.8.
Primary inapparent dengue infection (Amended 
16 October 2017) .......................................................59
5.7.1.9.
Probable dengue case (Amended 16 October 
2017)..........................................................................59
5.7.1.10.
Negative Dengue case ...............................................59
5.7.1.11.
Indeterminate dengue case (Amended 16 
October 2017) ............................................................59
5.7.2.
Chikungunya................................................................................60
5.7.2.1.
Suspected symptomatic chikungunya case ................60
5.7.2.2.
Laboratory-confirmed symptomatic 
chikungunya case.......................................................61
5.7.2.3.
Virologically confirmed symptomatic 
chikungunya infection.................................................61
5.7.2.4.
Previous chikungunya infection at baseline ................61
5.7.2.5.
Inapparent chikungunya infection ...............................61
5.7.2.6.
Probable chikungunya case........................................61
5.7.2.7.
Negative chikungunya case........................................61
5.7.2.8.
Indeterminate chikungunya case ................................62
 
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5.7.3.
Zika..............................................................................................62
5.7.3.1.
Suspected symptomatic Zika case .............................62
5.7.3.2.
Laboratory-confirmed symptomatic Zika case.............63
5.7.3.3.
Virologically confirmed symptomatic Zika 
infection......................................................................63
5.7.3.4.
Previous Zika infection at baseline .............................63
5.7.3.5.
Inapparent Zika infection ............................................63
5.7.3.6.
Probable Zika case.....................................................63
5.7.3.7.
Negative Zika Case ....................................................63
5.7.3.8.
Indeterminate Zika case .............................................64
5.8.
Biological sample handling and analysis.....................................................64
5.8.1.
Use of specified study materials ..................................................64
5.8.2.
Biological samples evaluation......................................................65
5.8.2.1.
Laboratory assays ......................................................65
5.8.2.2.
Laboratory read-outs ..................................................66
6.
SERIOUS ADVERSE EVENTS..............................................................................72
6.1.
Serious adverse events ..............................................................................72
6.1.1.
Definition of a serious adverse event ...........................................72
6.2.
Detecting and recording serious adverse events.........................................73
6.2.1.
Evaluation of serious adverse events related to a study 
procedure ....................................................................................73
6.2.2.
Prompt reporting of SAEs related to a study procedure ...............74
6.2.3.
Contact information for reporting serious adverse events 
and other events to GSK Biologicals............................................75
6.2.3.1.
Back-up system in case the electronic SAE 
reporting system does not work..................................75
6.2.3.2.
Updating of SAE information after freezing of 
the subject’s eCRF .....................................................76
6.2.4.
Regulatory reporting requirements for serious adverse 
events..........................................................................................76
6.3.
Follow-up of SAEs related to study procedure ............................................76
6.4.
Treatment of adverse events ......................................................................76
6.5.
Subject Cards.............................................................................................76
7.
SUBJECT COMPLETION AND WITHDRAWAL.....................................................77
7.1.
Subject completion .....................................................................................77
7.2.
Subject withdrawal......................................................................................77
7.3.
Subject replacement (Amended 16 October 2017) .....................................78
8.
DATA EVALUATION: CRITERIA FOR EVALUATION OF OBJECTIVES...............78
8.1.
Endpoints....................................................................................................78
8.1.1.
Primary endpoint..........................................................................78
8.1.2.
Secondary endpoints ...................................................................78
8.1.3.
Tertiary endpoints (Amended 16 October 2017) ..........................79
8.2.
Sample size consideration ..........................................................................79
8.2.1.
Assumptions:...............................................................................79
8.2.2.
Estimating design effect and precision.........................................80
8.2.3.
Expected precision of incidence rate estimates............................81
8.3.
Study cohorts to be evaluated.....................................................................86
8.3.1.
Total cohort..................................................................................86
8.3.2.
According-To-Protocol cohort ......................................................86
 
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8.4.
Conduct of analyses ...................................................................................86
8.4.1.
Sequence of analyses..................................................................86
8.5.
Statistical methods......................................................................................86
8.5.1.
Analysis of demographics/baseline characteristics ......................86
8.5.2.
Analysis of primary and secondary endpoints..............................87
8.5.3.
Analysis of symptoms and spectrum of dengue disease..............89
8.5.4.
Analysis of tertiary objectives (Amended 16 October 2017) .........89
8.5.5.
Statistical considerations for interim analyses..............................90
9.
ADMINISTRATIVE MATTERS ...............................................................................90
9.1.
Remote Data Entry instructions ..................................................................91
9.2.
Monitoring by GSK Biologicals....................................................................91
9.3.
Archiving of data at study sites ...................................................................92
9.4.
Audits .........................................................................................................93
9.5.
Posting of information on public registers....................................................93
9.6.
Ownership, confidentiality and publication ..................................................93
9.6.1.
Ownership ...................................................................................93
9.6.2.
Confidentiality ..............................................................................93
9.6.3.
Publication...................................................................................94
9.6.4.
Provision of study results to investigators and publication............94
10. COUNTRY SPECIFIC REQUIREMENTS...............................................................94
11. REFERENCES (AMENDED 16 OCTOBER 2017)..................................................95
 
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LIST OF TABLES
PAGE
Table 1
Study groups and epochs foreseen in the study.....................................34
Table 2
List of study procedures for initial cohort subjects (Amended 16 
October 2017)........................................................................................41
Table 3
List of study procedures for expansion cohort subjects (Amended 
16 October 2017) ...................................................................................44
Table 4
List of study procedures for replacement subjects (Amended 16 
October 2017)........................................................................................46
Table 5
Intervals between study visits/contacts for INITIAL cohort......................48
Table 6
Intervals between study visits/contacts for EXPANSION cohort 
(Amended 16 October 2017)..................................................................48
Table 7
Interval between visits for suspected dengue/chikungunya/Zika
case (Amended 16 October 2017) .........................................................48
Table 8
Humoral Immunity (Amended 16 October 2017) ....................................65
Table 9
Virology (Amended 16 October 2017) ....................................................65
Table 10
Haematology..........................................................................................66
Table 11
Laboratory read-outs at each time point, and priority ranking for 
scheduled visits (Amended 16 October 2017)........................................67
Table 12
Laboratory read-outs at each time point, and priority ranking for 
suspected dengue, chikungunya and/or Zika visits for EARLY 
presenters (Amended 16 October 2017) ................................................69
Table 13
Laboratory read-outs at each time point, and priority ranking for 
suspected dengue, chikungunya and/or Zika visits for LATE 
presenters..............................................................................................71
Table 14
Timeframes for submitting SAE reports to GSK .....................................74
Table 15
Precision of the expected incidences of dengue for a single 
cohort of subjects in Brazil .....................................................................82
Table 16
Numerators and Denominators within each strata (DENV type, 
study site, gender and age-group and previous dengue exposure 
(primary or secondary) ...........................................................................88
 
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LIST OF FIGURES
PAGE
Figure 1
Schematic representation of the scheduled visits...................................34
Figure 2
Active tracking algorithm ........................................................................54
Figure 3
Classification of dengue cases...............................................................56
 
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LIST OF APPENDICES
PAGE
APPENDIX A STUDY LABORATORIES (Amended 16 October 2017).........................97
APPENDIX B STRUCTURED SCRIPT ........................................................................98
APPENDIX C AMENDMENTS AND ADMINISTRATIVE CHANGES TO THE 
PROTOCOL...........................................................................................99
 
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LIST OF ABBREVIATIONS
ATP
According-To-Protocol
CBC
Complete blood count
CI
Confidence interval
CRADA
Cooperative Research and Development Agreement
DENV
Dengue virus 
eCRF 
electronic Case Report Form 
ELISA
Enzyme-linked Immunosorbent Assay
FHP
Family Health Physician Program
Fiocruz
Fundação Oswaldo Cruz 
GCP
Good Clinical Practice
GSK
GlaxoSmithKline
HCT
Hematocrit
ICF
Informed Consent Form
ICH
International Conference on Harmonization
IEC
Independent Ethics Committee
IgG
Immunoglobulin type G
IgM
Immunoglobulin type M 
IRB
Institutional Review Board
LIRA
Larval Index Rapid Assay 
LAR
Legally Acceptable Representative
MoH
Ministry of Health
NS1
Non Structural 1
RDE
Remote Data Entry
RT-qPCR
Reverse Transcriptase quantitative Polymerase Chain Reaction
SAE
Serious Adverse Event
SDV
Source Document Verification
SPM
Study Procedures Manual
WHO
World Health Organization
YF
Yellow Fever
 
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GLOSSARY OF TERMS
Anonymization:
Information that identifies a specific individual 
(including, but not limited to name, address and national 
identification number such as social security number, date 
of birth) has been removed and no link to the donor, 
through a code number for example, is maintained.
Block
A group of dwellings (buildings or residential units); 
dwellings may include one or more households. Blocks 
are used as sampling units in the LIRA strategy and serve 
as unit for spatial analysis. 
Child in care:
A child who has been placed under the control or 
protection of an agency, organization, institution or entity 
by the courts, the government or a government body, 
acting in accordance with powers conferred on them by 
law or regulation. The definition of a child in care can 
include a child cared for by foster parents or living in a 
care home or institution, provided that the arrangement 
falls within the definition above. The definition of a child 
in care does not include a child who is adopted or has an 
appointed legal guardian.
Coded:
Information is associated with a subject number i.e. a 
code number. Coded information can only be linked back 
to the individual via a key code i.e. a listing of the 
research participants and their code. Within the 
pharmaceutical industry coding data is the usual 
mechanism used for protecting an individual’s research 
data. The key code is kept secure, usually by the 
investigator, and GSK researchers cannot identify the 
research individual other than in exceptional and 
controlled circumstances.
Cohort study:
A form of epidemiology 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).
 
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Diary log:
A diary log will be used in this study. The diary log is 
given to subjects to record body temperatures and 
symptoms in the event that a suspected dengue, Zika 
and/or chikungunya symptom occurs (see Section 
5.7.1.1). The information collected in the diary log will be 
provided to the study physician as a tool in the medical 
evaluation of the subject. If the physician considers that 
the subject meets the criteria for suspected dengue, Zika 
and/or chikungunya he/she will use the information 
contained in the diary log to complete the description of 
first clinical symptoms in the eCRF.
Early presenter
A suspected dengue, Zika and/or chikungunya case 
presenting at the health care facility within 5 days 
following the onset of fever and/or rash (for Zika virus).
Eligible:
Qualified for enrolment into the study based upon strict 
adherence to inclusion/exclusion criteria.
Epidemiology study:
An observational study or an interventional study without 
administration of medicinal product(s) as described in a 
research protocol.
Epoch:
An epoch is a well-defined part of a protocol that covers a 
set of consecutive time points. Generally, an epoch is
self-contained and allows to perform a data analysis to 
address some of the trial objectives (e.g. primary, booster, 
yearly follow-ups, retrospective data collection, 
prospective data collection).
eTrack:
GSK’s tracking tool for clinical/epidemiology trials.
Evaluable:
Meeting all eligibility criteria, complying with the 
procedures defined in the protocol, and, therefore, 
included in the according-to-protocol (ATP) analysis (see 
Section 8.3 for details on criteria for evaluability).
Expansion cohort subjects
Subjects recruited in the additional sites for study 
expansion
Initial cohort subjects
Subjects enrolled at the start of the study.
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.
 
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Late presenter
A suspected dengue, Zika and/or chikungunya case 
presenting at the health care facility 6 to 30 days after the 
onset of fever and/or rash (for Zika virus).
Lost to follow-up
A subject who moved outside study area or cannot be 
reached after several attempts and cannot perform the 
scheduled visit within the low dengue season period.
Prematurely terminate 
participation
A subject who is withdrawn from the study by the 
investigator or who decides to stop participation before 
the time he/she had to be in the study.
Primary dengue infection
The first dengue infection experienced by a subject is 
qualified as primary while subsequent infections (second, 
third or fourth) are qualified as secondary.
Prospective study:
A study in which the subjects/cases are identified and 
then followed forward in time in order to address one or 
more study endpoints
Replacement subjects
Subjects who will be enrolled to compensate for subjects:

from the initial cohort: who will not want to extend
participation for three additional years or

from the initial and expansion cohorts: who 
prematurely terminate participation or subjects lost to 
follow-up. 
Research protocol:
A document that describes the objective(s), design, 
methodology, statistical considerations, and organization 
of a trial. The protocol usually also gives the background 
and rationale for the trial, but these could be provided in 
other protocol referenced documents
Seroconversion: 
Documented positive antibody test with previous 
documented negative antibody test.
Site Monitor:
An individual assigned by the sponsor who is responsible 
for assuring proper conduct of clinical/ epidemiology 
studies at one or more investigational sites
Study population:
Enrolled members of a sample of the population of 
interest
 
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Subject:
Term used throughout the protocol to denote an 
individual who has been contacted in order to participate 
or participates in the clinical/ epidemiology study, or a 
person about whom some medical information has been 
recorded in a database
Subject number:
A unique number identifying a subject, assigned to each 
subject consenting to participate in the study
Surveillance:
Surveillance is defined as the on-going 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 (Amended 16 October 2017)
Dengue, the most common arthropod-borne viral disease worldwide, is caused by four 
types of dengue viruses (DENV 1-4), transmitted primarily by Aedes aegypti, a mosquito 
that is highly adapted to urban environments. Dengue infection can cause a range of 
clinical illnesses, from inapparent to a life threatening hemorrhagic disease, often 
associated with pre-existing heterotypic dengue virus antibodies. 
An estimated 2.5 billion people are at risk of infection in tropical and subtropical 
countries worldwide (WHO, 2009) where major urban epidemics are responsible for 
substantial social and economic burden. Dengue has drastically increased in the last two 
decades in Central and South America with Brazil being one of the most affected 
countries (Siqueira-Junior, 2008). In Central Brazil, an almost 20-fold increase in 
reported cases of dengue have been reported from 1990 (1,660 cases) to 2000 (20,552 
cases) (Siqueira, 2004). In all of Brazil, the number of yearly reported cases increased 
from 124,827 in 1995 to more than 1 million in 2010 (PAHO, 2011)
Currently, strategies to reduce disease burden rely mainly on mosquito control and 
human preventive behaviour modification, as treatment is limited to supportive care. The 
World Health Organization (WHO) has, therefore, considered the development of a 
vaccine a priority research area (Brandt, 1990), and a number of vaccine candidates are 
under development or being tested in clinical trials (Thomas, 2011).
In preparation for phase III trials, the WHO recommends that the primary efficacy 
endpoint be the presence of DENV in a patient with signs and/or symptoms of dengue 
disease (WHO, 2009; Edelman, 2008). However, mild cases of dengue are largely 
underreported, as shown in dengue cohort studies and capture-recapture studies 
(Wichmann, 2011; Vong, 2011; Suaya, 2007). For example, in Nicaragua an active 
dengue surveillance system in children was able to detect approximately 14 to 28 
(average 21.3) times more dengue cases each year per 100,000 persons than passive 
surveillance among similar paediatric populations (Standish, 2010). 
Zika is a flavivirus and chikungunya virus is a member of the alphavirus genus, and 
Togaviridae family. These are emerging mosquito-borne viral diseases in Latin 
America. Zika virus infections have been laboratory confirmed in 19 states in Brazil 
between April and December 2016 [Boletim Epidemiológico, 2017]. In 2016, until 
epidemiological week 52, 215,319 cases were reported and 130,701 were confirmed 
[Boletim Epidemiológico, 2017]. 
In 2015, 20,661 autochthonous suspected chikungunya cases were reported in Brazil 
[Boletim Epidemiológico, 2017]. In 2016, 271,824 reported cases and 151,318 were 
confirmed up to epidemiological week 47 [Boletim Epidemiológico, 2017]. Zika and 
chikungunya viruses cause symptoms often similar to dengue during the acute phase 
of illness.
 
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Another challenge when conducting vaccine trials is to select geographic settings where 
the incidence of dengue would allow for a reliable assessment of vaccine efficacy.
Endemicity for more than one dengue virus type is also highly desirable. Thus, selection 
of geographically diverse sites can potentially maximize the likelihood of having a 
sufficient number of exposed individuals for the conduct of a vaccine trial. Finally, 
identifying all other coexisting flaviviruses circulating at potential study sites is 
important, as infection by such viruses might, in theory, modulate immune response and 
clinical course of dengue infection. 
In this study, we will estimate the incidence of dengue infection and disease in a cohort 
of subjects recruited from different geographic areas over time in Brazil. The study will 
also identify and train potential sites for the conduct of phase III studies in the future.
1.2.
Rationale for the study
The estimation of  dengue incidence is critical when designing an efficacy trial for a 
dengue vaccine. In a given city or region, dengue incidence is highly variable from one 
year to another (variations in geography, season, year-to-year variations in mosquito 
vector populations in different areas, etc.,) and cannot yet be predicted with sufficient 
accuracy in many locations being considered for clinical trials. In a dengue vaccine 
efficacy trial, volunteers are recruited from geographically defined sites. Some of these 
sites may experience high dengue transmission during the trial duration while others may 
have limited transmission. Therefore, making a valid assumption regarding the average 
incidence over time and across sites in order to assess the efficacy of an investigational 
vaccine is important.
In addition, a critical factor for the success of an efficacy trial is training staff at the 
various sites to perform study procedures such as febrile illness detection, dengue 
laboratory diagnosis and data collection in a cohort of subjects.
The proposed study will estimate the incidence of dengue infection and disease across 
geographically distinct locations in order to validate attack rate assumptions in future 
efficacy trials. We will enrol individuals from a random sample of households from 
selected communities of geographically different sites in Brazil and assess incidence of 
dengue infection and disease, by conducting scheduled serosurveys and an enhanced 
surveillance of febrile disease.
This study will also prepare potential sites for future clinical trials by setting up the 
logistics and training staff on site to enrol a cohort of subjects, perform dengue 
surveillance and other study procedures. 
Finally, in line with the overarching strategy of building a strong partnership with the 
Ministry of Health (MoH), this study will serve as a useful comparison for evaluation of 
dengue surveillance systems
 
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2.
OBJECTIVES
2.1.
Primary objective

To estimate the incidence of laboratory-confirmed symptomatic dengue infection in 
the study population by year/season.
Refer to Section 8.1.1 for the primary endpoint.
2.2.
Secondary objectives

To estimate the serotype-specific incidence of virologically-confirmed symptomatic 
dengue infection in the study population overall and by season.

To estimate the incidence of symptomatic dengue infection (including laboratory-
confirmed and probable cases) in the study population by study site, gender, age-
group, previous dengue exposure (primary or secondary), overall and by season.

To estimate the prevalence of previous dengue infection (dengue seroprevalence) in 
the study population overall and by study site, gender and age-group at enrolment.

To estimate the incidence of primary inapparent, dengue infection, in the study 
population overall and by study site, gender age-group and by season.

To describe symptoms and spectrum of dengue disease in the study population.
Refer to Section 8.1.2 for the secondary endpoints.
2.3.
Tertiary objectives (optional) (Amended 16 October 2017)

To investigate potential risk factors for symptomatic dengue infection.

To investigate the role of dengue neutralizing antibodies elicited through natural 
infection in protection against subsequent infection.

To estimate the degree of underreporting of dengue cases using the passive national 
notification surveillance.

To estimate the incidence of symptomatic Zika virus infection (including 
laboratory-confirmed and probable cases) in the study population by study site, 
gender, age-group, previous flavivirus exposure, overall and by season.

To estimate the incidence of symptomatic chikungunya virus infection (including 
laboratory-confirmed and probable cases) in the study population by study site, 
gender, age-group, overall, and by season.

To describe previous Yellow Fever (YF) antibody profile in selected dengue cases to 
investigate the role of previous YF immunity on the dengue neutralizing antibody 
profile following dengue infection.

To describe the spatial and temporal distribution of dengue cases among cohort 
participants in the study areas.
 
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
To describe the spatial and temporal distribution of Zika and chikungunya cases 
among cohort participants in the study areas.

To describe other infectious aetiologies related to differential diagnosis of dengue 
(chikungunya and Zika) in subjects with episodes of febrile illness referred to as 
“suspected dengue case”.

To estimate the seroprevalence of antibodies against Zika and chikungunya virus 
at selected timepoints.
Refer to Section 8.1.3 for tertiary endpoints
3.
STUDY DESIGN OVERVIEW (AMENDED 16 OCTOBER 
2017)

Type of design: Prospective, multicenter, community-based, household-sampling, 
cohort study in Brazil.

The study period, initially planned to be one year, has been extended by additional
three years (overall four years) to cover three additional dengue seasons
(Amendment 2).

Initial cohort subjects were invited to extend their participation. 

(See glossary of terms for the definition of initial and expansion cohort subjects).

In each cohort: replacement subjects may be enrolled in order to compensate for 
subjects who did not wish to extend their participation for three additional years (for 
subjects from the initial cohort – Amendment 2), subjects who prematurely terminate 
participation or subjects who are lost to follow-up. 
This will be done to maintain a cohort size of at least 500 subjects per site, in the 
Initial and Expansion cohorts, at the beginning of each additional study year/season.
In last three months of the of penultimate year of the study, each site will review 
the number of active subjects and drop-out rate.  If the risk of having fewer than 
the required number of active participants at the end of the study is high, 
recruitment for participants into the replacement cohort may be performed with 
sponsor approval. If the risk is high, recruitment into the replacement cohort 
should begin even if the number of active subjects is 500 or above 500.
(See glossary of terms for the definition of replacement subjects, subjects lost to 
follow-up and subjects who prematurely terminate participation ).

Informed consent (and assent, if applicable) will be obtained:

From the subject (and parents/LAR of the subjects,  if applicable) at the study 
start for the initial cohort.

From the subject (and parents/LAR of the subjects,  if applicable) of the initial 
cohort prior to participation in the additional three years of the study.

From the subject (and parents/LAR of the subjects,  if applicable) at the study 
start for the expansion cohort. 
 
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
From the subject (and parents/LAR of the subjects,  if applicable) prior to 
enrolment in the study for the replacement subjects.

Study population: Subjects six months of age and older at the time of enrolment, 
who either live in households in study areas with support from the Family Health 
Physician Program (FHP) or the Larval Index Rapid Assay (LIRA) or with field 
research experience in the community (preferred) or where a similar system of 
mapped communities with potential for surveillance exists.

There will be 2 waves of enrolment for recruiting 3600 subjects:

Initial Cohort = about 1800 subjects – up to 6 visits (Amendment 2) – INITIAL 
sites

Expansion Cohort = about 1800 subjects – up to 4 visits – NEW sites

Study visits: The visits will be as follows and further shown in Figure 1:

Initial cohort subjects consenting to extend participation for the three additional 
years will have:
o
three scheduled visits in the first year (Visit 1, Visit 2 and Visit 3) at 
approximately six months intervals (+/- 28 days), 
o
and thereafter, one scheduled visit per year preferably during a period of 
low dengue transmission for the three additional years; i.e., one visit at 
study Year 2, Year 3 and Year 4 (Visit 4, Visit 5 and Visit 6 respectively).

Initial cohort subjects who do not consent to extend participation for three 
additional years will:
o
have three scheduled visits (Visit 1, Visit 2 and Visit 3) at approximately 
six months intervals (+/- 28 days). 
o
conclude their participation in the study at Visit 3 or the last follow up 
visit if the subject is suspected of having a dengue case on-going at Visit 
3.

Subjects from the expansion cohort will have only three years of follow-up, with 
at most 4 scheduled visits.

For replacement subjects, the number of scheduled visits will depend on the 
study year in which they are enrolled. Replacement subjects will not be enrolled 
until after the third visit of the first study year is concluded for the initial cohort. 
The number of scheduled visits will range between two and four.
The yearly scheduled visits for the initial and expansion cohorts and 
replacement subjects will preferably occur during the period of usually low 
dengue transmission.
In last three months of the of penultimate year of the study, each site will 
review the number of active subjects and drop-out rate.  If the risk of having 
fewer than the required number of active participants at the end of the study is 
high, recruitment for participants into the replacement cohort may be 
performed with sponsor approval. If the risk is high, recruitment into the 
 
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replacement cohort should begin even if the number of active subjects is 500 
or above 500.
Figure 1
Schematic representation of the scheduled visits
V=Visit
The scheduled visits for initial cohort subjects are shown in black.
New enrolments will be done yearly (as needed) till the end of study Year 3.
Enrollment of expansion cohort subjects will start preferably during study Year 2
For replacement subjects, the number of visits will depend on the year enrolled. 

Type of study: self-contained

Data collection: Electronic Case Report Form (eCRF )

Duration of the study: Four years for the overall study. For an individual subject, it 
will range between one and four years.  

Epoch 001: prospective data collection starting at Visit 1, Day 0 and ending at 
the last subject last visit .
Table 1
Study groups and epochs foreseen in the study
Study Group
Number of subjects*
Age 
(Minimum)
Epoch
Prospective study 
cohort
Initial Cohort:
1800 at enrolment (first year)
At least 1500 in each 
subsequent year 
6 months 
and older
Epoch 001 
Expansion Cohort: 
1800 at enrolment (first 
year).
At least 1500 in each 
subsequent year
*The final number of participants will take into account the active subjects, subjects who prematurely terminate 
participation and subjects lost to follow up.
 
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3.1.
Discussion of study design
3.1.1.
Selection of study sites
Selection of study sites
At least six sites covering different regions of the country will be selected for this study. 
Selection criteria of the sites include support from the Family Health Physician Program 
(FHP), the Larval Index Rapid Assay (LIRA) or with field research experience in the 
community. The FHP is a Brazilian government public health strategy that consists of 
multidisciplinary teams of health care workers that cover geographically defined areas to 
provide basic/preventive care to community members. The teams include a doctor, a 
nurse, a nurse auxiliary, and community health care workers who pay regular visits to the 
household under its responsibility to deliver health promotion activities and facilitate 
triage and referral into the health unit, among other activities. Community health care 
workers are local residents who are trained for basic tasks.
LIRA is an alternative vector surveillance strategy that consists of random sampling of a 
number of dwellings in which surveillance of Aedes-positive breeding places is carried 
out, and has been widely implemented in Brazil (Pontes, 2000). 
At least one community of households within each site will be selected.
3.1.2.
Rationale for study design
A single cohort study was chosen to estimate the incidence of dengue in the community. 
Data will be collected from a population of individuals aged 6 months and older for 
maximum four dengue seasons. Inclusion of individuals of various ages will further allow 
for a better understanding of disease dynamics, clinical spectrum, and validate attack 
rates for potential subgroups to be included in future dengue vaccine trials.
This study will use cluster (household) sampling to take into account the correlation that 
may exist within households. Individuals within a household are expected to have a more
similar risk of infection compared to individuals from different households. For example, 
individuals living in the same household are more likely to share similar risk of being 
exposed to DENV infected mosquitoes. The sample size has been adjusted to account for
this between-cluster variability.
A possible bias may arise from the non-response rate. To reduce the possibility of this 
potential bias, visits will be scheduled on weekends or at times more convenient to the 
subject. Where applicable, FHP community health care workers will facilitate 
communication between study staff and participants.
 
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4.
STUDY POPULATION
Subjects six months of age and older at the time of enrolment who live in the selected 
study sites in Brazil, covering different regions of the country. 
4.1.
Selection of communities
This study plans to enrol a sample of subjects from randomly selected households from 
different communities within the selected sites. Community selection is based on the 
following characteristics: 

preferably areas where the Brazilian FHP or LIRA has been fully implemented or 
where access to the community is already established by a government program, e.g., 
registry of families by Secretary of Health, or other institutional program, such as 
university, in order to allow for random sampling of households; 

safe access by study personnel; 

high population density; and 

low migration rate. 
4.2.
Selection of households
Households will be randomly selected through LIRA strategy or from a list provided by 
the FHP or equivalent. 
The FHP or LIRA will provide an exhaustive list of households to the study sites and
will serve as the liaison between the study personnel and the household, and will not have 
any other role in this study.
Sites with access to LIRA will use LIRA’s specific sampling strategy. LIRA’s sampling 
strategy (preferred strategy) consists of dividing the administrative neighbourhoods of the 
city into blocks in which each building is enumerated by a unique code number. Within 
each block a corner is chosen and moving leftward, one in every four houses is 
systematically selected for vector inspection.
Where LIRA is not implemented but has FHP coverage, a random sample of households 
will be selected from the FHP database. 
Where another type of household registry offers the only access to the community, a 
random sample will be drawn from the registry database. 
Refer to Section 8.2 for a detailed description of the criteria used in the estimation of 
sample size. 
 
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4.3.
Overview of the recruitment plan
At least 3600 subjects will be recruited in the study in 2 waves of enrolment. Each wave, 
initial and expansion cohorts, will enrol about 600 subjects/site in order to maintain at 
least 500 subjects/site at the beginning of each dengue season.
Subjects will be recruited from randomly selected households originating from pre-
selected mapped communities. Preferably the recruitment period will occur outside of the 
peak dengue transmission season, and will continue until each site has reached its 
foreseen target. The expected period for recruiting the target sample size is approximately 
three months. Recruitment of replacement subjects will be done during the low dengue 
transmission and the recruitment period will depend on the number of subjects that need 
to be replaced.
Recruitment will be organized by study staff at participating sites according to the 
appropriate strategy for each site. Community agents or equivalent will serve as the 
liaison to schedule visits and may accompany study staff during the visits. The study will 
be explained to the individuals living in the household, and if any individual is interested 
in study participation, informed consent (and assent when applicable) will be obtained, 
eligibility criteria will be checked and subjects will be enrolled and interviewed for 
potential study participation.
If the target household is found empty or all members refuse to participate, the first 
household to the left will be approached. Those households refusing to participate will be 
recorded as such. A household refusal will be characterized when all individuals in the 
household refuse to participate in the study. Individual refusals in a given household will 
not preclude inclusion of other individuals living in the households, and will be recorded 
as such.
In the subsequent study years/seasons, the cohort size will be maintained at a minimum 
of 500 subjects per site. Since subjects who prematurely terminate participation and/or
lost to follow-up will be replaced, the final number of participants across the four study 
years may exceed 3600 subjects. Yearly, there will be an evaluation of active subjects. If 
the number of active subjects/site becomes < 500, there will be enrolment of replacement 
subjects to maintain a cohort size of at least 500 subjects/site. Enrolment will preferably 
take place during the low dengue transmission. Recruitment of replacement subjects 
could occur at the end of study year 1, 2 or 3. The recruitment approach will be the same 
as for the initial enrolment.
In order to better represent the Brazilian population distribution, subjects enrolled in the 
expansion cohort should be composed of at least 30% of subjects younger than 18 years 
old and should not exceed 20 % of adults 50 years or older. Subjects enrolled as 
replacement subjects for the initial and expansion cohort will be recruited to approach as 
much as possible a minimal representation of 30% of subjects younger than 18 years old 
and a maximal representation of 20% for adults 50 years or older.
Household visits may be scheduled to occur during weekends or at convenient times (like 
evenings) if deemed necessary.
 
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4.4.
Inclusion criteria for enrolment (Amended 16 October 2017)
All subjects must satisfy ALL the following criteria at study entry.

Written, signed or thumb-printed informed consent (and assent when applicable) 
must be obtained from the subject or subject’s parent(s)/LAR(s). If the 
subject/subject’s parent(s)/LAR(s) are illiterate the consent form will be 
countersigned by a witness.

Male or female at least 6 months of age at the time of enrolment.

Subject and/or subject’s parent(s)/LAR(s) who the study staff believes can comply 
with the requirements of the protocol (e.g., willingness to go to the hospital/clinic for 
visit/s if dengue, Zika and/or chikungunya is suspected, able to observe the signs of 
dengue, Zika and/or chikungunya and to understand how to take and report body 
temperature, etc.).

Subject who plans, at the time of enrolment, to remain at same residence/study area 
during their study participation period.
4.5.
Exclusion criteria for enrolment
The following criteria should be checked at the time of study entry. If ANY exclusion 
criterion applies, the subject must not be included in the study.

Child in care. (see the glossary for full definition)

Participation (current or planned) in another epidemiological study or in a clinical 
trial that would conflict with the current study, based on investigator’s judgement.

Terminal illness or severe mental incapacity.
 
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5.
STUDY CONDUCT 
5.1.
Regulatory and ethical considerations, including the 
informed consent process
The study will be conducted in accordance with all applicable regulatory requirements.
The study will be conducted in accordance with the ICH Guideline for Good Clinical 
Practice (GCP), any applicable local guidelines, including the Document of the Americas, 
all applicable subject privacy requirements and the guiding principles of the Declaration 
of Helsinki. 
The study has been designed and will be conducted in accordance with the ICH 
Harmonized Tripartite Guideline for clinical investigation of medicinal products in the 
paediatric population (ICH E11) and all other applicable ethical guidelines.
GSK will obtain favourable opinion/approval to conduct the study from the appropriate 
regulatory agency, in accordance with applicable regulatory requirements, prior to a site 
initiating the study in that country or will document that neither a favourable opinion nor 
an approval to conduct the study is needed.
Conduct of the study includes, but is not limited to, the following:

Institutional Review Board (IRB)/Independent Ethics Committee (IEC) review and 
favourable opinion/approval of study protocol and any subsequent amendments

Subject/ subject’s parent(s)/legally acceptable representative (LAR[s])  informed 
consent and subject informed assent, as appropriate

Investigator reporting requirements as stated in the protocol.
GSK will provide full details of the above procedures to the investigator, either verbally, 
in writing, or both.
Freely given and written informed consent must be obtained from each subject and/or 
each subject’s parent(s)/LAR(s) and subject informed assent as appropriate, prior to 
participation in the study. 
GSK Biologicals will prepare a model Informed Consent Form (ICF) which will embody 
the applicable ICH GCP 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 Harmonized Tripartite Guidelines for Good Clinical Practice, 
those subjects who can only be enrolled in the study with the consent of the subject’s 
parent(s)/LAR(s) (e.g. minors), should be informed about the study to the extent 
 
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compatible with the subject’s understanding and, if capable, the subject should sign and 
personally date a written informed assent. It is required to be signed by each subject, if 
capable, in addition to the informed consent that is to be signed by his/her parent or legal 
representative. Requirement of assent should be assessed based on the age of the study 
population and the local requirements.
GSK Biologicals strongly recommends that if the subject reaches the age of consent 
during the study they will be asked to provide consent at the next study visit (if 
applicable). This procedure should be applied according to local laws and regulations.
If a subject is unable to read or if a legally acceptable representative is unable to read, the 
informed consent will be obtained in the presence of an impartial witness, according to 
the International Conference on Harmonization (ICH) guidelines. For all participants 
unable to read for whatever reason, the subject’s fingerprint must be obtained on the ICF. 
The investigator has the final responsibility for the final presentation of the ICF, 
respecting the mandatory requirements of local regulations. The ICF generated by the 
investigator with the assistance of the sponsor’s representative must be acceptable to 
GSK Biologicals and be approved (along with the protocol, and any other necessary 
documentation) by the IRB/IEC.
5.2.
Subject identification
Subject numbers will be assigned sequentially to subjects consenting to participate/to be 
included in the study, according to the range of subject numbers allocated to the/each 
study centre. Household numbers will also be assigned.
5.3.
General study aspects
Supplementary study conduct information not mandated to be present in this protocol is 
provided in the accompanying Study Procedures Manual (SPM). The SPM provides the 
investigator and the site personnel with administrative and detailed technical information 
that does not impact the safety of the subjects.
 
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5.4.
Outline of study procedures
Study procedures are outlined in Table 2 and Table 3 for initial and expansion cohorts, respectively in Table 4 for replacement subjects.
Table 2
List of study procedures for initial cohort subjects (Amended 16 October 2017)
Surveillance
Suspected 
Dengue/Zika/Chikungunya
Visits (at hospital/clinic)
as applicable
Procedure
Visit 1 
Day 0
Monthly 
contact 
between 
visits
Visit 2 
Month 6
Monthly 
contact 
between 
visits
Visit 3
Month 
12 (Year 
1)
Monthly 
contact 
between 
visits
Visit 4
Month 
24 (Year 
2)
Monthly 
contact 
between 
visits
Visit 5
Month 
36 (Year 
3)
Monthly 
contact 
between 
visits
Visit 6
Month 
48 (Year 
4)
First visit
Day 0
Return 
Visit 2
Follow-
up visit
Day 21
Informed consent (and assent if 
applicable)
●
Re-consent (and assent if applicable)8
●
●
●
●
●
●
●
●
Subject number and initial household 
number attribution
●
Check inclusion/ exclusion criteria
●
Record socio-demographic information
or updates (including household 
characteristics if applicable)
●
●
●
●
Medical history including YF and 
dengue vaccination history or updates
●
●
●
●
●
●
Distribute subject ID card distribution 
and suspected dengue instruction kit
O
Blood sample for serology (5 mL)
●
●
●
●
●
●
Instruct/remind subjects/subject's 
parent(s)/LAR(s) on suspected 
dengue, chikungunya and Zika
assessment procedures (where 
applicable)
O
O
O
O
O
O
O
O
O
O
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Surveillance
Suspected 
Dengue/Zika/Chikungunya
Visits (at hospital/clinic)
as applicable
Procedure
Visit 1 
Day 0
Monthly 
contact 
between 
visits
Visit 2 
Month 6
Monthly 
contact 
between 
visits
Visit 3
Month 
12 (Year 
1)
Monthly 
contact 
between 
visits
Visit 4
Month 
24 (Year 
2)
Monthly 
contact 
between 
visits
Visit 5
Month 
36 (Year 
3)
Monthly 
contact 
between 
visits
Visit 6
Month 
48 (Year 
4)
First visit
Day 0
Return 
Visit 2
Follow-
up visit
Day 21
Issue diary logs to parents/LARs to be 
filled out in the event of a suspected 
dengue, chikungunya and Zika case1
O
O
O
O
O
O
Contact subject regarding any dengue, 
chikungunya and Zika symptoms 
and remind subject of procedures for 
suspected dengue, chikungunya and 
Zika
O
O
O
O
O
Physical examination/record current 
medical conditions(see Section 5.6)
●
●
●
Blood sample for 
dengue/zika/chikungunya infection
differential diagnosis3
●
●
Blood sample for CBC and hematocrit
(mandated for subjects presenting 
within 14 days following symptoms6
onset)
●
Record body temperature
●
Report SAEs related to study 
procedures4
●
●
●
●
●
●
●
●
●
Collect or verify diary logs if applicable
O
O
O
Study conclusion5
●
●
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● 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.
1 Diary logs given at scheduled visits are only to be filled out in the event that the subject has  fever and/or rash according to the case definition for suspected 
dengue/chikungunya/Zika. See Section 5.5.2.5 for details with regard to the diary log.
2 A return visit is a visit linked to suspected dengue/chikungunya/Zika, occurring if the evolution of the subject’s physical condition necessitates medical evaluation, according to local 
medical practice. The investigator will determine whether this return visit is necessary and whether it is considered as part of the same episode. See Section 5.6 for details.
3Blood sampling for dengue diagnosis which includes testing for RT-qPCR, dengue IgG and IgM ELISA, NS1 ELISA, neutralizing antibodies to dengue and virological/humoral,
testing for Zika and chikungunya infection.
4 SAE = Serious adverse event. See Section 6.1.1 for definition.
5 The study conclusion information in the eCRF is collected once for each subject at the last scheduled visit or at the last follow-up contact for a suspected dengue/zika/chikungunya
case, should this case occur during the last scheduled visit or earlier if the subject terminates study participation or is lost to follow-up.
6Symptoms are fever for dengue, Zika and chikungunya, and rash for Zika.
8The field staff will re-consent at the first opportunity
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Table 3
List of study procedures for expansion cohort subjects (Amended 16 October 2017)
Surveillance
Suspected 
Dengue/Zika/Chikungunya Visits (at 
hospital/clinic)
as applicable
Procedure
Visit 1 # 
Day 0
Monthly 
contact 
between 
visits
Visit 2
Monthly 
contact 
between 
visits 
Visit 3
Monthly 
contact 
between 
visits
Visit 4
First visit
Day 0
Return visit 2 Follow-up 
visit21
Informed consent (and assent if applicable)
● 
Re-consent (and assent if applicable)@
●
●
●
●
●
●
Subject number and initial household 
number attribution 
● 
Check inclusion/ exclusion criteria 
● 
Record socio-demographic information or 
updates (including household 
characteristics if applicable)
● 
●
●
●
Medical history, including YF and dengue
vaccination history or updates 
● 
● 
● 
● 
Distribute subject ID card distribution and 
suspected dengue instruction kit 
O 
Blood sample for serology (5 mL)
● 
● 
● 
● 
Instruct/remind subjects/subject's 
parent(s)/LAR(s) on suspected dengue, 
chikungunya and Zika assessments
procedures (where applicable)
O 
O 
O 
O
O
O 
Issue diary logs to parents/LARs to be filled 
out in the event of a suspected dengue, 
chikungunya and Zika case 1
O
O
O
O
Contact subject regarding any dengue 
chikungunya and Zika symptoms and 
remind subject of procedures for suspected 
dengue, chikungunya and Zika
O
O
O
Physical examination/record current 
medical conditions (see Section 5.6)
●
●
●
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Surveillance
Suspected 
Dengue/Zika/Chikungunya Visits (at 
hospital/clinic)
as applicable
Procedure
Visit 1 # 
Day 0
Monthly 
contact 
between 
visits
Visit 2
Monthly 
contact 
between 
visits 
Visit 3
Monthly 
contact 
between 
visits
Visit 4
First visit
Day 0
Return visit 2 Follow-up 
visit21
Blood sample for dengue,
Zika/chikungunya infection differential 
diagnosis3
●
●
Blood sample for CBC and hematocrit 
(mandated for subjects presenting 
within 14 days following symptoms6
onset)
●
Record body temperature 
●
Report SAEs related to study procedures 4
●
●
●
●
●
●
●
Collect or verify diary logs if applicable 
O
O
O
Study conclusion 5
●
●
● 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.
# Enrolment of expansion cohort subjects will start preferably during study Year 2.
1 Diary logs given at scheduled visits are only to be filled out in the event that the subject has fever and/or rash according to the case definition for suspected 
dengue/chikungunya/Zika. 
2 A return visit is a visit linked to suspected dengue/Zika/chikungunya, occurring if the evolution of the subject’s physical condition necessitates medical evaluation, according to local 
medical practice. The investigator will determine whether this return visit is necessary and whether it is considered as part of the same episode. See Section 5.6 for details.
3 Blood sampling for dengue diagnosis which includes testing for RT-qPCR, dengue IgG and IgM ELISA, NS1 ELISA, neutralizing antibodies to dengue and virological/humoral, 
testing for Zika and chikungunya infection.
4 SAE = Serious adverse event. See Section 6.1.1 for definition.
5 The study conclusion information in the eCRF is collected once for each subject at the last scheduled visit or at the last follow-up contact for a suspected dengue/Zika/chikungunya
case, should this case occur during the last scheduled visit or earlier if the subject terminates study participation or is lost to follow-up. 
6Symptoms are fever for dengue, Zika and chikungunya, and rash for Zika.
@ The field staff will re-consent at the first opportunity
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Table 4
List of study procedures for replacement subjects (Amended 16 October 2017)
Surveillance
Suspected 
Dengue/Zika/Chikungunya Visits (at 
hospital/clinic)
as applicable
Procedure
Visit 1 
Day 0
Monthly 
contact 
Year 2
Visit 2 
Monthly 
contact 
between 
visits
Visit 3 as 
applicable
Monthly 
contact 
between 
visits
Visit 4 as 
applicable
First visit 
Day 0
Return visit2 Follow-up 
visit Day 21
Informed consent (and assent if applicable)
●
Re-consent (and assent if applicable)@
●
●
●
●
●
●
Subject number and initial household 
number attribution 
●
Check inclusion/ exclusion criteria 
●
Record/ update socio-demographic 
information or updates (including 
household characteristics if applicable)
●
●
●
●
Medical history, including YF and dengue
vaccination history or updates 
●
●
●
●
Distribute subject ID card distribution and 
suspected dengue instruction kit 
O
O
O
Blood sample for serology (5 mL) 
●
●
●
●
Instruct/remind subjects/subject's 
parent(s)/LAR(s) on suspected dengue, 
chikungunya and Zika assessment 
procedures (where applicable) 
O
O
O
O
O
O
Issue diary logs to parents/LARs to be filled 
out in the event of a suspected dengue, 
chikungunya and Zika case 1
O
O
O
O
Contact subject regarding any dengue,
chikungunya and Zika symptoms and 
remind subject of procedures for suspected 
dengue, chikungunya and Zika
O
O
O
Physical examination/record current 
medical conditions (see Section 5.6)
●
●
●
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Surveillance
Suspected 
Dengue/Zika/Chikungunya Visits (at 
hospital/clinic)
as applicable
Procedure
Visit 1 
Day 0
Monthly 
contact 
Year 2
Visit 2 
Monthly 
contact 
between 
visits
Visit 3 as 
applicable
Monthly 
contact 
between 
visits
Visit 4 as 
applicable
First visit 
Day 0
Return visit2 Follow-up 
visit Day 21
Blood sample for dengue, 
Zika/chikungunya infection differential 
diagnosis3
●
●
Blood sample for CBC and hematocrit 
(mandated for subjects presenting 
within 14 days following symptoms6
onset)
●
Record body temperature 
●
Report SAEs related to study procedures 4
●
●
●
●
●
●
●
Collect or verify diary logs if applicable 
O
O
O
Study conclusion 5
●
●
● 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.
1 Diary logs given at scheduled visits are only to be filled out in the event that the subject has a fever and/or rash according to the case definition for suspected dengue, 
Zika/chikungunya symptoms. See Section 5.5.2.5 for details with regard to the diary log. 
2 A return visit is a visit linked to suspected dengue/Zika/chikungunya, occurring if the evolution of the subject’s physical condition necessitates medical evaluation, according to local 
medical practice. The investigator will determine whether this return visit is necessary and whether it is considered as part of the same episode. See Section 5.6 for details.
3 Blood sampling for dengue diagnosis which includes testing for RT-qPCR, dengue IgG and IgM ELISA, NS1 ELISA, neutralizing antibodies to dengue and virological/humoral, 
testing for Zika and chikungunya infection.
4 SAE = Serious adverse event. See Section 6.1.1 for definition.
5 The study conclusion information in the eCRF is collected once for each subject at the last scheduled visit or at the last follow-up contact for a suspected dengue/Zika/chikungunya
case, should this case occur during the last scheduled visit or earlier if the subject terminates study participation or is lost to follow-up. 
6Symptoms are fever for dengue, Zika and chikungunya, and rash for Zika.
@ The field staff will re-consent at the first opportunity
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Table 5
Intervals between study visits/contacts for INITIAL cohort 
Study Year*
Visits Interval
Optimal length of interval *
Allowed interval**
Year 1
Visit 1 (Day 0) Visit 2 (Month 6)
6 months 
± 28 days
Visit 2 (Month 6) Visit 3 (Month 12)
6 months
The interval may be adapted to 
allow scheduling the visit in a 
period of generally low dengue 
transmission
Year 2
Visit X Visit X+1¶
Note: Visit X is visit 3 for subjects 
enrolled in study year 1. For subjects 
enrolled in study year 2, visit X would 
be visit 1.
12 months 
The interval may be adapted to 
allow scheduling the visit in a 
period of generally low dengue 
transmission
Year 3
Visit X+1 Visit X+2
12 months 
The interval may be adapted to 
allow scheduling the visit in a 
period of generally low dengue 
transmission
Year 4
Visit X Visit X+3
12 months 
The interval may be adapted to 
allow scheduling the visit in a 
period of generally low dengue 
transmission
*Whenever possible the investigator should arrange study visits/contacts within this interval
**Subjects might not be eligible for inclusion in the ATP cohort for analysis if they make the study visit/contact outside 
this interval
Table 6
Intervals between study visits/contacts for EXPANSION cohort
(Amended 16 October 2017)
Interval
Optimal length of interval*
Allowed interval**
Visit 1 (Day 0)   Visit 2 (Year 1)
12 months
The interval may be adapted to allow scheduling the 
visit in a period of generally low dengue transmission
±28 days
Visit 2 (Year 1) Visit 3 (Year 2)
12 months
The interval may be adapted to allow scheduling the 
visit in a period of generally low dengue transmission
±28 days
Visit 3 (Year 2) Visit 4 (Year 3)
12 months
The interval may be adapted to allow scheduling the 
visit in a period of generally low dengue transmission
±28 days
*Whenever possible the investigator should arrange study visits/contacts within this interval
**Subjects might not be eligible for inclusion in the ATP cohort for analysis if they make the study visit/contact outside 
this interval
Table 7
Interval between visits for suspected dengue/chikungunya/Zika case
(Amended 16 October 2017)
Optimal length of time
Allowed interval
Suspected dengue/chikungunya/Zika
case: Interval between first and follow-
up visit 
21 days
+ 7 days
Note that this interval applies to all subjects enrolled regardless of the year of enrolment.
 
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5.5.
Detailed description of study procedures
5.5.1.
Procedures prior to study participation
See Section 4.2 for household selection
5.5.1.1.
Informed consent
Before performing any other study procedure, the signed/thumb printed informed consent 
of the subject or subject’s parent(s)/LAR(s) must be obtained. The signed/thumb printed 
assent form from any subject who is below the age of consent but at the age for assent 
should also be obtained in addition to the signed informed consent by his/her 
parent(s)/LAR(s) according to local rules and regulations. Refer to Section 5.1 for the 
requirements on how to obtain informed consent and assent, as appropriate.

Initial cohort subjects will be required to have a new consent/assent (where 
applicable) form signed for continuous participation in the study to allow coverage 
of three additional dengue seasons.

Consent/assent (where applicable) will also be obtained prior to participation for 
replacement subjects.
5.5.1.2.
Check inclusion and exclusion criteria 
Check all applicable inclusion and exclusion criteria as described in Sections 4.4 and 4.5
before enrolment. 
5.5.2.
Procedures at scheduled Visit 1 (enrolment visit) (Amended 16 
October 2017)
Ideally, Visit 1 (the enrolment visit) will be scheduled before the peak incidence of 
dengue based on previous years.
A subject number and initial household number will be attributed at Visit 1.
5.5.2.1.
Collect socio-demographic data
Socio-demographic data such as age, gender and household conditions will be recorded 
in the subject’s source document for subsequent recording in the eCRF. The geographical 
location of the household (block) will also be recorded in the eCRF.
 
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5.5.2.2.
Collect specific medical history data (Amended 16 October 2017)
A baseline medical history, including history of dengue infection, Zika and chikungunya 
infection, and YF and dengue vaccination history will be taken. Results will be 
recorded in the subject’s source document for subsequent recording in the eCRF.
YF and dengue vaccination status will be recorded, along with the source of the 
information (either written or oral record). A vaccination record, if available, is 
preferable but self-reported history will be recorded if the vaccination record is not 
available. 
Information on medical history (e.g., diabetes, cardiovascular diseases, asthma, cancers, 
hematologic diseases, genetic disorders) will be collected.  
5.5.2.3.
Collect  blood sample

A blood sample for serology will be collected from all enrolled subjects (see Section 
5.8 for details regarding sample collection). For children <2 years of age, the total 
amount of blood collected will not exceed 5 mL at any visit.
5.5.2.4.
Instruction on enhanced passive dengue, chikungunya and Zika
surveillance by the subject or subject’s parent(s)/LAR(s) (Amended 16 
October 2017)
Study personnel will train subjects/subject’s parent(s)/LAR(s) to recognize dengue, Zika
and chikungunya symptoms and will instruct them to contact the study personnel or 
come to a designated study hospital/clinic for medical evaluation within 5 days of the 
occurrence of fever and/or rash that may be associated with suspected dengue, 
chikungunya or Zika (defined in Section 5.7.1, 5.7.2 and 5.7.3).  
Each household will also be given at least one dengue kit, as applicable, which includes 
thermometers, study contact information (phone numbers) and an instruction card with 
information about dengue, Zika and chikungunya symptoms. This card will also provide 
instructions on who to contact and what to do if any of these infections are suspected.
Participants or LAR will be instructed on how to take body temperature and how to 
record the temperatures in a diary log. 
See Section 5.6 for details regarding management of suspected dengue, chikungunya
and Zika cases.
5.5.2.5.
Diary log distribution (Amended 16 October 2017)
A diary log will be given to all subjects in the household to be used in the event of 
suspected dengue, chikungunya and/or Zika illness. Diary logs will be distributed at the 
first scheduled visit and if they are needed, at subsequent visits (if the subject no longer 
has one, e.g., the previous one was lost or used).  A new diary log will be issued by the 
study staff whenever necessary.
 
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The subject will be instructed to start recording any symptoms, including rash and body 
temperature at any time dengue, chikungunya and/or Zika infections are suspected. 
Refer to Section 5.6.1 for details.
5.5.2.6.
Recording of serious adverse events related to a study procedure
The subject/subject’s parent(s)/LAR(s) will be instructed to contact the study staff should 
any serious adverse event related to a study procedure occur during the study. 
Refer to Section 6.2 for procedures for the Investigator to record SAEs that are related to 
study participation and for guidelines on how to report these SAEs to GSK Biologicals.
5.5.3.
Procedures at subsequent scheduled visits (2-6, as applicable) 
(Amended 16 October 2017)

For initial cohort subjects, Visit 2 will occur approximately 6 months after Visit 1
and Visit 3 approximately 6 months after Visit 2. Initial cohort subjects willing to 
extend their participation to three additional years will be re-consented at Visit 3 (i.e.
at the end of study Year 1).

For expansion cohort subjects, scheduled visits should ideally be performed at yearly 
intervals, preferably during periods of usually low dengue transmission. Expansion 
cohort subjects will have at most 4 scheduled visits, depending on the time of 
enrolment.

For replacement subjects enrolled in the subsequent study years, scheduled visits 
should ideally be performed at yearly intervals, preferably during periods of usually 
low dengue transmission.

A blood (serum) sample will be collected for serology.
Note: Indirect DENV IgG assay is repeated in the following scheduled visits (after 
enrolment) only if the subject was negative for DENV IgG at the previous scheduled 
visit.
Zika IgG assay is repeated in the following scheduled visits (after enrolment) only 
if the subject was negative for Zika IgG at the previous scheduled visit.
Chikungunya IgG assay is repeated in the following scheduled visits (after 
enrolment) only if the subject was negative for chikungunya IgG at the previous 
scheduled visit.
Even if the tests are not repeated, the volume of the blood sample remains 
unchanged. 

Socio-demographic and household information will be updated in eCRF (for e.g. 
change of home location).

Medical and vaccination histories will be updated in the eCRF.

The investigator will remind the subjects about the procedures in case of signs and 
symptoms of dengue, Zika and chikungunya.
 
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
A new diary log will be distributed for continued dengue, chikungunya and Zika
surveillance if needed, and any completed diary logs will be verified, as applicable. 
Subjects will be asked to contact the study staff in the event of the occurrence of a 
symptom that may be associated with suspected dengue, chikungunya or Zika (see 
Section 5.7.1.1, 5.7.2 and 5.7.3 for suspected dengue, chikungunya or Zika
definition).

The subject will be instructed to start recording any symptoms, including rash and 
body temperature any time dengue, chikungunya and/or Zika is suspected. Refer to 
Section 5.6.1 for details.

Any SAEs related to study procedures will be recorded.
5.5.4.
Study conclusion (Amended 16 October 2017)
The Study Conclusion screen page in the eCRF will be completed at the last study 
contact. This last contact could occur at the last scheduled visit or follow up visit for a 
suspected dengue, chikungunya or Zika case (if it is on-going at the last scheduled visit) 
or earlier if the subject terminates study participation or is lost-to-follow-up.
The study staff will review data collected to ensure accuracy and completeness and will 
complete the Study Conclusion screen page 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. If so, the investigator will ask each 
subject/subject’s parent(s)/LAR(s) if he or she would be willing to participate (or let their 
child participate) in a long-term follow-up study and subject will be asked to sign a new 
consent form.
5.5.5.
Dengue, chikungunya and Zika case detection (Amended 16 
October 2017)
Suspected cases (defined in Section 5.7.1, 5.7.2 and 5.7.3) in the study cohort may arise 
from three sources: 1) referred by study personnel during scheduled home visits; 2) 
through enhanced passive surveillance; and 3) as a result of active surveillance between 
scheduled visits.
5.5.5.1.
Case detection during scheduled home visits (Amended 16 October 
2017)
If a subject is identified as suspected case of dengue, chikungunya and/or Zika during 
any home visit, he or she will be referred to the designated study hospital/clinic for 
medical evaluation (refer to Section 5.6).
 
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5.5.5.2.
Case detection through enhanced passive surveillance (Amended 16 
October 2017)
The subject/subject’s parent(s)/LAR(s) will be instructed to contact the study staff (the 
local study coordinator) at any time dengue or chikungunya is suspected (i.e., body 
temperature 38°C measured (by any route) for at least two consecutive days) or if Zika
is suspected (fever [i.e., body temperature 38°C measured (by any route) for at least 
two consecutive days] and/or rash). 
The subject/subject’s parent(s)/LAR(s) will be instructed to contact the hospital or study 
clinic should the subject manifest any signs or symptoms they/the subject’s 
parent(s)/LAR(s) perceive as an emergency or severe.
The phone contact will be preferably made during working hours/days for logistical 
reasons. A reasonable schedule for phone contacts to the local study coordinator is from 
Monday to Saturday, 7:00 AM to 8:00 PM. In the case of an emergency outside of the 
defined hours, the patient should contact or present to their local emergency room, and 
contact the study staff point of contact as soon as convenient to the subject/subject’s 
parent(s)/LAR(s).
When a subject/subject’s parent(s)/LAR(s) contacts the study staff, the local study 
coordinator will then arrange for an appointment at the designated study hospital/clinic. 
The subject/subject’s parent(s)/LAR(s) will be instructed to record any symptom 
including rash and body temperature on the diary log daily until the appointment.  
The visit should be arranged as soon as possible, preferably during week days for 
logistical reasons. If the subject contacts the study staff during the weekend, the visit 
should be scheduled for the following Monday. All efforts should be made to guarantee 
that the subject be seen by the fifth day of disease onset, at latest.
During holidays, arrangements will be made with study physicians and the subjects 
should be seen at the designated study hospital/clinic. 
Although subjects will be instructed to contact the study staff in the event of suspected 
dengue, chikungunya and/or Zika, there may be cases where the subject is taken directly 
to the hospital or clinic. If this occurs when the study physician is not available, the staff 
at the designated hospital should notify the local study coordinator and the study 
physician. 
The study staff should ensure that:

the acute sample will be properly collected,

all clinical information will be retrievable, 

a return visit (if applicable) will be scheduled

and a follow-up visit will be scheduled.
See Section 5.6 for cases that present to a non-study hospital/clinic.
 
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5.5.5.3.
Case detection through active surveillance (Amended 16 October 2017)
Telephone calls (or home visits when applicable, if a phone call is not feasible) will be 
conducted at least monthly and more frequently if needed. During the phone call or visit, 
a structured script will be used to inquire about dengue, chikungunya and Zika
symptoms since the last contact.
An example of questions to be included in the structured script is provided in 
APPENDIX B.
If dengue, chikungunya and/or Zika is suspected during active surveillance, an 
appointment will be arranged at the designated study hospital/clinic, and at least one 
additional visit will be required for case follow-up (see Section 5.6).
Subjects whose status cannot be ascertained during active surveillance will be tracked by 
the study personnel through an active tracking algorithm.
Figure 2
Active tracking algorithm
5.6.
Management of suspected dengue, chikungunya and Zika
cases (Amended 16 October 2017)
All study subjects with suspected dengue, chikungunya and/or Zika should be seen at a 
designated study hospital or clinic by the study physician.
The first visit to the study hospital/clinic will include a detailed clinical examination to 
assess the subject’s general condition, body temperature, height and weight, cardiac and 
respiratory rates, blood pressure, dengue, chikungunya and Zika associated clinical 
signs/symptoms and relevant clinical signs/symptoms (e.g., fatigue, headache, pain 
behind the eyes, abdominal pain, nausea, vomiting, muscle ache, joint ache, diffuse rash, 
photophobia and pruritus).
A blood sample should be collected by the hospital/clinic for laboratory confirmation of 
dengue, chikungunya, Zika and differential diagnosis. This sample is referred to as the 
‘acute’ serum sample. In addition, a blood sample for haematology (CBC and HCT) will 
be collected (mandated procedure for subjects presenting within the first 14 days 
 
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following onset of fever and/or rash). If blood chemistry is conducted according to 
local clinical practice, these data may also be collected.
If dengue, chikungunya or Zika is still suspected after the first visit to the study 
hospital/clinic, a return visit for suspected dengue, chikungunya and/or Zika may be 
needed and will be conducted as directed by National guidelines by the study physician. 
A return visit is a visit linked to suspected dengue, chikungunya and/or Zika, occurring 
if the evolution of 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. If a return visit 
occurs, the diary log will be verified and possibly used by the physician to complete the 
clinical symptoms in the eCRF. 
Medical data related to any suspected dengue, chikungunya and/or Zika case will be 
collected for hospitalized study subjects and for those followed in the outpatient setting at 
the return visit. Treatment will be given according to local standard routines, following 
national guidelines.
Approximately 21(+7) days after the first visit for suspected dengue, chikungunya 
and/or Zika, a follow-up (convalescent) visit will be required. A physical examination 
will be conducted and a ‘convalescent’ serum sample (i.e., a blood sample collected after 
the acute phase for dengue, chikungunya and/or Zika) should be collected at this follow-
up visit. Ideally, this collection should be done at the study hospital/clinic but may be 
done at the subject’s home if directed by the study physician or delegated study staff, if 
the subject is unable to come to the hospital/ clinic.
Subjects who, for any reason, seek medical assistance at any non-study health care 
facility, will be identified through active surveillance, and clinical data will be 
retrospectively collected on the eCRF. The study physician will be responsible for 
collecting the retrospective data and informing the local study coordinator for appropriate 
follow-up. 
5.6.1.
Diary log instructions for suspected dengue, chikungunya and/or
Zika cases (Amended 16 October 2017)
The subject/subject’s parent(s)/LAR(s) will be instructed to record body temperature and 
rash on the diary log daily until the appointment. See Section 5.6 for subjects who do not 
reach the study staff and go directly to the hospital/clinic. The subject will be instructed 
to start recording any symptoms, including rash and body temperature any time dengue, 
chikungunya and/or Zika virus infection is suspected. 
The information collected in the diary log will be provided to the study physician as a 
tool in the medical evaluation of the subject. If the physician considers that the subject 
meets the criteria for suspected dengue, chikungunya and/or Zika, he could use the 
information contained in the diary log to complete the description of first clinical 
symptoms in the eCRF.
 
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If dengue, chikungunya and/or Zika is suspected, the  diary log will be verified by the 
investigator or designee at the follow-up visit if the subject is not hospitalized. If the 
subject is hospitalized, information with regard to dengue, chikungunya and/or Zika
infection will be recorded in the hospital record. Information from diary log and hospital 
records can be used to update clinical data in the eCRF.
5.7.
Case definitions
5.7.1.
Dengue
The following classification of dengue cases will be used in this study.
Figure 3
Classification of dengue cases
5.7.1.1.
Suspected symptomatic dengue case (Amended 16 October 2017)
Febrile illness with body temperature 38°C measured (by any route) on at least two 
consecutive days and less than 14 days with or without the presence of other dengue 
symptoms or signs, without an obvious aetiology unrelated to dengue, based on 
investigator’s judgement.
Although subjects are asked to come to a study hospital if fever (body temperature 
38°C) is sustained for two consecutive days, a subject could present on the first day of 
fever. In this case the physician may still consider the subject as a suspected dengue case 
based on medical judgement and collect a blood sample for laboratory diagnosis.
Subjects should be examined at the health care facility during the 5 days following 
fever onset, as this is a requirement for RT-qPCR diagnosis of dengue infection and 
serotype identification.
However, subjects presenting until 30th (included) day of fever onset will be considered 
for investigation. After the 30th day, subjects will not be considered as suspected 
dengue cases. 
A suspected dengue case presenting at the health care facility within 5 days following the 
onset of fever will be defined as an ‘early presenter’. 
 
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Subjects presenting for care between sixth and 30th day of fever onset will be defined as 
‘late presenters’.
An example of other signs and symptoms of dengue, associated with fever, include but 
are not limited to: fatigue, headache, pain behind the eyes, abdominal pain, nausea, 
vomiting, muscle ache, joint ache, diffuse rash on the trunk, abnormal sensitivity to light 
(photophobia) and itching of skin (pruritus).
5.7.1.2.
Laboratory-confirmed symptomatic dengue case (Amended 16 October 
2017)
Early presenter:
At least one of the following findings must be met for a laboratory-confirmed dengue 
case: 

Dengue virus identification through RT-qPCR on the acute serum sample 

Dengue virus NS1 positive on acute serum sample through ELISA.

Anti-dengue IgM seroconversion between acute and convalescent serum samples 
through ELISA.
Late presenter:

Anti-dengue IgM seroconversion between acute and convalescent serum samples 
through ELISA.
5.7.1.3.
Virologically confirmed symptomatic dengue infection 
A virologically confirmed symptomatic dengue infection is defined as a dengue case 
confirmed by RT-qPCR. 
5.7.1.4.
Moderate to severe dengue
A moderate to severe case of dengue is defined as follows (in accordance to the “dengue 
with warning signs” and “severe dengue” definitions in the 2009 WHO guidelines for 
dengue).
 
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One or more of the WHO 2009 warning signs or one or more of the WHO 2009 criteria 
for severe dengue are met: 
Warning signs

Abdominal pain or tenderness

Persistent vomiting

Clinical fluid accumulation

Mucosal bleed

Lethargy, restlessness

Liver enlargement >2 cm

Laboratory: increase in hematocrit (HCT) concurrent with rapid decrease in platelet 
count
Criteria for severe dengue

Severe plasma leakage leading to:

Shock (Dengue Shock Syndrome)

Fluid accumulation with respiratory distress

Severe bleeding as evaluated by clinician

Severe organ involvement

Liver: Alanine aminotransferase (ALT) or Aspartate aminotransferase (AST) 
≥1000

Central Nervous System (CNS): Impaired consciousness

Heart and other organs
5.7.1.5.
Previous dengue infection (Amended 16 October 2017)
A subject will be considered as having previous dengue infection at visit 1(baseline) if:

Dengue IgG positive at visit 1 (baseline)
or

Laboratory-confirmed symptomatic dengue case detected at visit 1 (baseline)
A subject will be considered as having previous dengue infection at any time after visit 
1 (baseline), if:

Dengue IgG positive at previous schedule visit(s)
or

Laboratory-confirmed symptomatic dengue case detected previously at study 
surveillance
 
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5.7.1.6.
Primary symptomatic dengue case (Amended 16 October 2017)
A primary symptomatic dengue case is a subject with laboratory confirmed or probable
symptomatic dengue infection without evidence of previous dengue infection (absence
of Ig G antibodies at the previous scheduled visit and absence of laboratory-confirmed 
symptomatic case detected previously at study surveillance).
5.7.1.7.
Secondary symptomatic dengue case (Amended 16 October 2017)
A secondary symptomatic dengue case is a subject with laboratory confirmed or 
probable symptomatic dengue infection with evidence of previous dengue infection
(presence of Ig G antibodies at the previous scheduled visit(s) or laboratory-confirmed 
symptomatic case detected previously at study surveillance).
5.7.1.8.
Primary inapparent dengue infection (Amended 16 October 2017)
This condition is defined as a documented seroconversion (anti-dengue IgG antibodies) 
between two sequential sera samples obtained during the scheduled visits without clinical 
suspicion of dengue (identified during the time period in which seroconversion occurred).
5.7.1.9.
Probable dengue case (Amended 16 October 2017)

For early presenters, a probable case will be that case without laboratory  
confirmation, presenting IgG positive in the convalescent sample.

For late presenters, a probable case will be the case without seroconversion of 
IgM, presenting at least one IgG positive in one sample (acute or convalescent).
5.7.1.10.
Negative Dengue case
For early and late presenters, a negative dengue case is a participant with a negative 
result for all planned laboratory tests on both acute and convalescent samples.
5.7.1.11.
Indeterminate dengue case (Amended 16 October 2017)
An indeterminate dengue case is a participant evaluated as an SDC (Section 5.7.1.1.) 
and not classified as laboratory confirmed case (Section 5.7.1.2), probably case
(Section  5.7.1.9) or negative case (Section 5.7.1.10).
 
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5.7.2.
Chikungunya
5.7.2.1.
Suspected symptomatic chikungunya case 
Febrile illness with body temperature 38°C measured (by any route) on at least two 
consecutive days and less than 14 days with or without the presence of other 
chikungunya symptoms or signs, without an obvious aetiology unrelated to 
chikungunya, based on investigator’s judgement.
Although subjects are asked to come to a study hospital if fever (body temperature 
38°C) is sustained for two consecutive days, a subject could present on the first day of 
fever. In this case the physician may still consider the subject as a suspected 
chikungunya case based on medical judgement and collect a blood sample for 
laboratory diagnosis.
Subjects should be examined at the health care facility during the 5 days following 
fever onset, as this is a requirement for RT-qPCR diagnosis of dengue infection in the 
case of co-infection.
However, subjects presenting until 30th (included) day of fever onset will be considered 
for investigation. After the 30th day, subjects will not be considered as suspected 
chikungunya cases. 
A suspected chikungunya case presenting at the health care facility within 5 days 
following the onset of fever will be defined as an ‘early presenter’. 
Subjects presenting for care between sixth and 30th day of fever onset will be defined as 
‘late presenters’.
An example of other signs and symptoms of chikungunya, associated with fever, 
include but are not limited to: 

Polyarthralgia is usually bilateral and symmetric, and can be debilitating

Other symptoms may include headache, myalgia, arthritis, conjunctivitis, 
nausea/vomiting, or maculopapular rash.
Rare complications include uveitis, retinitis, myocarditis, hepatitis, nephritis, bullous 
skin lesions, hemorrhage, meningoencephalitis, myelitis, Guillain-Barré syndrome, 
and cranial nerve palsies.
 
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5.7.2.2.
Laboratory-confirmed symptomatic chikungunya case
Early presenter:
At least one of the following findings must be met for a laboratory-confirmed 
chikungunya case: 

Chikungunya virus identification through RT-qPCR on the acute serum sample 

Anti-chikungunya IgM seroconversion between acute and convalescent serum 
samples through ELISA.
Late presenters:

Anti-chikungunya IgM seroconversion between acute and convalescent serum 
samples through ELISA.
5.7.2.3.
Virologically confirmed symptomatic chikungunya infection 
A virologically confirmed symptomatic chikungunya infection is defined as a 
chikungunya case confirmed by RT-qPCR. 
5.7.2.4.
Previous chikungunya infection at baseline
A subject will be considered as having previous chikungunya infection at baseline 
(enrolment visit/Visit 1), if:

Chikungunya IgG positive at baseline visit OR

Laboratory-confirmed symptomatic chikungunya case at baseline
5.7.2.5.
Inapparent chikungunya infection 
This condition is defined as a documented seroconversion (anti-chikungunya IgG 
antibodies) between two sequential sera samples obtained during the scheduled visits 
without clinical suspicion of chikungunya (identified during the time period in which 
seroconversion occurred).
5.7.2.6.
Probable chikungunya case
For early and late presenters, a probable chikungunya case will be that case without a 
lab-confirmed criteria, presenting at least one IgG positive in one sample (acute or 
convalescent).
5.7.2.7.
Negative chikungunya case
For early and late presenters, a negative chikungunya case is a participant with a 
negative result for all planned laboratory tests on both acute and convalescent samples.
 
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5.7.2.8.
Indeterminate chikungunya case
An indeterminate chikungunya case is a participant evaluated as an suspected 
chikungunya case (Section 5.7.2.1.) and not classified as laboratory confirmed case 
(Section 5.7.2.2), probably case (Section  5.7.2.6) or negative case (Section 5.7.2.7).
5.7.3.
Zika
5.7.3.1.
Suspected symptomatic Zika case
Rash and/or febrile illness on at least two consecutive days and less than 14 days with 
or without the presence of other Zika symptoms or signs, without an obvious aetiology 
unrelated to Zika, based on investigator’s judgement.
Although subjects are asked to come to a study hospital if fever and/or rash is 
sustained for two consecutive days, a subject could present on the first day of fever 
and/or rash. In this case the physician may still consider the subject as a suspected 
Zika case based on medical judgement and collect a blood sample for laboratory 
diagnosis.
Subjects should be examined at the health care facility during the 5 days following 
fever and/or rash onset, as this is optimal for RT-qPCR diagnosis of Zika infection.
However, subjects presenting until 30th (included) day of fever and/or rash onset will 
be considered for investigation. After the 30th day, subjects will not be considered as 
suspected Zika cases. 
A suspected Zika case presenting at the health care facility within 5 days following the 
onset of fever and/or rash will be defined as an ‘early presenter’. Subjects presenting 
for care between sixth and 30th day of fever and/or rash onset will be defined as ‘late 
presenters’. At the clinical presentation of the first visit, fever will take priority in the 
classification of “early” or “late” presenter if the participant reported  both fever and 
rash are present.  If the participant reported the rash without fever, the date of onset of 
rash will determine classification.
An example of other signs and symptoms include arthralgia, arthritis, or conjunctivitis 
(non-purulent/hyperaemic). 
 
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5.7.3.2.
Laboratory-confirmed symptomatic Zika case
Early presenters:
At least one of the following findings must be met for a laboratory-confirmed Zika 
case.

Zika virus identification through RT-qPCR on the acute serum sample 

Anti-Zika IgM seroconversion between acute and convalescent serum samples 
through ELISA and confirmed by Zika neutralization antibody testing.
Late presenters:

Anti-Zika IgM seroconversion between acute and convalescent serum samples 
through ELISA and confirmed by Zika neutralization antibody testing.
5.7.3.3.
Virologically confirmed symptomatic Zika infection 
A virologically confirmed symptomatic Zika infection is defined as a Zika suspected 
case confirmed by RT-qPCR. 
5.7.3.4.
Previous Zika infection at baseline
A subject will be considered as having previous Zika infection at baseline (enrolment 
visit/Visit 1), if:

Zika IgG positive at baseline visit as confirmed by neutralizing antibody tests OR

Laboratory-confirmed symptomatic Zika case at baseline
5.7.3.5.
Inapparent Zika infection 
This condition is defined as a documented seroconversion and confirmed by Zika 
neutralising antibody testing between two sequential sera samples obtained during the 
scheduled visits without clinical suspicion of Zika (identified during the time period in 
which seroconversion occurred).
5.7.3.6.
Probable Zika case

For early and late presenters, a probable Zika case will be that case without a lab-
confirmed criteria, presenting at least one IgM/IgG positive in one sample (acute 
or convalescent).
5.7.3.7.
Negative Zika Case
For early and late presenters, a negative Zika case is a participant with a negative 
result for all planned laboratory tests on both acute and convalescent samples.
 
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5.7.3.8.
Indeterminate Zika case
An indeterminate Zika case is a participant evaluated as an suspected Zika case 
(Section 5.7.3.1.) and not classified as laboratory confirmed case (Section 5.7.3.2), 
probably case (Section 5.7.3.6) or negative case (Section 5.7.3.7).
5.8.
Biological sample handling and analysis
The biological samples collected in this study are whole blood.
Please refer to the SPM for details of biospecimen management (handling, storage and 
shipment).
Samples will not be labelled with information that directly identifies the subjects but will 
be coded with the identification number for the subject (subject number). 
Collected samples may be used for future testing, according to local regulations, in other 
assays, for test improvement or test development of analytical methods related to the 
pathogens under study. Under these circumstances, additional testing on the samples may 
be performed by GSK Biologicals or a GSK designated laboratory outside the scope of 
this protocol. Further research on the sample will only be conducted by GSK Biologicals 
after further assessment and approval(s) by Ethics Committee(s) and the National 
Commission of Ethics in Research (when applicable) for the test(s) to be done and also 
after the subjects’ informed consent. Information on further investigations and their 
rationale can be obtained from GSK Biologicals.
Any sample testing will be done in line with the consent of the individual 
subject/subject’s parent(s)/LAR(s).
Any human pharmacogenetic testing will require specific consent from the individual 
subjects/subject’s parent(s)/LAR(s) and the ethics committee approval. 
Refer also to the Investigator Agreement, the Investigator cannot perform any other 
biological assays except those described in the protocol or its amendment(s). 
Collected samples will be stored for up to 10 years (counting from when the last subject 
performed the last study visit/contact), with the option to extend the retention time.
5.8.1.
Use of specified study materials
When materials are provided by GSK Biologicals, that all samples (including serum 
samples) must be collected and stored exclusively using those materials in the appropriate 
manner; this is MANDATORY. The use of other materials could result in the exclusion 
of the subject from the ATP analysis (See Section 8.3 for the definition of study cohorts 
to be evaluated). The investigator must ensure that his/her personnel and the 
laboratory(ies) under his/her supervision comply with this requirement. However, when 
GSK Biologicals does not provide material for collecting and storing samples, 
appropriate materials from the investigator’s site must be used. Refer to the SPM.
 
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5.8.2.
Biological samples evaluation
A detailed description of the assays performed and laboratory addresses are provided in 
the appendices.
Assays will be performed using standardized and validated procedures.
5.8.2.1.
Laboratory assays 
Table 8
Humoral Immunity (Amended 16 October 2017)
System
Component
Method
Kit / Manufacturer
Laboratory
Serum
IgM to Dengue 
ELISA
Commercial kit Panbio or 
equivalent 
Local# or GSK designated 
laboratory
IgG to Dengue 
ELISA
Indirect IgG Panbio or 
equivalent (for scheduled 
visits)
IgG Capture PanBio or 
equivalent (for unscheduled 
visits)
Local# or GSK designated 
laboratory
Local# or GSK designated 
laboratory
Serum
Dengue virus types 
1-4 neutralizing 
antibodies
Dengue 
neutralization 
assay
In-house
Laboratório de Tecnologia 
Virológica, Bio-Manguinhos, 
Fiocruz  Rio de Janeiro or 
GSK designated laboratory
Serum
Antibodies against 
Zika and 
chikungunya
To be 
determined
To be determined
GSK designated laboratory
Serum
YF virus neutralizing 
antibody*
YF
neutralization 
assay
To be determined
GSK designated laboratory
*For subjects residing in a yellow fever endemic region or vaccinated against yellow fever
#Testing performed according to local practices
Table 9
Virology (Amended 16 October 2017)
System
Component
Method
Kit/Manufacturer
Laboratory
Serum
DENV RNA
RT-qPCR
In-house
Laboratório de Tecnologia 
Virológica, Bio-Manguinhos, 
Fiocruz  Rio de Janeiro (GSK 
as back-up)
Serum
Zika RNA
RT-PCR
To be determined
GSK designated laboratory
Serum
Chikungunya RNA
RT-PCR
To be determined
GSK designated laboratory
Serum
NS1 
ELISA 
Commercial kit 
Biorad,Platelia 
Local
Although not a specific study endpoint, viral isolation and sequencing may be performed 
if deemed necessary for future research (the aliquot reserved for RT-qPCR will be used in 
this case).
 
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Table 10
Haematology
System
Component
Method
Kit/Manufacturer
Laboratory
Whole 
Blood
CBC
Per local 
standard 
practice
Per local standard 
practice
Local
Hematocrit
Local 
Assays performed on an as needed basis if dengue is suspected
The GSK Biologicals’ and Fiocruz 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 should also 
have a Quality System established and supported by procedures.  
Aliquots for PCR and the neutralization assay will be sent to Fiocruz with the possibility 
of sending reserve aliquots to GSK for retesting if necessary. Aliquots for re-testing will 
be sent from Fiocruz
5.8.2.2.
Laboratory read-outs
In case of insufficient blood sample volume to perform all assays, the samples will be 
analyzed according to priority ranking provided in Table 11, Table 12 and Table 13.
 
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Table 11
Laboratory read-outs at each time point, and priority ranking for 
scheduled visits (Amended 16 October 2017)
SCHEDULED VISITS
Blood sampling time 
point
No. Subjects
(anticipated) Subset*
Possible assays 
Serum 
aliquot
volume
Aliquot
priority 
rank
Test 
priority 
rank
Aliquot 
number
Type of 
contact/ 
Amount of 
whole blood
Sampling 
time point
Scheduled 
Visit 1
whole blood 5 
mL
(Day 0)
3600 
(1800 from 
wave 1 &
2***)
-
Dengue IgG ELISA**
250 µL
1
1
1
yes
Dengue virus 1-4 
neutralizing 
antibodies
1 mL
2
1
2
YF neutralization
4
Zika neutralization
3
Zika serology (IgG) #
2
Chikungunya 
serology (IgG) #
yes
Chikungunya 
neutralization
remaining 
volume
3
1
3
Scheduled 
Visit 2 
5 mL whole 
blood
Visit 1+ 6 
months for 
subject 
enrolled in 
Study Year 
1; Visit 1+ 
12 months 
for subject 
enrolled in 
beginning 
of  Study 
Year 2, 3 or 
4)
3190 (1660 
from wave 1 
and 1530 
from wave 
2) 
yes
Dengue IgG ELISA** 
(only for subjects IgG 
negative at the 
previous visit)
250 µL
1
1
1
yes
Dengue virus 1-4 
neutralizing 
antibodies
1 mL
2
1
2
YF neutralization
4
Zika neutralization
3
Zika serology (IgG) #
2
Chikungunya 
serology (IgG) #
yes
Chikungunya 
neutralization
remaining 
volume
3
1
3
 
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SCHEDULED VISITS
Blood sampling time 
point
No. Subjects
(anticipated) Subset*
Possible assays 
Serum 
aliquot
volume
Aliquot
priority 
rank
Test 
priority 
rank
Aliquot 
number
Type of 
contact/ 
Amount of 
whole blood
Sampling 
time point
Scheduled 
Visit 3 
5 mL whole 
blood
(Visit 2+ 6 
months for 
subject 
enrolled in 
Study Year 
1; Visit 
2+12 
months for 
subject 
enrolled in 
beginning 
of  Study 
Year 2 or 3)
3030 (1530 
from wave 1 
and 1500 
from wave 
2)
yes
Dengue IgG ELISA**
(only for subjects IgG 
negative at the 
previous visit)
250 µL
1
1
1
yes
Dengue virus 1-4 
neutralizing 
antibodies
1 mL
2
1
2
YF neutralization
4
Zika neutralization
3
Zika serology (IgG) #
Chikungunya 
serology (IgG) #
2
yes
Chikungunya 
neutralization
remaining 
volume
3
1
3
Subsequent 
Yearly Visit(s) 
as applicable
5 mL whole 
blood
Yearly 
3000 for 
Visit 4 and 
1500 for visit 
5 and 6 
which 
applies only 
to subjects 
of the wave 
1
yes
Dengue IgG ELISA**
(only for subjects IgG 
negative at the 
previous visit)
250 µL
1
1
1
yes
Dengue  virus 1-4 
neutralizing 
antibodies
1 mL
2
1
2
YF neutralization
4
Zika neutralization
3
Zika serology (IgG) #
2
Chikungunya 
serology (IgG) #
yes
Chikungunya 
neutralisation
remaining 
volume
3
1
3
*tertiary (exploratory) analysis, number of subjects in the subset will be determined during analysis 
-  not applicable, 
** Dengue IgG indirect ELISA
*** Wave 1 enrolment = initial cohort / Wave 2 enrolment = Expansion cohort
# Indirect DENV IgG assay is repeated in the following scheduled visits (after enrolment) only if the subject 
was negative for DENV IgG at the previous scheduled visit. Zika IgG assay is repeated in the following 
scheduled visits (after enrolment) only if the subject was negative for Zika IgG at the previous scheduled 
visit; chikungunya IgG assay is repeated in the following scheduled visits (after enrolment) only if the 
subject was negative for chikungunya IgG at the previous scheduled visit
 
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Table 12
Laboratory read-outs at each time point, and priority ranking for 
suspected dengue, chikungunya and/or Zika visits for EARLY 
presenters (Amended 16 October 2017)
SUSPECTED DENGUE, CHIKUNGUNYA AND/OR ZIKA VISITS – EARLY PRESENTERS
Blood sampling time point
No. 
Subjects
Subset
Component
Serum 
aliquot 
volume
Aliquot 
priority 
rank
Test 
priority 
rank
Aliquot 
number
Type of 
contact/ 
Amount of 
whole blood
Sampling time 
point
First visit 
(amount of 
blood= 5 mL for 
< 2 years of 
age and 10 mL 
for all other 
subjects)
Sample collected 5 
days of fever and/or 
rash
unknown
-
Dengue RT-
qPCR
750 µL
1
1
1
Zika and 
chikungunya 
RT-qPCR
2
Dengue virus 
1-4 
neutralizing 
antibodies
750 µL
3
1
2
Dengue IgM 
and NS1 
ELISA
500 µL
2
1
3
Zika 
neutralising 
antibodies
1 mL
4
1
4
Chikungunya 
neutralizing 
antibodies
and Serology 
for Zika 
(IgG/IgM) and 
chikungunya 
(IgG/IgM)
remaining 
volume
5
1
5
Optional for severe cases
PCR 
200 µL
-
First visit (total 
amount of 
blood per local 
practice 
Sample collected 
before day 14 of 
onset of fever/rash
unknown
CBC, HCT
No aliquot
-
 
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SUSPECTED DENGUE, CHIKUNGUNYA AND/OR ZIKA VISITS – EARLY PRESENTERS
Blood sampling time point
No. 
Subjects
Subset
Component
Serum 
aliquot 
volume
Aliquot 
priority 
rank
Test 
priority 
rank
Aliquot 
number
Type of 
contact/ 
Amount of 
whole blood
Sampling time 
point
Suspected 
dengue follow-
up visit (total 
amount of 
blood 5 mL)
Approximately 21 
days after first visit
unknown
-
Dengue IgM 
and Dengue 
capture IgG 
ELISA
500 µL
1
1
1
Dengue  virus 
1-4 
neutralizing 
antibodies
1 mL
2
1
2
Serology for 
Zika 
(IgM/IgG) and 
chikungunya 
(IgM/IgG )
2
Zika 
neutralizing 
antibodies
3
Chikungunya 
neutralizing
antibodies
remaining 
volume
3
1
3
-  not applicable
 
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Table 13
Laboratory read-outs at each time point, and priority ranking for 
suspected dengue, chikungunya and/or Zika visits for LATE 
presenters
SUSPECTED DENGUE, CHIKUNGUNYA AND/OR ZIKA VISITS – LATE PRESENTERS
Blood sampling time point
No. 
Subjects
Subset
Component
Serum 
aliquot 
volume
Aliquot 
priority 
rank
Test 
priority 
rank
Aliquot 
number
Type of 
contact/ 
Amount of 
whole blood
Sampling 
time point
First visit (total 
amount of 
whole blood 5 
mL)
Sample 
collected > 5 
days or more  
after fever
and/or rash
unknown
-
Dengue IgM 
and Dengue 
capture IgG 
ELISA 
500 µL
1
1
1
Dengue  virus 
1-4 neutralizing 
antibodies
1 mL
2
1
2
Zika and 
chikungunya  
RT-qPCR
2
Serology for 
Zika (IgG/IgM)
and 
chikungunya 
(IgG/IgM)
remaining 
volume
3
1
3
First visit (total 
amount of blood 
per local 
practice 
Sample 
collected 
before day 14 
of onset of 
fever/rash
unknown
CBC, HCT
No aliquot
-
Suspected 
dengue, 
chikungunya 
and Zika follow-
up visit (total 
amount of blood 
5 mL)
Approximately 
21 days after 
first visit
unknown
-
Dengue IgM 
and Dengue 
capture IgG 
ELISA
500 µL
1
1
1
Dengue  virus 
1-4 neutralizing 
antibodies
1 mL
2
1
2
Serology for 
Zika (IgG/IgM)
and 
chikungunya 
(IgG/IgM)
2
Zika 
neutralizing 
antibodies
3
Chikungunya 
neutralizing 
antibodies
remaining 
volume
3
1
3
- not applicable
 
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6.
SERIOUS ADVERSE EVENTS
In this prospective cohort study no test product/vaccine will be given. However blood 
samples will be collected at scheduled visits and unscheduled visits for suspected 
dengue, chikungunya and/or Zika. 
In order to fulfil international reporting obligations, serious adverse events (SAEs) that 
are related to study procedures ( blood collection) will be collected and recorded from the 
time the subject consents to participate in the study until the study conclusion for each 
subject.
The investigators or site staff 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 or 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.
6.1.
Serious adverse events
6.1.1.
Definition of a serious adverse event
A serious adverse event is any AE that:
a.
Results in death
b.
Is life-threatening
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 hospitalization or prolongation of existing hospitalization,
In general, hospitalization 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 hospitalization are also considered AEs. If a 
complication prolongs hospitalization or fulfils any other serious criteria, the event 
will also be considered serious. When in doubt as to whether ‘hospitalization’ 
occurred or was necessary, the AE should be considered serious.
Hospitalization 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.
 
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d.
Results in disability/incapacity, OR
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.
e.
Is a congenital anomaly/birth defect in the offspring of a study subject
f.
Medical or scientific judgement should be exercised in deciding whether reporting is 
appropriate in other situations, such as important medical events that may not be 
immediately life-threatening or result in death or hospitalisation but may jeopardise 
the subject or may require medical or surgical intervention to prevent one of the 
other outcomes listed in the above definition. These should also be considered 
serious. Examples of such events are invasive or malignant cancers, intensive 
treatment in an emergency room or at home for allergic bronchospasm, blood 
dyscrasias or convulsions that do not result in hospitalisation.
6.2.
Detecting and recording serious adverse events
6.2.1.
Evaluation of serious adverse events related to a study 
procedure
Any SAE either observed by the investigator or his/her staff or reported by the 
subject/subject’s parents/LAR(s) related to study procedures will be evaluated by the 
investigator. The nature of each event, data and time (where appropriate) of onset, 
outcome, intensity and relationship to the study procedures should be established.
When an SAE related to a study procedure 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 on the eCRF or SAE Report screens as applicable. It is 
not acceptable for the investigator to send photocopies of the subject’s medical records to 
GSK Biologicals instead of the appropriate completed SAE screens in the eCRF. 
However, there may be instances when copies of medical records for certain cases are 
requested by GSK Biologicals. In this instance, all subject identifiers will be blinded on 
the copies of the medical records prior to submission to GSK Biologicals.
The investigator will attempt to establish a diagnosis pertaining to the event based on 
signs, symptoms, and/or other clinical information. In such cases, the diagnosis should be 
documented as the SAE and not the individual signs/symptoms.
The investigator should assess the causality of each reportable SAE. The investigator will 
use clinical judgement to determine the relationship of SAEs to study procedures. 
Alternative causes, such as natural history of the underlying diseases, concomitant 
therapy and other risk factors will be considered and investigated.
 
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There may be situations when a SAE has occurred and the investigator has minimal 
information to include in the initial report to GSK Biologicals. However it is very 
important that the investigator always makes an assessment of causality for every event 
prior to submission of the SAE to GSK Biologicals. The investigator may change his/her 
opinion of causality in light of follow-up information, amending the SAE information 
accordingly. 
An SAE related to a study procedure will be examined by the investigator to the extent to 
enable determination of all contributing factors applicable to each SAE. 
Possible contributing factors include:

Medical history

Concomitant medication

Other procedure not required by the protocol

Other cause (specify).
Outcome of any reportable SAE during the entire study will be assessed as:

Recovered/resolved

Recovering/resolving

Not recovered/not resolved

Recovered with sequelae/resolved with sequelae

Fatal
6.2.2.
Prompt reporting of SAEs related to a study procedure
SAEs related to a study procedure that occurs at any time during the study will be 
reported promptly to GSK within the timeframes described in Table 14, once the 
investigator determines that the event meets the protocol definition of a SAE.
Table 14
Timeframes for submitting SAE reports to GSK 
Type of Event
Initial Reports
Follow-up of Relevant Information
on a Previous Report
Timeframe
Documents
Timeframe
Documents
SAEs related to a study procedure 
24 hours*
SAE screen
24 hours*
SAE screen
* Timeframe allowed after receipt or awareness of the information
 
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6.2.3.
Contact information for reporting serious adverse events and 
other events to GSK Biologicals
Study Contact for Reportable SAEs
During office hours:
Local 
Name: Dr.
GlaxoSmithKline Biologicals
, 
, 
, Brazil CEP 22783-110
Email: 
Phone: 
Cell: 
Name: 
Clinical Safety Analyst
Pharmacovigilance Department
GlaxoSmithKline Brazil
, 
, 
, 
 Brazil 22783-110
Email: 
Phone: 
Back-up Study Contact for Reporting SAEs
24/24 hour and 7/7 day availability:
GSK Biologicals Clinical Safety & Pharmacovigilance
Fax: 
 or 
Once an investigator becomes aware that a study procedure related SAE has occurred in a 
study subject, the investigator (or designate) must complete the information in the SAE 
screens of the eCRF WITHIN 24 HOURS. The SAE screens will always be completed as 
thoroughly as possible with all available details of the event. Even if the investigator does 
not have all information regarding a SAE, the SAE screens should still be completed 
within 24 hours. Once additional information is received, the SAE screens in the eCRF 
should be updated WITHIN 24 HOURS.
The investigator will always provide an assessment of causality at the time of the initial 
report.
6.2.3.1.
Back-up system in case the electronic SAE reporting system does not 
work
If the electronic SAE reporting system does not work, the investigator (or designate) must 
complete, then date and sign a SAE Report Form and fax it to the GSK Biologicals 
Clinical Safety and Pharmacovigilance department within 24 hours. 
This back-up system should only be used if the electronic SAE reporting system is not 
working and NOT if the system is slow. As soon as the electronic SAE reporting system 
is working again, the investigator (or designate) must complete the SAE screens in the 
eCRF within 24 hours. The final valid information for regulatory reporting will be the 
information reported through the electronic SAE reporting system. 
 
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PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD

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6.2.3.2.
Updating of SAE information after freezing of the subject’s eCRF
When additional information is received on a SAE after freezing of the subject’s eCRF, 
new or updated information is to be recorded on a SAE Report Form, with all changes 
signed and dated by the investigator. The updated form should be faxed to the GSK 
Biologicals Clinical Safety and Pharmacovigilance department or to the Study Contact 
for Reporting SAEs (refer to the Sponsor Information Sheet) WITHIN 24 HOURS of 
receipt of the follow-up information.
6.2.4.
Regulatory reporting requirements for serious adverse events
The investigator will promptly report all SAEs to GSK in accordance with the procedures 
detailed in Section 6.2.2. Prompt notification of SAEs by the investigator to the Study 
Contact for Reporting SAEs is essential so that legal obligations and ethical
responsibilities towards the safety of other subjects are met.
6.3.
Follow-up of SAEs related to study procedure
After the initial SAE report, the investigator is required to proactively follow each subject 
and provide further information on the subject’s condition to GSK Biologicals.
All SAEs documented at a previous visit/contact and designated as not recovered/not 
resolved or recovering/resolving will be reviewed at subsequent visits/contacts until the 
end of the study.
Investigators will follow-up subjects with an SAE until the event has resolved, subsided, 
stabilized, disappeared, or until the event is otherwise explained, or the subject is lost to 
follow-up. 
If a subject dies during participation in the study or during a recognized follow-up period, 
GSK Biologicals will be provided with a copy of any available post-mortem findings, 
including histopathology.
6.4.
Treatment of adverse events
Treatment of any SAE related to a study procedure is at the sole discretion of the 
investigator and according to current good medical practice. Any medication 
administered for the treatment of an SAE should be recorded in the subject’s eCRF.
6.5.
Subject Cards
Study subjects will be provided with the address and telephone number of the main 
contact for information about the epidemiological study.
Investigator/delegate should therefore provide a “subject card” to each subject. The aim 
of this card is to inform any physician having contact with a study subject, that the patient 
is participating in this epidemiology study.
Subjects must be instructed to keep these cards in their possession at all times.
 
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7.
SUBJECT COMPLETION AND WITHDRAWAL
7.1.
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.
7.2.
Subject withdrawal
Subjects who are withdrawn because of an SAE must be clearly distinguished from 
subjects who are withdrawn for other reasons. Investigators will follow subjects who are 
withdrawn as result of a SAE until resolution of the event.
All data collected until the date of withdrawal/last contact of the subject will be used for 
the analysis. Withdrawals/drop-outs will be replaced (see Section 7.3 for further details).
A subject is considered a ‘withdrawal’ from the study when no study procedure has 
occurred, no follow-up has been performed or no further information has been collected 
for this subject from the date of withdrawal/last contact.
Investigators will make an attempt to contact those subjects who do not return for 
scheduled visits or follow-up. 
Information relative to the withdrawal will be documented in the eCRF. The investigator 
will document whether the decision to withdraw a subject from the study was made by 
the subject himself/herself by the subject’s parent(s) or LAR(s) or by the investigator, as 
well as which of the following possible reasons was responsible for withdrawal:

Serious adverse event.

Non-serious adverse event.

Protocol violation (specify).

Consent withdrawal, not due to an adverse event*

Moved from the study area.

Lost to follow-up.

Other (specify).
*If a subject is withdrawn from the study because he/she/the subject’s parent(s) has 
withdrawn consent, the investigator will document the reason for withdrawal of consent, 
if specified by the subject, in the eCRF.
 
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7.3.
Subject replacement (Amended 16 October 2017)
Enrolment of new subjects to replace:

subjects not wishing to continue after amendment 2 (initial cohort), or

subjects who prematurely terminate participation (initial and expansion cohorts) or

subjects lost to follow-up will be done (initial and expansion cohorts). 
The aim is to maintain a critical cohort size of 500 subjects/site. This replacement will 
be done at the beginning of each study year: during the scheduled visits starting at the end 
of study Year 1 up till the end of study Year 3.
The number of subjects to be recruited as replacements will be defined based on the 
number of subjects still active in the study. If at the end of each study year we have 500
subjects/site, no replacements will be needed. 
In last three months of the of penultimate year of the study, each site will review the 
number of active subjects and drop-out rate..  If the risk of having fewer than the 
required number of active participants at the end of the study is high, recruitment for 
participants into the replacement cohort may be performed with sponsor approval. If 
the risk is high, recruitment into the replacement cohort should begin even if the 
number of active subjects is 500 or above 500.
The time window for the recruitment of replacements will be during the low dengue 
transmission, preferably.
8.
DATA EVALUATION: CRITERIA FOR EVALUATION OF 
OBJECTIVES
8.1.
Endpoints
8.1.1.
Primary endpoint

Laboratory-confirmed symptomatic dengue infection (all DENV types).
Symptomatic dengue infection is defined in Section 5.7.1.2
8.1.2.
Secondary endpoints

DENV-type specific primary laboratory-confirmed symptomatic dengue infection.

DENV-type specific secondary laboratory-confirmed symptomatic dengue infection.

Primary symptomatic dengue infection (including laboratory-confirmed and probable 
cases).
 
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
Secondary symptomatic dengue infection (including laboratory-confirmed and 
probable cases).

Previous dengue infection(s) (dengue seroprevalence) at baseline (defined in Section 
5.7.1.5)

Primary inapparent dengue infection..

Severity of symptoms of symptomatic dengue (using the 2009 WHO guidelines).
8.1.3.
Tertiary endpoints (Amended 16 October 2017)
Exploratory objectives may be assessed based on, but not limited to the following 
endpoints:

Symptomatic laboratory-confirmed Zika virus infection

Symptomatic Zika virus infection (including laboratory-confirmed and probable 
cases).

Symptomatic laboratory-confirmed chikungunya infection

Symptomatic chikungunya infection (including laboratory-confirmed and 
probable cases).

Risk factors for dengue infection and disease.

Neutralizing antibodies titers against DENV 1-4.

Neutralizing antibody titers against YF virus.

Spatial and temporal distribution of dengue cases (confirmed and probable 
symptomatic cases and inapparent infections).

Spatial and temporal distribution of laboratory confirmed and probable Zika virus 
cases

Spatial and temporal distribution of patients with laboratory confirmed and 
probable chikungunya virus infection

Occurrence of laboratory confirmed or probable Zika and/or chikungunya 
infection in suspected dengue cases (differential diagnosis of dengue), 
retrospectively

Antibody titre against Zika and chikungunya virus at the scheduled visits
8.2.
Sample size consideration
8.2.1.
Assumptions:
A target to obtain at least 500 subjects/site still active at the end of the first year of the 
study is planned. Since subjects might leave the study due to a variety of reasons, e.g. 
moving away geographically, sample size is increased to allow for this. On the basis of 
 
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an annual drop-out rate between 15 and 20%, approximately 600 subjects/site will be 
enrolled at the beginning of this study. The recruitment will be cluster sampling , i.e. the 
unit of sampling will be the households. Different areas where the FHP has been fully 
implemented or where access to the community is already established by a government 
program will be first selected, followed by a selection of households in the respective 
areas. All subjects in each household will be invited for recruitment to the study.
New sites will start enrolling subjects preferably during study Year 2. The target is to 
enrol approximately 600 subjects per additional site. Replacement subjects may be 
recruited in order to maintain at least 500 subjects/site still active at the beginning of the 
next dengue season.
In order to better represent the Brazilian population distribution, subjects enrolled in the 
expansion cohort should be composed of at least 30% of subjects younger than 18 years 
old and should not exceed 20 % of adults 50 years or older. Subjects enrolled as 
replacement subjects for the initial and expansion cohort will be recruited to approach as 
much as possible a minimal representation of 30% of subjects younger than 18 years old 
and a maximal representation of 20% for adults 50 years or older.
The total study duration per site will last approximately 4 years for the initial cohort and 
approximately 3 years for the expansion cohort, and new subjects will be enrolled if the 
number of subjects still active in the study is below 500/site after year 1 and at the 
beginning of each additional study year/season.
Between 2007 and 2010, the average incidence of reported dengue cases in the Brazilian 
population was about 0.3% per year (or 0.003 cases per person per year). Assuming an 
underreporting factor of 3 (Brazil MoH, 2011) the average incidence of dengue is 
expected to be about 1% per year (or 0.01 cases per person per year).
8.2.2.
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 1.8 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 
3. Although in theory rho can have a value up to 1, in practice values higher than 0.4 are 
uncommon. We used a conservative estimate of 0.4 for this study (Bennett, 1991).The 
design effect is then estimated to 1.8.
 
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8.2.3.
Expected precision of incidence rate estimates
Table 15 shows the 95% CI for a range of expected incidences based on the Poisson 
distribution (exact method, normal approximation and the normal approximation 
accounting for a design effect of 1.8) in different scenarios. The scenarios were defined 
according to the following criteria:

Number of subjects enrolled at the beginning of the study (for some sites it will be 
the second year). At season level and for all sites: 2000, 4000, 1800, 3600, 1500, 
3000. For all seasons and for all sites: 4000, 3600 and 3000. At season level and site 
level: 600 and 500.

Number of years of follow-up. At study level: 1 or 4 years, but half of the sites will 
be followed for 3 years and this is taken into account for the calculations.  At site
level: 1, 3 or 4 years.

Overall incidences: 3, 6, 9, 12 or 15 events per 1000 person-years.
With an overall sample size of a minimum of about 3,600 subjects enrolled and a follow-
up period of one year, the 95% confidence interval (CI) for an expected incidence of 
dengue of 9 cases per 1000 person-years using a cluster design is [4.7 ; 13.3]. With 3600 
subjects enrolled, 4 years of follow-up and new enrolments to keep the number of 
subjects above 3000 at the beginning of each subsequent season, the 95% CI is [6.3 ; 
11.7].
 
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Table 15
Precision of the expected incidences of dengue for a single cohort of subjects in Brazil
Cohort 
size
Number of
years
Number of
person-years
Number 
of
cases
Incidence of dengue
(per 1000 person-years)
95% CI of incidence rate (n / 1000 P-Y)
New enrolment
Exact (Poisson)
Normal approx
Normal approx
with design effect
After 
Y1
After 
Y2
After 
Y3
Lower
Upper
Lower
Upper
Lower
Upper
Overall
2000
1
1845.9
6
3.0
1.0
6.7
0.5
5.5
-0.4
6.4
2000
1
1845.9
11
6.0
3.0
10.7
2.5
9.5
1.3
10.7
2000
1
1845.9
17
9.0
5.2
14.5
4.7
13.3
3.2
14.8
2000
1
1845.9
22
12.0
7.5
18.1
7.0
17.0
5.3
18.7
2000
1
1845.9
28
15.0
9.9
21.7
9.4
20.6
7.5
22.5
4000
1
3691.9
11
3.0
1.5
5.4
1.2
4.8
0.6
5.4
4000
1
3691.9
22
6.0
3.8
9.1
3.5
8.5
2.6
9.4
4000
1
3691.9
33
9.0
6.2
12.6
5.9
12.1
4.9
13.1
4000
1
3691.9
44
12.0
8.7
16.1
8.5
15.5
7.3
16.7
4000
1
3691.9
55
15.0
11.3
19.5
11.0
19.0
9.7
20.3
4000
4
9086.6
27
3.0
2.0
4.4
1.9
4.1
1.5
4.5
327
4000
4
9086.6
55
6.0
4.5
7.8
4.4
7.6
3.9
8.1
327
4000
4
9086.6
82
9.0
7.2
11.2
7.0
11.0
6.4
11.6
327
4000
4
9086.6
109
12.0
9.9
14.5
9.7
14.3
9.0
15.0
327
4000
4
9086.6
136
15.0
12.6
17.7
12.5
17.5
11.6
18.4
327
1800
1
1661.3
5
3.0
0.9
7.2
0.4
5.6
-0.5
6.5
1800
1
1661.3
10
6.0
2.8
11.1
2.3
9.7
1.0
11.0
1800
1
1661.3
15
9.0
5.0
14.9
4.4
13.6
2.9
15.1
1800
1
1661.3
20
12.0
7.3
18.6
6.7
17.3
4.9
19.1
1800
1
1661.3
25
15.0
9.7
22.1
9.1
20.9
7.1
22.9
3600
1
3322.7
10
3.0
1.4
5.6
1.1
4.9
0.5
5.5
3600
1
3322.7
20
6.0
3.6
9.3
3.4
8.6
2.5
9.5
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Cohort 
size
Number of
years
Number of
person-years
Number 
of
cases
Incidence of dengue
(per 1000 person-years)
95% CI of incidence rate (n / 1000 P-Y)
New enrolment
Exact (Poisson)
Normal approx
Normal approx
with design effect
After 
Y1
After 
Y2
After 
Y3
Lower
Upper
Lower
Upper
Lower
Upper
3600
1
3322.7
30
9.0
6.1
12.9
5.8
12.2
4.7
13.3
3600
1
3322.7
40
12.0
8.6
16.3
8.3
15.7
7.0
17.0
3600
1
3322.7
50
15.0
11.1
19.8
10.8
19.2
9.4
20.6
3600
4
8611.3
26
3.0
2.0
4.4
1.8
4.2
1.4
4.6
170
450
3600
4
8611.3
52
6.0
4.5
7.9
4.4
7.6
3.8
8.2
170
450
3600
4
8611.3
78
9.0
7.1
11.2
7.0
11.0
6.3
11.7
170
450
3600
4
8611.3
103
12.0
9.8
14.5
9.7
14.3
8.9
15.1
170
450
3600
4
8611.3
129
15.0
12.5
17.8
12.4
17.6
11.5
18.5
170
450
1500
1
1384.5
4
3.0
0.8
7.6
0.1
5.9
-0.9
6.9
1500
1
1384.5
8
6.0
2.6
11.7
1.9
10.1
0.5
11.5
1500
1
1384.5
12
9.0
4.7
15.6
4.0
14.0
2.3
15.7
1500
1
1384.5
17
12.0
6.9
19.3
6.2
17.8
4.3
19.7
1500
1
1384.5
21
15.0
9.2
22.9
8.5
21.5
6.3
23.7
3000
1
2768.9
8
3.0
1.3
5.9
1.0
5.0
0.3
5.7
3000
1
2768.9
17
6.0
3.5
9.7
3.1
8.9
2.1
9.9
3000
1
2768.9
25
9.0
5.8
13.3
5.5
12.5
4.3
13.7
3000
1
2768.9
33
12.0
8.3
16.8
7.9
16.1
6.5
17.5
3000
1
2768.9
42
15.0
10.8
20.3
10.4
19.6
8.9
21.1
3000
4
8306.7
25
3.0
1.9
4.4
1.8
4.2
1.4
4.6
225
450
450
3000
4
8306.7
50
6.0
4.4
7.9
4.3
7.7
3.8
8.2
225
450
450
3000
4
8306.7
75
9.0
7.1
11.3
7.0
11.0
6.3
11.7
225
450
450
3000
4
8306.7
100
12.0
9.8
14.6
9.6
14.4
8.8
15.2
225
450
450
3000
4
8306.7
125
15.0
12.5
17.9
12.4
17.6
11.5
18.5
225
450
450
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Cohort 
size
Number of
years
Number of
person-years
Number 
of
cases
Incidence of dengue
(per 1000 person-years)
95% CI of incidence rate (n / 1000 P-Y)
New enrolment
Exact (Poisson)
Normal approx
Normal approx
with design effect
After 
Y1
After 
Y2
After 
Y3
Lower
Upper
Lower
Upper
Lower
Upper
Site
600
1
553.8
2
3.0
0.2
12.3
-1.6
7.6
-3.1
9.1
600
1
553.8
3
6.0
1.2
17.0
-0.5
12.5
-2.7
14.7
600
1
553.8
5
9.0
2.7
21.4
1.1
16.9
-1.6
19.6
600
1
553.8
7
12.0
4.6
25.1
2.9
21.1
-0.2
24.2
600
1
553.8
8
15.0
6.5
29.2
4.8
25.2
1.3
28.7
600
3
1486.0
4
3.0
0.8
7.5
0.2
5.8
-0.7
6.7
67
600
3
1486.0
9
6.0
2.7
11.5
2.1
9.9
0.7
11.3
67
600
3
1486.0
13
9.0
4.8
15.3
4.2
13.8
2.5
15.5
67
600
3
1486.0
18
12.0
7.1
19.0
6.4
17.6
4.5
19.5
67
600
3
1486.0
22
15.0
9.4
22.6
8.8
21.2
6.6
23.4
67
600
4
1947.5
6
3.0
1.0
6.7
0.6
5.4
-0.3
6.3
67
75
600
4
1947.5
12
6.0
3.1
10.6
2.6
9.4
1.4
10.6
67
75
600
4
1947.5
18
9.0
5.3
14.3
4.8
13.2
3.3
14.7
67
75
600
4
1947.5
23
12.0
7.6
17.9
7.1
16.9
5.5
18.5
67
75
600
4
1947.5
29
15.0
10.1
21.5
9.6
20.4
7.7
22.3
67
75
500
1
461.5
1
3.0
0.1
14.3
-2.0
8.0
-3.7
9.7
500
1
461.5
3
6.0
1.0
18.6
-1.1
13.1
-3.5
15.5
500
1
461.5
4
9.0
2.5
22.7
0.3
17.7
-2.6
20.6
500
1
461.5
6
12.0
4.2
26.8
2.0
22.0
-1.4
25.4
500
1
461.5
7
15.0
5.8
31.2
3.8
26.2
0.0
30.0
500
3
1384.5
4
3.0
0.8
7.6
0.1
5.9
-0.9
6.9
75
75
500
3
1384.5
8
6.0
2.6
11.7
1.9
10.1
0.5
11.5
75
75
500
3
1384.5
12
9.0
4.7
15.6
4.0
14.0
2.3
15.7
75
75
500
3
1384.5
17
12.0
6.9
19.3
6.2
17.8
4.3
19.7
75
75
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Cohort 
size
Number of
years
Number of
person-years
Number 
of
cases
Incidence of dengue
(per 1000 person-years)
95% CI of incidence rate (n / 1000 P-Y)
New enrolment
Exact (Poisson)
Normal approx
Normal approx
with design effect
After 
Y1
After 
Y2
After 
Y3
Lower
Upper
Lower
Upper
Lower
Upper
500
3
1384.5
21
15.0
9.2
22.9
8.5
21.5
6.3
23.7
75
75
500
4
1845.9
6
3.0
1.0
6.7
0.5
5.5
-0.4
6.4
75
75
75
500
4
1845.9
11
6.0
3.0
10.7
2.5
9.5
1.3
10.7
75
75
75
500
4
1845.9
17
9.0
5.2
14.5
4.7
13.3
3.2
14.8
75
75
75
500
4
1845.9
22
12.0
7.5
18.1
7.0
17.0
5.3
18.7
75
75
75
500
4
1845.9
28
15.0
9.9
21.7
9.4
20.6
7.5
22.5
75
75
75
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8.3.
Study cohorts to be evaluated
8.3.1.
Total cohort
The Total cohort will include all subjects enrolled in the study. 
8.3.2.
According-To-Protocol cohort
The ATP cohort will include all evaluable subjects, that is, subjects who meet all 
eligibility criteria, complying with the procedures defined in the protocol and for whom 
data of at least one follow-up contact are available. The surveillance period for one 
subject will be defined as the duration from date of enrolment until the date of the last 
follow-up contact for this subject. Reasons for elimination from ATP analyses will be 
established at the time of data cleaning and documented.
8.4.
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.
8.4.1.
Sequence of analyses
An interim analysis will be performed when subjects from the initial cohort of first two 
sites have completed two years of surveillance (Visit 4) and the third site have completed 
at least one year of surveillance (Visit 3). All analyses of primary and secondary 
objectives will be performed on data as cleaned as possible.
The final analysis will be performed when all prospective data have been collected and 
cleaned.
8.5.
Statistical methods
Any deviation or change from the original statistical plan will be described and justified 
in the final study report. 
All the analyses of primary objectives will be performed on the ATP cohort. Analysis of 
demographics will be performed on the total cohort.
8.5.1.
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 summarized 
overall and by region, and at the beginning of each season using descriptive statistics.
 
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8.5.2.
Analysis of primary and secondary endpoints
The following analyses will be performed overall and by DENV type, study site, gender 
and age-group previous dengue exposure (Yes = DENV IgG antibodies at previous visit 
and No = no DENV IgG antibodies at previous visit):

Incidence rate of laboratory-confirmed symptomatic dengue infection with 95% CI 
for each season separately: the numerator will be the number of subjects with lab-
confirmed symptomatic dengue infection during the season (between the visits 
scheduled before and after the season). The denominator will be the total person-
years at risk, i.e. from the visit scheduled before the season until the first RT-qPCR 
confirmed symptomatic dengue infection during the season, the next scheduled visit 
or the subject’s withdrawal, whichever comes first.

Incidence rate of laboratory-confirmed symptomatic dengue infection with 95% CI 
all seasons combined: the numerator will be the number of subjects with lab-
confirmed symptomatic dengue infection during the study period. The denominator 
will be the total person-years at risk, i.e. from enrolment until the first RT-qPCR 
confirmed symptomatic dengue infection, the end of the study or the subject’s 
withdrawal, whichever comes first.
The following analyses will be performed by study site, gender and age-group previous 
dengue exposure (primary or secondary):

Incidence rate of laboratory confirmed or probable symptomatic dengue infections 
with 95% CI.for each season separately and overall. The numerator will be the 
number of subjects with symptomatic dengue infection (including laboratory-
confirmed and probable cases) during the period. The denominator will be the total 
person-years at risk.

Incidence rate of probable symptomatic dengue infection with 95% CI for each 
season separately and overall. The numerator will be the number of subjects with 
probable symptomatic dengue infection. The denominator will be the total person-
years at risk.
The following analyses will be performed by study site, gender and age-group:

The proportion of subjects with primary inapparent dengue infection at each 
subsequent scheduled visit (not at enrolment) with 95% CI will be calculated as the 
number of dengue IgG positive cases and at the visit divided by the total number of 
subjects IgG negative at the previous visit. 

The proportion of subjects with secondary symptomatic dengue infection (probable 
or confirmed) at each subsequent scheduled visit (not at enrolment) with 95% CI will 
be calculated as the number of subjects with secondary symptomatic infection since 
the previous visit divided by the total number of IgG positive subjects at the previous 
visit.
 
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
The (crude) seroprevalence of dengue infection will be calculated at enrolment and 
at each scheduled visit as a proportion (i.e., the number of dengue IgG positive 
subjects tested positive at this visit or known to be positive from previous visits 
divided by the total number of subjects for whom dengue IgG serostatus is known). 

The clinical characteristics of symptomatic dengue infection (symptoms, 
hospitalizations, severity) will be presented as proportions of dengue cases overall 
and per sub-group (type (DENV 1-4), study site, gender and age group).
Endpoint numerators and denominators are described in Table 16 below:
Table 16
Numerators and Denominators within each strata (DENV type, study 
site, gender and age-group and previous dengue exposure (primary 
or secondary) 
Analysis
Numerator
Denominator
Incidence rate of laboratory 
confirmed symptomatic 
infection by year
All confirmed acute cases during the 
season (between the visits scheduled 
before and after the season)
Total person-years at risk, i.e. from 
the visit scheduled before the season 
until the first RT-qPCR confirmed 
symptomatic dengue infection during 
the season, the next scheduled visit 
or withdrawal, whichever comes first.
Incidence rate of laboratory-
confirmed symptomatic 
dengue infection for all 
seasons combined
All confirmed acute cases during the 
study period
Total person-years at risk, i.e. from 
enrolment until the first RT-qPCR 
confirmed symptomatic dengue 
infection, the end of the study or 
withdrawal, whichever comes first.
Incidence rate of laboratory 
confirmed or probable 
symptomatic dengue 
infections 
All symptomatic dengue infections 
(including laboratory-confirmed and 
probable cases) during the period.
Total person-years at risk*.
Incidence rate of probable 
symptomatic dengue infection 
All probable symptomatic dengue 
infection
Total person-years at risk*.
Proportion of subjects with 
primary inapparent dengue 
infection
All dengue IgG positive cases at the 
considered visit
Total number of IgG negative 
subjects at the previous visit
Proportion of subjects with 
secondary symptomatic 
dengue infection
All symptomatic infection since the 
previous visit
Total number of IgG positive subjects 
at the previous visit
Seroprevalence of dengue 
infection
All dengue IgG positive cases
Total number of subjects tested or 
previously positive
* Time at risk: In analyses by season the time at risk is calculated as the time from the visit scheduled before the 
season until the first event during the season, the next scheduled visit or the subject’s withdrawal, whichever comes 
first.
In the analyses of all seasons combined the time at risk is calculated as the time from enrolment until the first event, 
the end of the study or the subject’s withdrawal, whichever comes first.
CIs for incidence rates and proportions will account for clustering of observations within 
households for the analyses by centre and also the stratification by centres for the overall 
analyses. Details of the methods will be described in the SAP.
 
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8.5.3. Analysis of symptoms and spectrum of dengue disease
The clinical characteristics of symptomatic dengue infection (symptoms, hospitalizations, 
severity) will be presented.
8.5.4. Analysis of tertiary objectives (Amended 16 October 2017)
Analysis of tertiary objectives is optional and may or may not be performed. If these 
analyses are performed, they will be conducted by the Research and Development 
Department. Statistical methods for tertiary objectives will be described in the SAP.
The neutralization assay, if performed, will only be done on blood samples from a subset 
of subjects. YF virus antibody testing will be performed retrospectively, for subjects in 
YF endemic region and who was tested positive for dengue.
Analysis of risk factors for symptomatic dengue
Poisson regression models will be used for exploring the risk factors (e.g. region, age and 
gender, etc.) for symptomatic dengue. The analyses will account for clustering of 
observations within households for the analyses by centre and also the stratification by 
centres for the overall analyses.
Incidence rates of symptomatic Zika infections
The following analyses will be performed by study site, gender and age-group, previous 
dengue exposure and previous Zika infection:

Incidence rate of laboratory-confirmed or probable symptomatic Zika infection 
with 95% CI for each season separately: the numerator will be the number of 
subjects with lab-confirmed or probable symptomatic Zika infection during the 
season (between the visits scheduled before and after the season). The 
denominator will be the total person-years at risk, i.e. from the visit scheduled 
before the season until the first lab-confirmed or probable symptomatic Zika
infection during the season, the next scheduled visit or the subject’s withdrawal, 
whichever comes first.

Incidence rate of laboratory-confirmed or probable symptomatic Zika infection 
with 95% CI all seasons combined: the numerator will be the number of subjects 
with lab-confirmed or probable symptomatic Zika infection during the study Zika
follow-up period. The denominator will be the total person-years at risk, i.e. from 
start of the study Zika follow-up period until the first lab-confirmed or probable 
symptomatic Zika infection, the end of the study or the subject’s withdrawal, 
whichever comes first.
 
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Incidence rates of symptomatic chikungunya infections
The following analyses will be performed by study site, gender and age-group, previous 
dengue exposure and previous chikungunya infection:

Incidence rate of laboratory-confirmed or probable symptomatic chikungunya 
infection with 95% CI for each season separately: the numerator will be the 
number of subjects with lab-confirmed or probable symptomatic chikungunya 
infection during the season (between the visits scheduled before and after the 
season). The denominator will be the total person-years at risk, i.e. from the visit 
scheduled before the season until the first lab-confirmed or probable symptomatic 
chikungunya infection during the season, the next scheduled visit or the subject’s 
withdrawal, whichever comes first.

Incidence rate of laboratory-confirmed or probable symptomatic chikungunya 
infection with 95% CI all seasons combined: the numerator will be the number of 
subjects with lab-confirmed or probable symptomatic chikungunya infection 
during the study chikungunya follow-up period. The denominator will be the total 
person-years at risk, i.e. from start of the study chikungunya follow-up period until 
the first lab-confirmed or probable symptomatic chikungunya infection, the end of 
the study or the subject’s withdrawal, whichever comes first.
Analysis for Zika and chikungunya viruses in suspected dengue cases
The proportions and associated exact 2-sided 95% confidence intervals (CI)of the Zika
and chikungunya viruses in suspected dengue cases will be summarized.
Analysis for seroprevalence of antibody titers against Zika and chikungunya viruses at 
selected timepoints
Seroprevalence at each selected timepoint will be calculated as a proportion i.e. the 
number of seropositive samples at each selected timepoint over the total number of 
samples with antibody results. An exact 95% CI will be computed.
8.5.5. Statistical considerations for interim analyses
Since there is no hypothesis testing, no adjustment of type I error is needed for the 
interim analyses. 
9.
ADMINISTRATIVE MATTERS
To comply with ICH GCP or other applicable guidelines administrative obligations 
relating to data collection, monitoring, archiving data, audits, confidentiality and 
publications must be fulfilled. This study will be sponsored by GSK and co-funded by 
GSK and Fiocruz. As study sponsor, GSK will delegate some activities to Fiocruz, 
according to the provisions in their Cooperative Research and Development Agreement 
(CRADA).
 
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9.1.
Remote Data Entry instructions
Inform, a validated computer application, will be used as the method for data collection
on electronic case report form (eCRF).
In all cases, subject initials will not be collected nor transmitted to GSK. Subject data 
necessary for analysis and reporting will be entered/transmitted into a validated database 
or data system. Clinical data management will be performed in accordance with 
applicable GSK standards and data cleaning procedures.
While completed eCRFs are reviewed by a GSK Biologicals’ Site Monitor at the study 
site, omissions or inconsistencies detected by subsequent eCRF review may necessitate 
clarification or correction of omissions or inconsistencies with documentation and 
approval by the investigator or appropriately qualified designate. In all cases, the 
investigator remains accountable for the study data. 
The investigator will be provided with a CD-ROM of the final version of the data 
generated at the investigational site once the database is archived and the clinical study 
report is complete and approved by all parties.
9.2.
Monitoring by GSK Biologicals
Monitoring visits by a GSK Site Monitor are for the purpose of confirming that GSK 
Biologicals’ sponsored studies are being conducted in accordance with the ethical 
principles that have their origins in the Declaration of Helsinki and that are consistent 
with Good Clinical practice (GCP) or other applicable guidelines and the applicable 
regulatory requirement(s) (verifying continuing compliance with the protocol, 
amendment(s), verifying that the site staff and facilities continue to be adequate to 
conduct the study). 
The investigator must ensure provision of reasonable time, space and qualified personnel 
for monitoring visits.
Direct access to all study-site related and source data is mandatory for the purpose of 
monitoring review. The monitor will perform an eCRF review and a Source Document 
Verification (SDV). By SDV we understand verifying eCRF entries by comparing them 
with the source data that will be made available by the investigator for this purpose. 
The Source Documentation Agreement Form describes the source data for the different 
data in the eCRF. This document should be completed and signed by the site monitor and 
investigator and should be filed in the monitor’s and investigator’s study file. Any data 
item for which the eCRF will serve as the source must be identified, agreed and 
documented in the source documentation agreement form. 
For eCRF, the monitor could validate the screens at each visit. 
 
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In accordance with applicable regulations, GCP or other applicable guidelines and GSK 
procedures, 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.
The study will be monitored to verify that, among others, the: 

Data are authentic, accurate, and complete.

Safety and rights of subjects are being protected.

Study is conducted in accordance with the currently approved protocol and any 
amendments, any other study agreements, GCP or other applicable guidelines and all 
applicable regulatory requirements. 
The investigator and the head of the medical institution (where applicable) agree to allow 
the monitor direct access to all relevant documents.
Upon completion or premature discontinuation of the study, the monitor will conduct site 
closure activities with the investigator or site staff, as appropriate, in accordance with 
applicable regulations, GCP or other applicable guidelines and GSK procedures.
9.3.
Archiving of data at study sites
Following closure of the study, the investigator must maintain all site study records in a 
safe and secure location. The records must be maintained to allow easy and timely 
retrieval, when needed (e.g. audit or inspection), and, whenever feasible, to allow any 
subsequent review of data in conjunction with assessment of the facility, supporting 
systems, and staff. Where permitted by applicable laws/regulations or institutional policy, 
some or all of these records can be maintained in a validated format other than hard copy 
(e.g. microfiche, scanned, electronic for studies with an eCRF); however, caution needs 
to be exercised before such action is taken. The investigator must assure that all 
reproductions are legible and are a true and accurate copy of the original and meet 
accessibility and retrieval standards, including re-generating a hard copy, if required. 
Furthermore, the investigator must ensure there is an acceptable back-up of these 
reproductions and that an acceptable quality control process exists for making these 
reproductions.
GSK will inform the investigator/institution of the time period for retaining these records 
to comply with all applicable regulatory requirements. However, the 
investigator/institution should seek the written approval of the sponsor before proceeding 
with the disposal of these records. The minimum retention time will meet the strictest 
standard applicable to that site for the study, as dictated by ICH GCP or other applicable 
guidelines any institutional requirements or applicable laws or regulations, or GSK 
standards/procedures; otherwise, the minimum retention period will default to 25 years. 
 
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The investigator/institution must notify GSK of any changes in the archival 
arrangements, including, but not limited to, the following: archival at an off-site facility, 
transfer of ownership of the records in the event the investigator leaves the site. 
9.4.
Audits
To ensure compliance with GCP or other applicable guidelines and all applicable 
regulatory requirements, GSK may conduct a quality assurance audit. Regulatory 
agencies may also conduct a regulatory inspection of this study. Such audits/inspections 
can occur at any time during or after completion of the study. If an audit or inspection 
occurs, the investigator and institution agree to allow the auditor/inspector direct access 
to all relevant documents and to allocate his/her time and the time of his/her staff to the 
auditor/inspector to discuss findings and any relevant issues.
9.5.
Posting of information on public registers
Study information from this protocol will be posted on public registers (e.g. GSK Clinical 
Study Register, clinicaltrials.gov) before enrolment of subjects begins as applicable.
9.6.
Ownership, confidentiality and publication
9.6.1.
Ownership
All information provided by GSK and all data and information generated by the site as 
part of the study (other than a subject’s medical records) are owned by GSK and Fiocruz  
who shall each be entitled to use the results according to the provisions of their 
Collaborative Research and Development Agreement (CRADA) on Dengue.
All rights, title, and interests in any inventions, know-how or other intellectual or 
industrial property rights which are conceived or reduced to practice by site staff during 
the course of or as a result of the study are the sole property of GSK, and are hereby 
assigned to GSK
9.6.2.
Confidentiality
Documented evidence that a potential investigator is aware and agrees to the confidential 
nature of the information related to the study must be obtained by means of a 
confidentiality agreement.
All information provided by GSK, and all data and information generated by the site as 
part of the study (other than a subject’s medical records) will be kept confidential by the 
investigator and other site staff. This information and data will not be used by the 
investigator or other site personnel for any purpose other than conducting the study. 
These restrictions do not apply to: (i) information which becomes publicly available 
through no fault of the investigator or site staff; (ii) information which is necessary to 
disclose in confidence to an IEC or IRB solely for the evaluation of the study; (iii) 
information which is necessary to disclose in order to provide appropriate medical care to 
 
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a study subject; or (iv) study results which may be published as described in the next 
paragraph. 
If a written contract for the conduct of the study which includes confidentiality provisions 
inconsistent with this statement is executed, that contract’s confidentiality provisions 
shall apply rather than this statement.
9.6.3.
Publication
Prior to submitting for publication, presentation, use for instructional purposes, or 
otherwise disclosing the study results generated by the site (collectively, a ‘Publication’), 
the investigator shall provide GSK and Fiocruz with a copy of the proposed Publication 
and allow GSK and Fiocruz a period to review the proposed Publication (at least twenty-
one working days, or at least fifteen working days for abstracts/posters/presentations). 
Proposed Publications shall not include either GSK confidential information other than 
the study results or personal data on any subject, such as name or initials.
At GSK’s request, the submission or other disclosure of a proposed Publication will be 
delayed a sufficient time to allow GSK to seek patent or similar protection of any 
inventions, know-how or other intellectual or industrial property rights disclosed in the 
proposed Publication.
If a written contract for the conduct of the study, which includes publication provisions 
inconsistent with this statement is executed, that contract’s publication provisions shall 
apply rather than this statement.
9.6.4.
Provision of study results to investigators and publication
Where required by applicable regulatory requirements, an investigator signatory will be 
identified for the approval of the study report. The investigator will be provided 
reasonable access to statistical tables, figures, and relevant reports and will have the 
opportunity to review the complete study results at a GSK or Fiocruz site or other
mutually-agreeable location.
A manuscript will be progressed for publication in the scientific literature if the results 
provide important scientific or medical knowledge.
10.
COUNTRY SPECIFIC REQUIREMENTS
Not applicable
 
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11.
REFERENCES (AMENDED 16 OCTOBER 2017)
Bennett S, Woods T, Liyanage W, Smith DL. A simplified general method for cluster-
sample surveys of health in developing countries World Health Stat Q. 1991; 44(3): 98-
106.
Boletim Epidemiológico - Volume 48 - n 05 - 2017 - Secretaria de Vigilância em Saúde 
− Ministério da Saúde. Accessed at : http://combateaedes.saude.gov.br/images/pdf/2017-
Dengue_Zika_Chikungunya-SE4.pdf. Accessed on 16 October 2017.
Brandt W.  From the World Health Organization: development of dengue and Japanese 
Encephalitis vaccines. J Inf Dis. 1990; 162(3): 577-83.
Brazil MoH (Ministry of Health) DATASUS, 2011 www.datasus.gov.br 
Edelman R, Hombach J, “Guidelines for the clinical evaluation of dengue vaccines in 
endemic areas”: Summary of a World Health Organization Technical Consultation, 
Vaccine, Volume 26, Issue 33, 5 August 2008, Pages 4113-4119, ISSN 0264-410X, 
10.1016/j.vaccine.2008.05.058
PAHO 17 Feb 2011, Epidemiological Alert: Dengue in the Americas 
http://new.paho.org/hq/index.php?option=com_content&task=view&id= 
264&Itemid=363&lang=es
Pontes RJ, Freeman J, Oliveira-Lima JW, Hodgson JC, Spielman A. Vector densities that 
potentiate dengue outbreaks in a Brazilian city. Am J Trop Med Hyg. 2000; 62: 378–383.
Siqueira, JB, Martelli CM, Maciel IJ, Oliveira RM, Ribeiro MG, Amorim FP, Moreira 
BC, Cardoso DD, Souza WV, Andrade AL. Household survey of dengue infection in 
Central Brazil: spatial point pattern analysis and risk factors assessment. Am J Trop Med 
Hyg. 2004; 71(4): 646-51.
Siqueira-Junior J, Maciel I, Barcellos C, Souza W, Carvalho M, Nascimento N, Oliveira 
R, Morais-Neto O,  Martelli C. Spatial point analysis based on dengue surveys at 
household level in central Brazil BMC Public Health, 2008; 8: 361-70.
Standish K, Kuan G, Aviles W, Balmaseda A, Harris E. High Dengue Case Capture Rate 
in Four Years of a Cohort Study in Nicaragua Compared to National Surveillance Data. 
PLoS Negl Trop Dis. 2010; 4(3): e633. doi:10.1371/journal.pntd.0000633
Suaya J, Shepard D, Beatty M. Dengue: burden of disease and costs of illness. WHO on 
behalf of the Special Programme for Research and Training in Tropical Diseases, 2007 
Geneva, 1–5 October 2006 
http://www.who.int/tdr/publications/publications/swg_dengue_2.htm
Thomas S.  The Necessity and Quandaries of Dengue Vaccine Development  J Inf Dis. 
2011; 203: 299–303.
 
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Vong S, Huy R, Ong S, Teurlai M, Ly S, Beauté J, Duong V, Buchy P. Epidemiology 
and burden of dengue in Cambodia 2011 BMC Proceedings. 2011; 5(Suppl 1):49.
Wichmann, O, Yoon I-K, Vong S, Limkittikul K, Gibbons R, Mammen M, Ly S, Buchy 
P, Sirivichayakul C, Buathong R, Huy R, Letson W, Sabchareon A. Dengue in Thailand 
and Cambodia: an assessment of the degree of under recognized disease burden based on 
reported cases PLoS Negl Trop Dis. 2011; 5(3) e996. 
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
STUDY LABORATORIES (Amended 16 October 
2017)
GSK Biologicals’ laboratories (for back up samples)
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
Non GSK laboratories
Laboratory
Address
Local laboratories
Details presented in a separated document
Laboratório de Tecnologia 
Virológica – LATEV
Bio-Manguinhos
Fundação Oswaldo Cruz - Fiocruz
Av. Brasil, 4365, Manguinhos
Pavilhão Rocha Lima, sala 403
Rio de Janeiro, RJ. CEP: 21.040-360
Brazil
 
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APPENDIX B
STRUCTURED SCRIPT
1.
Have you/has your child been sick in the past month?
(    ) Yes
(    ) No
2.
If yes, did you/your child have:
(    ) Fever 
(    ) Headache
(    ) retro-orbital pain
(    ) rash
(    ) muscle or joint pain
(    ) bleeding
3.
Have you sought medical care due to your/your child’s symptoms?
4.
If yes, what was the diagnosis and where did you seek medical care?
5.
Are you aware of dengue, chikungunya or Zika cases in your neighborhood during 
the past month?
Any subject that meets the case definition for suspected dengue, chikungunya or Zika
the subject (and subject’s parent(s)/LAR(s) as applicable) will be referred to the study 
hospital according to protocol
 
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APPENDIX C
AMENDMENTS AND ADMINISTRATIVE 
CHANGES TO THE PROTOCOL
GlaxoSmithKline Biologicals
Clinical Research & Development
Protocol Amendment 1 
eTrack study number and 
Abbreviated Title
116606 (EPI-DENGUE-006 BOD BR)
Amendment 1 version and date:
Final: 14 June 2013
Coordinating author:
, Scientific Writer
Rationale/background for changes: 
The Brazilian IRB requested that the protocol be amended to address the shared 
responsibilities of Fiocruz and GSK with regard to the following statement in the 
protocol Section 9.6.1. Ownership. According to the Brazilian resolution CNS 292/99, 
“The onus and benefits came from the investigation process and research results must 
be distributed in a fair way between the involved parts, and must be described in the 
protocol.” 
After discussions between GSK and Fiocruz legal, it was decided that Fiocruz should 
also have the property of study data. GSK will continue to be the only sponsor but 
since the study will be funded by both institutions, both will have access to study data 
as in accordance with CRADA.
Additional changes related to this request, and minor unrelated changes have also been 
made. A detailed list of changes is provided below:
Section 9.6.1 Ownership:All information provided by GSK and all data and 
information generated by the site as part of the study (other than a subject’s medical 
records) are owned by GSK and Fiocruz  who shall each be entitled to use the results 
according to the provisions of their Collaborative Research and Development 
Agreement (CRADA) on Dengue.  
Section 9.6.3 Publication
Prior to submitting for publication, presentation, use for instructional purposes, or 
otherwise disclosing the study results generated by the site (collectively, a 
‘Publication’), the investigator shall provide GSK and Fiocruz with a copy of the 
proposed Publication and allow GSK and Fiocruz a period to review the proposed 
Publication (at least twenty-one working days, or at least fifteen working days for 
abstracts/posters/presentations).
Section 9.6.4 Provisions of study results to investigators and publication
Where required by applicable regulatory requirements, an investigator signatory will be 
identified for the approval of the study report. The investigator will be provided 
reasonable access to statistical tables, figures, and relevant reports and will have the 
opportunity to review the complete study results at a GSK or Fiocruz site or other 
mutually-agreeable location. 
 
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Other minor changes: 
Title page: 
Added line for Amendment 1 version and date
Changed 2012 to 2012-2013 in this line: Copyright 2012-2013 the GlaxoSmithKline 
group of companies
Signature pages: 
Changed heading from Protocol Sponsor Signatory Approval to Protocol Amendment 1  
Sponsor Signatory Approval and same change for Investigator Agreement
Page 3: Protocol Amendment Rationale added 
List of Abbreviations: added CRADA and Fiocruz
Section 5.5.6.3 An example of questions to be included in the The telephone script is 
provided in …
Added Appendix C: Amendments and Administrative Changes to the protocol.
 
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GlaxoSmithKline Biologicals
Clinical Research & Development
Protocol Amendment 2
eTrack study number and Abbreviated 
Title
116606 (EPI-DENGUE-006 BOD BR)
Amendment number:
Amendment 2
Amendment 2 Final Date:
Final: 23 July 2014
Coordinating author:
, Scientific Writer, Out-
contractor for GSK
Rationale/background for changes:
The protocol is amended to allow for extension of the study by three years to cover 
three additional dengue seasons. There will be three scheduled visits (Visit 1, 2 and 3) 
during the first study year and one scheduled visit per year during study Year 2, Year 3 
and Year 4 (Visit 4, 5 and 6). The scheduled visits will preferably take place during the 
low transmission period. A blood sample will be collected at each scheduled visit. The 
objectives have been updated to reflect the study extension.
This protocol amendment will also allow enrolment of new subjects to replace the 
subjects not wishing to extend their participation for three additional years, subjects 
who prematurely terminate participation and subjects lost to follow-up in order to 
maintain a cohort size of at least 1500 subjects during each year/season. A section on 
how subjects will be replaced was added.
An interim analyses is also planned.
Additional minor corrections have been made.
Amendment 2 List of Changes: deleted text shown with strikethrough, new text 
shown with bold italics
Title page: 
Added line for Amendment 2 version and date.
The word “multiyear” was added to the Detailed Title as follows: A prospective, 
multicentre, multiyear cohort study to determine dengue incidence in children and adults 
in communities from endemic regions in Brazil.
Co-ordinating and contributing authors for this amendment 2 were as follows: 
Co-ordinating author:
, Scientific Writer / 
 
, Scientific Writer, XPE working for GSK 
Biologicals 
 
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GSK Contributing authors:
 
Epidemiologist, Brazil
Epidemiologist, Senior Manager
Epidemiologist 
Project Statistician
Project Statistician
Senior Manager Lead statistician
Study Manager
 
Study Manager
Study Delivery Lead
Global Study Manager (advisory role)
Global Vaccine Clinical Laboratories Manager 
Local Delivery Lead
Clinical Laboratory Project Manager
Senior Manager, Global Study Management 
Clinical Operations Manager
Clinical Laboratory Project Manager
Global Vaccine Clinical Laboratories Manager 
Clinical Safety representative 
Study Data Manager
Study Data Manager
Medical Affairs Director, Brazil
Medical Affairs Director, Brazil
Regional Health Outcomes Director
Regional Health Outcomes Director
Director, Clinical Research & Translational Science
Non-GSK Contributing 
authors:
Bio-Manguinhos, Fiocruz
Bio-Manguinhos, Fiocruz/ad hoc
Principal Investigator, Manaus, Fundação de 
Medicina Tropical Dr. Heitor Vieira Dourado
Principal Investigator, Salvador de Bahia, Fundação 
Oswaldo Cruz, Ministério da Saúde
Principal Investigator, Rio de Janeiro, Fundação 
Oswaldo Cruz, Ministério da Saúde
Changed 2012-2013 to 2012-2014 in this line: Copyright 2012-2014 the 
GlaxoSmithKline group of companies
Signature pages: 
Changed heading from Protocol Amendment 1 to Protocol Amendment 2 for Sponsor 
Signatory Approval and same change for Investigator Agreement.
Page 4: Protocol Amendment 2 Rationale added 
Additional minor corrections like bulleting and colouring the table headings grey were 
done.
 
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GLOSSARY OF TERMS: added Block, Initial cohort subjects, Replacement subjects, 
Lost to follow-up, Prematurely terminate participation and Primary dengue infection.
Section 1.1 Background: The first and the last paragraphs were changed as follows: 
Dengue, the most common arthropod-borne viral disease worldwide, is caused by four 
types of dengue viruses (DENV 1-4), transmitted primarily by Aedes aegypti, a mosquito 
that is highly adapted to urban environments. Dengue infection can cause a range of 
clinical illnesses, from inapparent to a life threatening hemorrhagic disease, often 
associated with pre-existing heterotypic dengue virus antibodies. 
In this study, we will estimate the incidence of dengue infection and disease in a cohort 
of subjects recruited from different geographic areas over time in Brazil. The study will 
also identify and train potential sites for the conduct of phase III studies in the future.
Section 2 Objectives: The objectives were changed as follows with the same changes 
effected in the Synopsis objectives:
Section 2.1
Primary objective

To estimate the incidence of laboratory-confirmed symptomatic dengue infection in 
the study population by year/season.
Section 2.2
Secondary objectives

To estimate the serotype-specific incidence of virologically-confirmed symptomatic 
dengue infection in the study population overall and by season.

To estimate the incidence of symptomatic dengue infection (including laboratory-
confirmed and probable cases) in the study population by DENV type study site, 
gender, age-group, previous dengue exposure (primary or secondary), overall and by 
season.

To estimate the prevalence of previous dengue infection (dengue seroprevalence) in 
the study population overall and by study site, gender and age-group at enrolment.

To estimate the incidence of primary inapparent, dengue infection, in the study 
population overall and by study site, gender age-group and by season.
Section 2.3
Tertiary objectives (optional): The following objective was added.

To describe the spatial and temporal distribution of dengue cases among cohort 
participants in the study areas.
Section 3
Study design overview
This section was changed as follows in the body and respective sections of the Synopsis :

Type of design: Prospective, multicenter, community-based, household-sampling, 
cohort study in Brazil.

The study period, initially planned to be one year, is extended by three years to 
cover three additional dengue seasons. 
 
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
Initial cohort subjects will be invited to extend their participation. 
(See glossary of terms for the definition of initial cohort subjects).

Replacement subjects may be enrolled in order to compensate for subjects who do 
not wish to extend their participation for three additional years, subjects who 
prematurely terminate participation or subjects who are lost to follow-up. This will 
be done to maintain a cohort size of at least 1500 subjects at the beginning of each 
additional study year/season.
(See glossary of terms for the definition of replacement subjects, subjects lost to follow-
up and subjects who prematurely terminate participation ).

Informed consent (and assent, if applicable) will be obtained:

From the parents/LAR of the subjects (and from the subjects, if applicable) at 
the study start for the initial cohort.

From the parents/LAR of the subjects (and from the subjects, if applicable) of 
the initial cohort prior to participation in the additional three years of the 
study. 

From the parents/LAR of the replacement subjects (and from the replacement  
subjects, if applicable) prior to enrolment in the study.

Study visits: The study will consist of three scheduled household visits over a period 
of approximately one year, per subject. Preferably, the recruitment period will occur 
before the peak incidence of dengue based on previous years
The visits will be as follows and further shown in Figure 1:

Initial cohort subjects consenting to extend participation for the three 
additional years will have:
o
three scheduled visits in the first year (Visit 1, Visit 2 and Visit 3) at 
approximately six months intervals, 
o
and thereafter, one scheduled visit per year during a period of low 
dengue transmission for the three additional years; i.e; one visit at study 
Year 2, Year 3 and Year 4 (Visit 4, Visit 5 and Visit 6 respectively).

Initial cohort subjects who do not consent to extend participation for three 
additional years will:
o
Have three scheduled visits (Visit 1, Visit 2 and Visit 3) at approximately 
six months intervals. 
o
Conclude their participation in the study at Visit 3 or the last follow up 
visit if the subject is suspected of having a dengue case on-going at Visit 
3.

For replacement subjects, the number of scheduled visits will depend on the 
study year in which they are enrolled. Replacement subjects will not be 
enrolled until after the third visit of the first study year is concluded for the 
initial cohort. The number of scheduled visits will range between two and 
four.
 
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The yearly scheduled visits for the initial cohort and replacement subjects will 
preferably occur during the period of usually low dengue transmission. 
(Synopsis) Figure 1 Schematic representation of the scheduled visits 
V=Visit
The scheduled visits for initial cohort subjects are shown in black.
New enrolments will be done yearly (as needed) till the end of study Year 3. 
For replacement subjects, the number of visits will depend on the year enrolled. These visits are shown in 
different colours for each year.

Duration of the study: approximately one year per subject from the time of consent
Four years for the overall study. For an individual subject, it will range between 
one and four years.

Epoch 001: prospective data collection starting at Visit 1 Day 0 and ending at 
Visit 3 Year 1 the last subject last visit 
(Synopsis)Table 2
Study groups and epochs foreseen in the study 
Study Group
Number of subjects*
Age 
(Minim
um)
Epoch
Prospective
1800 (planned enrollment) at 
enrolment (first year)
At least 1500 in each 
subsequent year 
6 
months 
Epoch 001
*The final number of participants will take into account the active subjects, subjects who prematurely 
terminate participation and subjects lost to follow up.
Section 3.1
Discussion of study design
3.1.1
Selection of study sites: Specific cities where the study will be conducted were 
deleted as follows:
At least three sites in Brazil covering at least 3 different regions of the country will be 
selected for this study, one each in Manaus, Salvador and Rio de Janeiro. 
3.1.2
Rationale for study design: The second sentence in the first paragraph was 
modified as follows:
Data will be collected from a population of individuals aged 6 months and older for at
least one maximum four dengue seasons.
 
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Section 4
Study population: Specific cities where the study will be conducted were 
deleted as follows: This change was also implemented in the Synopsis.
Subjects six months of age and older at the time of enrolment who live in the selected 
study sites in Brazil, covering at least 3 different regions of the country.: North (Manaus), 
Northeast (Salvador) and Southeast (Rio de Janeiro). 
4.2
Selection of households; Some specifics in the last paragraph were deleted and 
some words added as follows:
Approximately 1800 subjects, 600 per site need to be recruited for this study in order to 
obtain an evaluable sample size of approximately 1500 subjects at the end of the first 
year of the study., 500 per site. Based on an average household size of 5 , approx. 125 
households should be selected per site.
4.3
Overview of the recruitment plan; This section was changed as follows:
Subjects will be recruited from randomly selected households originating from pre-
selected mapped communities. Preferably the recruitment period will occur outside of the 
peak dengue transmission season, and will continue until each site has reached its 
foreseen target. The expected period for recruiting the target sample size is approximately 
three months. Recruitment of replacement subjects will be done during the low dengue 
transmission and the recruitment period will depend on the number of subjects that 
need to be replaced.
Recruitment will be organized by study staff at participating sites according to the 
appropriate strategy for each site. Community agents or equivalent will serve as the 
liaison to schedule visits and may accompany study staff during the visits. The study will 
be explained to the individuals living in the household, and if any individual is interested 
in study participation, informed consent (and assent when applicable) will be obtained, 
eligibility criteria will be checked and subjects will be enrolled and interviewed for 
potential study participation.
If the target household is found empty or all members refuse to participate, the first 
household to the left will be approached. Those households refusing to participate will be 
recorded as such. A household refusal will be characterized when all individuals in the 
household refuse to participate in the study. Individual refusals in a given household will 
not preclude inclusion of other housemates, and will be recorded as such.
In the subsequent study years/seasons, the cohort size will be maintained at, at least 
1500 subjects. Since subjects who prematurely terminate participation and/or lost to 
follow-up will be replaced, the final number of participants across the four study years 
will exceed 1800. Yearly, there will be an evaluation of active subjects. If the number of 
active subjects becomes < 1500, there will be enrolment of replacement subjects to 
maintain a cohort size of 1500 subject. Enrolment will preferably take place during the 
low dengue transmission. Recruitment of replacement subjects could occur at the end 
of study year 1, 2 or 3. The recruitment approach will be the same as for the initial 
enrolment.
Household visits may be scheduled to occur during weekends or at convenient times 
(like evenings) if deemed necessary.
 
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4.4
Inclusion criteria for enrolment; This section was changed as follows:

Subject and/or subject’s parent(s)/LAR(s) who the investigator study staff believes 
can comply with the requirements of the protocol (e.g., willingness to go to the 
hospital/clinic for visit/s if dengue is suspected, able to observe the signs of dengue 
and to understand how to take and report body temperature, etc.)

Subject who plans, at the time of enrolment, to remain at same residence/study area 
during their study participation period.
Section 5
Study conduct
Section 5.4
Outline of study procedures; Table 2 was modified to procedures for 
initial cohort subjects including the three years extension and procedures for replacement 
subjects were included in a separate table, Table 3. Symbols for footnotes were changed 
to numbers.
Study procedures are outlined in Table 2 and Table 3 for initial cohort and replacement 
subjects respectively
 
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Table 2
List of study procedures for initial cohort subjects 
Surveillance
Suspected Dengue 
Visits (at 
hospital/clinic)
as applicable*3
Procedure
Visit 1
Day 0
Monthly 
contact
between 
visits
Visit 2
Month 6
Monthly 
contact 
between 
visits
Visit 3
Month 
12 (Year 1)
Monthly 
contact 
between 
visits
Visit 4
Month 
24 (Year 
2)
Monthly 
contact 
between 
visits
Visit 5
Month 
36 (Year 
3)
Monthly 
contact 
between 
visits
Visit 6
Month 
48 (Year 
4)
First 
visit 
Day 0
Return 
visit **2
Follow-
up visit
Day 21
Informed consent (and assent if 
applicable)
●
Re-consent (and assent if 
applicable)
●bullet 
added
Subject number and household number 
attribution
●
Check inclusion/exclusion criteria
●
Record socio-demographic information
●
Medical history and YF vaccination 
history or updates 
●
●
●
●
●
●
Distribute subject ID card distribution 
and suspected dengue instruction kit 

Blood sample for serology 5 mL
●
●
●
●
●
●
Instruct/ remind subjects/subject’s 
parent(s)/LAR(s) on  suspected dengue 
assessment procedures (where 
applicable)










Issue diary logs to parents/LARs to be 
filled out in the event of a suspected 
dengue case†1






Contact subject regarding any dengue 
symptoms and remind subject of 
procedures for suspected dengue





Physical examination/ record current 
medical conditions (see Section 5.6)
●
●
●
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Surveillance
Suspected Dengue 
Visits (at 
hospital/clinic)
as applicable*3
Procedure
Visit 1
Day 0
Monthly 
contact
between 
visits
Visit 2
Month 6
Monthly 
contact 
between 
visits
Visit 3
Month 
12 (Year 1)
Monthly 
contact 
between 
visits
Visit 4
Month 
24 (Year 
2)
Monthly 
contact 
between 
visits
Visit 5
Month 
36 (Year 
3)
Monthly 
contact 
between 
visits
Visit 6
Month 
48 (Year 
4)
First 
visit 
Day 0
Return 
visit **2
Follow-
up visit
Day 21
Blood sample for dengue infection 
diagnosis*3
●
●
Blood sample for CBC and hematocrit
●
Record body temperature
●
Report SAEs related to study 
procedures††4
●
●
●
●
●
●
●
●
●
Collect or verify diary logs if applicable



Study conclusion§5
●
●
● 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.
† 1Diary logs given at scheduled visits are only to be filled out in the event that the subject has a fever or suspected dengue symptoms. See Section 5.5.2.5 for details with regard to the 
diary log.
**2 A return visit is a visit linked to suspected dengue, occurring if the evolution of the subject’s physical condition necessitates medical evaluation, according to local medical practice. 
The investigator will determine whether this return visit is necessary and whether it is considered as part of the same episode. See Section 5.6 for details.
*3 May include testing for RT-qPCR, dengue IgG and IgM ELISA, NS1 ELISA, neutralizing antibodies to dengue and Yellow fever, as well as dengue virus isolation if deemed 
necessary.
††4SAE = Serious adverse event- see Section 6.1.1 for definition.
§5 The study conclusion information in the eCRF is collected once for each subject at the last scheduled visit or at the last follow-up contact for a suspected dengue case, should this 
case occur during the last scheduled visit or earlier if the subject terminates study participation or is lost to follow-up. 
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Table 3
List of study procedures for replacement subjects 
Surveillance
Suspected Dengue Visits (at 
hospital/clinic)
as applicable3
Procedure
Visit 1
Day 0
Monthly 
contact
between 
visits
Visit 2*
Monthly 
contact 
between 
visits
Visit 3*
as applicable
Monthly 
contact 
between 
visits
Visit 4 
as applicable
First visit 
Day 0
Return visit2 Follow-up 
visit
Day 21
Informed consent (and assent if 
applicable)
●
Subject number and household 
number attribution
●
Check inclusion/exclusion criteria
●
Record/update socio-demographic 
information
●
●*
Medical history and YF vaccination 
history or updates 
●
●
●
Distribute subject ID card 
distribution and suspected dengue 
instruction kit 



Blood sample for serology 5 mL
●
●
●
●
Instruct/ remind subjects/subject’s 
parent(s)/LAR(s) on  suspected 
dengue assessment procedures 
(where applicable)







Issue diary logs to parents/LARs to 
be filled out in the event of a 
suspected dengue case1





Contact subject regarding any 
dengue symptoms and remind 
subject of procedures for suspected 
dengue



Physical examination/ record 
current medical conditions (see 
Section 5.6)
●
●
●
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Surveillance
Suspected Dengue Visits (at 
hospital/clinic)
as applicable3
Procedure
Visit 1
Day 0
Monthly 
contact
between 
visits
Visit 2*
Monthly 
contact 
between 
visits
Visit 3*
as applicable
Monthly 
contact 
between 
visits
Visit 4 
as applicable
First visit 
Day 0
Return visit2 Follow-up 
visit
Day 21
Blood sample for dengue infection 
diagnosis3
●
●
Blood sample for CBC and 
hematocrit
●
Record body temperature
●
Report SAEs related to study 
procedures4
●
●
●
●
●
●
●
Collect or verify diary logs if 
applicable



Study conclusion5
●
●
● 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.
* This could be the first visit (enrolment) for some replacement subjects and the procedures for Visit 1 will be done as applicable.
1 Diary logs given at scheduled visits are only to be filled out in the event that the subject has a fever or suspected dengue symptoms. See Section 5.5.2.5 for details with 
regard to the diary log. 
2 A return visit is a visit linked to suspected dengue, occurring if the evolution of the subject’s physical condition necessitates medical evaluation, according to local 
medical practice. The investigator will determine whether this return visit is necessary and whether it is considered as part of the same episode. See Section 5.6 for 
details.
3 May include testing for RT-qPCR, dengue IgG and IgM ELISA, NS1 ELISA, neutralizing antibodies to dengue and Yellow fever, as well as dengue virus isolation if deemed 
necessary.
4 SAE = Serious adverse event- see Section 6.1.1 for definition.
5 The study conclusion information in the eCRF is collected once for each subject at the last scheduled visit or at the last follow-up contact for the dengue case, should 
this case occur during the last scheduled visit or earlier if the subject prematurely terminates participation or is lost to follow-up.
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Table with intervals between visits was deleted and separate tables made for scheduled 
visits and suspected dengue visits as follows:
Interval
Optimal length of interval *
Allowed interval**
Visit 1  (Day 0)   Visit 2 (Month 6)
6 months
±28 days 
Visit 2 (Month 6) Visit 3 (Year 1)
6 months
±28 days
Interval between visit for a suspected 
dengue case and the final follow-up 
visit for a case
21 days
+7 days 
*Whenever possible the investigator should arrange study visits/contacts within this interval
**Subjects will not be eligible for inclusion in the ATP cohort for analysis if they make the study visit/contact outside 
this interval
Table 4
Intervals between study visits 
Study Year*
Visits Interval
Optimal length of interval *
Allowed interval**
Year 1
Visit 1 (Day 0)   Visit 2 (Month 6)
6 months
±28 days
Visit 2 (Month 6) Visit 3 Month 12
6 months
The interval may be adapted 
to allow scheduling the visit 
in a period of generally low 
dengue transmission
Year 2
Visit X Visit X+1¶
Note: Visit X may be visit 3 for 
subjects enrolled in study year 1. 
For subjects enrolled in study year 
2, visit X would be visit 1.
12 months
The interval may be adapted 
to allow scheduling the visit 
in a period of generally low 
dengue transmission
Year 3
Visit X+1 Visit X+2
12 months
The interval may be adapted 
to allow scheduling the visit 
in a period of generally low 
dengue transmission
Year 4
Visit X Visit X+3
12 months
The interval may be adapted 
to allow scheduling the visit 
in a period of generally low 
dengue transmission
*Whenever possible the investigator should arrange study visits/contacts within this interval
**Subjects might not be eligible for inclusion in the ATP cohort for analysis if they make the study visit/contact outside 
this interval
Table 5
Interval between visits for suspected dengue case 
Optimal length of time
Allowed interval
Suspected dengue case: Interval 
between first and follow-up visit 
21 days
+7 days 
Note that this interval applies to all subjects enrolled regardless of the year of enrolment.
Section 5.5.1.1
Informed consent; The following text was added:

Initial cohort subjects will be required to have a new consent/assent (where 
applicable) form signed for continuous participation in the study to allow coverage 
of three additional dengue seasons.
 
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
Consent/assent (where applicable)will also be obtained prior to participation for 
replacement subjects.
The sections below were deleted and merged into section 5.5.3 with the following 
additions:
5.5.3 Procedures at scheduled Visit 2
5.5.4 Procedures at scheduled Visit 3 

Visit 3 will occur approximately 12 months after Visit 1. 

A blood (serum) sample will be collected 

Medical and vaccination histories will be updated in the eCRF

Any SAEs related to study procedures will be recorded.
Section 5.5.3 Procedures at subsequent scheduled visits (2-6, as applicable);
following text was added: 

For initial cohort subjects, Visit 2 will occur approximately 6 months after Visit 1 
and Visit 3 approximately 6 months after Visit 2. Initial cohort subjects willing to 
extend their participation to three additional years will be re-consented at Visit 3 
(ie at the end of study Year 1).

For replacement subjects enrolled in the subsequent study years, scheduled visits 
should ideally be performed at yearly intervals, preferably during periods of 
usually low dengue transmission
Section 5.5.4 Study conclusion; The following text was added to the first paragraph:
The study conclusion screen page in the eCRF will be completed at the last study contact. 
This last contact could occur at the last scheduled visit or follow up visit for a suspected 
dengue case (if it is on-going at the last scheduled visit) or earlier if the subject 
terminates study participation or is lost to follow-up.
Section 5.5.5.2
Case detection through enhanced passive surveillance
The last paragraph was bulleted as follows:
The study staff should ensure that:

the acute sample will be properly collected,

all clinical information will be retrievable, 

a return visit (if applicable) will be scheduled

and a follow-up visit will be scheduled.
 
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Section 5.5.5.3
Case detection through active surveillance
The second paragraph was changed as follows: 
An example of questions to be included in the telephone structured script is provided in 
APPENDIX B
Section 5.6
Management of suspected dengue cases; The first and last paragraphs 
were changed as follows:
All study subjects with suspected dengue should be seen at a designated study hospital or 
clinic by the study physician. 
Subjects who, for any reason, seek medical assistance at any non-study health care 
facility, will be identified through active surveillance, and clinical data will be 
retrospectively collected on the eCRF. The study physician will be responsible for 
collecting the retrospective data and informing the local study coordinator for appropriate 
follow-up. 
Section 5.7.1 Suspected symptomatic dengue case; The first paragraph was changed 
as follows:
Febrile illness with body temperature 38°C measured (by any route) on at least two 
consecutive days and less than 14 days with or without the presence of other dengue 
symptoms or signs, without an obvious etiology unrelated to dengue, based on 
investigator’s judgement.
Section 5.7.2 Laboratory-confirmed symptomatic dengue case:
At least one of the following findings must be met for a laboratory-confirmed dengue 
case: 

Dengue virus identification through RT-qPCR on the acute serum sample 

Dengue virus NS1 positive on acute serum sample through ELISA.

Anti-dengue IgM seroconversion between acute and convalescent serum samples 
through ELISA.
Section 5.8.2.1
Laboratory assays; The footnote to Table 6 (Humoral immunity) 
was changed as follows: 
*For subjects residing in Manaus a yellow fever endemic region or vaccinated against 
yellow fever
Section 5.8.2.2
Laboratory read-outs; The initial Table 7: Laboratory read-outs 
at each time point, and priority ranking was split into scheduled visits, early presenters 
and late presenters as follows:
 
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Table 7
Laboratory read-outs at each time point, and priority ranking
SCHEDULED VISITS 
Blood sampling time point
No. 
Subjects
(planned
enrollment)
Subset*
Component
Serum aliquot Components 
priority rank
Type of contact/ 
Amount of whole 
blood
Sampling time 
point
Scheduled Visit  1
5 mL whole blood 
(Day 0)
1800
-
Dengue IgG ELISA**
250 µL
1
yes
Dengue virus 1-4 and YF 
neutralizing antibodies
2 X 1 mL
2
Scheduled Visit  2 
5 mL whole blood
(Month  6)
1800
-
Dengue IgG ELISA**
250 µL
1
yes
Dengue virus 1-4 and YF 
neutralizing antibodies
2 X 1 mL 
2
Scheduled Visit  3 
5 mL whole blood
(Month 12)
1800
-
Dengue IgG ELISA**
250 µL
1
yes
Dengue  virus 1-4 and YF 
neutralizing antibodies
2 X 1 mL 
2
SUSPECTED DENGUE VISITS
First visit- EARLY 
presenter
(amount of blood= 
5 mL for < 2 years 
of age and 10 mL 
for all other 
subjects)
Sample 
collected 5 
days of fever 
and/or rash
unknown
-
RT-qPCR
750 µL
1
RT-qPCR (reserve) Viral 
isolation and sequencing
750 µL
3
Dengue IgM and NS1 
ELISA
500 µL 
2
Dengue  virus 1-4 
neutralizing antibodies
2 X 1 mL or 
remaining 
volume
4
Optional for severe cases***
PCR 
200 µL
-
First visit- LATE 
presenter
(total amount of 
whole blood 5 mL)
Sample 
collected > 5 
days or more  
after 
fever/symptom/s
unknown
-
Dengue IgM and Dengue 
capture IgG ELISA 
NS1 ELISA
500 µL
1
Dengue  virus 1-4 
neutralizing antibodies
2 x 1 mL or 
remaining 
volume
2
First visit (total 
amount of blood 
per local practice 
unknown
CBC, HCT
No aliquot
-
Suspected dengue 
follow-up visit
(total amount of 
blood 5 mL)
Approximately 
21 days after 
first visit
unknown
-
Dengue IgM and Dengue 
capture IgG ELISA
500 µL
1
Dengue  virus 1-4 
neutralizing antibodies
2 x 1 mL or 
remaining 
volume
2
*tertiary (exploratory) analysis, number of subjects in the subset will be determined during analysis 
-  not applicable
** Dengue IgG indirect ELISA
*** Sample taken per routine practice for standard patient care management 
 
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Table 9
Laboratory read-outs at each time point, and priority ranking for 
scheduled visits 
SCHEDULED VISITS
Blood sampling time point
No. 
Subjects
(anticipated
)
Subset
*
Possible assays 
Serum 
aliquot
volume
Component
s priority 
rank
Aliquot 
number
Type of 
contact/ 
Amount of 
whole blood
Sampling time 
point
Scheduled 
Visit 1
5 mL whole 
blood 
(Day 0)
1800
-
Dengue IgG ELISA**
250 µL
1
1
yes
Dengue virus 1-4 and 
YF neutralizing 
antibodies
1 mL
2
2
yes
Dengue  virus 1-4 
and YF neutralizing 
antibodies
remain
ing 
volume
3
3
Scheduled 
Visit 2 
5 mL whole 
blood
Visit 1+ 6 months 
for subject 
enrolled in Study 
Year 1; Visit 1+ 12 
months for subject 
enrolled in 
beginning of  
Study Year 2, 3 or 
4)
1800 
yes
Dengue IgG ELISA** 
(only for subjects 
IgG negative at the 
previous visit)
250 µL
1
1
yes
Dengue virus 1-4 and 
YF neutralizing 
antibodies
1 mL
2
2
yes
Dengue  virus 1-4 
and YF neutralizing 
antibodies
remain
ing 
volume
3
3
Scheduled 
Visit 3 
5 mL whole 
blood
(Visit 2+ 6 months 
for subject 
enrolled in Study 
Year 1; Visit 2+12 
months for subject 
enrolled in 
beginning of  
Study Year 2 or 3)
1500 
yes
Dengue IgG ELISA**
(only for subjects 
IgG negative at the 
previous visit)
250 µL
1
1
yes
Dengue virus 1-4 and 
YF neutralizing 
antibodies
1 mL
2
2
yes
Dengue  virus 1-4 
and YF neutralizing 
antibodies
remain
ing 
volume
3
3
Subsequent 
Yearly Visit(s) 
as applicable
5 mL whole 
blood
Yearly 
1500
yes
Dengue IgG ELISA**
(only for subjects 
IgG negative at the 
previous visit)
250 µL
1
1
yes
Dengue  virus 1-4 
and YF neutralizing 
antibodies
1 mL
2
2
yes
Dengue  virus 1-4 
and YF neutralizing 
antibodies
remain
ing 
volume
3
3
*tertiary (exploratory) analysis, number of subjects in the subset will be determined during analysis 
-  not applicable, 
** Dengue IgG indirect ELISA
 
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Table 10
Laboratory read-outs at each time point, and priority ranking for 
suspected dengue visits for EARLY presenters
SUSPECTED DENGUE VISITS – EARLY PRESENTERS
Blood sampling time point
No. 
Subjects
Subset
Component
Serum 
aliquot 
volume
Components 
priority rank
Aliquot 
number
Type of contact/ 
Amount of whole 
blood
Sampling time 
point
First visit (amount 
of blood= 5 mL for 
< 2 years of age 
and 10 mL for all 
other subjects)
Sample 
collected 5 
days of fever 
and/or rash
unknown
-
RT-qPCR
750 µL
1
1
RT-qPCR 
(reserve) Viral 
isolation and 
sequencing
750 µL
3
2
Dengue IgM 
and NS1 ELISA
500 µL
2
3
Dengue  virus 
1-4 neutralizing 
antibodies
1 mL
4
4
Dengue  virus 
1-4 
neutralizing 
antibodies
remaining 
volume
5
5
Optional for severe cases
PCR 
200 µL
-
First visit (total 
amount of blood 
per local practice 
unknown
CBC, HCT
No aliquot
-
Suspected 
dengue follow-up 
visit (total amount 
of blood 5 mL)
Approximately 
21 days after 
first visit
unknown
-
Dengue IgM 
and Dengue 
capture IgG 
ELISA
500 µL
1
1
Dengue  virus 
1-4 neutralizing 
antibodies
1 mL
4
4
Dengue  virus 
1-4 
neutralizing 
antibodies
remaining 
volume
5
5
-  not applicable
 
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Table 11
Laboratory read-outs at each time point, and priority ranking for 
suspected dengue visits for LATE presenters
SUSPECTED DENGUE VISITS – LATE PRESENTERS
Blood sampling time point
No. 
Subjects
Subset
Component
Serum 
aliquot 
volume
Components 
priority rank
Aliquot 
number
Type of contact/ 
Amount of whole 
blood
Sampling time 
point
First visit (total 
amount of whole 
blood 5 mL)
Sample 
collected > 5 
days or more  
after 
fever/symptom/s
unknown
-
Dengue IgM and 
Dengue capture 
IgG ELISA 
NS1 ELISA
500 µL
1
1
Dengue  virus 1-4 
neutralizing 
antibodies
1 mL
2
2
Dengue  virus 1-
4 neutralizing 
antibodies
remaining 
volume
3
3
First visit (total 
amount of blood 
per local practice 
unknown
CBC, HCT
No aliquot
-
Suspected 
dengue follow-up 
visit (total amount 
of blood 5 mL)
Approximately 
21 days after 
first visit
unknown
-
Dengue IgM and 
Dengue capture 
IgG ELISA
500 µL
1
1
Dengue  virus 1-4 
neutralizing 
antibodies
1 mL
2
2
Dengue  virus 1-
4 neutralizing 
antibodies
remaining 
volume
3
3
-  not applicable
In case of insufficient blood sample volume to perform all assays, the samples will be 
analyzed according to priority ranking provided in Table 9, Table 10 and Table 11
 
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Figure 4
Flow chart of serum aliquot testing for suspected dengue cases;
 
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Section 6.2.3 Contact information for reporting serious adverse events and other 
events to GSK Biologicals
Study Contact for Reportable SAEs
During office hours:
Local 
Name: Dr.
GlaxoSmithKline Biologicals
, 
, 
, Brazil CEP 22783-110
Email: 
Phone: 
Cell: 
Fax: 
 
Name: 
GlaxoSmithKline Biologicals
 
, 
, 
, Brazil 22783-110
Email: 
Phone: 
Fax: 
Back-up Study Contact for Reporting SAEs
24/24 hour and 7/7 day availability:
GSK Biologicals Clinical Safety & Pharmacovigilance
Fax: 
 or 
Section 7.2
Subject withdrawal; The second paragraph was changed as follows: 
All data collected until the date of withdrawal/last contact of the subject will be used for 
the analysis. Withdrawals/drop-outs will not be replaced (see section 7.3 for further 
details). 
Section 7.3 on subject replacement was added as follows:
Section 7.3 
Subject replacement 
Enrolment of new subjects to replace: 

subjects not wishing to continue after this amendment, or

subjects who prematurely terminate participation or

subjects lost to follow-up will be done. 
The aim is to maintain a critical cohort size of 1500 subjects. This replacement will 
be done before each surveillance year during scheduled visits starting at the end of 
study Year 1 (Visit 3) up till the end of study Year 3 (Visit 5).
The number of subjects to be recruited as replacements will be defined based on the 
number of subjects still active in the study. If at the end of each study year we have 
 
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PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD

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1500 subjects, no replacements will be needed. The time window for the recruitment 
of replacements will be during the low dengue transmission.
Section 8.1
Endpoints
Section 8.1.2 Secondary endpoints; The endpoints were updated as follows:

DENV-type specific by study site, gender,  age-group, and previous dengue exposure 
(primary or secondary) primary laboratory-confirmed symptomatic dengue 
infection

DENV-type specific secondary laboratory-confirmed symptomatic dengue 
infection

Primary symptomatic dengue infection (including laboratory-confirmed and 
probable cases)

Secondary symptomatic dengue infection (including laboratory-confirmed and 
probable cases)
Section 8.1.3 Tertiary endpoints
The following endpoint was added:

Spatial and temporal distribution of dengue cases (confirmed and probable 
symptomatic cases and inapparent infections).
Section 8.2 
Sample size consideration: This was re-written and formatted into sub 
sections with some rewording as follows:
Table 19
Precision for expected incidences of dengue for a single cohort of subjects 
in Brazil
95% CI of incidence rate (n / 100 P-Y)
Poisson Exact 
CI  
Normal approx.
Normal approx 
with design effect
Number 
of person-
years
Number 
of cases
Incidence 
of dengue 
(per 100 
person-
years)
lower
upper
lower
upper
lower
upper
Overall
2000
3
0.15
0.03
0.44
0.00
0.32
0.00
0.38
2000
6
0.30
0.11
0.65
0.06
0.54
0.00
0.62
2000
9
0.45
0.21
0.85
0.16
0.74
0.06
0.84
2000
12
0.60
0.31
1.05
0.26
0.94
0.14
1.06
2000
15
0.75
0.42
1.24
0.37
1.13
0.24
1.26
2000
18
0.90
0.53
1.42
0.48
1.32
0.34
1.46
2000
21
1.05
0.65
1.61
0.60
1.50
0.45
1.65
1800
3
0.17
0.03
0.49
0.00
0.36
0.00
0.42
1800
6
0.33
0.12
0.73
0.07
0.60
0.00
0.69
1800
9
0.50
0.23
0.95
0.17
0.83
0.06
0.94
1800
12
0.67
0.34
1.16
0.29
1.04
0.16
1.17
1800
15
0.83
0.47
1.37
0.41
1.26
0.27
1.40
 
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95% CI of incidence rate (n / 100 P-Y)
Poisson Exact 
CI  
Normal approx.
Normal approx 
with design effect
Number 
of person-
years
Number 
of cases
Incidence 
of dengue 
(per 100 
person-
years)
lower
upper
lower
upper
lower
upper
1800
18
1.00
0.59
1.58
0.54
1.46
0.38
1.62
1800
21
1.17
0.72
1.78
0.67
1.67
0.50
1.84
1500
3
0.20
0.04
0.58
0.00
0.43
0.00
0.50
1500
6
0.40
0.15
0.87
0.08
0.72
0.00
0.83
1500
9
0.60
0.27
1.14
0.21
0.99
0.07
1.13
1500
12
0.80
0.41
1.40
0.35
1.25
0.19
1.41
1500
15
1.00
0.56
1.65
0.49
1.51
0.32
1.68
1500
18
1.20
0.71
1.90
0.65
1.75
0.46
1.94
1000
3
0.30
0.06
0.88
0.00
0.64
0.00
0.76
1000
6
0.60
0.22
1.31
0.12
1.08
0.00
1.24
1000
9
0.90
0.41
1.71
0.31
1.49
0.11
1.69
1000
12
1.20
0.62
2.10
0.52
1.88
0.29
2.11
1000
15
1.50
0.84
2.47
0.74
2.26
0.48
2.52
1000
18
1.80
1.07
2.84
0.97
2.63
0.68
2.92
site
500
3
0.60
0.12
1.75
0.00
1.28
0.00
1.51
500
6
1.20
0.44
2.61
0.24
2.16
0.00
2.49
500
9
1.80
0.82
3.42
0.62
2.98
0.22
3.38
500
12
2.40
1.24
4.19
1.04
3.76
0.58
4.22
500
15
3.00
1.68
4.95
1.48
4.52
0.96
5.04
500
18
3.60
2.13
5.69
1.94
5.26
1.37
5.83
*If three study sites and same number of subjects enrolled per region.
Section 8.2.1 Assumptions:
It is planned to obtain approximately 1500 subjects still active at the end of the first 
year of the study. Because subjects might leave the study due to a variety of reasons, e.g. 
moving away geographically, sample size is increased to allow for this., If we assume a
an annual drop-out rate of about between 15 and 20% during the one-year follow-up 
period then we will need to recruit a total of about 1800 subjects to this study will be 
considered. About Therefore, approximately 1800 subjects will be selected in enrolled at 
the beginning of this study using cluster sampling , i.e. the unit of sampling will be the 
households. At least T three areas where the FHP has been fully implemented or where 
access to the community is already established by a government program will be first 
selected, followed by a selection of households in the respective areas. All subjects in 
each household will be recruited invited for recruitment to the study. The study will last 
approximately 4 years, and new subjects will be enrolled between seasons if the number
of subjects still active in the study is below 1500.
Between 2007 and 2010, the average incidence of reported dengue cases in the Brazilian 
population was about 0.3% per year (or 0.003 cases per person per year). Assuming an 
underreporting factor of 3 (Brazil MoH, 2011) the average incidence of dengue is 
expected to be about 1% per year (or 0.01 cases per person per year).
 
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Section 8.2.2 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 1.8 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 
3. Although in theory rho can have a value up to 1, in practice values higher than 0.4 are 
uncommon. We used a conservative estimate of 0.4 for this study (Bennett, 1991).The 
design effect is then estimated to 1.8.
Section 8.2.3 Expected precision of incidence rate estimates
Table 13 shows the 95% CI for a range of expected incidences based on the Poisson 
distribution (exact method, normal approximation and the normal approximation 
accounting for a design effect of 1.8). With an overall sample size of a minimum of about 
1,800 subjects enrolled and a follow-up period of one year, the 95% confidence interval 
(CI) for an expected incidence of dengue of 9 cases per 1000 person-years using a cluster 
design is [2.9 ; 15.1]. With 4 years of follow-up and new enrolments to keep the number 
of subjects above 1500 at the beginning of each subsequent season, the 95% CI is [5.7 ; 
12.3].
 
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Table 13
Precision for expected incidences of dengue for a single cohort of subjects in Brazil
Cohort 
size
Number of
years
Number of
person-years
Number 
of
cases
Incidence of dengue
(per 1000 person-years)
95% CI of incidence rate (n / 1000 P-Y)
Exact (Poisson)
Normal approx
Normal approx
with design effect
Lower
Upper
Lower
Upper
Lower
Upper
Overall
2000
1
1845.9
6
3.0
1.0
6.7
0.5
5.5
-0.4
6.4
2000
1
1845.9
11
6.0
3.0
10.7
2.5
9.5
1.3
10.7
2000
1
1845.9
17
9.0
5.2
14.5
4.7
13.3
3.2
14.8
2000
1
1845.9
22
12.0
7.5
18.1
7.0
17.0
5.3
18.7
2000
1
1845.9
28
15.0
9.9
21.7
9.4
20.6
7.5
22.5
2000
4
6183.9
19
3.0
1.8
4.7
1.6
4.4
1.2
4.8
2000
4
6183.9
37
6.0
4.2
8.3
4.1
7.9
3.4
8.6
2000
4
6183.9
56
9.0
6.8
11.7
6.6
11.4
5.8
12.2
2000
4
6183.9
74
12.0
9.4
15.0
9.3
14.7
8.3
15.7
2000
4
6183.9
93
15.0
12.1
18.4
11.9
18.1
10.9
19.1
1800
1
1661.3
5
3.0
0.9
7.2
0.4
5.6
-0.5
6.5
1800
1
1661.3
10
6.0
2.8
11.1
2.3
9.7
1.0
11.0
1800
1
1661.3
15
9.0
5.0
14.9
4.4
13.6
2.9
15.1
1800
1
1661.3
20
12.0
7.3
18.6
6.7
17.3
4.9
19.1
1800
1
1661.3
25
15.0
9.7
22.1
9.1
20.9
7.1
22.9
1800
4
5842.4
18
3.0
1.8
4.8
1.6
4.4
1.1
4.9
1800
4
5842.4
35
6.0
4.2
8.3
4.0
8.0
3.3
8.7
1800
4
5842.4
53
9.0
6.7
11.8
6.6
11.4
5.7
12.3
1800
4
5842.4
70
12.0
9.4
15.1
9.2
14.8
8.2
15.8
1800
4
5842.4
88
15.0
12.0
18.5
11.9
18.1
10.8
19.2
1500
1
1384.5
4
3.0
0.8
7.6
0.1
5.9
-0.9
6.9
1500
1
1384.5
8
6.0
2.6
11.7
1.9
10.1
0.5
11.5
1500
1
1384.5
12
9.0
4.7
15.6
4.0
14.0
2.3
15.7
1500
1
1384.5
17
12.0
6.9
19.3
6.2
17.8
4.3
19.7
1500
1
1384.5
21
15.0
9.2
22.9
8.5
21.5
6.3
23.7
1500
4
5537.8
17
3.0
1.7
4.8
1.6
4.4
1.1
4.9
1500
4
5537.8
33
6.0
4.1
8.4
4.0
8.0
3.3
8.7
1500
4
5537.8
50
9.0
6.7
11.9
6.5
11.5
5.6
12.4
1500
4
5537.8
66
12.0
9.3
15.2
9.1
14.9
8.1
15.9
1500
4
5537.8
83
15.0
12.0
18.6
11.8
18.2
10.7
19.3
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Cohort 
size
Number of
years
Number of
person-years
Number 
of
cases
Incidence of dengue
(per 1000 person-years)
95% CI of incidence rate (n / 1000 P-Y)
Exact (Poisson)
Normal approx
Normal approx
with design effect
Lower
Upper
Lower
Upper
Lower
Upper
Site
600
1
553.8
2
3.0
0.2
12.3
-1.6
7.6
-3.1
9.1
600
1
553.8
3
6.0
1.2
17.0
-0.5
12.5
-2.7
14.7
600
1
553.8
5
9.0
2.7
21.4
1.1
16.9
-1.6
19.6
600
1
553.8
7
12.0
4.6
25.1
2.9
21.1
-0.2
24.2
600
1
553.8
8
15.0
6.5
29.2
4.8
25.2
1.3
28.7
600
4
1947.5
6
3.0
1.0
6.7
0.6
5.4
-0.3
6.3
600
4
1947.5
12
6.0
3.1
10.6
2.6
9.4
1.4
10.6
600
4
1947.5
18
9.0
5.3
14.3
4.8
13.2
3.3
14.7
600
4
1947.5
23
12.0
7.6
17.9
7.1
16.9
5.5
18.5
600
4
1947.5
29
15.0
10.1
21.5
9.6
20.4
7.7
22.3
500
1
461.5
1
3.0
0.1
14.3
-2.0
8.0
-3.7
9.7
500
1
461.5
3
6.0
1.0
18.6
-1.1
13.1
-3.5
15.5
500
1
461.5
4
9.0
2.5
22.7
0.3
17.7
-2.6
20.6
500
1
461.5
6
12.0
4.2
26.8
2.0
22.0
-1.4
25.4
500
1
461.5
7
15.0
5.8
31.2
3.8
26.2
0.0
30.0
500
4
1845.9
6
3.0
1.0
6.7
0.5
5.5
-0.4
6.4
500
4
1845.9
11
6.0
3.0
10.7
2.5
9.5
1.3
10.7
500
4
1845.9
17
9.0
5.2
14.5
4.7
13.3
3.2
14.8
500
4
1845.9
22
12.0
7.5
18.1
7.0
17.0
5.3
18.7
500
4
1845.9
28
15.0
9.9
21.7
9.4
20.6
7.5
22.5
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Section 8.4.1 Sequence of analysis; The following text was added:
An interim analysis will be performed when all active subjects have achieved the visit 4. 
All analyses of primary and secondary objectives will be performed on data as cleaned 
as possible. 
The final analysis will be performed when all prospective data have been collected and 
cleaned. An interim analysis will not be performed.
Section 8.5.1 Analysis of demographics/baseline characteristics; The following 
words were added:
Socio-demographic and patient characteristics (e.g., age at study enrollment, gender, 
household conditions, medical history and vaccination history) will be summarized 
overall and by region, and at the beginning of each season using descriptive statistics. 
Section 8.5.2 Analysis of primary and secondary endpoints
The following analyses will be performed overall and by DENV type, study site, gender 
and age-group and previous dengue exposure (Yes = DENV IgG antibodies at previous 
visit and No = no DENV IgG antibodies at previous visit):

Incidence rate of laboratory-confirmed symptomatic dengue infection with 95% CI 
for each season separately: the numerator will be the number of subjects with lab-
confirmed symptomatic dengue infection during the study period season (between 
the visits scheduled before and after the season). The denominator will be the total 
person-years at risk, i.e. from the visit scheduled before the season until the first 
RT-qPCR confirmed symptomatic dengue infection during the season, the next 
scheduled visit or the subject’s withdrawal, whichever comes first.

Incidence rate of laboratory-confirmed symptomatic dengue infection with 95% CI 
all seasons combined: the numerator will be the number of subjects with lab-
confirmed symptomatic dengue infection during the study period. The 
denominator will be the total person-years at risk, i.e. from enrolment until the 
first RT-qPCR confirmed symptomatic dengue infection, the end of the study or 
the subject’s withdrawal, whichever comes first.
The following analyses will be performed by study site, gender and age-group and 
previous dengue exposure (primary or secondary) :

Incidence rate of laboratory confirmed or probable symptomatic dengue infections
with 95% CI by type (DENV 1-4), study site, gender and age group and previous 
dengue exposure (primary or secondary).for each season separately and overall. 
The numerator will be the number of subjects with symptomatic dengue infection 
(including laboratory-confirmed and probable cases) during the period. The 
denominator will be the total person-years at risk.

Incidence rate of probable symptomatic dengue infection with 95% CI for each 
season separately and overall. The numerator will be the number of subjects with 
probable symptomatic dengue infection. The denominator will be the total person-
years at risk.
 
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The following analyses will be performed by study site, gender and age-group:

The proportion of subjects with primary inapparent dengue infection at at 6 and 12 
months each subsequent scheduled visit (not at enrolment) with 95% CI will be 
calculated as the number of dengue IgG positive cases and acute cases at enrollment
at the visit divided by the total number of subjects enrolled at baseline IgG negative 
at the previous visit. 

Incidence rate of primary inapparent dengue infection with 95% CI : the numerator 
will be the number of cases of inapparent dengue infection and the denominator will 
be the total person-years at risk.

The proportion of subjects with secondary symptomatic dengue infection (probable 
or confirmed) at each subsequent scheduled visit (not at enrolment) with 95% CI 
will be calculated as the number of subjects with secondary symptomatic infection 
since the previous visit divided by the total number of IgG positive subjects with 
confirmed symptomatic dengue infection and IgG positive at visit 1 or baseline), 
with 95% CI.at the previous visit.

The (crude) seroprevalence of dengue infection will be calculated at enrolment and
at each scheduled visit as a proportion (i.e., the number of dengue IgG positive cases 
and acute cases at enrollment divided by the total number of subjects enrolled 
subjects tested positive at this visit or known to be positive from previous visits 
divided by the total number of subjects for whom dengue IgG serostatus is known). 

The clinical characteristics of symptomatic dengue infection (symptoms, 
hospitalizations, severity) will be presented as proportions of mild, moderate and 
severe dengue cases overall and per sub-group (type (DENV 1-4), study site, gender 
and age group).
Table 10
Endpoint Numerators and Denominators
Endpoint
Numerator
Denominator
Prevalence of past dengue infection 
at enrollment
Dengue IgG positive + acute cases at 
enrolment
All individuals enrolled
Incidence rate of laboratory 
confirmed symptomatic infection
All confirmed acute cases after enrollment
Total person-years at risk
Incidence rate of primary inapparent 
infection
Dengue IgG positive at 6 months or 12 months 
without history of fever
Total person-years at 
risk*
Cumulative incidence of primary 
inapparent infection over 6 months
Dengue IgG positive at 6 months and IgG 
negative at baseline (enrollment), without 
history of fever
Number of subjects IgG  
negative at baseline
Cumulative incidence of primary 
inapparent infection over 12 months
Dengue IgG positive at 6 months or at 12 
months, and IgG negative at baseline without 
history of fever
Number of subjects 
IgG/IgM negative at 
baseline
Cumulative incidence of secondary 
symptomatic infection
All secondary symptomatic cases
Individuals with dengue 
IgG at baseline
* Time at risk is calculated as the time during which a subject is in the study population and remains dengue IgG 
negative at baseline (Visit 1) and/or at 6 months, and therefore at risk of seroconversion in 6 months (Visit 2) or 12 
months (Visit 3), respectively.
 
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Table 14
Numerators and Denominators within each strata (DENV type, study 
site, gender and age-group and previous dengue exposure (primary or secondary)
Analysis
Numerator
Denominator
Incidence rate of laboratory 
confirmed symptomatic 
infection by year
All confirmed acute cases during the 
season (between the visits scheduled 
before and after the season)
Total person-years at risk, i.e. from 
the visit scheduled before the 
season until the first RT-qPCR
confirmed symptomatic dengue 
infection during the season, the 
next scheduled visit or withdrawal, 
whichever comes first.
Incidence rate of laboratory-
confirmed symptomatic 
dengue infection for all 
seasons combined
All confirmed acute cases during the 
study period
Total person-years at risk, i.e. from 
enrolment until the first RT-qPCR 
confirmed symptomatic dengue 
infection, the end of the study or 
withdrawal, whichever comes first.
Incidence rate of laboratory 
confirmed or probable 
symptomatic dengue 
infections 
All symptomatic dengue infections 
(including laboratory-confirmed and 
probable cases) during the period.
Total person-years at risk*.
Incidence rate of probable 
symptomatic dengue 
infection 
All probable symptomatic dengue 
infection
Total person-years at risk*.
Proportion of subjects with 
primary inapparent dengue 
infection
All dengue IgG positive cases at the 
considered visit
Total number of IgG negative 
subjects at the previous visit
Proportion of subjects with 
secondary symptomatic 
dengue infection
All symptomatic infection since the 
previous visit
Total number of IgG positive 
subjects at the previous visit
Seroprevalence of dengue 
infection
All dengue IgG positive cases
Total number of subjects tested or 
previously positive
* Time at risk: In analyses by season the time at risk is calculated as the time from the visit scheduled before 
the season until the first event during the season, the next scheduled visit or the subject’s withdrawal, 
whichever comes first.
In the analyses of all seasons combined the time at risk is calculated as the time from enrolment until the first 
event, the end of the study or the subject’s withdrawal, whichever comes first. 
CIs for incidence rates and proportions will account for clustering of observations 
within households and will be calculated using generalized estimating equations (GEE) 
assuming an exchangeable correlation matrix and robust variance estimation. for the 
analyses by centre and also the stratification by centres for the overall analyses. Details 
of the methods will be described in the SAP.
Section 8.5.4 Analysis of tertiary objectives
Analysis of tertiary objectives is optional and may or may not be performed. If these 
analyses are performed, they will be conducted by the Research and Development 
Department. Statistical methods for tertiary objectives will be described in the SAP.
The neutralization assay, if performed, will only be done on blood samples from a subset 
of subjects. YF virus antibody testing will be performed retrospectively, for subjects in 
Manaus YF endemic region and who tested positive for dengue. 
 
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Analysis of risk factors for symptomatic dengue
In this study, we will use Generalized Estimating Equations (GEE) to obtain population-
averaged estimates for clustered data (so-called marginal model). The GEE estimates the 
within-cluster similarity of the residuals, and then uses this estimated correlation to re-
estimate the regression parameters and to calculate robust standard errors. To use GEE, 
we need to specify how we think observations in our data are correlated with each other. 
Here, we assume an exchangeable correlation structure/matrix also known as compound 
symmetry. This means that within a cluster (house-hold) any two observations are equally 
correlated, but that there is no correlation between observations from different clusters 
(house-holds). This structure is fully characterized by one correlation parameter which is 
the intra class correlation coefficient. 
A Poisson regression models using the GEE approach (using the GENMOD procedure in 
SAS) will be used for exploring the risk factors (e.g. region, age and gender, etc.) for 
symptomatic dengue. The saturated model (including all potential risk factors) and the 
model obtained after backward selection at P<0.05 level will be presented. Analyses will 
be performed using SAS 9.2. The analyses will account for clustering of observations 
within households for the analyses by centre and also the stratification by centres for 
the overall analyses. 
Section 8.5.5 Statistical considerations for interim analyses
No interim analysis is planned. Since there is no hypothesis testing, no adjustment of 
type I error is needed for the interim analyses. 
Section 9.1
Remote Data Entry instructions; The first paragraph was changed as 
follows: 
Remote Data Entry (RDE)Inform (electronic case report form ,eCRF), a validated 
computer application, will be used as the method for data collection.
Section 9.2
Monitoring by GSK Biologicals: RDE was replaced with eCRF 
including other changes as follows:
Monitoring visits by a GSK Site Monitor are for the purpose of confirming that GSK 
Biologicals’ sponsored studies are being conducted in accordance with the ethical 
principles that have their origins in the Declaration of Helsinki and that are consistent 
with Good Clinical practice (GCP) or other applicable guidelines and the applicable 
regulatory requirement(s) (verifying continuing compliance with the protocol, 
amendment(s), verifying that the site staff and facilities continue to be adequate to 
conduct the study). 
The investigator must ensure provision of reasonable time, space and qualified personnel 
for monitoring visits.
Direct access to all study-site related and source data is mandatory for the purpose of 
monitoring review. The monitor will perform an RDE eCRF review and a Source 
Document Verification (SDV). By SDV we understand verifying RDE eCRF entries by 
 
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comparing them with the source data that will be made available by the investigator for 
this purpose. 
The Source Documentation Agreement Form describes the source data for the different 
data in the RDE 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 RDE eCRF will serve as the source must be identified, 
agreed and documented in the source documentation agreement form. 
For RDE eCRF, the monitor could validate the screens at each visit. 
In accordance with applicable regulations, GCP or other applicable guidelines and GSK 
procedures, 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.
The study will be monitored to verify that, among others, the: 
APPENDIX A 
STUDY LABORATORIES: Non GSK laboratories were updated 
as follows:
Non GSK  laboratories 
Laboratory
Address
Fundação de Medicina Tropical Dr. 
Heitor Vieira Dourado - FMT
Gerência de Virologia
Dr. Heitor Vieira Dourado - FMT
Gerência de Virologia Av. Pedro Teixeira, 25, 
Dom Pedro
Manaus, AM. CEP: 69.040-000
Brazil
Centro de Pesquisas Gonçalo 
Moniz - CPqGM Fundação 
Oswaldo Cruz - FiocruzLaboratório 
de Patologia e Biologia Molecular -
LPBM
Rua Waldemar Falcão, 121, Candeal 
Salvador, BA. CEP: 40.296-710
Brazil
Instituto de Pesquisa Clínica 
Evandro Chagas - IPEC, 
Fundação Oswaldo Cruz -
Fiocruz
Laboratório de Imunodiagnóstico
Bio-Manguinhos
LATEV
Av. Brasil, 4365, Manguinhos
Pavilhão Rocha Lima sala 403
Rio de Janeiro, 21040-360-RJ. CEP: 21.040-900
Brazil
Laboratório de Tecnologia 
Virológica – LATEV
Bio-Manguinhos
Fundação Oswaldo Cruz - Fiocruz
Av. Brasil, 4365, Manguinhos
Pavilhão Rocha Lima, sala 403
Rio de Janeiro, RJ. CEP: 21.040-360
Brazil
APPENDIX B 
TELEPHONE STRUCTURED SCRIPT 
 
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GlaxoSmithKline Biologicals
Clinical Research & Development
Protocol Amendment 3 
eTrack study number and 
Abbreviated Title
116606 (EPI-DENG-006 BOD BR)
Amendment 3 version and date:
Final: 09 January 2015
Rationale/background for changes: 

The protocol is amended to increase the size of the study population and expand 
the number of sites. The target number of subjects to be recruited in the new 
expansion sites is 1800: per site, it is set at 600 subjects at initiation and should be 
maintained to at least 500 subjects at the beginning of each subsequent study year. 
This improved regional coverage should allow to better capture the diversity of 
dengue epidemiology in the different regions of Brazil and strengthen the 
operational preparation of sites for potential future dengue vaccine efficacy trials.

In order to better represent the Brazilian population distribution, a criteria for the 
age stratification of the population has been defined. In the first two sites (initial 
cohort) having completed enrolment, it has been observed that children were 
slightly less represented than in the Brazilian population (19% in the sites versus 
33% in the country) and that older adults over 50 years of age were slightly over-
represented (28.8% in the sites versus 20% in the Brazilian population). Thus, we 
proposed that subjects enrolled in the expansion cohort should be composed of at 
least 30% of children and should not exceed 20 % of adults 50 years or older. 
Subjects enrolled as replacement subjects for the initial and expansion cohort will 
be recruited to approach as much as possible a minimal representation of 30% of 
children and a maximal representation of 20% for adults 50 years or older.

Clarification: the determination of whether a subject with dengue suspicion is 
either an early or a late presenter is based on the time of onset of fever.

It is highlighted within the document that the indirect DENV IgG assay is repeated 
in the following scheduled visits (after enrolment) only if the subject was negative 
for DENV IgG at the previous scheduled visit. A footnote was added in the study 
procedures tables and a note in the Procedures at subsequent scheduled visits 
section.

The criteria to determine the trigger of the interim analysis have been further 
defined. The differences in the study start in the initial sites have led to the 
necessity of updating the scope of the interim analysis. 

The local laboratories details from APPENDIX A were deleted since new 
laboratories will be identified for testing study samples in the additional sites. The 
information with the details for all the local laboratories will be submitted in a 
separated document.
Coordinating author:
, Scientific Writer, XPE 
Pharma & Science for GSK Biologicals
 
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Amended text has been included in bold italics and deleted text in strikethrough in 
the following sections:
Cover page
GSK Contributing authors:
Global Vaccine Clinical Laboratories Manager
Global Vaccine Clinical Project Manager
Study Data Manager
Non-GSK Contributing authors
Bio-Manguinhos, Fiocruz
Synopsis
Study design

2 waves of enrolment for recruiting 3600 subjects:

Initial Cohort = about 1800 subjects – up to 6 visits 
(Amendment 2) – INITIAL sites

Expansion Cohort = about 1800 subjects – up to 4 visits 
(Amendment 3) – NEW sites 

The study period, initially planned to be one year, has beenis
extended by three years to cover three additional dengue seasons 
(Amendment 2). 

Initial cohort subjects will bewere invited to extend their 
participation. 
(See glossary of terms for the definition of initial and expansion
cohort subjects)

In each cohort: rReplacement subjects may be enrolled in order 
to compensate for subjects who dido not wish to extend their 
participation for three additional years (for subjects from the 
initial cohort – Amendment 2), subjects who prematurely 
terminate participation or subjects who are lost to follow-up. 
This will be done to maintain a cohort size of at least 500 subjects 
per site, in the Initial and Expansion cohorts,1500 subjects at 
the beginning of each additional study year/season.

Informed consent (and assent, if applicable) will be obtained:

From the parents/LAR of the subjects (and from the 
subjects, if applicable) at the study start for the expansion 
cohort. 

Subjects from the expansion cohort will have only three 
years follow-up, with at most 4 scheduled visits. 

For replacement subjects, the number of scheduled visits will 
depend on the study year in which they are enrolled. 
Replacement subjects will not be enrolled until after the third 
 
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visit of the first study year is concluded for the initial cohort. 
The number of visits for the replacement subjects will range 
between two and four.
The yearly scheduled visits for the initial and expansion
cohorts and replacement subjects will preferably occur during 
the period of usually low dengue transmission.
Synopsis Figure 1
Schematic representation of the scheduled 
visits
V= Visit
The scheduled visits for initial cohort subjects are shown in black.
New enrolments will be done yearly (as needed) till the end of Study Year 3.
Enrollment of expansion subjects will start preferably during study Year 2. 
For replacement subjects, the number of visits will depend on the year enrolled. These visits 
are shown in different colours for each year.
Synopsis Table 1
Study group and epoch foreseen in the study
Study Group
Number of subjects*
Age (Min/Max)
Epoch
Prospective study cohort 
Initial cohort:
1800 at enrolment (first year)
At least 1500 in each 
subsequent year 
6 months of age and older
Epoch 001 
Expansion Cohort: 
1800 at enrolment (first year).
At least 1500 in each 
subsequent year
* The final number of participants will take into account the active subjects, subjects who prematurely terminate 
participation and subjects lost to follow-up. 
Discussion 
of study 
design
Selection of study sites
At least three six sites in Brazil will be selected for this study. Selection 
criteria include support from the Family Health Physician Program 
(FHP), the Larval Index Rapid Assay (LIRA) or with field research 
experience in the community. The FHP is a Brazilian government public 
health strategy that consists of multidisciplinary teams of health care 
workers that cover geographically defined areas to provide 
basic/preventive care to community members. The teams include a 
doctor, a nurse, a nurse auxiliary, other health professionals and 
community health care workers who pay regular visits to the household 
 
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under its responsibility to deliver health promotion activities and 
facilitate triage and referral into the health unit, among other activities. 
Community health care workers are local residents who are trained for 
basic tasks.
Number of 
subjects
Approximately 1800 3600 subjects will be initially recruited in the study 
in 2 waves of enrolment. Each wave, initial and expansion cohorts, will 
enrol about 1800 subjects. Replacement subjects may be recruited 
afterwards to compensate for subjects not willing to extend participation 
for the three additional years, subjects who prematurely terminate 
participation and subjects lost to follow-up. The recruitment of 
replacement subject aims at maintaining the number of subjects at a 
minimum of 1500 subjects/site at the beginning of study Year 2, 3 and 4. 
The total number of subjects enrolled in the study will may exceed 
18003600.
In order to better represent the Brazilian population distribution, 
subjects enrolled in the expansion cohort should be composed of at 
least 30% of subjects younger than 18 years old and should not exceed 
20 % of adults 50 years or older. Subjects enrolled as replacement 
subjects for the initial and expansion cohort will be recruited to 
approach as much as possible a minimal representation of 30% of 
subjects younger than 18 years old and a maximal representation of 
20% for adults 50 years or older.
GLOSSARY OF TERMS
Early presenter
A suspected dengue case presenting at the health care 
facility within 5 days following the onset of fever.
Expansion cohort subjects
Subjects recruited in the additional sites for study 
expansion
Late presenter
A suspected dengue case presenting at the health care 
facility 6 days or more after the onset of fever
Replacement subjects
These are subjects that will be enrolled to compensate for 
subjects:

from initial cohort: who will not want to extend 
participation for three additional years or

from initial and expansion cohorts: subjects who 
prematurely terminate participation or subjects lost to 
follow-up. 
 
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Section 3.
STUDY DESIGN OVERVIEW

The study period, initially planned to be one year, has beenis extended by three 
years to cover three additional dengue seasons (Amendment 2).

Initial cohort subjects will bewere invited to extend their participation. 

(See glossary of terms for the definition of initial and expansion cohort subjects).

In each cohort: rReplacement subjects may be enrolled in order to compensate for 
subjects who do did not wish to extend their participation for three additional years
(for subjects from the initial cohort – Amendment 2), subjects who prematurely 
terminate participation or subjects who are lost to follow-up. 
This will be done to maintain a cohort size of at least 500 subjects per site, in the 
Initial and Expansion cohorts,1500 subjects at the beginning of each additional 
study year/season.

Informed consent (and assent, if applicable) will be obtained:

From the parents/LAR of the subjects (and from the subjects, if applicable) at 
the study start for the expansion cohort.

From the parents/LAR of the replacement subjects (and from the replacement  
subjects, if applicable) prior to enrolment in the study.

2 waves of enrolment for recruiting 3600 subjects:

Initial Cohort = about 1800 subjects – up to 6 visits (Amendment 2) –
INITIAL sites

Expansion Cohort = about 1800 subjects – up to 4 visits (Amendment 3) –
NEW sites

Study visits: The visits will be as follows and further shown in Figure 1:

Subjects from the expansion cohort will have only three years follow-up, with 
at most 4 scheduled visits.

For replacement subjects, the number of scheduled visits will depend on the 
study year in which they are enrolled. Replacement subjects will not be enrolled 
until after the third visit of the first study year is concluded for the initial cohort. 
The number of scheduled visits will range between two and four.
The yearly scheduled visits for the initial and expansion cohorts and 
replacement subjects will preferably occur during the period of usually low 
dengue transmission.
 
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Figure 1
Schematic representation of the scheduled visits
V=Visit
The scheduled visits for initial cohort subjects are shown in black.
New enrolments will be done yearly (as needed) till the end of study Year 3.
Enrollment of expansion cohort subjects will start preferably during study Year 2. 
For replacement subjects, the number of visits will depend on the year enrolled. These visits are shown in different 
colours for each year.
Table 1
Study groups and epochs foreseen in the study
Study Group
Number of subjects*
Age 
(Minimum)
Epoch
Prospective study 
cohort
Initial cohort:
1800 at enrolment (first year)
At least 1500 in each 
subsequent year 
6 months 
and older
Epoch 001 
Expansion Cohort: 
1800 at enrolment (first 
year).
At least 1500 in each 
subsequent year
*The final number of participants will take into account the active subjects, subjects who prematurely terminate 
participation and subjects lost to follow up. 
Section 3.1.1. Selection of study sites
At least three six sites covering at least 3 different regions of the country will be selected 
for this study. Selection criteria of the sites include support from the Family Health 
Physician Program (FHP), the Larval Index Rapid Assay (LIRA) or with field research 
experience in the community. The FHP is a Brazilian government public health strategy 
that consists of multidisciplinary teams of health care workers that cover geographically 
defined areas to provide basic/preventive care to community members. The teams include 
a doctor, a nurse, a nurse auxiliary, and community health care workers who pay regular 
visits to the household under its responsibility to deliver health promotion activities and 
facilitate triage and referral into the health unit, among other activities. Community 
health care workers are local residents who are trained for basic tasks.
 
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Section 4.
STUDY POPULATION
Subjects six months of age and older at the time of enrolment who live in the selected 
study sites in Brazil, covering at least 3 different regions of the country. 
Section 4.2.
Selection of households
Approximately 1800 subjects need to be recruited for this study in order to obtain an 
evaluable sample size of approximately 1500 subjects at the end of the first year of the 
study. 
Section 4.3.
Overview of the recruitment plan
At least 3600 subjects will be recruited in the study in 2 waves of enrolment. Each 
wave, initial and expansion cohorts, will enrol about 600 subjects/site in order to 
maintain at least 500 subjects/site at the beginning of each additional study 
year/season.
In the subsequent study years/seasons, the cohort size will be maintained at, at least 1500 
subjects per site. Since subjects who prematurely terminate participation and/or lost to 
follow-up will be replaced, the final number of participants across the four study years 
will may exceed 18003600 subjects. Yearly, there will be an evaluation of active 
subjects. If the number of active subjects/site becomes < 1500, there will be enrolment of 
replacement subjects to maintain a cohort size of at least 1500 subjects/site. Enrolment 
will preferably take place during the low dengue transmission. Recruitment of 
replacement subjects could occur at the end of study year 1, 2 or 3. The recruitment 
approach will be the same as for the initial enrolment.
In order to better represent the Brazilian population distribution, subjects enrolled in 
the expansion cohort should be composed of at least 30% of subjects younger than 18 
years old and should not exceed 20 % of adults 50 years or older. Subjects enrolled as 
replacement subjects for the initial and expansion cohort will be recruited to approach 
as much as possible a minimal representation of 30% of subjects younger than 18 
years old and a maximal representation of 20% for adults 50 years or older. 
Section 5.4.
Outline of study procedures
Study procedures are outlined in Table 2 and Table 3 for initial and expansion cohorts, 
respectively in Table 4 for replacement subjects.
 
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Table 2 List of study procedures for initial cohort subjects 
Surveillance
Suspected Dengue Visits (at 
hospital/clinic)
as applicable3
Procedure
Visit 1 
Day 0
Monthly 
contact 
between 
visits
Visit 2 
Month 6
Monthly 
contact 
between 
visits
Visit 3
Month 
12 (Year 
1)
Monthly 
contact 
between 
visits
Visit 4 
Month 
24 (Year 
2)
Monthly 
contact 
between 
visits
Visit 5 
Month 
36 (Year 
3)
Monthly 
contact 
between 
visits
Visit 6 
Month 
48 (Year 
4)
First 
visit 
Day 0
Return 
visit 2
Follow-
up visit 
Day 21
Blood sample for serology (5 mL)6
●
●
●
●
●
●
6 Indirect DENV IgG assay is repeated in the following scheduled visits (after enrolment) only if the subject was negative for DENV IgG at the previous scheduled visit.
Table 3 List of study procedures for expansion cohort subjects 
Surveillance
Suspected Dengue Visits (at 
hospital/clinic)
as applicable3
Procedure
Visit 1* 
Day 0
Monthly 
contact 
between 
visits
Visit 2
Monthly 
contact 
between 
visits 
Visit 3
Monthly 
contact 
between 
visits
Visit 4 
First visit 
Day 0
Return visit2 Follow-up 
visit Day 21
Informed consent (and assent if 
applicable) 
●
Subject number and household 
number attribution 
●
Check inclusion/ exclusion criteria 
●
Record/ update socio-demographic 
information
●
●*
Medical history and YF vaccination 
history or updates 
●
●
●
Distribute subject ID card distribution 
and suspected dengue instruction kit 
O
O
O
Blood sample for serology (5 mL)6 
●
●
●
●
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Surveillance
Suspected Dengue Visits (at 
hospital/clinic)
as applicable3
Procedure
Visit 1* 
Day 0
Monthly 
contact 
between 
visits
Visit 2
Monthly 
contact 
between 
visits 
Visit 3
Monthly 
contact 
between 
visits
Visit 4 
First visit 
Day 0
Return visit2 Follow-up 
visit Day 21
Instruct/remind subjects/subject's 
parent(s)/LAR(s) on suspected 
dengue assessment procedures 
(where applicable) 
O
O
O
O
O
O
O
Issue diary logs to parents/LARs to 
be filled out in the event of a 
suspected dengue case 1
O
O
O
O
O
Contact subject regarding any 
dengue symptoms and remind 
subject of procedures for suspected 
dengue 
O
O
O
Physical examination/record current 
medical conditions (see Section 5.6)
●
●
●
Blood sample for dengue infection 
diagnosis3,
●
●
Blood sample for CBC and 
hematocrit
●
Record body temperature 
●
Report SAEs related to study 
procedures 4
●
●
●
●
●
●
●
Collect or verify diary logs if 
applicable 
O
O
O
Study conclusion 5
●
●
● 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.
* Enrollment of expansion cohort subjects will start during Year 2, at the earliest.
1 Diary logs given at scheduled visits are only to be filled out in the event that the subject has a fever or suspected dengue symptoms. See Section 5.5.2.5 for details with 
regard to the diary log. 
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2 A return visit is a visit linked to suspected dengue, occurring if the evolution of the subject’s physical condition necessitates medical evaluation, according to local 
medical practice. The investigator will determine whether this return visit is necessary and whether it is considered as part of the same episode. See Section 5.6 for 
details.
3 May include testing for RT-qPCR, dengue IgG and IgM ELISA, NS1 ELISA, neutralizing antibodies to dengue and Yellow fever, as well as dengue virus isolation if deemed 
necessary.
4 SAE = Serious adverse event. See Section 6.1.1 for definition.
5 The study conclusion information in the eCRF is collected once for each subject at the last scheduled visit or at the last follow-up contact for the dengue case, should 
this case occur during the last scheduled visit or earlier if the subject prematurely terminates participation or is lost to follow-up.
6 Indirect DENV IgG assay is repeated in the following scheduled visits (after enrolment) only if the subject was negative for DENV IgG at the previous scheduled visit.
Table 4 List of study procedures for replacement subjects
Surveillance
Suspected Dengue Visits (at 
hospital/clinic)
as applicable3
Procedure
Visit 1 
Day 0
Monthly 
contact 
between 
visits
Visit 2 *
Monthly 
contact 
between 
visits 
Visit 3* as 
applicable
Monthly 
contact 
between 
visits
Visit 4 as 
applicable
First visit 
Day 0
Return visit2 Follow-up 
visit Day 21
Blood sample for serology (5 mL) 6 
●
●
●
●
6 Indirect DENV IgG assay is repeated in the following scheduled visits (after enrolment) only if the subject was negative for DENV IgG at the previous scheduled visit.
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Table 5 Intervals between study visits/contacts for INITIAL cohort
Study Year*
Visits Interval
Optimal length of interval *
Allowed interval**
Year 1
Visit 1 (Day 0) Visit 2 (Month 6)
6 months 
± -28 days
Visit 2 (Month 6) Visit 3 (Month 12)
6 months
The interval may be adapted 
to allow scheduling the visit in 
a period of generally low 
dengue transmission
Year 2
Visit X Visit X+1¶
Note: Visit X may be visit 3 for subjects 
enrolled in study year 1. For subjects 
enrolled in study year 2, visit X would 
be visit 1.
12 months 
The interval may be adapted to 
allow scheduling the visit in a 
period of generally low dengue 
transmission
Year 3
Visit X+1 Visit X+2
12 months 
The interval may be adapted 
to allow scheduling the visit in 
a period of generally low 
dengue transmission
Year 4
Visit X Visit X+3
12 months 
The interval may be adapted 
to allow scheduling the visit in 
a period of generally low 
dengue transmission
*Whenever possible the investigator should arrange study visits/contacts within this interval
**Subjects might not be eligible for inclusion in the ATP cohort for analysis if they make the study visit/contact outside 
this interval
Table 6 Intervals between study visits/contacts for EXPANSION cohort 
Interval
Optimal length of interval*
Allowed interval**
Visit 1 (Day 0)   Visit 2 (Year 1)
12 months 
The interval may be adapted to allow scheduling 
the visit in a period of generally low dengue 
transmission
±28 days 
Visit 2 (Year 1) Visit 3 (Year 2)
12 months 
The interval may be adapted to allow scheduling 
the visit in a period of generally low dengue 
transmission
±28 days
Visit 3 (Year 2) Visit 4 (Year 3)
12 months 
The interval may be adapted to allow scheduling 
the visit in a period of generally low dengue 
transmission
±28 days
Interval between visit for a 
suspected dengue case and the 
final follow-up visit for a case
21 days
+7 days 
*Whenever possible the investigator should arrange study visits/contacts within this interval
**Subjects might not be eligible for inclusion in the ATP cohort for analysis if they make the study 
visit/contact outside this interval
 
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Section 5.5.3. Procedures at subsequent scheduled visits (2-6, as applicable)

For expansion cohort subjects, scheduled visits should ideally be performed at 
yearly intervals, preferably during periods of usually low dengue transmission. 
Expansion cohort subjects will have at most 4 scheduled visits.

A blood (serum) sample will be collected for serology
Note: Indirect DENV IgG assay is repeated in the following scheduled visits (after 
enrolment) only if the subject was negative for DENV IgG at the previous 
scheduled visit. Even if the test is not repeated, the volume of the blood sample 
remains unchanged.
Section 5.7.1. Suspected symptomatic dengue case
A suspected dengue case presenting at the health care facility within 5 days following 
the onset of fever and/or rash will be defined as an ‘early presenter’. 
Subjects presenting for care after the fifth day of fever and/or rash onset will be defined 
as ‘late presenters’.
An example of other signs and symptoms of dengue, associated with fever, include but 
are not limited to: fatigue, headache, pain behind the eyes, abdominal pain, nausea, 
vomiting, muscle ache, joint ache, diffuse rash on the trunk, abnormal sensitivity to light 
(photophobia) and itching of skin (pruritus).
 
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Section 5.8.2.2.
Laboratory read-outs
Table 11
Laboratory read-outs at each time point, and priority ranking for 
scheduled visits
SCHEDULED VISITS
Blood sampling time point
No. Subjects
(anticipated)
Subset
*
Possible assays 
Serum 
aliquot
volume
Components 
priority rank
Aliquot 
numbe
r
Type of 
contact/ 
Amount of 
whole blood
Sampling time 
point
Scheduled 
Visit 1
5 mL whole 
blood 
(Day 0)
3600 
(1800 from 
wave 1 & 
2***)1800
-
Dengue IgG ELISA**
250 µL
1
1
yes
Dengue virus 1-4 and 
YF neutralizing 
antibodies
1 mL
2
2
yes
Dengue  virus 1-4 and 
YF neutralizing 
antibodies
remaining 
volume
3
3
Scheduled 
Visit 2 
5 mL whole 
blood
Visit 1+ 6 
months for 
subject enrolled 
in Study Year 
1; Visit 1+ 12 
months for 
subject enrolled 
in beginning of  
Study Year 2, 3 
or 4)
3190 (1660 
from wave 1 
and 1530 
from wave 
2)1800
yes
Dengue IgG ELISA** 
(only for subjects IgG 
negative at the 
previous visit)
250 µL
1
1
yes
Dengue virus 1-4 and 
YF neutralizing 
antibodies
1 mL
2
2
yes
Dengue  virus 1-4 and 
YF neutralizing 
antibodies
remaining 
volume
3
3
Scheduled 
Visit 3 
5 mL whole 
blood
(Visit 2+ 6 
months for 
subject enrolled 
in Study Year 
1; Visit 2+12 
months for 
subject enrolled 
in beginning of  
Study Year 2 or 
3)
3030 (1530 
from wave 1 
and 1500 
from wave 
2)1500 
yes
Dengue IgG ELISA**
(only for subjects IgG 
negative at the 
previous visit)
250 µL
1
1
yes
Dengue virus 1-4 and 
YF neutralizing 
antibodies
1 mL
2
2
yes
Dengue  virus 1-4 and 
YF neutralizing 
antibodies
remaining 
volume
3
3
Subsequent 
Yearly Visit(s) 
as applicable
5 mL whole 
blood
Yearly 
3000 for Visit 
4 and 1500 
for visit 5 
and 6 which 
applies only 
to subjects 
of the wave 
11500
yes
Dengue IgG ELISA**
(only for subjects IgG 
negative at the 
previous visit)
250 µL
1
1
yes
Dengue  virus 1-4 and 
YF neutralizing 
antibodies
1 mL
2
2
yes
Dengue  virus 1-4 and 
YF neutralizing 
antibodies
remaining 
volume
3
3
*tertiary (exploratory) analysis, number of subjects in the subset will be determined during analysis 
-  not applicable, 
** Dengue IgG indirect ELISA
*** Wave 1 enrolment = initial cohort / Wave 2 enrolment = Expansion cohort
 
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Table 12
Laboratory read-outs at each time point, and priority ranking for 
suspected dengue visits for EARLY presenters
SUSPECTED DENGUE VISITS – EARLY PRESENTERS
Blood sampling time point
No. 
Subjects
Subset
Component
Serum 
aliquot 
volume
Components 
priority rank
Aliquot 
number
Type of contact/ 
Amount of whole 
blood
Sampling time 
point
Suspected 
dengue follow-up 
visit (total amount 
of blood 5 mL)
Approximately 
21 days after 
first visit
unknown
-
Dengue IgM 
and Dengue 
capture IgG 
ELISA
500 µL
1
1
Dengue  virus 
1-4 neutralizing 
antibodies
1 mL
42
42
Dengue  virus 
1-4 neutralizing 
antibodies
remaining 
volume
53
53
- not applicable
 
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Figure 4: Flow chart of serum aliquot testing for suspected dengue cases
Section 7.3.
Subject replacement
Enrolment of new subjects to replace: 

subjects not wishing to continue after this amendment 2 (initial cohort), or

subjects who prematurely terminate participation (initial and expansion cohorts) or

subjects lost to follow-up will be done (initial and expansion cohorts). 
The aim is to maintain a critical cohort size of 1500 subjects/site. This replacement will 
be done at the beginning ofbefore each studysurveillance year: during the scheduled 
visits starting at the end of study Year 1 (Visit 3) up till the end of study Year 3 (Visit 5).
The number of subjects to be recruited as replacements will be defined based on the 
number of subjects still active in the study. If at the end of each study year we have 
1500 subjects/site, no replacements will be needed. The time window for the 
recruitment of replacements will be during the low dengue transmission.
 
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Section 8.2.1. Assumptions:
It is planned to obtain approximately at least 1500 subjects/site still active at the end of 
the first year of the study. Because subjects might leave the study due to a variety of 
reasons, e.g. moving away geographically, sample size is increased to allow for this., On 
the basis of an annual drop-out rate between 15 and 20%, will be considered. 
Therefore,approximately 1800 600 subjects/site will be enrolled at the beginning of this 
study. The recruitment will be using cluster sampling , i.e. the unit of sampling will be 
the households. At least threeDifferent areas where the FHP has been fully implemented 
or where access to the community is already established by a government program will 
be first selected, followed by a selection of households in the respective areas. All 
subjects in each household will be invited for recruitment to the study.
New sites will start enrolling subjects preferably during study Year 2. The target is to 
enrol approximately 600 subjects per additional site. Replacement subjects may be 
recruited in order to maintain at least 500 subjects/site still active at the beginning of 
the next dengue season.
In order to better represent the Brazilian population distribution, subjects enrolled in 
the expansion cohort should be composed of at least 30% of subjects younger than 18 
years old and should not exceed 20 % of adults 50 years or older. Subjects enrolled as 
replacement subjects for the initial and expansion cohort will be recruited to approach 
as much as possible a minimal representation of 30% of subjects younger than 18 
years old and a maximal representation of 20% for adults 50 years or older.
The total study duration per site will last approximately 4 years for the initial cohort 
and approximately 3 years for the expansion cohort, and new subjects will be enrolled 
between seasons if the number of subjects still active in the study is below 1500/site after 
year 1 and at the beginning of each additional study year/season.
Section 8.2.3. Expected precision of incidence rate estimates
Table 13 shows the 95% CI for a range of expected incidences based on the Poisson 
distribution (exact method, normal approximation and the normal approximation 
accounting for a design effect of 1.8) in different scenarios. The scenarios were defined 
according to the following criteria:

Number of subjects enrolled at the beginning of the study (for some sites it will be 
the second year). At season level and for all sites: 2000, 4000, 1800, 3600, 1500, 
3000. For all seasons and for all sites: 4000, 3600 and 3000. At season level and 
site level: 600 and 500.

Number of years of follow-up. At study level: 1 or 4 years, but half of the sites will 
be followed for 3 years and this is taken into account for the calculations.  At site 
level: 1, 3 or 4 years.

Overall incidences: 3, 6, 9, 12 or 15 events per 1000 person-years.
With an overall sample size of a minimum of about 1,8003,600 subjects enrolled and a 
follow-up period of one year, the 95% confidence interval (CI) for an expected incidence 
of dengue of 9 cases per 1000 person-years using a cluster design is [2.94.7 ; 15.113.3]. 
With 3600 subjects enrolled, 4 years of follow-up and new enrolments to keep the 
number of subjects above 1500 3000 at the beginning of each subsequent season, the 
95% CI is [5.76.3 ; 12.311.7]. 
 
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Table 15 Precision forof the expected incidences of dengue for a single cohort of subjects in Brazil
Cohort 
size
Number of
years
Number of
person-years
Number 
of
cases
Incidence of dengue
(per 1000 person-years)
95% CI of incidence rate (n / 1000 P-Y)
New enrolment
Exact (Poisson)
Normal approx
Normal approx
with design effect
After 
Y1
After 
Y2
After 
Y3
Lower
Upper
Lower
Upper
Lower
Upper
Overall
2000
1
1845.9
6
3.0
1.0
6.7
0.5
5.5
-0.4
6.4
2000
1
1845.9
11
6.0
3.0
10.7
2.5
9.5
1.3
10.7
2000
1
1845.9
17
9.0
5.2
14.5
4.7
13.3
3.2
14.8
2000
1
1845.9
22
12.0
7.5
18.1
7.0
17.0
5.3
18.7
2000
1
1845.9
28
15.0
9.9
21.7
9.4
20.6
7.5
22.5
4000
1
3691.9
11
3.0
1.5
5.4
1.2
4.8
0.6
5.4
4000
1
3691.9
22
6.0
3.8
9.1
3.5
8.5
2.6
9.4
4000
1
3691.9
33
9.0
6.2
12.6
5.9
12.1
4.9
13.1
4000
1
3691.9
44
12.0
8.7
16.1
8.5
15.5
7.3
16.7
4000
1
3691.9
55
15.0
11.3
19.5
11.0
19.0
9.7
20.3
4000
4
9086.6
27
3.0
2.0
4.4
1.9
4.1
1.5
4.5
327
4000
4
9086.6
55
6.0
4.5
7.8
4.4
7.6
3.9
8.1
327
4000
4
9086.6
82
9.0
7.2
11.2
7.0
11.0
6.4
11.6
327
4000
4
9086.6
109
12.0
9.9
14.5
9.7
14.3
9.0
15.0
327
4000
4
9086.6
136
15.0
12.6
17.7
12.5
17.5
11.6
18.4
327
1800
1
1661.3
5
3.0
0.9
7.2
0.4
5.6
-0.5
6.5
1800
1
1661.3
10
6.0
2.8
11.1
2.3
9.7
1.0
11.0
1800
1
1661.3
15
9.0
5.0
14.9
4.4
13.6
2.9
15.1
1800
1
1661.3
20
12.0
7.3
18.6
6.7
17.3
4.9
19.1
1800
1
1661.3
25
15.0
9.7
22.1
9.1
20.9
7.1
22.9
3600
1
3322.7
10
3.0
1.4
5.6
1.1
4.9
0.5
5.5
3600
1
3322.7
20
6.0
3.6
9.3
3.4
8.6
2.5
9.5
3600
1
3322.7
30
9.0
6.1
12.9
5.8
12.2
4.7
13.3
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Cohort 
size
Number of
years
Number of
person-years
Number 
of
cases
Incidence of dengue
(per 1000 person-years)
95% CI of incidence rate (n / 1000 P-Y)
New enrolment
Exact (Poisson)
Normal approx
Normal approx
with design effect
After 
Y1
After 
Y2
After 
Y3
Lower
Upper
Lower
Upper
Lower
Upper
3600
1
3322.7
40
12.0
8.6
16.3
8.3
15.7
7.0
17.0
3600
1
3322.7
50
15.0
11.1
19.8
10.8
19.2
9.4
20.6
3600
4
8611.3
26
3.0
2.0
4.4
1.8
4.2
1.4
4.6
170
450
3600
4
8611.3
52
6.0
4.5
7.9
4.4
7.6
3.8
8.2
170
450
3600
4
8611.3
78
9.0
7.1
11.2
7.0
11.0
6.3
11.7
170
450
3600
4
8611.3
103
12.0
9.8
14.5
9.7
14.3
8.9
15.1
170
450
3600
4
8611.3
129
15.0
12.5
17.8
12.4
17.6
11.5
18.5
170
450
1500
1
1384.5
4
3.0
0.8
7.6
0.1
5.9
-0.9
6.9
1500
1
1384.5
8
6.0
2.6
11.7
1.9
10.1
0.5
11.5
1500
1
1384.5
12
9.0
4.7
15.6
4.0
14.0
2.3
15.7
1500
1
1384.5
17
12.0
6.9
19.3
6.2
17.8
4.3
19.7
1500
1
1384.5
21
15.0
9.2
22.9
8.5
21.5
6.3
23.7
3000
1
2768.9
8
3.0
1.3
5.9
1.0
5.0
0.3
5.7
3000
1
2768.9
17
6.0
3.5
9.7
3.1
8.9
2.1
9.9
3000
1
2768.9
25
9.0
5.8
13.3
5.5
12.5
4.3
13.7
3000
1
2768.9
33
12.0
8.3
16.8
7.9
16.1
6.5
17.5
3000
1
2768.9
42
15.0
10.8
20.3
10.4
19.6
8.9
21.1
3000
4
8306.7
25
3.0
1.9
4.4
1.8
4.2
1.4
4.6
225
450
450
3000
4
8306.7
50
6.0
4.4
7.9
4.3
7.7
3.8
8.2
225
450
450
3000
4
8306.7
75
9.0
7.1
11.3
7.0
11.0
6.3
11.7
225
450
450
3000
4
8306.7
100
12.0
9.8
14.6
9.6
14.4
8.8
15.2
225
450
450
3000
4
8306.7
125
15.0
12.5
17.9
12.4
17.6
11.5
18.5
225
450
450
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Cohort 
size
Number of
years
Number of
person-years
Number 
of
cases
Incidence of dengue
(per 1000 person-years)
95% CI of incidence rate (n / 1000 P-Y)
New enrolment
Exact (Poisson)
Normal approx
Normal approx
with design effect
After 
Y1
After 
Y2
After 
Y3
Lower
Upper
Lower
Upper
Lower
Upper
Site
600
1
553.8
2
3.0
0.2
12.3
-1.6
7.6
-3.1
9.1
600
1
553.8
3
6.0
1.2
17.0
-0.5
12.5
-2.7
14.7
600
1
553.8
5
9.0
2.7
21.4
1.1
16.9
-1.6
19.6
600
1
553.8
7
12.0
4.6
25.1
2.9
21.1
-0.2
24.2
600
1
553.8
8
15.0
6.5
29.2
4.8
25.2
1.3
28.7
600
3
1486.0
4
3.0
0.8
7.5
0.2
5.8
-0.7
6.7
67
600
3
1486.0
9
6.0
2.7
11.5
2.1
9.9
0.7
11.3
67
600
3
1486.0
13
9.0
4.8
15.3
4.2
13.8
2.5
15.5
67
600
3
1486.0
18
12.0
7.1
19.0
6.4
17.6
4.5
19.5
67
600
3
1486.0
22
15.0
9.4
22.6
8.8
21.2
6.6
23.4
67
600
4
1947.5
6
3.0
1.0
6.7
0.6
5.4
-0.3
6.3
67
75
600
4
1947.5
12
6.0
3.1
10.6
2.6
9.4
1.4
10.6
67
75
600
4
1947.5
18
9.0
5.3
14.3
4.8
13.2
3.3
14.7
67
75
600
4
1947.5
23
12.0
7.6
17.9
7.1
16.9
5.5
18.5
67
75
600
4
1947.5
29
15.0
10.1
21.5
9.6
20.4
7.7
22.3
67
75
500
1
461.5
1
3.0
0.1
14.3
-2.0
8.0
-3.7
9.7
500
1
461.5
3
6.0
1.0
18.6
-1.1
13.1
-3.5
15.5
500
1
461.5
4
9.0
2.5
22.7
0.3
17.7
-2.6
20.6
500
1
461.5
6
12.0
4.2
26.8
2.0
22.0
-1.4
25.4
500
1
461.5
7
15.0
5.8
31.2
3.8
26.2
0.0
30.0
500
3
1384.5
4
3.0
0.8
7.6
0.1
5.9
-0.9
6.9
75
75
500
3
1384.5
8
6.0
2.6
11.7
1.9
10.1
0.5
11.5
75
75
500
3
1384.5
12
9.0
4.7
15.6
4.0
14.0
2.3
15.7
75
75
500
3
1384.5
17
12.0
6.9
19.3
6.2
17.8
4.3
19.7
75
75
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Cohort 
size
Number of
years
Number of
person-years
Number 
of
cases
Incidence of dengue
(per 1000 person-years)
95% CI of incidence rate (n / 1000 P-Y)
New enrolment
Exact (Poisson)
Normal approx
Normal approx
with design effect
After 
Y1
After 
Y2
After 
Y3
Lower
Upper
Lower
Upper
Lower
Upper
500
3
1384.5
21
15.0
9.2
22.9
8.5
21.5
6.3
23.7
75
75
500
4
1845.9
6
3.0
1.0
6.7
0.5
5.5
-0.4
6.4
75
75
75
500
4
1845.9
11
6.0
3.0
10.7
2.5
9.5
1.3
10.7
75
75
75
500
4
1845.9
17
9.0
5.2
14.5
4.7
13.3
3.2
14.8
75
75
75
500
4
1845.9
22
12.0
7.5
18.1
7.0
17.0
5.3
18.7
75
75
75
500
4
1845.9
28
15.0
9.9
21.7
9.4
20.6
7.5
22.5
75
75
75
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Section 8.4.1. Sequence of analyses
An interim analysis will be performed when all active subjects from the initial cohort of 
first two sites have completed two years of surveillance (Vachieved the visit 4) and the 
third site have completed at least one year of surveillance (Visit 3). All analyses of 
primary and secondary objectives will be performed on data as cleaned as possible.
APPENDIX A
Non GSK laboratories
Laboratory
Address
Fundação de Medicina Tropical Dr. 
Heitor Vieira Dourado - FMT
Gerência de Virologia
Av. Pedro Teixeira, 25, Dom Pedro
Manaus, AM. CEP: 69.040-000
Brazil
Centro de Pesquisas Gonçalo 
Moniz - CPqGM Fundação 
Oswaldo Cruz - FiocruzLaboratório 
de Patologia e Biologia Molecular -
LPBM
Rua Waldemar Falcão, 121, Candeal 
Salvador, BA. CEP: 40.296-710
Brazil
Instituto de Pesquisa Clínica 
Evandro Chagas - IPEC, Fundação 
Oswaldo Cruz - Fiocruz
Laboratório de Imunodiagnóstico
Av. Brasil, 4365, Manguinhos
Rio de Janeiro, RJ. CEP: 21.040-900
Brazil
Local laboratories
Details presented in a separated document
Laboratório de Tecnologia 
Virológica – LATEV
Bio-Manguinhos
Fundação Oswaldo Cruz - Fiocruz
Av. Brasil, 4365, Manguinhos
Pavilhão Rocha Lima, sala 403
Rio de Janeiro, RJ. CEP: 21.040-360
Brazil
 
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GlaxoSmithKline Biologicals
Clinical Research & Development
Protocol Amendment 4 
eTrack study number and 
Abbreviated Title
116606 (EPI-DENGUE-006 BOD BR)
Amendment 4 version and date:
Final: 16 October 2017
Coordinating author:
, Scientific Writer
Rationale/background for changes: 
Additional exploratory objectives have been added in this protocol amendment to 
explore the seroprevalence of antibodies to Zika and chinkungunya viruses in the study 
population, as well as the potential aetiological role of these viruses in episodes of 
febrile illness detected during follow-up .
Zika is a flavivirus and chikungunya virus is a member of the alphavirus genus, and 
Togaviridae family. These cause emerging mosquito-borne viral diseases in Latin 
America especially in Brazil. Zika virus infections have been laboratory confirmed in 
19 states in Brazil between April and December 2015 [Boletim Epidemiológico n5, 
2016]. In 2016, until epidemiological week 47, 196.976 cases were reported and 
101,851 were confirmed [Boletim epidemiológico n 33, 2016]. 
In 2015, 20,661 autochthonous suspected chikungunya cases were reported in Brazil 
[Boletim Epidemiológico n5, 2016]. In 2016, 216,102 reported cases and 102,638 were 
confirmed up to epidemiological week 47 [Boletim epidemiológico n33, 2016]. Zika 
and chikungunya viruses cause symptoms often similar to dengue during the acute 
phase of illness.
The interpretation of the serological tests used to determine the seroprevalence of 
dengue antibodies or for diagnostic of acute dengue infection should take into account 
the possibility of cross-reaction with antibodies elicited by exposure to the Zika virus. 
The public health importance of Zika and chikungunya virus emergence, as well as the 
potential impact of these viruses on the interpretation of the laboratory assays, supports 
the inclusion of the exploratory objectives related to Zika and chikungunya infection.
Fever is the triggering event for a subject to be evaluated as a suspect dengue case in 
the study. While fever is common in suspected chikungunya cases, fever does not 
always present in suspected Zika cases.  For this reason, rash is being added as a 
triggering event, in addition to fever, for Zika virus disease evaluation. A prospective 
surveillance will be maintained for febrile cases and added for rash cases. . Also, 
considering the epidemiological situation in Brazil for both Zika and chikungunya 
infection, a retrospective testing for these infections in subjects considered as suspect 
dengue case since the beginning of the study will also be done.
The current case definition for suspected dengue does not include a criteria specifying 
the maximum interval between fever onset and the initial medical visit. This is 
 
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specified in section 5.7.1.1.
Currently, haematology testing (HCT, CBC) is planned to be performed for all 
suspected dengue cases at the initial visit, regardless of the time between fever onset 
and the visit. However, for patients presenting more than 14 days after fever and/or 
rash onset, the relevance of these tests is low. Therefore, a maximum interval will be 
defined in this amendment.
The geographical distribution of the participants may impact the risk of infection. 
Therefore, updating information on the household of the study participants and their 
location at yearly visits is important during the follow-up period (which may extend to 
4 years). Hence, socio-demographic and household information will be collected at 
yearly visits.
Some sites may have more than 500 active participants enrolled in the penultimate year 
of the study.  However, their site may have experience with high drop-out rate and can 
expect to drop below 500 participants during the last year of the study.  To address this 
challenge, criteria to allow for replacement cohort recruitment was clarified.
Also, the NS1 test has been removed for the late presenters since this test does not have 
clinical relevance in the case of late presenters.
Collection of dengue vaccination history has been added since a competitor dengue 
vaccine is currently available in the Brazilian market.
Amended text has been included in bold italics and deleted text in strikethrough in 
the following sections:
Cover page:
GSK Contributing authors:
Clinical Laboratory Sciences Manager
Non-GSK Contributing authors:
Bio-Manguinhos, Fiocruz
GSK Contributing authors for current version:
Epidemiologist, Brazil
Epidemiologist, Director
Local Delivery Lead
Local Delivery Lead
Clinical Laboratory Sciences Manager 
Clinical Laboratory Study Management
Lead Statistician
Statistician
Study Delivery Lead
Clinical Read-out Team Leader
Study statistician
Medical Affairs Director, Brazil
 
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Copyright 2012-2017 the GlaxoSmithKline group of companies. All rights reserved. Unauthorized 
copying or use of this information is prohibited.
© [2017] GSK group of companies or its licensor.
Sponsor signatory approval:
Sponsor signatory
,
CEPL, Early Development, 
Vaccines R&D,
GSK Biologicals US
Synopsis:
Tertiary 
(optional) 
objectives

To investigate potential risk factors for symptomatic dengue 
infection.

To investigate the role of dengue neutralizing antibodies 
elicited through natural infection in protection against 
subsequent infection.

To estimate the degree of underreporting of dengue cases 
using the passive national notification surveillance.

To estimate the incidence of symptomatic Zika virus 
infection (including laboratory-confirmed and probable 
cases) in the study population by study site, gender, age-
group, previous flavivirus exposure, overall and by season.

To estimate the incidence of symptomatic chikungunya 
virus infection (including laboratory-confirmed and 
probable cases) in the study population by study site, 
gender, age-group, overall, and by season.

To describe previous Yellow Fever (YF) antibody profile in 
selected dengue cases to investigate the role of previous YF 
immunity on the dengue neutralizing antibody profile 
following dengue infection.

To describe the spatial and temporal distribution of dengue 
cases among cohort participants in the study areas.

To describe the spatial and temporal distribution of Zika 
and chikungunya cases among cohort participants in the 
study areas.

To describe other infectious aetiologies related to 
differential diagnosis of dengue (chikungunya and Zika) in 
subjects with episodes of febrile illness referred to as 
“suspected dengue case”.
 
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
To estimate the seroprevalence of antibodies against Zika 
and chikungunya virus at selected timepoints.
Study design

Type of design: Prospective, multicenter, community-based, 
household-sampling, cohort study in Brazil.

Study population: Subjects six months of age and older at 
the time of enrolment who live in the selected study sites in 
Brazil, covering different regions of the country.

There will be 2 waves of enrolment for recruiting 3600 
subjects:

Initial Cohort = about 1800 subjects – up to 6 visits 
(Amendment 2) – INITIAL sites

Expansion Cohort = about 1800 subjects – up to 4 visits  
– NEW sites 

The study period, initially planned to be one year, has been 
extended by additional three years (overall 4 years) to cover 
three additional dengue seasons (Amendment 2). 

Initial cohort subjects were invited to extend their 
participation. 
(See glossary of terms for the definition of initial and 
expansion cohort subjects)
In each cohort: replacement subjects may be enrolled in order 
to compensate for subjects who did not wish to extend their 
participation for three additional years (for subjects from the 
initial cohort – Amendment 2), subjects who prematurely 
terminate participation or subjects who are lost to follow-up. 
This will be done to maintain a cohort size of at least 500 
subjects per site, in the Initial and Expansion cohorts, at the 
beginning of each additional study year/season.
At the end of the penultimate year of the study, the cohort 
size will be reviewed to address the potential need for 
replacement cohort recruitment.
(See glossary of terms for the definition of replacement 
subjects, subjects who prematurely terminate participation 
and subjects lost to follow-up ) 

Informed consent (and assent, if applicable) will be obtained:

From the subject (and parents/LAR of the subjects,  if 
applicable) at the study start for the initial cohort.

From the subject (and parents/LAR of the subjects,  if 
applicable) of the initial cohort prior to participation in 
the additional three years of the study.
 
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
From the subject (and parents/LAR of the subjects,  if 
applicable) at the study start for the expansion cohort. 
From the subject (and parents/LAR of the subjects, if applicable) 
prior to enrolment in the study for the replacement subjects.

Study visits: The visits will be as follows and shown in 
Synopsis Figure 1:

Initial cohort subjects consenting to continue 
participation for the three additional years will have:
o
three scheduled visits in the first year (Visit 1, 
Visit 2 and Visit 3) at six months intervals (+/- 28 
days), 
o
and thereafter, one scheduled visit per year during 
a period of low dengue transmission for the three 
additional years; i.e; one visit at study Year 2, Year 
3 and Year 4 (Visit 4, Visit 5 and Visit 6 
respectively).

Initial cohort subjects who do not consent to extend 
participation for three additional years will:
o
have three scheduled visits (Visit 1, Visit 2 and 
Visit 3) at six months interval (+/- 28 days). 
o
conclude their participation in the study at Visit 3 
or the last follow up visit if the subject is suspected 
of having a dengue case on-going at Visit 3.

Subjects from the expansion cohort will have only three 
years of follow-up, with at most 4 scheduled visits.

For replacement subjects, the number of scheduled visits 
will depend on the study year in which they are enrolled. 
Replacement subjects will not be enrolled until after the 
third visit of the first study year is concluded for the 
initial cohort. The number of visits for the replacement 
subjects will range between two and four.

The yearly scheduled visits for the initial and expansion 
cohorts and replacement subjects will preferably occur during 
the period of usually low dengue transmission.

Suspected dengue, chikungunya and/or Zika case detection 
(Amended 16 October 2017): Suspected dengue, Zika
and/or chikungunya cases will be detected using three 
sources: 
1) referred by study personnel during scheduled home visits; 
2) through enhanced passive surveillance; and 
3) as a result of active surveillance between scheduled visits; 
(see Section 5.5.3 for details). 

If dengue, Zika and/or chikungunya is/are suspected, a 
medical appointment with a physician at a designated study 
 
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hospital/clinic will be arranged (see Section 5.6 for 
management of suspected dengue, Zika and chikungunya
cases). 

If the suspicion is confirmed during the visit: 
iv. A blood sample for dengue, Zika and/or chikungunya
diagnosis and for haematology (CBC and HCT) (mandated 
procedure for subjects presenting within the first 14 days 
following onset of fever and/or rash) will be collected at the 
first visit for suspected dengue, Zika and/or chikungunya
(acute blood sample) and 
v.
a second blood sample (convalescent blood sample) will be 
collected approximately 21 (maximum 28 days) days later. 
vi. In between, a return visit may be scheduled as needed.

Data collection: eCRF

Type of study: self-contained

Duration of the study: Four years for the overall study. For 
an individual subject, it will range between one and four 
years.  

Epoch 001: prospective data collection starting at Visit 1 
Day 0 and ending at the last subject last visit.
Endpoints:
Tertiary (optional)
Exploratory objectives may be assessed based on, but not limited to 
the following endpoints:

Symptomatic laboratory-confirmed Zika virus infection

Symptomatic Zika virus infection (including laboratory-
confirmed and probable cases).

Symptomatic laboratory-confirmed chikungunya infection

Symptomatic chikungunya infection (including laboratory-
confirmed and probable cases).

Risk factors for dengue infection and disease

Neutralizing antibodies titers against DENV 1-4

Neutralizing antibody titers against YF virus

Spatial and temporal distribution of dengue cases (confirmed and 
probable symptomatic cases and inapparent infections)

Spatial and temporal distribution of laboratory confirmed and 
probable Zika virus cases
 
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
Spatial and temporal distribution of patients with laboratory 
confirmed and probable chikungunya virus infection

Occurrence of laboratory confirmed or probable Zika and/or 
chikungunya infection in suspected dengue cases (differential 
diagnosis of dengue), retrospectively

Antibody titre against Zika and chikungunya virus at the 
scheduled visits
Glossary of terms
Diary log:
A diary log will be used in this study. The diary log is 
given to subjects to record body temperatures and 
symptoms in the event that a suspected dengue, Zika 
and/or chikungunya symptom occurs (see Section 
5.7.1.1). The information collected in the diary log will be 
provided to the study physician as a tool in the medical 
evaluation of the subject. If the physician considers that 
the subject meets the criteria for suspected dengue, he/she 
will use the information contained in the diary log to 
complete the description of first clinical symptoms in the 
eCRF.
Early presenter
A suspected dengue, Zika and/or chikungunya case 
presenting at the health care facility within 5 days 
following the onset of fever and/or rash for Zika virus.
Late presenter
A suspected dengue, Zika and/or chikungunya case 
presenting at the health care facility 6 to 30 days or more
after the onset of fever and/or rash for Zika virus.
List of abbreviations:
DHF
Dengue Hemorrhagic Fever
ICU:
Intensive Care Unit
SST
Serum Separator Tube
 
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Glossary of terms
Diary log:
A diary log will be used in this study. The diary log is 
given to subjects to record body temperatures and 
symptoms in the event that a suspected dengue, Zika 
and/or chikungunya symptom occurs (see Section 
5.7.1.1). The information collected in the diary log will be 
provided to the study physician as a tool in the medical 
evaluation of the subject. If the physician considers that 
the subject meets the criteria for suspected dengue, Zika 
and/or chikungunya, he/she will use the information 
contained in the diary log to complete the description of 
first clinical symptoms in the eCRF.
Early presenter
A suspected dengue, Zika and/or chikungunya case 
presenting at the health care facility within 5 days 
following the onset of fever and/or rash for Zika virus.
Late presenter
A suspected dengue, Zika and/or chikungunya case 
presenting at the health care facility 6 to 30 days after the 
onset of fever and/or rash for Zika virus.
Seroconversion: 
Documented positive antibody test with previous 
documented negative antibody test.
Section 1.1
Background
Dengue, the most common arthropod-borne viral disease worldwide, is caused by four 
types of dengue viruses (DENV 1-4), transmitted primarily by Aedes aegypti, a mosquito 
that is highly adapted to urban environments. Dengue infection can cause a range of 
clinical illnesses, from inapparent to a life threatening hemorrhagic disease, often 
associated with pre-existing heterotypic dengue virus antibodies. 
An estimated 2.5 billion people are at risk of infection in tropical and subtropical 
countries worldwide (WHO, 2009) where major urban epidemics are responsible for 
substantial social and economic burden. Dengue has drastically increased in the last two 
decades in Central and South America with Brazil being one of the most affected 
countries (Siqueira-Junior, 2008). In Central Brazil, an almost 20-fold increase in 
reported cases of dengue have been reported from 1990 (1,660 cases) to 2000 (20,552 
cases) (Siqueira, 2004). In all of Brazil, the number of yearly reported cases increased 
from 124,827 in 1995 to more than 1 million in 2010 (PAHO, 2011)
Currently, strategies to reduce disease burden rely mainly on mosquito control and 
human preventive behaviour modification, as treatment is limited to supportive care. The 
World Health Organization (WHO) has, therefore, considered the development of a 
vaccine a priority research area (Brandt, 1990), and a number of vaccine candidates are 
under development or being tested in clinical trials (Thomas, 2011).
 
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In preparation for phase III trials, the WHO recommends that the primary efficacy 
endpoint be the presence of DENV in a patient with signs and/or symptoms of dengue 
disease (WHO, 2009; Edelman, 2008). However, mild cases of dengue are largely 
underreported, as shown in dengue cohort studies and capture-recapture studies 
(Wichmann, 2011; Vong, 2011; Suaya, 2007). For example, in Nicaragua an active 
dengue surveillance system in children was able to detect approximately 14 to 28 
(average 21.3) times more dengue cases each year per 100,000 persons than passive 
surveillance among similar paediatric populations (Standish, 2010). 
Zika is a flavivirus and chikungunya virus is a member of the alphavirus genus, and 
Togaviridae family. These are emerging mosquito-borne viral diseases in Latin 
America. Zika virus infections have been laboratory confirmed in 19 states in Brazil 
between April and December 2016 [Boletim Epidemiológico, 2017]. In 2016, until 
epidemiological week 52, 215,319 cases were reported and 130,701 were confirmed 
[Boletim Epidemiológico, 2017]. 
In 2015, 20,661 autochthonous suspected chikungunya cases were reported in Brazil 
[Boletim Epidemiológico, 2017]. In 2016, 271,824 reported cases and 151,318 were 
confirmed up to epidemiological week 47 [Boletim Epidemiológico, 2017]. Zika and 
chikungunya viruses cause symptoms often similar to dengue during the acute phase 
of illness.
Another challenge when conducting vaccine trials is to select geographic settings where 
the incidence of dengue would allow for a reliable assessment of vaccine efficacy. 
Endemicity for more than one dengue virus type is also highly desirable. Thus, selection 
of geographically diverse sites can potentially maximize the likelihood of having a 
sufficient number of exposed individuals for the conduct of a vaccine trial. Finally, 
identifying all other coexisting flaviviruses circulating at potential study sites is 
important, as infection by such viruses might, in theory, modulate immune response and 
clinical course of dengue infection. 
In this study, we will estimate the incidence of dengue infection and disease in a cohort 
of subjects recruited from different geographic areas over time in Brazil. The study will 
also identify and train potential sites for the conduct of phase III studies in the future.
Section 2.3
Tertiary Objectives

To investigate potential risk factors for symptomatic dengue infection.

To investigate the role of dengue neutralizing antibodies elicited through natural 
infection in protection against subsequent infection.

To estimate the degree of underreporting of dengue cases using the passive national 
notification surveillance.

To estimate the incidence of symptomatic Zika virus infection (including 
laboratory-confirmed and probable cases) in the study population by study site, 
gender, age-group, previous flavivirus exposure, overall and by season.
 
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
To estimate the incidence of symptomatic chikungunya virus infection (including 
laboratory-confirmed and probable cases) in the study population by study site, 
gender, age-group, overall, and by season.

To describe previous Yellow Fever (YF) antibody profile in selected dengue cases to 
investigate the role of previous YF immunity on the dengue neutralizing antibody 
profile following dengue infection.

To describe the spatial and temporal distribution of dengue cases among cohort 
participants in the study areas.

To describe the spatial and temporal distribution of Zika and chikungunya cases 
among cohort participants in the study areas.

To describe other infectious aetiologies related to differential diagnosis of dengue 
(chikungunya and Zika) in subjects with episodes of febrile illness referred to as 
“suspected dengue case”.

To estimate the seroprevalence of antibodies against Zika and chikungunya virus 
at selected timepoints.
Section 3
Study design overview

Type of design: Prospective, multicenter, community-based, household-sampling, 
cohort study in Brazil.

The study period, initially planned to be one year, has been extended by additional 
three years (overall four years) to cover three additional dengue seasons 
(Amendment 2).

Initial cohort subjects were invited to extend their participation. 

(See glossary of terms for the definition of initial and expansion cohort subjects).

In each cohort: replacement subjects may be enrolled in order to compensate for 
subjects who did not wish to extend their participation for three additional years (for 
subjects from the initial cohort – Amendment 2), subjects who prematurely terminate 
participation or subjects who are lost to follow-up. 
This will be done to maintain a cohort size of at least 500 subjects per site, in the 
Initial and Expansion cohorts, at the beginning of each additional study year/season.
In last three months of the of penultimate year of the study, each site will review 
the number of active subjects and drop-out rate.  If the risk of having fewer than 
the required number of active participants at the end of the study is high, 
recruitment for participants into the replacement cohort may be performed with 
sponsor approval. If the risk is high, recruitment into the replacement cohort 
should begin even if the number of active subjects is 500 or above 500.
(See glossary of terms for the definition of replacement subjects, subjects lost to 
follow-up and subjects who prematurely terminate participation ).

Informed consent (and assent, if applicable) will be obtained:
 
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
From the subject (and parents/LAR of the subjects,  if applicable) at the study 
start for the initial cohort.

From the subject (and parents/LAR of the subjects,  if applicable) of the initial 
cohort prior to participation in the additional three years of the study.

From the subject (and parents/LAR of the subjects,  if applicable) at the study 
start for the expansion cohort. 

From the subject (and parents/LAR of the subjects,  if applicable) prior to 
enrolment in the study for the replacement subjects.

Study population: Subjects six months of age and older at the time of enrolment, 
who either live in households in study areas with support from the Family Health 
Physician Program (FHP) or the Larval Index Rapid Assay (LIRA) or with field 
research experience in the community (preferred) or where a similar system of 
mapped communities with potential for surveillance exists.

There will be 2 waves of enrolment for recruiting 3600 subjects:

Initial Cohort = about 1800 subjects – up to 6 visits (Amendment 2) – INITIAL 
sites

Expansion Cohort = about 1800 subjects – up to 4 visits – NEW sites

Study visits: The visits will be as follows and further shown in Figure 1:

Initial cohort subjects consenting to extend participation for the three additional 
years will have:
o
three scheduled visits in the first year (Visit 1, Visit 2 and Visit 3) at 
approximately six months intervals (+/- 28 days), 
o
and thereafter, one scheduled visit per year preferably during a period of 
low dengue transmission for the three additional years; i.e., one visit at 
study Year 2, Year 3 and Year 4 (Visit 4, Visit 5 and Visit 6 respectively).

Initial cohort subjects who do not consent to extend participation for three 
additional years will:
o
have three scheduled visits (Visit 1, Visit 2 and Visit 3) at approximately 
six months intervals (+/- 28 days). 
o
conclude their participation in the study at Visit 3 or the last follow up 
visit if the subject is suspected of having a dengue case on-going at Visit 
3.

Subjects from the expansion cohort will have only three years of follow-up, with 
at most 4 scheduled visits.

For replacement subjects, the number of scheduled visits will depend on the 
study year in which they are enrolled. Replacement subjects will not be enrolled 
until after the third visit of the first study year is concluded for the initial cohort. 
The number of scheduled visits will range between two and four.
 
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The yearly scheduled visits for the initial and expansion cohorts and 
replacement subjects will preferably occur during the period of usually low 
dengue transmission.
In last three months of the of penultimate year of the study, each site will 
review the number of active subjects and drop-out rate.  If the risk of having 
fewer than the required number of active participants at the end of the study is 
high, recruitment for participants into the replacement cohort may be 
performed with sponsor approval. If the risk is high, recruitment into the 
replacement cohort should begin even if the number of active subjects is 500 
or above 500.
Section 3.2.1 Rationale for study design:
A single cohort study was chosen to estimate the incidence of dengue in the community. 
Data will be collected from a population of individuals aged 6 months and older for 
maximum four dengue seasons. Inclusion of individuals of various ages will further allow 
for a better understanding of disease dynamics, clinical spectrum, and validate attack 
rates for potential subgroups to be included in future dengue vaccine trials.
This study will use cluster (household) sampling to take into account the correlation that 
may exist within households. Individuals within a household are expected to have a more 
similar risk of infection compared to individuals from different households. For example, 
individuals living in the same household are more likely to share similar risk of being 
exposed to DENV infected mosquitoes. The sample size has been adjusted to account for 
this between-cluster variability.
A possible bias may arise from the non-response rate. To reduce the possibility of this 
potential bias, visits will be scheduled on weekends or at times more convenient to the 
subject. Where applicable, FHP community health care workers will facilitate 
communication between study staff and participants.
Section 4.3
Overview of the recruitment plan:
At least 3600 subjects will be recruited in the study in 2 waves of enrolment. Each wave, 
initial and expansion cohorts, will enrol about 600 subjects/site in order to maintain at 
least 500 subjects/site at the beginning of each dengue season.
Subjects will be recruited from randomly selected households originating from pre-
selected mapped communities. Preferably the recruitment period will occur outside of the 
peak dengue transmission season, and will continue until each site has reached its 
foreseen target. The expected period for recruiting the target sample size is approximately 
three months. Recruitment of replacement subjects will be done during the low dengue 
transmission and the recruitment period will depend on the number of subjects that need 
to be replaced.
Recruitment will be organized by study staff at participating sites according to the 
appropriate strategy for each site. Community agents or equivalent will serve as the 
liaison to schedule visits and may accompany study staff during the visits. The study will 
be explained to the individuals living in the household, and if any individual is interested 
 
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in study participation, informed consent (and assent when applicable) will be obtained, 
eligibility criteria will be checked and subjects will be enrolled and interviewed for 
potential study participation.
If the target household is found empty or all members refuse to participate, the first 
household to the left will be approached. Those households refusing to participate will be 
recorded as such. A household refusal will be characterized when all individuals in the 
household refuse to participate in the study. Individual refusals in a given household will 
not preclude inclusion of other individuals living in the households, and will be recorded 
as such.
In the subsequent study years/seasons, the cohort size will be maintained at a minimum 
of 500 subjects per site. Since subjects who prematurely terminate participation and/or 
lost to follow-up will be replaced, the final number of participants across the four study 
years may exceed 3600 subjects. Yearly, there will be an evaluation of active subjects. If 
the number of active subjects/site becomes < 500, there will be enrolment of replacement 
subjects to maintain a cohort size of at least 500 subjects/site. Enrolment will preferably 
take place during the low dengue transmission. Recruitment of replacement subjects 
could occur at the end of study year 1, 2 or 3. The recruitment approach will be the same 
as for the initial enrolment.
In order to better represent the Brazilian population distribution, subjects enrolled in the 
expansion cohort should be composed of at least 30% of subjects younger than 18 years 
old and should not exceed 20 % of adults 50 years or older. Subjects enrolled as 
replacement subjects for the initial and expansion cohort will be recruited to approach as 
much as possible a minimal representation of 30% of subjects younger than 18 years old 
and a maximal representation of 20% for adults 50 years or older.
Household visits may be scheduled to occur during weekends or at convenient times (like 
evenings) if deemed necessary.
Section 4.4
Inclusion criteria for enrolment
All subjects must satisfy ALL the following criteria at study entry.

Written, signed or thumb-printed informed consent (and assent when applicable) 
must be obtained from the subject or subject’s parent(s)/LAR(s). If the 
subject/subject’s parent(s)/LAR(s) are illiterate the consent form will be 
countersigned by a witness.

Male or female at least 6 months of age at the time of enrolment.

Subject and/or subject’s parent(s)/LAR(s) who the study staff believes can comply 
with the requirements of the protocol (e.g., willingness to go to the hospital/clinic for 
visit/s if dengue, Zika and/or chikungunya is suspected, able to observe the signs of 
dengue, Zika and/or chikungunya and to understand how to take and report body 
temperature, etc.).

Subject who plans, at the time of enrolment, to remain at same residence/study area 
during their study participation period.
 
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Section 5.1
Regulatory and ethical considerations, including the informed consent 
process
The study will be conducted in accordance with all applicable regulatory requirements.
The study will be conducted in accordance with the ICH Guideline for Good Clinical 
Practice (GCP), any applicable local guidelines, including the Document of the Americas, 
all applicable subject privacy requirements and the guiding principles of the Declaration 
of Helsinki. 
The study has been designed and will be conducted in accordance with the ICH 
Harmonized Tripartite Guideline for clinical investigation of medicinal products in the 
paediatric population (ICH E11) and all other applicable ethical guidelines.
GSK will obtain favourable opinion/approval to conduct the study from the appropriate
regulatory agency, in accordance with applicable regulatory requirements, prior to a site 
initiating the study in that country or will document that neither a favourable opinion nor 
an approval to conduct the study is needed. 
Conduct of the study includes, but is not limited to, the following:

Institutional Review Board (IRB)/Independent Ethics Committee (IEC) review and 
favourable opinion/approval of study protocol and any subsequent amendments

Subject/ subject’s parent(s)/legally acceptable representative (LAR[s])  informed 
consent and subject informed assent, as appropriate

Investigator reporting requirements as stated in the protocol.
GSK will provide full details of the above procedures to the investigator, either verbally, 
in writing, or both.
Freely given and written informed consent must be obtained from each subject and/or 
each subject’s parent(s)/LAR(s) and subject informed assent as appropriate, prior to 
participation in the study. 
GSK Biologicals will prepare a model Informed Consent Form (ICF) which will embody 
the applicable ICH GCP 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 Harmonized Tripartite Guidelines for Good Clinical Practice, 
those subjects who can only be enrolled in the study with the consent of the subject’s 
parent(s)/LAR(s) (e.g. minors), should be informed about the study to the extent
compatible with the subject’s understanding and, if capable, the subject should sign and 
personally date a written informed assent. It is required to be signed by each subject, if 
capable, in addition to the informed consent that is to be signed by his/her parent or legal 
 
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representative. Requirement of assent should be assessed based on the age of the study 
population and the local requirements.
GSK Biologicals strongly recommends that if the subject reaches the age of consent 
during the study they will be asked to provide consent at the next study visit (if 
applicable). This procedure should be applied according to local laws and regulations.
If a subject is unable to read or if a legally acceptable representative is unable to read, the 
informed consent will be obtained in the presence of an impartial witness, according to 
the International Conference on Harmonization (ICH) guidelines. For all participants 
unable to read for whatever reason, the subject’s fingerprint must be obtained on the ICF. 
The investigator has the final responsibility for the final presentation of the ICF, 
respecting the mandatory requirements of local regulations. The ICF generated by the 
investigator with the assistance of the sponsor’s representative must be acceptable to 
GSK Biologicals and be approved (along with the protocol, and any other necessary 
documentation) by the IRB/IEC.
 
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Section 5.4
Outline of study procedures
Table 2
List of study procedures for initial cohort subjects 
Surveillance
Suspected 
Dengue/Zika/Chikungunya
Visits (at hospital/clinic)
as applicable
Procedure
Visit 1
Day 0
Monthly 
contact 
between 
visits
Visit 2
Month 6
Monthly 
contact 
between 
visits
Visit 3
Month 
12 (Year 
1)
Monthly 
contact 
between 
visits
Visit 4 
Month 
24 (Year 
2)
Monthly 
contact 
between 
visits
Visit 5 
Month 
36 (Year 
3)
Monthly 
contact 
between 
visits
Visit 6 
Month 
48 (Year 
4)
First visit 
Day 0
Return
visit 2
Follow-
up visit 
Day 21
Informed consent (and assent if 
applicable)
●
Re-consent (and assent if 
applicable)@
●
●
●
●
●
●
●
●
Subject number and initial
household number attribution 
●
Check inclusion/ exclusion criteria 
●
Record socio-demographic 
information or updates (including 
household characteristics if 
applicable)
●
●
●
●
Medical history including YF and 
dengue vaccination history or 
updates
●
●
●
●
●
●
Distribute subject ID card 
distribution and suspected dengue 
instruction kit
O
Blood sample for serology (5 mL)
●
●
●
●
●
●
Instruct/remind subjects/subject's 
parent(s)/LAR(s) on suspected 
dengue, chikungunya and Zika
assessment procedures (where 
applicable)
O
O
O
O
O
O
O
O
O
O
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Surveillance
Suspected 
Dengue/Zika/Chikungunya
Visits (at hospital/clinic)
as applicable
Procedure
Visit 1
Day 0
Monthly 
contact 
between 
visits
Visit 2
Month 6
Monthly 
contact 
between 
visits
Visit 3
Month 
12 (Year 
1)
Monthly 
contact 
between 
visits
Visit 4 
Month 
24 (Year 
2)
Monthly 
contact 
between 
visits
Visit 5 
Month 
36 (Year 
3)
Monthly 
contact 
between 
visits
Visit 6 
Month 
48 (Year 
4)
First visit 
Day 0
Return
visit 2
Follow-
up visit 
Day 21
Issue diary logs to parents/LARs to 
be filled out in the event of a 
suspected dengue, chikungunya 
and Zika case1
O
O
O
O
O
O
Contact subject regarding any 
dengue, chikungunya and Zika
symptoms and remind subject of 
procedures for suspected dengue, 
chikungunya and Zika
O
O
O
O
O
Physical examination/record 
current medical conditions(see 
Section 5.6)
●
●
●
Blood sample for 
dengue/zika/chikungunya
infection differential diagnosis3
●
●
Blood sample for CBC and 
hematocrit (mandated for 
subjects presenting within 14 
days following symptoms7
onset)
●
Record body temperature
●
Report SAEs related to study 
procedures4
●
●
●
●
●
●
●
●
●
Collect or verify diary logs if 
applicable
O
O
O
Study conclusion5
●
●
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● 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.
1 Diary logs given at scheduled visits are only to be filled out in the event that the subject has fever and/or rash according to the case definition for suspected 
dengue/chikungunya/Zika. See Section 5.5.2.5 for details with regard to the diary log.
2 A return visit is a visit linked to suspected dengue/chikungunya/Zika, occurring if the evolution of the subject’s physical condition necessitates medical evaluation, according to local 
medical practice. The investigator will determine whether this return visit is necessary and whether it is considered as part of the same episode. See Section 5.6 for details.
3 Blood sampling for dengue diagnosis which includes testing for RT-qPCR, dengue IgG and IgM ELISA, NS1 ELISA, neutralizing antibodies to dengue and virological/humoral,
testing for Zika and chikungunya infection.
4 SAE = Serious adverse event. See Section 6.1.1 for definition.
5 The study conclusion information in the eCRF is collected once for each subject at the last scheduled visit or at the last follow-up contact for a suspected dengue/zika/chikungunya
case, should this case occur during the last scheduled visit or earlier if the subject terminates study participation or is lost to follow-up.
7 Symptoms are fever for dengue, Zika and chikungunya, and rash for Zika.
@ The field staff will re-consent at the first opportunity
Table 3
List of study procedures for expansion cohort subjects 
Surveillance
Suspected 
Dengue/Zika/Chikungunya Visits (at 
hospital/clinic)
as applicable
Procedure
Visit 1
# 
Day 0
Monthly 
contact 
between 
visits
Visit 2
Monthly 
contact 
between 
visits 
Visit 3
Monthly 
contact 
between 
visits
Visit 4
First visit 
Day 0
Return visit 2 Follow-up 
visit21
Informed consent (and assent if applicable) 
● 
Re-consent (and assent if applicable)@
●
●
●
●
●
●
Subject number and initial household 
number attribution 
● 
Check inclusion/ exclusion criteria 
● 
Record socio-demographic information or 
updates (including household 
characteristics if applicable)
● 
●
●
●
Medical history, including YF and dengue
vaccination history or updates 
● 
● 
● 
● 
Distribute subject ID card distribution and 
suspected dengue instruction kit 
O 
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Surveillance
Suspected 
Dengue/Zika/Chikungunya Visits (at 
hospital/clinic)
as applicable
Procedure
Visit 1
# 
Day 0
Monthly 
contact 
between 
visits
Visit 2
Monthly 
contact 
between 
visits 
Visit 3
Monthly 
contact 
between 
visits
Visit 4
First visit 
Day 0
Return visit 2 Follow-up 
visit21
Blood sample for serology (5 mL)6 
● 
● 
● 
● 
Instruct/remind subjects/subject's 
parent(s)/LAR(s) on suspected dengue, 
chikungunya and Zika assessments
procedures (where applicable)
O 
O 
O 
O
O
O 
Issue diary logs to parents/LARs to be filled 
out in the event of a suspected dengue, 
chikungunya and Zika case 1
O
O
O
O
Contact subject regarding any dengue 
chikungunya and Zika symptoms and 
remind subject of procedures for suspected 
dengue, chikungunya and Zika
O
O
O
Physical examination/record current 
medical conditions (see Section 5.6)
●
●
●
Blood sample for dengue
Zika/chikungunya infection differential 
diagnosis3,
●
●
Blood sample for CBC and hematocrit 
(mandated for subjects presenting 
within 14 days following symptoms7
onset)
●
Record body temperature 
●
Report SAEs related to study procedures 4
●
●
●
●
●
●
●
Collect or verify diary logs if applicable 
O
O
O
Study conclusion 5
●
●
● 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.
# Enrolment of expansion cohort subjects will start preferably during study Year 2.
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1 Diary logs given at scheduled visits are only to be filled out in the event that the subject has fever and/or rash according to the case definition for suspected 
dengue/chikungunya/Zika. 
2 A return visit is a visit linked to suspected dengue/Zika/chikungunya, occurring if the evolution of the subject’s physical condition necessitates medical evaluation, according to local 
medical practice. The investigator will determine whether this return visit is necessary and whether it is considered as part of the same episode. See Section 5.6 for details.
3 Blood sampling for dengue diagnosis which includes testing for RT-qPCR, dengue IgG and IgM ELISA, NS1 ELISA, neutralizing antibodies to dengue and virological/humoral, 
testing for Zika and chikungunya infection.
4 SAE = Serious adverse event. See Section 6.1.1 for definition.
5 The study conclusion information in the eCRF is collected once for each subject at the last scheduled visit or at the last follow-up contact for a suspected dengue/Zika/chikungunya
case, should this case occur during the last scheduled visit or earlier if the subject terminates study participation or is lost to follow-up. 
7 Symptoms are fever for dengue, Zika and chikungunya, and rash for Zika.
@ The field staff will re-consent at the first opportunity
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Table 4
List of study procedures for replacement subjects
Surveillance
Suspected 
Dengue/Zika/Chikungunya Visits (at 
hospital/clinic)
as applicable
Procedure
Visit 1 
Day 0
Monthly 
contact 
between 
visits
Visit 2 
Monthly 
contact 
between 
visits 
Visit 3 as 
applicable
Monthly 
contact 
between 
visits
Visit 4 as 
applicable
First visit 
Day 0
Return visit2 Follow-up 
visit Day 21
Informed consent (and assent if 
applicable) 
●
Re-consent (and assent if applicable)@
●
●
●
●
●
●
Subject number and initial household 
number attribution 
●
Check inclusion/ exclusion criteria 
●
Record/ update socio-demographic 
information or updates (including 
household characteristics if applicable)
●
●
●
●
Medical history, including YF and 
dengue vaccination history or updates 
●
●
●
●
Distribute subject ID card distribution and 
suspected dengue instruction kit 
O
O
O
Blood sample for serology (5 mL) 
●
●
●
●
Instruct/remind subjects/subject's 
parent(s)/LAR(s) on suspected dengue, 
chikungunya and Zika assessment 
procedures (where applicable) 
O
O
O
O
O
O
Issue diary logs to parents/LARs to be 
filled out in the event of a suspected 
dengue, chikungunya and Zika case 1
O
O
O
O
Contact subject regarding any dengue,
chikungunya and Zika symptoms and 
remind subject of procedures for 
suspected dengue, chikungunya and 
Zika
O
O
O
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Surveillance
Suspected 
Dengue/Zika/Chikungunya Visits (at 
hospital/clinic)
as applicable
Procedure
Visit 1 
Day 0
Monthly 
contact 
between 
visits
Visit 2 
Monthly 
contact 
between 
visits 
Visit 3 as 
applicable
Monthly 
contact 
between 
visits
Visit 4 as 
applicable
First visit 
Day 0
Return visit2 Follow-up 
visit Day 21
Physical examination/record current 
medical conditions (see Section 5.6)
●
●
●
Blood sample for dengue, 
Zika/chikungunya infection differential 
diagnosis3
●
●
Blood sample for CBC and hematocrit 
(mandated for subjects presenting 
within 14 days following symptoms7
onset)
●
Record body temperature 
●
Report SAEs related to study procedures 4
●
●
●
●
●
●
●
Collect or verify diary logs if applicable 
O
O
O
Study conclusion 5
●
●
● 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.
1 Diary logs given at scheduled visits are only to be filled out in the event that the subject has fever and/or rash according to the case definition for suspected dengue
chikungunya/Zika. See Section 5.5.2.5 for details with regard to the diary log. 
2 A return visit is a visit linked to suspected dengue/Zika/chikungunya, occurring if the evolution of the subject’s physical condition necessitates medical evaluation, according to local 
medical practice. The investigator will determine whether this return visit is necessary and whether it is considered as part of the same episode. See Section 5.6 for details.
3 Blood sampling for dengue diagnosis which includes testing for RT-qPCR, dengue IgG and IgM ELISA, NS1 ELISA, neutralizing antibodies to dengue and virological/humoral, 
testing for Zika and chikungunya infection.
4 SAE = Serious adverse event. See Section 6.1.1 for definition.
5 The study conclusion information in the eCRF is collected once for each subject at the last scheduled visit or at the last follow-up contact for a suspected dengue/Zika/chikungunya
case, should this case occur during the last scheduled visit or earlier if the subject terminates study participation or is lost to follow-up. 
@ The field staff will re-consent at the first opportunity
.
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Table 5
Intervals between study visits/contacts for INITIAL cohort
Study Year*
Visits Interval
Optimal length of interval *
Allowed interval**
Year 1
Visit 1 (Day 0) Visit 2 (Month 6)
6 months 
± 28 days
Visit 2 (Month 6) Visit 3 (Month 12)
6 months
The interval may be adapted to 
allow scheduling the visit in a 
period of generally low dengue 
transmission
Year 2
Visit X Visit X+1¶
Note: Visit X is visit 3 for subjects 
enrolled in study year 1. For subjects 
enrolled in study year 2, visit X would 
be visit 1.
12 months 
The interval may be adapted to 
allow scheduling the visit in a 
period of generally low dengue 
transmission
Year 3
Visit X+1 Visit X+2
12 months 
The interval may be adapted to 
allow scheduling the visit in a 
period of generally low dengue 
transmission
Year 4
Visit X Visit X+3
12 months 
The interval may be adapted to 
allow scheduling the visit in a 
period of generally low dengue 
transmission
*Whenever possible the investigator should arrange study visits/contacts within this interval
**Subjects might not be eligible for inclusion in the ATP cohort for analysis if they make the study visit/contact outside 
this interval
Table 7
Interval between visits for suspected dengue case (Amended 16 
October 2017)
Interval
Optimal length of interval*
Allowed interval**
Visit 1 (Day 0)   Visit 2 (Year 1)
12 months
The interval may be adapted to allow scheduling the 
visit in a period of generally low dengue transmission
±28 days
Visit 2 (Year 1) Visit 3 (Year 2)
12 months
The interval may be adapted to allow scheduling the 
visit in a period of generally low dengue transmission
±28 days
Visit 3 (Year 2) Visit 4 (Year 3)
12 months
The interval may be adapted to allow scheduling the 
visit in a period of generally low dengue transmission
±28 days
Interval between visit for a suspected 
dengue case and the final follow-up 
visit for a case
21 days
+7 days
Table 7
Interval between visits for suspected dengue/chikungunya/Zika case 
Optimal length of time
Allowed interval
Suspected dengue/chikungunya/Zika
case: Interval between first and follow-
up visit 
21 days
+ 7 days
Note that this interval applies to all subjects enrolled regardless of the year of enrolment.
 
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Section 5.5.2 Procedures at scheduled Visit 1 (enrolment visit)
Ideally, Visit 1 (the enrolment visit) will be scheduled before the peak incidence of 
dengue based on previous years.
A subject number and initial household number will be attributed at Visit 1.
Section 5.5.2.2
Collect specific medical history data
A baseline medical history, including history of dengue infection, YF vaccination history 
will be taken and results , of dengue, Zika and chikungunya infection, and YF and 
dengue vaccination history will be taken. Results will be recorded in the subject’s source 
document for subsequent recording in the eCRF.
YF and dengue vaccination status will be recorded, along with the source of the 
information (either written or oral record). A vaccination record, if available, is 
preferable but self-reported history will be recorded if the vaccination record is not 
available. 
Information on medical history (e.g., diabetes, cardiovascular diseases, asthma, cancers, 
hematologic diseases, genetic disorders) will be collected.  
Section 5.5.2.3
Collect blood sample

A blood sample for serology will be collected from all enrolled subjects (see Section 
5.8 for details regarding sample collection). For children <2 years of age, the total 
amount of blood collected will not exceed 5 mL at any visit.
Section 5.5.2.4
Instruction on enhanced passive dengue, chikungunya and Zika
surveillance by the subject or subject’s parent(s)/LAR(s)
Study personnel will train subjects/subject’s parent(s)/LAR(s) to recognize dengue Zika
and chikungunya symptoms and will instruct them to contact the study personnel or 
come to a designated study hospital/clinic for medical evaluation within 5 days of the
occurrence of fever and/or rash that may be associated with suspected dengue, 
chikungunya or Zika (defined in Section 5.7.1, 5.7.2 and 5.7.3).  
Each household will also be given at least one dengue kit, as applicable, which includes 
thermometers, study contact information (phone numbers) and an instruction card with 
information about dengue, Zika and chikungunya symptoms. This card will also provide 
instructions on who to contact and what to do if any of these infections are suspected.
Participants or LAR will be instructed on how to take body temperature and how to 
record the temperatures in a diary log. 
See Section 5.6 for details regarding management of suspected dengue, chikungunya
and Zika cases.
 
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5.5.2.4.1
Diary log distribution
A diary log will be given to all subjects in the household to be used in the event of 
suspected dengue, chikungunya and/or Zika illness. Diary logs will be distributed at the 
first scheduled visit and if they are needed, at subsequent visits, if needed (if the subject 
no longer has one, e.g., the previous one was lost or used).  A new diary log will be 
issued by the study staff whenever necessary. 
The subject will be instructed to start recording any symptoms and body temperature any 
time dengue, chikungunya and/or Zika infections are suspected. Refer to Section 5.6.1 
for details.
5.5.2.4.2
Recording of serious adverse events related to a study procedure
The subject/subject’s parent(s)/LAR(s) will be instructed to contact the study staff should 
any serious adverse event related to a study procedure occur during the study. 
Refer to Section 8.2 for procedures for the Investigator to record SAEs that are related to 
study participation and for guidelines on how to report these SAEs to GSK Biologicals.
5.5.2.4.2
Procedures at subsequent scheduled visits (2-6, as applicable)

For initial cohort subjects, Visit 2 will occur approximately 6 months after Visit 1 
and Visit 3 approximately 6 months after Visit 2. Initial cohort subjects willing to 
extend their participation to three additional years will be re-consented at Visit 3 (i.e. 
at the end of study Year 1).

For expansion cohort subjects, scheduled visits should ideally be performed at yearly 
intervals, preferably during periods of usually low dengue transmission. Expansion 
cohort subjects will have at most 4 scheduled visits, depending on the time of 
enrolment.

For replacement subjects enrolled in the subsequent study years, scheduled visits 
should ideally be performed at yearly intervals, preferably during periods of usually 
low dengue transmission.

A blood (serum) sample will be collected for serology.
Note: Indirect DENV IgG assay is repeated in the following scheduled visits (after 
enrolment) only if the subject was negative for DENV IgG at the previous scheduled 
visit. Even if the test is not repeated, the volume of the blood sample remains 
unchanged. 

Socio-demographic and household information will be updated in eCRF (for e.g. 
change of home location) (for all scheduled visits planned after approval of 
protocol amendment 4).

Medical and vaccination histories will be updated in the eCRF.

The investigator will remind the subjects about the procedures in case of signs and 
symptoms of dengue, Zika and chikungunya.
 
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
A new diary log will be distributed for continued dengue, chikungunya and Zika
surveillance if needed, and any completed diary logs will be verified, as applicable. 
Subjects will be asked to contact the study staff in the event of the occurrence of a 
symptom that may be associated with suspected dengue or Zika (see Section 5.7.1.1 
for suspected dengue definition).

The subject will be instructed to start recording any symptoms and body temperature 
any time dengue, chikungunya and/or Zika is suspected. Refer to Section 5.6.1 for 
details.

Any SAEs related to study procedures will be recorded.
Section 5.5.2.5
Diary log distribution
A diary log will be given to all subjects in the household to be used in the event of 
suspected dengue, chikungunya and/or Zika illness. Diary logs will be distributed at the 
first scheduled visit and if they are needed, at subsequent visits (if the subject no longer 
has one, e.g., the previous one was lost or used).  A new diary log will be issued by the 
study staff whenever necessary.
Section 5.5.3 Procedures at subsequent scheduled visits (2-6, as applicable)

For initial cohort subjects, Visit 2 will occur approximately 6 months after Visit 1 
and Visit 3 approximately 6 months after Visit 2. Initial cohort subjects willing to 
extend their participation to three additional years will be re-consented at Visit 3 (i.e. 
at the end of study Year 1).

For expansion cohort subjects, scheduled visits should ideally be performed at yearly 
intervals, preferably during periods of usually low dengue transmission. Expansion 
cohort subjects will have at most 4 scheduled visits, depending on the time of 
enrolment.

For replacement subjects enrolled in the subsequent study years, scheduled visits 
should ideally be performed at yearly intervals, preferably during periods of usually 
low dengue transmission.

A blood (serum) sample will be collected for serology.
Note: Indirect DENV IgG assay is repeated in the following scheduled visits (after 
enrolment) only if the subject was negative for DENV IgG at the previous scheduled 
visit.
Zika IgG assay is repeated in the following scheduled visits (after enrolment) only 
if the subject was negative for Zika IgG at the previous scheduled visit.
Chikungunya IgG assay is repeated in the following scheduled visits (after 
enrolment) only if the subject was negative for chikungunya IgG at the previous 
scheduled visit.
Even if the tests are not repeated, the volume of the blood sample remains 
unchanged. 
 
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
Socio-demographic and household information will be updated in eCRF (for e.g. 
change of home location).

Medical and vaccination histories will be updated in the eCRF.

The investigator will remind the subjects about the procedures in case of signs and 
symptoms of dengue, Zika and chikungunya.

A new diary log will be distributed for continued dengue, chikungunya and Zika
surveillance if needed, and any completed diary logs will be verified, as applicable. 
Subjects will be asked to contact the study staff in the event of the occurrence of a 
symptom that may be associated with suspected dengue, chikungunya or Zika (see 
Section 5.7.1.1, 5.7.2 and 5.7.3 for suspected dengue, chikungunya or Zika
definition).

The subject will be instructed to start recording any symptoms, including rash and 
body temperature any time dengue, chikungunya and/or Zika is suspected. Refer to 
Section 5.6.1 for details.

Any SAEs related to study procedures will be recorded.
Section 5.5.4 Study conclusion
The Study Conclusion screen page in the eCRF will be completed at the last study 
contact. This last contact could occur at the last scheduled visit or follow up visit for a 
suspected dengue, chikungunya or Zika case (if it is on-going at the last scheduled visit) 
or earlier if the subject terminates study participation or is lost-to-follow-up.
The study staff will review data collected to ensure accuracy and completeness and will 
complete the Study Conclusion screen page 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. If so, the investigator will ask each 
subject/subject’s parent(s)/LAR(s) if he or she would be willing to participate (or let their 
child participate) in a long-term follow-up study and subject will be asked to sign a new 
consent form.
Section 5.5.5 Dengue, chikungunya and Zika case detection
Suspected cases (defined in Section 5.7.1, 5.7.2 and 5.7.3) in the study cohort may arise 
from three sources: 1) referred by study personnel during scheduled home visits; 2) 
through enhanced passive surveillance; and 3) as a result of active surveillance between 
scheduled visits.
Section 5.5.5.1
Case detection during scheduled home visits
If a subject is identified as suspected case of dengue, chikungunya and/or Zika during 
any home visit, he or she will be referred to the designated study hospital/clinic for 
medical evaluation (refer to Section 5.6).
 
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Section 5.5.5.2
Case detection through enhanced passive surveillance
The subject/subject’s parent(s)/LAR(s) will be instructed to contact the study staff (the 
local study coordinator) at any time dengue or chikungunya is suspected (i.e., body 
temperature 38°C measured (by any route) for at least two consecutive days) or if Zika 
is suspected (fever [i.e., body temperature 38°C measured (by any route) for at least 
two consecutive days] and/or rash). 
The subject/subject’s parent(s)/LAR(s) will be instructed to contact the hospital or study 
clinic should the subject manifest any signs or symptoms they/the subject’s 
parent(s)/LAR(s) perceive as an emergency or severe.
The phone contact will be preferably made during working hours/days for logistical 
reasons. A reasonable schedule for phone contacts to the local study coordinator is from 
Monday to Saturday, 7:00 AM to 8:00 PM. In the case of an emergency outside of the 
defined hours, the patient should contact or present to their local emergency room, and 
contact the study staff point of contact as soon as convenient to the subject/subject’s 
parent(s)/LAR(s).
When a subject/subject’s parent(s)/LAR(s) contacts the study staff, the local study 
coordinator will then arrange for an appointment at the designated study hospital/clinic. 
The subject/subject’s parent(s)/LAR(s) will be instructed to record any symptom 
including rash and body temperature on the diary log daily until the appointment.  
The visit should be arranged as soon as possible, preferably during week days for 
logistical reasons. If the subject contacts the study staff during the weekend, the visit 
should be scheduled for the following Monday. All efforts should be made to guarantee 
that the subject be seen by the fifth day of disease onset, at latest.
During holidays, arrangements will be made with study physicians and the subjects 
should be seen at the designated study hospital/clinic. 
Although subjects will be instructed to contact the study staff in the event of suspected 
dengue, chikungunya and/or Zika, there may be cases where the subject is taken directly 
to the hospital or clinic. If this occurs when the study physician is not available, the staff 
at the designated hospital should notify the local study coordinator and the study 
physician. 
The study staff should ensure that:

the acute sample will be properly collected,

all clinical information will be retrievable, 

a return visit (if applicable) will be scheduled

and a follow-up visit will be scheduled.
See Section 5.6 for cases that present to a non-study hospital/clinic.
 
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Section 5.5.5.3
Case detection through active surveillance
Telephone calls (or home visits when applicable, if a phone call is not feasible) will be 
conducted at least monthly and more frequently if needed. During the phone call or visit, 
a structured script will be used to inquire about dengue, chikungunya and Zika
symptoms since the last contact.
An example of questions to be included in the structured script is provided in 
APPENDIX B. 
If dengue, chikungunya and/or Zika is suspected during active surveillance, an 
appointment will be arranged at the designated study hospital/clinic, and at least one 
additional visit will be required for case follow-up (see Section 5.6).
Subjects whose status cannot be ascertained during active surveillance will be tracked by 
the study personnel through an active tracking algorithm.
Figure 2
Active tracking algorithm
Section 5.6
Management of suspected dengue, chikungunya and Zika cases 
All study subjects with suspected dengue, chikungunya and/or Zika should be seen at a 
designated study hospital or clinic by the study physician.  
The first visit to the study hospital/clinic will include a detailed clinical examination to 
assess the subject’s general condition, body temperature, height and weight, cardiac and 
respiratory rates, blood pressure, dengue, chikungunya and Zika associated clinical 
signs/symptoms and relevant clinical signs/symptoms (e.g., fatigue, headache, pain 
behind the eyes, abdominal pain, nausea, vomiting, muscle ache, joint ache, diffuse rash, 
photophobia and pruritus).
A blood sample should be collected by the hospital/clinic for laboratory confirmation of 
dengue, chikungunya, Zika and differential diagnosis. This sample is referred to as the 
‘acute’ serum sample. In addition, a blood sample for haematology (CBC and HCT) will 
be collected (mandated procedure for subjects presenting within the first 14 days 
following onset of fever and/or rash). If blood chemistry is conducted according to 
local clinical practice, these data may also be collected.
 
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If dengue, chikungunya or Zika is still suspected after the first visit to the study 
hospital/clinic, a return visit for suspected dengue, chikungunya and/or Zika may be 
needed and will be conducted as directed by National guidelines by the study physician. 
A return visit is a visit linked to suspected dengue, chikungunya and/or Zika, occurring 
if the evolution of 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. If a return visit 
occurs, the diary log will be verified and possibly used by the physician to complete the 
clinical symptoms in the eCRF. 
Medical data related to any suspected dengue, chikungunya and/or Zika case will be 
collected for hospitalized study subjects and for those followed in the outpatient setting at 
the return visit. Treatment will be given according to local standard routines, following 
national guidelines.
Approximately 21(+7) days after the first visit for suspected dengue, chikungunya 
and/or Zika, a follow-up (convalescent) visit will be required. A physical examination 
will be conducted and a ‘convalescent’ serum sample (i.e., a blood sample collected after 
the acute phase for dengue, chikungunya and/or Zika) should be collected at this follow-
up visit. Ideally, this collection should be done at the study hospital/clinic but may be 
done at the subject’s home if directed by the study physician or delegated study staff, if 
the subject is unable to come to the hospital/ clinic.
Subjects who, for any reason, seek medical assistance at any non-study health care 
facility, will be identified through active surveillance, and clinical data will be 
retrospectively collected on the eCRF. The study physician will be responsible for 
collecting the retrospective data and informing the local study coordinator for appropriate 
follow-up. 
Section 5.6.1 Diary log instructions for suspected dengue and Zika cases
The subject/subject’s parent(s)/LAR(s) will be instructed to record body temperature and 
rash on the diary log daily until the appointment. See Section 5.6 for subjects who do not 
reach the study staff and go directly to the hospital/clinic. The subject will be instructed 
to start recording any symptoms, including rash and body temperature any time dengue, 
chikungunya and/or Zika virus infection is suspected. 
The information collected in the diary log will be provided to the study physician as a 
tool in the medical evaluation of the subject. If the physician considers that the subject 
meets the criteria for suspected dengue, chikungunya and/or Zika, he could use the 
information contained in the diary log to complete the description of first clinical 
symptoms in the eCRF.
If dengue, chikungunya and/or Zika is suspected, the  diary log will be verified by the 
investigator or designee at the follow-up visit if the subject is not hospitalized. If the 
subject is hospitalized, information with regard to dengue chikungunya and/or Zika
infection will be recorded in the hospital record. Information from diary log and hospital 
records can be used to update clinical data in the eCRF.
 
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Section 5.7
Case definitions
Section 5.7.1 Dengue
The following classification of dengue cases will be used in this study.
Figure 3
Classification of dengue cases
Section 5.7.1.1
Suspected symptomatic dengue case 
Febrile illness with body temperature 38°C measured (by any route) on at least two 
consecutive days and less than 14 days with or without the presence of other dengue 
symptoms or signs, without an obvious aetiology unrelated to dengue, based on 
investigator’s judgement.
Although subjects are asked to come to a study hospital if fever (body temperature 
38°C) is sustained for two consecutive days, a subject could present on the first day of 
fever. In this case the physician may still consider the subject as a suspected dengue case 
based on medical judgement and collect a blood sample for laboratory diagnosis.
Subjects should be examined at the health care facility during the 5 days following 
fever onset, as this is a requirement for RT-qPCR diagnosis of dengue infection and 
serotype identification.
However, subjects presenting until 30th (included) day of fever onset will be considered 
for investigation. After the 30th day, subjects will not be considered as suspected 
dengue cases. 
A suspected dengue case presenting at the health care facility within 5 days following the 
onset of fever will be defined as an ‘early presenter’. 
Subjects presenting for care between sixth and 30th day of fever onset will be defined as 
‘late presenters’.
An example of other signs and symptoms of dengue, associated with fever, include but 
are not limited to: fatigue, headache, pain behind the eyes, abdominal pain, nausea, 
vomiting, muscle ache, joint ache, diffuse rash on the trunk, abnormal sensitivity to light 
(photophobia) and itching of skin (pruritus).
 
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Section 5.7.1.2
Laboratory-confirmed symptomatic dengue case
Early presenter:
At least one of the following findings must be met for a laboratory-confirmed dengue 
case: 

Dengue virus identification through RT-qPCR on the acute serum sample 

Dengue virus NS1 positive on acute serum sample through ELISA.

Anti-dengue IgM seroconversion between acute and convalescent serum samples 
through ELISA.
Late presenter:
Anti-dengue IgM seroconversion between acute and convalescent serum samples 
through ELISA.
Section 5.7.1.5
Previous dengue infection
Dengue seroprevalence measured at baseline will be defined as:
A subject will be considered as having previous dengue infection at visit 1(baseline) if:

Dengue IgG positive at visit 1 (baseline)
or

Laboratory-confirmed symptomatic dengue case detected at visit 1 (baseline)
A subject will be considered as having previous dengue infection at any time after visit 
1 (baseline), if:

Dengue IgG positive at previous schedule visit(s)
or
Laboratory-confirmed symptomatic dengue case detected previously at study 
surveillance
Section 5.7.1.7
Primary symptomatic dengue case
A primary symptomatic dengue case is a subject with laboratory confirmed or probable
symptomatic dengue infection without evidence of past previous dengue infection
(presence of absence of Ig G antibodies at the previous scheduled visit (s) or absence of 
laboratory-confirmed symptomatic case at baseline detected previously at study 
surveillance).
Section 5.7.1.10
Secondary symptomatic dengue case
A secondary symptomatic dengue case is a subject with laboratory confirmed or 
probable symptomatic dengue infection with evidence of past previous dengue infection 
 
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(presence of Ig G antibodies at the previous scheduled visit or laboratory-confirmed 
symptomatic case at baseline detected previously at study surveillance).
Section 5.7.1.6
Primary inapparent dengue infection
This condition is defined as a documented seroconversion (anti-dengue IgG antibodies) 
to one or more dengue types between two sequential sera samples obtained during the
scheduled visits without clinical suspicion of dengue (identified during the time period in 
which seroconversion occurred).
Section 5.7.1.9
Probable dengue case

A suspected dengue case, based on strong clinical suspicion or a clinical diagnosis, 
in a late presenter (i.e., RT-qPCR not performed), and: 

Anti-dengue IgM or anti-dengue IgG* positivity on at least one sample (on either the 
acute or convalescent blood sample)
and 

NS1 negative on the acute sample
and 

No evidence of anti-dengue IgM seroconversion between the acute and the 
convalescent sample
*The ELISA test allows the detection of dengue IgG levels characteristic of acute 
secondary infections.

For early presenters, a probable case will be that case without laboratory  
confirmation, presenting IgG positive in the convalescent sample.

For late presenters, a probable case will be the case without seroconversion of 
IgM, presenting at least one IgG positive in one sample (acute or convalescent).
Section 5.7.1.10
Negative dengue case
For early and late presenters, a negative dengue case is a participant with a negative 
result for all planned laboratory tests on both acute and convalescent samples.
Section 5.7.1.11
Indeterminate dengue case
A case is considered as indeterminate when RT-PCR or NS1 is negative (early 
presenters) or not performed (late presenters) and a convalescent serum is missing to 
confirm or rule out dengue.
An indeterminate dengue case is a participant evaluated as an SDC (Section 5.7.1.1.) 
and not classified as laboratory confirmed case (Section 5.7.1.2), probably case 
(Section  5.7.1.9) or negative case (Section 5.7.1.11).
 
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Section 5.7.2 Chikungunya
Section 5.7.2.1
Suspected symptomatic chikungunya case 
Febrile illness with body temperature 38°C measured (by any route) on at least two 
consecutive days and less than 14 days with or without the presence of other 
chikungunya symptoms or signs, without an obvious aetiology unrelated to 
chikungunya, based on investigator’s judgement.
Although subjects are asked to come to a study hospital if fever (body temperature 
38°C) is sustained for two consecutive days, a subject could present on the first day of 
fever. In this case the physician may still consider the subject as a suspected 
chikungunya case based on medical judgement and collect a blood sample for 
laboratory diagnosis.
Subjects should be examined at the health care facility during the 5 days following 
fever onset, as this is a requirement for RT-qPCR diagnosis of dengue infection in the 
case of co-infection
However, subjects presenting until 30th (included) day of fever onset will be considered 
for investigation. After the 30th day, subjects will not be considered as suspected 
chikungunya cases. 
A suspected chikungunya case presenting at the health care facility within 5 days 
following the onset of fever will be defined as an ‘early presenter’. 
Subjects presenting for care between sixth and 30th day of fever onset will be defined as 
‘late presenters’.
An example of other signs and symptoms of chikungunya, associated with fever, 
include but are not limited to: 

Polyarthralgia is usually bilateral and symmetric, and can be debilitating

Other symptoms may include headache, myalgia, arthritis, conjunctivitis, 
nausea/vomiting, or maculopapular rash.
Rare complications include uveitis, retinitis, myocarditis, hepatitis, nephritis, bullous 
skin lesions, hemorrhage, meningoencephalitis, myelitis, Guillain-Barré syndrome, 
and cranial nerve palsies.
Section 5.7.2.2
Laboratory-confirmed symptomatic chikungunya case
Early presenter:
At least one of the following findings must be met for a laboratory-confirmed 
chikungunya case: 

Chikungunya virus identification through RT-qPCR on the acute serum sample 

Anti-chikungunya IgM seroconversion between acute and convalescent serum 
samples through ELISA.
 
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Late presenters:

Anti-chikungunya IgM seroconversion between acute and convalescent serum 
samples through ELISA.
Section 5.7.2.3
Virologically confirmed symptomatic chikungunya infection 
A virologically confirmed symptomatic chikungunya infection is defined as a 
chikungunya case confirmed by RT-qPCR. 
Section 5.7.2.4
Previous chikungunya infection at baseline
A subject will be considered as having previous chikungunya infection at baseline 
(enrolment visit/Visit 1), if:

Chikungunya IgG positive at baseline visit OR

Laboratory-confirmed symptomatic chikungunya case at baseline
Section 5.7.2.5
Inapparent chikungunya infection 
This condition is defined as a documented seroconversion (anti-chikungunya IgG 
antibodies) between two sequential sera samples obtained during the scheduled visits 
without clinical suspicion of chikungunya (identified during the time period in which 
seroconversion occurred).
Section 5.7.2.7
Probable chikungunya case
For early and late presenters, a probable chikungunya case will be that case without a 
lab-confirmed criteria, presenting at least one IgG positive in one sample (acute or 
convalescent).
Section 5.2.7.8
Negative chikungunya case
For early and late presenters, a negative chikungunya case is a participant with a 
negative result for all planned laboratory tests on both acute and convalescent samples.
Section 5.7.2.6
Indeterminate chikungunya case
An indeterminate chikungunya case is a participant evaluated as an suspected 
chikungunya case (Section 5.7.2.1.) and not classified as laboratory confirmed case 
(Section 5.7.2.2), probably case (Section  5.7.2.6) or negative case (Section 5.7.2.7).
 
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Section 5.7.3 Zika
Section 5.7.3.1
Suspected symptomatic Zika case
Rash and/or febrile illness on at least two consecutive days and less than 14 days with 
or without the presence of other Zika symptoms or signs, without an obvious aetiology 
unrelated to Zika, based on investigator’s judgement.
Although subjects are asked to come to a study hospital if fever and/or rash is 
sustained for two consecutive days, a subject could present on the first day of fever 
and/or rash. In this case the physician may still consider the subject as a suspected 
Zika case based on medical judgement and collect a blood sample for laboratory 
diagnosis.
Subjects should be examined at the health care facility during the 5 days following 
fever and/or rash onset, as this is optimal for RT-qPCR diagnosis of Zika infection.
However, subjects presenting until 30th (included) day of fever and/or rash onset will 
be considered for investigation. After the 30th day, subjects will not be considered as 
suspected Zika cases. 
A suspected Zika case presenting at the health care facility within 5 days following the 
onset of fever and/or rash will be defined as an ‘early presenter’. Subjects presenting 
for care between sixth and 30th day of fever and/or rash onset will be defined as ‘late 
presenters’. At the clinical presentation of the first visit, fever will take priority in the 
classification of “early” or “late” presenter if the participant reported  both fever and 
rash are present.  If the participant presents the rash without fever, the date of onset of
rash will determine classification.
An example of other signs and symptoms include arthralgia, arthritis, or conjunctivitis 
(non-purulent/hyperaemic). 
Section 5.7.3.2
Laboratory-confirmed symptomatic Zika case
Early presenters:
At least one of the following findings must be met for a laboratory-confirmed Zika 
case.

Zika virus identification through RT-qPCR on the acute serum sample 

Anti-Zika IgM seroconversion between acute and convalescent serum samples 
through ELISA and confirmed by Zika neutralization antibody testing.
Late presenters:

Anti-Zika IgM seroconversion between acute and convalescent serum samples 
through ELISA and confirmed by Zika neutralization antibody testing.
 
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Section 5.7.3.3
Virologically confirmed symptomatic Zika infection 
A virologically confirmed symptomatic Zika infection is defined as a Zika suspected 
case confirmed by RT-qPCR.
Section 5.7.3.4
Previous Zika infection at baseline
A subject will be considered as having previous Zika infection at baseline (enrolment 
visit/Visit 1), if:

Zika IgG positive at baseline as confirmed by neutralizing antibody tests visit OR

Laboratory-confirmed symptomatic Zika case at baseline
Section 5.7.3.5
Inapparent Zika infection 
This condition is defined as a documented seroconversion and confirmed by Zika 
neutralising antibody testing between two sequential sera samples obtained during the 
scheduled visits without clinical suspicion of Zika (identified during the time period in 
which seroconversion occurred).
Section 5.7.3.7
Probable Zika case

For early and late presenters, a probable Zika case will be that case without a lab-
confirmed criteria, presenting at least one IgG positive in one sample (acute or 
convalescent).
Section 5.7.3.7
Negative Zika case
For early and late presenters, a negative Zika case is a participant with a negative 
result for all planned laboratory tests on both acute and convalescent samples.
Section 5.7.3.6
Indeterminate Zika case
An indeterminate Zika case is a participant evaluated as an suspected Zika case 
(Section 5.7.3.1.) and not classified as laboratory confirmed case (Section 5.7.3.2), 
probably case (Section 5.7.3.6) or negative case (Section 5.7.3.7).
 
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Section 5.8.2.1
Laboratory assays
Table 8
Humoral Immunity
Syste
m
Component
Method
Kit / Manufacturer
Laboratory
Serum
IgM to Dengue 
ELISA
Commercial kit Panbio or 
equivalent 
Local# or GSK designated 
laboratory
IgG to Dengue 
ELISA
Indirect IgG Panbio or 
equivalent (for scheduled 
visits)
IgG Capture PanBio or 
equivalent (for unscheduled 
visits)
Local# or GSK designated 
laboratory
Local# or GSK designated 
laboratory
Serum
Dengue virus types 1-
4 neutralizing 
antibodies
Dengue 
neutralizati
on assay
In-house
Laboratório de Tecnologia 
Virológica, Bio-Manguinhos, 
Fiocruz  Rio de Janeiro or GSK 
designated laboratory.
Seru
m
Antibodies against 
Zika and 
chikungunya
To be 
determine
d
To be determined
GSK designated laboratory
Serum
YF virus neutralizing 
antibody*
YF 
neutralizati
on assay
To be determined
GSK designated laboratory
*For subjects residing in a yellow fever endemic region or vaccinated against yellow fever
#Testing performed according to local practices
Table 9
Virology 
System
Component
Method
Kit/Manufacturer
Laboratory
Serum
DENV RNA
RT-qPCR
In-house
Laboratório de Tecnologia 
Virológica, Bio-Manguinhos, 
Fiocruz  Rio de Janeiro (GSK 
as back-up)
Serum
Zika RNA
RT-PCR
To be determined
GSK designated laboratory
Serum
Chikungunya RNA
RT-PCR
To be determined
GSK designated laboratory
Serum
NS1 
ELISA 
Commercial kit Biorad,Platelia 
Local
Section 5.8.2.2
Laboratory read-outs
In case of insufficient blood sample volume to perform all assays, the samples will be 
analyzed according to priority ranking provided in Table 11, Table 12 and Table 13.
 
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Table 11
Laboratory read-outs at each time point, and priority ranking for 
scheduled visits 
SCHEDULED VISITS
Blood sampling time 
point
No. Subjects
(anticipated) Subset*
Possible assays 
Serum 
aliquot
volume
Aliquot
priority 
rank
Test 
priority 
rank
Aliquot 
number
Type of 
contact/ 
Amount of 
whole 
blood
Sampling 
time point
Scheduled 
Visit 1
5 mL whole 
blood 
(Day 0)
3600 
(1800 from 
wave 1 & 
2***)
-
Dengue IgG ELISA**
250 µL
1
1
1
yes
Dengue virus 1-4 
neutralizing 
antibodies
1 mL
2
1
2
YF neutralization
4
Zika neutralization
3
Zika serology (IgG) #
2
Chikungunya 
serology (IgG) #
yes
Chikungunya 
neutralization
remaining 
volume
3
1
3
Scheduled 
Visit 2 
5 mL whole 
blood
Visit 1+ 6 
months for 
subject 
enrolled in
Study Year 
1; Visit 1+ 12 
months for 
subject 
enrolled in 
beginning of  
Study Year 
2, 3 or 4)
3190 (1660 
from wave 1 
and 1530 
from wave 
2) 
yes
Dengue IgG ELISA** 
(only for subjects IgG 
negative at the 
previous visit)
250 µL
1
1
1
yes
Dengue virus 1-4 
neutralizing 
antibodies
1 mL
2
1
2
YF neutralization
4
Zika neutralization
3
Zika serology (IgG) #
2
Chikungunya 
serology (IgG) #
yes
Chikungunya 
neutralization
remaining 
volume
3
1
3
Scheduled 
Visit 3 
5 mL whole 
blood
(Visit 2+ 6 
months for 
subject 
enrolled in 
Study Year 
1; Visit 2+12 
months for 
3030 (1530 
from wave 1 
and 1500 
from wave 
2)
yes
Dengue IgG ELISA**
(only for subjects IgG 
negative at the 
previous visit)
250 µL
1
1
1
yes
Dengue virus 1-4 
neutralizing 
antibodies
1 mL
2
1
2
 
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SCHEDULED VISITS
Blood sampling time 
point
No. Subjects
(anticipated) Subset*
Possible assays 
Serum 
aliquot
volume
Aliquot
priority 
rank
Test 
priority 
rank
Aliquot 
number
Type of 
contact/ 
Amount of 
whole 
blood
Sampling 
time point
subject 
enrolled in 
beginning of  
Study Year 2 
or 3)
YF neutralization
4
Zika neutralization
3
Zika serology (IgG) #
Chikungunya 
serology (IgG) #
2
yes
Chikungunya 
neutralization
remaining 
volume
3
1
3
Subsequent 
Yearly 
Visit(s) as 
applicable
5 mL whole 
blood
Yearly 
3000 for 
Visit 4 and 
1500 for visit 
5 and 6 
which 
applies only 
to subjects 
of the wave 
1
yes
Dengue IgG ELISA**
(only for subjects IgG 
negative at the 
previous visit)
250 µL
1
1
1
yes
Dengue  virus 1-4 
neutralizing 
antibodies
1 mL
2
1
2
YF neutralization
4
Zika neutralization
3
Zika serology (IgG) #
2
Chikungunya 
serology (IgG) #
yes
Chikungunya 
neutralisation
remaining 
volume
3
1
3
*tertiary (exploratory) analysis, number of subjects in the subset will be determined during analysis 
-  not applicable, 
** Dengue IgG indirect ELISA
*** Wave 1 enrolment = initial cohort / Wave 2 enrolment = Expansion cohort
# Indirect DENV IgG assay is repeated in the following scheduled visits (after enrolment) only if the subject 
was negative for DENV IgG at the previous scheduled visit. Zika IgG assay is repeated in the following 
scheduled visits (after enrolment) only if the subject was negative for Zika IgG at the previous scheduled 
visit; chikungunya IgG assay is repeated in the following scheduled visits (after enrolment) only if the 
subject was negative for chikungunya IgG at the previous scheduled visit
 
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Table 12
Laboratory read-outs at each time point, and priority ranking for 
suspected dengue, chikungunya and/or Zika visits for EARLY presenters (Amended 
16 October 2017)
SUSPECTED DENGUE, CHIKUNGUNYA AND/OR ZIKA VISITS – EARLY PRESENTERS
Blood sampling time point
No. 
Subjects
Subset
Component
Serum 
aliquot 
volume
Aliquot 
priority 
rank
Test 
priority 
rank
Aliquot 
number
Type of 
contact/ 
Amount of 
whole blood
Sampling time 
point
First visit 
(amount of 
blood= 5 mL for 
< 2 years of 
age and 10 mL 
for all other 
subjects)
Sample collected 5 
days of fever and/or 
rash
unknown
-
Dengue RT-
qPCR
750 µL
1
1
1
Zika and 
chikungunya 
RT-qPCR
2
Dengue virus 
1-4 
neutralizing 
antibodies
750 µL
3
1
2
Dengue IgM 
and NS1 
ELISA
500 µL
2
1
3
Zika 
neutralising 
antibodies
1 mL
4
1
4
Chikungunya 
neutralizing 
antibodies
and Serology 
for Zika 
(IgG/IgM) and 
chikungunya 
(IgG/IgM)
remaining 
volume
5
1
5
Optional for severe cases
PCR 
200 µL
-
First visit (total 
amount of 
blood per local 
practice 
Sample collected 
before day 14 of 
onset of fever/rash
unknown
CBC, HCT
No aliquot
-
Suspected 
dengue follow-
up visit (total 
amount of 
blood 5 mL)
Approximately 21 
days after first visit
unknown
-
Dengue IgM 
and Dengue 
capture IgG 
ELISA
500 µL
1
1
1
Dengue  virus 
1-4 
neutralizing 
antibodies
1 mL
2
1
2
Serology for 
Zika 
(IgM/IgG) and 
chikungunya 
(IgG/IgM )
2
 
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SUSPECTED DENGUE, CHIKUNGUNYA AND/OR ZIKA VISITS – EARLY PRESENTERS
Blood sampling time point
No. 
Subjects
Subset
Component
Serum 
aliquot 
volume
Aliquot 
priority 
rank
Test 
priority 
rank
Aliquot 
number
Type of 
contact/ 
Amount of 
whole blood
Sampling time 
point
Zika 
neutralizing 
antibodies
3
Chikungunya 
neutralizing
antibodies
remaining 
volume
3
1
3
-  not applicable
 
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Table 13
Laboratory read-outs at each time point, and priority ranking for 
suspected dengue, chikungunya and/or Zika visits for LATE presenters
SUSPECTED DENGUE, CHIKUNGUNYA AND/OR ZIKA VISITS – LATE PRESENTERS
Blood sampling time point
No. 
Subjects
Subset
Component
Serum 
aliquot 
volume
Aliquot 
priority 
rank
Test 
priority 
rank
Aliquot 
number
Type of 
contact/ 
Amount of 
whole blood
Sampling 
time point
First visit (total 
amount of 
whole blood 5 
mL)
Sample 
collected > 5 
days or more  
after fever 
and/or rash
unknown
-
Dengue IgM 
and Dengue 
capture IgG 
ELISA 
500 µL
1
1
1
Dengue  virus 
1-4 neutralizing 
antibodies
1 mL
2
1
2
Zika and 
chikungunya  
RT-qPCR
2
Serology for 
Zika (IgG/IgM)
and 
chikungunya 
(IgG/IgM)
remaining 
volume
3
1
3
First visit (total 
amount of blood 
per local 
practice 
Sample 
collected 
before day 14 
of onset of 
fever/rash
unknown
CBC, HCT
No aliquot
-
Suspected 
dengue, 
chikungunya 
and Zika follow-
up visit (total 
amount of blood 
5 mL)
Approximately 
21 days after 
first visit
unknown
-
Dengue IgM 
and Dengue 
capture IgG 
ELISA
500 µL
1
1
1
Dengue  virus 
1-4 neutralizing 
antibodies
1 mL
2
1
2
Serology for 
Zika (IgG/IgM)
and 
chikungunya 
(IgG/IgM)
2
Zika 
neutralizing 
antibodies
3
Chikungunya 
neutralizing 
antibodies
remaining 
volume
3
1
3
- not applicable
 
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Figure 4: Flow chart of serum aliquot testing for suspected dengue cases 
 
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Section 6
Serious adverse events
In this prospective cohort study no test product/vaccine will be given. However blood 
samples will be collected at scheduled visits and unscheduled visits for suspected 
dengue, chikungunya and/or Zika. 
In order to fulfil international reporting obligations, serious adverse events (SAEs) that 
are related to study procedures ( blood collection) will be collected and recorded from the 
time the subject consents to participate in the study until the study conclusion for each 
subject.
The investigators or site staff 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 or 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.
 
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Section 7.2.3 Contact information for reporting serious adverse events and other 
events to GSK Biologicals
Study Contact for Reportable SAEs
During office hours:
Local 
Name: Dr. 
GlaxoSmithKline Biologicals
, 
, 
, Brazil CEP 22783-110
Email: 
Phone: 
Cell: 
Name: 
Clinical Safety Analyst
Pharmacovigilance Department 
GlaxoSmithKline Biologicals
GlaxoSmithKline Brazil
, 
, 
, 
, Brazil 22783-110
Email: 
 
Phone: 
Fax: 
Back-up Study Contact for Reporting SAEs
24/24 hour and 7/7 day availability:
GSK Biologicals Clinical Safety & Pharmacovigilance
Fax: 
 or 
 
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Section 8.3
Subject replacement
Enrolment of new subjects to replace: 

subjects not wishing to continue after amendment 2 (initial cohort), or

subjects who prematurely terminate participation (initial and expansion cohorts) or

subjects lost to follow-up will be done (initial and expansion cohorts). 
The aim is to maintain a critical cohort size of 500 subjects/site. This replacement will 
be done at the beginning of each study year: during the scheduled visits starting at the end 
of study Year 1 up till the end of study Year 3.
The number of subjects to be recruited as replacements will be defined based on the 
number of subjects still active in the study. If at the end of each study year we have 500 
subjects/site, no replacements will be needed. 
In last three months of the of penultimate year of the study, each site will review the 
number of active subjects and drop-out rate..  If the risk of having fewer than the 
required number of active participants at the end of the study is high, recruitment for 
participants into the replacement cohort may be performed with sponsor approval. If 
the risk is high, recruitment into the replacement cohort should begin even if the 
number of active subjects is 500 or above 500.
The time window for the recruitment of replacements will be during the low dengue 
transmission, preferably.
Section 9.1.3 Tertiary endpoints
Exploratory objectives may be assessed based on, but not limited to the following 
endpoints:

Symptomatic laboratory-confirmed Zika virus infection

Symptomatic Zika virus infection (including laboratory-confirmed and probable 
cases).

Symptomatic laboratory-confirmed chikungunya infection

Symptomatic chikungunya infection (including laboratory-confirmed and 
probable cases).

Risk factors for dengue infection and disease.

Neutralizing antibodies titers against DENV 1-4.

Neutralizing antibody titers against YF virus.

Spatial and temporal distribution of dengue cases (confirmed and probable 
symptomatic cases and inapparent infections). 
 
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
Spatial and temporal distribution of laboratory confirmed and probable Zika virus 
cases

Spatial and temporal distribution of patients with laboratory confirmed and 
probable chikungunya virus infection

Occurrence of laboratory confirmed or probable Zika and/or chikungunya 
infection in suspected dengue cases (differential diagnosis of dengue), 
retrospectively

Antibody titre against Zika and chikungunya virus at the scheduled visits
Section 8.5.4 Analysis of tertiary objectives
Analysis of tertiary objectives is optional and may or may not be performed. If these 
analyses are performed, they will be conducted by the Research and Development 
Department. Statistical methods for tertiary objectives will be described in the SAP.
The neutralization assay, if performed, will only be done on blood samples from a subset 
of subjects. YF virus antibody testing will be performed retrospectively, for subjects in 
YF endemic region and who was tested positive for dengue. 
Analysis of risk factors for symptomatic dengue
Poisson regression models will be used for exploring the risk factors (e.g. region, age and 
gender, etc.) for symptomatic dengue. The analyses will account for clustering of 
observations within households for the analyses by centre and also the stratification by 
centres for the overall analyses.
Incidence rates of symptomatic Zika infections
The following analyses will be performed by study site, gender and age-group, previous 
dengue exposure and previous Zika infection:

Incidence rate of laboratory-confirmed or probable symptomatic Zika infection 
with 95% CI for each season separately: the numerator will be the number of 
subjects with lab-confirmed or probable symptomatic Zika infection during the 
season (between the visits scheduled before and after the season). The 
denominator will be the total person-years at risk, i.e. from the visit scheduled 
before the season until the first lab-confirmed or probable symptomatic Zika 
infection during the season, the next scheduled visit or the subject’s withdrawal, 
whichever comes first.

Incidence rate of laboratory-confirmed or probable symptomatic Zika infection 
with 95% CI all seasons combined: the numerator will be the number of subjects 
with lab-confirmed or probable symptomatic Zika infection during the study Zika 
follow-up period. The denominator will be the total person-years at risk, i.e. from 
start of the study Zika follow-up period until the first lab-confirmed or probable 
symptomatic Zika infection, the end of the study or the subject’s withdrawal, 
whichever comes first.
 
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Incidence rates of symptomatic chikungunya infections
The following analyses will be performed by study site, gender and age-group, previous 
dengue exposure and previous chikungunya infection:

Incidence rate of laboratory-confirmed or probable symptomatic chikungunya 
infection with 95% CI for each season separately: the numerator will be the 
number of subjects with lab-confirmed or probable symptomatic chikungunya 
infection during the season (between the visits scheduled before and after the 
season). The denominator will be the total person-years at risk, i.e. from the visit 
scheduled before the season until the first lab-confirmed or probable symptomatic 
chikungunya infection during the season, the next scheduled visit or the subject’s 
withdrawal, whichever comes first.

Incidence rate of laboratory-confirmed or probable symptomatic chikungunya 
infection with 95% CI all seasons combined: the numerator will be the number of 
subjects with lab-confirmed or probable symptomatic chikungunya infection 
during the study chikungunya follow-up period. The denominator will be the total 
person-years at risk, i.e. from start of the study chikungunya follow-up period until 
the first lab-confirmed or probable symptomatic chikungunya infection, the end of 
the study or the subject’s withdrawal, whichever comes first.
Analysis for Zika and chikungunya viruses in suspected dengue cases
The proportions and associated exact 2-sided 95% confidence intervals (CI)of the Zika 
and chikungunya viruses in suspected dengue cases will be summarized.
Analysis for seroprevalence of antibody titers against Zika and chikungunya viruses at 
selected timepoints
Seroprevalence at each selected timepoint will be calculated as a proportion i.e. the 
number of seropositive samples at each selected timepoint over the total number of 
samples with antibody results. An exact 95% CI will be computed.
Section 9.3
Archiving of data at study sites
Following closure of the study, the investigator must maintain all site study records in a 
safe and secure location. The records must be maintained to allow easy and timely 
retrieval, when needed (e.g. audit or inspection), and, whenever feasible, to allow any 
subsequent review of data in conjunction with assessment of the facility, supporting 
systems, and staff. Where permitted by applicable laws/regulations or institutional policy, 
some or all of these records can be maintained in a validated format other than hard copy 
(e.g. microfiche, scanned, electronic for studies with an eCRF); however, caution needs 
to be exercised before such action is taken. The investigator must assure that all 
reproductions are legible and are a true and accurate copy of the original and meet 
accessibility and retrieval standards, including re-generating a hard copy, if required. 
Furthermore, the investigator must ensure there is an acceptable back-up of these 
reproductions and that an acceptable quality control process exists for making these 
reproductions.
 
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GSK will inform the investigator/institution of the time period for retaining these records 
to comply with all applicable regulatory requirements. However, the 
investigator/institution should seek the written approval of the sponsor before proceeding 
with the disposal of these records. The minimum retention time will meet the strictest 
standard applicable to that site for the study, as dictated by ICH GCP 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 25
years. 
The investigator/institution must notify GSK of any changes in the archival 
arrangements, including, but not limited to, the following: archival at an off-site facility, 
transfer of ownership of the records in the event the investigator leaves the site.
Section 11
References
Boletim Epidemiológico - Volume 48 - n 05 - 2017 - Secretaria de Vigilância em Saúde 
− Ministério da Saúde. Accessed at : http://combateaedes.saude.gov.br/images/pdf/2017-
Dengue_Zika_Chikungunya-SE4.pdf. Accessed on 22 August 2016.
Siqueira, JB, Martelli CM, Maciel IJ, Oliveira RM, Ribeiro MG, Amorim FP, Moreira 
BC, Cardoso DD, Souza WV, Andrade AL. Household survey of dengue infection in 
Central Brazil: spatial point pattern analysis and risk factors assessment. Am J Trop Med 
Hyg. 2004; 71(4): 646-51.
 
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Appendix A
Study laboratories
GSK Biologicals’ laboratories (for back up samples)
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, North America, 
Laval NEOMED Labs
Biospecimen Reception - Clinical Serology
525 Cartier blvd West - Laval - Quebec - Canada -
H7V 3S8
GSK Biologicals Global Vaccine 
Clinical Laboratory, Wavre-Nord 
Noir Epine
Avenue Fleming, 20 - B-1300 Wavre - Belgium
Appendix B
Structured script
Are you aware of dengue, chikungunya or Zika cases in your neighborhood during the 
past month?
Any subject that meets the case definition for suspected dengue, chikungunya or Zika
the subject (and subject’s parent(s)/LAR(s) as applicable) will be referred to the study 
hospital according to protocol
 
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