Geneva Dengue

TDR/SWG/08
Report on Dengue
Special Programme for Research & Training

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TDR/SWG/08
Report of the Scientific Working Group

meeting on Dengue
Geneva, 15 October 2006
TDR/SWG/08
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Executive summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1. Dengue as a public health problem and efforts to increase understanding
and control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2. Ongoing dengue research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
3. Purpose and objectives of the Scientific Working Group . . . . . . . . . . . . . . . . . . . 12
4. Global research agenda recommended by the Scientific Working Group . . . . . . 13
Annex 1
AGENDA: Scientific Working Group on Dengue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Annex 2
Dengue
Contents
LIST OF PARTICIPANTS: Scientific Working Group on Dengue . . . . . . . . . . . . . . . . . . . . . . . . 23
Annex 3
WORKING PAPERS: Epidemiological trends and disease burden
3.1 Recent epidemiological trends, the global strategy and public health advances
in dengue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.2 Dengue: burden of disease and costs of illness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
3.3 Dengue in Africa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Annex 4
WORKING PAPERS: Pathogenesis, vaccines, drugs, diagnostics
4.1 Understanding pathogenesis, immune response and viral factors . . . . . . . . . . . . . . . . 54
4.2 Opportunities in the development of dengue vaccines . . . . . . . . . . . . . . . . . . . . . . . . 61
4.3 Opportunities in the development of anti-dengue drugs . . . . . . . . . . . . . . . . . . . . . . 66
4.4 Laboratory tests for the diagnosis of dengue virus infection . . . . . . . . . . . . . . . . . . . 74
Annex 5
WORKING PAPERS: Clinical management
5.1 Research needs related to dengue case management in the health system . . . . . . . . . 86
Annex 6
WORKING PAPERS: Transmission dynamics and vector control
6.1 Dengue transmission dynamics: assessment and implications for control . . . . . . . . . . 92
6.2 Control of dengue vectors: tools and strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
6.3 Insecticide resistance in Aedes aegypti . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
Repor t of the Scientif ic Working Group on Dengue, 2006 TDR/SWG/08
iii
Dengue
iv
Annex 7
WORKING PAPERS: Surveillance and delivery issues
7.1 Dengue research needs related to surveillance and emergency response . . . . . . . . . . 124
7.2 Geographic information system for dengue prevention and control . . . . . . . . . . . . . 134
7.3 Achieving behaviour change for dengue control: methods, scaling-up,
and sustainability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
7.4 Delivery issues related to vector control operations: a special focus on the Americas . 150
Dengue is the most rapidly spreading vector borne disease. An estimated 50 million
dengue infections occur annually and approximately 2.5 billion people live in dengue
endemic countries. Because of the rapidly increasing public health importance of this
disease, in 1999 dengue was incorporated in the portfolio of the UNICEF, UNDP,
World Bank, WHO Special Programme for Research and Training in Tropical Diseases
(TDR). The 2002 World Health Assembly Resolution WHA55.17 urged greater
commitment to dengue among Member States and WHO; of particular significance is
the 2005 Revision of the International Health Regulations (WHA58.3), which includes
dengue as an example of a disease that may constitute a public health emergency of
international concern.
Dengue
Executive summary
It was against this background that the Dengue Scientific Working Group of 60 experts
from 20 countries including WHO staff from four Regions and Headquarters met in
Geneva in October 2006 to review existing knowledge on dengue and establish priorities
for future dengue research aimed at improving dengue treatment, prevention and control.
The goal of the Scientific Working Group was to outline a research agenda by identifying
bottlenecks and making detailed and specific research recommendations. The SWG
wanted to identify areas of research that could lead to tangible benefits for people in
disease endemic countries within the coming years as well as outline a strategic vision for
applied and basic research from which benefits would be felt in the medium to long term.
As a result of major demographic changes, rapid urbanization on a massive scale,
global travel and environmental change, the world, particularly the tropical world, faces
enormous future challenges from emerging infectious diseases. Dengue epitomizes these
challenges. In the early years of the 21st Century we are collectively failing to meet the
challenge posed by dengue as the disease spreads unabated and almost 40% of the worlds
population now lives at risk of contracting the disease. There is currently no specific
clinically useful diagnostic test, no drugs, and no vaccine, and we have failed to widely
or effectively implement existing vector control and clinical management measures that
we know would help to reduce the vector population and reduce case fatality rates. Yet
there has never been a more optimistic time to be involved in dengue and dengue research,
and interest in the disease has attracted a new generation of talented and committed
clinicians and scientists. Modern science, from clinical medicine to basic research on
pathophysiology, drug and vaccine discovery, through to the social and behavioural
sciences and vector biology and control, offers a unique opportunity to make a tangible
and substantial impact on dengue over the next decade. But in order to achieve what
Dengue
is possible, a paradigm shift is required in our current approach. The dengue research
community needs to: push for much greater implementation of existing knowledge to
reduce case fatality rates, extend basic and clinical research to understand the underlying
pathophysiology, aid diagnostics and drug discovery and further improve clinical
outcome, speed up the development of vaccine candidates including moving as quickly as
possible to efficacy trials, and gather evidence for implementing best practices for control
of the vector.
All of this is possible in the next ten years. But to achieve this, dengue needs a much
stronger voice within dengue endemic countries and within the global public health
community to persuade society, funding agencies and policy-makers of the importance
of the disease. We are at a critical epidemiological juncture in infectious, particularly
viral, emerging diseases at the start of the 21st Century, and in many ways dengue serves
as a model for how we might meet that challenge. The lessons learned from dengue will
have implications for a number of other diseases and our approach to their control. The
implementation of the best of existing knowledge and practice supplemented by future
research applied in an integrated, holistic fashion can be expected to significantly change
the lives of individuals living in dengue-endemic countries in the coming years. The
Scientific Working Group hopes this research agenda will help provide a strategic plan for
how we might collectively achieve the aims of reducing morbidity and mortality based on
better understanding of the pathophysiology associated with dengue, on implementation
strategy, and on reduction of virus transmission.
Global dengue research agenda

The priority dengue research areas are organized around four major research streams,
which will provide evidence and information for policy-makers and control programmes
and lead to more cost-effective strategies that will reverse the epidemiological trend.
Stream 1: Research related to reducing disease severity and case fatality

OPTIMIZATION OF CLINICAL MANAGEMENT
An efficient out-patient system and clinical and laboratory indicators of early dengue
virus infection, plasma leakage and shock, as well as an effective and safe method of
managing severe haemorrhage, dengue in pregnancy, and patients with co-morbidity, need
to be validated in order to scale up the use of improved treatment guidelines.
Dengue
It is recommended to analyse:
New methods and guidelines for triage andout-patient care.
The validity, role and accessibility of available and new diagnostics.
The predictive value of prognostic markers (host and viral, non-invasive measurement
of vascular leakage).
Standardized approaches to determining and documenting severe disease and response
to treatment.
Best practices for the treatment of dengue including early treatment to reduce severity,
treatment of established shock, and effective and safe management of haemorrhage.
The impact of co-morbidities on disease severity, and the effect of pregnancy.
The causes of dengue deaths (including treatment failures).
Process and impact evaluation of staff training

Training programmes in case management can help to rapidly reduce case fatality.
However, training has to be standardized and adapted to the prevailing local
health care system and best practice has to be identified and implemented in
dengue-endemic countries.
The process and impact of existing/future training programmes.
Critical issues in dengue pathogenesis

A better understanding of dengue pathogenesis will provide a foundation for future
rational clinical interventions. In particular we need to understand the changes
underlying: endothelial permeability in plasma leakage, dengue virus diversity, and
immune response to dengue viruses.
The physiological and molecular mechanisms leading to vascular leak and haemorrhage.
The host genetic factors associated with dengue severity.
The dengue viral factors and antigenic subtypes related to tropism, epidemic potential,
and virulence.
The mechanisms of antibody-mediated enhancement and protection.
The mechanisms of virus entry and cellular/tissue tropism.
T and B cell responses and their relation to immunopathology and protection.
Genetic predisposition to dengue.
Dengue
Stream 2: Research related to transmission control through improved vector management

Development and evaluation of vector control tools and strategies
The effectiveness of powerful vector control tools has been compromised by issues
of delivery, coverage and acceptability.Promising new tools and approaches need to
be evaluated.
The efficacy of new vector control tools and strategies in different contexts.
Combinations of new and/or existing tools in different contexts.
The scaling up of successful pilot projectsto state or national level.
Surveillance and response

Strengthening of surveillance systems through development and validation of reliable
risk indicators and the application of information technologyis needed for improved
decision-making.
Improved methods and their application/standardization in operational contexts.
The development and utilization of early warning and response systems.
The triggers that will allow effective response to incipient epidemics.
The contribution of information technology to decision-making.
Stream 3: Research related to primary and secondary prevention

Vaccines
Vaccines offer the greatest hope for dengue prevention and there are several candidates
in clinical development. The identification of vaccine components that are suitably safe
and immunogenic, and of immune correlates of protection, should accelerate successful
vaccine development and regulatory approval.
New vaccine candidates, adjuvants, and vaccination strategies.
Correlates of protective immunity for use as an endpoint in vaccine trials.
Immune responses in vaccine trials and natural infections.
Prospectsfor phase 3 and 4 vaccine evaluation trials in multiple field sites.
Drugs
Anti-dengue drugs may have prophylactic (e.g. outbreak prevention) and therapeutic (e.g.
prevention of severe disease) uses, including potential for impact on incidence and severity
of ensuing disease. Anti-viral drug discovery for dengue has accelerated in recent years
along with our knowledge of drugable targets in the virus.
Viral-encoded proteins for drug, diagnostics and vaccine design.
New (including natural) products or existing licensed drugs.
Dengue
Issues associated with future vaccine usage and coverage, including cost-effectiveness
studies of implementation.
Stream 4: Health policy research contributing to adequate publichealth response

There is a contradiction between the high priority afforded to dengue at political level
and the low level of resources allocated to dengue prevention and control program
activities.Health policy research will facilitate a redress of this imbalance.
Tools for rational decision-making and adequate prioritization of dengue, such as
studies of dengue burden and costs of illness.
Factors leading to success or failure of national programmes.
Decision-making that results in declaration of state of emergency.
The importance and burden of dengue in less studied regions (e.g. Africa).
Conclusion
The Scientific Working Group hopes this research agenda will help provide a strategic
plan for how we might collectively achieve the aims of reducing dengue morbidity and
mortality and its negative socioeconomic impact. Donors and the research community
are encouraged to take part in this major programme and to contribute through timely
information to the database TDR is establishing for keeping track of research activities
and relevant findings.
Dengue
1. Dengue as a public health problem and efforts

to increase understanding and control
This document provides an overview of the state of the art of dengue research as well as
recommendations for a global priority research agenda aiming to provide information
for policy and practice and allow evidence-based decisions to be made in order to reverse
the ongoing exponential increase in dengue, including in its severe forms. Throughout
the document, dengue is used as a generic term which includes uncomplicated and
severe forms of the disease such as dengue haemorrhagic fever, dengue shock syndrome,
dengue fever with haemorrhage, and, in some contexts, the silent or unapparent forms of
dengue infection.
Dengue as an expanding public health problem

Dengue is the most rapidly spreading arboviral disease in the tropics and subtropics.
The global burden of dengue has increased at least four-fold over the last three decades
and almost half the worlds population is estimated to be at risk (see Nathan and Dayal
Drager, working paper [WP] 3.1).
The primary vectors Aedes aegypti and the probably less important Aedes albopictus have
spread throughout the tropics. An estimated 50 million dengue infections now occur
annually, particularly in South-East Asia, the Americas, and the Western Pacific islands.
About 500000 severe dengue cases are reported annually and some 19000 dengue-related
deaths were registered in 2002. Despite the fact that dengue is accepted as an important
public health problem in the WHO South-East Asia Region (SEAR), Western Pacific
Region (WPR) and Americas Region (AMR), without reliable data about the burden of
disease in the African Region (AFR) (see Sang, WP 3.3) and the Eastern Mediterranean
Region (EMR), no detailed documentation of the real burden of dengue exists. Estimates
starting from the 1980s (see Suaya, Shepard and Beatty, WP 3.2) show dengue as
an emerging disease with disastrous consequences for peoples health and household
economy, and for society in general. As the registration and reporting of dengue varies
from country to country and region to region, and surveillance systems are generally
weak (see Ooi, Gubler and Nam, WP 7.1), it is difficult to make comparisons between
countries, monitor international trends, and measure the impact of interventions.
International efforts to control dengue

Because of the rapidly increasing public health importance of this disease, a 2002
World Health Assembly Resolution (WHA55.17)1 urged greater commitment to dengue
among Member States and WHO, while the 2005 Revision of the International Health
Regulations (WHA 58.3)2 included dengue as a disease that may constitute a public health
emergency of international concern. In 1999 dengue was incorporated in the portfolio of
Research activities supported through WHO, including through its regional offices in the
Americas (PAHO), South-East Asia (SEARO) and Western Pacific (WPRO), emphasize
three major research lines: improved vector control, case management, and primary
prevention through vaccine discovery and development (Kroeger et al. 2006).
Other major dengue initiatives include the Pediatric Dengue Vaccine Initiative (PDVI)
and the Innovative Vector Control Consortium (IVCC), both supported by the Bill
and Melinda Gates Foundation and others. DENFRAME and DENCO are research
consortia supported by the European Commission. Clinical dengue research in Asia is
supported by the Wellcome Trust; ecosystems research for dengue control is supported
by the Canadian International Development Research Centre (IDRC) and TDR; dengue
drug discovery, accelerated by the Novartis Institute for Tropical Diseases, is supported
by Novartis and the Singapore Economic Development Board and the academic sector
(see Selisko et al, WP 4.3); dengue control in the Mekong Delta is supported by the Asian
Development Bank and, in the Americas, by the Inter-American Development Bank.
Major initiatives in the discovery and development of dengue vaccines and diagnostics
as well as of insecticides come from the industrial sector supported and/or coordinated
by WHO and other partners (see Barrett and Hombach, WP 4.2; Buchy, WP 4.4).
Major publications have been produced in relation to dengue vector control and clinical
management (see Nathan and Dayal Drager, WP 3.1).
Dengue
the Special Programme for Research and Training in Tropical Diseases (TDR) and the
WHO Initiative for Vaccine Research (IVR). Other WHO departments are also directly
or indirectly involved in dengue research and an inter-departmental working group on
prevention and control of dengue was established, which is chaired by the department for
control of Neglected Tropical Diseases (WHO/NTD).
Dengue
2. Ongoing dengue research

Towards improved vector control
New methods for identifying and targeting the most productive larval habitats of the
vector are needed to improve the cost-effectiveness of vector control. A multicountry
project to determine the practicality and reliability of using the pupal/demographic survey
of A. aegypti in water containers as a method for identifying breeding sites producing
the majority of adult mosquitoes was based on the recommendations of a commissioned
report.3 The efficacy of targeted intervention in the most productive container habitats is
now being examined in a multicentre study.4,5 The efficacy of new vector control tools is
also being analysed in multicentre studies6,7 (also see McCall and Kittayapong, WP 6.2),
while the usefulness of new information technology (IT) tools for decision-making, such
as spatial analysis of vector breeding using geographical information systems (GIS) and
determination of dengue vector threshold levels using mathematical modelling, will be
explored by the IVCC.
Community participation is a vital component in the delivery and sustainability of
effective dengue vector control, and WHO commissioned the development of a step-bystep guide on social mobilization and communication for dengue prevention and control.8
This communication-for-behavioural impact (COMBI) approach to social mobilization
has been field tested in several countries in Asia, Latin America and the Caribbean,
where it contributed to the challenging task of engaging communities in more focused
application of vector control measures against Ae. aegypti and other important vectors
(see Elder and Lloyd, WP 7.3).
The TDR Steering Committee on Strategic Social, Economic and Behavioural Research
provides the major coordinating input for interdisciplinary projects on vector ecology
and its relation to biological and social aspects of disease transmission and control. This
programme is supported by IDRC and is focused on six Asian study sites. TDR also
funded several mosquito genome-based research projects that are potentially relevant to
vector control, following which work to complete the A. aegypti genome and refine vector
transformation techniques continued, leading to techniques for the stable transformation
of Aedes into mosquitoes that are refractory to dengue infection.
The testing and evaluation of pesticides and pesticide application technologies for public
health use, including for dengue prevention and control, is coordinated by the WHO
Pesticide Evaluation Scheme (WHOPES, at: www.who.int/WHOPES). The Vector
Ecology and Management unit of the WHO department for control of Neglected Tropical
Diseases (NTD) has provided additional technical support and guidance for developing
Towards improved case management

Effective management of dengue patients requires rapid diagnosis. Current diagnostic
tests are however of variable quality and their performance and accuracy have not been
validated; they are based on viral genome detection and are complex and expensive.
Since 2004, at joint meetings between TDR and the Paediatric Dengue Vaccine Initiative
(PDVI), product characteristics for dengue diagnostic tests for different indications have
been defined, an inventory of existing tests drawn up, and a TDR-led strategy for dengue
test development and evaluation presented.9 Establishment of laboratory networks in
South-East Asia and Latin America, and field trials of the performance of selected
tests, are under way. Plans for facilitating the development of new tests with higher
performance characteristics are also being developed by the European Commission
supported DENFRAME consortium.
Dengue
promising applications of technologies for dengue vector control, such as those associated
with long-lasting insecticidal materials (see McCall and Kittayapong, WP 6.2).
Dengue has different clinical manifestations that, according to the present classification
system, fall into categories such as dengue fever (DF), dengue haemorrhagic fever (DHF)
and dengue shock syndrome (DSS). Other severe manifestations include dengue fever with
unusual haemorrhage and dengue with severe organ complications. The variety of clinical
pictures and complexity of the classification system are confusing for many clinicians
and complicate triage and case management of patients as well as reporting10,11,12 (also
see Lum et al, WP 5.1). While the pathogenesis of dengue is gradually better understood,
key areas such as the pathways of immune enhancement or the virological/immunological
factors that lead to DHF and other severe forms of dengue (see Simmons et al, WP
4.1) are as yet not understood at all. Such understanding is, however, critical for vaccine
development. Life-threatening complications are more likely to occur when individuals
who are already immune to one of the four dengue virus serotypes become infected
with another virus serotype, although the variations between different ethnic, age and
nutritional groups as well as between regions are poorly understood.
Towards improved primary prevention through support

of vaccine development and drug discovery
IVR in close collaboration with PDVI provides guidance on the evaluation and testing
of dengue vaccines and contributes to shaping the underpinning agenda on research
issues. This facilitating role is critical since considerable challenges remain for the
Dengue
successful clinical development of a dengue vaccine. Specific issues relate to the complex
disease epidemiology and concerns over vaccine-induced disease enhancement through
immunopathological processes (see Barrett and Hombach, WP 4.2). To increase the
number of candidate dengue vaccines in the pipeline, research has been carried out to
validate monkey models, characterize humoral immune responses to dengue virus and
vaccine candidates, and explore novel strategies for attenuating dengue virus. To facilitate
vaccine testing, international reference materials have been established and a consultation
process initiated to define correlates for protection by dengue vaccines. Ongoing activities
emphasize support for the clinical evaluation of dengue vaccines, in step with candidate
vaccines being progressed by industry.
Dengue drug discovery has attracted the attention of industry (Novartis Institute for
Tropical Diseases in Singapore) and academic institutions. Better understanding of the
virus encoded proteins will lead to new opportunities for targeted drug design (see Selisko
et al, WP 4.3).
Towards improved epidemiological surveillance

DengueNet is an open-access web-based information system that allows public health
officials and researchers to share a common database and foster collaboration (http://
www.who.int/denguenet) (see Nathan and Dayal Drager, WP 3.1). It was created by the
WHO unit of Epidemic and Pandemic Alert and Response (EPR) for collecting and
analysing global standardized epidemiological data on dengue and DHF cases and deaths,
and circulating virus serotypes, by geographical area and time. DengueNet is implemented
by the WHO regional offices, by ministries of health of endemic countries, and by
laboratories. Rapid analysis of indicators such as case fatality rates allows monitoring
of the current situation during disease outbreaks and the possibility of targeting
training to improve early recognition, referral and hospital case management for severe
dengue.13 Standardized global dengue surveillance data,14 the principal result expected
from full implementation of DengueNet, are critical for the public health community
to define the burden of dengue and to assist governments and other stakeholders to
make investment decisions about applied research and development. However, the
establishment of national surveillance systems is still an enormous challenge (see Ooi,
Gubler and Nam, WP 7.1).
10
Most of the research programmes mentioned above have inbuilt elements of policy
analysis and implementation research related to service delivery issues including studies
about the usefulness of modern information technology (see Martinez, WP 7.2) and the
sustainability of strategies (see San Martin and Brathwaite, WP 7.4). While capacity
strengthening components are an overarching theme in most research programmes,
more systematic approaches to individual and institutional capacity building for dengue
research is desirable.
Dengue
Policy research, implementation research related to

delivery issues, and research capacity strengthening
11
Dengue
3. Purpose and objectives of the

12
The main purpose of the SWG meeting was to set the global agenda for future dengue
research in terms of describing what is known and which priority research areas should
be covered in the future. As a starting point, a review of the existing evidence on dengue
prevention and control was needed as well as an update of the ongoing dengue studies
and strategies for translating research findings into policy and practice (see working
papers in annexes 37). This, as well as interaction with dengue control and management,
provides a basis for defining further priorities in dengue research.
The specific objectives of the meeting were to:
Provide an overview of the state of the art of dengue prevention and control and
research needs.
Identify key research areas for the next few years that can inform policy and improve
dengue prevention and research.
The priority dengue research areas are organized along four major research streams that
will provide evidence and information for policy-makers and control programmes and
lead to more cost-effective strategies to reverse the epidemiological trend.
Stream 1: Research related to reducing disease severity and case fatality

Optimization of clinical management
An efficient out-patient system, and clinical and laboratory indicators of early dengue,
plasma leakage and shock, as well as an effective and safe method of managing severe
haemorrhage, dengue in pregnancy, and patients with co-morbidity, need to be identified
and validated in order to scale up improved and standardized treatment guidelines.
Dengue
4. Global research agenda recommended

by the Scientific Working Group
New methods and guidelines for triage andout-patient care of dengue patients, and to
validatetheir feasibility and results at different levels.
The validity, role and accessibility of available and new diagnostics for dengue.
The predictive value of prognostic markers (host/viral early warning signs) of disease
severity, and to validate procedures for early recognition and treatment of plasma
leakage and shock including non-invasive measurement of vascular leakage.
Standardized approaches to determining the clinical signs of shock in children and
adults, including the role and techniques of: measuring blood pressure in shock patients;
diagnosing severe dengue through ultrasound, other non-invasive technology and
laboratory markers (albumin, cholesterol); and response to treatment
Best practices for effective and safe management of dengue, including early treatment
to reduce severity, treatment of established shock (including using oral re-hydration
therapy), and effective and safe management of haemorrhage.
The impact of co-morbidities such as obesity, diabetes mellitus, hypertension and
chronic heart diseases, and the effect of pregnancy, on severity.
The causes of dengue deaths (including treatment failures) in order to learn lessons from
negative outcomes.
Process and impact evaluation of staff training

Implementation of training programmes in case management has an immediate impact on
case fatality (which is reduced), as has been shown in many countries. However, training
has to be standardized and adapted to the prevailing local health care system. A review
of dengue morbidity and mortality targeted at resolving the major problems in case
management could include reorganization and reallocation of resources.
13
Dengue
It is recommended to analyse the:

Process and impact of existing/future training programmes to determine how best we
can implement improved dengue case management in different health care systems and
achieve greatest impact.
Critical issues in dengue pathogenesis

Better understanding of dengue pathogenesis will be the foundation for future
rational clinical interventions. In particular, we need to understand the molecular and
pathophysiological changes underlying: endothelial permeability in plasma leakage
syndrome, dengue virus diversity (which may account for heterogeneity in virus biology
including virulence and epidemic potential), and immune response to dengue viruses
(which may paradoxically predispose individuals to severe disease).
Physiological and molecular causes of vascular leak and haemorrhage.
Host genetic factors associated with dengue severity.
Dengue viral factors and antigenic subtypes related to tropism, epidemic potential,
and virulence.
Mechanisms of antibody-mediated enhancement and protection.
Mechanisms of virus entry and cellular/tissue tropism.
T and B cell responses and their relation to immunopathology and protection in primary
and secondary infections.
Genetic predisposition to dengue.
Stream 2: Research related to transmission control through improved vector management

Development and evaluation of vector control tools and strategies
Although powerful vector control tools are available, in practice their effectiveness has
been compromised by issues of delivery, coverage and acceptability.Promising new tools
and approaches need to be evaluated for: efficacy in reducing dengue transmission, cost
effectiveness, community acceptance, and prospects for preventing and controlling other
vector-borne diseases, while it needs to be determined how best to take promising pilot
community interventions to scale in a cost-effective way.
14
Surveillance and response

Disease and vector surveillance are fundamental to effective programme
management.Strengthening of surveillance systems through development and validation
of reliable risk indicators and the application of information technologyis needed for
improved decision-making.
Dengue

Efficacy of new vector control tools and strategies in different contexts.
Effectiveness, cost, community acceptance, and feasibility of combinations of new and/
or existing tools, including integrated vector management and ecosystems interventions,
with a range of partners in different contexts.
Scaling up of pilot projectsto state or national level in order to identify and disseminate
best practices.
Improved methods such as the pupal demographic survey and its application
in operational contexts as indicators of risk for outbreak and for informing
targeted intervention.
The development and utilization of early warning and response systems.
The triggers (factors and information) that will allow effective response to
incipient epidemics.
The contribution of information technology (e.g. GIS, bioinformatics, DengueNet,
mathematical models) to decision-making.
Stream 3: Research related to primary and secondary prevention

Vaccines
Vaccines offer the greatest hope for dengue prevention and several candidates are
in clinical development. The challenge has been to identify vaccine components
and immunization strategies that are suitably safe and broadly immunogenic. The
identification of immune correlates of protection should accelerate successful vaccine
development and regulatory approval.
Through discovery and pre-clinical research, new vaccine candidates, adjuvants, and
vaccination strategies.
15
Dengue
Correlates of protective immunity for use as an endpoint in vaccine trials.

Immune responses in vaccine trials and natural infections.
Through the development of further field sites, including sites free of non-dengue
flaviviruses, the prospectsfor phase 3 and 4 vaccine evaluation.
The issues associated with future vaccine usage and coverage through vaccine
implementation research.
Drugs
Anti-dengue drugs may have prophylactic (e.g. outbreak prevention) and therapeutic
(e.g. prevention of severe disease) uses, with an ensuing impact on disease incidence and
severity. Anti-viral drug discovery for dengue has accelerated in recent years along with
our knowledge of drugable targets in the virus.
The structure of viral-encoded proteins to aid rational drug, diagnostics and vaccine design.
New (including natural) products or existing licensed drugs with good safety profiles
and to foster drug discovery efforts.
Stream 4: Health policy research contributing to adequate publichealth response

There is a contradiction between the high priority afforded at political level to dengue
and the low level of resources actually allocated to dengue prevention and control.Health
policy research will facilitate a redress of this imbalance.
The issues and events that will, through adequate dissemination of information
(including of burden of disease [DALYS] and costs of illness), elevate dengue to a high
priority at national and international levels.
Case studies of national programmes to identify factors leading to success or failure of
dengue prevention and control programmes in order to develop a set of best practices.
The decision-making that results in a declaration of state of emergency to allow
more timely and effective political response, and to identify the data triggers used in
this process.
The importance and burden of dengue in less studied regions, particularly Africa, and
its role as contributor to fevers of unknown origin.
16
1. World Health Assembly. Dengue fever

and dengue haemorrhagic fever prevention
and control (WHA 55.17). http://www.
who.int/gb/ebwha/pdf_files/WHA55/
ewha5517.pdf (accessed Nov 7, 2006).
2. World Health Assembly. Revision of the
International Health Regulations (WHA
58.3). http://www.who.int/gb/ebwha/
pdf_files/WHA58/WHA58_3-en.pdf
(accessed Nov 7, 2006).
3. Focks DA. A review of entomological sampling methods and indicators for dengue
vectors. Geneva, TDR, 2003 (TDR/IDE/
Den/03.1)
4. Focks DA, Alexander N. Multicountry
study of Aedes aegypti pupal productivity survey methodology: findings and recommendation. Geneva, Special Programme
for Research and Training in Tropical
Diseases, 2006 (TDR/IRM/DEN/06.1).
http://www.who.int/tdr/publications/
publications/pdf/aedes_aegypti.pdf
5. Nathan MB, Focks DA, eds. Pupal/
demographic surveys for the targeted
control of denude vectors and the estimation of transmission risk. Annals of
Tropical Medicine and Parasitology, 2006,
100:supplement no.1.
6. Seng et al. Inhibition of adult emergence
of Aedes aegypti in simulated domestic
water-storage containers by using a controlled -release formulation of pyriproxyfen. Journal of the American Mosquito
Control Association, 2007, 23:(in press).
7. Kroeger A et al. Effective control of dengue vectors with curtains and water container covers treated with insecticide
in Mexico and Venezuela: cluster randomised trials. British Medical Journal,
2006, 332:12471252.
8. Parks W, Lloyd L. Planning social mobilization and communication for dengue fever
prevention and control. A step-by-step guide.
Geneva, World Health Organization,
2004 (ISBN 92 4 159107 2; WHO/
CDS/WMC/2004.2; TDR/STR/SEB/
DEN/04.1)
9. TDR. Dengue diagnostics: proceedings of an
international workshop. Geneva, Special
Programme for Research and Training
in Tropical Diseases, 2005 (TDR/IRM/
DIAG/DEN/05.1).
10. Deen JL et al. The WHO dengue classification and case definitions: time for a
reassessment. Lancet, 2006, 368:170173.
11. Rigau-Perez J, Laufer MK. Dengue-related deaths in Puerto Rico, 1922-1996:
Diagnosis and clinical alarm signals. Clinical Infectious Diseases, 2006,
42:12411246.
1 2. Bandyopadhyay S, Lum LC, Kroeger A.
Classifying dengue: a review of the difficulties in using the WHO case classification for dengue haemorrhagic fever.
Tropical Medicine and International Health,
2006, 11:12381255.
13. WHO. Dengue haemorrhagic fever: early
recognition, diagnosis and hospital management. An audiovisual guide for health care
workers responding to outbreaks. Geneva,
World Health Organization, 2006 (WHO/
CDS/EPR/2006.4).
14. DengueNet: http://www.who.int/csr/
disease/dengue/denguenet/en/index.
html (accessed Nov 7, 2006).
Dengue
References
17
Dengue
18
Dengue
Annex 1
AGENDA: Scientific Working Group on Dengue
19
Monday
2 October
09.0009.30
09.3009.45
09.4510.10
10.1010.30
10.3011.00
Item
Speaker
Welcome address
Overview of TDRs research strategy and vision
Introduction of participants
Overview of activities for the SWG meeting and TDR/WHO
research streams
Recent epidemiological trends, the global strategy and public
health advances
Global dengue burden: the known and the unknown
Coffee break
Dr D.L. Heymann, Acting ADG/CDS

Dr R. Ridley, Director TDR
SWG Chair/Secretariat
A. Kroeger, TDR
M. Nathan, R. Dayal-Drager
J.A. Suaya
Issues and challenges for dengue research

11.0011.30
Dengue research needs related to surveillance and emergency

response
Research needs related to delivery issues and
behavioural change
Research needs related to dengue case management in the
health system
D. Gubler, Vu Sinh Nam,

Eng Eong Ooi
L. Lloyd, J.L. San Martin,
R. Martinez, J. Elder
L. Lum, N. Hung
M.G. Guzman, C. Simmonds
15.0015.30
Understanding pathogenesis, immune response and

virus factors
Lunch break
Opportunities in the evaluation and development of
dengue diagnostics
Opportunities in the development of dengue vaccines
A. Barrett
15.3016.00
16.0016.30
Coffee break
Opportunities in the development of dengue drugs
B. Canard
16.3017.00
17.0017.30
Dengue vector control: tool development and strategies

Dengue vector control: resistance and resistance
management
P. McCall, P. Kittayapong
J. Hemingway
17.3018.00
Summary: dengue research needs

Closure day 1 and reception
SWG Chair
11.3012.00
12.0012.30
12.3013.00
13.0014.30
14.3015.00
20
P. Buchy, S. Yoksan, E. Hunsperger
Tuesday
3 October
09.0009.30
Item
Name
Dengue transmission dynamics: assessment

and implications for control
D. Focks, R. Barrera
Introduction to working groups:

objectives and expected outcomes
Working groups I, II, III, IV (meet
individually)
Coffee break
Working groups: continued
Lunch break
Coffee break
Working groups: presentation of research priorities, rationale
for selection, and recommendations
SWG Chair/Rapporteur
Working groups
09.3009.45
09.4510.30
10.3011.00
11.0012.30
12.3014.00
14.0015.30
15.301600
16.0017.30
17.3018.00
Working groups, chairs, and TDR/WHO secretariat

Closure day 2
Wednesday
4 October
Item
Working groups
Working groups
Working groups: plenary session
Name
Consolidating research needs and setting priorities

09.009.15
09.1510.30
Objectives and outcomes for day 3

Small group to review the overall prioritization, harmonize
recommendations, and outline strategic plan
Working groups to finalize reports
10.3011.00
11.0012.30
Coffee break
Small group: continued
Lunch break
Small group: report on overall priorities and draft strategic
plan (in plenary)
Working groups: comments on summary recommendations
(in plenary)
Discussion and amendment of conclusions,
recommendations, and draft strategic plan (in plenary)
Concluding remarks and closure of meeting
Closure day 3
12.3014.00
14.0014.20
14.2015.00
15.0016.30
16.3017.00
Small group (meets separately):
SWG chair, Rapporteur and TDR/
WHO members
Working groups (meet individually)
Small group
Working groups
21
Thursday
5 October
08.3010.30
10.3011.00
11.0012.30
12.3014.00
14.0015.30
15.3016.00
16.0017.30
Item
Name
SWG and updated dengue guidelines

Coffee break
Finalization of SWG Report
Lunch break
Coffee break
Guidelines working group
Parallel meeting of TDR/PDVI diagnostic group from 10.30 hrs.
22
Chairs, rapporteurs
Chairs, rapporteurs
Chairs, rapporteurs
Dengue
Annex 2
LIST OF PARTICIPANTS:
Scientific Working Group on Dengue
23
Advisers: pathogenesis, vaccines, diagnostics, drugs
Maria G. Guzman
Instituto de Medicina Tropical Pedro Kouri
Autopista Novia del Mediodia, Km 6
PO Box 601, Marianao 13
Ciudad de la Habana, CUBA
Tel: + 53 7 202 0450
Fax: + 53 7 202 0651
Email: Lupe@ipk.sld.cu
Philippe Buchy
Head, Virology Unit, Institut Pasteur
in Cambodia
5 Monivong bld, PO Box 983
Phnom Penh, CAMBODIA
Tel: + 855 (0) 12 802982
Fax: + 855 (0) 23 725606
Email: pbuchy@pasteur-kh.org
Jose Pelegrino
PO Box 601, Marianao 13
Tel: + 53 7 202 0648
Fax: + 53 7 202 0651
Email: jlpele05@yahoo.es
Scott Halstead
Research Director, Paediatric Dengue
Vaccine Initiative
5824 Edson Lane
North Bethesda, MD 20852,USA
Tel: + 1 301 984 8704
Alan Barrett
University of Texas, Medical Branch
at Galveston
Department of Pathology
Galveston, Texas 77555-0609, USA
Tel: + 1 409 772 6662
Fax: + 1 409 772 2500
Email: abarrett@utmb.edu
Prida Malasit
Medical Molecular Biology Unit, Office for
Research and Development
12th Floor, Adulyadej Vikrom Building
Faculty of Medicine Siriraj Hospital,
Mahidol University
2 Prannok Road, Bangkoknoi
Bangkok 10700, THAILAND
Tel: + 66 2 4184793
Fax: + 66 2 4184793
Email: sipml@mahidol.ac.th
David Vaughn
Director Military Infectious Diseases
Research Program
United States Army Medical Research and
Material Command
Building 722, Room 42, 504 Scott Street
Fort Detrick, MD 21702-5012, USA
Tel: + 1 301 619 7567
Fax: + 1 301 619 2416
Email: david.vaughn@amedd.army.mil
Cameron Simmons
Center for Tropical Diseases, University
of Oxford
190 Ben Ham Tu Qan 5
Ho Chi Minh City, VIET NAM
Tel: + 84 8 835 3954
Fax: + 84 8 835 3904
Email: camsimmons1@gmail.com
Bruno Canard
AFMB UMR 6098 CNRS/UI/UII ESIL
Case 932, 163 Avenue de Luminy
13288 Marseille, cedex 9, FRANCE
Tel: + 33 4 91 82 86 44
Fax: + 33 4 91 82 86 46
Email: Bruno.Canard@afmb.univ-mrs.fr
24
Fax: + 1 301 984 4423
Email: halsteads@erols.com
Elizabeth Hunsperger
Centers for Disease Control and Prevention
Division of Vector Borne Infectious Diseases,
1324 Calle Caada
San Juan, PR 00920, PUERTO RICO
Tel: + 1 787 706 2472
Fax: + 1 787 706 2496
Email: enh4@cdc.gov
Bill Letson
851 Niagara Street
Denver, Colorado, 80220 USA
Tel: + 1 303 399 6474
Fax: + 1 303 782 5576
Email: bletson@pdvi.org
Harvey Artsob
National Microbiology Laboratory, Public
Health Agency of Canada
1015 Arlington Street, Winnipeg,
MB R3E 3R2, CANADA
Tel: + 1 204 789 2134
Fax: + 1 204 789 2082
E-mail: Harvey_Artsob@phac-aspc.gc.ca
WHO staff:
Joachim Hombach, Initiative for Vaccine
Research (IVR); Renu Dayal Drager, Office
for Alert and Response Operations (ARO);
Tikki Elka Pangestu, Research Policy and

Cooperation (RPC) (see full list of WHO
participants below)
Advisers: clinical
Ivo Castelo Branco

Ncleo de Medicina Tropical da Universedade
Federal do Ceara
Rua Carolina Sucupira, 770/202
Aldeota, Fortaleza, CE, CEP: 60140-120, BRAZIL
Tel: + 55 85 33669319 and + 55 85 8876 4782
Fax: + 55 85 33 66 9316
E-mail: ivo@ufc.br or ivocastelo@uol.com.br
Jeremy Farrar
Center for Tropical Diseases
University of Oxford
190 Ben Ham Tu Qan 5
Tel: + 84 8 835 3954
Fax: + 84 8 835 3904
Email: jfarrar@oucru.org
Lucy Lum
Department of Paediatrics, Faculty of Medicine
University of Malaya
Kuala Lumpur, MALAYSIA
Tel: + 662 246 6780
Fax: + 662 246 6750
Email: LUMCS@ummc.edu.my and lumcs@
em.edu.my
Eva Harris
Division of Infectious Diseases,
School of Public Health
University of California, Berkeley,
140 Warren Hall
Berkeley, CA 94720-7360, USA
Tel: + 1 510 642 4845
Fax: + 1 510 642 6350
Email: eharris@berkeley.edu
Nguyen Thanh Hung

Department of Dengue Hemorrhagic Fever
Childrens Hospital N1 Ho Chi Minh City
341 Su Van Hanh Street, District 10
Tel: + 84 89271119 9270355
Fax: + 84 892 70053
Email: hungdhf@hcm.fpt.vn
Siripen Kalayanarooj
Queen Sirikit National Institute of
Child Health,
420/8 Rajavithi Road
Tel: + 662 3548333
Fax: + 662 3548434
Email: sirip@health.moph.go.th
Eric Martinez
PO Box 601 , Marianao 13
Tel: + 53 7 202 0450
+ 53 7 832 5863 (Home)
Fax: + 53 7 202 6051
Email: emartinez@infomed.sld.cu
or ericm@ipk.sld.cu
Nidia Rizzo
CDC Regional Office for Central America
and Panama
Center for Health Studies
Universidad del Valle, 18 Av.11-95, Zona 15
Guatemala City, GUATEMALA
Tel: + 502 3690791 5, ext.330
Fax: + 502 3697539
Email: nrrz@cdc.gov
Gavin Screaton
Professor of Medicine
Imperial College, Hammersmith Hospital
Du Cane Road, London W12 0NN, UK
Tel: + 44 208 383 2002 or + 44 208 383 3201
Fax: + 44 208 383 3203
Email: g.screaton@imperial.ac.uk
WHO:
Olaf Horstick, TDR; Martin Weber, Child and
Adolescent Health and Development (CAH);
Shibani Bandyopadhyay, TDR (see full list of
WHO participants below)
25
Advisers: vector ecology, transmission dynamics

Roberto Barrera
Dengue Branch, Division of Vector-Borne
Infectious Diseases
Centers for Disease Control and Prevention,
1324 Calle Caada
San Juan, PR 00920, PUERTO RICO
Tel: + 1 787 706 2399
Fax: + 1 787 706 2496
Email: rbarrera@cdc.gov
Janet Hemingway
Director, Liverpool School of Tropical Medicine
Liverpool L3 5QA, UK
Tel: + 44 151 705 3261
Fax: + 44 151 707 0155
Email: hemingway@liverpool.ac.uk
Philip McCall
Vector Research Group
Liverpool School of Tropical Medicine
Liverpool L3 5QA, UK
Tel: + 44 151 705 3261
Fax: + 44 151 707 0155
Email: p.mccall@liv.ac.uk
Dana Focks
Infectious Disease Analysis
PO Box 12852, Gainesville, FL 32604, USA
Tel: + 1 352 375 3520
Fax: + 1 352 372 1838
Email: dafocks@id-analysis.com
Sutee Yoksan
Centre for Vaccine Development,
Mahidol University
Institute of Science & Technology for
Research & Development
25/25 Phutthamonthon 4
Salaya, Nakhonpathom 73170, THAILAND
Tel: + 662 441 0190
Fax: + 662 441 9336
Email: grsys@mahidol.ac.th
26
Luke Alphey
Department of Zoology
University of Oxford
South Parks Road
Oxford, OX1 3PS, UK
Tel: + 44 1865 271157
Fax: + 44 1865 271157
Email: luke.alphey@zoo.ox.ac.uk
Didier Fontenille
Director IRD Montpellier
Institut de Recherche pour le Dveloppement
Laboratoire de Lutte Contre les Insectes
Nuisibles
Agropolis BP 64501, 34394 Montpellier,
Cedex 5, FRANCE
Tel: + 33 4 6704 3222
Fax: + 33 4 6754 2044
Email: fontenil@mpl.ird.fr
Rosemary Sang
Kenya Medical Research Institute (KEMRI),
Centre for Virus Research
P.O. Box 54628, Nairobi, KENYA
Tel: + 254 02 2722541
Fax: + 254 02 2720030
Email: Rsang@kemri.org
WHO:
Chusak Prasittisuk, WHO Regional Office for
South-East Asia (SEARO); Chang Moh Seng,
WHO Regional Office for the Western Pacific
(WPRO); Robert Bos, Protection of the Human
Environment (PHE); Kazuyo Ichimori, Vector
Ecology and Management (VEM); Lucien
Manga, WHO Regional Office for Africa (AFRO)
(see full list of WHO participants below)
Advisers: strategy development, surveillance,

outbreak management, behavioural change
Duane Gubler
Director, Asia-Pacific Institute of Tropical
Medicine and Infectious Diseases
Department of Tropical Medicine and
Medical Microbiology
John A. Burns School of Medicine
651 Ilalo Street, BSB 3rd Floor
Honolulu, HI 96813, USA
Tel: + 1 808 6921606
Fax: + 1 808 6921979
Email: dgubler@hawaii.edu
John Elder
Professor of Behavioral Sciences
Graduate School of Public Health
San Diego State University
San Diego, CA 92182, USA
Tel: + 1 619 594 2997 or + 1 619 895 7385
Fax: + 1 619 594 2998
Email: jelder@projects.sdsu.edu
Jose A Suaya
Senior Research Associate
Schneider Institute for Health Policy.
Brandeis University
Heller School, MS 035, 415 Street
Waltham, Massachusetts, 02454-9110, USA
Tel: + 1 781 736 3904
Fax: + 1 781 736 3928
Email: suaya@brandeis.edu
Pattamaporn Kittayapong
Center for Vectors and Vector-Borne Diseases,
Faculty of Science
Department of Biology, Faculty of Science
Mahidol University, Rama 6 Road
Tel: + 66 2 2015935
Fax : + 66 2 354 7161
Email: grpkt@mucc.mahidol.ac.th
Vu Sinh Nam
National Institute of Hygiene and Epidemiology
1 Yersin Street,
Hanoi 10000, VIET NAM
Tel. + 844 971 5679
Fax: + 844 971 6497
Email: vusinhnam@hn.vnn.vn
Chaiporn Rojansirivet
Director, Bureau of Vector-Borne Diseases
Department of Disease Control
Ministry of Public Health
88/7 Tiwanont Road
Nonthaburi 11000, THAILAND
Email: chaiporn@health.moph.go.th
Eng Eong Ooi
Defence Medical and Environmental
Research Institute
DSO National Laboratories
27, Medical Drive, #09-01
SINGAPORE 117510
Tel: + 65 6485 7238
Fax: + 65 6485 7262
Email: oengeong@dso.org.sg
Linda Lloyd
3443 Whittier St.
Tel: + 1 619 226 4651
Fax: + 1 619 226 4651
Email: lsl@ix.netcom.com
WHO:
Jose Luis San Martin, WHO Regional
Office for the Americas (AMRO); Michael
Nathan, Neglected Tropical Diseases (NTD);
Elil Renganathan, Health Technology and
Pharmaceuticals (HTP) (see full list of WHO
participants below)
27
WHO Regional Office staff

Chang Moh Seng, WPRO
Chusak Prasittisuk, SEARO
Jose Luis San Martin, AMRO
Ramon Martinez, AMRO

Lucien Manga, AFRO
WHO Headquarters staff

Michael Nathan, NTD
Joachim Hombach, IVR
Renu Dayal Drager, ARO
Martin Weber, CAH

Elil Renganathan, HTP
Robert Bos, PHE
Kazuyo Ichimori, VEM

Tikki Elka Pangestu, RPC
TDR staff
Robert Ridley
Hans Remme
Jane Kengeya
Axel Kroeger
Janis Lazdins
Yeya Toure
Ayoade Oduola
Fabio Zicker
Johannes Sommerfeld
Shibani Bandyopadhyay
Rosanna Peeling
Nina Mattock
Olaf Horstick
* unable to attend
28
EPIDEMIOLOGICAL TRENDS AND DISEASE BURDEN

3.1 R
ECENT EPIDEMIOLOGICAL TRENDS, THE GLOBAL STRATEGY AND PUBLIC
HEALTH ADVANCES IN DENGUE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
3.2 DENGUE: BURDEN OF DISEASE AND COSTS OF ILLNESS. . . . . . . . . . . . . . . . . . 35
3.3 DENGUE IN AFRICA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Dengue
Annex 3
WORKING PAPERS:
29
WORKING PAPER 3.1. Recent

Epidemiological Trends,
the Global Strategy
and Public Health
Advances in Dengue
World Health Organization (WHO) continues to

grow exponentially. For the first 5 years of the current decade, the annual average number of cases
was 925896, almost double the figure for 19901999
(479848 cases) (figure 1). In 2001, a record 69 countries from the WHO Regions of South-East Asia,
Western Pacific, and the Americas reported dengue
activity (figure 1). In 2002, the WHO Region of the
Americas reported more than 1 million cases for the
first time. Although there is poor surveillance and
no official reporting of dengue to WHO from countries in the WHO African Region and the Eastern
Mediterranean Region, recent outbreaks of suspected dengue have been recorded in Madagascar,
Pakistan, Saudi Arabia, Sudan and Yemen, 2005
2006. For reviews of dengue history in these regions,
see the paper by Rosemary Sang, in the present
report, and EMRO (2005).
M.B. Nathan 1 and R. Dayal-Drager 2

1
Department of Control of Neglected Tropical Diseases,
Communicable Diseases Cluster, World Health
Organization, Geneva, Switzerland
2
Department of Epidemic and Pandemic Alert and
Response, Communicable Diseases Cluster, World
Health Organization, Geneva, Switzerland
Epidemiological highlights
We conservatively estimate that, since the last
meeting of the Scientific Working Group (SWG)
on Dengue in 2000 (TDR, 2000), there has been an
increase of 110 million in the number of persons living in urban areas of the world with a high burden
of dengue (United Nations, 2002). Approximately
975 million people now live in these urban areas,
that is, almost half the global population estimated
to be at risk of dengue infection.
Geographical extension of areas with dengue transmission or resurgent dengue activity have been documented in Bhutan, Hawaii (USA), the Galapagos
Islands (Ecuador), Easter Island (Chile), Hong Kong
Special Administrative Region and Macao Special
Administrative Region (China) between 2001 and
2004 (figure 2). All four dengue viruses are circulating in Asia, Africa and the Americas. Perhaps the
only comforting news is that reported case-fatality
rates have been lower in recent years than in the
decades before 2000.
By decade, the annual average number of cases

of dengue fever or severe dengue reported to the
Figure 1. Average annual number of cases of dengue or severe dengue reported to WHO, and average annual number of
countries reporting dengue
1 000 000
925 896
900 000
Number of cases
50
700 000
600 000
40
479 848
500 000
30
400 000
295 554
300 000
20
122 174
200 000
15 497
100 000
10
908
0
5
00
00
20
19
9
0
9
19
9
19
8
9
19
8
19
7
0
9
19
7
19
6
0
19
6
19
5
19
5
Number of countries
60
800 000
30
70
Figure 2. Countries and areas at risk of dengue transmission, 2006
Isoth.: Isotherm; the 10C January and July isotherms represent the approximate northern and southern geographic limits, respectively, at which Aedes aegypti can survive
the coolest months of the year. It has been found at higher latitudes (45 degrees N) but has not been able to survive the winter.
Chikungunya virus, Alphavirus (Togaviridae), has

received considerable attention recently, because of
severe outbreaks in areas of known endemicity, notably India and Indonesia, and in islands of the Indian
Ocean, where it has seldom or never been reported
before. Mention is made of these outbreaks because
chikungunya and dengue share common vectors in
Aedes aegypti and Ae. albopictus mosquitoes.
The Global and

Regional Strategies
The Global Strategy for Dengue Fever/Dengue
Haemorrhagic Fever Prevention and Control is
more than ten years old (WHO, 1996), but remains
essentially unchanged. It comprises five major elements: selective integrated vector control, with
community and intersectoral participation; active
disease surveillance based on a strong health-information system; emergency preparedness, capacity
building and training; and vector-control research.
Efforts have since focused on three fundamental
aspects: surveillance for planning and response;
reducing the disease burden; and changing behaviours. The Fifty-fifth World Health Assembly in 2002
(resolution WHA55.19) urged greater commitment

among Member States and WHO to implement
the strategy.
Of particular significance is the resolution (WHO,
2006c), Revision of the International Health
Regulations, made by the Fifty-eighth World Health
Assembly in 2005 (WHO, 2005), which includes mention of dengue fever (and yellow fever) as an example
of a health event that may constitute a public health
emergency of international concern, and of which,
under such circumstances, WHO should be notified
under the International Health Regulations.
Advances since the last SWG

Since the last SWG, several new, improved or validated tools and strategies have been developed and
are available to public health practitioners and clinicians. They include:
Rapid commercial diagnostic tests in use in
endemic countries.
Pocket book of hospital care for children. Guidelines
for the management of common illnesses with limited
31
Figure 3. Dengue research and training supported and encouraged by the UNICEF-UNDP-World Bank-WHO Special Programme
for Research and Training in Tropical Diseases or the Initiative for Vaccine Research
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*Topics that are currently under investigation, or will soon be under investigation, in multicountry studies.
Modified from Kroeger et al. (2006) Annals of Tropical Medicine and Parasitology, 100(Suppl. 1):S98, with the permission of the Liverpool School of Tropical Medicine.
resources (inclusion of dengue in the management

of fever) (WHO, 2006b).
An audiovisual guide and transcript for
health-care workers responding to outbreaks
(WHO, 2006a).
Guidelines for planning social mobilization
and communication.
Global strategic framework for integrated vector
management (WHO, 2004).
Dengue CD-ROM, Wellcome Trust,
Topics in International Health series
(Wellcome Trust et al., 2006).
Entomological survey technique to identify the most productive container habitats of
the vector(s).
Seven insecticide products evaluated by WHO
for mosquito larviciding (five insect growth regulators, two bacterial larvicides), four of which are
approved for use in drinking-water (methoprene
EC, pyriproxyfen GR, Vectobac DT and GR);
three insecticide products evaluated by WHO for
space spray applications to control mosquitoes
(all pyrethroids).
The sequence of the Ae. aegypti genome.
Advances in the development and operational deployment of DengueNeta for global
dengue surveillance.
International Health Regulations 2005: voluntary

compliance in effect.
New initiatives contributing to the development of
new tools and strategies include:
Paediatric Dengue Vaccine Initiativeb
(Bill & Melinda Gates Foundation).
The Innovative Vector Control Consortiumc
(Bill & Melinda Gates Foundation).
Asia-Pacific Dengue Partnership.
DENFRAMEd and DENCO (European Union).
Novartis Institute for Tropical Diseasese
(Novartis and the Singapore Economic
Development Board).
Regional Development Banks (Asian
Development Bank, Inter-American
Development Bank).
Streams of dengue research and training that have
been supported by the UNICEF-UNDP-World
Bank-WHO Special Programme for Research and
Training in Tropical Diseases (TDR) and by the
Initiative for Vaccine Research, or which will soon
be the subject of investigation, are summarized in
b
Paediatric Dengue Vaccine Initiative: http://www.pdvi.org/
Innovative Vector Control Consortium: http://www.ivcc.com/
DENFRAME: http://www.denframe.org/
D
engueNet: http://www.who.int/csr/disease/dengue/
denguenet/en/index.html
32
N
ovartis Institute for Tropical Diseases:
http://www.nitd.novartis.com/
Table 1. Control strategy, major challenges and research needs for the prevention and control of dengue
Main control strategy
Major problems and challenges
Major research needs
Reduction/interruption of
transmission through vector control
Lifestyles that provide abundant larval

habitats
Water infrastructure and solid waste
management
Sustainability of vector control
Vector-control targets/thresholds to reduce/interrupt

transmission
Behavioural changes conducive to the prevention and
control of dengue
Patient management and

supportive treatment
Early diagnosis and treatment
Patient management and supportive treatment
Vaccine and drug development
Pathogenesis and disease prognosis

Vaccine and drug development
Modified from Cattand et al. (2006), reproduced with permission from the World Bank.
figure 3 (from Kroeger et al., 2006). They involve the

development and efficacy testing of tools for vector
control, case management and primary prevention
(vaccine development).
of budgets and programme operations offer opportunities for strengthening and expanding this
integrated vector management approach for transmission control.
Cattand et al. (2006) perceive a growing consensus

that community-based approaches to transmission
control are desirable and necessary (see table 1), and
that they improve prospects for the sustainability
of vector-control programmes, although few such
interventions have expanded beyond the pilot stage.
However, Cattand et al. suggest that decentralization
The biggest challenge today appears to be how to

maximize the cost-effective deployment of tools and
interventions that are already available. For this,
we need clearly-directed operational research. For
tomorrow, we must pursue the development of new
tools and strategies, including better diagnostics,
treatments and the elusive public-health vaccine.
33
References
Cattand P et al. (2006). Tropical diseases lacking adequate control measures: dengue, leishmaniasis and
African trypanosomiasis. Chapter 6. In: Jamison
Dean T, ed. Disease control priorities in developing countries, 2nd ed. New York, World Bank and Washington,
Oxford University Press, pp. 451466.
EMRO (2005). Division of Communicable Disease Control,
Regional Office for the Meditteranean, Newsletter, Issue
No. 6, pp. 78 (http://www.emro.who.int/pdf/
dcdnewsletter6.pdf).
Kroeger A et al. (2006). Dengue research and training
supported through the World Health Organization.
Annals of Tropical Medicine and Parasitology, 100
(Suppl.1):S97S101.
WHO (2006a). An audiovisual guide and transcript for health care workers responding to outbreaks. Geneva, World Health Organization
(WHO/CDS/EPR/2006.4 and 4a).
WHO (2006b). Pocket book of hospital care for children.
Guidelines for the management of common illnesses with
limited resources. Geneva, Department of Child and
Adolescent Health, World Health Organization.
WHO (2006c). World Health Assembly resolution WHA59.2. Application of the International
Health Regulations (http://www.who.
int/gb/ebwha/pdf_files/WHA59/WHA59_2-en.pdf).
WHO (2005). World Health Assembly resolution WHA58.3. Revision of the International
Health Regulations (http://www.who.
int/csr/ihr/IHRWHA58_3-en.pdf).
TDR (2000). Scientific Working Group, 34 April

2000, Geneva, Switzerland: recommendations.
Geneva, UNICEF-UNDP-World Bank-WHO
Special Programme for Research and Training in
Tropical Diseases/World Health Organization
(TDR/DEN/SWG/00.1).
WHO (2004). Global strategic framework for integrated vector management. Geneva, World
Health Organization (WHO/CDS/CPE/
PVC/2004.10; http://whqlibdoc.who.
int/hq/2004/WHO_CDS_CPE_PVC_2004_10.pdf).
United Nations (2002). World urbanization prospects. The 2001 revision. New York, United Nations,
Department of Economic and Social Affairs
(ESA/P/WP.173).
WHO (2002). World Health Assembly resolution

WHA55/19. Dengue prevention and control. (http://
www.who.int/gb/ebwha/pdf_files/WHA55/
ea5519.pdf).
Wellcome Trust/UNICEF-UNDP-World BankWHO Special Programme for Research and Training

in Tropical Diseases (2006). Dengue [CD-ROM].
Wellcome Trust (Topics in International Health series)
(http://www.who.int/tdr/media/multimedia/
dengue.htm).
WHO (1996) Report of the Consultation on Key Issues in

Dengue Vector Control: toward the operationalization of a
global strategy, 610 June 1995, Geneva. Geneva, World
Health Organization (CTD/FIL(DEN)/IC/96.1).
34
WORKING PAPER 3.2.

Dengue: burden of disease
and costS of illness
Jose A. Suaya,1 Donald S. Shepard,1 Mark E Beatty2
1
Schneider Institute for Health Policy, Heller School,
Brandeis University, Waltham, MA, USA
2
Pediatric Dengue Vaccine Initiative, Seoul, Republic
of Korea
Summary
This paper provides a framework for considering the global burden of illness attributable to dengue, and its cost, and
describes challenges encountered in the estimation of these
values. Major challenges include: lack of uniform application
of the World Health Organization (WHO) case definition,
limited capabilities and standards of dengue laboratories,
limited accuracy of rapid tests, misdiagnosis, lack of uniform
criteria to report cases of dengue to WHO, limited role of
surveillance and reporting systems, under-reporting of fatal
and non-fatal dengue, misclassification in reporting, limited
public knowledge about major regions at risk and travellers.
While the scientific literature contains few studies on the burden of dengue and cost of illness, available results suggest
that the actual number of cases of dengue may range from
3 to 27 times the reported number. We propose a conceptual
framework for research.
Introduction
Dengue is a rapidly growing public health problem in tropical and subtropical countries where the
majority of the worlds population resides and is
increasing most rapidly. However, most of these
nations are economically disadvantaged and are
faced with multiple public health problems, and
therefore may not have the resources to combat the
continued emergence of dengue.1-3
With approximately two billion people living in
tropical and subtropical regions of the world, and
an additional roughly 120 million people each
year4 travelling to these regions, a large share of the
worlds population is at risk of contracting dengue.
Two estimates have suggested that between 50 and
100 million cases of dengue fever (DF) occur annually,5-7 corresponding to an incidence rate of 2.55.0%
of the two billion people worldwide at risk. These
cases result in hundreds of thousands of hospitalizations, and about 20000 deaths each year.2 The
spectrum of dengue infection ranges from asymptomatic infection to death. Clinical presentations
of the febrile phase include a milder non-localizing
fever syndrome, or influenza-like illness, and classic
dengue, or break-bone fever. Immediately after the
febrile phase, the disease may progress to the more

severe but less common forms of disease, which
include dengue haemorrhagic fever (DHF; a febrile
illness followed by abnormally low platelet counts,
egress of plasma into the pleural and abdominal
cavities and haemorrhagic symptoms), and dengue
shock syndrome (DSS; DHF with evidence of systemic hypoperfusion). Although death occurs rarely
in the febrile phase, it is most commonly the result
of hypoperfusion after the development of DHF.
Between 250000 and 500000 people develop severe
dengue each year.8
Mildly symptomatic dengue is usually treated in
an ambulatory care setting, while the more severe
forms require inpatient management. Although
more than 90% of patients who develop severe dengue have serological evidence of a previous dengue
infection, it is not possible to predict which patients
will progress to these more serious forms, complicating the triage and medical management of patients.
Given the scope of the disease and the large numbers of persons with symptomatic infection, dengue infection may have a tremendous impact on the
health-care systems of the countries involved and
on household and labour economies, especially during epidemics.
Quantifying the epidemiological and economic burden of dengue is key to formulating policy decisions
on research priorities, prevention programmes, clinical training for management of the disease, and the
introduction of new technology. Reliable diagnosis,
testing, and reporting of dengue would allow better
understanding of the true burden posed by dengue
in a world of competing public health issues. Reports
should capture seasonal and cyclical (annual) variations in disease incidence. Such reports can be used
for multiple purposes. First, they provide the basis
for comparisons of the burden of dengue in different
geographical locations and time periods. Second,
they help country-level, regional, and global public health authorities to make informed decisions on
resource allocations. Decisions can be based on a
comparison of the burden of dengue with that of
other health problems. Accurate estimates of the
magnitude of dengue can serve to justify donor
funding, setting priorities for research, and accelerating the development of dengue vaccines. Third,
such reports will serve as important baselines for
assessing the impact of any intervention (e.g. a
larvicide campaign or vaccine) that could alter the
burden of dengue, and will also provide a key ingredient in costeffectiveness analyses of a single or
multiple interventions and technologies.
The prospect of introducing vaccines against dengue
35
Figure 1. Dengue cases: global annual number of cases reported and number of countries reporting to WHO by year,
1955 to 2005

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makes the importance of understanding the burden

of dengue especially important. Substantial investments are required in preclinical research, human
testing, manufacturing, distribution, etc. These
activities will involve many stakeholders, from
donors to manufacturers and governments to consumers. This paper seeks to make these decisions
better informed.
The epidemiological
burden of dengue
The burden of illness caused by dengue refers to the
amount of disease imposed by dengue and measured using a set of epidemiological indicators, such
as number of clinical cases classified by severity
(DF, DHF, and DSS), duration of the illness episode,
quality of life during the illness episode, case-fatality rate, and absolute number of deaths during a
period of time. All of these epidemiological indicators can be combined into a single health indicator,
such as quality-adjusted life years (QALY)9 or disability-adjusted life years (DALY).10 Internationally,
DALYs are most often used. The burden imposed
by dengue and the potential benefits of any intervention, such as vaccination, can then be expressed
in terms of DALYs lost or saved and cost per DALY
lost or saved.
Current knowledge of the burden of dengue

Dengue is endemic in all WHO Regions except the
36
WHO European Region.11 Figure 1 shows the global

number of cases reported by year and the characteristic cyclical variations attributable to high epidemic and low post-epidemic years. In each year
until 1976, fewer than 40000 cases from fewer than
15 countries were reported annually to WHO. In
1974, WHO developed the first guidelines for the
diagnostic and management of dengue.8 Figure 1
shows that the lowest number of cases was reported
in 1979 and the highest number in 2002, with 110000
and 1300000 cases reported, respectively. Figure 2
shows the annual global number of deaths attributable to dengue and the number of countries reporting. There were similar cyclical variations in the
number of deaths coinciding with epidemic and
non-epidemic years. After 1977, the year with the
lowest reported number of dengue deaths was 1978
(with 807 fatalities) and the year with the highest
number was 1983 (with 6031 fatalities). The majority
of the cases and deaths were reported from southeast Asia and the western Pacific.
A number of studies have attempted to estimate the
burden of dengue in terms of DALYs. For example, the burden was estimated for Puerto Rico in
19841994.12 Estimates were based on the numbers
of cases and deaths reported to the national surveillance system, and on age-group expansion factors
used to control for under-reporting. The authors
estimated an average loss of 658 DALYs per million
population per year, and concluded that this burden was comparable to that attributed to meningitis,
Figure 2. Dengue deaths: global annual number of deaths reported and number of countries reporting to WHO by year,
1955 to 2005

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hepatitis, malaria, tuberculosis, the childhood cluster of diseases (polio, measles, pertussis, diphtheria, and tetanus), or intestinal helminthiasis, and of
the same order of magnitude as tuberculosis in Latin
America and the Caribbean. A study in south-east
Asia estimated a loss of 420 DALYs per million population per year, comparable to that of meningitis
(390 DALYs per million population per year), twice
the burden of hepatitis, and one third of the burden imposed by HIV/AIDS in the region.13 A study
for Thailand estimated that countrys burden at a
loss of 427 DALYs per million population per year
for 2001.14
The Disease Control Priorities Project has recently
published the global burden of disease for 2001 to
200315. It estimated the global burden of dengue as
528000 DALYs for the year 2001. This corresponds
to a burden of 264 DALYs per million population
per year for two billion people living worldwide in
areas at risk of dengue.
Major gaps in knowledge of dengue burden

Current global estimations of the burden of dengue
are considered to be uncertain because of a number
of factors, discussed below. The under-reporting of
dengue cases (both fatal and non-fatal) is probably
the most important barrier to obtaining an accurate
assessment.
Lack of uniform application of the WHO

case definition
WHO has published guidelines for the diagnosis,
classification, and management of dengue, which
have been adapted by WHO regional offices.16
Investigators have reported difficulties in applying the case definition because of its complexity and
the limited ability to explain observed patterns of
disease.8, 17, 18 Because of these difficulties and the
need to use categories relevant to their own needs
for planning and management, some countries have
instituted their own case definitions.8
Limited capabilities and standards of

dengue laboratories
Tests for anti-dengue IgM antibodies are commercially available. Their accuracy may vary with the
situation in which they are used. Tests for virus detection by cell culture or nucleic acid detection (e.g.
polymerase chain reaction) require the capabilities
of sophisticated research laboratories to produce test
materials and perform quality testing.19 Laboratory
capabilities, infrastructure, technical expertise and
research capacity need to be improved.20
Scientists at a recent WHO-sponsored meeting
pointed out limitations in laboratory standards,
quality control, and dengue serological diagnosis and virus isolation, as well as for reporting and
information exchange in south-east Asian and western Pacific countries.21 Similar considerations may
37
apply to laboratories in the Americas as suggested

by an evaluation of quality control of serological
diagnostic tests in major national reference laboratories responsible for dengue surveillance and diagnosis in the region.22 Although the majority of the
participating laboratories had a high level of performance in detecting IgG and IgM antibodies to
dengue, only 63% of 86 laboratories that received
samples for testing between 1996 and 2001 decided
to participate in quality control. This study also highlighted the challenges faced by participating laboratories, such as interruptions in the availability of
antigens, the low sensitivity of testing for IgM antibody by enzyme-linked immunosorbent assay, and
the lack of alternative tests and techniques for the
diagnosis of dengue. There is no information about
quality control for the isolation or identification of
dengue virus. Regions with imported dengue, such
as the USA and Europe, may face additional challenges. Since dengue is not common in these areas,
laboratory testing for dengue may not be available and the disease may be overlooked as a cause
of symptoms among ill travellers. For example, as
part of an external quality assurance evaluation, 20
serum samples were sent in 2002 to 18 European
participating laboratories to be tested for the presence of dengue virus-specific IgM and IgG antibodies.23 Laboratories reported concurrent and correct
results for 71% of the IgG-positive samples and 89%
of the IgG-negative samples. However, though 97%
of the IgM-negative samples showed concurrent
and correct results, only 58% of the IgM-positive
samples had concurrent and correct results. These
findings highlight the need for quality controls and
improvements in testing for dengue in countries
with imported dengue; worldwide laboratory capabilities and quality control are not adequate.
Limited accuracy of rapid tests

In a recent meta-analysis of 11 studies of rapid diagnostic assays for dengue, the authors evaluated the
performance of an immunochromatographic test
(ICT) manufactured by a leading company in its
field. The meta-analysis showed that these assay
had a wide spectrum of sensitivity (0.45 to 1.0) and
specificity (0.57 to 1.0), suggesting that such tests
may have limited accuracy.24 Also, the cost of rapid
tests is a barrier to their systematic use in developing countries. Little is known about the performance
of other rapid diagnostic tests sold in the market.
Misdiagnosis
Despite the available clinical guidelines, dengue can
be misdiagnosed (by under-diagnosis or over-diagnosis). Given the lack of specificity of the symptoms
of dengue, clinicians can confuse dengue with other
38
infections, such as influenza, enterococus, chikungunya, viral haemorrhagic fevers, leptospirosis,

malaria, or typhoid.25 Moreover, dengue infection
as an undifferentiated febrile illness may represent a large proportion of all the symptomatic cases
of dengue.26
A study reviewing medical records for a period
of 6 months in Laredo, Texas, in 1999 showed that
only 50% of patients with clinical suspicion of dengue were diagnosed as such.27 Misdiagnosis is more
likely if other febrile diseases with similar clinical
characteristics occur concomitantly. For example, in
Barbados in 1995 and 1997, the majority of patients
with suspected leptospirosis actually had dengue.
Conversely, some of the cases of suspected dengue
were actually leptospirosis. 28 In another study of
an outbreak of dengue in Bangladesh, about 18% of
cases of suspected dengue gave negative results in
laboratory tests for dengue and proved to have leptospirosis.29 In a study in a dengue-endemic province
in Viet Nam, among 697 patients with acute undifferentiated fever visiting primary care facilities, for
whom paired serum samples were collected, acute
dengue was diagnosed in 33.6% cases.26
Misdiagnosis can be influenced by treatment guidelines. For example, although WHO guidelines for
the treatment of febrile children aged 2 months
to 5 years are useful to ensure that children with
fever and no alternative explanation are empirically
treated for malaria, this guideline may contribute
to misdiagnosis of dengue, particularly in areas of
low malaria transmission or where physicians are
not routinely using malaria smears to confirm the
diagnosis.30, 31 In addition, drawing blood and sending it for testing for dengue may not be viewed by
the health-service provider as a worthwhile expenditure of time or money. In the absence of rapid testing, the result of the test will not be available for
days or weeks, and the provider may be left to diagnose the patient with a viral syndrome or fever of
no known source and treat the patient empirically.
Rapid testing may improve the situation, but the
cost of the test may be a disincentive. Finally, even in
those countries with adequate laboratory facilities,
cross-reactivity between anti-dengue antibodies and
antibodies to other flaviviruses (West Nile virus, St
Louis Encephalitis) and the dynamics of the immune
response to flavivirus (boosting of antibodies to the
primary infecting flavivirus during a second flavivirus infectionoriginal antigenic sin) further contributes to the problem of confirming diagnosis of
dengue in areas of the world where multiple flaviviruses exist.
Lack of uniform criteria to report cases of dengue to WHO

In the WHO Region of the Americas, cases of dengue are reported to WHO stratified by severity: DF
and DHF. However, in the WHO South-East Asia
Region and the Western Pacific Region, cases are
reported without distinction of severity, though most
reported cases involve hospitalization for DHF.11
Additionally, while some countries only report cases
of severe dengue, others report all cases, and still
others report only confirmed suspected cases.32 This
lack of uniform reporting makes it difficult to perform meaningful international comparisons and
aggregations.
Limited role of surveillance and

reporting systems
Surveillance systems depend mainly on the capacity of the hospital to record, monitor, and report
dengue statistics. Less information is obtained from
clinics, and still more limited data are reported by
private-sector medical practices. In south Asia, for
example, notification is barely enforced and the
number of cases of communicable diseases (such as
dengue) dealt with in the private sector is usually
unknown.33 In the Americas, surveillance systems
are also generally passive and considered to be ineffective in defining the full scope of transmission in a
given community.34
Underreporting of non-fatal dengue

There are a number of factors that contribute to the
under-reporting of dengue. Firstly, notification of
suspected dengue to public health authorities (communicable diseases units) is legally required in most
of the affected countries, but rarely enforced. Since
the results will often not be available for days or
weeks after the visit that led to the sample collection, the results may be viewed as having little clinical value to the treating physician. Complicated
reporting or lengthy requirements may be additional factors that reduce the motivation of healthcare providers to routinely report cases, since
reporting may not be of intrinsic value to the patient
or busy health-care providers. Surveillance systems
usually have logistic and budgetary constraints.
Consequently, the reporting of dengue is likely to
be fragmented, incomplete, inconsistent, and unreliable. Thus, under-reporting of dengue cases, and
probably even of deaths attributable to dengue, is a
major concern to be addressed.
Evidence of under-reporting is evident in studies conducted in Brazil, Indonesia, Puerto Rico,
and Thailand. In Belo Horizonte, south-eastern
Brazil, the level of reporting of hospitalized suspected cases of dengue was estimated to be 63%
between 1997 and 2002.35 As the cases recorded in
the reporting system were the more severe, the overall case-fatality rate may have been consequently
overestimated. In Indonesia, the number of reported
cases was compared with medical records of hospitalized DHF cases admitted in four major hospitals
in Bandung during 1994. Only 31% of these cases
were captured in the report.36 Similar under-reporting was found in Puerto Rico, where only 28.4% of
hospitalized cases of DHF were detected by any of
the surveillance and reporting systems.37 In another
study, the same author tried to measure the burden of dengue in Puerto Rico during 19841994.12 To
deal with the existing under-reporting, it was estimated that for every case of dengue reported among
children, there were about 10 additional cases not
reported. Among adults, it was estimated that for
every case reported, 27 cases went unreported. In a
recent study in Thailand, under-reporting was recognized and the true number was estimated as 10fold the number reported.14
Misclassification in reporting of dengue

Cases of dengue can be misclassified at the time of
diagnosis because of the lack of familiarity of some
medical providers with dengue as a disease, or difficulties with using the WHO classification system.8
Correct classification is important clinically, because
death is associated with the more severe form of the
disease. Correct classification is important epidemiologically because WHO has suggested that the
dengue case-fatality rate can be computed by dividing the number of deaths by the number of cases
of DHF.16 If classification is not uniform, comparisons of case-fatality rate between countries can be
misleading. The severity of dengue is also a predictor of the use of health-care services and of the costs
of medical care.38 In a study in Puerto Rico, only 17
DHF and 3 DSS cases were identified among 986
hospitalized cases of dengue reported via the surveillance system in 19901991. A review of the hospital records of those patients, however, found that
88 and 14 of them had a clinical diagnosis of DHF
and DSS, respectively.39 Reviews of medical records
identified about five times more cases of severe dengue than were reported to the routine surveillance
system. If appropriate allocation of resources to
address the dengue problem is to occur, better recognition of the severity of dengue and improved
reporting is needed. Another review of the medical records of patients with dengue during the 2002
epidemic in Taiwan, China, found that 71% of DHF
patients were discharged without such a diagnosis.40
39
A consequence of this misclassification is the underreporting of severe dengue.
reported cases and deaths in India is only a fraction

of that reported in south-east Asia,
Underreporting of fatal dengue
In many regions of India, an increasing number of

suspected cases of dengue are seropositive for IgM
and IgG antibodies.43 The existence of IgG antibodies in a patient demonstrates prior infection with
dengue and an increased risk of the severe forms of
the disease. Outbreaks of dengue are increasingly
reported in rural areas, implying that the population
at risk is increasing, since dengue is considered to be
a predominantly urban disease.44-47
Reports of deaths caused by dengue are intuitively

assumed to be more accurate than reports for nonfatal cases. During the 1998 epidemic of dengue
in Puerto Rico, there were 17000 reported cases of
dengue and 19 deaths for which dengue was confirmed or probable. For the same year, however,
only five deaths attributable to dengue are shown
on WHO DengueNet, the WHO-sponsored internet-based system for the global surveillance of DF
and DHF.41 This single time-point finding indicates
a four-fold under-reporting of laboratory-positive
dengue deaths. In addition, there were another 37
deaths for which dengue was initially suspected but
could not be confirmed because the virus was identified by virus isolation or immunohistochemcial
staining of tissue, the patient died before seroconverting, and no other explanation for the death was
identified. However, dengue was ruled out in six of
these cases.42An analysis of paired samples gathered
during routine surveillance in Puerto Rico demonstrated that roughly half of the cases that were
initially indeterminate based on testing of the acutephase sample could be reclassified as confirmed
owing to seroconversion identified by testing a convalescent sample (Garcia, unpublished data), suggesting that an additional 15 deaths suspected to be
from dengue may actually may have been caused
by dengue. If correct, this raises the under-reporting
factor to seven in Puerto Rico. Difficulties in reporting and classification that occurred in Puerto Rico
are likely to occur in other countries where dengue is endemic. Additional factors that may further
interfere with confirmation and reporting of dengue
deaths to WHO include political and economic disincentives raised by concerns regarding the possible
impact on tourism.
Limited public knowledge from major regions

at risk
Data on the transmission of dengue is limited for
dengue-endemic regions that have a significant portion of the worlds populationIndia, China, and
sub-Saharan Africa.
India
One billion people (15% of the worlds population)
reside in India. Indias population is twice that of
south-east Asia, the region that currently reports
the most dengue-related deaths. Despite comparable environmental risk conditions, the number of
40
Surveillance for dengue has been very limited in

India and reporting to the central government has
not been mandatory.48 A recent study concerning
the epidemic of dengue in Chennai in 2001 has suggested that the surveillance system was unlikely to
generate proper information on the epidemiology of
the disease.49 In 2004, a WHO initiative called for promoting improvement of dengue surveillance as part
of the Integrated Disease Surveillance Programme
in India, strengthening laboratory networking and
quality assurance, and reviewing case definitions.50
Although improvements are being made, the current gaps in epidemiological data and surveillance
mean that the burden of dengue in India is uncertain. However, dengue is recognized as one of the
leading causes of death and hospitalization among
children in India.51
China
One billion three hundred million people, 20% of the
worlds population, live in China Roughly one fifth
of Chinas land mass, including some of the more
densely populated regions, lies in tropical climes
where dengue transmission could occur all year
round. Published reports on outbreaks of dengue
detailed the re-emergence of dengue in the 1980s
and 1990s.52-54 However, since 2003, public data from
WHO does not include cases in China41, making the
documentation of the current burden of dengue in
this country very difficult.
Sub-Saharan Africa
The burden of dengue in Africa remains poorly
understood. Travellers and military personnel visiting or stationed in Africa have been identified as
having laboratory-confirmed dengue infections,
indicating that the virus is circulating.55, 56 Several
studies of seroprevalence and fever in sub-Saharan
Africa have identified evidence for the presence of
the dengue virus in many sub-Saharan countries,
including Cameroon, Djibouti, Kenya, Senegal, the
Sudan and Burkino Faso. These studies reported
lower seroprevalence rates than those seen in other
tropical countries, such Haiti, Brazil, or Thailand,

ranging from 10% to 34% among persons tested.5759
As expected, higher levels of prevalence are noted
among urban residents than rural residents.60-65
In addition, periodic outbreaks of DF have been
reported in the region.63-66 Although genetic factors
could provide some protection, without systematic surveillance and serosurveys with appropriate
sample schemes to give a fair representation of the
disease burden in the population, the past and current burden of dengue in Africa may remain poorly
understood. Moreover, if dengue is an endemic
problem in sub-Saharan Africa, more urbanization
will only increase the burden of dengue.
Limited knowledge about dengue in travellers

According to the World Tourism Organization, in
2004, 125.4 million international tourists visited
countries where they might be at risk for acquiring dengue infection.4 Depending on the population
studied and the laboratory methods used, serological evidence of recent dengue infection was found
in between 7% and 45% of cases of febrile travellers
returning from areas where dengue is endemic,6769
confirming that dengue is an important cause of
fever among returning travellers. The increasing
number of cases of dengue creates a significant economic burden owing to working days lost. However,
given the spectrum of clinical illness, not all patients
may seek medical attention or receive diagnostic
testing. As a result, under-reporting of dengue infection occurs even in developed countries. Moreover,
of those patients who are diagnosed with dengue not
all may be reported to public health authorities. For
example, between 1 January 2001 and 31 December
2004 seven residents of the USA were diagnosed with
dengue after returning from Thailand.56 According
to the World Tourism Organization, 2012077 USA
tourists visited Thailand during the same period,70
giving a rate of 3.5 dengue infections per 1 million
visitors to Thailand. However, among a prospective
cohort of Dutch travellers, 0.7% of travellers returning from south-east Asia experienced symptomatic,
laboratory-confirmed (anti-dengue IgM seroconversion) dengue infections.71 If the risk of infection is
similar for travellers from the Netherlands and the
USA visiting south-east Asia, for each case reported
to the United States Centers for Disease Control and
Prevention there may be 5000 additional unreported
clinical dengue infections. This is a conservative estimate since the USA, unlike the Netherlands, shares
a border with a country where dengue is endemic
Mexico. As a result, USA residents have a higher
potential exposure to dengue.
Personnel deployed in dengue-endemic areas during humanitarian emergencies and conflicts are
at a higher risk of dengue infection than are regular travellers, since they usually live in areas without vector-control activities or air conditioning, and
usually stay in those areas longer than do tourists.
For example, during a 5-month deployment as part
of the United Nations Mission in Haiti, 32% of 249
personnel with febrile illness had dengue.72
The economic burden of

dengue, or costS of illness
Terminology
Cost-of-illness calculations generally distinguish
direct and indirect costs.
Direct costs are those within the health-care system.
They comprise the cost of diagnosis, treatment and
prevention of dengue. There are three major direct
cost categoriesmedical care, surveillance and
reporting, and prevention. The cost of medical care
includes the cost for ambulatory and inpatient care.
Surveillance and reporting costs take into account
efforts by governments and international organizations to monitor and disseminate information
about cases, outbreaks, and deaths. Prevention costs
include activities to prevent dengue, such as vector control (e.g. inspections, management of disposables, use of larviciding and fumigation, education,
media campaigns, and community mobilization).
Indirect costs are the economic value lost by households and society in general owing to illness and
premature mortality of dengue patients and productivity losses of household members and
friends affected.
The estimation of direct and indirect costs is complex because it must take into account different payers or economic sectors (public sector, household,
third party, employers, society), different levels of
government (district, regional, national), different
national government agencies (Ministries of Health,
Education, Environment; the Armed Forces), and
different international organizations (e.g. WHO,
United Nations Development Programme).
The system of national health accounts (NHA) provides a framework for examining costs within the
health sector (i.e. direct costs).73 This framework
helps countries to assess the totality of financial
resources available to the health sector (from the
public, private, and donor sectors), to identify the
financing agencies through which these funds flow,
and to analyse how these funds are used (by type
41
of provider, function, geographic region or population group). NHA also provides analysts and policy-makers with a tool that not only assists in the
analysis of current use of resources, but also helps
in the planning of future resource needs and tracking to determine whether resources are reaching
the target population. In countries where dengue
is endemic, NHA can help analyse current expenditure (public, private, and by donor) on treating
dengue. In turn, this information can be used to
analyse the costeffectiveness of any new vaccine
and understand who will derive the most benefit. Understanding patterns of health-care use and
expenditure may contribute to the development of
policies that will improve the allocation of resources
to the poorer segments of society, who might not
be able to pay for a vaccine or other dengue-related
interventions. WHO published the Guide to producing national health accounts in 2003.73
The most important government activities related
to dengue include vector control, educational activities, mass media programmes, and ambulatory
and inpatient care. Knowledge about spending on
these activities by district, regional or national governments is fragmented. Government-sponsored
health-care activities include care at clinics and hospitals. In hospitals, patients can receive ambulatory
care (outpatient department and emergency room)
or inpatient care (general, intermediate, or intensive
care). Information about the use of hospital services
can be obtained by following a cohort of people for
a given period of time (community-based study), or
obtained from a hospital itself (facility-based study).
Hospital costs include tests, drugs, supplies, healthcare personnel and medical facilities. To estimate the
hospital costs of dengue patients, two approaches
can be adopted: micro- or macro-costing. Microcosting consists of a detailed inventory of the different services available and used in the hospital,
the quantity used and the unit cost for each of the
services. Macro-costing estimates the average unit
cost for each output (e.g. hospital day of care or
emergency room visit) rather than cost for each of
its components (each laboratory test, drug administered, or procedure carried out by medical personnel). Macro-costing is simpler than micro-costing, as
it requires access only to the hospital annual budget
or spending and its breakdown by departments,
and the total number of output units (such as hospitalizations, average length of stay, outpatient visits,
emergency room visits, etc).
Productivity losses and school absenteeism as a
result of dengue infections have not been accurately
evaluated in most countries. Similarly, care-seeking
behaviour, household out-of-pocket spending on
42
treatment for dengue, caregivers time, and family

and psychological disruption have not been systematically or consistently measured.
Current knowledge and gaps concerning

economic burden
Data on costs (in US dollars) of treatment are limited
to the impact of outbreaks in a few countries. A few
examples follow.
Costs of dengue in Thailand

In Thailand, a cost study done on DHF in 1994 estimated the weighted average direct patient cost
(including travel, food and lodging and opportunity) at US$63.60, plus US$45.56 borne by the
government for routine service costs in hospitals,
totalling US$109.16. The per-capita cost of vector
control in Thailand in that year was US$0.081 74 A
more recent study calculated a similar cost per case
of US$61.14
Costs of dengue in South-East Asia

In another study in south-east Asia that assessed the
potential costeffectiveness of a paediatric vaccine
for dengue, it was estimated that the societal cost
per case of treating dengue (including ambulatory
visits, hospitalization, medications, travel expenses
and parents time seeking treatment) was US$139
for DHF, and US$4.29 for DF, with a baseline cost of
treatment equal to US$99 per 1000 population per
year. For comparison, the population-weighted average gross national income per capita was US$1083
for south-east Asia in 2001.13
The same study reported the cost of dengue vector
control per capita in other Asian countries: US$0.015
in Indonesia in 1998, US$0.081 and US$0.188 in
Thailand in 1994 and 1998, respectively, US$0.240
in Malaysia in 2002 and US$2.40 in Singapore in
2000. On the other hand, per-capita spending on
vector control in 14 Latin American countries in
1997 ranged from US$0.020 to US$3.560, compared
with US$0.140 to US$8.490 in 17 Caribbean islands
in 1990.
Costs of dengue in Puerto Rico

According to a study on the impact of an outbreak of
DF in rural Puerto Rico, the loss of income attributable to the disease,(either from illness or from loss of
time in caring for ill family members) was estimated
to be equal to US$305 per household or US$125
per person.75
Worldwide summary by WHO

The UNICEF-UNDP-World Bank-WHO Special
Programme for Research and Training in Tropical
Diseases (TDR) has summarized the costs of epidemic outbreaks of DF/DHF in several countries.11
In Cuba, for example, the cost per treated case is
US$299 compared with US$44 in Nicaragua, and
US$44 in Puerto Rico.
Facility-based studies in eight countries

With support from the Pediatric Dengue Vaccine
Initiative, a team from Brandeis University is coordinating researchers in eight countries to implement
facility-based studies to measure the socioeconomic
impact of dengue on households and the local or
national health system. These multi-country studies use a common protocol for data collection and
analysis. Three of the countries are in south-east
Asia (Cambodia, Malaysia, and Thailand), while the
other five are in Central and South America (Brazil,
El Salvador, Guatemala, Panama, and Venezuela).
Each study identified treated cases of dengue via
one or more health institutions in the country (hospital, clinic, national laboratory, or public insurance
system). For hospitalized patients, the researchers
abstracted data from the patients medical record.
One or two rounds of interviews were conducted
with the patient or guardian (if the patient was a
child). About 60% of patients were interviewed
twice (generally once during treatment and again
after recovery), and the remainder interviewed once.
About 2000 patients from 62 health facilities (both
public and private) were recruited. Of the eight
studies, five cover adults and seven cover children.
While analysis is still underway, data are feeding
into efforts to work with ministries of health and
other critical agencies in the collaborating countries
to judge the potential benefit of dengue vaccines.
For example, the investigators in Malaysia found
that a hospitalized patient received an average of 10
visits from household members. Data from Thailand
showed that family members invested the equivalent of 23 working days caring for one hospitalized
patient. The Cambodian team documented substantial family disruption when a mother had to spend
her day beside her sick child in the hospital while
the grandmother provided food and other relatives
cared for the other children at home. The breadth
of involvement by household members illustrates
that the impact of each case goes well beyond
medical spending.
The data will allow careful comparisons to be made
between the cost-of-illness for dengue and other
diseases, such as rotavirus and pneumonia, which
commonly cause hospitalization of children. The

data suggest that the cost to the family is a severe
burden and is as high as or greater than that of
other diseases.
Models of disease burden and costs

Disease modelling is often used to estimate the burden of disease and costs at the country or regional
(multi-country) level. A model for the economics of
a disease comprises a group of mathematical relationships among disease states, such relationships
being able to provide estimates of infections, clinical cases of varying levels of severity, treatments
received, use of health resources, and costs. While
an original study may seek to describe the burden
of disease in one population at one time (e.g. a specific district), a model can potentially cover a large
geographical area and span of time periods. Disease
models typically incorporate data from a multitude
of sources. By pooling studies, best estimates can be
derived and refined to achieve internal consistency.
For example, staff at WHO have developed a mathematical model of disease, DISMOD, to check the
internal consistency of epidemiological estimates
of incidence, prevalence, duration and case fatality.76 Disease models not only describe the current
or historical situation, but can also be used to project
future situations under current policies, as well as
the impact of new policies and technologies for prevention or treatment.
In 1993, Shepard & Halstead published a disease
model to examine the benefits and cost-offsets of
improved case management of dengue (medical
treatment, environmental control, such as reduction
of mosquito breeding sites, and the potential development of a vaccine).77 This study demonstrated that
a model can be used not only to examine individual
proposed policies, but also on any combination of
proposed policies. A study on the costeffectiveness
of a potential paediatric tetravalent dengue vaccine13 modelled the burden of disease attributable
to dengue in south-east Asia in 2000, and projected
how that burden would be reduced by the proposed
vaccine. Figure 3 shows an updated version of the
studys state-transition model of dengue infection
and illness. The studys cost projections were based
on estimated vaccine costs and projected savings in
treatment costs. The net cost per 1000 population
was estimated to be only US$17 (89% less than the
gross cost). Also, the cost per DALY saved by a paediatric vaccine would be US$50, making the potential vaccine highly cost-effective.
Estimates of the costs of vector control

Several researchers have developed estimates of the
43
Figure 3. State-transition model of dengue illness
Population
Infection
Asymptomatic
Clinical cases
Unspecified
symptoms
Dengue fever
Severe dengue
Deaths
Reprinted from Vaccine, 22, Shepard DS et al., Costeffectiveness of a pediatric dengue vaccine, pp. 12751280, copyright (2004), with permission from Elsevier
costs of vector control programmes for dengue. In

1993, a team sponsored by WHO developed guidelines for assessing the costeffectiveness of vector
control, which included procedures for ascertaining
the costs of such programmes.78 They presented case
studies on vector-control programmes for malaria
and schistosomiasis, but not dengue. In the same
year, another study reported the costs of Singapores
intensive vector-control programme.77
Two of the authors of this paper recently developed a model for estimating the cost of vector-control programmes at a country level and applied
the model to Malaysia.79 The procedure identifies
the two major components of a vector-control programme: inspections and fumigation. Aggregate
costs were estimated by determining the volume of
each activity per year (inspecting and fumigating
premises and neighbourhoods near a location where
dengue had been found) and the unit cost of each
activity, and deriving the total cost. The study estimated the national cost of vector control in 2002 at
US$5.8 million or US$0.24 per capita. Of this total,
74% of costs were attributed to inspection and 24%
to fumigation.
Estimates of population-based costs

Building on selected facility-based studies and
studies of the costs of vector control, the costs of
44
ospitalized cases and vector-control activities were

h
estimated for Malaysia using a NHA perspective.80
The study found that these costs (which exclude
ambulatory cases) to the health system in Malaysia
was US$12.8 million or US$0.53 per capita, of
which 54% was for treatment of illness and 46% was
for vector control.
Standardization of protocols
Standardized protocols for the collection of epidemiological and cost data, for analysis and interpretation can make study results both complete and
comparable across countries, as recently noted for
another disease.81
Research priorities
Conceptual framework
The challenge in estimating the epidemiological and
economic burden of dengue can be encapsulated
in an imaginary dialogue between the user and the
producer of this information. The user of information, nicknamed InfoNeed, is the director of a programme or the developer of a policy around dengue.
InfoNeed needs information for his policy-making and managerial responsibilities, such as planning or revising a control programme, developing a
treatment plan, or considering the development or
purchase of some new technology, such as a better

diagnostic test or a dengue vaccine. The producer
of information, nicknamed InfoGive, is an analyst
with access to the scientific literature, public databases, and possibly additional data. InfoGive knows
that research studies may have high precision but
limited generalizability, while databases such as
reported numbers of cases can be subject to underreporting, misclassification, and other limitations
discussed above. Data may be virtually absent for
some regions of the world or times.
As the imaginary dialogue begins, InfoGive may
plead that the data do not exist to answer the policymakers questions definitively. Continuing the dialogue, the policy-maker acknowledges the problem,
but responds that the questions cannot wait until
the ideal data become available. Policy-makers need
guidance now. Understanding that need, InfoGive
seeks to generate the most accurate answer possible
based on existing data. For purposes of generating
research priorities, this paper also seeks to identify
the types of studies which could be done within a
few years and with limited resources that would
contribute most to strengthening the worlds understanding of the burden of dengue.
To address the policy-makers needs while acknowledging the limitations of existing data, it is helpful
to generate a conceptual framework for the burden of dengue. Table 1 describes the three domains
of epidemiological and economic burden. For each
domain, it is important to describe quantities (numbers of surveillance and prevention activities, and
numbers of cases treated) and aggregate costs. The
last column, illnesses, comprises both epidemiological and economic burdens.
The cases of illness are the most complicated domain.
In view of the earlier discussion in this paper about
under-reporting and misdiagnosis, studying dengue illness reported via a surveillance system or
diagnosed in a specific health facility is analogous
to viewing an iceberg. At first sight, an analyst sees
only the part above the water, yet 90% of the iceberg
is hidden below the water. In the case of dengue,
at first sight, the analyst may study only reported
cases. The full burden of dengue includes a spectrum of types of services and reporting:
Confirmed dengue, seen and correctly labelled
by a health professional and confirmed by laboratory diagnosis.
Suspected dengue, seen by a health provider and
classified as suspected dengue, without laboratory confirmation. Note that the absence of laboratory confirmation may be due to many factors:
the health provider did not order the test, the
cost of testing is high, the time window was not
appropriate, or, occasionally, the patient passed
away before the biological marker would be
applicable. Depending on the location of treatment and the completeness of the reporting system, the case may or may not be reported in the
countrys epidemiology system.
Fever treated by a health provider, but attributed
to an illness other than dengue. The patient did
not receive a laboratory test for dengue because
providers did not consider a diagnosis of dengue
to be likely, the patient was diagnosed with a disease that is treatable and should not be missed
(e.g. malaria) as well as the factors above.
Nevertheless, in aggregate these cases are numerous and some are likely to be dengue.
Fever or other symptoms experienced by the
patient and managed by self-treatment. The
fear of dengue may cause these undifferentiated fevers to be treated more intensively (and
perhaps hospitalized) than they would have
otherwise if the risk of dengue infection were
not present.
In addition, illness caused by the dengue virus falls
within a spectrum of severity, ranging from asymptomatic infection to death. Figure 3, adapted from
the authors earlier model,13 shows this spectrum
of the disease. Conceptually, the epidemiogical burden could be computed by estimating the number
of patients who reach each stage in the diagram by
Table 1. Domains for estimating the epidemiologic and economic burden of dengue
Location
Surveillance and reporting
Prevention
Illness (confirmed and

suspected cases)
Inside the health systema
Laboratory testing and

personnel; operation of
surveillance system
Costs of vector control by

government
Cost of care faced by medical-care

providers, out-of-pocket expenses by
households, and payments by third
parties
Outside the health system
Negligible items
Value of community resources

and participation in vector
control
Travel expense, time lost, and quality

of life lost by patients and their
family and friends
Would be included in a countrys national health accounts
45
assigning probabilities to each stage, their duration

with dengue, and the associated loss in DALYs per
case. Similarly, the economic burden can be obtained
by multiplying the number of people at each stage
by the cost to the health-care system per person at
each stage and the other costs of illness per patient
at each stage. The greatest practical challenge is
obtaining the number of cases in each branch and
their associated unit burdens.
Implications for the evaluation of vaccines

The literature as discussed above indicates how
important it is that all stakeholders involved in the
development of a dengue vaccine understand the
burden of dengue, as well as the reasons why current data substantially underestimate the burden. To
some extent, existing data can generate expansion
factors that correct for under-diagnosis, misdiagnosis, and other limitations of existing information.
More importantly, additional data are needed to fill
the gaps. Studies are starting to focus on health facilities in which severe dengue is treated and concentrated, while population-based studies indicate the
full spectrum of the disease. As dengue varies by
locality, such studies are being conducted in many
continents in which the disease is endemic. By linking burden to the cost of treatment and the loss
of time and productivity, the economic burden of
dengue is assessed together with the human burden. Donors such as the European Commission, the
Pediatric Dengue Vaccine Initiative, and WHO are
supporting work on these important gaps and contributing to the information base for vaccines and
other approaches to controlling the disease.
Specific recommendations for research

Improve knowledge of the burden of dengue
Standardize the reporting of cases among reporting counties to allow comparability.
Improve documentation of reporting procedures
and sites used by reporting countries to provide
clarity in the application of the case definition.
Develop and offer a system for quality assurance
in laboratories in reporting countries, to maintain
consistency and comparability in case reporting.
46
Temporarily, and if possibly periodically, employ

alternative but complimentary methods of case
ascertainment in reporting countries to evaluate the effectiveness and representativeness
of existing surveillance and reporting systems
(e.g. population-based serosurveys and facility-based studies oriented towards capturerecapture methods).
Promote comparative studies with other diseases
to provide points of reference for competing public-health priorities.
Increase knowledge of costs

Develop and apply standard methods for economic studies.
Add economic components to epidemiological and clinical studies and to outbreak
investigations.
Measure the costs of vector-control programmes.
Measure the costs of community efforts for vector control.
Determine the costeffectiveness of strategies for
the prevention and management of dengue.
Estimate the reduction in dengue burden, costs
and costeffectiveness of new diagnostic, preventive, and therapeutic technologies.
Generate data for under-studied regions

and populations
Initiate studies of epidemiological and economic
burden in India, China, and Africa.
Initiate studies of epidemiological and economic
burden for international travellers.
Acknowledgments
This review funded by the Pediatric Dengue Vaccine
Initiative. For comments and assistance, the authors
are indebted to: Chrisann Newransky, Clare Hurley,
Rana Sughayyar and Binod Sah (Brandeis), Axel
Kroger (WHO), and Richard Mahoney (PDVI).
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EN/Section3/Section218_842.htm).
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52. Fan WF, Yu SR, Cosgriff TM. The reemergence
of dengue in China. Reviews of Infectious Diseases,
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53. Qiu FX et al. The first epidemic of dengue hemorrhagic fever in the Peoples Republic of China.
American Journal of Tropical Medicine and Hygiene,
1991, 44:364370.
54. Qiu FX et al. Dengue in China: a clinical review.
71:349359.
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Travel-associated dengue infections, United States,
20012004. MMWR Morbidity Mortality Weekly
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57. Halstead SB et al. Haiti: absence of dengue hemorrhagic fever despite hyperendemic dengue virus
transmission. American Journal of Tropical Medicine
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58. Tuntaprasart W et al. Seroepidemiological survey

among schoolchildren during the 20002001 dengue outbreak of Ratchaburi Province, Thailand.
Southeast Asian Journal of Tropical Medicine and
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Tropical Medicine and International Health, 2002,
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60. Collenberg E et al. Seroprevalence of six different
viruses among pregnant women and blood donors
in rural and urban Burkina Faso: a comparative
analysis. Journal of Medical Virology, 2006, 78:683
692.
61. Kuniholm MH et al. Seroprevalence and distribution of Flaviviridae, Togaviridae, and Bunyaviridae
arboviral infection in rural Cameroonian adults.
2006, 74:10781083.
62. Morrill JC et al. Serological evidence of arboviral
infections among humans of coastal Kenya. Journal
of Tropical Medicine and Hygiene, 1991, 94:166168.
63. Rodier GR et al. Epidemic dengue 2 in the city of
Djibouti 19911992. Transactions of the Royal Society
for Tropical Medicine and Hygiene, 1996, 90:237240.
45. Ibrahim SA et al. Measles in suburban Khartoum:
an epidemiological and clinical study. Tropical
Medicine and International Health, 2002, 7:442449.
65. Saluzzo JF et al. Isolation of dengue 2 and dengue
4 viruses from patients in Senegal. Transactions of
the Royal Society for Tropical Medicine and Hygiene,
1986, 80:5.
66. Johnson BK et al. Epidemic dengue fever caused
by dengue type 2 virus in Kenya: preliminary
results of human virological and serological studies. East African Medical Journal, 1982, 59:781784.
67. Lpez-Vlez R et al. Dengue in Spanish travelers returning from the tropics. European Journal of
Clinical Microbiology and Infectious Diseases, 1996,
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probable dengue virus infection among Dutch
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78. Philips M, Mills A, Dye C. Guidelines for costeffectiveness analysis of vector control. Geneva, World
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93.4.pdf).
79. Suaya JA, Shepard DS. A cost model for vector control programs against dengue. Abstract No. 62527,
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Annual Meeting of the American Public Health
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80. Shepard DS, Suaya JA, Lum L. Expenditure tracking
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81. WHO. Costeffectiveness of rotavirus vaccines
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(WER), 2006, 81:349356.
49
WORKING PAPER 3.3.

Dengue in Africa
Rosemary C. Sang
Arbovirology/Viral Haemorrhagic Fever Laboratory,
Centre for Virus Research, Kenya Medical Research
Institute, PO Box 54628, Nairobi, Kenya
Introduction
Although the history of dengue in Africa is poorly
documented, it is known that dengue has been on
the continent since the start of the 20th century. A
retrospective serosurvey by Kokernot et al. (1956)
suggests that dengue in Africa existed as far back as
19261927, when it caused an epidemic in Durban,
South Africa. Despite poor surveillance for dengue in Africa, it is clear that epidemic dengue fever
caused by all four dengue serotypes has increased
dramatically since 1980, with most epidemics occurring in eastern Africa and to a smaller extent, in
western Africa. The threat of the disease in South
Africa has been evaluated and documented.
Eastern Africa
Although dengue epidemics are infrequent in eastern Africa when compared with south-east Asia, the
Americas and the Caribbean, all four serotypes of
dengue have caused outbreaks in this region.
In 1982, an outbreak of dengue fever caused by dengue virus 2 (DEN-2) was reported in the Kenyan
coastal towns of Malindi and Kilifi (Johnson et al.,
1982); clinical presentation was consistent with classical dengue fever, with no severe dengue reported.
Since then there have been sporadic cases of dengue
reported in Kenya (unpublished observations), and
a serology survey carried out in 2005 (unpublished)
revealed the occurrence of dengue transmission in
coastal and inland parts of Kenya.
The 1982 outbreak in Kenya is believed to have
spread from the Seychelles outbreak that occurred
between 1977 and 1979 (Metselaar et al., 1980). The
islands of Comoros, in the Indian Ocean, experienced an epidemic in 1993 that affected more than
56000 people. Serology surveys revealed that there
had been previous outbreaks on the islands in 1948
and 1984.
In 19841985, an outbreak of dengue was reported
in Pemba, Mozambique. Two deaths were reported
to be associated with this epidemic. During this outbreak, most patients appeared to be experiencing
50
secondary infection with flavivirus, and the two

deaths were attributed to severe dengue (Gubler et
al., 1986). Another outbreak of dengue caused by
DEN-2 was reported in the city of Djibouti, on the
horn of Africa, in 19911992, with cases of severe
dengue being reported mainly among tourists and
expatriates (Rodier et al., 1996). In 19921993, an outbreak of dengue was reported among United States
troops engaged in the mission Operation Restore
Hope in Somalia, which lies to the north of Kenya. It
was found that 2% of cases were caused by DEN-3
and 41% by DEN-2 (Sharp et al., 1995). During the
outbreak, only cases of classical dengue were seen,
and there were no cases of severe dengue.
A dengue outbreak attributed to DEN-2 was
reported to have occurred in Sudan, also north of
Kenya, in 1986. The clinical presentation of patients
seen during the outbreak was consistent with dengue fever and there was no evidence of severe dengue. One infection with DEN-1 and 17 infections
with DEN-2 were diagnosed by viral isolation
(Hyams et al., 1986).
The available evidence so far indicates that
DEN-1, -2 and -3 appear to be a common cause of
acute fever in eastern Africa, and that the frequency
of epidemics continues to increase, with emergence
of other serotypes since the Seychelles outbreak in
1977 (see table 1).
Table 1. Past epidemics and reported cases of dengue and
severe dengue in Eastern Africa
Epidemic/
detection
Country
Dengue
serotypes
Reference
19771979
Seychelles
DEN-2
Metselaar et al.
(1980)
1982
Kenya
DEN-2,
Johnson et al. (1982)
19841985
Mozambique
DEN-3
Gubler et al. (1986)
19851986
Sudan
DEN-1, DEN-2
Hyams et al. (1986)
19911992
Djibouti
DEN-2
Rodier et al. (1996)
19921993
Somalia
DEN-2, DEN-3
Kanesa-Thesan et al.
(1994); Sharp et al.
(1995)
1948, 1984
and 1993
Comoros
DEN-1, DEN-2
Boisier et al. (1994)
2005
Eritrea
Not
determined
Unpublished
Although outbreaks and sporadic cases of dengue have continued to occur in eastern Africa, little
effort has been made to identify vectors and transmission cycles (sylvatic, periurban or urban). It has
been assumed that the outbreaks are most likely
to be transmitted by Aedes aegypti, which is widely
istributed in the region. Most of the reports on dend

gue in eastern Africa arise from outbreak investigations that are carried out by visiting scientists and,
in most instances, no entomological studies are performed, and no serology surveys are done to determine the extent of the outbreaks.
Western Africa
In the 1960s, DEN-1, -2 and -3 were isolated for
the first time from samples taken from humans in
Nigeria (Carey et al., 1971). Subsequently, dengue
has been found to occur in Senegal and Burkina
Faso (predominantly being transmitted in sylvatic
cycles), and possibly in other tropical rainforests in
western Africa (see table 2).
Table 2. Past epidemics and reported cases of dengue and
severe dengue in western Africa
Epidemic/
detection
Country
Dengue
serotypes
Reference
19641968
Nigeria
DEN-1, DEN-2
Carey et al.
(1971)
197485
Senegal
DEN-2
Saluzzo et al.
(1986)
198386
Burkina Faso
DEN-2
Robert et al.
(1993), Hervy et
al. (1984)
1982
Burkina Faso
DEN-2
Gonzalez et al.
(1985)
1980, 1990
Senegal
DEN-2, DEN-4
Saluzzo et al.
(1986), TraoreLaminaza et al.
(1994)
19992000
Senegal
DEN-2
Diallo et al.
(2003)
Fewer outbreaks have been documented than in

eastern Africa. Most reports are associated with
transmission in vectors and some sporadic cases
in humans. A number of entomological surveillance activities (motivated by the ambition to identify sylvatic cycles of dengue and the evolutionary
relationship between sylvatic dengue and endemic/
epidemic dengue) have been undertaken. Through

virus isolation, a number of Aedes species have been
associated with dengue transmission in western
Africa; these include Ae. taylori, Ae. furcifer, Ae. luteocephalus, Ae. vittatus and Ae. aegypti (Diallo et al.,
2003). Interestingly, there are more outbreaks of yellow fever in western Africa than in eastern Africa,
although these two viruses have common vectors.
Southern Africa
Since the first outbreak of dengue in South Africa in
19261927, cases of the disease imported from India
have been detected in the 1980s (Blackburn & Rawat,
1987). The threat of dengue in South Africa has been
evaluated in vector studies, and competent vectors
have been identified (Jupp & Kemp, 1993). This,
together with the occurrence of imported cases, led
to recommendations for continuous surveillance to
obvert outbreaks in the country.
Conclusion
Although dengue appears to be spreading in Africa,
the funding received for surveillance and other
research activities pertaining to dengue has been
very limited. This has been mainly owing to the
assumption that dengue is not a significant health
problem on the continent, and this is largely attributed to the fact that severe forms of dengue illness
are rarely reported. As most dengue infections are
subclinical or present as dengue fever, they go undiagnosed and are commonly treated as malaria or
other endemic fevers, such as typhoid and leptospirosi,. This has resulted in an underestimation of
the magnitude of the dengue problem in Africa.
Acknowledgments
The author would like to thank the WHO Special
Diseases (TDR) for the invitation to participate in
the Dengue Scientific Working Group (SWG) and to
contribute to this report.
51
References
Blackburn NK, Rawal R. Dengue fever imported from
India: a report of 3 cases. South African Medical Journal,
1987, 21:386287.
Boisier P et al. [Dengue 1 epidemic in the Grand
Comoro Island (Federal Islamic Republic of the
Comores). March-May 1993]. Annales de la Socit
Belge de Mdecine Tropicale, 1994, 74:217229.

Carey DE et al. Dengue virus from febrile patients in
Nigeria 196468. Lancet, 1971, 1:105106.
Diallo M et al. Amplification of the sylvatic cycle of
dengue virus type 2, Senegal, 19992000: entomologic
findings and epidemiologic considerations. Emerging
Infectious Diseases. 2003, 9:362366.
Gonzalez JP et al. Dengue in Burkina Faso (exUpper Volta): seasonal epidemics in the urban area
of Ouagadougu. Bulletin de la Socit de Pathologie
Exotique et de Ses Filiales. 1985, 78:714.
Gubler DJ et al. Dengue virus transmission in Africa.
American Journal of Tropical Medicine and Hygiene, 1986,
35:12801284.
Hervy JP et al. Circulation du virus dengue 2 dans
plusieurs milieux boiss des savanes soudaniennes
de la rgion de Bobo-Dioulasso (Burkina Faso). Cahier
ORSTOM Srie Entomologie Mdicale et Parasitologie
1984, 22:135143.
Hyams KC et al. Evaluation of febrile patients
in Port Sudan, Sudan: isolation of dengue virus.
35:860865.
Johnson BK et al. Epidemic dengue fever caused by
dengue type 2 virus in Kenya: preliminary results of
human virological and serological studies. East African
Medical Journal, 1982, 59:781784.
52
Jupp PG, Kemp A. The potential for dengue in South

Africa: vector competence tests with dengue 1 and 2
viruses and six mosquito species. Transactions of the
Royal Society of Tropical Medicine and Hygiene, 1993,
67:639643.
Kanesa-Thesan N et al. Dengue serotype 2 and 3 in
US forces in Somalia. Lancet, 1994, 3343:678.
Kokernot RH, Smithburn KC, Weinbren MP.
Neutralising antibodies to arthropod-borne viruses
in human and animals in the Union of South Africa.
Journal of Immunology, 1956, 77:313322
Metselaar D et al. An outbreak of dengue 2 fever in
the Seychelles, probably transmitted by Aedes albopictus. (Skuse). Bulletin of the World Health Organization,
1980 58:937943.
Robert V et al. Yellow fever virus, dengue 2 and
other arboviruses isolated from mosquitoes, in
Burkina Faso, from 1983 to 1986. Entomological and
Epidemiological Considerations, 1993, 86:90100.
Rodier GR et al. Epidemic dengue 2 in the city of
Djibouti 19911992. Transactions of the Royal Society of
Saluzzo JF et al. Isolation of dengue 2 and dengue 4
viruses from patients in Senegal. Transactions of the
Royal Society of Tropical Medicine and Hygiene, 1986,
80:85.
Sharp TW et al. Dengue fever in US troops during Operation Restore Hope, Somalia, 19921993.
53:8994.
Traore-Laminaz M et al. Dengue 2 outbreak in
South-East Senegal during 1990: virus isolation from
mosquitoes. Journal of Medical Entomology, 1994,
31:623627.
PATHOGENESIS, VACCINES, DRUGS, DIAGNOSTICS

4.1 UNDERSTANDING PATHOGENESIS, IMMUNE RESPONSE AND VIRAL FACTORS . . . 54
4.2 OPPORTUNITIES IN THE DEVELOPMENT OF DENGUE VACCINES . . . . . . . . . . . . . 61
4.3 OPPORTUNITIES IN THE DEVELOPMENT OF ANTI-DENGUE DRUGS. . . . . . . . . . . 66
4.4 LABORATORY TESTS FOR THE DIAGNOSIS OF DENGUE VIRUS INFECTION . . . . . . 74
Dengue
Annex 4
WORKING PAPERS:
53
WORKING PAPER 4.1.

Understanding
pathogenesis,
immune response
and viral factors
Cameron P. Simmons,1 Scott B. Halstead,2 Allan
Rothman,3 Eva Harris,4 Gavin Screaton,5 Rebecca
Rico-Hesse,6 David Vaughn,7 Eddie Holmes,8
Maria Guzman9
1
Center for Tropical Diseases, University of Oxford,
190 Ben Ham Tu Qan 5, Ho Chi Minh City, Viet Nam
2
Paediatric Dengue Vaccine Initiative, 5824 Edson
Lane, North Bethesda, MD, 20852, USA
3
University of Massachusetts Medical School, 55 Lake
Avenue North, Worcester, MA 01655, USA
4
Division of Infectious Diseases, School of Public
Health, University of California, Berkeley, 140 Warren
Hall, Berkeley, CA 94720-7360, USA
5
Imperial College, Hammersmith Hospital, DuCane
Road, London W12 0NN, UK
6
Department of Virology & Immunology, Southwest
Foundation for Biomedical Research, San Antonio,
Texas, USA
7
Military Infectious Diseases Research Program,
United States Army Medical Research and Materiel
Command, Building 722, Room 42, 504 Scott Street,
Fort Detrick, MD 21702-5012, USA
8
Center for Infectious Disease Dynamics, Department
of Biology, The Pennsylvania State University,
Mueller Laboratory, University Park, PA 16802, USA
9
Instituto de Medicina Tropical Pedro Kouri,
Autopista Novia del Mediodia, Km 6, PO Box 601,
Marianao 13, Ciudad de la Habana, Cuba
The problem
Dengue is the most important human viral disease
transmitted by arthropod vectors, yet no effective
and sustainable control measures exist. The recognition of severe dengue in Bangkok and Manila in the
1950s, and the spread of the disease to the Asian and
Pacific regions, and more recently to the Americas,
represents a major public health challenge posed
by a disease previously characterized as a debilitating but mild illness. Currently, dengue fever and
severe dengue are important causes of morbidity in
many tropical and subtropical regions of the world.
More than half of the worlds population lives in
areas at risk of infection and the incidence of the
disease has grown 30-fold in the past 50 years.1 It
has been estimated that the baseline burden of disease in south-east Asia is 0.42 disability-adjusted life
years (DALYs) per 1000 population, of which 52%
54
was attributable to premature mortality and 48% to

acute morbidity, comparable to that attributable to
meningitis in the Western Pacific, twice the burden
of hepatitis and one third that of HIV/AIDS.2 Casefatality rates for dengue shock syndrome range
from 0.1% to 10%, depending on the level of clinical
expertise for fluid management.
The Scientific Working Group on Dengue Research
organized by the UNICEF-UNDP-World BankWHO Special Programme for Research and Training
in Tropical Diseases (TDR)/WHO from 24 October
2006 discussed the global agenda for future research
on dengue and identified priority research areas for
future years. Dengue pathogenesis was one of the
topics widely discussed, given its implications for
case management, and the development of vaccines
and drugs.
The objectives:
To briefly summarize the state-of-the-art of
research on the pathogenesis of dengue.
To identify avenues of research with implications for the treatment, control and prevention
of dengue.
Pathophysiology of dengue
The definition of severe dengue is currently under
review.3 Nevertheless, there is a consensus that mild
and severe dengue syndromes generally represent
a disease continuum that is differentiated physiologically by the degree of vascular permeability.
Vascular leakage begins during the febrile phase but
at a time when the dengue virus (DENV) load is in
steep decline. Systemic leakage often becomes clinically apparent around the time of defervescence,
when haemoconcentration, ascites, pleural effusion or cardiovascular hypotension are present. In
children and adults, severe dengue is most often
(although not exclusively) associated with anaemnestic immune responses (secondary DENV infections). Severe dengue can also occur during primary
DENV infection of infants born to dengue-immune
mothers. An immunological basis for the pathogenesis of endothelial permeability and vascular leakage is widely accepted, but definitive data that
explain the mechanism of endothelial permeability are lacking. Thus, the biggest challenge in the
field is to understand the molecular mechanisms
of vascular leakage. In addition to plasma leakage, haemostatic abnormalities including marked
thrombocytopenia and bleeding are observed. It has
been suggested that depression of platelet synthesis
resulting in thrombocytopenia, and the production
of anti-platelet auto-antibodies occur during dengue
infection. Also, cross-reactivity of antibodies to the

envelope glycoprotein of the virus with plasminogen has been associated with bleeding. Finally, liver
involvement with mild elevation of serum transaminase activity is common.
Research priorities:
1. To acquire a better understanding of fluid physiology during severe dengue, and thus support
interventions based on rational fluid selection
and resuscitation in severe dengue.
2. To compare the pathophysiology of severe dengue during primary and secondary DENV infections among children and adults.
3. To identify changes in the endothelial layer that
are associated with severe dengue, e.g. through
biopsy studies or appropriate animal models.
4. To identify molecular mediators of endothelial permeability and vascular leakage during
severe dengue.
5. To acquire a better understanding of thrombocytopenia and bleeding manifestations.
6. To define the role of molecular mimicry
between dengue virus and endogenous host
proteins in pathogenesis.
7. To study the possible hepatotropic capacity
of DENV.
DENV genotypes and virulence

Humans are the major host of DENVs, with Aedes
mosquitoes, particularly Ae. aegypti and Ae. albopictus, being the principal vectors. At the genetic level,
DENVs exist as four antigenically distinct serotypes
that exhibit up to 30% divergence across their polyproteins. There is also considerable genetic variation
within each serotype in the form of phylogenetically distinct subtypes or genotypes.4,5 Currently,
three subtypes can be identified for DENV-1, six for
DENV-2 (one of which is only found in non-human
primates), four for DENV-3 and four for DENV-4,
with another DENV-4 being exclusive to non-human
primates. DENV subtypes often have differing geographical distributions, with some being more
widespread than others, indicating that both population subdivision and gene flow are important in
structuring genetic diversity. Phylogenetic analyses have also revealed that (i) subtypes frequently
co-circulate within the same locality; (ii) south-east
Asia harbours the greatest amount of DENV genetic
diversity, suggesting that it acts as a viral source
population; and (iii) there are periodic fluctuations
in genetic diversity, including lineage extinction.6
Sequence surveillance of DENV genotypes has suggested that some subtypes are more commonly associated with severe dengue. For example, DENV-2
and DENV-3 with Asian origins have been associated with epidemics (or cases) of severe dengue.7,8
These genotypes have established endemic cycles
in other continents, and in some cases, seem to have
displaced the autochtonous genotypes that did not
cause severe dengue. More recent studies, in both
human primary targets of infection (e.g. dendritic
cells) and in whole mosquitoes, have also shown
that these viruses produce higher viral titre.9 It is
not well understood why some viruses have greater
replicative fitness than others. Therefore, it is important to more broadly monitor the transmission of
these potentially virulent genotypes, to define if
these associations hold up in diverse human genetic
backgrounds and immune histories. The evolution
of DENV during an epidemic and its association
with epidemic severity also warrant further study.10
Finally, the sequence of primary and secondary
infection may be important, with sequential infection, e.g. DENV-1/DENV-2 and DENV-1/DENV-3,
suggested to give a high risk of severe disease.11,12
A technical manual, prepared by the Pan American
Health Organization (PAHO), outlines protocols
that could be used in many laboratories to standardize approaches to sequencing and phylogenetics
and obtain comparable results.
1. To move towards genotyping, in addition to
serotyping, of epidemic/case samples.
2. To acquire more quantitative measures of virulence factors to use in new, in-silico models of
dengue population dynamics.
3. To investigate the potential role of intrahost
diverstiy (quasispecies) in viral evolution and
disease severity.
4. To understand whether some sequences of
DENV are associated with greater severity
of disease.
Viral pathogenesis
Current evidence points to skin dendritic cells, tissue macrophages, peripheral blood monocytes and
hepatocytes, but not endothelial cells, as host cells
for DENV replication,13,14 although more work is
needed to define all target cells for DENV infection
in humans. The dominant host cell receptor(s) for
virus entry into these cell types has not been identified, although co-receptors such as DC-SIGN on
dendritic cells have been reported. In the monkey
model, DENV inoculated into skin rapidly moved
to macrophages in regional lymph nodes and other
lymphatic organs including spleen and liver.13 The
magnitude of viraemia and NS1 antigenaemia has
been associated with disease severity, including
55
complement activation, although these factors

alone may not explain all aspects of disease pathogenesis or clinical outcomes.15,16 Antiviral drugs to
inhibit DENV replication or cell adherence are being
developed by several groups, and if administered
early enough, might modulate the course of infection and possibly improve or shorten clinical illness.
Cytokines and chemokines generated by immune
activation correlate with disease severity.17 Animal
models (not all dengue-related) have established the
potential for these cytokines to contribute to the clinical manifestations of dengue, e.g. vascular permeability. Inflammatory cytokines alone may account
for systemic vascular leakage in severe dengue, but
unequivocal evidence for this is lacking.
1. To identify early prognostic markers of disease
severity (host or viral) for use at point of care,
e.g. antigen detection in urine or blood.
2. To understand the very early events in DENV
infection, from mosquito inoculation (dose, site)
to peak viraemia.
3. To understand the role of NS1 in complement activation, immune evasion and anti-NS1
responses in immunity.
4. To identify the human cells infected after mosquito inoculation and the cellular receptors.
5. To identify cell types infected by DENV in
humans, both early in infection (peripheral
blood mononuclear cells, biopsies) and late in
infection via autopsy studies.
Antibodies involved in the

development of immunity
There is a consensus that antibodies (IgM and
IgG) are likely to be critical effectors in the resolution of dengue viraemia and long-term immunity. Antibodies may provide immune protection
by blocking cellular attachment, viral fusion or by
antibody-dependent cellular cytotoxicity (ADCC).
ADCC has been associated with severe dengue.18 It
is not certain that existing in-vitro assays accurately
characterize the anti-dengue activity of antibodies as they exist in vivo. For example, some studies
have suggested that pre-infection neutralizing antibody titres measured in the conventional plaquereduction neutralization assay (PRNT) are poorly
predictive of subsequent viraemia or disease severity caused by certain DENV serotypes.19,20 These
are important observations given that neutralizing antibody in PRNT-type assays is being used as
a primary end-point in trials of dengue vaccines.
Investigations into the structural organization and
folding of DENV and West Nile virus Env proteins
56
have identified epitopes and protein domains targeted by neutralizing monoclonal antibodies, and
in some cases have described their mechanisms of
action.21,22 A better understanding of the relevance
of anti-NS1 antibodies in the resolution of viraemia
and immunity is also an important goal. The characterization of DENV-specific human monoclonal
antibodies from immune donors will provide further insights into the molecular basis of antibody
function in neutralization or infection enhancement.
The Pediatric Dengue Vaccine Initiative is providing leadership and funding for basic science aimed
at improving our understanding of the role of antibody in immunity to and pathogenesis of dengue.
1. To acquire a better understanding of in-vitro
neutralizing antibodies as correlates of protection against different DENV serotypes
and genotypes.
2. To understand the role of ADCC during
dengue infection.
3. To develop better assays to measure antibodymediated immunity and assess their predictive
value in prospective field studies.
4. To assess the memory B-cell response over
time in vaccinated individuals versus in
natural infections.
Antibody enhancement
of infection
Classical severe dengue accompanies a first DENV
infection in some infants aged less than 1 year who
are born to mothers who are immune to dengue.23,24
Severe dengue also occurs in second (occasionally
third) heterotypic DENV infections in older individuals, but the overall incidence is low given the
total number of heterotypic infections.25 Severe dengue can occur after a long interval between primary
and secondary DENV infection (2025 years), and
indeed might be more severe after long intervals.26,27
Infants born to dengue-immune mothers and previously-infected children or adults have in common
a single immune factorIgG dengue-reactive antibodies. The phenomenon of antibody-dependent
enhancement (ADE), whereby dengue antibodies
at subneutralizing concentrations enhance DENV
infections in Fc-receptor bearing cells,28 provides a
unifying basis for these epidemiological findings.
One explanation for the low incidence of severe dengue given the total number of heterotypic infections
is that cross-reactive dengue-neutralizing antibodies may block productive infection.29 Heterotypic
Pediatric Dengue Vaccine Initiative: www.pdvi.org
neutralization may explain why American genotype DENV-2 viruses failed to produce severe dengue in DENV-1-immune individuals.30 However,
the clinical significance of ADE in potentiating disease severity remains controversial. In part, this is
because the in-vitro infection-enhancing activity of
pre-infection plasma has not always correlated with
subsequent clinical severity of disease.20,29 The natural history of the antibody response to dengue,
and particularly the characteristics of homotypic
and heterotypic neutralizing antibodies, e.g. avidity,
cross-reactivity to different genotypes and their role
in protection or pathogenesis, remain poorly studied. A better understanding of the clinical importance of ADE and a comprehensive study of human
antibodies raised by dengue infection will be crucial
as candidate dengue vaccines advance into larger
clinical trials.
1. To thoroughly characterize the natural history
of the antibody response to dengue infection.
2. Large prospective studies are required to determine whether in-vitro assays that measure
infection enhancement can predict subsequent
clinical severity of disease.
3. Studies in infants are needed to understand the
pathogenesis of severe dengue in hosts possessing anti-dengue antibody, but not cellular
immunity.
4. Acquire a better understanding of the
role of ADE after short and long intervals between infection with different DENV
serotypes/genotypes.
T cell-mediated immunity
and pathogenesis
The similarity between DENVs accounts for crossreactivity in the humoral and cellular immune
response. In Thai children with acute secondary dengue, there is massive activation, proliferation and
programmed cell death of dengue-specific T cells.31
There is also evidence that the response is dominated by cross-reacting memory T-cell clones generated during previous infections (original antigenic
sin).31 Finally, there is a correlation between the
magnitude of the peripheral blood T-cell response
and disease severity, although in many cases this
association is observed well after the acute symptoms have resolved.31,32 In vitro, many serotypecross-reactive T-cell clones respond in a different
fashion to stimulation in vitro by antigens from different DENV serotypes. This effect results in a modified profile of cytokine production.33 Collectively,
these observations have led to the suggestion that
profound T-cell activation, cytokine release and cell

death may contribute to the systemic disturbances
leading to severe dengue, and original antigenic
sin in the T-cell responses may suppress/delay
viral elimination, leading to higher viral loads and
increased immunopathology. These events may act
in concert with phenomena such as ADE. Pathogenic
memory T-cell responses cannot explain severe dengue in infants with primary infections. However, it
is conceivable that severe dengue in infants occurs
via a different mechanism than in more immunologically experienced subjects like children and adults
with secondary DENV infections. A challenge for
the interpretation of T-cell responses during dengue haemorrhagic fever (DHF) is that often the flavivirus-infection history of the patient is unknown.
Furthermore, dengue-specific T cells are not usually
detectable in the peripheral blood during the febrile
phase of the illness.
1. To determine how the primary and secondary
T-cell responses to dengue differ.
2. To identify the predominant site of T-cell reactivity in DENV infection.
3. To assess whether T-cell responses can be protective in dengue.
4. To define the roles of CD4 versus CD8 T-cell
responses in immunopathology in dengue.
5. To explore whether dengue vaccines should
aim to induce T-cell responses to nonstructural
antigens or whether they should be tailored to
concentrate purely on antibody responses to
envelope determinants.
6. To study the short- and long-term T-cell
response against DENV serotypes/genotypes.
Innate immunity
Host and viral events during early DENV infection remain poorly understood. Type I and type II
interferons can contribute to control of viral replication in vitro and in laboratory mice.34,35 Studies in
vitro suggest that DENV nonstructural proteins can
attenuate the antiviral effects of type I interferon.36
Natural killer cells are activated in acute dengue
and may contribute to killing of infected cells by
cytokine release or ADCC. Complement is also activated in acute dengue and soluble NS1 may be
important in this process.37 Dendritic cells play significant roles in antigen presentation and the regulation of acquired immune responses. Dendritic cells,
either in the dermis or in lymphoid tissue, may be
receptive to DENV replication in vivo.13 CD209, or
DC-SIGN, a dendritic cell-expressed lectin, is an efficient coreceptor for viral entry into dendritic cells.38
57
DENV infection modulates dendritic cell maturation, but is associated with secretion of inflammatory and immune-modulating cytokines.39 Mice that
are transplanted with human haematopoeitic cells
and that develop functional dendritic cells display
clinical parameters (viraemia, fever, thrombocytopaenia, rash) that reflect similar events in dengue
fever in humans.
1. A
nimal models and studies of skin explant
infection could accelerate our understanding of
early hostvirus interactions.
Genetic susceptibility
and resistance
A genetic basis to the regulation of disease expression is likely, a remarkable example being resistance
to DHF observed in Cubans of African descent.40
More recently, the prevalence of Negroid anthropometric characteristics was associated with a lower
incidence of DHF (2%), while individuals with a
predominance of Caucasoid anthropometric characteristics had a higher incidence of DHF (30%).41
Numerous small casecontrol studies have identified disease associations, e.g. in human leukocyte
antigen (HLA) alleles, the vitamin D receptor and
FcIIa. A functional mutation in the promoter region
of DC-SIGN has been associated with susceptibility to mild dengue, but not DHF.42 Surprisingly, this
data suggested that dengue fever and DHF were
not a continuum, but discrete clinical entities. With
the exception of studies on DC-SIGN, casecontrol
association studies in dengue have been small, have
58
not been replicated in multiple populations, and

lack functional data to support the genetic association. Interpretation of these existing data therefore
requires caution.
1. Large, well-powered casecontrol or family
trio studies that investigate genome-wide polymorphisms that are associated with the clinically important events in dengue, e.g. dengue
shock syndrome versus DHF, symptomatic versus asymptomatic, that can be replicated in
different populations.
2. Functional investigations that support and help
explain genetic associations.
Conclusions
For simplicity, we have presented brief research
backgrounds and priorities in digestible segments. However, we recognize that the outcome of
a DENV infection is dictated by a complex interaction between host and viral factors. This implies
that, wherever possible, future research investigations should take a balanced approach by comparing the infection outcome in symptomatic cases as
well as in asymptomatic individuals, and considering the afferent (phenomena that promote the survival of the virus) and efferent (phenomena that
promote the elimination of the virus) mechanisms.
Large, longitudinal studies of populations at risk in
areas where dengue is endemic could provide the
foundation for this balanced approach to dengue
pathogenesis.
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WORKING PAPER 4.2.

Opportunities in
the development of
dengue vacCines
Alan DT Barrett1 and Joachim Hombach2
1
Department of Pathology and Sealy Center for Vaccine
Development, University of Texas Medical Branch,
Galveston, Texas, USA
2
Initiative for Vaccine Research, World Health
Organization, Geneva, Switzerland
Introduction
It has been said that no modality, with the exception of clean drinking-water, has had a greater effect
on the control of infectious diseases than vaccines.
Dengue vaccine research was initiated in the 1940s
with Sabin and colleagues (Sabin & Schlesinger,
1945) working on the development of a live attenuated vaccine. In the mid 1970s, many stakeholders
came together to develop a dengue vaccine; it was
stated that this objective would be achieved within
10 years. Although great strides have been made in
our understanding of the virus and disease, no vaccine has yet been licensed and no candidate vaccine has progressed beyond phase II clinical trials.
While some manufacturers remain optimistic that
a dengue vaccine will be launched within the next
5 years, it should be remembered that it takes an
average of at least 15 years for a candidate vaccine
to advance from the discovery phase to licensing.
There are multiple reasons why the development
of a dengue vaccine is complex, and these are discussed below.
The complexity of dengue

vaccine development
Dengue disease is caused by four serologically
related flaviviruses designated DEN-1, DEN-2,
DEN-3 and DEN-4. The first major problem in developing a dengue vaccine is thus the need to develop
not just one but four immunogens that will give a
balanced response such that a protective immune
response is induced against all four viruses simultaneously, i.e. the vaccine has to be tetravalent.
Another factor behind the need for a tetravalent
vaccine is the problem of immune enhancement,
including antibody-dependent enhancement. It is
clear that infection by one dengue virus leads to
lifelong protective immunity to the infecting virus,
i.e. homotypic immunity; however, many studies
have demonstrated that secondary infections (i.e.

infection by one dengue virus followed by infection by a second dengue virus, e.g. DEN-2 followed
by DEN-3) can lead to severe and occasionally fatal
dengue. Thus, there is the theoretical danger that a
dengue vaccine would have the potential to cause
severe dengue in vaccinees, if solid immunity was
not established against all four viruses. However,
it should be emphasized that, to date, there is no
evidence that any vaccinee receiving a candidate
vaccine has subsequently succumbed to severe disease. It is rather the case that vaccinees have shown
evidence of immunity for varying lengths of time
depending on the candidate vaccine administered
(Pengsaa et al., 2006). Nonetheless, the potential risk
of immune enhancement by a candidate vaccine
must be evaluated.
The third problem is that the mechanism by which
protective immunity against dengue infection is
induced is poorly understood. At the present time,
vaccines are licensed for use in humans against
three diseases caused by other members of the genus
Flavivirus: yellow fever, Japanese encephalitis and
tick-borne encephalitis. All the available data indicate that neutralizing antibodies are the major contributor to protective immunity; it is thus assumed
that neutralizing antibodies are the main effector
of protection against infection by dengue viruses.
Evidence in support of this hypothesis comes from
studies on passive transfer and maternal antibodies
(Pengsaa et al., 2006). However, this does not prove
that neutralizing antibodies are the only mechanism
of protective immunity against infection by dengue.
Further studies are necessary to establish correlates
of protection to demonstrate that candidate vaccines
induce a protective immune response. Evaluating
immunity is difficult as it must be demonstrated (via
clinical data or immunological correlates) that each
of the four components of the tetravalent vaccine is
able to induce immunity. The situation is complicated by the fact that dengue viruses co-circulate
in geographical areas where other flaviviruses are
found; distinguishing immunity to disease caused
by a particular dengue virus is consequently a difficult and complex problem.
The fourth major problem is the lack of a suitable
animal model in which candidate vaccines can be
evaluated. The lack of animal models has severely
hindered progress in identifying the determinants
of attenuation, virulence and immunogenicity of
dengue viruses that can be applied to vaccine development. The dengue viruses are also arboviruses
(arthropod-borne viruses) and normally have a
transmission cycle involving mosquitoes (predominantly Aedes aegypti), with humans as the vertebrate
61
host and, unlike most other arboviruses, they have

lost the need of an enzootic cycle for maintenance.
Clearly, it is not ethical to undertake experiments on
humans unless they are justified. Two animal models (mice and non-human primates) are used to evaluate candidate vaccines but, unfortunately, neither
displays the clinical symptoms seen in humans.
Mice are often used as a small-animal model to
make an initial evaluation of the ability of candidate
vaccines to induce a protective immune response.
Although this information is useful, all strains of
dengue virus investigated to date cause encephalitic disease in the mouse model, which is not representative of the disease in humans. Furthermore,
the majority of strains of the four viruses are only
able to cause disease in newborn or very young
mice. Some strains cause disease in weanling mice,
but only when inoculated directly into the brain.
Not surprisingly, it has been found that the mouse
model is not always predictive of the situation in
species of higher animals, i.e. a candidate vaccine
that protects mice may not be effective in another
animal. The second animal model is the non-human
primate, of which several species have been used
to evaluate candidate dengue vaccines. Non-human
primates do not get clinical disease but do demonstrate viraemia (originally measured as infectivity,
but now normally measured by real-time reversetranscriptase polymerase chain reaction, RT-PCR,
to determine genomic equivalents of virus, and
termed viral load), and immunological parameters are used as a proxy to measure efficacy. Clearly,
the mouse and non-human primate models must be
used to evaluate candidate vaccines before they are
tested in humans. However, the only true model for
dengue disease is in humans and, not surprisingly,
candidate vaccines fail at different stages of evaluation in mice, monkeys or humans.
Acambis/Sanofi Pasteur) are in phase IIb clinical trials, while other candidates are in phase I or
advanced preclinical development.
Overview of
candidate vaccines
Live attenuated vaccines
Since the 1940s, several live, attenuated vaccine candidates have been developed on the basis of classical
virology techniques, of which one, developed by the
Walter Reed Army Institute of Research (WRAIR),
has involved passage of dengue virus isolates in primary dog kidney (PDK) and primary African green
monkey kidney cells for attenuation, and the candidate vaccine is produced in fetal rhesus lung (FRhL)
cells. Although monovalent candidates have proved
to be safe and immunogenic in studies in humans,
interference was seen when the candidate strains
were combined into tetravalent formulations. In
collaboration with GlaxoSmithKline (GSK), a total
of 18 different tetravalent formulations have been
empirically derived, to select for high and balanced
immunogenicity and low reactogenicity, and evaluated in human volunteers (Edelman et al., 2004).
At present, formulation 17 is undergoing phase II
clinical trials.
Recombinant vaccines
Overall, there is limited understanding concerning which parameters can be used to demonstrate
the development of protective immunity (although
it is assumed that seroconversion of antibodies is
important). Owing to the limitations of current animal models, clinical trials are a critical component
of vaccine development in terms of the information
they provide on immunity and reactogenicity, while
long-term evaluation of volunteers is required to
demonstrate lack of evidence for immune enhancement/severe disease.
A number of research groups have used genetic engineering to develop a dengue vaccine; site-directed
mutagenesis or exchange of genes from different
sources is used to make a full-length complementary DNA (cDNA) of the virus genome in a plasmid that can be transcribed in vitro to generate viral
RNA that is transfected into cells from which the
virus can be recovered. The most advanced candidate to be based on this approach is the ChimeriVax
platform, which uses the yellow fever 17D vaccine
as a backbone. The development of chimeric yellow fever vaccinedengue vaccine viruses is being
undertaken by Acambis in collaboration with Sanofi
Pasteur. In this case, the yellow fever 17D vaccine
virus is used as a vector for insertion of the DEN prM
and E structural genes to replace the corresponding yellow fever genes. Four candidates have been
developed, one for each DEN virus (Guirakoo et al.,
2004). Like the WRAIR/GSK candidate described
above, the Acambis/Sanofi Pasteur candidate is in
phase IIb clinical trials.
In spite of the limits of our knowledge, there has

been significant progress in the development of candidate dengue vaccines in the past 5 years, indicating promise for a commercial vaccine in coming
years. Two candidate vaccines (WRAIR/GSK and
Of the other candidate vaccines being investigated,

one is produced using a platform technology developed by a group at the United States National
Institutes of Health and is based on a recombinant
DEN-4 virus backbone that contains a 30 nucleotide
62
deletion in the 3 non-coding region of the viral

genome (termed rDEN430 3NCR), which attenuates dengue viruses The production of the candidate
vaccine involved two approaches: (1) introduction
of the 30 3NCR deletion into wildtype DEN-1,
DEN-2 and DEN-3 viruses i.e. resulting in rDEN130,
rDEN230, and rDEN330 attenuated viruses; and
(2) construction of chimeric DEN-4/DEN-1, -2, -3
viruses, which expressed the prM/E gene region of
DEN-1, -2, or -3 virus in the attenuated genetic background of the rDEN430 virus (Blaney et al., 2006).
These vaccine candidates are currently in phase I
and phase II clinical trials.
developed by Hawaii Biotech Inc., and is based on

recombinant E and NS1 proteins produced in the
Drosophila S2 expression system and purified by
immuno-affinity. The vaccine contains the ectodomain of the E protein (called 80%E, i.e. it comprises
N-terminal 80% of the E protein) plus the DEN-2
NS1 protein, and has been formulated in a number
of proprietary adjuvants. The rationale for including
DEN-2 NS1 was the aim of induction of a cell-mediated immune response in addition to the induction
of neutralizing antibodies by the E protein. This
candidate vaccine has been demonstrated to show
promise in non-human primates.
A group at the United States Food and Drug Agency

is using a similar approach. This involves introducing several attenuating mutations into the stemloop structure at the 3 terminus of the dengue virus
genome (termed MutF). This group has successfully tested a DEN-1 candidate vaccine in monkeys
(Markoff et al., 2002).
A purified inactivated virus (PIV) vaccine formulation, similar to the successful inactivated Japanese
encephalitis and tick-borne encephalitis vaccines,
and using DEN-2 virus, has been investigated by the
WRAIR group on a proof-of-principle basis. This
vaccine demonstrated promise in non-human primates, but was not as efficacious as live DEN-2 vaccine (Putnak et al., 2005).
The United States Centers for Disease Control and

Prevention has developed a platform based on the
empirical attenuated DEN-2 PDK53 virus developed by researchers at Mahidol University in
Thailand. The significance of this platform is that it
is based on attenuating mutations in the 5 non-coding region and nonstructural proteins NS1 and NS3
(Huang et al., 2003), with no mutations in the envelope E protein, making it a potential platform for
tetravalent vaccine development. Chimeric recombinant DEN-2 PDK53 with substitution of membrane protein precursor (prM) and E protein genes
from wildtype DEN-1, -3 and -4 viruses have been
generated and produced promising results in nonhuman primates.
Alternative approaches
Although the development of recombinant viruses
generated by genetic engineering is a very promising approach, potential complications remain: the
molecular basis of attenuation of dengue viruses
is poorly understood and it is difficult to evaluate
the attenuated phenotype of these candidate vaccines without studies in humans. This has encouraged other research groups to investigate alternative
approaches. A number of groups have investigated
candidate DNA vaccines, the most promising candidate having been developed by the United States
Naval Medical Research Center. Using DEN-1 for
proof-of-principle, this DNA candidate vaccine is
based on the prM and E protein genes; it has been
shown to induce immunity in non-human primates, and is currently undergoing phase I clinical
trials. The most advanced subunit vaccine is being
Although the DNA, 80%E and PIV approaches have

their limitations, there is currently much interest in
primeboost approaches (i.e. the sequential use of
different immunogens) for a candidate DEN vaccine. It is thus possible that live vaccines used in
conjunction with the one or more of the alternative approaches may yield a vaccine with improved
safety and immunogenicity profiles. Finally, there
are recent developments in adjuvant technology
that may have applicability to improved immunogenicity of subunit vaccines.
Prospects and problems

The current candidate vaccine pipeline appears to
be sufficiently advanced such that licensing of one
or more dengue vaccines may be expected within
57 years. We note that the most advanced candidates are live vaccines. These have the advantages
of inducing a broad immune response that has the
highest chance of being protective and long-lasting, while also being relatively cheap to produce.
However, the issues involved in licensing and use
of live vaccines are well-known, relating to safety
follow-up and use in special target groups (people infected with HIV, pregnant women, etc).
Accordingly, there is value in continuing research
on alternative strategies, such as subunit vaccines,
which may become the product of choice for travellers or other special target groups, such as those for
whom live vaccines are not appropriate.
A major challenge in the future concerns the criteria
that should be used to evaluate candidate vaccines
63
in population-based efficacy trials in exposed populations. This will require consideration of a number
of technical, operational and regulatory issues.
Methodological obstacles to the addressing of these
issues remain considerable, particularly in the context of a tetravalent response (i.e. the need to discriminate between the four different dengue viruses
simultaneously), and neutralization needs to be correlated with protection in vivo. The role of heterotypic antibody in protection appears to be limited,
but further studies are desirable. These antibodies,
however, might be less relevant for vaccines, but
current methods do not allow for discrimination
between homotypic and heterotypic antibodies in a
tetravalent response. The demonstration of tetravalent priming remains therefore an important goal in
vaccine evaluation. In the case of dengue, particular
attention needs to be given to the definition of protection, which should be scientifically sound and
reasonable from a public health perspective. Apart
from clinical definitions, virological parameters
in particular, viraemianeed careful consideration.
Also to be considered are means to demonstrate efficacy in areas in which dengue activity and circulating dengue virus vary, and in a background of
other circulating flaviviruses that will complicate
measurement of the immune response in vaccinees.
Multicentric vaccine trials will be required in different geographical settings, including Asia and
the Americas. Vaccine developers and the Pediatric
Dengue Vaccine Initiative (PDVI) are establishing
such field sites.
How will vaccine efficacy be measured? Clinical
end-points need to be defined for DEN vaccine efficacy trials. This could be dengue fever, severe dengue, or another disease entity. The WHO Initiative
for Vaccine Research has set up a study group to
address this and other considerations, in order to
produce a guidance document for the planning of
efficacy trials. This will include directions for safety

assessment of dengue vaccines.
As stated above, there is currently no established
immunological correlate for protection from dengue, although there is evidence that points towards
the protective role of neutralizing antibodies. The
availability of a correlate would greatly facilitate the
evaluation of candidate vaccines, and WHO has recommended that comprehensive immunology studies to be done in conjunction with future trials for
the definition of correlates (Hombach et al., submitted). In this context, there is also need for harmonization and validation of key immunological assays, in
particular for the assessment of functional, neutralizing antibodies. WHO, in collaboration with PDVI,
is developing tools and reagents for this purpose.
PDVI is also investing in research on tools to enable the exact characterization of antibody responses,
with the aim of distinguishing neutralizing antibodies from potentially enhancing antibodies. Such a
tool would be of immense value for the safety characterization of candidate vaccines, and would complement long-term safety follow up.
In addition, immunological correlates will be of critical utility in facilitating interpretation of immune
response data in relation to protection of an immunized individual; facilitating bridging clinical
studies; enabling comparison between vaccine candidates; defining the relevant parameter to establish
vaccine potency tests; enabling comparison of the
efficacy of components of candidate tetravalent vaccines, and supporting the assessment of vaccine efficacy in different settings and population groups.
In conclusion, we are living at an exciting time with
regard to the development of a dengue vaccine, but
much has yet to be achieved before such a vaccine
will be licensed.
* PDVI: http://www.pdvi.org/default.asp
64
References
Blaney JE Jr et al. Development of a live attenuated dengue virus vaccine using reverse genetics. Viral
Immunology, 2006, 19:1032.
Edelman R et al. Phase I trial of 16 formulations
of a tetravalent live-attenuated dengue vaccine.
2003, 69(Suppl):4860. Erratum in: American Journal of
Guirakhoo F et al. Safety and efficacy of chimeric yellow fever-dengue virus tetravalent vaccine formulations in nonhuman primates. Journal of Virology, 2004,
78:47614775.
Hombach J et al. Scientific consultation on immunological
correlates of protection induced by dengue vaccines. Report
from a meeting held at in Geneva, World Health
Organization. Manuscript submitted for publication.
Markoff L et al. Derivation and characterization of a

dengue type 1 host range-restricted mutant virus that
is attenuated and highly immunogenic in monkeys.
Journal of Virology, 2002, 76:33183328.
Pengsaa K et al. Dengue virus infections in the first
two years of life and the knetics of transplacentally transferred dengue neutralizing antibodies in
Thai children. Journal of Infectious Diseases, 2006,
194:15701576.
Putnak RJ et al. An evaluation of dengue type-2 inactivated, recombinant subunit, and live-attenuated vaccine candidates in the rhesus macaque model. Vaccine,
2005, 23:44424452.
Sabin AB, Schlesinger RW. Production of immunity to dengue with virus modified by propagation in
mice. Science, 1945, 101:640642.
Huang CY et al. Dengue 2 PDK-53 virus as a chimeric carrier for tetravalent dengue vaccine development.
Journal of Virology, 2003, 77:1143611447.
65
WORKING PAPER 4.3.

Opportunities in the
development of ANTIdengue drugs
Barbara Selisko, Jean-Claude Guillemot, Karine
Alvarez, Bruno Canard
Laboratoire Architecture et Fonction des
Macromolcules Biologiques (AFMB), Centre National
de la Recherche Scientifique (CNRS), Unit Mixte de
Recherche (UMR) 6098, Marseille, France
Objectives
The aim of this paper is to review opportunities in
the development of anti-dengue drugs. First, the
timeliness and potential use of such drugs will be
addressed in the context of the growing dengue
problem, evolving diagnostic technologies, and the
difficulties of the task. Second, an analysis of current
sources of drugs will be provided in order to position future treatments for dengue in a realistic economic context. Finally, dengue virus (DENV) drug
targets will be considered, with a particular emphasis on nonstructural (NS) viral proteins. We will
list and describe state-of-the-art methods that are
expected to lead to the discovery, design, and characterization of drugs that are effective against the
four DENV serotypes.
Drug therapy for Dengue

There are three main ways to address the problem of
dengue disease control. The first and currently only
available strategy is to eradicate the mosquito (Aedes
spp.) vector. Although appropriate vector control is
effective, it may be difficult for political, geographical and logistical reasons, as evidenced by the resurgence of both the mosquito vector and DENV in
areas where both had been previously eradicated.
The second is vaccination, as exemplified by the
success of the vaccine against yellow fever virus.
The development of vaccines against dengue is an
active area of research complicated by the presence
of four DENV serotypes and the previous lack of
a suitable animal model for dengue disease. The
third way, drug therapy, is complementary to the
other approaches. There are currently no anti-DENV
drugs available.
The timeliness of anti-DENV drug discovery

Successful antiviral drug design and chemotherapy
depends on answers to several questions such as:
66
Is the disease detectable and treatable in a

timely fashion?
Are there enough patients to conduct clinical
trials?
Does a drug-mediated decrease of viral load
affect a viral disease outcome?
Are drug-discovery and drug-design technologically and scientifically feasible?
Can cost-effective drugs be made available in
afflicted countries and regions?
Today, based on our current understanding of
DENV and related viruses, and the diagnostic methods currently available and under development,
the answers to these questions argue positively in
favour of dengue drug discovery and design.
The appropriateness of antiviral chemotherapy

Several lines of research or recent developments
argue in favour of the development of drugs to treat
dengue. First, it has been demonstrated that there
is a direct correlation between high viral load and
the development of the more severe, life-threatening form of the disease. Thus, a drug able to reduce
viral load at an early stage would potentially prevent dengue fever and the life-threatening severe
form of dengue. Second, diagnostic tests to rapidly detect DENV infection at an early stage are currently under development by a number of research
organizations, and should soon be available. Third,
viral replication may be occurring in cell reservoirs,
tissues, and body compartments other than the circulatory system where an antiviral could reach and
target them. Fourth, in endemic outbreaks, prophylactic mass treatment around index cases would be
essential. Rapid diagnostics would detect infected
yet asymptomatic people. Decreasing viraemia in
humans should result in a decrease in the numbers of infected vectors and thus interruption of the
transmission chain. Therefore, an efficient and safe
drug, delivered early in the course of the disease,
might not only save lives but also curb potential epidemics. Fifth, an on-the-shelf drug would allow a
rapid response in the case of a sudden outbreak, and
should not require cold storage (unlike current prospective vaccines), a benefit for use in developing
countries. Sixth, it is also possible, at a very early
stage in drug design, to guide research on molecules
that will be cheap to produce. Costeffectiveness is
a crucial issue for this poverty-linked disease. Cost
effectiveness can be achieved by selecting appropriate, easy-to-synthesize chemical scaffolds or, once
active natural compounds are found, by pursuing
research on those that represent major chemical constituents of appropriately available plants. This last
point has been totally neglected for a number of
r easons, among which is an excessive faith in highthroughput screening (HTS) techniques and the fact
that the success of the natural-product screening
approach in cancer is not translatable to drugs to
combat infectious agents, as described later in this
paper.
Novel scientific and technological

developments promote modern drug design
Twenty years of research on the human immunodeficiency virus (HIV) and, more recently, hepatitis
C virus (HCV) have boosted the antiviral chemotherapy field.13 Viral polymerases and proteases
are targets par excellence, as validated by the use of
inhibitors of HIV reverse transcriptase and protease,
hepatitis B polymerase and herpes virus polymerase
as antiviral drugs. Anti-HCV protease inhibitors are
in various stages of clinical trials. HCV belongs to
the Hepacivirus genus within the Flaviviridae family.
Knowledge and strategies gained from the successful three-dimensional structure-based drug-design
process for HCV can now be translated to the DENV
research field.
Research on Flavivirus and Flaviviridae has led to the
characterization of an increasing number of virusencoded proteins and enzymes, including envelope
and capsid proteins, polymerases, helicases and proteases. Processes involved in the entry of DENV into
cells (virusreceptor binding, E protein conformational changes, virus internalization and membrane
fusion) are becoming better understood at the molecular level. For DENV, whose RNA genome is decorated by a type-1 cap structure, enzymes involved in
cap formation such as the RNA triphosphatase, guanylyltransferase (still unknown) and methyltransferase are additional potential targets; considerable
progress has been made in their characterization.
Chemical libraries containing molecules of natural
and synthetic origins can now be screened against
these novel pathways and targets. In this review we
intend to give an overview on the current situation
of dengue drug development.
Screening and design of anti-DENV drugs

HTS techniques allow large numbers of compounds
to be screened against a given target, on a fasterthan-ever timescale. Computer-aided studies on
structureactivity relationships facilitate a responsive and efficient management of research results
and programmes. Drug resistance must be considered as part of the drug-design process, as drugresistance mechanisms are being increasingly
characterized and drug combinations optimized, in
order to avoid or delay resistance. Perhaps the first
large-scale effort to discover anti-DENV drugs is to
be credited to researchers at the Novartis Institute of

Tropical Diseases, Singapore, who conducted a complete screen of their proprietary library against the
DENV protease domain from nonstructural protein
NS3 (see below).
The advent of drug discovery and design in

an academic environment
The availability of molecules of potential therapeutic interest has long been hampered by their proprietary origin. Pharmaceutical companies have
developed their own chemical libraries over the
years. Access to these libraries is either restricted or
difficult for academic laboratories; this represents a
major problem for research into neglected diseases,
as academic and industrial interests may not coincide. Several countries have generated libraries of
synthetic chemicals that are fully accessible to academic researchers working in drug discovery. The
National Cancer Institute in the USA has set up a
programme to freely distribute chemical libraries for
screening purposes, with an agreement on subsequent potential intellectual properties. Several similar free-access chemical libraries exist.a The existence
of such libraries renders drug discovery and design
possible for academic groups that do not have strong
collaborations with chemists, or an internal source of
compounds. In such academic environments, a network of international academic institutions would
thus control the whole chain of events and processes
required, from the discovery of a drug up to its preclinical evaluation.
New challenges from natural products can be

met in a sustainable manner
In the last 20 years, the advent of HTS techniques
has led to decreased emphasis on natural products as drug sources, with a subsequent decrease in
the discovery of new active substances. However,
the Convention on Biodiversity signed in Rio de
Janeiro, 1992,b facilitates fair, reciprocal, and legal
collaborations to use natural resources for research
purposes. The ongoing progress in structure determination of viral targets presents novel avenues for
research. One can now infer how a crude extract of
natural substances should be pre-cleaned so as to
select and bulk pre-fractionated extracts, ensuring
that their molecular diversity will be fully usable.
For example, examination of the surface potential
of a protein target discriminates the chemical characteristics of a drug target site (e.g. active site) from
secondary sites that are of no use for drug selection
Free-access chemical libraries are listed in: http://www.univorleans.fr/icoa/eposter/eccc10/monge.
The Convention on Biological Diversity: http://www.biodiv.

org/convention/default.shtml
67
(e.g. positively-charged grooves to which bind sulfated polysaccharides that non-specifically inhibit
the reaction assay). It is thus possible to exploit the
as-yet unknown, potentially great, antiviral properties of natural products.
Natural products have previously been neglected
in antiviral research because of difficulties that can
now be overcome. Many plant-derived compounds
are cytotoxic. From the antiviral point of view, cytotoxicity is not desirable and complicates screening
efforts. By jeopardizing selectivity, cytotoxicity has
stopped many compounds or extracts on their way
towards antiviral preclinical trials. It masks the putative antiviral property of a plant extract. Such an
extract, that is both cytotoxic and contains a potent
antiviral molecule, cannot be easily selected using
infected cell-based assays. This difficulty can obviously be circumvented by assays making use of isolated (or purified) viral targets.
The chemical space provided by natural products is
nearly infinite. It has been evaluated that up to 1060
molecules with the potential to be used as drugs exist
in nature,4 and could be made available, provided
that the current biodiversity (much present in tropical, dengue-afflicted countries) is not lost but wisely
used. This astonishing number is to be compared to
the current number of synthetic, pure chemical compounds, approximately 2 1010, present in laboratories around the world, most of them in proprietary
libraries unavailable to most scientists.
Finally, plants are natural and cheap factories for
the production of costly drugs. Basing the design
of an anti-DENV drug on natural active substances
has the potential to provide and disseminate knowledge, growth, and economic benefits in a more sustainable way, to countries afflicted by a specific
disease such as dengue.
Screening Federal Drug Administrationapproved drugs

We believe that a screening facility making use of
the more than 1000 Federal Drug Administrationapproved drugs should be established. These drugs
should be tested as first-line strategy, for dengue as for any other emerging virus, because one
might find a cheap and already approved drug
that is active against dengue, a discovery process that would greatly speed-up approval. If we
draw experience from the chikungunya epidemics in the Indian Ocean in 2006, a cheap antimalarial drug has shown potent anti-chikungunya virus
in vitro, and is currently ongoing clinical trials (X.
de Lamballerie, Universit de la Mditerrane,
68
Marseille, France, personal communication). The

Federal Drug Administration-approved status of
this drug against malaria has certainly greatly accelerated and facilitated clinical trials. Such an investigation has not so far been performed for dengue,
and should be a priority.
DENV targets
The NS3 protein
The protease domain
Initial work on enzymatic and structural characterization of the N-terminal serine protease
domain of NS3 (NS3pro) was carried out by
groups led by Radhakrishnan Padmanabhan
(Georgetown University, Washington, USA),
Paul Young (University of Queensland, Brisbane,
Australia), and Krishna Murthy (University of
Alabama Birmingham, USA). They defined a minimal active protease domain,5 determined the structure of NS3pro6,7 and revealed that a conserved
hydrophilic domain of 40 amino acids within NS2B
conveys full activity to NS3pro.8 They, and in parallel, the groups of Paul Young and of David Farlie
(University of Queensland, Australia) were able to
generate soluble, active single-chain NS2B/NS3
constructs allowing studies on protease/substrate
and protease/cofactor interactions conducted by
these groups9,10 and the group of Gerd Katzenmeier
(Mahidol University, Nakornpathom, Thailand.e.g.11
These studies in turn paved the way for the identification and characterization of inhibitors. Non-substrate-based inhibitors were identified that bind into
the P1 sub-pocket of the catalytic site of the dengue NS3 protease and it was found that they are of
equal potency in West Nile virus (WNV) NS3 protease.12 groups headed by Thomas Keller (Novartis
Institute for Tropical Diseases, Singapore), and
Gerd Katzenmeier explored substrate- or transitionstate-based synthetic peptide inhibitors.1315 Smallmolecule inhibitors purified from a plant were
identified16 by a group led by Noosaadah Abd
Rahman (University of Malaya, Kuala Lumpur,
Malaysia. Novartis launched HTS with 1.4 million
compounds and a lead compound was identified.c
Evaluation work with proteases from all four DENV
serotypes17 is in progress. Novartis is complementing this approach by virtual docking, speed-screen
and fragment-based screening.d The most recent
contribution to dengue anti-NS3pro drug research
is the release of the three-dimensional structures
See Dengue Digest 3-1, March 2006 (http://www.nitd.novartis.
com/includes/teasers/teaser_attaches/dengue_diguest/
Dengue%20Digest%20vol%203%20no%201.pdf)
See Dengue Digest 2-1, April 2005 (http://www.nitd.novartis.com/

downloads/dengue_digest_vol_2_no_1_april.pdf)
of the NS2b/NS3 construct of DENV2 and WNV,18

the latter in complex with a peptide inhibitor, by
the group of Ulrich Hommel (Novartis Institutes
of Biomedical Research, NIBP, Basel, Switzerland).
The Hommel group showed that DENV NS3pro in
complex with NS2B deviates substantially from the
uncomplexed structure. The cofactor is stabilizing
the NS3pro structure by contributing additional
strands, completes the substrate-binding site and is
thus involved in substrateinhibitor binding. This
was evidenced by the WNV NS2b/NS3inhibitor
complex, which explained the strong activation of
NS3pro by NS2b and revealed key interactions for
substrate recognition that accounted for structure
activity relationships for substrate-based inhibitors.
The helicase/RNA triphosphatase domain

Research on the molecular characterization of the
helicase/NTPase/RNA triphosphatase domain
(NS3hel) of DENV and WNV was initiated mainly
by groups led by Padmanabhan and by Gerd and
Gisela Wengler (Institut fr Virologie, Justus-LiebigUniversitat Giessen, Germany). The latter demonstrated the existence of NTPase (providing the
necessary energy for the unwinding helicase activity) and RNA triphosphatase (RTPase) activities in
NS3hel of WNV.19,20 Padmanabhan and colleagues
demonstrated helicase activity and defined a minimal helicase domain.21 They and our own group
showed evidence that all three activities share the
same active centre, including the characteristic
Walker A (P-loop GxGK) and B (DExH) motifs.21,22
Accordingly, all activities are strictly dependent on
magnesium ions.22
The RTPase of DENV represents a new class of
RTPases that use a helicase scaffold to exert their
activity. Additional elements for RTPase activity
might in part be provided by the C-terminal subdomain. This was evidenced by the recent determination of the three-dimensional structure of DENV
NS3hel23 by Julien Lescar and colleagues (Nanyang
Technological University, Singapore), which shows
the same features as yellow fever virus (YFV)
NS3hel24 determined in parallel by a group led by
Richard Kuhn and Janet Smith (Purdue University,
West Lafayette, USA). Flavivirus NS3hel shares the
fold of the NTP-hydrolysing molecular motor, the
catalytic core, consisting of a tandem repeat (subdomains I and II) of a RecA-like fold with HCV NS3
helicase devoid of the RTPase activity. The C-terminal
subdomain (III) is completely different. A great deal
of research is still required to elucidate the molecular mechanism of RTPase activity and the contribution of the C-terminal subdomain. A HTS-adaptable
assay of RTPase activity has not yet been described.
The helicase catalyses the unwinding of doublestranded DNA and RNA substrates with overhanging 3 and 5 ends in vitro.2123 Full-length
NS3 showed an activity that was 30 times higher
than that of the isolated NS3hel domain. A mutation study by the group of Subhash Vasudevan
(NITD, Singapore) used therefore full-length NS3
to elucidate critical residues for helicase activity.25
Interestingly, they found that the decrease of the
energy-supplying NTPase activity does not always
lead to a concomitant decrease of the helicase activity. Thus NTPase inhibitors might not necessarily
inhibit helicase activity. Additionally, they proposed
the existence of a surface pocket on subdomain II
that might act as a helix opener and thus be an
attractive target for small molecules. Consequently,
Novartis is currently using an assay for helicase
rather than NTPase to identify a lead compound by
HTS of small-molecule libraries.c Other strategies are
to try ATP-competitive or nucleic acid-competitive
inhibitors. The groups of Peter Borowski (Bernhard
Nocht Institute of Tropical Medicine, Hamburg,
Germany) and Ramachandra Hosmane (University
of Maryland, Baltimore, USA) have identified ringexpanded nucleoside analogues as inhibitors of the
helicase but not NTPase activity of WNV NS3.25
The NS5 protein

The methyltransferase domain
In 1993, sequence analysis by Eugene Koonin27
predicted the presence of a binding site for S-adenosyl-L-methionine (AdoMet), the cofactor of
AdoMet-dependent methyltransferases (MTase) in
the N-terminus of Flavivirus NS5. In 2002, our group
was able to decipher the three-dimensional structure and activity of an N-terminal methyltransferase
domain of DENV NS5.28 The DENV NS5MTase
domain acted as a (nucleoside-2-O)-MTase
(2OMTase) on small RNA GpppAC5 substrates, thus
being apparently responsible for the ribose methylation of the conserved adenosine of Flavivirus cap-1
structure (7MeGppp2OMeAGU). NS5MTase adopts the
conserved fold of a vast family of AdoMet-dependent MTases being complemented by a subdomain
that seems to be responsible to bind the cap guanosine of the RNA substrate during 2O-methylation. A
structure of the complex of DENV NS5MTase with
weak inhibitor (IC50, approximately 101mol/l])
ribavirin-triphosphate demonstrated binding to this
site, which thus represents an inhibitor-binding
site that should be explored further. Another site is
the AdoMet-binding site itself. The structure of the
complex of DENV NS5MTase with the coproduct
methyltransfer S-adenosyl-L-homocysteine28 is thus
a starting point for drug design.
69
Very recently, the group of Pei-Yong Shi (State

University of New York, Albany, USA) disclosed
that the NS5MTase domain of WNV can act as
the 2OMTase but also as the (guanine-N7)-MTase
(N7MTase) on specific RNA substrates with the
sequence of the 5 of the WNV genome.29 Thus, the
active centre of the WNV MTase seems to be fairly
flexible, being able to support the methylation of
two chemically very different acceptor groups. If
this holds true for DENV MTase, inhibitors binding
to the cofactor-binding site should be able to inhibit
the two methylation steps within DENV cap formation. They also showed that the presence of the
polymerase domain did not show any influence on
the MTase activites in vitro. Thus the MTase domain
alone can be used for HTS. Novartis is currently
setting up an HTS-adaptable assay to explore the
MTase as a target.c Within the European academic
collaborative project VIZIER on structural genomics
of RNA viruses, our group in collaboration with the
group of Alwyn Jones (Uppsala University, Uppsala,
Sweden) have been using the DENV NS5MTase in a
virtual screening process of AdoMet analogues that
led to the discovery on one active molecule as confirmed in biochemical tests (unpublished results).
The polymerase domain

The C-terminal domain of NS5 bears the primerindependent (de novo) RNA-dependent RNA
polymerase (RdRp) activity. This is a key activity
within viral replication, and DENV RdRp is thus a
prime target for drug discovery. Nevertheless, its
exploitation has been hampered by difficulties in
defining the limits of a functional RdRp domain
and producing sufficient amounts of protein for
HTS and structure studies. Concerning the characterization of the RdRp activity of recombinant fulllength DENV NS5, important contributions were
made by the group of Padmanabhan.30,31 They presented evidence for conformational changes of NS5
necessary to initiate de-novo synthesis and elongate
nascent RNA. This invites the investigator to search
for allosteric non-nucleoside inhibitors (NNIs) that
trap the enzyme in one of these conformations and
in this way inhibit RNA synthesis. There are many
examples of such inhibitors for HCV RdRp that
might be used as a base for inhibitor discovery and
design. Nevertheless, the elucidation of the threedimensional structure is a vital point. One step in
this direction was the definition and high-yield
expression of a functional RdRp domain (NS5Pol)
by our group.32 The functional comparison with fulllength NS5 showed that the presence of the MTase
domain does not significantly influence RdRp activity. DENV NS5Pol has been used in a recent study
See http://www.vizier-europe.org/
70
by the group of Andrea Gamarnik (Institut Leloir,

Buenos Aires, Argentina) who explored RNA synthesis on specific templates, especially minus strand
synthesis, and the interaction of NS5Pol with a newly
defined, essential promoter element in the 5 end of
the genomic RNA template.33 Since NS5Pol by itself
is suitable for HTS, we have adapted a radioactive
assay on homopolymeric template poly(rC)32 for the
screening of several chemical libraries (20000 compounds) accessible to academic laboratories (French
National Chemical Library, Prestwick library, NCI
diversity library) giving rise to various hits (unpublished results). Novartis has launched HTS of DENV
NS5 and identified various hits that are in the process of being evaluated.c
The structure of DENV NS5Pol has been studied
intensively, and has now been determined at 2.4
resolution by Julien Lescars group in Singapore.
It will soon be released thanks to the collaboration
between this group, Novartis, and our own group,
and thus will give a boost to DENV anti-RdRp drug
design. The structural elucidation was greatly facilitated, through molecular replacement techniques,
by recently available crystal structures of cognate
WNV RdRp domains, obtained by our group at
2.4 resolution (Malet et al., 2006, submitted, pdb
codes 2HFZ, 2HCN and 2HCS).
The replicative complex in its

cellular environment
Proteinprotein interactions within the DENV replicative complex have been proposed as potential
drug targets.34 Our knowledge on the formation and
composition of the replicative complex of DENV
and the actual interactions between the NS proteins as well as cross-modulations of their activities is still rudimentary, but progress is being made.
It was shown in a recent paper from the group of
Padmanabhan that the interaction with protein
NS5 was important for nucleoside triphosphatase/
RNA helicase and 5-RNA triphosphatase activities of NS3.35 Additionally, the group of Subash
Vasudevan showed that NS4B modulated NS3 helicase activity.36 Further insight into the structural
organization of the NS proteins and putative cellular partners within the replicative complex will
provide new possibilities in the control of DENV
replication. The induction of a number of cellular
proteins during DENV infection has been reported
by several authors.3739 A key step in the evolution
of the infection towards a disease is the down-regulation of the interferon (IFN) response. NS protein
4B and possibly 2A and 4A were identified as candidate IFN antagonists.40 Research on drug therapies
aiming at boosting the IFN response or other yet-to-
be discovered mechanisms governing innate immunity will certainly blossom in the near future.
Other targets, and inhibitors without

identified target
In addition to the NS3 or NS5 enzymes described
above, the DENV envelope protein represents an
alternate target whose crystal structure, published
by the group of Stephen Harrison41,42 revealed a ligand-binding pocket that has not yet been targeted in
structure-based studies of drug design.
A blind screening assay of drugs is based on
infected-cell or replicon assays. For example, inhibition of DENV replication by 5-Aza-7-deazaguanosine was demonstrated in an infected-cell-based
assay43 by the group of Robert Sidwell (ZymeTx Inc.,
Oklahoma City, USA). Moreover, a group from the
Pasteur Institute in Noumea (New Caledonia) has
shown that natural metabolites isolated from marine
invertebrates were potent anti-dengue agents.44 The
search for anti-flavivirus compounds is currently
also focusing on WNV. Recent reports by the groups
of Pei-Yong Shi and David Ferguson (University of
Minnesota, Minneapolis, USA) described new inhibitors of WNV RNA synthesis identified by luciferaseexpressing WNV-infection assay in HTS format,45,46
which also showed activity in a DENV replicon system. The group of Timothy Block (Drexel University,
Dylestown, USA) found that screening using a subgenomic-replicon system allowed the identification
of new compounds to combat WNV.47
Conclusion and
research priorities
The design of drugs to treat dengue is at a very exciting crossroads, not only because scientific progress
on DENV targets has been tremendous in the last
five years, but also because the dengue problem
is huge and is increasing, such that dengue might
soon join the list of diseases with economically viable drug markets. Lessons from the arboviral disease chikungunya in the Indian Ocean indicate that,
in addition to the morbidity issue, economies can
be harshly affected by sudden epidemics, and that
antivirals will certainly play a role complementary

to that of vaccines and vector control in the near
future. The problem of anti-DENV clinical trials
will probably remain a challenge in terms of patient
recruitment and follow-up, but the field of antivirals against dengue is expected to be the first of its
kind focusing on neglected diseases, paving the
way for other neglected or unexpected potentially
pandemic diseases.
We would like to propose the following re-
search priorities:
1. To continue structural elucidation, for the four
DENV serotypes, of major players in the replication complex (mainly full-length NS3 and NS5,
and their assembled complex), and envelope
proteins. Together with screening programmes,
accelerated drug design will obviously and crucially depend on these structural models.
2. To unravel the intracellular traffic and interactome of DENV proteins in the infected cell,
in order to provide novel cellular and/or viral
interactors, or to characterize antiviral innate
responses suitable as targets for drug design.
3. To support the use of natural-product chemical
libraries in drug discovery for several key reasons: sustainability, access to substances in dengue-afflicted countries, independent economic
development provided by natural substance
cultivation and exploitation, and most importantly, great chances for successfully discovering
active drugs.
4. To support the development of suitable animal
models for preclinical evaluation of drugs.
5. To support global reflection on the prequisites and settings of clinical trials for seasonal,
emerging/re-emerging, or pandemic diseases
for which it is now beyond doubt that antivirals
must be developed in the margin of the current
market- and corporate-driven initiatives.
Acknowledgments
Our work is supported by the Direction Gnrale
des Armes, the Rgion Provence-Alpes-Cte
dAzur-Corse, and the Centre National de la
Recherche Scientifique.
71
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73
WORKING PAPER 4.4.

Laboratory tests for
the diagnosis of dengue
VIRUS INFECTION
Philippe Buchy,1 Sutee Yoksan,2 Rosanna W.
Peeling,3 Elizabeth Hunsperger4
1
Institut Pasteur in Cambodia, Virology
Unit, 5 Monivong boulevard, PO Box 983,
Phnom Penh, Cambodia
2
Mahidol University, Centre for Vaccine Development,
Bangkok, Thailand
3
UNICEF/UNDP/World Bank/WHO Special
Diseases (TDR), World Health Organization,
Geneva, Switzerland
4
Centers for Disease Control and Prevention (CDC),
Dengue Branch, San Juan, Puerto Rico
1. Introduction
Dengue, a mosquito-transmitted viral disease that
produces variable symptoms, ranging from asymptomatic infection to life-threatening disease, is present
in about 110 tropical and subtropical countries. As
dengue is increasing in incidence, improved diagnosis, early detection of severe cases, and efficient
medical management are of primary importance in
all areas where dengue is endemic. Traditionally,
dengue has been diagnosed by virus isolation or
serological methods, but with recent advances in
molecular techniques and in rapid detection technology, a range of novel diagnostic tests will soon be
commercially available that will improve case management and aid disease control efforts.
The goal of this paper is to review the diagnostic
tools that are currently available or in development
and their potential role in case detection, identification of prognostic markers of severe disease, surveillance and outbreak investigations.
2. The immunological response

to infection with dengue virus
The acquired immune response to infection with
dengue virus consists of the production of IgM
and IgG antibodies primarily directed against the
virus envelope proteins. The immune response
varies depending on whether the individual has
a primary or a secondary infection (Vorndam &
Kuno, 1997). In general, serodiagnosis of dengue
is dependent on the stage of the infection. Figure 1
depicts the general time-line of a primary infection
74
from virus isolation/identification to detection of

IgM and IgG.
A primary infection with dengue is characterized by
a slow and low-titre antibody response. IgM antibody is the first immunoglobulin isotype to appear.
Anti-dengue IgG at low titre is detectable at the
end of the first week of illness, increasing slowly
thereafter. In contrast, during a secondary infection
(a dengue infection in a host that had been previously infected by a dengue virus or other flavivirus)
antibody titres rise extremely rapidly and antibody
reacts broadly with many flaviviruses (Innis et al.,
1989). High levels of IgG are detectable even in the
acute phase and they rise dramatically over the following 2 weeks. The kinetics of the IgM response
are more variable. Since IgM levels are significantly
lower in secondary dengue infections, some falsenegative results in tests for anti-dengue IgM are
observed during secondary infections. According
to the Pan American Health Organization (PAHO)
guidelines (PAHO, 1994), IgM antibody is detectable by day 5 of illness in 80% of all dengue cases, and
by day 610 of illness in 9399% of cases, and may
then remain detectable for more than 90 days. IgM
antibody-capture enzyme-linked immunosorbent
assay (MAC-ELISA) has become an important tool
in the routine diagnosis of dengue; this technique
has a sensitivity and specificity of approximately
90% and 98%, respectively, but only when used 5 or
more days after the onset of fever. Different formats,
such as capture ELISA, capture ultramicroELISA,
dot-ELISA, AuBioDOT IgM capture and dipsticks,
have been developed. Serum, blood on filter paper,
and saliva, but not urine, can be used for detection
of IgM if samples are taken within the appropriate time frame (5 days or more after the onset of
fever) (Vasquez et al., 2006). The different commercial kits available have variable sensitivity and specificity (Guzman et al., 2004; Blacksell et al., 2006).
A further challenge in the diagnosis of dengue is
the fact that anti-dengue IgM antibodies also crossreact to some extent with other flaviviruses, such as
Japanese encephalitis, St Louis encephalitis and yellow fever.
3. A review of diagnostic
methods for the detection
of dengue infection
3.1 Techniques for virus isolation and
identification
In the early stages of infection, isolation and identification of dengue virus is traditionally the only
way to diagnose a current dengue infection. In this
Figure 1. General time-line of a primary infection with dengue virus, from identification and
isolation of the virus to detection of IgM and IgG.
14 days
Years
Detection of IgG
Isolation of virus
Onset of symptoms
3 months
Detection of IgM
technique, serum from patients is applied to mosquito cell lines. After amplification of the virus in
infected cells, the serotype is identified using monoclonal antibodies specific to each dengue serotype.
This technique is only sensitive when there is a relatively high level of infectious particles in the serum.
Dengue viraemia is short, typically starting 2 or 3
days before the onset of fever and lasting until day 4
or 5 of illness. Serum is the sample of choice for routine diagnosis by virus detection, although dengue
virus can also be detected in plasma, leukocytes and
in some tissues obtained at autopsy.
The intrathoracic inoculation of mosquitoes (Ae.
aegypti, Ae. albopictus, Toxorhynchites splendens, Tx.
amboinensis) is the most sensitive system for the isolation of dengue virus, but because of the particular technical skill and special containment facilities
required for direct inoculation of mosquitoes, cell
culture is preferable for routine diagnosis. The mosquito cell line C6/36 (clone obtained from Ae. albopictus) has become the host cell of choice for routine
isolation of dengue virus, although the Ae. pseudoscutellaris cell line AP61 has also been used successfully (Singh et al., 1968; Race et al., 1979). Mammalian
cell cultures such as Vero cells, LLCMK2 and others
have also been employed, with less efficiency.
Identification of the dengue virus is generally accomplished using immunofluorescence techniques with
serotype-specific monoclonal anti-dengue antibodies on mosquito head squashes or infected cells
(Henchal et al., 1983). Some strains are not easily
identified because of a low concentration of virus.
Plaque assay is the gold standard methodology
for the quantification of dengue virus. An indirect
immunofluorescence assay was proposed by Payne
et al. (2006) as an alternative to this test. Flow cytometry has recently been reported as a useful method
for the identification of dengue virus 1 (DEN-1),
and allows the virus to be identified 10 hours earlier
than with an immunofluorescence assay, using an
anti-nonstructural glycoprotein (NS1) monoclonal
antibody (Kao et al., 2001)
3.2 Serological methods

3.2.1 MAC ELISA
Classic serological testing for dengue includes
MAC-ELISA. This assay uses dengue-specific antigens from all four serotypes (DEN 14) for the capture of anti-dengue IgM-specific antibodies in serum
samples. Most of the antigens used for this assay
are derived from the dengue virus envelope protein. The limitations of this test include the specificity of these antigens and cross-reactivity with other
circulating flaviviruses. These limitations have to be
taken into account when working in regions where
multiple flaviviruses co-circulate. IgM detection is
not useful for the determination of dengue serotypes owing to cross-reactivity of the antibody, even
during primary infections.
3.2.2 IgG ELISA

The classic IgG ELISA used for the detection of a
past infection with dengue uses the same antigens
as the MAC-ELISA. The assay is usually performed
with multiple dilutions of the sera tested to determine an end-point dilution. This assay correlates
with the haemagglutination assay used in the past.
The higher the end-point dilution, the more robust
the response obtained after the infection. In general, IgG ELISA lacks specificity within the flavivirus sero-complex groups; however, Cardosa et al.
(2002) demonstrated that the IgG response to premembrane protein is specific to individual flaviviruses. No cross-reaction was observed when sera
were tested from individuals infected with dengue
virus or Japanese encephalitis virus. An excellent
specificity of anti dengue-specific IgG was obtained
by Baretto Dos Santos et al. (2004) in an assay using
a recombinant polypeptide located in the N-terminal
portion of the envelope protein. Although the detection of specific IgG has been superseded in the diagnosis of acute infection, seroepidemiological studies
are best carried out using ELISAs to detect specific
IgG. IgG avidity ELISAs can be used to determine
whether an infection is primary or secondary, and
can be more useful than the haemagglutination inhibition test for this purpose (Matheus et al., 2005).
75
3.2.3 IgM/IgG ratio

The IgM/IgG ratio is also used to distinguish primary from secondary infections with dengue. A
dengue virus infection has been defined as primary
if the capture IgM/IgG ratio is greater than 1.2, or as
secondary if the ratio is less than 1.2. This ratio testing system has been adopted by commercial vendors such as PanBio. However, Falconar et al. (2006)
have recently shown that the ratios vary depending
on whether the patient has a serological non-classical or a classical dengue infection, and redefined the
ratios, taking into consideration the four subgroups
of classical infection with dengue. The adjusted
ratios of greater than 2.6 and less than 2.6, established by these authors, correctly classified 100% of
serologically classical dengue infections and 90% of
serologically non-classical infections.
3.2.4 Plaque reduction and neutralization test

(PRNT) and the microneutralization assay
PRNT is the most specific serological tool for the
determination of dengue antibodies (Calisher et al.,
1989) and is used to determine the infecting serotype in convalescent sera. This assay measures the
titre of neutralizing antibodies in the serum of the
infected individual and determines the level of protection the individual had against the infecting virus.
The assay is based on the principle of interaction of
virus and antibody, resulting in inactivation of virus
such that it is no longer able to infect and replicate
in cell culture. Some of the variability found in this
assay is attributable to differences in interpretation
of the results. The cell lines and virus seeds used
as well as the dilution of the sera accounts for these
differences.
The microneutralization assay is based on the same
principle as PRNT; however, instead of counting the
number of plaques per well, this assay uses a colorimetric measurement of virus-induced cell lysis to
determine the end-point dilution. This assay was
designed to use small amounts of reagents and to be
suitable for the high-throughput testing of large numbers of samples. Some of the limitations of the assay
include a poor correlation with PRNT results with
samples from people with secondary infections.
3.3 Molecular methods

3.3.1 Reverse-transcriptase polymerase chain
reaction (RT-PCR) and real-time RT-PCR
The PCR assay routinely used by some laboratories
for the identification of dengue virus is the nested
RT-PCR assay developed by Lanciotti et al. (1992).
This comprises a two-step PCR reaction involving an initial reverse transcription and amplifica-
76
tion step using universal dengue primers targeting

a region of the virus genome (C-prM) followed by
a second amplification that is serotype specific. The
products of these reactions are separated by electrophoresis on an agarose gel, and the differentsized bands observed are compared with a standard
marker for the relative molecular mass of nucleic
acids. Dengue serotypes are identified by the size of
their bands.
The real-time RT-PCR assay is a one-step assay system using primer pairs and probes that are specific
to each dengue serotype. The use of a fluorescent
probe enables the detection of the reaction products in real time without need for electrophoresis.
Many real-time RT-PCR assays have been developed either as singleplex (only detecting one serotype at a time) or multiplex (able to identify all
four serotypes from a single sample). The multiplex
assays have the advantage that a single reaction can
be used to determine all four serotypes without the
potential for introduction of contamination during
manipulation of the sample (Johnson et al., 2005b;
Chien et al., 2006). The fourplex real-time RT-PCR
assays are often less sensitive than nested RT-PCR
assay but are faster. An advantage of this assay is
the ability to determine viral load in a given sample,
which is believed to be important in determining
the severity of dengue disease (Vaughn et al., 2000).
4. Innovation in the
development of dengue
diagnostics
4.1 NS1 assays
The NS1 gene product is a glycoprotein produced by
all flaviviruses and is essential for viral replication
and viability. During viral replication, NS1 is localized to cellular organelles. The protein is secreted
by mammalian cells, but not by insect cells. The
secreted form of the protein is a hexamer composed
of dimer subunits. Glycosylation of this protein is
believed to be important for secretion. NS1 antigen
appears as early as day 1 after the onset of fever and
declines to undetectable levels after day 56. NS1 is
also a complement-fixing antigen and it produces a
very strong humoral response. Because this protein
is secreted, many studies have been dedicated to
the utility of NS1 as a tool for the diagnosis of infection with dengue virus. These studies focus on various aspects of diagnosis, including antigen-capture
enzyme-linked immunosorbent assay (ELISA), and
NS1-specific IgM and IgG responses.
In the last 6 years there have been several studies
addressing the use of NS1 antigen and anti-NS1
antibodies as a tool for the diagnosis of dengue. An

antigen-capture ELISA test was described, with sensitivities ranging from 4 to 1ng/ml (Young et al.,
2000; Libraty et al., 2002). These studies identified a
correlation between disease severity and the quantity of NS1 antigen in the serum. However, another
study did not find this correlation and in fact could
not differentiate between a primary and a secondary
infection (Alcon et al., 2002). Recently, a serotypespecific monoclonal antibody-based NS1 antigencapture ELISA that showed good serotype specificity
has been developed (Xu et al., 2006). Shu et al. (2003)
have standardized an NS1 serotype-specific IgG
indirect ELISA to differentiate primary and secondary dengue virus infections and demonstrated
a good correlation between anti-NS1 serotype-specific IgG (determined by ELISA) and PRNT results.
The NS1 serotype-specific IgG ELISA worked reliably for the serotyping of dengue virus in convalescent-phase sera from patients with primary infection
and in acute-phase sera from patients with secondary infection (which would detect the serotype that
caused the first infection), but not so with convalescent-phase sera from patients with secondary
infections. Because the results of these studies were
varied, results correlating with IgM and IgG assays
as well as disease severity and predictors of viraemia, further evaluation of this assay should be performed to determine the main differences between
each study.
Commercial kits for the detection of NS1 antigen in
serum samples are available. These assays do not
differentiate between the serotypes. As NS1 antigen
appears early in infection and before the appearance of antibody, such assays are useful for early
case detection and for outbreak investigations.
Evaluations of these assays should be performed to
assess their utility and costeffectiveness.
4.2 The DENFRAME project

The DENFRAME project, funded by the European
Economic Community in 2006, proposes to develop
novel tools for the diagnosis of dengue based on
the use of chemiluminescent optical-fibre biosensors
to detect virions, genome and anti-dengue antibodies. Dengue-specific recombinant antigen and new
monoclonal antibodies are also used to produce new
ELISA tests for the detection of anti-dengue IgM,
IgG, IgA and IgE in the serum and saliva of infected
patients, in order to increase the sensitivity and specificity of these assays and to facilitate their standardization and automation.
4.2.1 Luminescence-based optical

fibre biosensor
Luminescence-based techniques are becoming
increasingly popular owing to their high sensitivity, low background, wide dynamic range and relatively inexpensive instrumentation. Luminometry is
up to five orders of magnitude more sensitive than
absorption spectroscopy and more than 1000 times
more sensitive than fluorometry (Salama et al.,
2004). A state-of-the-art luminometer can detect as
little as 0.6pg of ATP or 0.1fg of luciferase (approximately 1100 molecules), two common luminescent
analytes (Turner et al., 1985). Compared with fluorescence, luminometry does not need an excitation
source or interference filters, luminescent analytes
do not undergo photobleaching, and remains the
technology that is most suitable for use on a DNA
microarray, or chip. Successful assays have already
been developed for monitoring of water (Polyak et
al., 2001) and diagnosis of bacterial (Leshem et al.,
2004) and viral (Konry et al., 2003) infections, thanks
to proprietary immobilization technologies (Marks
et al., 2002) and a photon-counting device based on
a photomultiplier tube.
4.2.2 Monoclonal antibody mAb4E11

This antibody is directed against the DEN-1 virus.
It binds domain III (residues 296400) of the viral
envelope glycoprotein E and neutralizes the virus
(Bedouelle et al, 2006). This monoclonal antibody
recognizes and neutralizes the four serotypes of the
virus and does not cross-react with other flaviviruses (Megret et al., 1992). The Fab and Fv fragments
of mAb4E11 and domain EDIII can be expressed in
Escherichia coli (Renard et al., 2003).
4.2.3 Natural cytotoxicity receptor immunoglobulins (NCR-Igs)

Natural cytotoxicity receptors (NCRs) are expressed
by natural killer (NK) cells. Different NCRs recognize viral haemagglutinins from different virus
families, including orthomyxoviruses, paramyxoviruses, pox viruses and flaviviruses. NCR-Igs are
recombinant molecules comprising the extracytoplasmic part of the NCR fused to the Fc portion of
human IgG1.
4.2.4 Recombinant proteins

A recombinant soluble form of E glycoprotein (sE)
of dengue virus can be expressed in Drosophila melanogaster S2 cell lines. S2 cell clones grow well in
serum-free medium at room temperature, without
the need for a carbon dioxide incubator (Muerhoff
et al., 2004). sE secretion is easily induced by addition of copper sulfate. Large amounts of purified sE
77
for each of the four serotypes of dengue virus can

be easily produced (Desprs P, personal communication). D. melanogaster S2 cell clones expressing the
domain III (EDIII) from glycoprotein E have been
developed. Purified soluble EDIII is immunoreactive
with neutralizing anti-E monoclonal antibodies.
An optical fibre immunosensor has been generated
by modifying the fibre tip with recombinant proteins or with a phage-display library of selected
epitopes/mimotopes. This methodology has been
shown to be highly sensitive, allowing the detection
of antibodies in a serum titer of as low as 1:2621440
(Marks et al., 1997). In the West Nile virus (WNV)
model, the optical-fibre immunosensor was found
recently to be at least 100 times more sensitive than
the typical ELISA methodology, and allowed the
assay time to be reduced to only 30 minutes (Marks
RS, personal communication). This assay will be
adapted for the detection of anti-dengue IgM, IgG,
IgA and IgE. Other samples (whole blood, plasma
and saliva) from patients infected with dengue will
also be tested.
A biosensor based on modified optical fibres
designed to detect the viral genome as well as an
optical fiber tip modified with monoclonal antibodies or NCR-Igs for virions capture will be built and
evaluated for the DENFRAME project.
Since the use of either monoclonal antibodies (Nawa
et al., 2001) or recombinant proteins (Cardosa et al.,
2002; Barreto Dos Santos et al., 2004; Videa et al.
2006) significantly improves the quality (sensitivity and specificity) of IgM- and IgG-specific ELISA
assays, we propose using both approaches simultaneously. The use of recombinant antigen eliminates
the problems and avoids the laborious procedures
associated with the standardization of dengue virus
antigen prepared in mouse brain or cell culture. By
using classical ELISA and MAC-ELISA approaches,
new assays for the detection of anti dengue-specific immunoglobulins (IgM, IgG, IgA, IgE) using
novel recombinant antigens and several appropriate antibodies are being developed. Serum, plasma
and saliva from infected patients will be screened in
parallel in different laboratories and results will be
compared with the gold standard diagnostic methods. Such ELISA tests can eventually be automated,
thus allowing large-scale screening during dengue
outbreaks or its use in sero-epidemiological studies.
4.2.5 Microsphere-based immunoassay (MIA)

The traditional serodiagnostic methods use the
MAC-ELISA and IgG-ELISA as principal tests.
Depending on the sample, this testing is followed
78
by a confirmatory PRNT for positive samples; however, this confirmation can only take place in laboratories with this capability. The MAC-ELISA is a
2-day test that requires about 4 hours of a technicians time. Therefore, a more rapid yet equally sensitive, single test to replace the dengue MAC-ELISA
would be of benefit.
Microsphere-based immunoassays (MIAs) are
becoming increasingly popular as a serological
option for the laboratory diagnosis of many diseases
(Kellar et al., 2001). This technology is based on the
covalent bonding of antigen or antibody to microspheres or beads. The detecting instrument is a simplified flow cytometer. The lasers simultaneously
identify the microsphere sets (bead sets) and measure the fluorescence associated with the reaction.
This methodology is particularly attractive because
it is faster than the MAC-ELISA and can be used to
identify many different antibody responses to multiple viruses. MIAs have the potential to be especially useful in arbovirus serology because tests for
infection by viruses of the same genus can share
similar formats.
At the Centers for Disease Control and Prevention
(CDC), USA, dengue-specific antigens are being
developed that could be combined with the current WNV and St Louis Encephalitis (SLE) platform
previously developed by Johnson et al. (2005a). In
this assay system, unique beads will contain covalently-bonded flavivirus-reactive antibody. The
dengue-specific antigen is allowed to bind to these
beads and the sample from the patient is then mixed
with the dengue antigen-coated beads. Using flow
cytometry, the microspheres or beads are sorted to
identify a sample as WNV, SLE or DEN14 in a single reaction.
4.3 Biosensor technology using

mass spectrometry
Rapid advances in biosensor technology using mass
spectrometry have led to the development of powerful systems that can provide rapid discrimination
of biological components in complex mixtures. The
mass spectra that are produced can be considered
to be a specific fingerprint or molecular profile of
the bacteria or virus analysed. The software system
built into the instrument identifies and quantifies
the pathogen in a given sample by comparing the
resulting mass spectra with those in a database of
infectious agents, and thus allows the rapid identification of many thousands of types of bacteria
and viruses. Additionally, these tools can recognize
a previously unidentified organism in the sample
and describe how it is related to others previously
encountered. This could be useful in determining

not only dengue serotypes, but dengue genotypes
during an outbreak. The infectious-agent identification kits can be designed to meet specific needs
and come in a 96-well format. Processing the samples involves four main steps of DNA extraction,
PCR amplification, mass spectrometry and computer analysis of results.
DNA extraction
The profile of the pathogen can be identified from
human or animal clinical samples (blood, throat
swabs, skin wipes, hair). Before amplification of
any genetic material present, a lysis/purification
protocol removes PCR inhibitors and concentrates
nucleic acids.
PCR
The sample is applied to a microtitre plate containing a pair of broad-range PCR primers. The
broad-range primers are designed to amplify the
DNA/RNA of an individual viral family or families, typically resulting in a mixture of amplicons of
about 100 base pairs in length that reflects the complexity of the original mixture of virus present in the
starting sample.
Analysis by electrospray mass spectrometry

The PCR products are desalted and electrosprayed
into a mass spectrometer, the fundamental component of the system. The mass spectrometer measures the precise weight of each nucleic acid
present. Signals, which appear as peaks, are
obtained for each of the amplified regions and for a
calibrant molecule.
Signal processing and identification

of organisms
Signal-processing software is used to process the
spectral signals to determine the mass of each of the
PCR products present with sufficient accuracy such
that the base composition of adenosines, guanosines, cytidines, and thymidines can be established.
Using the unique fingerprint represented by combined base compositions from multiple PCR reactions, it is possible to identify the organisms present
in the starting sample.
This technology has been successfully used to identify 24 bacterial contaminants in food (Mazzeo et al.,
2006). One of the major advantages of this system
is the ability to identify the pathogen in a relatively
short period of time compared with standard methods, and to develop an arbovirus-testing algorithm
that could be specifically designed for the needs of
each country. In addition, identification of the agent

responsible for an outbreak of an emerging infectious disease could be determined more rapidly than
by conventional methods.
5. Evaluation of diagnostic
tests for dengue
5.1 UNICEF/UNDP/World Bank/WHO
Special Programme for Research and
Training in Tropical Diseases (TDR)
5.1.1 Background
The mission of the Diagnostics Research and
Development unit within TDR is to promote
and facilitate the development, evaluation and
deployment of diagnostic tools for the control of
tropical diseases.
Accurate diagnostic tests have a key role in patient
management and the control of most infectious diseases. Unfortunately, in many developing countries
clinical care is often critically compromised by the
lack of regulatory controls on the quality of these
tests. As a result, in many developing countries
diagnostic tests are sold and used without evidence
of effectiveness. There is an urgent need for evaluation of commercially available diagnostic tests
for dengue.
5.1.2 Identifying priorities

TDR, in collaboration with the Pediatric Dengue
Vaccine Initiative (PDVI), convened a meeting of
experts to determine the ideal test specifications for
diagnostic tools for case management, epidemiological surveillance or vaccine efficacy trials. An inventory of antigen- and antibody-detection tests for the
diagnosis of dengue was compiled. A consensus
was reached that the highest priority was to evaluate assays for the detection of anti-dengue IgM, in
either a rapid test or ELISA format. The next priority
was the evaluation of antigen detection tests, such
as the NS1 assays, for early case detection.
The group also identified the need to establish a
bank of well-characterized specimens from different
endemic areas for facilitating test evaluations.
5.1.3 Establishing a dengue specimen bank/

evaluation network
TDR/PDVI identified a network of laboratories
in Latin America and Asia that have the capacity
and expertise to perform diagnostic evaluations as
well as participate in the collection of well-characterized specimens. A reference laboratory has been
identified in each region to which specimens from
79
participating laboratories in the region are sent and

stored.
5.2 Evaluation of dengue IgM tests
In preparation for the evaluation, standardized

evaluation protocols are developed and panel composition is determined according to the type of evaluation required. Evaluation panels are drawn from
the two regional specimen banks and are validated
by the reference laboratories. The two regional reference laboratories collaborate in the formation of
final panel that will be used for the evaluations.
The operational characteristics of the IgM tests

under evaluation are described in Table 2.
Table 1. TDR/PDVI dengue specimen bank/evaluation sites

Selected
site
WHO Region
Reference
centre
Evaluation
laboratories
South-East Asia
Americas
Dr Sutee Yoksan
Centre for Vaccine
Development, Mahidol
University, Bangkok,
Thailand
Dr Elizabeth Hunsperger
Dengue Branch, Centers
for Disease Control and
Prevention (CDC), San
Juan, Puerto Rico
Dr Vinh Chau Nguyen

Hospital for Tropical
Medicine (Cho Quan
Hospital), Ho Chi Minh
City, Viet Nam
Dr Susana Vzquez
Instituto Medicina
Tropical Pedro Kouri,
Havana, Cuba
Dr Philippe Buchy
Institut Pasteur in
Cambodia, Phnom Penh,
Cambodia
Dr Pedro Vasconcelos
Instituto Edvandro
Chagas, Belem, Brazil
Dr Shamala Devi Sekaran

Department of Medical
Microbiology, Faculty of
Medicine, University of
Malaya. Kuala Lumpur,
Malaysia
Dr Delia Enria
Instituto Nacional
Enfermedades Virales
Humanas Dr Julio I
Maiztegui Pergamino,
Argentina
5.2.1 Tests under evaluation
Table 2. Dengue IgM tests under evaluation

Description
ELISA tests
Rapid tests
Country of origin
Australia, USA, UK
Australia, Japan,
India, Republic of
Korea, USA
Antibodies
detected
IgM/IgG or IgM only
IgM/IgG or IgM only
Solid phase
Antigen adsorbed into

plastic wells sold as
8-well strips 12 set
into a plastic tray
Nitrocellulose strips
sold as dipsticks or
encased in plastic
cassettes
Specimen type
Sera
Whole blood, sera or

plasma
Number of tests, by 96- well or 192

package
1096
Antigen
Purified virus or
recombinant antigen
Purified dengue
antigen or
recombinant antigen,
four serotypes
Volume of sample
required
110 l
15 l
Supplies required
but not provided
ELISA reader, pipettes
Some may require a

micropipette
Time taken to
obtain results
14 hours
1590 minutes
Price per kit (US

dollars)
Depends on volume
of order
Depends on volume
of order
Storage (C)
28C
28C or 430C
Names of the principal investigators are given in italic type
ELISA, enzyme-linked immunosorbent assay; UK, United Kingdom; USA, United

States of America
5.1.4 Engagement with industry
5.2.2 Composition of the IgM evaluation panel
A letter was sent from TDR to manufacturers of commercially available dengue IgM tests describing the
evaluation process, to explore their interest in participating in the evaluation. Companies that indicated interest were sent a copy of the standard WHO
Confidentiality and Material Transfer Agreement
that describes in detail their obligations in providing a sufficient number of tests for the evaluation
and explains that they would be given courtesy
review of the evaluation results before publication.
Companies can visit the evaluation sites and send
queries but are not in a position to alter or block
the publication of the evaluation results. All results
will be available to WHO Member States. They will
also be posted on the TDR website and published
in peer-reviewed journals. Companies that agree to
the terms of the evaluation will be included in the
evaluation. Table 2 describes the operational characteristics of the IgM tests that are currently under
evaluation.
A panel of 350 well-characterized serum specimens

will be used for the performance evaluation. The
specimens will be assembled by the reference laboratories from archived collections from geographically diverse areas, provided by the evaluating
laboratories. These samples will be validated by the
reference laboratories and assigned panel codes.
80
The serological performance panel is intended to

offer a comprehensive assessment of the performance of existing diagnostic laboratory tests, to ensure
that such tests are specific to dengue viruses and do
not give false positive results when tested against
sera from patients infected with a related flavivirus or with other etiological agents causing acute
febrile illness. The inclusion of dengue sera at different titres is intended to determine whether the kits
being evaluated are capable of detecting sera with
medium and low titres of antibody.
To evaluate sensitivity, a total of 200 serum specimens from patients with primary and secondary
infections and by different serotypes are tested, as
described in Table 3.
For the evaluation of specificity, the panel contains
samples of sera that are negative for dengue, as
described in Table 3, including those from patients
infected with other flaviviruses, with acute febrile
illness attributable to other causes, with clinical conditions such as rheumatoid arthritis that may interfere with the assay causing false positives.
Table 3. Proposed composition of a serological panel to be
used for evaluating the performance of diagnostic tests for
dengue
No. of
samples
200
combined
Source of sera
Dengue types
Primary infection serotypes 1,

2, 3, 4
Secondary infection serotypes 1,
2, 3, 4
Primary infection high, medium,
low titre
Secondary infection high,
medium, low titre
Up to 50
Cross-reactive
flavivirus
(acute or
convalescence)
West Nile virus, yellow fever,

Japanese encephalitis, St Louis
encephalitis, tick-borne flavivirus
Up to 100
Syndromic
acute febrile
illnesses
Leptospira, malaria, scrub typhus,

mayaro, enterviruses
Interference
panel
Rheumatoid arthritis, myeloma,

hypergammaglobulinaemia,
medications (e.g. steroids)
Negatives
Negative for dengue antibodies
Up to 25
20
5.2.4 Reference standard test

The CDC ELISA and the Armed Forces Research
Institute of Medical Sciences (AFRIMS) ELISA are
the reference methods for the IgM test evaluation
and the results for all test kits will be compared to
those for these assays.
5.2.5 Summary of the evaluation scheme
Element
Antibody titre
panel components. A simple questionnaire and

tracking records for specimen management will be
developed, which should include a unique specimen number, date and site of specimen collection,
demographic and epidemiology information. After
receiving informed consent from the blood donor,
samples can be collected by venepuncture or by
plasmaphoresis. The sera should be coded in such a
way that they cannot be traced to the donor.
5.2.3 Prospective sample collection for

serum banking
Since a reasonably large amount of serum is needed
to meet the panel requirements, guidelines were
developed for the collection process. Institutional
review board (IRB) approval details were summarized as well as possible sources to locate specific
A schematic of the evaluation scheme is shown in

Figure 2. The roles and responsibilities of the reference laboratories, the network laboratories and
industry are outlined for every stage of the evaluation process.
6. Conclusions and
recommendations
To improve case management, surveillance, outbreak investigations and to ensure the success of
dengue vaccine trials, quality diagnostic tools are
essential. However, current diagnostic tools available for dengue are not practical for point-of-care
use or during the febrile phase of the disease. Many
tools are commercially available but their performance and operational characteristics have not been
widely evaluated. More novel diagnostic techniques
need to be developed for patient management. The
goal of a new diagnostic tool would combine antigen (e.g. NS1 antigen) and IgM/IgG detection in a
single test and ideally prognostic markers of disease severity would be paired with etiologic diagnosis. The recommended new tools, reference material
collection and specimen banks discussed within this
document address these needs.
81
Figure 2. Schematic for laboratory-based evaluation of dengue diagnostics

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84
CLINICAL MANAGEMENT
5.1 RESEARCH NEEDS RELATED TO DENGUE CASE MANAGEMENT IN THE HEALTH SYSTEM. . . . . . . 86
Dengue
Annex 5
WORKING PAPERS:
85
WORKING PAPER 5.1.

Research needs related to
dengue case management
in the health system
Lucy Lum,1 Nguyen Thanh Hung,2 Siripen
Kalayanarooj,3 Eric Martinez,4 Jeremy Farrar,5 Eva
Harris,6 Ivo Castelo Branco Coelho,7 Nidia Rizzo,8
Martin Weber,9 Olaf Horstick,10 Susanne Carai9
1
Department of Paediatrics, Faculty of Medicine,
University of Malaya, Kuala Lumpur, Malaysia
2
Department of Dengue Hemorrhagic Fever, Childrens
Hospital N1, Ho Chi Minh City, Viet Nam
3
Queen Sirikit National Institute of Child Health,
Rajavithi Road, Bangkok, Thailand
4
Instituto de Medicina Tropical Pedro Kouri,
Autopista Novia del Mediodia, Havana, Cuba
5
Centre for Tropical Diseases, University of Oxford, Ho
Chi Minh City, Viet Nam
6
Division of Infectious Diseases, School of Public
Health, University of California, Berkeley,
California, USA
7
Ncleo de Medicina Tropical, Universidade Federal
do Ceara, Rua Carolina Sucupira, 770/202, Aldeota,
Fortaleza, CE, CEP 60140-120, Brazil
8
Centers for Disease Control, Regional Office
for Central America and Panama, Center
for Health Studies, Universidad del Valle,
Guatemala City, Guatemala
9
Department of Child and Adolescent Health
and Development, World Health Organization,
Geneva, Switzerland
10
UNICEF-UNDP-World Bank-WHO Special
Diseases, Geneva, Switzerland
Objectives
The objectives of this paper are to summarize the
current state of dengue management, and to identify areas of clinical management needing further
improvement and research, in order to improve the
clinical outcome of dengue.
The mechanisms of pathogenesis in severe dengue
are complex and remain incompletely understood,
but it is clear that the critical abnormality that differentiates severe dengue from its mild form is the
presence of increased vascular permeability. This
phenomenon begins during the febrile phase, but
at a time when the viral load and body temperature are declining. This period, known as defervescence, is defined by an axillary temperature of
less than 38oC, and usually occurs between day 3
86
and day 5 of fever. An early sign of increased vascular permeability is haemoconcentration but, with
continued plasma leakage, pleural effusion, ascites
and depletion of the intravascular volume leading
to hypovolemic shock become apparent. It is at the
time of defervescence that the disease manifests its
severity, unlike other viral illnesses for which a clinical improvement is to be expected with a decline
in body temperature. Treatment that is focused on
the early recognition of plasma leakage and shock,
and replacement of intravascular fluids and restoration of haemodynamic stability is associated with a
good clinical outcome (WHO, 1997; Bridget, 2005).
In contrast, when the early state of shock is not diagnosed and the consequent delay in administration of
intravenous fluid therapy leads to prolonged shock,
multi-organ dysfunction and significant haemorrhage set in to complicate the clinical picture (Deen,
2000; Lum et al., 2002). Thus, in order to reduce mortality and morbidity caused by dengue, the goals of
dengue management should be:
To recognize dengue infection at an early stage;
To detect the early onset of plasma leakage in
these patients; and
To appropriately manage dehydration
and hypovolemia.
Recognizing infection
at an early stage
There are no currently accepted guidelines for the
recognition of early dengue infection. Kalayanarooj
et al.(Kalayanarooj et al., 1997) have demonstrated
that when children were recruited having had a
fever for less than 72 hours, those with dengue infection were more likely to have marked gastrointestinal symptoms of nausea, vomiting and anorexia, a
positive tourniquet test and leukopenia than those
with non-dengue viral illnesses. Additionally, children with raised levels of liver enzymes were more
likely to have severe dengue than those whose levels of liver enzymes were normal at recruitment.
The mean age of the children in this study was
68 years.
How applicable are these clinical features in distinguishing dengue from non-dengue infection in a
wider population including adults, who are increasingly bearing the burden of illness? Current laboratory confirmation of dengue is largely by dengue
IgM serology. In most patients with dengue, IgM
serology begins to become positive at the time of
defervescence, and hence is not helpful in identifying an early infection.
1. To acquire a better understanding of the clinical and laboratory features of the early stage
of infection with dengue in both children
and adults.
2. To determine the early indicators of
severe disease.
3. To validate the early warning signs and symptoms (for clinician and caregivers).
4. To identify criteria for hospital admission.
5. To identify high-risk populationsinfants,
patients with obesity, bronchial asthma, underlying diseases, adults with comorbid conditions.
6. To identify early viral markers of dengue infection that can be applied in the field
(Libraty, 2002).
7. To characterize the spectrum of dengue infections and unusual manifestations, such as myocarditis, encephalitis (Nimmannityav et al.,
1987; Lum et al., 1996; Solomon et al., 2000).
Outpatient management
of patients with dengue
Once a clinician suspects or is able to confirm that
his patient is infected with dengue, what is the
best way to provide the care that will determine a
good outcome?
Early dengue
An international study on the economic burden of
dengue has shown that about 80% of patients with
dengue in Cambodia, Malaysia and Thailand make
the first visit to a medical doctor within the first 2
days of fever (LCS Lum, personal communication).
This early contact could be provoked by generalized
body pains or marked gastrointestinal symptoms
that may cause dehydration.
Research questions:
1. To validate the early warning signs and symptoms of severe disease.
What is the best system for caring for outpatients
with dengue? A patient could be given a follow-up card that has information including the
diagnosis of suspected dengue, serial full-bloodcount results (to include, at least, haematocrit
[erythrocyte volume fraction]) and indicators for
admission, at the first medical contact. This card
would be shown to the doctor providing the subsequent care, thus providing some degree of continuity. Would this practice reduce the incidence
of patients being turned away from admission,
resulting in late presentation of shock?
2. Would this follow-up card system result

in a more effective triage system at the
emergency department?
Physicians at the emergency department who
may not be familiar with the disease presentation might be alerted to the possibility of a severe
disease and thus initiate prompt referral and
treatment.
Harris and colleagues (Harris et al., 2003) have
demonstrated that ingestion of fluids such as
water, fruit juices and lemonade in the 24 hours
before being seen by a clinician is protective
against hospitalization. What is the best fluid
that could be tolerated by patients with marked
gastrointestinal symptoms? A possible candidate is the reduced-osmolarity oral rehydration
solution (sodium, 75 mEq/l; chloride, 65 mEq/l;
potassium, 20 mEq/l; citrate, 290 mg/l; glucose
or rice powder as carbohydrate) recently recommended by WHO and the United Nations
Childrens Fund (UNICEF) (Alam, 2006). This
formulation has been demonstrated to be associated with an extremely low incidence of symptomatic hyponatremia in patients with dehydrating
diarrhoeal diseases, irrespective of their age and
the cause of diarrhoea.
3. Would this reduced-osmolarity oral rehydration
solution be tolerated by ambulatory or inpatient
dengue patients? Would it prevent dehydration
and hyponatremia?
4. Would the reduced-osmolarity oral rehydration
solution reduce the severity of shock and avoid
hyponatremia and metabolic acidosis?
Patients in the critical phase of dengue

Patients with severe dengue do present for the
first time in a busy emergency department and are
attended by a junior physician who may not be
familiar with the disease (Simoes et al., unpublished
data). In the context of the Integrated Management
of Childhood Illness, most countries in south-east
Asia have adopted guidelines to recognize severe
dengue in first-level health facilities (WHO, 2005).
Patients may be triaged based on certain parameters, such as temperature, heart rate and blood pressure. Patients in whom blood pressure is thought
to be normal and the temperature normal or below
normal, as would be the case in dengue shock syndrome, would receive the lowest priority in a triage
based on presence or absence of fever. Furthermore,
in patients in a state of shock, blood pressure measurements using the automated oscillometric method,
in which the systolic and diastolic pressures and
pulse rate are displayed digitally, have been found to
be higher than measurements with the conventional
87
sphygmomanometer. A literature search suggests

that this technology has been validated in children
and adults with haemodynamic stability (Gurdial
et al., 2004; Ni et al., 2006). There has been no study
to compare blood pressure readings taken with the
oscillometric method and those taken using the conventional sphygmomanometer in patients in different states of haemodynamic instability.
Research questions:
1. To compare the automated oscillometric blood
pressure method with the conventional sphygmomanometer in patients with severe dengue in
various states of haemodynamic instability.
2. To compare capillary refill time, quality of pulse,
cold extremities and other signs of shock with
blood pressure and pulse pressure as early signs
of shock, to monitor progress and fluid therapy.
3. What are the clinical features of shock in children
and adults?
4. Will early oral fluid therapy while waiting in the
outpatient department reduce severity of shock?
5. Will intravenous fluid therapy prevent dengue
shock and when should it be started?
6. Will the prescription of 0.9% saline solution at a
dose of 5 to 10 ml/kg body weight given intravenously during 1 hour to all patients with a history of more than 3 days of fever and any one of
the following signsrepeated vomiting, severe
abdominal pain, lethargy (regardless of the intravascular status)reduce the severity of subsequent shock, i.e. reduce the need for subsequent
fluid replacement?
7. How useful is ultrasound examination of the
chest and abdominal cavity in detecting subclinical plasma leakage? To validate this against
clinical signs of significant plasma leakage.
Gall bladder wall-thickening and or fluid perigall bladder wall as measured by ultrasound
(Srikiatkhachorn et al., 2005, and Srikiatkhachorn
et al., in press).
8. Additional laboratory markers, such as cholesterol, albumin, aspartate amino transferase, and
alanine aminotransferase, could be useful for differentiating dengue and severe dengue and for
guiding management, e.g. where baseline haematocrit [erythrocyte volume fraction] is not
known, or cases of bleeding where erythrocyte
volume fraction may not be elevated, a low albumin level may alert the physician to possibility
of dengue. Would any of the markers apart from
haematocrit alert the physician to the possibility of increased capillary permeability and hence
prompt the early initiation of fluid therapy?
Treatment of severe dengue that is entirely orientated
88
towards prompt assessment and replacement of

fluid needs is live-saving. Comprehensive guidelines for dengue case management published by
the WHO Regional Office for South-East Asia have
shown that early volume replacement of lost plasma
with isotonic salt solution can modify the severity of
disease and prevent shock.
Indications for intravenous fluid therapy for dengue haemorrhagic fever grade I and II are developed in Viet Nam for one or more of the following
signs/symptoms:
Repeated vomiting;
Acute, severe abdominal pain; rapid liver
enlargement;
Haematemesis, melaena, frank gingival bleeding,
severe epistaxis;
Lethargy;
Rapid pulse;
High degree of haemoconcentration, rapidly rising haematocrit.
Among thousands of patients treated each year in
Viet Nam, around one quarter of the patients with
dengue haemorrhagic fever grade I and II need
intravenous fluid therapy during 2448 hours in the
critical phase of the illness. Early volume replacement of lost plasma by intravenous fluid therapy in
these patients can modify the severity of disease.
Haemorrhagic manifestations in severe dengue are
to be expected but are usually minor. Severe and
clinically significant haemorrhages are however,
unusual, despite the severe thrombocytopenia and
prolonged coagulation profile. In severe dengue,
significant haemorrhage is a complication of the disease rather than an integral to it and usually accompanies prolonged shock. However, there have been
several reports of severe bleeding in patients who
did not have or had minimal evidence of plasma
leakage (Sumarmo et al., 1983; Chan, 1987; Hayes et
al., 2000; Qiu et al., 1991; Pushpa et al., 1998). The latter phenomenon has a high morbidity and mortality
and pathophysiology is still poorly understood.
There are no studies on the management of severe
bleeding in severe dengue. The administration of prophylactic transfusions of platelets or blood products
is still widely practiced. Although there is evidence
that these practices are not useful in the prevention of significant haemorrhage (Lum et al., 2003),
demonstrating their harmfulness to the patient with
dengue might further deter the use of prophylactic transfusions of platelets and fresh frozen plasma.
How useful is platelet transfusion in a patient with
severe dengue and significant haemorrhage?
Research questions:
1. Are platelet transfusions harmful for patients
with non-bleeding severe dengue?
2. How useful is platelet transfusion in a patient with
severe dengue and significant haemorrhage?
3. What is the optimal method of management of
bleeding in a patient with severe denguefresh
whole blood or packed cells, with or without
platelets, etc?
4. How can significant but occult haemorrhage be
detected? Is there a formula that suggests that
significant haemorrhage has occurred?
5. Is hormonal therapy useful in reducing the
severity of per-vaginal bleeding in menstruating
patients with dengue?
6. To identify the cause of death in denguee.g.
delayed recognition of dengue, fluid overload.
Severe dengue in adults

Previously an almost exclusively childhood disease,
severe dengue is now increasingly being observed
in adults, especially young adults, in countries
with intermediate economies, such as Malaysia,
Singapore and some parts of Thailand. While childhood dengue is well described, severe dengue in
adults is a relatively unexplored area, and nowhere
is the challenge greater for physicians than in adults
during pregnancy or with comorbid conditions such
as diabetes mellitus, hypertension, and asthma.
Training in case management

of dengue and severe dengue
Successful management of severe dengue demands
the highest clinical acumen from the physician.
Reorganizing the delivery of care to patients with
dengue may enhance the acquisition of knowledge and skills. Teams dedicated to the case management of dengue have been successfully formed
in countries such as Thailand (Kalayanarooj, 2000),
Viet Nam and, more recently, Malaysia. If reorganizing delivery of care to patients can have a positive impact on the outcome (e.g. reducing length of
stay, reducing use of blood products, more uniform
care, economic burden, morbidity and mortality),
perhaps hospital management will be motivated to
support the establishment of such a team.
Dr Suchitra Nimmannitya and Dr Siripen
Kalayanarooj (Queen Sirikit National Institute of
Child Health, Childrens Hospital, Department
of Medical Services, Ministry of Public Health,
Bangkok, Thailand) have conducted an effective
training programme on case management for

medical staff in Thailand and other countries for
many years.
In Viet Nam; the National Control Program for dengue has developed and organized a training programme on the standard case management of
dengue and severe dengue for medical staff, including physicians, nurses, medical students, and health
workers at all levels of the health-care system. The
programme also focuses on health education on
dengue for mothers/caregivers. Training of trainers
has been organized and on-site intervention teams
have been set up in provincial and referral hospitals. A dengue-management team has been set up in
each hospital. Staff of the teams can consult together
regarding the management of severe cases. They
can also discuss directly with experienced teams in
regional and central hospitals via a hotline connecting all health-care facilities by telephone, fax and email, which has been set up in order to exchange
information and experience on the case management of severe dengue. These measures have a good
impact on reducing the fatality of severe dengue in
southern Viet Nam (Hung & Lan, 2003). Such programmes should be extended to countries where
case fatality for severe dengue is high.
Evaluation of impact
of training
(translational research)
Evaluating the impact of training will require research
into costeffectiveness, better-trained nurses, capacity building, how best to deliver health care via the
system, organization of health care, implementation
research in the context of a health-care system. How
can the medical knowledge acquired in Malaysia,
Nicaragua, Thailand, and Viet Nam be transferred
to other countries with less experience in dengue
management?
Conclusion
In summary, research priorities in clinical dengue
research include studies on optimization of clinical management in the outpatient system, clinical
and laboratory indicators of early dengue infection,
plasma leakage and shock, as well as a safe method
of managing severe bleeding, dengue in pregnancy and patients with comorbidity. The impact
of existing and future training programmes should
be evaluated.
89
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reduced osmolarity oral rehydration solution. Journal
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Bridget AW et al. Comparison of three fluid solutions
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Chan VF Virological and epidemiological studies of dengue haemorrhagic fever in the Philippines.
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Deen JL. Late presentation and increased mortality
in children with dengue haemorrhagic fever. Tropical
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Gurdial SM, Balraj SH, James MW. Comparison of
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sphygmomanometer in a paediatric population. Blood
Pressure Monitoring, 2004, 9:3945.
Lum LCS et al. Risk factors for hemorrhage in

severe dengue infection. Journal of Pediatrics, 2002,
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Dengue haemorrhagic fever with unusual manifestations. Southeast Asian Journal of Tropical Medicine and
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Solomon T et al. Neurological manifestations of dengue infection. Lancet, 2000, 355:10531059.
Hung NT, Lan NT. Improvement of case managementa key factor to reduce case fatality rate of dengue hemorrhagic fever in southern Viet Nam. Dengue
Bulletin, 2003, 27:144148.
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and Towers, Washington DC, USA, 2005.
Kalayanarooj S. Capacity building in case management of dengue/DHF. Thai Pediatric Journal, 2000,
7:178179.
Srikiatkhachorn A et al. Detection of plasma leakage in dengue hemorrhagic infected patients by serial
ultrasonographic studies. In press.
Kalayanarooj S, Nimmannitya S. Clinical and laboratory presentations of dengue patients with different
serotypes. Dengue Bulletin, 2000, 24:5359.
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Indonesia. Bulletin of the World Health Organization,
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Kalayanarooj S et al. Early clinical and laboratory indicators of acute dengue illness. The Journal of
Libraty DH et al. High circulating levels of dengue
virus non-structural protein NSI early in dengue illness correlate with the development of dengue hemorrhagic fever. The Journal of Infectious Diseases, 2002,
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prevention and control, 2nd ed. Geneva, World Health
Organization, 1997.
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and Development. Dengue, dengue hemorrhagic fever
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Management of Childhood Illness. Geneva, World Health
Organization, 2005.
TRANSMISSION DYNAMICS AND VECTOR CONTROL

6.1 DENGUE TRANSMISSION DYNAMICS: ASSESSMENT AND IMPLICATIONS FOR CONTROL . . . . . . 92
6.2 CONTROL OF DENGUE VECTORS: TOOLS AND STRATEGIES . . . . . . . . . . . . . . . . . . . . . . . . . 110
6.3 INSECTICIDE RESISTANCE IN AEDES AEGYPTI. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
Dengue
Annex 6
WORKING PAPERS:
91
WORKING PAPER 6.1.

DENGUE TRANSMISSION
DYNAMICS: ASSESSMENT AND
IMPLICATIONS FOR CONTROL
Dana A. Focks1 and Roberto Barrera2
1
Infectious Disease Analysis, PO Box 12852,
Gainesville, FL 32604, USA
2
National Center for Infectious Diseases, Dengue
Branch, DVBID, 1324 Calle Caada, San Juan,
Puerto Rico 00920-3860
values are lower. This phenomenon produces a lag (temporal autocorrelation) between conditions promoting transmission and the subsequent realization in the epidemic when the
number of infections is high.
The final section, viral factors, investigates the role that virus
titre and variation in viraemic periods play in transmission
dynamics. Also covered is the often underappreciated role
that stochastic events play in the dengue system. The section
on co-circulation of multiple serotypes includes the following topics: 1) spatial and temporal variation in serotype abundance; 2) the founder or stochastic effect; 3) the influence of
herd immunity on serotype abundance; 4) the interaction of
different serotypes through the mechanism of heterologous
immunity; and 5) the potential influence of antibody-dependent enhancement on the dynamics and persistence of multiple serotypes of virus.
ABSTRACT
This paper is essentially a mathematical treatment of the
epidemiology of dengue with a view to control. The paper
begins with two important mathematical insights central to
the development of mathematical epidemiology: the mass
action principal the course of an epidemic is dependent on
the rate of contact between susceptible hosts and infectious
vectors, and threshold theory the introduction of a few infectious individuals into a community of susceptible individuals
will not give rise to an outbreak unless the density of vectors
exceeds a certain critical level. These insights lie at the heart
of two mathematical, mechanistic, and weather-driven models (CIMSiM and DENSiM) used to elucidate the non-linear
relationships influencing the dengue system. Transmission
thresholds in terms of Ae. aegypti pupae per person are discussed in the context of dengue and control. A method, the
pupal demographic survey, is described whereby the productivities of various classes of water-holding container can
be measured, permitting the development of targeted control
strategies that have estimates of endpoints in terms of transmission thresholds, e.g. Ae. aegypti pupae per person.
Next, the role of weather is discussed noting that daily, seasonal, and interannual variability in temperature, atmospheric moisture, and rainfall all influence the dengue system
in a variety of ways. Whether a particular aspect of weather
can exert a controlling influence depends on the state of the
system. Several cities are contrasted in terms of rainfall being
or not being a driver of the dynamics of Ae. aegypti and dengue. Atmospheric moisture is shown under rare conditions to
adversely influence egg and adult survival, and transmission
dynamics. Under the heading of temperature, the influence
of temperature-driven variation on the extrinsic incubation
period and gonotrophic cycle length is discussed and examples are given where these two temperature-driven variables are responsible for much of the interannual variability
in transmission. Finally, the influence of weather anomalies
associated with El Nio/Southern Oscillation (ENSO) is discussed. The section concludes with a discussion of the possible use and potential of early warning systems (EWS) for
dengue control.
The section on lags between factors favouring transmission
and cases presents examples of how increasingly high initial
values of R0 in the months preceding an epidemic can result
in substantially more infections in the subsequent epidemic
phase when conditions may have actually moderated and R0
92
INTRODUCTION
A cursory scan of the chapter titles in a text on dengue epidemiology highlights the many facets of
the dengue story. Similar to work in other vectorborne infectious systems, as the understanding of
the biology became more detailed, our reductionist
efforts have necessarily involved a growing variety
of highly specialized researchers working on particular problems ranging from molecular to cellular to the whole animal on three entities, the virus,
the insect vector, and the human. This has led, on a
larger scale, to studies on the dynamics of the dengue system as also influenced by human behaviour,
climate, and the movement of viruses and humans.
Epidemiology, the branch of medicine that investigates the causes and control of epidemics, involves,
implicitly or explicitly, all of the elements contributing to the occurrence or non-occurrence of a disease
in a population in a word, epidemiology deals
with the ecology of the disease. Given the many
aspects of dengue which interact directly or indirectly and at different temporal and spatial scales
and usually in a nonlinear fashion, it is not surprising that it is difficult to identify a single key factor
responsible for the particular dynamics of the system as a whole. Yet it is understandable that each
specialist tends to see the overall behaviour of the
disease from the perspective of his/her discipline.
The virologist sees variation in viral virulence as an
important determinant of dynamics. Some entomologists are fairly certain that the density and dynamics of the vector population are major influences,
yet others uncritically believe that any density of
the vector is sufficient for epidemics and causes
are to be sought elsewhere. Climatologists suspect
that interannual climate variability is a facet that is
underappreciated by others in understanding epidemics. Herein, then, lies the utility of mathematical
epidemiologythe building of models of infectious
diseases to integrate the interacting components of

the systemso that its behaviour and causes can be
understood. It is simply another tool, a tool potentially and historically useful in understanding the
dynamics and control of infectious disease.
Mathematical epidemiology, the application of
mathematics to the investigation of infectious disease, was probably begun by Daniel Bernoulli in
1760; he used mathematical techniques to evaluate
the effectiveness of variolation against smallpox in
an attempt to influence public policy.1 Today, mathematical epidemiology has evolved, from simply
providing quantitative tools useful in the description of incidence of infectious diseases, to statistical
models attempting to correlate incidence with various determinants, and more recently, to dynamic,
mechanistic, first principal models that serve as
tools to investigate the role of one variable against
the background of other factors, which in combination, are involved in the dynamics and control.
Two important mathematical insights were central
to the development of mathematical epidemiology.
In 1906, Hamer postulated that the course of an epidemic depended on the rate of contact between susceptible and infectious individuals;2 this notion,
the mass action principal, has become a central concept in mathematical epidemiologythe rate of
spread of an infection within a population is proportional to the product of the density of susceptible and infectious people. Ross used this principal
in his pioneering work on the dynamics of malaria
transmission.3 The insight of Hamer and Ross was
further developed by Kermack and McKendrick in
1927 into an understanding of the concept of thresholds.4 Anderson and May consider this threshold theory, coupled with the mass action principal, to be the
cornerstone upon which modern epidemiological
theory is built.1 This notion of thresholds indicates
that the introduction of a few infectious individuals into a community of susceptibles will not give
rise to an epidemic outbreak unless the density of
susceptibles (or vectors) is above a certain critical
level. Threshold theory has important control ramifications. More recent advances in the rapid growth
of mathematical epidemiology have recognized that
spatial aspects cannot be ignored and that variation
and the elements of chance are important determinants of the spread and persistence of infection.
It is unfortunate that little use of epidemiological
models has been made in empirical studies and in
the development of public health policy regarding
infectious human diseases Anderson and May
relate this directly to the abstract nature of much of
the theoretical work and the lack of ties to field data.1
They find that in view of the successes achieved
by combining empirical and theoretical work in the

physical sciences, it is surprising that many people
still question the potential usefulness of mathematical models in epidemiology.
This paper on the epidemiology of dengue differs a bit from the traditional approach of documenting epidemics and their spread by use of
insights gained from a pair of weather-driven simulation models that were developed to provide
insight into dengue dynamics and control, CIMSiM
(Container-Inhabiting Mosquito Simulation Model)
and DENSiM (Dengue Simulation Model). The
models incorporate the theoretical principals outlined above, but in a computer simulation environment that permits use of site-specific information
on human demographics, herd immunity, and the
breeding habitat of Ae. aegypti and related mosquitoes. Descriptions and validation studies of these
models have been presented earlier.5,6,7 CIMSiM is
used to integrate a host of factors pertaining to vector dynamics and provides the entomological inputs
to DENSiM. Site parameterization of CIMSiM
requires conducting a pupal and demographic survey described below. Whereas CIMSiM is essentially
an accounting program of vector dynamics, DENSiM
is the corresponding account of the dynamics of
human population and virus transmission between
hosts and vectors. Both models are weather-driven
and stochastic with a daily time step.
Specifically, the models take into account the following aspects of the dengue system: the development
rates and survival rates of Ae. aegypti eggs, larvae,
pupae, and adults are functions of temperature and
atmospheric moisture (saturation deficit); the extrinsic incubation period of the virus within the mosquito is a function of temperature and the titre of
virus within the host, the titre being a characteristic of the particular type of virus circulating; human
age structure and density are dynamic, reflecting
country-specific demographic patterns in age-specific birth, fecundity, and death rates; the type-specific immune status of individuals is maintained
with maternally-acquired antibody of newborns
reflecting the mothers immune status; age-specific
and type-specific ratios of cases to infections or of
DHF/DSS to cases, if known, can be used to model
incidence of clinical illness in addition to infection.
TRANSMISSION THRESHOLDS
We begin with a brief discussion of transmission
thresholds as they pertain to the Ae. aegypti/dengue system because they will be a useful measure in
subsequent sections; a fuller description was published earlier.8 The phenomenon of thresholds is
93
based on two concepts: the mass action principal

the course of an epidemic is dependent on the rate
of contact between susceptible hosts and infectious
vectors, and threshold theorythe introduction of a
few infectious individuals into a community of susceptibles will not give rise to an outbreak unless the
density of vectors exceeds a certain critical level. In
practice, both of these concepts require knowing the
ratio of humans to vectors in absolute numbers. In
contrast to other mosquito-borne systems such as
malaria where it is essentially impossible to quantify adult production or density, the strict preference
of Ae. aegypti for artificial containers in the domestic and peridomestic environment allows estimation
of the required ratio with a high degree of accuracy.
Before presenting estimates of dengue thresholds,
we need to look at the quantification tool, the pupal
and demographic survey, and at a definition of what
constitutes an epidemic.
some large-mouth pipettes, a white enamel pan,

and small shell vials. The inspectors request permission to examine the water-holding containers
and enquire as to the number of people living at
the house (or sleeping there the previous night).
With permission, they proceed to strain each container at the location, re-suspending the sieved contents in a small amount of clean water in the enamel
pan, from where the containers pupae are pipetted into a labelled vial. If there are other containerinhabiting species in the area besides Ae. aegypti, the
contents of each vial are transferred to small cups
covered with bridal veil secured with a rubber band;
these are held in the lab (or hotel room) until adult
emergence occurs and identification can be made.11
A key for identification of container-inhabiting mosquito pupae from South-East Asia has recently been
published.12 Data are usually summarized by container type in a spreadsheet.
Pupal and demographic survey
Definition of epidemics
Dengue control programmes today are most commonly based on the suppression of Ae. aegypti and
not on eradication. With the trend away from a strict
reliance on insecticides, current efforts largely focus
on reducing the number of larval breeding habitats.9,10 Several authors have recently made the case
that the traditional Stegomyia indices, as epidemiologic indicators of dengue transmission risk, should
be abandoned as they have a number of serious
shortcomings.11 These authors instead argue that a
pupal and demographic survey, providing an estimate of the number of pupae per person in a community by type of container, e.g. drums, flower vases,
pots, cisterns, discarded tyres, is more appropriate
for assessing risk and directing control operations.11
This method uses the ratio of pupae per person for
several reasons: 1) unlike any of the other life stages,
it is possible to actually count the absolute number
of Ae. aegypti pupae in most domestic environments;
2) container-inhabiting Stegomyia pupae are easily
and inexpensively separated from other genera and
identified to species as emerged adults or pupae; 3)
because pupal mortality is slight and well-characterized, the number of pupae is highly correlated
with the number of adults; 4) the statistic of pupae
per person can be related to transmission risk and
provide target levels of reduction required in control efforts.
A definition of an epidemic that was arbitrary but

useful from a public health point of view was used
in defining transmission thresholds: any single year
where seroprevalence rises by at least 10% was to be
considered to be an epidemic year. Ten per cent was
selected because any disease involving that proportion of the population would be considered an epidemic and this level of transmission would result
in slightly more than 1% of the population being
infected during the peak of the epidemic a minimum value that has been suggested as sufficient for
the detection of transmission.13 Just how many mosquitoes per person are required to support this level
of transmission is a function of many factors, but the
ones considered key determinants are seroprevalence of dengue antibody and temperature.8 In these
assessments, several important assumptions that
are likely to be true in most tropical locations were
made: 1) vector competence is adequate; 2) blood
feeding by Ae. aegypti occurs primarily (>90%) on
humans; 3) essentially all hosts are at risk of being
bitten. The conditions in the southeastern United
States are an obvious exception to these assumptions. The catholic feeding preferences of some of
the other Aedes dengue vectors, e.g. Aedes albopictus, would preclude using the thresholds developed
specifically for Ae. aegypti presented here.
In practice, conducting the pupal and demographic

survey involves visiting 50 or more residences, usually with a pair of inspectors equipped with nothing more than a few litres of clean water, a sieve,
*
USA Standard Sieve Series Number 30 sieve (equivalent to ASTM

designation E11, 600 m (0.0243) opening)
94
Transmission thresholds
The dengue models were used to estimate thresholds as a function of pre-existing antibody levels in
human populations, ambient air temperatures, and
size and frequency of viral introduction (table 1).8
Because the dengue system (both models and biology) is stochastic, at threshold, as defined above,
Table 1. Transmission thresholds in terms of Ae. aegypti

pupae per person as a function of ambient temperature and
prevalence of dengue antibody.
Specifically, this table contains the estimated number of Ae. aegypti pupae
per person required to result in a 50% probability of a 10% or greater rise in
seroprevalence of dengue antibody during the course of a year resulting from 12
monthly viral introductions of a single viraemic individual.8
Temperature
(oC)
Table 2. Comparison of observed numbers of Ae. aegypti

pupae per person in various dengue-endemic or denguereceptive locations with estimated transmission thresholds
based on average summertime temperatures and an initial
seroprevalence of 33%.8
Temp
(oC)a
Pupae
per
personb
Reynosa,
Mexicof
29.4
2.75
Mayaguez,
Puerto Ricof
26.6
Trinidad
(20 sites)11
Ratiod
%
Controle
0.26
10.4
90
1.73
1.05
1.7
40
27.0
22.7g
0.86
26.4
96
El Progreso,
Honduras7
29.1
0.34
0.31
1.1
10
San Juan,
Puerto Ricof
27.8
2.75
0.58
4.7
79
Bangkok,
Thailand6,20
29.2
1.69
0.29
5.8
83
Location
Transmission threshold by initial

seroprevalence of antibody
0%
33%
67%
22
7.13
10.70
23.32
24
2.20
3.47
7.11
26
1.05
1.55
3.41
28
0.42
0.61
1.27
30
0.10
0.15
0.30
32
0.06
0.09
0.16
the probability of a viral introduction leading to an

epidemic is 50%. And obviously, therefore, 50% of
introductions would lead to situations ranging from
the complete loss of virus to situations where there
was less than a 10% rise in seroprevalence. In other
words, the threshold is the break or tipping point.
Threshold levels were estimated to range between
about 0.5 and 1.5 Ae. aegypti pupae per person for
ambient air temperatures of 28oC and initial seroprevalences ranging between 0% to 67%. The size of
the viral introduction used in these studies, ranging
between 1 and 12 infectious individuals per year,
was not seen to significantly influence the magnitude of the threshold. The development of transmission thresholds has given us a new and important
tool for monitoring targets for source reduction/
control efforts. Moreover, in terms of risk assessment, transmission thresholds provide estimates of
the level of elimination or control that are necessary
to preclude transmission (table 2).
It should go without saying that we see exceeding
threshold as being a necessary but not sufficient
cause of transmission. Using a table of transmission
thresholds (table 1) takes into account the degree
of susceptibility in the human population (and can
give you an appreciation of the possible consequences of inadequately knowing herd immunity
levels), but there is obviously no statement about
the presence or type of viruses that may or may not
be circulating or introduced. As presented below,
transmission thresholds are useful for risk assessment and risk reduction. In the absence of virus
and a control programme, they speak of receptivity
to virus; in the endemic state, they provide targets
and end points for targeted source reduction/control programmes. Because for a given level of herd
Thresholdc
a
Temp refers to average temperature during the months of JuneAugust or

DecemberFebruary in locations above and below the equator, respectively.
b
Pupae per person refers to the average number of Ae. aegypti pupae per person
observed in survey.
c
Threshold refers to the estimated transmission threshold for 12 monthly
introductions, assuming an initial seroprevalence of 33%.
d
Ratio is the ratio of observed pupae per person and the estimated temperature
and seroprevalence-specific threshold.
e
% Control is the degree of reduction in pupae per person necessary to reduce
observed field level to that of the threshold.
f
Unpublished studies conducted by Focks in collaboration with others. Surveys in
Puerto Rico and Mexico were limited and preliminary.
g
Observed range: 1.463.4 pupae per person; the island-wide average is used for
calculation.11
immunity transmission thresholds are so strongly

influenced by temperature (table 1), there is the possibility of developing early warning systems for
dengue in regions such as parts of South-East Asia
where predictable El Nio/Southern Oscillation
(ENSO) events are associated with known temperature anomalies.
IMMATURE HABITAT
The primary habitat of immature Ae. aegypti in the
domestic and peridomestic environment is manmade containers. Breeding in natural containers
such as leaf axils in the domestic environment is
thought possible only in so far as adults from nearby
artificial containers can supply oviposition. For surveillance and control programmes, containers have
been classified by a number of schemes: indoors/
outdoors, essential/non-essential, presence of active
immatures, etc. However, the notion of using the
product of productivity and abundance of each type
of container has been shown to more useful from
the perspective of adult dynamics, risk assessment
and control.8,11
95
Productivity
Initial efforts based on counting
positive containers
During the initial efforts to control urban yellow
fever in South America, control specialists discovered that a substantial reduction in the number Ae.
aegypti breeding sites would often eliminate transmission. This observation became the basis of efforts,
organized in 1923 by the Rockefeller Foundation, to
eradicate yellow fever in coastal cities of northern
Brazil.14 Improved methods developed subsequently
under Fred Soper resulted, quite unexpectedly, in the
eradication of Ae. aegypti in several cities in 1933. The
goal of vector eradication arose later in Brazil, not as
a requirement for yellow fever eradication but rather
from a desire to protect Ae. aegypti-free zones from reinfestation.15 To monitor vector control progress and
to determine if prophylactic levels had been achieved,
Stegomyia indices were developed.16.17 The initial indices, described in 1923, were the House (Premises)
Index (HI) the percentage of houses infested with
larvae and/or pupae, and the Container Index (CI)
the percentage of water-holding containers infested
with active immatures; 30 years later, the Breteau
Index (BI) the number of positive containers per
100 houses became a common measure.
Inadequacy of traditional measures

Today, most dengue control efforts are based on suppression of Ae. aegypti and not eradication.18,19 The
Stegomyia indices, as epidemiologic indicators of
dengue transmission, have recently been shown to
be inadequate. The traditional indices have a number
of serious shortcomings. The CI is probably the poorest since it reflects only the proportion of containers
positive in an area and does not take into account
the number of containers per area, per house, or per
person. The HI is perhaps better, but this index fails
to give the number of positive containers per positive house or person. Of the indices, the BI is arguably the best, combining information on containers
and houses. Ostensibly these measures are, in some
undefined sense, related to risk of transmission; surprisingly, however, the statistics HI, CI, and BI do not
correlate well with one another.11 Moreover, all three
indices fail to take into account the fact that containers vary in the production of adult Ae. aegypti. For
example, two very different containers, an indoor
flower vase, commonly found with larvae but seldom producing an adult because of frequent water
changes and, say, an uncovered, outdoor 55-gallon
(207-litre) drum under a fig tree which supports a
standing crop of 10 or 20 or 50 pupae, are for the
purposes of calculating the indices, equally positive.
Field observations bear this out: Southwood et al.
96
reported, for a temple area in Bangkok, about a 23fold difference in the most and the least productive
types of container.20 A six-fold difference was seen
in Honduras.8 Connor and Monroe, in their original paper on indices, recognized these shortcomings
and, in 1923, pointed out that herd immunity was
an additional and important epidemiologic factor
not considered by the Stegomyia indices.16 We would
add an additional shortcoming the indices fail to
adequately provide information about Ae. aegypti
density on a per area or, more importantly, a per
person basis. This latter statistic, Ae. aegypti pupae
per person, can be used to estimate, for each type of
container (drum, tyre, vase, etc.), what proportion of
the transmission threshold it accounts for. Pupae per
person, through the use of the table of transmission
thresholds (table 1), permits specifying the epidemiological significance if that particular type is eliminated or controlled (table 3).
Threshold estimates in risk assessment and

targeted source control programmes
The underlying notion of targeted source reduction
is one of selectively attacking the most important
types of container. Field observations cited above
suggest the rationale is sound in that containers
vary significantly in their production of Ae. aegypti.
The actual epidemiologic significance of any particular type of container, say discarded tyres, is a function of the average standing crop of pupae found in
that type and the abundance of that container. Table
3 is an example of how transmission thresholds and
the pupal and demographic survey could provide
guidance to a targeted source reduction effort. The
estimate of the transmission threshold provides an
overall target, an upper limit to the number of pupae
per person for the environment that ensures viral
introductions would result in very little or no transmission. The survey permits estimating the contribution of each type of container and allows, using
nothing more than a spreadsheet, conducting whatif analyses of various strategies designed to selectively attack different types of containers at various
rates of elimination or control based on their epidemiologic importance and how amenable they are to
elimination and/or control.
Our example is based on surveys conducted during June 1995 in urban areas of central St. George
County in northern Trinidad.11 Based on average
temperatures for this period (27.8C) and assuming a seroprevalence rate of 33%, the estimate of
the transmission threshold is ca. 0.71 pupae per
person (interpolation of table 1). The surveys estimated human densities to be ca. 160 per hectare and
provided data on the nine major types of breeding
Table 3. An example of pupal/demographic survey results from urban sites in St. George County of Trinidad conducted during
the rainy season of 1995 and incorporating a transmission threshold estimate of 0.71 pupae per person.11
The threshold estimate is based on interpolating values in table 1 using an average
June temperature of 27.7C and an overall seroprevalence of 33%. Pupae per ha is the
product of containers per ha and pupae per container. Pupae per person is the ratio of
pupae per hectare and the average human density of 160 per ha. Portion of threshold
Containers per
hectare
Pupae per
container
Pupae per
hectare
Pupae per
person
Portion of
threshold
Relative
importance
Saucer
3.9
0.20
0.8
0.005
0.007
0.004
Tyre
0.8
1.00
0.8
0.005
0.007
0.004
Small miscellaneous
1.2
1.10
1.3
0.008
0.012
0.006
40.0
0.05
2.0
0.013
0.018
0.010
9.5
0.40
3.8
0.024
0.034
0.018
Container typea
Indoor vase
Tank
Bucket
1.1
10.90
12.0
0.075
0.106
0.057
13.5
3.80
51.3
0.321
0.452
0.245
8.3
6.70
55.6
0.348
0.490
0.266
Indoor drum
19.4
4.20
81.5
0.509
0.719
0.390
Totals
97.7
209.1
1.307
1.844
1.000
Tub
Outdoor drum
is the ratio of pupae per person and the threshold estimate. Relative importance is
the ratio of pupae per person for each container type and the total number of pupae
per person, 1.307. In the dry season, the rain-filled containers dry out and cease to
produce adult mosquitoes.a
Container names in bold indicate rain-filled containers.
c ontainer, their abundance and average standing

crop of Ae. aegypti pupae (table 3). In this environment, there was an average of ca. 98 water-filled
containers and 209 pupae per hectare; the number
of pupae per person was 1.31 or 184% of the threshold. Numerically, the two most common types were
indoor containers, the flower vase and water storage drum. Notice, however, that because these types
differed significantly in productivity, the epidemiologic significance of the indoor drum, based on contribution to the number of pupae per hectare or per
person, is some 40-times that of the vase. Dividing
the estimate of pupae per person for each type by
the threshold of 0.71 yields an estimate of what proportion of the threshold is contributed by each; this
indicates the vases contribute <2% of the threshold whereas the indoor drum accounts for >70%.
Obviously, as eradication is not in mind, targeting
the more important types based on this logic would
suggest a focus on indoor and outdoor drums and
perhaps on tubs. If table 3 is put into a spreadsheet,
evaluating various targeted strategies becomes easy.
We see that an overall reduction of ca. 50% of all
containers, the control or elimination of about 50 of
the 100 containers, would result in the number of
pupae per person being about 92% of the threshold.
We also can see that a targeted approach that eliminated about 55% of the three most important types,
the drums and the tubs, would put the population at
about 93% of threshold, and would require the control or elimination of only about 23 containers per
hectare. This approach would also take into account
specific container types for the required reduction
in pupae, given some types were uncontrollable by
virtue of their location, ownership, use, etc. Below,

we return to this site, and will look at the consequences of lack of rain during the dry period of
MarchMay.
WEATHER
Daily, seasonal, and interannual variability in temperature, atmospheric moisture, and rainfall all
influence the dengue system in a variety of ways.
Whether a particular aspect of weather can exert
a controlling influence depends on the state of
the system.
Rainfall and the immature habitat

The response to seasonal and interannual variation
in amount of rainfall is a function of the proportion
of the transmission threshold that arises from rainfilled containers. The following examples illustrate
the role of rainfall in the dynamics of vector and
transmission, and how precipitation seasonality can
interact with other parameters.
Bangkok, Thailand
In response to the emerging problem of epidemic
dengue haemorrhagic fever (DHF) in South-East
Asia in the late 1950s, the World Health Organiza
tion, at the request of the government of Thailand,
set up the Aedes Research Unit (ARU) to study the
ecology and control of Ae. aegypti. At the time it was
fairly well established that dengue viruses were
responsible for DHF and that Ae. aegypti was the epidemic vector.21 Moreover, the initial hypothesis was
97
that seasonal changes in the density of the vector

and the incidence of DHF were correlated, primarily because the disease was associated strongly
with the wet season, when rainfall would presumably increase the number of breeding sites and/or
increase adult survival.22,23 In light of these conjectured relationships, a series of year-long studies was
conducted between 1966 and 1968 on the larval habitat,23 survival, density, and dynamics of immature20
and adult populations,24 and on the gonotrophic
cycle of Ae. aegypti.25
With one exception, these studies were conducted
in the residential compound of a Buddhist temple,
the Wat Samphaya. The Wat was chosen because of
its convenient size and because the type of housing and human density were representative of much
of Bangkok. Also typical was the water supply of
standpipes and the types of water-filled containers
present, primarily large 100200-litre water-storage
jars, flower pot plates, and ant traps located under
the legs of food cupboards, tables, etc.24 Southwood
et al. and Tonn et al. reported finding that in the Wat,
as in the rest of Bangkok, Ae. aegypti was the only
mosquito breeding in the great majority of containers.20,23 Throughout the study period there were ca.
100 jars, 50 flower plates, and 50 ant traps present in
the Wat and ca. 53% of these were occupied by Ae.
aegypti; the number of water-filled containers and
the proportion with mosquitoes were remarkably
constant, and with the exception of a portion of the
ant traps, all containers were filled manually and
not influenced by rainfall.20,23 The initial conclusion
from the larval studies was that the key factor(s)
responsible for the seasonality of DHF in Bangkok
was not fluctuation in adult production and density
in response to rainfall. The models demonstrate this
as well: in 20-year runs, there is only a slight, and
not statistically significant, variation in the number
of Ae. aegypti females per person and this and its
lags going back several months do not correlate with
cases. The initial field results led to the next hypothesis, that seasonality in transmission was due to seasonality in adult survival due to temperature and/or
atmospheric dryness associated with the hot season.
We will come back to the Bangkok story when the
role of these variables is discussed.
South-western Puerto Rico

In contrast with Bangkok, longitudinal studies in
southwestern Puerto Rico show a positive correlation between rainfall and vector abundance, with the
correlation being strongest in the dry, south coastal
portions of the island.26 Also in marked contrast with
Bangkok is the fact that most breeding occurs outdoors and in rain-filled containers, the primary ones
98
being animal watering dishes and discarded tyres.

Moore describes the relationship between rainfall,
vector abundance, and transmission as follows:
At least in southern Puerto Rico, Ae. aegypti densities rise quickly with the onset of rains in July and
August. This relationship further leads to a rather
close correspondence between seasonal rainfall and
dengue fever incidence, the peak of which occurs
about six or eight weeks after the peak in rainfall. In
1973 the rains began in June, and dengue therefore
also appeared earlier than usual. In 1974, however,
when the rains began later than usual, the peak dengue fever incidence did not occur until December
1974 and January 1975.26 The authors concluded
under their conditions of principally outdoor breeding that rainfall patterns seem to be a reasonably
effective predictor of time of peak dengue transmission. That is not to say that they understood only
rainfall to be necessary, as they certainly appreciated
the need for viruses and susceptible human populations. So here we have an example, especially
in the south coastal areas, of where rainfall is the
key factor influencing temporal and spatial patterns
of transmission.
St. George County of Trinidad

To put the influence of seasonal variation in rainfall
into a more quantitative context, that of pupae per
person, contrast the total number of pupae per person in table 3 (wet season) with that of table 4 representing the dry season. Note that the rain-filled
containers (tyres, small miscellaneous, and outdoor drums) cease to support breeding and the total
number of pupae per person falls from 1.31 to 0.95;
in the uncontrolled situation, the respective proportions of the transmission threshold are 1.84 and 1.33
respectively. Note also that in the dry season when
the populations are a bit lower, only a single class of
container, either the tub or the indoor drum, would
have to be controlled or eliminated to bring the community below threshold.
Atmospheric moisture
The drying power of the atmosphere, as measured in
saturation deficit (mBars pressure), reflects the combined influence of temperature and relative humidity. The dengue system (and models) are influenced
by saturation deficit in several ways. In CIMSiM,
atmospheric moisture influences evaporation rates
from containers along with certain characteristics of
the container, including their size, shape, and exposure to direct sunlight. Also, deficits greater than ca.
10 mBars progressively reduce survival of newlylaid eggs and adults. The impact on egg survival
under very dry conditions is minimal and, with the
possible rare exception of breeding in exposed lime
Table 4. Pupal/demographic survey results from urban sites in St. George County of Trinidad, conducted during the dry season
(MarchMay) and incorporating a transmission threshold estimate of 0.71 pupae per person.11
Container typea
Saucer
Tyre
Small miscellaneous
Indoor vase
3.9
40.0
Pupae per
container
0.20
0.05
Pupae per
hectare
0.8
-
Pupae per
person
Portion of
threshold
0.005
0.007
Relative
importance
0.005
-
2.0
0.013
0.018
0.013
Tank
9.5
0.40
3.8
0.024
0.033
0.025
Bucket
1.1
10.90
12.0
0.075
0.106
0.079
13.5
3.80
51.3
0.321
0.452
0.339
Tub
Outdoor drum
Containers per
hectare
Indoor drum
19.4
4.20
81.5
0.509
0.717
0.538
Totals
87.4
151.4
0.946
1.332
1.000
Container names in bold indicate rain-filled containers.
rock solution holes adjacent to beaches, is easily compensated by subsequent density-dependent larval
survival. Based on CIMSiM, only at particularly hot
and dry continental locations such as Ouahigouya,
Burkina Faso, are conditions such that adult survival
is reduced by excessive temperatures and high saturation deficits. Here, the dynamics and abundance
of adults and immatures would not be materially
different under milder conditions, again, primarily
due to resilience in the entomological system from
density-dependent larval survival. However, the
shortened adult lifespan significantly reduces transmission in simulation studies (not shown).
Temperature
Vector dynamics
In temperate locations, Ae. aegypti overwinters in
the immature stages, and seasonal variation in
adult abundance clearly reflects the key role of temperature on the development of immature stages.
However, under tropical conditions, adult abundance varies not with temperature but with variation
in the abundance and productivity of water-holding
containers; container productivity is limited, not by
temperature or oviposition, but by density-dependent larval survival which is ultimately driven by the
amount of food falling into or formed photosynthetically within the container. This is consistent with
both CIMSiMs rather constant estimates of adult
abundance from manually-filled containers under
conditions of constant temperatures of 22C to
32C (not shown). In light of this, going back to the
Bangkok story of the investigation into the cause(s)
of seasonal variation in incidence of dengue, we
should not be surprised to read that the field work
of Sheppard et al. indicated no seasonal trends in
adult survival.24
The influence of temperature-driven variation

on the extrinsic incubation period and gonotrophic cycle length
Under moist, tropical field conditions, for example
Bangkok, where the major mortality sources are accidents such as encountering a spiders web, the probability of surviving a single day (Sa) is constant and
independent of temperature. Experiments to measure this parameter in the field are notoriously noisy
but a consensus value is somewhere between 0.87
and 0.91 per day under conditions without temperature or moisture deficit extremes such as for most
locations in dengue-endemic regions, e.g. SouthEast Asia.5 The integral of Sat provides the average lifespan of the female; for Sa = 0.89, the average
lifespan is ca. 8.6 days. Keeping in mind that the
resulting age distribution declines exponentially
with age, it is easy to see that numerically, while
most emerging females die at an early age, the tail
of this age distribution contains the rather rare but
older individuals with the potential to transmit.*
The length of time required for a newly infected
female to become infectious, the extrinsic incubation period (EIP), is a non-linear function of temperature; the same can be said regarding the length
of the gonotrophic cycle (table 5). Notice that if a
female takes an infectious bite on her first day of
life, the length of time required for her to have a
disseminated infection is EIP plus one daya substantial portion of the average lifespan, so most will
not pass on virus before death. Moreover, once disseminated, the probability of transmitting virus will
vary with how often the mosquito bites, which is
related to the duration of gonotrophic development.
* t=
Sattd
t=1
99
Figure 1 presents an estimate of the average number

of potentially infectious replete feeds per newlyemerged female as a function of temperature and
daily survival probability. This figure makes, for the
purpose of comparisons, the unrealistic assumption
that all mosquitoes take an infectious blood at one
day of age. The actual number of potentially infectious bites per replete feed is unknown and may be
as high 2 or 3 or more interrupted feeding attempts
with resumption on the same or different host.7
From an epidemiological perspective, it is important to realize that a temperature-related doubling
of expected number of potentially-replete feeds,
the consequence of 2 or 3 degrees warmer temperatures, is equivalent to a doubling of the density of
Ae. aegypti. While temperature plays a role in most
facets of transmission dynamics, its influence on
the speed of viral dissemination and frequency of
biting is a key regulating force entraining seasonal
variability. And indeed, the field work of Pant et al.
allowed them to conclude that the source of the seasonality seen in dengue in Bangkok was due not to
rainfall variability leading to adult abundance seasonality, nor to excessively high temperatures and/
or dryness leading to reduced adult survival, but
to temperature-related variability in the infectiousness of Ae. aegypti females through the agency of
EIP and gonotrophic development rates.25 This is the
same conclusion reached through an analysis of the
Bangkok situation made with CIMSiM/DENSiM.6
The influence of interannual

climate variation
The El Nio Southern Oscillation (ENSO) is an
atmosphereocean coupled system which produces
quasi-periodic short-term climate and sea surface
temperature changes over the Pacific region with
impacts on weather worldwide including many
countries in the Americas, Africa, and Asia. The system oscillates between two extremes known as El
Nio and La Nia, which are associated with approximately opposite disturbances to climate worldwide. A chief phenomenon of an El Nio phase is
eastward extension of warm surface waters situated
off northwestern Australia towards the west coast of
equatorial South America. During the cool phase, La
Nia, equatorial westerlies result in an upwelling of
cold abyssal water which is transported to the west
creating a tongue of abnormally cool surface waters
extending towards Indonesia. Because convection
rainfall in this region is limited to sea surface temperatures greater than ca. 2627C, the spatial distribution of rainfall is associated with equatorial
sea surface temperature anomalies associated with
ENSO state. The Southern Oscillation refers to the
oscillation of atmospheric pressures between the
100
Table 5. Lengths and daily rates of the extrinsic incubation

period of virus within Ae. aegypti and the gonotrophic
development cycle.5
Temperature
Extrinsic
incubation period
Gonotrophic cycle
Rate per daya
Days
Rate per daya
22
0.04
24.0
0.14
7.3
24
0.05
20.0
0.17
6.1
26
0.07
14.0
0.24
4.2
28
0.08
11.8
0.29
3.5
30
0.10
9.9
0.34
2.9
32
0.12
8.4
0.41
2.4
Days
T he daily rate is that proportion of total development occurring on a particular

day at the specified temperature
eastern and western Pacific. One of the summary

or indicator statistics of ENSO state is the southern
oscillation index (SOI), the normalized difference in
pressure between Darwin and Tahiti. El Nio and
La Nia are associated with negative and positive
values of the SOI respectively. It is not surprising
that much innerannual variability in climate in the
central Pacific is attributable to the state and intensity of ENSO.
Because many infectious disease systems are influenced by weather, ENSO state and associated
anomalies in rainfall, atmospheric moisture, and
temperature have become topics of considerable
interest. An excellent review of ENSO and health
has been provided by Menno Bouma and others.27 Largely through the use of numerical simulation models of the ocean and atmosphere, forecasts
of anticipated ENSO state are increasingly skilful,
such that it is reasonable to expect that useful early
warning systems (EWSs) will be developed.28 Initial
efforts involved nothing more that simple attempts
to demonstrate correlations between ENSO state
and outbreaks. More sophisticated development
of ENSO-based EWSs will require addressing two
related problems. The first of these is that, while skill
in forecasting an ENSO event is currently adequate,
predicting the strength of the oscillation is problematic and leads to lack of skill in predicting regional
weather anomalies. The second area in need of much
attention is elucidation of the mechanisms whereby
weather anomalies lead to anomalies in the disease
system.29 Several recent studies have shown temporal correlations between malaria epidemics and various indices of ENSO state.30,31,32
Given that dengue incidence is a function of interaction of the many factors outlined above, it is not
surprising that dengue activity has been correlated
Figure 1. Average number of potentially infectious replete feeds per newly-emerged female as a function of temperature and
daily survival probability.
This figure makes, for the purpose of comparisons, the unrealistic assumption that all mosquitoes take an infectious blood meal at one day of age. The actual number of
potentially infectious bites per replete feed is unknown and may be as high 2 or 3 or more interrupted feeding attempts with resumption on the same or different host.7
1.8
1.6
1.4
0.91
1.2
0.89
1.0
0.87
0.8
0.6
0.4
0.2
0
22
24
26
28
30
32
Temperature (C)
with ENSO state or one of its statistics, SOI, in

regions (most clearly in the South Pacific) where
ENSO or SOI is correlated with temperature and/
or rainfall anomalies.28,33 Unfortunately this study
does not identify the environmental risk factors
unequivocally.
At a recent World Health Organization dengue
workshop in the South-East Asian region, directors
of national anti-dengue programmes in Thailand,
Vietnam, and Indonesia expressed the operational
need for an early warning system (EWS) that would
provide sufficient lead time (one to three months)
to permit mobilization of control operations. In
response, Focks et al. have attempted to develop practical EWSs for Yogyakarta, on the island of Java in
Indonesia, and Bangkok, Thailand, based on logistic
regression.34 The predictor variables are sea surface
temperature (SST) anomalies (a five-month running
mean of spatially averaged SST anomalies over the
tropical Pacific: 4S4N, 150W90W as measured
by the Japanese Meteorological Association) and
past monthly cases of dengue in each city. Previous
incidence of anomalously high or low cases is an
indication of interaction between the types of virus
currently circulating and the nature of the immune
status of the human population. SST anomalies are
highly correlated with subsequent surface air temperature anomalies and may be correlated with
atmospheric moisture as well. The predicted variable
is the probability of an epidemic year forecast one to
three months before peak transmission season. The
skill level for three-month predictions for Bangkok
were inadequate for an operational system (6 errors
in 35 years); the two and one-month forecasts had
error rates of 3 and 2 per 35 years, respectively. The
Java EWS, however, was sufficiently skilful to be
put into use in Yogyakarta, Indonesia; one, two and
three-month forecasts were without error for the 14year period of record. Note that this system does not
use ENSO state directly, but rather, one of its indicators, sea surface temperature anomalies.
A recent National Research Council report on the
subject of early warning systems is cautiously
optimistic but concludes that substantially more
research is needed to understand the relationships
between climate, human behaviour, and infectious
diseases. The report states that one goal of such
research should be to support a transition from
the current practice of surveillance and response
towards a more proactive prediction and prevention strategy.29
101
LAGS BETWEEN FACTORS

FAVOURING TRANSMISSION
AND CASES
Dengue epidemics obviously involve one persons
infection leading to anothers; the number of infections resulting in the next cycle from a single individual is commonly referred to as R0. As long as R0 is
greater than one, the epidemic grows exponentially
at a rate proportional to this ratio. The magnitude of
R0 is dynamic, reflecting integration of the host of
factors influencing dengue dynamics. Higher temperatures shorten EIP and the gonotrophic cycle and
are thus a factor tending to increase R0, as would
rainfall lead to more Ae. aegypti in the case of southwestern Puerto Rico. High levels of herd immunity, effective spraying that shortens adult survival,
or window screens limiting host access would
favour reductions in R0. Dynamically accounting
for the influences of the various factors through
time is a chief activity of accounting software such
as CIMSiM/DENSiM. Table 6 and figure 2 present
three examples of how increasingly high initial values of R0 in the months preceding an epidemic can
result in substantially more infections in the subsequent epidemic phase when conditions may have
actually moderated and R0 values are lower. This
produces a lag between conditions promoting transmission and the subsequent realization of the epidemic when the number of infections is high.
Table 7 provides correlation coefficients between
monthly cases and lagged monthly cases, average temperature, rainfall, length of gonotrophic
cycle, and EIP for dengue in Bangkok, Thailand,
19661994. It is not surprising that cases are highly
auto-correlated going back at least four months;
anomalously high (or low) prevalence this month
reflects unusually large (or small) prevalence last
month through the agency of Ae. aegypti giving rise
to subsequent cases. The lack of substantive correlation between current cases and current weather,
temperature and rainfall and their lags going back
one, two or three months may come as a bit of a surprise to people who are not acquainted with dengue
data. This phenomenon reflects the fact that epidemics take several months to develop to a level where
they are recognized to be a result of antecedent conditions as described above.13,35 To be truthful, there
are some non-trivial correlations between cases and
the preceding two months rainfall, suggesting that,
in contrast to the Wat Samphaya temple area, not
all containers are manually-filled in the metropolitan area.20 With regard to collaborating the story
of antecedent conditions being key determinants of
epidemics, the peaks in correlations between cases
and temperature, gonotrophic cycle length, and EIP
102
(as estimated with CIMSiM and DENSiM using historical weather data) three and four months earlier,
are important. Epidemics, under these conditions
of constantly endemic virus, are entrained by environmental determinants at play months before the
health community is aware that a nascent epidemic
is building. And, epidemics can and do occur under
weather conditions less than optimal for intense
transmission.
VIRAL FACTORS
Virus titre and variation in viraemic periods
The size of the virus inoculum, that is, the product
of viral titre and quantity of blood, influences the
probability of the vector subsequently developing
a disseminated infection with virus in the salivary
glands.7 It has been suggested, and there is some
evidence to support the notion, that the titre of virus
in the blood meal alone could influence the probability of subsequent infection.36,37,38 Moreover, duration of the dissemination period, EIP, can vary with
titre. Watts et al. reported that the EIP for dengue in
Ae. aegypti at 30C was 12 and 25 days for mosquitoes infected with high and low doses respectively.39
Provision has been made in DENSiM to allow evaluation of the consequences of these relationships.
Simulation studies regarding the nature of epidemic and endemic transmission

Studies comparing the consequences of viral titre
on the dynamics of endemic dengue suggest that
titre, through the agency of probability of dissemination and EIP, does indeed play a role. In studies
by Focks et al,7 the titres evaluated were 105 (low)
and 106 (high) median infective dose (MID50), the
human population was low, and the number of Ae.
aegypti pupae per person was ca. 150% of threshold.
Using 105 MID virus, the initial virgin soil epidemic
was acute leaving only 20% of the population uninfected. For the next five or six years after the initial epidemic, additional introductions resulted in
few locally-contracted infections due to herd immunity and the relatively low abundance of vector.
As the immune population aged, the younger age
classes progressively became more susceptible and,
as a consequence, most of the subsequent infections
occurred primarily in these classes. If this scenario
is run for decades, the age-specific distribution of
seroprevalence settles down to one of rising prevalence with age, with only small epidemics involving
at most a few hundred (primarily young) individuals, and with the overall prevalence of antibody
averaging ca. 70%. If this scenario is run again with
the titre of the introduced virus increased from 105
to 106 MID50, the initial epidemic is more acute and
Table 6 and Figure 2. Projected numbers of infections over time as a function of Ro.
the magnitude of the event. Note in each example that the ratio of new infections
in each cycle after day 81 is the same, 1.5, but the absolute numbers of infections
after additional cycles in the epidemic phase is larger as a function of the number of
infected in the pre-epidemic period. This then is a mechanism whereby environmental
conditions that promote increased intensity of transmission but before there are
large numbers of infections can become manifest months later as an epidemic under
conditions that are less conducive to transmission.
For illustration, we assume the periods of time between the onset of viraemia in the
first and subsequent infection cycles are multiples of 17 days. In each example, the
epidemic is initiated with a single viraemic individual. During the first four cycles,
the pre-epidemic period (up to day 81), Ro is set to a constant value of 1.5, 2.0,
or 2.5 for lines labelled infections (1.5, 1.5), infections (2.0, 1.5), and infections
(2.5, 1.5), respectively. For cycles 510, Ro is set to a constant 1.5 in each case.
The purpose of this illustration is to demonstrate that conditions several months
before the appearance of a large number of cases (the epidemic) significantly affect
Example 1
Cycle
Days
Months
Ro
Example 2
Infections
(1.5, 1.5)
Ro
Infections
(2.0, 1.5)
Example 3
Ro
Infections
(2.5, 1.5)
0.03
1.5
2.0
2.5
17
0.56
1.5
2.0
2.5
33
1.08
1.5
2.0
2.5
19
49
1.61
1.5
2.0
16
2.5
47
65
2.14
1.5
2.0
32
2.5
117
81
2.66
1.5
1.5
48
1.5
176
97
3.19
1.5
11
1.5
72
1.5
264
113
3.72
1.5
17
1.5
108
1.5
396
129
4.24
1.5
26
1.5
162
1.5
593
10
145
4.77
1.5
38
1.5
243
1.5
890

*OGFDUJPOT

$BTFT
$BTFT

$BTFT

%BZT
Table 7. Correlations between monthly cases and lagged cases, monthly average temperature (C), rainfall, length of
gonotrophic cycle (Gono) and extrinsic incubation period (EIP) for dengue in Bangkok, Thailand, from 19661994.
The length of the gonotrophic cycle and EIP were estimated using CIMSiM and DENSiM with historical weather data from metropolitan Bangkok.
Correlations greater than 0.30 are highlighted.
Correlations between current and past monthly averages

Lag (months)
Gonotrophic
cycle
Cases
Temperature
Rain
EIP
1.00
0.07
0.22
-0.06
-0.09
0.91
0.18
0.28
-0.16
-0.20
0.74
0.29
0.24
-0.25
-0.29
0.57
0.37
0.09
-0.32
-0.36
0.41
0.37
-0.04
-0.31
-0.35
0.27
0.26
-0.14
-0.21
-0.25
0.17
0.08
-0.22
-0.05
-0.07
103
shorter in duration and involves ca. 95% of the population. The nature of transmission following the
primary is different as well, with transmission being
more intense, the small ensuing epidemics sporadic
and more frequent, and with fewer people involved
but producing higher levels of immunity than those
associated with the lower-titre virus.
Simulation studies evaluating combinations of titres
and viraemic periods clearly indicate that combinations favouring transmission, e.g. higher titres and
longer periods, lead to more acute initial epidemics
followed by more frequent smaller epidemics that
ultimately involve a larger portion of the population
and higher seroprevalences.
Simulation studies on the role of

stochastic events
The dengue models have been used to estimate the
probability of an epidemic following a single introduction.7 Obviously, any number of factors combine
to determine the fate of introduced virus temperature, herd immunity, virus and vector characteristics, to name a few. However, under conditions near
transmission threshold, the outcome of an introduction is highly unpredictable for stochastic reasons.
An interesting question could be: how receptive is
a small village to a single introduction occurring at
various times of the year? Would this be modified
by titre of the virus, given the influence of titre on
the probability of infection and EIP in the mosquito?
Less ambitiously, we could frame the questions in
terms of parameter sensitivity if conditions are
near threshold, would factors such as seasonality in
mosquito abundance, size, and temperature be sufficiently influential against the backdrop of other
factors to significantly alter the probability of an epidemic, and would we expect this to be substantially
modified by the titre of introduced virus?
Simulation results for the eastern coastal region of
Honduras indicate that, at a low titre (105 MID50),
seasonal changes in weather result in an almost
three-fold difference in probability of an epidemic
resulting from a single introduction (30%35% in
December and January vs. 80% in AprilMay). That
is to say, a wintertime introduction is about onethird as likely to cause an epidemic as one occurring
in the spring or summer. The results, while suggesting that many introductions into a naive population would be lost and not produce an epidemic,
also indicate that a single introduction is capable
of producing an epidemic at any time of the year.
Simulations also indicate that introductions of hightitre virus more frequently lead to epidemics than
introductions of the lower-titre type. Associated
104
with the higher-titre virus is a reduction in magnitude of the role played by seasonal influences
summer introductions are only ca. 1.5 times more
likely to cause an epidemic than wintertime introductions of the same virus. The difference between
the ability of the two viruses to cause an epidemic is
most pronounced during the cooler months when
the high-titre virus is about two times more likely to
result in an epidemic than introduction of the lowertitre type. These results are typical of others (unpublished), where different factors or combinations of
factors become key regulatory factors under different conditions of weather, antibody presence, demographics, and mosquito characteristics.
CO-CIRCULATION OF
MULTIPLE SEROTYPES
The current pandemic of dengue and DHF/DSS
originated in the Pacific and South-East Asia in the
1940s, and has subsequently spread to the Americas
and Africa. Today, most urban centres of SouthEast Asia and many in Central and South America
are hyperendemic for dengue, frequently with all
four serotypes circulating simultaneously.40 Given
the significance of sequential infections in developing serious illness via antibody-dependent enhancement, factors regulating or influencing the spatial
and temporal distributions of dengue serotypes
may be important in regulating or influencing the
age-specific dynamics of infection and illness.41
An example of spatial and temporal variation

in serotype abundance
Figures 3 and 4 are presented as examples of serotype variability on a country scale; the figures are
based on ca. 1200 virus isolations from human sera
in North and South Viet Nam between 1990 and
1999.42 The data provide an indication of the relative
frequency and dynamics of dengue serotypes in circulation over a ten-year period. These estimates are
not necessarily unbiased or highly correlated with
the real picture, considering the possible differences
in virulence among serotypes and low number (<30
40) of isolations in several years. Keeping in mind
the nature of the data, and that dengue activity is
usually confined to roughly JuneNovember in the
North and is continuous but seasonal in the South,43
what can be said regarding some of the possible factors influencing the dynamics of serotypes spatially
and temporally?
Founder or stochastic effect

In some regions, the mix of serotypes found each
year simply reflects the mix in other endemic areas.
It is likely that dengue virus is lost during the cool

season in the North and is annually reintroduced
from more southerly locations each year;43 hence it
is not unexpected that the mix of serotypes circulating in the North bares some correspondence to
those in the South. A similar situation is reported for
small relatively isolated Pacific islands that are too
small to remain endemic; when virus does arrive,
the introduced strain was usually active earlier on
other larger islands.33
The influence of herd immunity on

serotype abundance
Another factor influencing the distribution of serotypes is the nature of herd immunity. During the
course of an epidemic not influenced by control
efforts or cooling temperatures, Ro ultimately falls
to less than one as a function of the rising proportion of immunes increasingly, potentially infectious bites fall on refractory individuals and the
epidemic dies out. The notion of herd immunity,
Figure 3. Circulation of dengue serotypes in North Viet Nam between 1990 and 1999 based on virus isolation from
febrile patients.42

%&/
%&/
%&/
%&/

Figure 4. Circulation of dengue serotypes in South Viet Nam between 1990 and 1999 based on virus isolation from
febrile patients.42

%&/
%&/
%&/
%&/

105
Figure 5. Output from DENSiM for a hypothetical site where dengue is endemic (see text for explanation).
The solid line is overall prevalence and mirrors the different age classes closely except for those of 14 and 59 years.

0WFSBMM

1FSDFOU

the proportion of individuals immune to a particular serotype of virus, therefore is a useful concept.
In acute, virgin soil epidemics, such as the DEN-1
outbreak in Cuba of 19771979 where some 44.5%
of the urban population experienced infection in a
single year, the level of herd immunity was roughly
identical with the prevalence of antibody in each
age class.44 However, in endemic areas where the
norm is ongoing circulation of multiple serotypes,
there is a general trend of increasing seropositivity
with age. As a result, not only is the nature of illness
and the age-specific distribution of serious illness
a function of current and previous dengue activity,
but the dynamics in abundance of dengue serotypes
is a function of previous dengue activity through the
proxy of herd immunity. Here past activity (or lack
of it) can influence the innate Ro of the same serotype through the agency of herd immunity.
Figure 5 is a plot of output from DENSiM for a
hypothetical site where dengue has historically
been endemic and seroprevalence to all four serotypes is high. During the 20 years displayed, virus
was not introduced from the outside but remained
endemic in the ca. 150000 people in the simulation.
The ca. 34 year periodicity of epidemics is driven
by waxing and waning herd immunity in the 09
year age classes. The volatility in prevalence of antibody in the 14 year age class, and to a lesser extent
among the 59 year age class, reflects two sources.
Reductions in prevalence through time come about
by children moving from the 14 year class to the
59 year class, with replenishment from predominantly uninfected infants from the 01 year age
class during years of low transmission. Prevalence
in the 14 year age class goes up sharply in epidemic
years because infection occurs in the age class and
106

the recruits from the infants class are more likely to

already be positive in epidemic years. In this example of hyper-endemicity, the variability in seroprevalence directly influences abundance of the
associated virus serotype. Note that in this example
it is ultimately the human birth rate that entrains the
34 year epidemic cycle.
The interaction of different serotypes

through the mechanism of heterologous
immunity
On the basis of simulation studies with DENSiM,
abundances of the different serotypes can influence each other through the phenomenon of shortlived heterologous immunity following infection. In
essence, an ongoing epidemic of a particular serotype temporally raises the effective herd immunity
to the other three serotypes by producing heterologous and cross-reacting titres in those recently
infected; once one epidemic is under way, it is somewhat less probable that a second epidemic (with
similar force of infection) will begin. This is a factor in the commonly observed phenomenon of asynchrony of epidemics of different serotypes (figure
6). But regarding the question of relative abundance
by serotype for any particular month, the proximate
determinant of abundance of each virus type in mosquitoes is simply a linear function of the frequency
of human infection of that type.
The potential influence of antibodydependent enhancement on the dynamics

and persistence of multiple serotypes of virus
Recently, another interesting hypothesis regarding the interaction of serotypes was made by
Figure 6. Output from DENSiM for hypothetical site where dengue has historically been endemic for all four serotypes.
Simulation begins with all age classes being 90% positive for antibody and an initial human population of 100000; the annual population growth is ca. 3.2%. Virus was
introduced only during the year preceding the results shown here, i.e. the results depict a situation where the viruses are not lost between epidemics.
%
%
%
%
7JSBFNJDQFSTPOTPWFSBMMWJFX

Ferguson et al.45 The authors note that antibodydependent enhancement (ADE) of dengue infection
involves cross-reactive antibodies from a previous
infection that serve to facilitate virus replication
within the host. They posit that the phenomenon of
enhanced infections in a subset of cells can result
in a higher probability of transmission of the virus
causing the secondary infection. Using a simple
set of differential equations, they demonstrate that
this linkage between serotypes via ADE can result
in persistent and complex cyclical patterns in the
relative abundance of serotypes of virus given the
assumption regarding ADE leading to a change in
transmission probabilities. The results of this study
suggest that this phenomenon of linkage via ADE
theoretically makes possible the co-existence of multiple serotypes, whereas without such linkages, one

or more serotypes would be expected to be lost due

to drift.
The recent work by Vaughn et al. has clearly demonstrated that peak viraemia is increased in at least
some secondary infections in humans.at Given that
virus titre is thought to influence both the probability of disseminated infection and the duration of
EIP within the female mosquito, a hypothesis linking ADE and increased probability of transmission
seems plausible.
Acknowledgment
This document was partially funded by a grant from
the US National Oceanographic and Atmospheric
Administrations Office of Global Programs.
107
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109
WORKING PAPER 6.2. Control

of dengue vectors:
tools and strategies
PJ McCall 1 and Pattamaporn Kittayapong 2
1
Vector Group, Liverpool School of Tropical Medicine,
Liverpool, England
2
Center for Vectors and Vector-Borne Diseases and
Department of Biology, Faculty of Science, Mahidol
University, Bangkok, Thailand
Introduction
Dengue is a vector-borne disease of tropical and
subtropical human populations, which occurs predominantly, but not only, in urban areas. The global increase in urbanization, such that the worlds
urban population of 1.7 billion in 1980 is expected to
double by 2010 (UNPP, 2006), is likely to lead to an
increase in dengue in the future. Control of peridomestic vectors is the only effective preventive measure currently available. With dengue occurring in a
range of different geographic, socioeconomic and
cultural contexts (high-density urban to lower-density periurban and rural situations), and with geographic variation in vector (sometimes two vectors)
and host behaviour, one intervention strategy is very
unlikely to suit all situations, and a range of control
methods and strategies is needed.
Dengue is transmitted by Aedes sp. mosquitoes that
breed in container habitats. The main vector, Aedes
aegypti, is a cosmotropical species that proliferates in
water containers in and around houses. Secondary
vectors include Ae. albopictus, an important vector in
south-east Asia and that has spread to the Americas,
western Africa and the Mediterranean rim, Ae.
mediovittatus in the Caribbean, and Ae. polynesiensis and Ae. scutellaris in the western Pacific region.
Although zoonotic cycles involving monkeys occur
in some forest areas of western Africa and southeastern Asia, there is no evidence that these play any
role in epidemics of human disease. Consequently,
control can be directed at Ae. aegypti breeding and
biting in the household and immediate vicinity.
Ae. aegypti breeds in many types of household containers, such as water storage jars, drums, tanks
and plant or flower containers. They do not fly far,
dispersing probably no more than 100 m beyond
the emergence location (Reiter et al., 1995; Muir &
Kay, 1998; Honorio et al., 2003; Harrington et al.,
2005) and are highly anthropophilic, rarely feeding on non-human hosts. Given these limits, control
or indeed elimination of peridomestic vector populations might appear feasible, but experience has
110
proven otherwise. Many eradication attempts have

failed, for various reasons: vertical programmes
were inefficient and unsustainable, outdoor space
spraying was ineffective; larviciding was often
rejected by communities and educational messages
to the population were often unsuccessful (Slosek,
1986; Winch et al., 1991; Gubler & Clark, 1994; Parks
& Lloyd, 2004). Today it is not unusual to find that
more than 50% of houses in many endemic areas are
infested with Aedes larvae, and the risk of epidemics is high (Nathan & Knudson, 1991; PAHO, 1994;
WHO, 2000). Eradication of Ae. aegypti populations
may be achievable, but it is rarely sustainable. The
present aim is to control, to below certain threshold
levels, rather than eliminate vector populations.
The previous UNICEF-UNDP-World Bank-WHO
Tropical Diseases (TDR) Scientific Working Group
on Dengue (Scientific Working Group on Dengue,
2000) stated the need for the development and
evaluation of new tools to reduce mosquito populations, including source reduction and to increase
evidence based vector control programmes and to
support state-of-the-art research on human behaviour and behaviour change in relation to mosquito
breeding. Partly in response to this call, a range of
promising intervention tools are now ready for further investigation for prevention of oviposition or
biting or suppression of vector populations, while
developments in molecular genetic research have
increased the likelihood that novel methods will
be developed.
Control of immature stages

Vector control, either by elimination of breeding
sites (clean-up campaigns) or by larviciding with
insecticides, has led many dengue programmes
in the past. Although still widely used and promoted, and successes have been achieved in many
contexts, clean-up campaigns are limited by the
level of compliance by the community and by the
fact that often the most productive containers (i.e.
those from which most Ae. aegypti emerge) cannot be disposed of or emptied (e.g. drinking-water
stores). Larvicidal treatment of such water is possible. Only temephos, permethrin, Bti (see below)
and pyriproxyfen are approved by WHO for use in
drinking-water (Chavasse, 1997). The organophosphate temephos (Abate) has been most widely used
and, although it is effective, acceptance levels are
often low and coverage poor. Placing additives with
persistent flavour into domestic water, particularly
drinking-water is, understandably, often viewed
with suspicion and not well accepted. Additionally,
regular water usage or emptying of containers (often
encouraged in simultaneous clean-up campaigns)

can reduce or negate the intended effect. The situation would be improved, first, if attention could be
limited to known productive, rather than all potential, breeding sites and secondly, if more acceptable
larvicides were available. A major new multi-country study using pupal/demographic surveys has
demonstrated that accurate identification of epidemiologically important breeding sites/containers
is possible and should enable targeting of control
(Focks & Alexander, 2006; Nathan et al., 2006). This
is a very important development as, although initial
surveys will have to be more intensive, the potential
increase in effectiveness gained by directing control
at productive containers is very significant. With
this strategy in mind, we have looked at the potential for a range of new larval control tools.
Insect growth regulator (IGR)pyriproxyfen

Pyriproxyfen is an insect juvenile-hormone analogue, which is active against many arthropods and
which has been in use for agricultural pest control
for about 15 years. It is extremely effective against
mosquitoes and can prevent the emergence of Ae.
aegypti at concentrations as low as 1 ppb or less,
while extremely high concentrations do not inhibit
oviposition (Itoh, 1994; Sihuincha et al., 2005).
Treated water does not taste tainted. Interestingly,
very low doses of pyriproxyfen can also sublethally
affect adults by decreasing fecundity or fertility, and
the contaminated adult female can transfer effective doses to any breeding site she subsequently visits (Itoh, 1994; Dell Chism & Apperson, 2003). New
pyriproxyfen formulations can retain efficacy for
6 months (Seng et al., 2006), reducing the need for
frequent reapplication, and new studies (Sihuincha
et al., 2005) are indicating its high level of efficacy.
Clearly further trials are now called for. Worryingly,
however, this intervention was not accepted by
communities in Mexico (Kroeger et al., 2006), suggesting that suspicion of domestic-water treatments
for dengue control remains an obstacle to larval control. Moreover, since pyriproxyfen only prevents
eclosion, larvae and pupae remain visibly active in
breeding sites, conveying the false impression of a
lack of efficacy and further compromising acceptance. A current trial in Thailand is investigating
these areas further.
Predatory copepods
Various predatory Mesocyclops spp. (Crustacea;
Eudecapoda) have been studied for their potential
to control mosquito larvae. Two species in particular, M. thermocyclopoides and M. aspericornis, have
proven effective against dengue vectors (Riviere et
al., 1987; Brown et al., 1991; Kay et al., 1992; Marten
et al., 1994; Mittal et al., 1997; Schaper 1999; Nam

et al., 2000). However, although copepods can survive up to 6 months in containers, they are often lost
when water is removed, containers are cleaned or
(copepod) food is limited, and reintroduction may
be necessary for sustainable control (Lardeux, 1992;
Marten et al., 1994; Schaper, 1999; Chansang et al.,
2004). However, in certain contexts Mesocyclops can
be very effective. A large-scale vector-control programme using copepods and clean-up campaigns
in Viet Nam successfully controlled dengue transmission for a number of years and, in this region at
least, appeared to be sustainable (Nam et al., 1998;
Kay et al., 2002; Nam et al., 2005). Attempts should
now be made to investigate the potential of this
method in other countries where vector-productive
containers could sustain copepod populations, and
where communities might be willing to accept such
a (depending on their experience) novel intervention. Furthermore, the efficacy of this method might
be improved by combining it with other compatible
tools, such as fish (Lardeux, 1992) or other methods
(see next section).
Bti toxins
The endotoxin produced by the entomopathogenic
bacteria Bacillus thuringiensis var. israelensis (Bti) has
high larvicidal activity in mosquitoes, but is nontoxic to important beneficial organisms. Various
formulations (i.e. wettable powders, granules and
briquettes) are effective, and newer slow-release
long-lasting formulations may reduce the need for
frequent reapplication. One recent field trial of slowrelease long-lasting Bti products in Thailand demonstrated that the treatments could persist for 3
months in utility water containers in rural villages
(Wiwat et al., personal communication).
Combination strategies have often yielded satisfactory results in terms of potency and long-term efficacy (Riviere et al., 1987; de Andrande & Modolo,
1991; Tietze et al., 1994; Neri-Barbosa et al., 1997).
When a combination of M. longisetus and Bti, Bacillus
sphaericus, or methoprene was evaluated (Tietze
et al., 1994), the combined agents were better than
either alone. The compatibility of the commercial
formulation Vectobac 12AS with certain chemical
insecticides for controlling Aedes larvae and adults
was demonstrated in Malaysia (Seleena et al., 1999).
A mixture of Vectobac 12AS and Actellic 50EC (pirimiphos-methyl) showed promise as a larvicide in
the laboratory (Chung et al., 2001). These combinations clearly warrant follow-up. An experiment to
integrate Bti with Mesocyclops and mosquito densovirus (AThDNV; see below), conducted in seminatural conditions, demonstrated that this complex
111
combination might also be effective (Wiwat et al.,

personal communication).
In Thailand it has been demonstrated that reductions of Ae. aegypti larvae of more than 90% could
be achieved for 3-month periods with combined
treatment of copepods and Bti, although supplementary food for the copepods (community-supplied products such as rice and leaves) was required
(Kosiyachinda et al., 2003; Chansang et al., 2004).
A pilot community-based intervention using integrated physical and biological control, including a
combination of copepods and Bti, in eastern Thailand
was successful and showed potential for expansion
into other areas (Kittayapong et al., 2006).
Mosquito densonucleosis viruses

Densonucleosis viruses or densoviruses (DNVs) are
invertebrate viruses in the genus Brevidensovirus
of the family Parvoviridae (Bergoin & Tijssen,
2000). The virions of the DNVs contain a 4 kb single-stranded DNA genome with terminal hairpins
packed in a non-enveloped particle (Bando et al.,
1990; Berns et al., 1996). Five strains of Aedes densoviruses have been identified to date (Buchatsky,
1989; Jousset et al., 1993; Barreau et al., 1994, 1996;
Kittayapong et al., 1999; Afanasiev & Carlson, 2003;
Chen et al., 2004). Current experiments show that
the efficiency in vector control could vary greatly
depending on both viral strains and geographic
origins of the mosquito vectors (Wiwat et al., personal communication). Application could involve
either direct lethal effects on treated populations,
like a biological insecticide, or shortening of adult
lifespan, much as described below for Wolbachia. A
recent finding has reported improved survival of
Ae. aegypti larvae from 15% to 58% after infecting
successive generations of mosquitoes with AThDNV,
the Thai strain densovirus (Roekring et al., 2006).
Future research to study interactions between the
pathogenic viruses and their mosquito hosts, with
respect to resistance and mode of action, will generate useful data. Field trials should be encouraged in
due course.
vector control of
adult mosquitoes
In the past, control directed at adult mosquitoes has
been limited to the use of ultra-low-volume (ULV)
application of insecticides, usually by vehiclemounted apparatus. There is controversy regarding
the efficacy of this type of control, with a number of
studies indicating that its effect is rarely, if ever, significant (Reiter & Gubler, 1997; Perich et al., 1990,
2000). This is likely to be partly the result of a failure
112
of outdoor sprayed insecticide to reach indoor populations of mosquitoes and failures of vertical programmes to deliver at community level. Despite
doubts about efficacy, such interventions remain
the last resort in combating epidemics. However,
new tools open the way to adult mosquito control at
community level.
Insecticide-treated materials
Insecticide-treated materials (ITMs; typically
deployed as insecticide-treated bednets [ITNs])
have proven highly effective in preventing diseases
transmitted by nocturnally-active vectors. Their efficacy in controlling diurnally-active Ae. aegypti is
now being evaluated. A cluster-randomized trial in
Latin America has demonstrated that insecticidetreated window curtains and/or insecticide-treated
domestic-water container covers can reduce dengue vector densities to low levels and potentially
have an impact on dengue transmission (Kroeger
et al., 2006). ITMs, particularly curtains, were well
accepted and their efficacy reinforced by the sight
of dead insects (cockroaches, houseflies and other
pests, as well as mosquitoes) beneath the treated
curtains. Importantly, a spill-over effect, whereby
the intervention reduced vector populations in
neighbouring control clusters, also occurred, such
that houses without ITMs that were located close to
treated houses were less likely to have infestations
than those further away. Presumably, as with other
vectors, the risk of a host-seeking mosquito contacting an ITM at some point during its frequent visits to
houses to blood-feed, means that its life expectancy
is reduced; in effect, the age structure of the vector
population is altered and few individuals are likely
to live long enough to become infective with dengue. This mass effect will reduce vectors throughout
the community (as shown for ITMs and malaria vectors; Gimnig et al., 2003; Hawley et al., 2003) and is
an excellent outcome, given that coverage with any
intervention tool is always less than 100%, sometimes markedly so. New trials are now underway in
Venezuela and Thailand to examine these interventions further and additional trials in yet more locations and contexts should be encouraged.
Indoor residual treatments are known to kill Ae.
aegypti, although such methods have rarely been
used, nor are they recommended today (WHO,
2006). Indeed, such is the strong endophagic/endophilic tendency of this vector (Christophers, 1960;
Perich et al., 2000) that the use of insecticidal aerosol cans in the household can also markedly affect
dengue transmission (Osaka et al., 1999). Could
other indoor treatments also work? The question of
whether ITNs might also affect Ae. aegypti has also
been considered in a pilot study in Haiti (Wiwat et al.,

personal communication). Although not a fully controlled trial, results indicated that bednets reduced
peridomestic dengue vector breeding (as measured
by standard indices), and may have helped reduce
seroconversion rates over 12 months. A communitywide effect was again observed, as dengue-vector
breeding also appeared to be reduced in neighbouring control areas. As dengue vectors bite during the
day, how could ITNs exert an effect? Either the presence of the insecticide reduced entry into houses by
repelling incoming mosquitoes or, most likely given
the community-level effect observed, the ITNs functioned as residually-treated resting surfaces with
which contact was made resulting in killing; an
effect that one might expect to be enhanced inside
the smaller houses typical in poorer districts. Effects
on Ae. aegypti of ITNs deployed widely for malaria
control have not been recorded despite the many trials that have been undertaken (presumably because
routine sampling for nocturnal malaria vectors
would not record Ae. aegypti), although low-level
Ae. aegypti biting was reduced to zero after introduction of ITNs in a village in the Democratic Republic
of the Congo (Karch et al., 1995). Sustainability of
many interventions against all mosquito vectors is a
problem in many contexts. However, use of ITNs is
invariably higher in urban areas (Gimnig et al., 2005;
Lindblade et al., 2005), precisely where dengue is
the greatest problem. That such an appropriate and
widespread intervention might also reduce dengue
transmission should be evaluated.
In considering ITM-based strategies for dengue control, new tools primarily developed for malaria control can also have applications for dengue control.
In the case of long-lasting insecticide-treated netting (LLIN), the netting is loaded with sufficient
insecticide during manufacture to avoid the need
for re-impregnation. Both LLIN window curtains
and water-container covers are effective (Kroeger
et al., 2006). The long-lasting formulation KO-Tab
1-2-3, which can be applied in the community to
any material, renders it as wash-resistant as LLIN
(Yates et al., 2005).Treatment of the existing window
curtains in a house might be possible and should
be investigated, although loss in efficacy over time,
resulting from the degradation of the insecticide
by ultraviolet rays in sunlight may be a problem.
Renewed interest from industry partners that perceive potential markets in this field, particularly for
malaria control, is likely to result in new products to
meet control needs in the coming years.
The mode of action of ITMs should be investigated:
do they repel Ae. aegypti (curtains, bednets or jar
covers), attract and kill them (during hostseeking or
oviposition) or exert their effect in some passive or

as yet unknown way? Finally, as is always the case
with insecticide-based strategies, resistance to existing insecticides is inevitable and the current global
state of insecticide resistance in Ae. aegypti is worrying (see Hemingway, in the present report). This
could be resolved by using alternatives to insecticides, as described in this article, by using different or by rotating insecticidesa real possibility, as
some of the ITMs that are proving effective in dengue control can be used with insecticides other than
the pyrethroids, which, because of their low mammalian toxicity have been first choice in the past.
Eventually, new insecticides will be needed, an
approach that is fundamental to the Gates-funded
Innovative Vector Control Consortium which
aims to develop a portfolio of chemical and technological tools suitable for vector control (Hemingway
et al., 2006).
Lethal ovitraps
The ovitrap or oviposition trap was first recommended by WHO for surveillance of Aedes vectors
(WHO, 1972; WHO, 1975), then modified to render
it lethal to adults or larvae of Ae. aegypti (Chan et al.,
1973; Chan, 1977; Zeichner & Perich, 1999; Perich et
al., 2003; Sithiprasasna et al., 2003; Kittayapong et
al., 2006). Notably, they were used to eradicate Aedes
vectors from Singapore International Airport (Chan,
1973). The autocidal ovitrap was designed and developed for the control of Aedes vectors in urban areas
with a high density of Aedes and a high incidence
of dengue haemorrhagic fever in Singapore (Chan
et al., 1977). In principle, ovitraps could kill adult
mosquitoes if the ovistrip was treated with insecticide (Zeichner & Perich, 1999) or destroy progeny
by using fine nylon netting for trapping the larvae
(Lok et al., 1977). In Brazil, lethal ovitraps with deltamethrin-treated ovistrips killed 89% of Ae. aegypti
adults and produced more than 99% larval mortality during 1-month field trials (Perich et al., 2003).
The advantages of lethal ovitraps for controlling
Aedes vectors include their simplicity, their specificity for and effectiveness against container breeders like Ae. aegypti and their potential for integration
with other chemical or biological control methodologies. Studies are still at an early stage (Perich
et al., 2003; Sithiprasasna et al., 2003) and should
be encouraged.
Innovative Vector Control Consortium:

http://www.ivcc.com/
113
The future: strategies based

on genetic modification
In the search for novel tools for vector control,
genetic modification of Aedes to resist infection with
dengue virus is one of the most seductive longterm approaches. Although some methodology has
already been shown to be effective in the laboratory
(Kokoza et al., 2000), the challenges here are technical (and potentially independent of any kind of
community participation in vector-control activities,
although initial acceptance of genetically-modified
mosquitoes may not always be certain) requiring
development of mosquito lines resistant to or unable to transmit dengue and replacement of the natural populations with these non-vector lines.
A major obstacle however, has been the slow development of strong driving systems to deliver dengue
virus-resistant genes into natural vector populations (Scott et al., 2002), although much research has
been devoted to identifying candidates such as
transposable elements, meiotic drive or endosymbiotic Wolbachia bacteria (Braig & Yan, 2001; James,
2005; Sinkins & Gould, 2006). Transposable elements have been shown to be efficient for transformation of mosquitoes using an external transposase
source, but further research is needed to investigate
the autonomous transposable elements in which the
effector gene is linked to the source of transposase
(Christophides, 2005). The use of meiotic drive in
genetic control also requires a greater understanding of the molecular mechanism, especially in mosquitoes. One of the proposed gene driving systems
which has potential is the use of maternally inherited
Wolbachia endosymbionts (Sinkins & Godfrey, 2004).
An advantage is the possibility for repeated spread
and invasion of naturally Wolbachia-infected populations via superinfections of different Wolbachia
strains. Much research is still needed.
Development of strategies based on genetic

modification of Ae. aegypti
One of the global collaborative Grand Challenges
in Global Health (GCGH)-funded projects aims to
use genetic-based strategies to prevent Ae. aegypti
from transmitting dengue viruses either by reducing densities of mosquito populations or by eliminating their ability to transmit dengue viruses
(James, personal communication). The main challenges and objectives of this project are summarized
in three topics: first, the effector genes for population replacement and reduction will be optimized;
secondly, safe and efficient drive systems for introgressing effector genes into mosquito populations
will be developed; and lastly, a field site for genetic
114
control trials will be established in a suitable developing country. Different directions are being pursued. Two approaches aim to reduce or eliminate
natural populations of Aedes vectors: densoviruses
could be developed as biological control agents of
Ae. aegypti larvae (Buchatsky, 1989), while release of
insects carrying dominant lethal mutations (RIDL)
are designed to reduce or eliminate mosquito populations, especially those that are infected with dengue viruses (Heinrich & Scott, 2000; Thomas et al.,
2000; Horn & Wimmer, 2003). Other methodssynthetic transposable elements (TEs) (Adelman et al.,
2002), meiotic drive (Mori et al., 2004) and underdominance (Davis et al., 2001)aim to modify vector populations by introgressing genes that eliminate
vector competence. These methods are currently at
various stages in development and readiness for
field-testing.
A different approach to is being taken via the development of a novel gene-based sterile insect technology (SIT) whereby genetically altered, rather than
irradiated, males are released into the environment
to mate with wild females, thereby reducing population numbers and ultimately preventing the
transmission of disease (Coleman & Alphey, 2004).
This strategy is very well advanced and likely to
lead to field trials in the very near future (Alphey,
personal communication).
Development of Aedes vectors with a

short lifespan
Another GCGH project aims to modify the age
structure of the population of dengue vectors such
that lifespan is reduced and mosquitoes die before
reaching the age when they could transmit dengue (ONeill et al., personal communication). The
strategy is based on introducing endosymbiotic
Wolbachia bacteria into wild vector populations,
these bacteria being capable of significantly shortening the adult mosquito lifespan (Min & Benzer, 1997;
McGraw et al., 2002). These bacteria are present naturally in most insects and widespread in mosquitoes (Werren et al., 1995; Kittayapong et al., 2000).
Both single and double strains of Wolbachia, naturally found in Ae. albopictus, have already been successfully transferred into Ae. aegypti (Xi et al., 2005;
Ruang-areerate & Kittayapong, 2006). Dengue
viruses need an extrinsic incubation period of about
1214 days in the vector before being transmitted
into humans; the Popcorn strain of Wolbachia could
shorten the life of its insect hosts to about half this
period. Theoretical models with realistic parameter
estimates suggest that an intervention using release
of Popcorn-transinfected vector lines could reduce
dengue transmission by 90100% (Brownstein et al.,

2003; Rasgon et al., 2003).
The primary objective of this project is to generate
stable lines of Ae. aegypti and Ae. albopictus that are
infected with the Popcorn Wolbachia. Once established, these Wolbachia-transinfected lines could
be characterized and tested in experimental cages
to determine virulence, transovarial transmission,
effect on host age and potential to spread in natural vector populations via cytoplasmic incompatibility. The project is ongoing and, if successful, a
field trial in large, confined field cages will be
attempted in an endemic country. Importantly, this
strategy has the potential to be implemented without the need to release transgenic mosquitoes into
the environment.
Any studies on post-genomic relationships between
mosquito hosts and their endosymbionts will benefit further development of genetic strategies to
control dengue vectors. Application of the population-replacement approach in field situations will
require detailed understanding of Wolbachia-transfected laboratory-generated strains compared with
the naturally uninfected strains of dengue vectors
in term of their expressed physiological and behavioural parameters at the post-genomic level. The
recently-completed sequencing of the Ae. aegypti
genome should facilitate identification of new effector genes, as well as potential gene-driving mechanisms.** Future research should include studies
related to novel genetic markers to investigate gene
flow and genetic diversity among different geographic populations, which will be crucial to the
success of population replacement strategies. Also
essential, in due course, will be the fostering of public understanding and support of such tools.
Priorities and opportunities

for research
A number of major initiatives have brought together
scientists from many disciplines. Current programmes include the WHO/TDR/International
Development Research Centre (IDRC) Research
Initiative on Eco-Bio-Social Research on Dengue in
Asia, a research initiative aimed at improving the

prevention of dengue by understanding its multifarious determinants and how they affect transmission
(and therefore influence the likelihood of successful
control) at community level. The European Union
is funding two major multidisciplinary studies on
dengue, one of which, DENCO, includes two large
cluster-randomized trials of ITMs and pyriproxyfen.
The Bill & Melinda Gates Foundation have funded a
range of initiatives in vector-borne diseases, three of
which at least directly target dengue (see above).
But many areas still require work. In addition to
those directions already mentioned, further studies are required to confirm the efficacy, acceptance,
costeffectiveness and sustainability of ITMs in controlled trials; trials of slow-release formulations of
Bti and pyriproxyfen; densovirus trials; the efficacy
of tools against Ae. albopictus should be investigated
wherever this is an important vector; the long-term
effects of interventions on mosquito populations
and the long-term effects and benefits to communities should also be investigated.
Ideal tools should be user-friendly, requiring little
additional work or behaviour change by householders. They should be affordable, safe and effective
in reducing vector densities below threshold levels
estimated by the pupae per person index; indeed,
as might be the case with insecticide-treated curtains in many societies, they could even be viewed
as desirable by householders. The ideal situation
that we must strive for is to have a suite of proven
effective, safe and environmentally friendly tools
available for intervention, from which the most
appropriate tool or combination of tools can be
selected to suit the specific biological and cultural
needs of each community.
Acknowledgements
The authors are grateful to the other members of
the Scientific Working Group for their discussions
throughout the meeting. In particular, we thank
Dana Focks, Roberto Barrera, Didier Fontenille,
Luke Alphey, Chusak Prasittisuk, Axel Kroeger and
Mike Nathan.
** A special issue of Science will be devoted to the Aedes genome in

early 2007.
115
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119
WORKING PAPER 6.3.

INSECTICIDE RESISTANCE
IN AEDES AEGYPTI
Janet Hemingway
Liverpool School of Tropical Medicine, Pembroke Place,
Liverpool L3 5QA
INTRODUCTION
Insecticide resistance in the vector Aedes aegypti is an
important but under-researched and poorly understood phenomenon. Several early reports of DDT
resistance, in the 1960s to 1980s, reported crossresistance between DDT and pyrethroids. Later literature suggests that organophosphate resistance is
also developing in some areas. The impact of this
resistance on operational activities such as larviciding and space spraying is largely unknown.
RESISTANCE DISTRIBUTION
Resistant populations of Ae. aegypti have been
detected in several countries throughout the geographical range of this species and, in some areas,
the evolution of insecticide resistance has been
linked to failure of the dengue control programme.
However there has been no systematic review of the
resistance status or of the impact of resistance on
insecticide-based vector control activities. The bulk
of the data available are from simple WHO susceptibility assays using insecticide impregnated papers;
very little is known about the molecular or biochemical basis of this resistance and yet such information is needed to identify the origin of resistance and
develop strategies to reduce the spread and minimize the impact of insecticide resistance mutations.
THE BIOCHEMICAL BASIS OF

RESISTANCE IN AEDES
There are two major routes by which insecticide
resistance develops. The insect can either change
the speed at which the insecticide is detoxified or it
can alter the target site in order to reduce sensitivity to poisoning. Both types of resistance have been
reported in Aedes. A number of simple biochemical
and molecular assays can be used to ascertain which
form of resistance occurs in any individual; further
work then needs to be undertaken on the implicated
metabolic class of enzymes or nervous system target site.
Three enzyme families are primarily implicated in
effecting increased levels of insecticide degradation:
120
the cytochrome P450 monooxygenase (P450), glutathione transferase, and esterase enzyme families,
which catalyse a wide range of detoxification reactions. These enzymes provide the first line of enzymatic defence against xenobiotics in most organisms;
the different classes are large and diverse in function, and consist of mixtures of highly specialized
enzymes, often with specific substrates and strictly
regulated expression profiles, and more generalist,
ubiquitously expressed enzymes. Many insect species show an amazing diversity of detoxification
enzymes. As insect genomes have been sequenced,
and the detoxification genes annotated, it has
become apparent that these gene families are evolving very rapidly and that each insect has a unique
complement of detoxification genes, with very few
orthologs across insect species.
In Ae. aegypti, the best understood of the metabolic
enzyme families are the glutathione S-transferases.
Grant and Hammock in the 1980s1 looked in detail
at a DDT-resistant strain of Ae. aegypti; the resistance was DDT specific and due to an elevation in
glutathione S-transferase (GST) activity. They isolated a single up-regulated GST from the strain.
Groups in Liverpool and Thailand took a more
holistic approach to looking at this enzyme class
and their pioneering work was later complemented
and extended when the draft Ae. aegypti genome
sequence became searchable.
Data from the draft genome sequence show that
there has been no expansion of the GST family in
Ae. aegypti. Indeed mosquitoes have considerably
fewer GST genes than Drosophila, although this deficit is partially rectified by alternative splicing of two
mosquito GST genes, which increases the number
of Ae. aegypti GST transcripts by three, from 26 to
29. Each of the GST classes found in An. gambiae is
represented in Ae. aegypti, including the two classes
which so far appear to be unique to mosquitoes.
Over half of the GSTs belong to two insect-specific
classes, the Delta and Epsilon classes, which include
the vast majority of GST enzymes with a defined
role in insecticide metabolism.
MOLECULAR ANALYSIS OF
TARGET SITE RESISTANCE
The clade of esterases associated with organophosphate (OP) resistance is the actetylcholinesterase
(ace) clade. These esterases provide the target sites
for both OP and carbamate insecticides; the toxins
act as irreversible inhibitors of the enzymes, blocking hydrolysis of the neurotransmitter acetylcholine.
Mutations in ace can reduce the binding of insecticides, resulting in resistance. As in An. gambiae, Ae.
aegypti contains two ace genes. Mutations in ace-1

have been associated with resistance to insecticides
in other mosquitoes, but no such mutations have
been reported in Ae. aegypti to date.
NEW INITIATIVES
Research in this area received a boost in the last
12 months with the provision of funding for the
Innovative Vector Control Consortium from the Bill
and Melinda Gates Foundation. This Consortium
is supporting three large-scale projects that should
positively impact on dengue control. The team,
which involves staff from Colorado State University,
the University of California Davis, and the Liverpool
School of Tropical Medicine, together with collaborators in many parts of the world, is developing:
A dengue decision support system to help operational staff at municipality, district or country
level to make rational decisions on where, when
and how to implement vector control.
Improved threshold modelling of dengue vector
control to help estimate the level of control that
will need to be achieved operationally to effect
control in different epidemiological settings (this
is reported in more detail in Focks and Barrera,
working paper 6.1).
Simpler and more accurate methods for operationally monitoring insecticide resistance in Ae.
aegypti in the field.
Extensive work has already been undertaken to
underpin this latter project, building on work initiated in Liverpool and funded by the Wellcome
Trust and others. The sequencing of the Ae. aegypti
genome, and the development of a robust microarray platform for detoxification genes, have greatly
facilitated the monitoring of insecticide-based control programmes and will enhance our ability to control this major disease vector in future. Searches of
the draft Ae. aegypti genome sequence, and comparison with other insects, have revealed that Ae. aegypti
has far more potential detoxification genes than any
other insect studied at the genome level to date.
Why Ae. aegypti has such an abundance of detoxification genes compared to other insect species is
unknown. The approximately one third higher
gene count in Ae. aegypti than in An. gambiae cannot be readily accounted for by differential exposure to xenobiotics. Both species have a preference
for breeding in clean water (as opposed to Culex
mosquitoes, which readily breed in water heavily contaminated with organic material), both are
highly anthropophilic and hence exposed to manmade pollutants, and both are frequently targeted
with insecticides.
GROWING EVIDENCE OF
RESISTANCE INVOLVING
CYTOCHROME P450S
Ae. aegypti contains a total of 158 full length, putatively catalytic P450 genes. This represents an expansion of approximately 55% compared to Anopheles
gambiae, and 86% compared to Drosophila melanogaster. Several large clusters of P450s are found in
the Ae. aegypti genome, the largest being a cluster of
18 CYP6 genes and a cluster of 16 CYP9 genes.
A total of thirty-seven CYP9 genes are present in
the Ae. aegypti genome compared with just eight in
An. gambiae. Why the CYP9 family is so abundant
in Ae. aegypti is unclear but a large subset of these
genes are over-expressed in one or more insecticideresistant strains, suggesting that this recent expansion may at least partially reflect an adaptation to
insecticide exposure.
THE POTENTIAL ROLE OF ESTERASES

The lack of clear orthology between Anopheles
and Aedes -esterases suggests that this is a rapidly evolving enzyme group. Alpha-esterases in
many other species are involved in metabolic resistance to insecticides; these include the Lucilia cuprina -E7 and the Culex quinquefasciatus and
genes (named according to substrate specificity as
opposed to phylogenetic relationships). These two
Culex genes are arranged in a head-to-head orientation, and amplification of one or both of them is
a major cause of OP resistance in Culex populations
worldwide. Interestingly, in contrast to the rapid
radiation of other -esterases, the two genes are well
conserved across Culex, Anopheles and Aedes, and the
head-to-head orientation is maintained in all species. It remains to be seen whether OP resistance in
Ae. aegypti (reported by Mourya2) is associated with
amplification of this genomic locus.
WHICH GENES ARE ASSOCIATED

WITH INSECTICIDE RESISTANCE?
A small-scale microarray, the Aedes Detox Chip, containing unique 70mer probes for 204 of the 233 Ae.
aegypti detoxification genes, has been constructed in
Liverpool and used to compare expression profiles
in susceptible and resistant populations. Twentyfive genes over-expressed in a resistant strain from
Thailand and 14 over-expressed in a resistant strain
from Mexico. Five of these genes, three CYP9 P450s
and two Epsilon GSTs, were over-expressed in both
resistant strains. Results of this preliminary analysis
will be reported in Science in 2007.
121
References
1. Grant DF, Hammock BD. Genetic and molecular
evidence for a trans-acting regulatory locus controlling glutathione S-transferase-2 expression

in Aedes aegypti. Molecular and General Genetics,
1992, 234(2):169-176.
2. Mourya DT et al. Insecticide susceptibility status
of certain populations of Aedes aegypti mosquito from rural areas of Maharashtra state. Indian
Journal of Medical Research, 1993, 97:87-91.
122
SURVEILLANCE AND DELIVERY ISSUES

7.1 D
ENGUE RESEARCH NEEDS RELATED TO SURVEILLANCE AND
EMERGENCY RESPONSE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
7.2 G
EOGRAPHIC INFORMATION SYSTEM FOR DENGUE PREVENTION
AND CONTROL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
7.3 A
CHIEVING BEHAVIOUR CHANGE FOR DENGUE CONTROL: METHODS,
SCALING-UP, AND SUSTAINABILITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
Dengue
Annex 7
WORKING PAPERS:
7.4 D
ELIVERY ISSUES RELATED TO VECTOR CONTROL OPERATIONS:
A SPECIAL FOCUS ON THE AMERICAS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
123
WORKING PAPER 7.1.

Dengue research needs
related to surveillance
and emergency response
Eng-Eong Ooi1, Duane J. Gubler2, Vu Sinh Nam3
1
DSO National Laboratories, 27 Medical Drive,
#09-01, Singapore 117510
2
Asia-Pacific Institute of Tropical Medicine and
Infectious Diseases, University of Hawaii, and
Department of Tropical Medicine and Medical
Microbiology, John A. Burns School of Medicine, 651
Ilalo Street, BSB 320, Honolulu, Hawaii 96813, USA
3
Vietnam Administration of Preventive Medicine,
Ministry of Health, 138A, Giang Vo, Ba Dinh, Hanoi,
Viet Nam
Introduction
Dengue fever/dengue hemorrhagic fever (DF/
DHF) is a disease that is endemic in the tropics, has
re-emerged to become the most common and most
important mosquito-borne viral disease in the world.
The current trend is a 4- to 6-yearly cycle of dengue
epidemics, with each cycle becoming larger in magnitude (Gubler, 2004). In the absence of an approved
vaccine, prevention of viral transmission through
public-health measures directed at controlling the
density of the vector mosquito population remains
the only viable preventive strategy. Effective prevention depends on a well-planned and operated
public-health, laboratory-based surveillance programme (Gubler, 1989; Gubler & Casta-Velez, 1991;
Rigau-Perez & Gubler, 1997).
A primary goal of public health surveillance in dengue is to monitor transmission to facilitate prevention
of the occurrence and spread of disease (RigauPerez & Gubler, 1997). Other goals for surveillance
include defining disease severity, determining the
costeffectiveness of public-health prevention programmes, and estimating the burden of disease in
the community (Teutsch, 2000). The ideal surveillance programme should thus be able to monitor
dengue cases accurately and predict impending epidemics from a background of endemic disease and
trigger the necessary preventive measures.
Recent research findings

In recent years, new findings have shed light on factors, other than incomplete immunity, that contribute to epidemic transmission for all four dengue
virus serotypes.
124
Viral factors
Phylogenetic studies have yielded interesting
insights into the selection and evolution of the dengue viruses, both temporal and geographical (Lewis
et al., 1993; Rico-Hesse et al., 1998; Twiddy et al.,
2002; Zhang et al., 2006; Imrie et al., 2006). Such
studies may also enable us to gain a better understanding of the viral factors that contribute to dengue virus virulence and epidemic potential (Gubler
et al., 1978; Gubler et al., 1981; Bennett et al., 2003;
Myat Thu et al., 2005). However, although current
technology allows us to monitor genetic changes in
viruses, the biological effect of these mutations has
yet to be elucidated. It is often difficult to attribute
genetic variation to specific phenotypic expression,
since dengue epidemics are associated with a host of
factors that could confound the analysis, and there
is no good animal model for dengue. Nonetheless,
further investigations in this field may yield fruitful results as genetic changes are likely to contribute to increased or decreased viral fitness and thus
epidemic potential and virulence. This includes
infectivity and ability to replicate in humans and
mosquitoes, thus resulting in increased or decreased
transmission. Genetic change in the virus could
also influence disease severity, and thus result in
more or less clinically overt disease. A critical area of
research, therefore, is to determine the influence of
viral factors on disease transmission dynamics and
disease severity.
Vector control
Various reports have shown that the inclusion of
community participation along with the use of old
and new vector-control tools (Chang et al., 2006;
Kroeger et al., 2006) as well as biological control
(Kay & Nam, 2005; Nam et al., 2005) have had positive effects on preventing disease transmission.
However, sustainability remains a problem, and
there is a need to establish proactive, laboratorybased disease- and vector-surveillance programmes
that provide a trigger for intensified vector control
to prevent epidemic transmission.
Experience gained in Singapore suggests that keeping the density of the vector population below a
threshold for epidemic transmission is a moving target. Lowered herd immunity after implementation
of effective control measures may paradoxically lead
to a rise in the number of cases of dengue, which in
turn, requires more intensive vector-control measures to prevent an epidemic (Chan, 1985; Goh, 1995;
Ooi et al., 2006). Identification of predictive entomological thresholds or markers for epidemic dengue, in combination with population susceptibility
would thus be a useful area of research. In addition,
experience gained recently in Viet Nam (Nam &

Kay, 2005) reinforces the lessons learned decades
ago in Singapore by Chan (1967), that without community involvement, vector control cannot be sustained. Subsequent experience in Singapore shows
that sustainability of the vector-control programme
is challenging, despite continued public education
and law enforcement.
While the role of Aedes aegypti is obvious, the role
of Ae. albopictus in maintaining dengue endemicity
is less clear. Although Ae. albopictus is an excellent
host and experimental vector for dengue viruses
(Gubler & Rosen, 1976; Rosen, et al., 1985), it has
not been frequently associated with epidemic dengue (Gubler, 2004). The reason for this is thought to
be related to this species ecology and blood-meal
seeking behaviour; Ae. albopictus has a broader host
range, and usually bites only once in obtaining a
blood meal. However, in most countries where dengue is endemic, both species of vectors can be found.
Furthermore, in many places such as Singapore, Ae.
albopictus outnumber Ae. aegypti and are more widespread geographically. Although Ae. albopictus may
not be an efficient vector for epidemic dengue, it
may play an important role in endemic transmission, maintaining dengue viruses in the population
until the population immunity is sufficiently lowered and the Ae. aegypti population is sufficiently
dense to support epidemic transmission (Rudnick,
1965; Gubler, 1987, 2004). Studies on the role that Ae.
albopictus plays in dengue transmission may thus be
useful in our understanding of the epidemiology of
dengue disease.
Population immunity
Several reports of large-scale serological surveys have been published in the last 5 years (Ooi
et al., 2001; Darcy et al., 2001; Hayes et al., 2003;
Tuntaprasart et al., 2003; Yamashiro et al., 2004; Thai
et al., 2005; Van Benthem et al., 2005; Balmaseda et
al., 2006). Although such studies may aid understanding of overall dengue activity in the area, few
have attempted to use these data to guide vectorcontrol operations. Serological surveys could be useful in elucidating the roles played by Ae. aegypti and
Ae. albopictus during epidemic and inter-epidemic
periods. Such a study would be entirely feasible in
places like Singapore where the geographical distribution of Ae. aegypti is different from that of Ae.
albopictus. With a combination of active virological
and entomological surveillance, such a study may
improve our understanding on the dynamics of dengue transmission and how these factors in turn contribute to endemic versus epidemic transmission.
Surveillance systems
A common theme that appears in reviews of the
areas where research on dengue is needed is that of
surveillance. Here, the literature suggests that much
could yet be done to improve on the sensitivity and
specificity of our surveillance programmes (Gubler
& Casta-Valez, 1991; Rigau-Perez & Gubler, 1997;
Gubler, 2002). Most countries continue to monitor dengue cases by using a passive surveillance
approach. An update of the table first published by
Gubler in 2002 (table 1) subjectively summarizes the
surveillance systems in countries where dengue is
endemic. Passive surveillance relies on disease notification by health-care professionals who have a
duty to report all suspected cases to public health
authorities. However, passive surveillance systems
are uniformly insensitive because of the low index
of suspicion for dengue during inter-epidemic periods (Gubler, 1989; Rigau-Perez & Gubler, 1997).
Limitation of passive surveillance

Two main problems are encountered in passive surveillance for dengue. These are:
Dengue infection leads to a wide range of

disease manifestations
Dengue infection results in a spectrum of clinical
outcomes: completely asymptomatic, undifferentiated viral syndrome, DF, DHF, dengue shock syndrome, and other severe manifestations such as
neurotropic disease and hepatic failure (George &
Lum, 1997). Passive surveillance using case definitions lacks specificity since many other infectious
diseases, such as influenza, chikungunya fever, the
viral haemorrhagic fevers, enterovirus infections,
leptospirosis, malaria, typhoid fever, etc., all present
with symptoms and signs that are similar to those
seen in patients with dengue in the acute phase of
illness (Halstead, 1997; George & Lum, 1997).
The use of passive surveillance alone also ignores
patients who present with undifferentiated febrile
illness or viral syndrome. This group of patients may
represent a large proportion of those with symptomatic dengue infection, depending on the age of the
patient and the strain of infecting virus (Hoang et
al., 2006). Any attempt to carry out passive surveillance among this group of cases will not be feasible.
However, mild viral syndrome may be of particular use in monitoring dengue transmission during
inter-epidemic periods when the incidence of classical DF and DHF is low (Gubler, 1998; Gubler &
Casta-Valez, 1991). In countries where dengue circulates hyperendemically, it is likely that emergence of
genetic variants with greater epidemic potential may
125
Table 1. Surveillance capabilities in the main countries where dengue fever or

dengue haemorrhagic fever is endemic
Country/
location
Surveillance
Passive
DF
Laboratory
capability
Active
DHF
DF/DHF
Serology
Epidemic
prediction
Virology
WHO South-East Asia Region

Bangladesh
++
India
Indonesia
+++
+a
Maldives
++
Myanmar
++
Sri Lanka
++
Thailand
+++
++
++a
WHO Western Pacific Region

Australia
+++
+++
++
+++
+++
++
Cambodia
++
+++
++
China
Lao Peoples
Democratic
Republic
Malaysia
++
+++
+++
+++
New Caledonia
++
++
+++
+++
Other South
Pacific islands
Philippines
++
Singapore
+++
+++
+++
+++
Tahiti
++
++
+++
+++
+++
++
+++
Viet Nam
WHO Region of the Americas

Argentina
++
++
Barbados
Belize
Bolivia
Brazil
++
++
+++
+++
Colombia
++
++
Costa Rica
++
++
Cuba
+++
+++
+++
+++
Dominican
Republic
Ecuador
El Salvador
French Guiana
Grenada
Guatemala
Haiti
Honduras
Jamaica
Lesser Antilles
Mexico
++
++
++
++
Nicaragua
++
++
+++
+++
Panama
(continued on next page)
126
Table 1 (continued)
Country/
location
Surveillance
Passive
Laboratory
capability
Active
Epidemic
prediction
DF
DHF
DF/DHF
Serology
Paraguay
Virology
-
Peru
Puerto Rico
++
++
Suriname
Trinidad
United States of
America
+++
Uruguay
Venezuela
++
++
++
++
+++
WHO African/Eastern Mediterranean Regions

Kenya
Senegal
Other African
countries
Pakistan
Saudi Arabia
+++
+++
+++
+++
Others
Province of
Taiwan (China)
Modified from Gubler (2002), with permission from SEARO/WPRO Dengue Bulletin.
DF, dengue fever; DHF, dengue haemorrhagic fever
The efficacy of the surveillance system and laboratory capability is rated as follows:
- does not exist ; + exists; ++ good; +++ best.
a
Does not include United States military, Centers for Disease Control, Institute Pasteur or WHO laboratories.
be partially responsible for the cyclical outbreaks

(Gubler et al., 1979; Gubler, 1987; Harris et al., 2000;
Gubler, 2004) since certain viral clades appear to be
more closely associated with increased transmission and severe disease outcomes (Rico-Hesse et al.,
1997; Wang et al., 2000; Bennett et al., 2003; Bennett
et al., 2006). Virological surveillance on cases that
present with mild viral syndrome may yield such
pre-epidemic isolates for comparative analysis.
Although more work will need to be done before
such data can be used for epidemic prediction, the
key to understanding dengue epidemiology lies in
better virological surveillance during the inter-epidemic periods (Gubler, 1989; Gubler & Casta-Valez,
1991; Gubler, 2004).
Variation in the case definitions used

The usefulness of the existing scheme for the classification of dengue and case definitions established according to the WHO guidelines has also
come under scrutiny (Sumarmo et al., 1983; Deen
et al., 2006). Experiences from various parts of the
world suggest that the usefulness of the case definition is not universal (Sumarmo et al., 1983; Dietz et
al., 1990; Harris et al., 2000; Balmaseda et al., 2005).

Perhaps more importantly, the WHO case definition underestimates the number of cases of severe
dengue among adults (Hammond et al., 2005). This
is a problem that needs to be addressed as dengue infection among travellers (Wilder-Smith &
Schwartz, 2005) and even in endemic countries like
Singapore, primarily affects the adult population
(Ooi et al., 2001; Ooi et al., 2006). Notwithstanding
the current debate over the WHO case definition,
there is also no consistency in the way these definitions are applied between countries where dengue
is endemic. Different countries classify DF/DHF
differently, and there is variation in the types of dengue cases that are included in surveillance reports,
countries adopting different criteria for classifying
dengue cases (Gubler, 2002; Deen et al., 2006). Some
countries report only DHF while others include DF
in their surveillance (Gubler, 2002). The existence of
all these different practices contributes to underestimation of the true extent of dengue transmission
and limits the ability to compare surveillance data
among countries and regions.
127
WHO and others have advocated active surveillance

since the 1980s (Gubler, 1989; PAHO, 1994; WHO,
1999). As previously recommended, virological
surveillance should be conducted on patients that
present with nonspecific viral syndrome, classical
DF, with haemorrhagic or neurological manifestation and on all patients with a fatal outcome following viral prodrome (Gubler et al., 1979; Gubler, 1989;
Gubler & Casta-Valez, 1991; Rigau-Perez & Gubler,
1997; Gubler, 1998). This approach, using sentinel
physicians, clinics, and hospitals, would result in a
more comprehensive surveillance for the transmission of dengue virus in the population. Yet, in southeast Asia where DF/DHF epidemics are reported
every 3 to 6 years, only Malaysia and Singapore
have an adequate laboratory capacity (Table 1). Most
other countries continue to rely on passive surveillance systems for DHF alone.
The long experience with dengue surveillance
and vector control in Singapore has recently been
reviewed (Ooi et al., 2006). One of the lessons learned
is the need for surveillance and vector control to be
carried out at the regional level (Ooi et al., 2006). If
it is not, countries that attempt to prevent this viral
disease are doomed to failure owing to re-importation of both virus and vector because of the rising
trend in global trade and travel.
In the Americas, proportionately more countries
report both DF and DHF, although good laboratory
support is still only available in Cuba, Brazil, Puerto
Rico, Nicaragua, and the USA (Table 1). However,
few countries carry out active surveillance for dengue disease. This is despite the efforts of PAHO in
the 1980s and 1990s to encourage Member States to
develop plans for disease prevention and control of
DF/DHF (Gubler, 2005; Panagos et al., 2005).
The situation in the Pacific has not changed since
2002; only Australia, Tahiti and New Caledonia have
good laboratory support for surveillance, although
active surveillance with epidemic prediction is carried out only in the state of Queensland, Australia.
This north-eastern state remains prone to dengue
outbreaks (Hanna et al., 2006), although very active
vector surveillance and control is in place to complement the existing epidemiological surveillance
(Montgomery & Ritchie, 2002; Ritchie et al., 2004).
To establish an active, laboratory-based surveillance
system, coupled with effective community-based,
integrated vector control requires both the necessary
public funds and political will. Unfortunately, most
countries where dengue is endemic have developing economies, and resources that could be channelled to prevention of disease transmission have
128
been directed to other more highly visible publichealth programmes. Although, the benefits derived
from an effective public-health approach to prevention and control (Gubler and Casta-Velez, 1991) are
significant, many countries have preferred to adopt
a spend-only-when-needed approach to vector control. This approach is often too little, too late, since
most emergency controls are only implemented
at the height of the epidemic (Gubler, 1989; Reiter
& Gubler, 1997; Reiter, 1998) and thus represent a
waste of public funds.
Solutions and issues to be addressed

Active surveillance
Given the type of information needed for dengue
surveillance, it is apparent that passive surveillance alone will not generate sufficient information
needed for the prediction of outbreaks. An active,
laboratory-based surveillance system and a better
understanding of dengue epidemiology are needed
for a more cost-effective prevention (Gubler 1989,
Gubler & Casta-Velez 1991, Rigau-Perez & Gubler,
1997). The universal use of the WHO case definitions, in the absence of new developments in this
field, is essential to enable the surveillance data to
be compared across countries and regions. In addition, the following will also be useful:
Standardization of the denominator
While the isolation of dengue virus has been
attempted in some parts of the world, the case definition used to select patients for virological surveillance varies from country to country. Furthermore,
there is often a lack of denominator, or the extent
to which the cases that fit the clinical entities are
sampled for dengue virus. This lack of denominator
limits the ability to make quantitative assessment
of dengue transmission and thus compare the effectiveness of various preventive measures when these
are applied in different places. As recommended
by Gubler (1979, 1989, 1991, 1998), virological surveillance should include patients that present with
nonspecific viral syndrome, in addition to classical
DF, with haemorrhagic or neurological manifestation and on all patients with a fatal outcome following viral prodrome. This approach would result in a
more comprehensive surveillance on dengue virus
transmission in the population.
Focus on urban centres
Since all public health authorities operate within
certain budget constraints, it would be important
to focus the surveillance effort on places where outbreaks are likely to emerge. The work by Cummings
et al. (2004) has provided good data to support
previous epidemiological observations that dengue

epidemics emerge from urban environments and
then spread to new areas (Gubler et al., 1979; Gubler,
2004). Thus, despite limited resources, focus on tropical urban centres, may enable a surveillance system
to be predictive of outbreaks.
agreement and standardization of the region of the

genome to be analysed will be useful in making the
sequence data more amenable to comparison across
countries and regions. Before this can be done, however, studies need to be conducted on the influence
of genetic changes on phenotypic expression (see
section on viral factors).
Laboratory support for dengue virus surveillance

Laboratory support is a critical component in surveillance (Gubler & Casta-Velez, 1991, Rigau-Perez
& Gubler, 1997). In particular, the laboratory should
be able to identify not only the presence of dengue
virus, but also its serotype, the severity of illness,
and whether the patient is experiencing a primary
or secondary infection. Furthermore, information
on the genetic sequence of the viruses circulating,
both during and between epidemics, will be of great
value to our eventual ability to predict epidemics.
Standardization of laboratory
methods
Currently, many countries where dengue is endemic
lack laboratory support for dengue surveillance. Among those that do have laboratory support, there exists variation in laboratory methods
used for virological surveillance. This is especially
true for molecular methods; the literature reports
a large number of real-time or end-point reversetranscriptase polymerase chain reactions (RT-PCRs)
for dengue virus. These assays vary in their sensitivity and specificity. Importantly, many of the new
serological assays have not been tested for crossreactivity to other viruses, especially co-circulating
flaviviruses like Japanese encephalitis, yellow fever
and West Nile viruses. Standardization of the laboratory methods used for virological and serological surveillance, along with the establishment of an
international quality assurance programme for such
laboratories would yield clear benefits.
Besides methods for the isolation or detection of
dengue viruses, standardization of the genes to be
sequenced for phylogenetic analysis should also
be determined and agreed upon such that the data
can be shared and compared within and between
regions. Currently, such studies have ranged
from partial to full genome sequences. Obviously
full genome sequences will be more informative,
although cost prohibits this from being done on a
large scale. New developments on mass spectrometry technology-based genomic sequencing (Liu et
al., 2005), chip-based high-throughput resequencing arrays (Wong et al., 2004) may lower the cost of
genetic sequencing when compared with the use of
capillary sequencers, and thus allow more viruses to
be analysed in this manner. If not, an international
lobal laboratory network for

G
dengue surveillance
It would be beneficial to establish WHO Collaborating
Centres for dengue virus surveillance. This would
complement and expand the DengueNet system,
where morbidity and mortality data are shared
among countries. Following the lead of the global
influenza and polio surveillance systems, establishing reference laboratories in different regions of the
world, which are responsible for more detailed analysis of the dengue virus isolates (such as phylogenetic analysis) may overcome the limitation posed
by the lack of public funds for such high-technology laboratory support in countries where dengue
is endemic. Countries with the necessary financial
and laboratory resources, such as Australia, Japan,
Singapore and the USA, could play such an important regional role. This, however, does not negate
the fact that every country where dengue is endemic
should have at least one laboratory that can support
virological surveillance.
Entomological surveillance
The clear need in entomological surveillance is an
index or a measure of vector population density that
may be predictive of epidemic dengue transmission.
Since eradication is not feasible, the goal of public-health preventive measures, in the absence of a
vaccine, is to maintain a vector population density
that is too low to support sustained viral transmission. It was thought from experience in Singapore
in the 1970s that a premises index (the percentage of
premises where Ae. aegypti larvae are found) of less
than 5% was sufficient to prevent epidemic dengue
(Chan, 1985). However, since the 1990s, it is obvious
that in Singapore, dengue incidence has increased
dramatically, despite an overall premise index of 2%
and below (Ooi et al., 2006). This, however, may be
owing to the insensitive nature of a national premise
index; despite the low national index, there are places
in Singapore where the density of the Ae. aegypti
population is high. Likewise, similar reports of limited ability to predict outbreaks have also been associated with the use of Breteau and container indices.
A complicating factor is the role of herd immunity.
Clearly, the vector-population densities required for
epidemic transmission are lower in regions with low
herd immunity (Newton & Reiter, 1992).
129
Emergency control
While vector population control in several instances,
such as in Singapore and Cuba, has clearly been successful although not completely sustainable, the
effectiveness of emergency control, as practiced in
most countries, is highly questionable. Most emergency control makes use of a combination of reducing the availability of larval habitats as well as
chemical adulticide . Chemical control, other than
using those chemicals with residual effect, has only
limited usefulness in either preventive or emergency
control. Yet, they continue to be used in many places
despite the lack of evidence of their effectiveness
(Gubler, 1989; Reiter & Gubler, 1997; Reiter, 1998). In
addition, their indiscriminate toxic activity may also
remove the natural predators of the dengue vectors.
In recent years, the development of powerful mathematical and computer tools allows for more sophisticated modelling of outbreaks of infectious disease.
Such models also allow for a theoretical assessment
of the ecological determinants of epidemic transmission, effectiveness of disease control and preventive
measures (Anderson & May, 1991; Focks et al., 1995;
Wearing & Rohani, 2006). Emergency control measures could perhaps benefit from the use of such a
tool to assess their efficacy. They might allow for
various control modalities to be assessed for their
effectiveness in reducing virus transmission, given a
range of likely scenarios. However, for such a tool to
be practically useful, validation of the mathematical
assumptions need to be carried out with actual epidemiological and entomological data.
A major problem with emergency control operations
is that, because of poor surveillance, they are usually implemented near peak epidemic transmission,
too late to have any impact on the epidemic (Gubler
130
1989, 1998). The reason for this is that implementing emergency control is a political decision, not a
public-health decision. To be fully effective, early
warning surveillance systems must have builtin triggers to automatically initiate the emergency
control programme.
In conclusion, DF/DHF has emerged as the most
important vector-borne viral illness in the tropical
world and this is likely to be the case well into the
21st century. In the absence of an approved vaccine,
the only means to control this disease is to interrupt the transmission of the virus. This will require a
sensitive and cost-effective disease- and vector-surveillance, coupled with a community-based larvalcontrol programme. Few countries where dengue
is endemic have such public-health infrastructure in place (Gubler, 2002). The challenge that lies
ahead is to put into operation these surveillance and
vector-control systems, while exploring how new
technologies in genetic sequencing can aid in our
understanding of dengue epidemics and capability
to predict outbreaks.
To establish an active laboratory-based multicentre surveillance system for dengue infection in
selected countries where dengue is endemic.
To establish a regional laboratory network to support virological surveillance and
information exchange.
To establish a system, coordinated by reference
centres, to share and genetically characterize
viruses isolated during epidemics and inter-epidemic periods.
To initiate research to understand the role of Ae.
albopictus in dengue transmission during epidemic and inter-epidemic periods.
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133
WORKING PAPER 7.2.

GEOGRAPHIC INFORMATION
SYSTEM FOR DENGUE
PREVENTION AND CONTROL
Ramon Martinez
WHO Regional Office for the Americas (AMRO)/Pan
American Health Organization (PAHO), Washington,
DC 20037, USA.
INTRODUCTION
The design and implementation of a sound information system is essential for the prevention and
control of dengue. The purpose of the information
system is to provide data and information needed
in any effort to prevent and control the transmission
of dengue, including in decision-making, planning,
evaluation and research. Owing to the complexity
of dengue transmission, access to data and information from all components of the control strategy,
including vector control, environmental and social
determinants, community behaviour, attitude and
practices, disease surveillance, the laboratory network, and health services at every level, is required.
Geographic information systems (GIS) and related
technologies have emerged as a new generation of
information systems with the capability to manage
spatial dimensions together with time, people and
other dimensions of interest, which is not possible
with the former information systems. GIS are currently recognized as a set of strategic and analytic
tools for public health, so the design and implementation of an information system for dengue control
with GIS capacity should be considered.
In this paper, a definition of GIS and some potentialities and limitations of GIS as components of the
information system for dengue prevention and control are presented.
DEFINITION
A generally well accepted definition of geographic
information system is an organized set of hardware,
software, spatial and non-spatial data, methods
and procedures, and personnel designed to input,
store, update, manage, analyse geographically referenced data, and display information in a synthetic and comprehensive way (Longley et al, 1999;
Castillo-Salgado et al, 2000). From a public health
perspective, GIS and related technologies such as
global positioning systems (GPS) and remote sensors (RS) facilitate: the locating of health events and
134
associated factors in space and time, the monitoring of risk factor behaviours and their relation to
health events, the identification and description of
distribution patterns of risk factors and health outcomes in time and space, health needs assessment,
the identification of vulnerable geographical areas
and population groups, priority-setting and monitoring and evaluation of the impact of health interventions, and assessment of health service response
(Castillo-Salgado et al, 2000).
The inclusion of people and procedures as part of
the definition is essential for GIS applications in a
public health context, given the need to link the science and methods of epidemiology to GIS mapping.
Without trained staff, one scenario is that GIS software will not be used at all, given the time and staff
constraints that exist in many public health agencies
and organizations. Alternatively, without trained
staff and standardized procedures, the technology may be used to develop maps that are invalid
or misleading.
POTENTIALITIES OF GIS FOR

DENGUE PROGRAMMES
GIS technology has potential for dengue prevention
and control programmes as follows:
GIS technology improves the ability of programme staff, planners, decision-makers and
researchers to organize and link datasets (e.g. by
using geocoded addresses, geographic boundaries,
or location coordinates) from different sources.
Geography provides a near-universal link for
integrating records from multiple information
sources into a more coherent whole. This ability to link datasets can help dengue programmes
integrate data from the five essential components
(epidemiology, entomology/vector control, community participation, laboratory, case management) and plan more cost-effective interventions.
For example, suppose that a dengue programme
could access the socio-demographic database of
the city, which is maintained by the local statistics
agency, and also the epidemiological dataset from
epidemiological surveillance, and the entomological dataset. Using GIS technology, the dengue
programme can combine these databases, mapping the demographic and social indicators by
block, pinpointing the location of breeding sites
and related entomological indicators, and including on the map the location of cases by residential
address to identify populations at higher risk of
dengue transmission and plan focalized action in
an efficient way. To take advantage of this potential, any dengue initiative and programme should
establish organizational changes and new collaborative links with institutions and departments in
the health sector, units of other sectors, governmental departments at regional, municipality and
community levels, and community organizations.
A review of the scope and necessary relationship
of the dengue programme information system
with other information systems based on a conceptual framework for reduction of dengue transmission is an essential step in improving it.
GIS, GPS and RS technologies provide dengue
programme staff with new types of data. For
example, with these technologies and when cartographic files (geo-referenced data) are not available, local programme staff and health workers
can use a GPS receiver to determine latitudelongitude coordinates for the locations of breeding
sites, cases and transmission sources, according to house lot, block and neighbourhood in the
city. Programme staff can also use digital imagery
from satellites and aerial photos to add details to
the map and improve the accuracy of information,
and help create/update cartographic databases.
For examples of digital imagery, see www.terraserver.com, www.spaceimaging.com and www.
ikonos.com. If a sequence of digital images for a
small area of interest is available, this can be used
to observe changes over time, such as in the development of housing, water bodies, roads, and landfills and other changes in land use and land cover.
GIS provides functions and procedures to facilitate the input of these types of data.
GPS receivers and personal data assistant (PDA)
technology used together can improve the collection of data in the field. For example, the entomological survey could be carried out using a
digital questionnaire implemented in a PDA. To
locate the breeding sites, a GPS receiver linked to
the PDA can feed in the longitudelatitude coordinates. Depending on the type of connection
and communication implemented, the collected
data can be entered into the GIS on return to the
office or can feed directly into the database using
internet services (web-based system). For a programme with fewer resources, the survey could
be carried out using paper forms to record, manually, the location coordinates read from the GPS
receiver. In this case, once in the office, each completed questionnaire should be entered into a digital format, spreadsheet or database table using
any well known and simple software. Experience
in implementing vector control information systems at local level in Central American countries
shows that vector control programme staff can
learn, in a one-hour demonstration, how to use
a GPS receiver as well as the complete procedure

for feeding collected data into the GIS.
GIS technology encourages the formation of data
partnerships and data sharing at the community
level. For example, to develop a map of coverage by, and frequency of, drinking water supply
to identify those areas in the city with no drinking water supply or with less frequent supply,
where people need to accumulate water for several days, the local dengue programme could
develop data partnerships with the local department in charge of drinking water supply. This is
only a simple example, but the same situation
pertains to obtaining data produced by other sectors. It is well recognized today that the inter-sectoral approach is key to success in controlling
dengue transmission. GIS technology facilitates
the linking of datasets from different sources, but
more important are the organizational aspects and
inter-sectoral agreements that guarantee access to
data from other organizations and help the processes of analysis, planning and decision-making
in dengue programmes.
Spatial analysis capability of GIS (distance,
proximity, containment measures) can be used
to improve entomology/vector control activities
and interventions such as focal treatment, and to
search for and destroy transmission sources. For
example, suppose the control programme, as part
of its continuous analysis, produces a map of the
city by block (the block as the unit of analysis). The
socio-demographic dataset is linked (geocoded) to
blocks, including the number of houses and population by age group. As a result of an entomological visit, some A. aegypti breeding sites are
found and their locations pinpointed on the map.
From the epidemiological component, the dengue cases are reported and their residence locations plotted on the same map. Creating a buffer
zone at a radius of 100 metres to the location of
breeding sites and dengue cases, programme staff
can determine the areas at higher risk of dengue
transmission and can answer questions such as
how many houses are within the high risk areas,
how many houses are within 100 metres of the
breeding sites, how many houses are within 100
metres of a house that has a dengue case, how
many houses are within an area close to the breeding sites and the house of the dengue case. The
answers provide information for deciding the
type of action and resources needed. How many
children are in these areas, and how many housewives live in the areas? The answers to these questions provide information about the people at risk,
and help to determine how soon action should
135
be taken. To complete this scenario, behavioural

indicators, community knowledge, attitudes and
practices, and availability of health services need
to be included. This leads to more evidence-based
decision-making in the dengue programme.
GIS technology enables work on multiple scales
in space and other dimensions (time, individual
and aggregated data). GIS allows the linking of
data for an individual with contextual information aggregated at a variety of geographic levels (e.g. household, block, neighbourhood, city,
municipality, state/department/province). This
capability enables the preparation of multi-level
spatial models to better evaluate and distinguish
biologic, contextual, and ecologic effects. For each
factor considered, a multi-level model can include
both individual predictors (data for each individual) and ecologic predictors (average or aggregate
measures) (Morgenstern, 1998). The information
system for the dengue programme should be
implemented as a multi-level approach, which at
the very least includes municipality, city/locality/community, block, and, if possible, house lot
and individual dengue cases.
GIS capabilities for spatial and spatialtemporal statistical analysis can improve the information system, giving better support to planning,
monitoring, evaluation, and decision-making in
the dengue control programme. For example, in
exploratory data analysis to assess the excess density of Aedes aegypti larvae across neighbourhoods,
blocks or vector-source sites in the city, tests for
spatial randomness could be used to evaluate if
a cluster is present, which would indicate there
are underlying factors (e.g. contextual, environmental, community practices). In a study in two
neighbourhoods in Iquitos, Per, spatial statistics
analysis was used to determine the spatial pattern
of Aedes aegypti and the containers in which they
develop (Getis, 2003). Spatial smoothing methods could be used to reveal the areas with high
and low risk of dengue transmission, producing maps of smoothed dengue incidence rates.
This method could also be used to stabilize the
rate when using small analytic units, a situation
in which unreliable estimations are due to small
numbers and/or small denominators.
GIS capability to synthesize and visualize information in maps. Compared with tables and
charts, maps developed using GIS technology
can be an extremely effective tool to help dengue programme technical staff synthesize, visualize and understand the problem. In addition,
action is more likely when the decision-maker can
see on a map that a problem is occurring in his/
136
her area of responsibility. GIS technology enables

detailed maps to be generated with relative speed
and ease. In turn, maps provide health workers,
dengue programme staff and community health
volunteers with useful information to advise community members, and health and other sector
decision-makers, about dengue as a health problem. For example, at a meeting with community
members including community health volunteers
and leaders, a map can be used to display the
neighbourhoods with high densities of Ae Aegypti
larvae, pupae and adults. The map can include the
location of potential vector sources (e.g. sites with
an accumulation of solid waste and containers,
houses identified as having domestic breeding
sites). With each click of the mouse, a point on the
map is selected and a box appears on the screen
showing a picture of the site and displaying the
conditions of this specific site. This dynamic feature of geographic information technology (the
ability to display information linked to a map) can
be very useful to programme staff and community members in identifying problems and searching for solutions. This GIS feature has been used
successfully by the dengue control programme in
Puntarenas, Costa Rica, in the process of advocacy with community leaders and health decisionmakers at local, regional and national levels.
LIMITATIONS OF GIS TECHNOLOGY

FOR DENGUE PROGRAMMES
Some of the current limitations of GIS technology
from a dengue prevention and control programme
perspective are as follows:
GIS technology is not yet a common tool in vector control programmes. In fact, few GIS applications can be found for prevention and control
of dengue and other vector-borne diseases. In
spite of the inclusion of a GIS component in
many health projects, few of these applications
have remained functional after the end of the
project. GIS being a young technology and the
users in need of training makes it difficult to integrate GIS technology into health programmes at
local/community level. Although there has been
enormous development of GIS software in the
last two decades, considerable room remains for
improvement and development of public health
applications. To this end, some national and
international institutions such as the US Centers
for Disease Prevention and Control (CDC) have
developed the mapping software EpiMap as part
of EpiInfo (Dean, 1999; CDC, 2006), the World
Health Organization (WHO) has developed the
software HealthMapper (WHO, 2006[a]), and the
Pan American Health Organization (PAHO) has

developed the software SIGEpi (Martinez-Piedra,
2001; PAHO, 2006). More details about these and
other softwares can be found in Martinez-Piedra,
2004. GIS technology could be linked with current information systems and specific information
tools at all levels, and specialized GIS software
products could be designed to support the new
challenges in prevention and control of dengue.
Accurate, low-cost street maps and other cartographic databases such as of neighbourhood, block
and house lot boundaries, are essential for dengue
control programmes. Without an up-to-date street
map, for example, control programme staff will
need to spend a lot of time and effort digitizing
the location of cases, or may not be able to map all
reports of cases and/or Ae. Aegypti breeding sites.
Accurate street maps are especially needed for
urban areas with fast growth. It is recommended
that dengue control programmes establish a coordination mechanism with the national institution that produces standard cartographic data.
It is also recommended that control programmes
develop basic capabilities for cartographic editing, including using GPS receivers and inputting
GPS data into GIS systems. Some countries do
not have a good structure or standard system of
addresses, even in urban areas, which makes the
geocoding process using residential address or
other references all the more difficult. In this case,
a specific standard coding scheme should be created (e.g. coding house lots and blocks).
Professionals, planners, technicians, and especially state/departmental/provincial and local
dengue prevention and control programme staff,
need training and user support in GIS technology, data, and epidemiologic methods in order
to use the technology appropriately and effectively. The cost of training programmes offered
by commercial GIS vendors can be a financial burden for any dengue programme, while GIS training programmes specifically custom designed for
public health professionals are still relatively limited. The GIS team at the Pan American Health
Organization (PAHO) has developed a course/
workshop on GIS applications in public health:
on each of four days, participants solve a public health problem using different epidemiological methods, including exploratory data analysis
and spatial analysis. This course/workshop has
been held in ten countries of the Americas Region
in the past two years as part of PAHO technical
cooperation to Member State institutions and professionals in epidemiology and analytic capacity.
Other successful training experience includes the
workshops for capacity building of vector control

programme staff at local level and for technology
transfer of the GIS model for malaria transmission control as part of the Regional programme of
sustainable alternatives for malaria vector control
without DDT in Mexico and Central America.
The cost of commercial GIS software is a barrier to extending the use and development of GIS
applications in public health and, particularly, in
dengue control programmes. GIS software for free
and/or low-cost distribution should be evaluated
under conditions of funding shortage, which is the
most frequent case in dengue endemic countries.
ADDITIONAL CONSIDERATIONS
Development of the information system for a dengue programme should take into account the
requirements derived from the Integrated Dengue
Management Strategy (EGI-Dengue) and its implementation in each specific country.
Every level of a dengue programme has its specific information needs. A significant number of
countries, at least in the Americas Region, have a
decentralized health system where more responsibility, in terms of resources and decisions for action,
is assigned to local level. The information system
should take these elements into consideration so that
the requirements of each level, including the global
initiatives, can be identified and redesigned if necessary to ensure data flow between levels according
to the requirements and availability of new information technology. As an example, the information system of the dengue programme should be articulated
with the global information system DengueNet
(WHO[b], 2006) and, at the same time, the experience and lessons derived from the implementation
of DengueNet should help in the design of information systems at national level.
The information system for dengue control should
provide solutions for increasing interoperability
among the information subsystems of each component (epidemiology, laboratory, entomology/vector
control, community participation). Similar solutions
should be implemented in the accessing of data from
the information systems of other sectors as needed.
This will require coordination and agreements with
institutions of the other sectors.
The information system/GIS and data model
should be multi-dimensional and provide a multilevel approach for responding to the complexity
of dengue transmission. As mentioned above, the
data model should allow analysis at the level of
137
municipality, city/locality/community, block, house

lot if possible, and the individual dengue case. With
micro (disaggregated) data, it is possible to identify
heterogeneity in the magnitude and frequency of
any measure across the area of analysis. The information system should help to create indicators at all
levels using standard procedures and methods.
Organizational, behavioural (motivation) and technical determinants of the performance of the information system should be taken into account to
guarantee its sustainability, data quality and information use for decision-making. Usually it takes a
lot of effort and resources of programme staff at local
level to capture data. Tons of papers (data forms) are
accumulated in health facilities/units of vector control, but transforming those data into useful information for analysis is very poor. A high proportion
of staff at local level even dont know the importance of correctly filling in a column of the weekly
aggregated report, nor have they ever seen guidelines for completing the data forms. Technical staff
are limited in their ability to analyse data, including
plotting/creating charts or calculating simple measures/indicators. The limited empowerment and
accountability of staff also contributes to their nonmotivation. More emphasis should be given to data
quality and the use of information for decision-making. At the same time, an effort is needed to build the
analytical capacity of staff at local and intermediate
levels. Improvement of the current information systems should help to make dengue programme procedures more efficient and cost effective.
EPIDEMIOLOGICAL STRATIFICATION
To be more cost effective in dengue control interventions, epidemiological stratification based on epidemiological, entomological and behavioural risk
indicators is needed. The information system should
help to provide data and/or indicators from these
138
components, as well as methods and procedures for

epidemiological stratification and priority-setting.
The methods should be implemented in a simplified
way and driven by the end-user in order to be used
on a continuous basis. Epidemiological stratification
has the main purpose of identifying groups of areas
(units of analysis) or strata which share the same
hierarchy/priority of risk factors, and consequently,
of applying specific and focalized interventions to
each stratum. Epidemiological stratification followed by priority-setting would be one solution to
staffing shortage and the impossibility of covering
large areas of housing when carrying out entomological surveys/inspections or source reduction/
elimination by limited vector control staff.
PRIORITY RESEARCH QUESTIONS

1. Does the current information system of the dengue control programme respond to the information needs and support planning, monitoring,
evaluation, and decision-making?
2. How should an information system for the dengue control programme be implemented/operationalized with the participation of all levels,
including the local/community level?
3. How can GIS, GPS and RS technologies be transferred to local/community level staff? How can
capacity be created at this level?
4. How can an information system be designed
taking into consideration the need to interoperate among the five essential components of
dengue programmes?
5. What are the core indicators of each component
of the dengue programme that the information
system should produce?
6. What indicators or risk factors needed by the
dengue control programme come from other sectors (e.g. water supply frequency and coverage,
water quality, climate measurements, land cover,
soil use)?
References
Castillo-Salgado C et al. Geographic information systems
in health: basic concepts. Washington DC, PAHO, 2000.
CDC. EpiInfo (http://www.cdc.gov/epiinfo/,
accessed September 16, 2006).
Dean AG. EpiInfo and EpiMap: current status
and plans for EpiInfo 2000. Journal of Public Health
Management Practice, 1999, 5(4):54-57.
Getis A et al. Characteristics of the spatial pattern
of dengue vector, Aedes aegypti, in Iquitos, Peru.
69(5):494-505.
Longley PA et al. Introduction. In: Longley PA et al,
eds. Geographical information systems: principles and
technical issues. Vol. 1, 2nd ed. New York, John Wiley
& Sons, 1999:1-27.
Martinez Piedra R et al. Software for mapping and

spatial analysis in epidemiology and public health.
Epidemiological Bulletin/PAHO, 2004, 25(4):1-9.
Morgenstern H. Ecologic studies. In: Rothman KJ,
Greenland S, eds. Modern epidemiology. 2nd ed.
Philadelphia (PA), Lippincott-Raven Publishers, 1998,
459-480.
Pan American Health Organization (PAHO). GIS
in public health. SIGEpi (http://ais.paho.org/sigepi,
accessed 16 September 2006).
World Health Organization (a). Public health mapping
and GIS. The HealthMapper (http://www.who.int/
health_mapping/tools/healthmapper/en/, accessed
16 September 2006).
World Health Organization (b). DengueNet
(http://www.who.int/denguenet, accessed 16
September 2006).
Martinez Piedra R et al. SIGEpi: Geographic information system in epidemiology and public health.
Epidemiological Bulletin/PAHO, 2001, 22(3):4-5.
139
WORKING PAPER 7.3.

ACHIEVING BEHAVIOUR
CHANGE FOR DENGUE
CONTROL: METHODS,
SCALING-UP, AND
SUSTAINABILITY
John Elder1 and Linda S. Lloyd2
1
Department of Behavioral Sciences, Graduate School
of Public Health, San Diego State University,
2
3443 Whittier St., San Diego, CA 92106, USA
INTRODUCTION
With the global resurgence of dengue and its more
severe form, dengue hemorrhagic fever (DHF), the
disease has re-emerged as a major threat to public
health. Typical approaches to dengue control and
vector control involve vertical programmes to reduce
the source of transmission. Physical (e.g. destruction
or other physical manipulation of water-holding
containers), biological (e.g. use of fish), and chemical (e.g. use of larvicides, spraying with systemic
insecticides) control methods can be successful if
substantial administrative and political support
is provided. However, such efforts often result in
short-term control as the areas become reinfested in
a fairly short period of time. Vertical vector control
programmes may be ineffective because communities are not active partners in the control actions but
rather are passive participants or recipients of the
control efforts (Gubler, 2002). In light of the restructuring efforts by ministries of health to decentralize
services, and of the generalized chronic underfunding of dengue control programmes, and in order to
provide effective control measures, it is critical to
address issues such as: (1) how to maintain quality of control in a decentralized system where decision-making takes place at regional, state, provincial
or municipal levels; (2) how to ensure that funding
is adequate to maintain programme infrastructure;
and (3) how to ensure, where traditionally staff have
been under the purview of the ministry of health
(e.g. communications, entomology) rather that the
regional or municipal health department, that there
are trained staff in technical areas at the local level.
Dengue may present as a mild illness episode, leading many people to underestimate its seriousness
and therefore the importance of controlling the mosquito vector. Some residents may be unaware of
how dengue is transmitted, and some may be unaware of the source of the vector mosquito; others
however may know where the Ae. aegypti mosquito
140
is produced and how the breeding sites can be controlled or eliminated but are not motivated to take
preventive action. Even those who do follow the recommended actions may still have Ae. aegypti or other
mosquitoes in their houses and, worse still, may suffer dengue infections if their neighbours do not participate in controlling domestic breeding sites, or
they may get bitten by an infected mosquito at their
place of work or study. Therefore, the issue for vector
control is not whether source reduction is effective,
but whether and how community participation can
be a part of that source reduction effort (Gubler and
Clark, 1996; Lloyd et al. 1994). Regardless of whether
the dengue control efforts take place through a centralized or decentralized system of care, the issues
are (1) how to meaningfully engage residents in sustained control actions; (2) how to effectively communicate with residents in ever-expanding urban
and semi-urban areas in light of reduced vector control staffing and chronic budget shortfalls; and (3)
how to measure the impact of residents actions
on Ae. aegypti breeding sites. This paper is divided
into two sections. The first will examine behaviour
change and dengue control efforts and the second
will examine delivery mechanisms for behaviour
change interventions in the community.
Behaviour change and dengue control

Although experts agree that community participation and modification of human behaviour at the
household level are crucial to effective control of Ae.
aegypti, the specific form that control efforts should
take continues to present a challenge to public
health officials. As the context for the present paper,
a review was conducted of research studies in community-based dengue control efforts published since
1995. This review was carried out through Internet
search engines (PubMed, Google Scholar, etc.) and
through reviews of existing paper files and libraryhoused journals. Given the nature of these searches,
most literature identified was published in English.
All behaviour change and/or health communication-oriented papers were reviewed with respect to
the following characteristics or variables:
country/setting
planning tool or approach used
level at which the intervention was directed (i.e.
household, school and other organizations, or
entire communities or regions)
person(s) who was(were) the source of communication or agent of change
dependent measure or outcome variable
research or evaluation design of the study or programme evaluation
results, and conclusions drawn by the authors.
The studies presented here were those for which

most of this information was evident in the article,
especially with regard to whether any evaluation
of the dengue control effort had been undertaken.
A summary of these studies (or programme evaluations) is presented in table 1.
In 2005, an evaluation of 11 WHO-supported dengue communication and mobilization programmes
using the communication for behavioural impact
(COMBI) planning tool (Parks and Lloyd, 2004) was
conducted in six South Asian and Latin America/
Caribbean countries (Elder, 2005). The conclusions
below are derived from this evaluation, as well as
from the review of recent programmes (table 1).
Progress and challenges

in community-based
behaviour change efforts
Multi-level behaviour and
community change
As can be seen in table 1, all programmes included
behaviour change efforts at the household level, and
some targeted the broader community and other
partners (schools were the most common partner).
However, vector control cannot be effective (or at
least, very effective) if carried out only on an individual basis. Thus, if mosquito breeding sites are
eliminated in one household but not in a neighbours or in public areas, the individuals in that
cleaner household are likely to receive some but little added protection against dengue. At some point,
however, a critical mass may be reached where a
sufficient number of vectors are eliminated in an
area or region, thereby reducing everyones risk for
contracting dengue. Thus, multi-level, vertically and
horizontally integrated programmes offer the best
solution to dengue control. For optimal effects, such
programmes would include not only communitywide (e.g. mass media) and house-by-house efforts,
but also those efforts of schools, worksites and other
organizations within the community.
At the community and regional levels, responsible agencies may need to identify programme
champions in order for their efforts to succeed,
while at other levels, groups of individuals may
share responsibility for maintaining programme
momentum and integrity. An assessment of different leadership modalities from the WHO evaluation
revealed that roughly half of the programmes were
led by strong, forceful individuals, while the others seemed to be more committee driven, with two
to several individuals sharing responsibility and
decision-making. Neither model seemed to have an
advantage over the other. In one case, the group of

individuals responsible for the effort seemed not to
agree on its key aspect, impeding progress towards
COMBI goals. In another, the programme champion
was such a strong individual that one would worry
about the future of the programme after leadership
turnover. But in most other cases, the model that
was chosen by the country or community seemed to
be the best one for them, a phenomenon consistent
with recent research on tailoring health communications. The advantages of a programme champion
are that their investment of energy and enthusiasm
will often achieve more results in the short term,
while the downside relates to the unclear implications for longer term sustainability and generalizability to regions without such individuals.
In any case, enthusiasm will at some point die out,
especially among non-paid volunteers, thus threatening sustainability. Setting limited periods of commitment or allowing the workers to move on at some
point to other health issues or even other communities (perhaps helping new neighbourhoods to start
their own front-line worker teams) could be among
the methods used to optimize commitment. Second,
plans must be developed to fade an effort out once
progress has been sufficient or nearly so. Booster
(or spot-check) home visits, for example, should be
increasingly infrequent, thus avoiding both health
worker and homeowner burn-out. Should entomological, epidemiological or behavioural data indicate
a need to renew a full intervention, this could then
be accomplished on a shorter-term scale, reverting
to spot-checks when needed.
While much has been written about social marketing
and many examples of successful social marketing
have been projects that took place over several years,
there are few examples of incorporation of social
marketing principles in dengue prevention and control programmes. According to the UK National
Social Marketing Centre (2006), social marketing is
the systematic application of marketing concepts
and techniques to achieve specific behavioural
goals, for a social or public good. In a comprehensive white paper on how to create a people-centred
public health strategy, the authors examine how
to improve disease prevention and health promotion within the British National Health System, and
they assess the potential of social marketing to move
beyond the prevention models currently in practice.
The authors state that social marketing can support
efforts to achieve an appropriate and effective balance between the role of individuals and the role of
the state and relevant bodies. Although an assumption might be that, in resource-rich countries, such
approaches would be systematically developed and
141
142
Examination of
existing cleaning
behaviours
El Progreso,
Honduras
KAP survey: revealed

knowledge, but lack
of action.
National KAP survey

+ formative research
to understand
household mosquito
behaviours
Fiji
Purwokerto
City, Central
Java,
Indonesia
Formative studies
with female heads
of household
revealed a desire for
clean water
Stages of change
in housewives
Bucaramanga,
Colombia
Dominican
Republic
Selection of
polypropylene
hoop with netting
after initial field
test of 19 different
jar covers
Planning tool
Cambodia:
three rural
villages
Country/
setting
vi
iv
iii
ii
Ref.
Table 1
C,O,H
C,O,H
C,O,H
C, H
C,O,H
Level
Social mobilization and communication via community

meetings, training, inspection role-modelling, and
identification/ endorsement of key stakeholders
Dasawisma: Ten-house alliance where house occupants
rotate inspection responsibility
Dissemination of source reduction kit with emphasis
on 3Ms: cleaning, covering, burying
Multi media campaign & community mobilization

Household visits by volunteers teaching effective
scrubbing of concrete basins, the primary water
storage device
Mass media component to support community effort
Multi media social mobilization to reduce breeding sites

- focus on control of tyres and drums
Breeding site reduction

Use of biological control (i.e. fish) to reduce larval
stages of the mosquito
Information transmission via national & local sources
Direct counselling from health educators
Mandatory community service for 11th grade students

with primary focus on door-to-door campaign
targeting housewives
Multi-level social marketing (print, theatre, radio,
social events)
Professional staff distributed deltamethrin-treated

polyester net covers for water
storage jars
Intervention/change agent
Monthly dasawisma evaluation to measure

level of preventive behaviour
(active, less active, not active).
Quarterly larval surveys
Tracking of dengue hemorrhagic fever
(DHF) hospital cases
House Index, incidence of dengue

cases, morbidity rates, exposure to
and understanding of key messages;
no. of households visited; media
exposure surveys
Entomological information,
epidemiological information,
household visits
Dasawisma activity
ratings every month
Pre- and postintervention
larval surveys
Pre- and postintervention tests
Pre- and 10-month

post-intervention test
on primary outcomes
among 100 randomly
sampled houses
Not indicated
Not indicated
Larval indices; no. of tyres in yard and

whether they were controlled
Pre- and postintervention: 8-month

knowledge test among
students; 5-year followup of households using
House Index
Pre- and postintervention crosssectional tests over

12 weeks
Evaluation design/
time period
Knowledge and awareness among students;

Household Index of mosquitoes in various
stages
Density of immature stages and adult

mosquitoes
Dependent measure/ outcome
House Index decreased from 20%

pre-intervention to 2%
with intervention
Surveys in 20022003
revealed awareness among
35% of housewives; need for
reinforcement of behaviour.
Increased control of tyres (from

34% to 61%)
Recommendations:
- Focus on behaviours rather
than only on knowledge; keep
behavioural targets simple
- Time-series evaluation rather
than pre- and post-intervention
basis
Simple and economical health

education messages
very beneficial
Knowledge increase
House Index dropped from 18% in
1998 to 5% in 2003
Recommend 3+ year intervention
to achieve impact
Effective at preventing growth in

larval stages; little impact on
adult mosquitoes
Results and comments
143
Pre-intervention
KAP and
entomological
survey
Examination of
failed prior attempts
In-depth interviews,
focus groups,
observation
of household
waste and water
management, KAP
and entomological
survey
Pilot efforts and

focus groups
Beyond-KAP
questionnaires
Entomological
surveys
Dengue virus
infections in
children 39
years old
Mrida,
Yucatn,
Mexico
Managua,
Nicaragua
Minimal success
with other
strategies
Planning tool
Colima,
Mexico
Johor
Bahru,
Johore,
Malaysia
Country/
setting
ix
viii
vii
Ref.
Table 1 (continued)
H, O
C, H
C, H
Level
Stratified cluster sample: 30 sentinel sites

Intervention: seven barrios in year 1, three more
added in year 2
Control: 20 barrios
Socializing Evidence for Participatory Action (SEPA)
communication strategy
Use of volunteers and brigadistas to encourage
further action
Communication/education campaign (interpersonal/

mass media):
- slogans
- strategically scheduled radio and television
broadcasts
- use of a spokes-puppet: Lela
- new behaviour introduced every 46 weeks (from
MayOct 1995), depending on complexity
Interpersonal, through activities in the home and
school environment (primarily focused on 4th graders).
1) Educational campaign to encourage community

participation in breeding ground elimination;
presentation, video, socio-drama; reinforcement
with prevention-related small gifts and printed
materials
2) Intervention with malathion spraying alone
3) Combination education & chemical
spraying treatment
COMBI approach:
1) Social mobilization (public relations campaign,
volunteers, youth)
2) Communication (buntings, self-evaluation
checklist, radio and newspaper advertisements,
point-of-service promotion, dengue-related radio
advertisements and talk shows)
Main emphasis on vector inspection/prevention and
early fever detection
Serological surveys among

young children
Beyond-KAP survey
Entomological indices to measure control

of vector Aedes aegypti
External evaluation
conducted at 25
months
Utilization of
evidence cycles
Containers positive
for Ae. aegypti
(requirement for
study participation)
marked for follow-up
and sampled over a
ten-month period
from June 1995 to
March 1996
Prospective evaluation
Pre- and postintervention KAP and
entomological surveys
(post-intervention
survey = after
6 months)
Prevalence of Aedes aegypti; KAP levels

regarding dengue and vector
KAP and entomological surveys

Composite behaviour score
Pre- and postintervention surveys

using multistage
stratified sample (926
of 1712 were paired)
Treatment-seeking
survey
Evaluation design/
time period
KAP survey results from household heads

or age 18+
Treatment-seeking behaviours in hospital
admitted dengue patients
By year 2, monitoring and

elimination of vector larvae
significantly more likely in
intervention barrios compared to
control barrios
Intervention barrios:
- Decreased use of insecticides,
increased knowledge sharing
and community leadership
- Decrease in entomological and
serological indices
Reported sense of personal
development in intervention
community
Decline in House, Container, and

Breteau indices
Positive increase in behaviour
scores after intervention
Significant increase in selfreported tyre behaviour
associated with no tyre-based
mosquito breeding.
Reduction in Basal House Index,

Basal Container Index, and Basal
Breteau Index
No significant changes in KAP
after the intervention treatment
99% of respondents in postintervention survey self-reported

Sunday household inspections vs.
71% in pre-intervention survey
59% of dengue-related hospital
admissions occurred within 24
hours of fever onset (42% in
control areas)
144
Viet Nam
Vanuatu
Puerto Rico
Country/
setting
Vector surveys,
KAP surveys (100
households in each
programme
commune)
Community-based
formative research
& audience
pre-tests
Increases in DHF
incidence prompted
need to determine
impact of pilot
community-based
programmes
Planning tool
Manples Community Project (est. 1998):

Establish community committees; workshop and
community meetings held to increase dengue fever
knowledge:
- Wan Smol Bag theater company model denguerelated preventive behaviours during performances at
community & school events.
Household donations to purchase plastic bags for small
water container disposal
Tyres: collection, disposal, and education (how to fill
with soil), mobilization of young people to drill water
drainage holes in immobile tyres
Volunteers apply temephos to larger water storage
containers not well received
Local manufacturer commissioned to create mosquitoproof water-storage containers
Vector control activities:
- Use of Mesocyclops in water storage containers to
prey on Aedes aegypti larvae.
- Institution of community clean-up campaigns to
collect and remove empty water containers
- Prevention education campaigns and interpersonal
reinforcement and motivation: church and
community meetings, media broadcasts, loudspeaker
announcements, drama performances, dengue
football competition, and print campaign
- Monitoring household mosquito prevention practices
& prevalence of mosquitoes
C, H
Community based prevention programmes:

Head Start:
- Children: mosquito-related activity book
- Parents: video on dengue fever prevention at the Head
Start centre and visit from Head Start personnel
Public school programme: 4th grade
science component
Posters and televised public service announcements
(PSAs): Dengue-Free Zone
Childrens museum interactive exhibit on Ae. aegypti
with guided tour
C, H
C,O,H
Level
i. Socheat et al., 2004.

ii. Luna et al., 2004.
iii. Leontsini et al., 2004; Leontsini, 2000; Chan et al., 1998;
Gordon, 1988; Gordon et al., 1990; Whiteford, 1997.
iv. Bera et al., 2004.

v. Fernndez, Martinez and Sherman, 2004.
vi. Kusriastuti et al., 2004.
vii. Suhaili et al., 2004.
C = community or regional; O = school, worksite or other organization; H = home; KAP = knowledge, attitudes and practices
xiii
xii
xi
Ref.
Table 1 (continued)
First project (2001) accomplished

full Ae. aegypti control in five out
of six communes
Second project (2003)
reduced larval populations by
99.6%100%
Small outbreak of dengue in 1998

f irst test of the intervention:
results = 100 cases recorded
with 0 mortality
Consultations with the community
were helpful in tailoring
intervention, especially with
respect to tyres
Higher levels of correct overall

knowledge on dengue and
decreased incorrect knowledge
among parents regarding the
mosquito life cycle
Greater impact on childrens
dengue knowledge than on
behaviour change and prevention
xi. Winch et al., 2002; Clark et al., 2004; Clark, 1992.

xii. Toaliu, Taleo, 2004.
xiii. Nam et al., 2004.
Annual evaluations
to monitor and
adjust activities
Quarterly vector
surveys
Analysis of dengue
prevention behaviours
in cross-sectional
sample of households
Frequent indices
recording throughout
programme
(19982001)
Comprehensive crosssectional evaluation
Evaluation design/
time period
viii. Espinoza-Gmez, Hernndez-Surez and

Coll-Crdenas, 2002.
ix. Galvn and Gutirrez, 2004.
x. Arostegui J et al., 2006; UBS Optimus Foundation, 2006.
Presence of mosquitoes, Mesocyclops, and

key breeding containers
Measurement of level of prevention
practices adoption through
standardized ratings
KAP survey results
Larval surveys measuring House and

Breteau indices; no formal evaluation of
behaviour change
Qualitative data: interviews and

focus groups
Impact of programmes on overall
infestation levels and on four specific
behaviours:
- Elimination of refuse
- Protecting used tyres from
the rain
- Maintaining larvae-free water
storage containers
- Use of commercial indoor
aerosol insecticides
Behavioural outcome variables: House
Index, Breteau Index, and containerspecific index
supported, in reality the public health and the health

care systems generally function as two separate entities with differing views on what prevention is and
who the target audience should be. It is important to
clarify that a national media campaign is not social
marketing, although it might be part of a social marketing programme. Regardless of the framework,
the following issues have been identified as essential to understanding prior to the development of
any community-based approaches.
Operationalization of behaviours
Few programmes provide clear definitions of specific human actions that can improve control as part
of the planning phase of community programmes,
and those that do often leap ahead to an examination of indications of mosquito breeding in the evaluation effort (table 1). A key element in any health
behaviour change or disease prevention programme
comprises the initial step of operationalization
of target behaviours. Operational definitions of
behaviours or the environments surrounding them
emphasize an objective observation of the physical
aspects of the behaviours. Thus, a behaviour can be
observed directly and reliably by examining the frequency, duration, or strength of the behaviour (e.g.
the frequency of applying larvicide, the duration of
cleaning used to reduce algae in a 55 gallon drum,
the intensity with which an individual appears to
scrub a cement basin), or the physical by-product of
that behaviour (e.g. the number of tyres left unprotected in a backyard vs. the number filled with dirt
or placed under a roof). Operational definitions,
therefore, go hand in hand with the nature of the
assessment used to arrive at those descriptions of
behaviour. Thus, phenomena such as knowledge
of breeding cycles or fear of mosquito contamination are not behaviours per se but inferred inner
causes of these behaviours. In practice it is often difficult for health education or vector control professionals to arrive at specific operational definitions of
behaviours, as they have frequently been trained to
emphasize these internal mechanisms.
In Ae. aegypti control efforts, it may be difficult to
observe the nature of the behaviour, and therefore
for monitoring purposes it is often necessary to
select physical by-products of the behaviour rather
than an observation of the behaviour. Nonetheless,
behaviours must still be operationally defined even
if their direct performance cannot be observed. It is
only through the operationalization of each behaviour that indicators can be developed to measure
whether or not the behaviour has taken place and to
what extent it has been carried out.
The operationalization of behaviour starts with the

selection of one or more specific target behaviours.
These behaviours are selected on a variety of criteria, the most important of which is whether the
behaviour itself seems to have an impact on the specific health problem. Nevertheless, many different
behaviours may potentially have such an impact
and the target behaviour must be narrowed down to
a manageable single or small group of behaviours.
Therefore, programme planners should also address
the following in the selection of target behaviours:
Feasibility: To what extent would the performance of the target behaviour result in negative
consequences for the individual performing it
(e.g. changing the taste of drinking water by adding temephos or fish)? Is the behaviour compatible with the persons current practice and with
sociocultural norms in the community? Does the
potential target behaviour require an unrealistic
rate or frequency or duration in order to be sufficient? What are the costs of the target behaviour
in terms of time, energy or other communityidentified expenditures?
One step at a time: Are there any existing
approximations to the target behaviour? Is the
behaviour already being performed perhaps
at a substandard but detectable level? Can this
behaviour be shaped to meet criteria? Are there
monitoring systems (see below) in place that
could be used to provide feedback to household
residents, health staff, and others who may gradually provide evidence of improvement in behaviour (Graeff, Elder and Booth, 1993)?
Some confusion about how to operationalize target behaviours derives from an inability to distinguish between whether the target behaviour
exists at all, or whether it does not exist in adequate strength due to performance deficit or
skill deficit. Performance deficit refers to a situation in which individuals may actually possess an existing skill but either do not receive
the reinforcement necessary for performing the
behaviour or receive inadequate reinforcement
and hence do not engage in adequate practice. As
part of the performance deficit analysis, understanding the functions served by containers (not
just their type and capacity) that are potential
breeding sites in the home is key to determining
the actions that can be implemented (Lloyd et
al., 1992; Galvn & Gutirrez, 2004). In contrast,
a skill deficit is simply that the individual does
not have the knowledge and practice associated
with adequate performance of a skill regardless
of whether he or she is motivated to engage in
that behaviour. Health communication and social
mobilization efforts take very different forms
145
depending on whether the bulk of the population evidences skill or performance deficit with
respect to the control of mosquito breeding.
Context of the behaviour: To further complicate
the definition and selection of target behaviours,
general community needs and capacity must
be examined simultaneously. In most studies,
both the target behaviours and the communities
selected evidence a range of difficulty. When
using the COMBI planning tool, programme
planners must focus on target behaviours that
will have a measurable impact on the specific
component of dengue prevention and control
being addressed through the communication/
social mobilization plan; the target behaviours,
however, are the result of a community-based
process through which the target population and
the programme planners identify and test behaviours for feasibility and effectiveness. Other programmes have used other models or processes to
define behaviours within a participatory community process (table 1). The target areas also evidence a range of characteristics in socioeconomic
status and accessibility, ranging from communities enjoying schools, roads in good condition,
utilities, general municipal services, and employment, to others with high crime and low employment, and buildings and roads in poor physical
condition. Some communities may be within a
few kilometres of their health centres while others are in remote locations. In other words, some
communities are relatively easy to work with,
while others are more difficult.
These independent dimensions of behavioural
and community difficulty may lead one to conclude that generally, when planners select a difficult community (e.g. poorer, with higher crime),
they may want to begin with an easier target behaviour (e.g. hermetic covering of containers). Should more accessible and prosperous
communities be selected, planners can be more
ambitious with respect to the choice of target
behaviours (e.g. frequent emptying and scrubbing of containers). It is axiomatic that poorer
communities need more resources to achieve
an equivalent result. Planners should focus on
what is truly practical for modest or resourcepoor environments (resource-poor referring to
both the programmatic environment and the
target community).
The monitoringfeedback loop

Related to the integration of efforts and the operationalization of specific, observable target behaviours is the need for an emphasis on information
146
sharing and feedback loops through monitoring and

evaluation. Few studies reported in the literature
(table 1) indicate that systematic monitoring and
evaluation have been carried out, and perhaps only
a few have used ongoing monitoring to improve or
reinforce efforts. Vector control and health education/communications staff seem to understand in
general what evaluation is, but how to conduct routine programme monitoring, and how to use those
data for programme adjustments throughout the
year, do not seem to be clearly understood. While
many national programmes can show that data are
collected for calculating entomological indices, few
can describe how these data are used during household visits since Aedes breeding sites are not prioritized, leading to the ongoing promotion of general
behavioural messages that have limited impact on
mosquito breeding as evidenced in continued high
larval indices.
Understanding of and enthusiasm for the COMBI
interventions among residents seems to be largely
a function of health workers giving individual, specific feedback at the household level. In fact, health
workers and volunteers seem to be more cognizant
of and capable when they use specific behaviourallybased feedback rather than more general exhortations to the community. Monitoring and evaluation
data must be accessible and apparent at higher levels as well. In the Nicaragua programme, maps with
colour-coded pins used to track neighbourhood outbreaks of dengue and malaria provided feedback to
all staff and community health volunteers regarding
epidemiological markers for programme progress,
and pinpointed specific blocks in the neighbourhoods where more intensive education and behaviour change work were needed. Staff and volunteers
met each month to discuss the neighbourhoods
and specific challenges, so that staff and volunteers
received continuous feedback and reinforcement for
their work, just as residents had received through
the self-retaining of records.
A weakness of all programmes examined to date
is the lack of behavioural indicators that have been
tested and validated for routine field use within the
context of national dengue programmes. Although
indicators have been created and tested in some
studies (e.g. in Mexico, Honduras), these indicators have not been operationalized within a dengue
prevention and control programme setting. There is
lack of staff with specific expertise in this area within
ministries of health, leading to ongoing, inappropriate use of entomological indicators as proxies for
human behaviours.
Delivery of behavioural interventions to

target populations
To date, special community-based projects may use
ministry of health staff, a combination of ministry
of health/externally funded staff, or may be completely externally funded. The ongoing challenge is
how to take promising results from a special project
and deliver them on a national scale, taking into consideration differences in vector ecology and in local
level capacity to manage programmes, lack of local
level staff with behaviour change expertise, political changes that impact programme services from
national to municipal level, staffing changes at all
levels, and chronic funding and staffing shortages.
Because most behaviour interventions have been
delivered through the existing structure of dengue programmes, for the most part, after a certain
period, the programme reverts to its original focus
and programming, that is, to entomological surveying and source reduction conducted by vector control staff. This is not only the case for behavioural
interventions, but laboratory and case management
also tend to function independently, even though the
need for integration of the five essential components
has been highlighted over the past years (PAHO,
1994 and 1999; WHO, 1999). In order to address
this issue, the Regional Program Office for Dengue
Prevention and Control of the Pan American Health
Organization developed a Strategy for Integrated
Dengue Management (EGI-Dengue), a process by
which countries functionally integrate the five key
components of a dengue prevention and control programme (epidemiology, entomology/vector control,
community participation, laboratory, case management) (PAHO, 2003). The EGI-Dengue process convenes a national technical expert group with two to
three experts in each of the five components to prioritize actions for each component area and then to
prioritize actions across the five areas. The national
group of experts monitors the implementation of
the national integration strategy via the logic framework (marco logico) developed as part of the process. The EGI-Dengue process has been under way in
the Americas since 2004.
BEHAVIOURAL RISK INDICATORS

Good programme planning is based on understanding who needs what service(s), when, and where.
Unfortunately, we do not have indicators by which
we can measure dengue behavioural risk, such as

blood pressure is used to indicate heart disease
and blood sugar to indicate diabetes. We need to be
able to stratify areas using epidemiological, entomological and behavioural risk indicators in order
to develop and then deliver an intervention mix
that will respond to the priority risk indicators of
that area.
KEY ISSUES FOR CONSIDERATION

IN BEHAVIOUR CHANGE
INTERVENTIONS
Programme leadership and planning for sustainable community participation and involvement.
Transfer of technical knowledge and skills in
planning participatory behavioural interventions
to health workers, community volunteers and
other partners at the local level.
Creation and maintenance of monitoring and feedback systems at the local and
national levels, including the development of
behavioural indicators.
Judicious mix of communication channels (interpersonal, mass media, publicity, etc.) to support
programme behavioural goals over time, based
not just on available funding but also on effectiveness for the local context.
PRIORITY RESEARCH QUESTIONS

How can indicators that measure behaviour change, and the extent of this change,
be operationalized?
What are the indicators of behavioural risk and
how can these indicators be part of a stratification process based on epidemiological, entomological and behavioural risk indicators?
Can the current, entrenched programme delivery
model, which is not, for the most part, achieving
the goals and objectives of controlling dengue
fever/DHF, be revamped, or do we need a new
programme model?
How can cost effectiveness be measured? Do
we need to measure the added benefit of each
individual component since we dont have a
fully integrated model that can be used as a
reference point?
How can we go to scale from pilot models of community-based communication/
mobilization efforts?
147
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149
WORKING PAPER 7.4.

DELIVERY ISSUES RELATED
to VECTOR CONTROL
OPERATIONS: A SPECIAL
FOCUS ON THE AMERICAS
Jos Lus San Martn and Olivia Brathwaite-Dick
Regional Dengue Program, Pan American Health
Organization/World Health Organization, Regional
Office for Dengue Prevention and Control, PAHO/
WHO Representation in Panama, Panama
Introduction
Vector control is an essential part of the control of
vector-borne diseases and effective preventive measures to reduce or interrupt their transmission. It also
plays a critical role in the prevention and containment of epidemics. With the gradual abandonment
of programmes for the eradication of malaria and
Aedes aegypti in the 1960s and 1970s and the decentralization of most vector control programmes,
capacity has diminished dramatically in many countries, although expenditure associated with vector
control is still responsible for a large share of the
budget of vector-borne disease control programmes
(Rodrguez Cruz, 2002).
While vaccines have been developed for other flaviviruses, such as yellow fever and Japanese encephalitis, the development of vaccines for dengue is
complicated by the need to incorporate all four virus
serotypes into a single preparation. An approved
vaccine is not likely to be available for 5 to 7 years;
the only way to prevent dengue transmission, therefore, is to reduce the population of its principal vector, Ae. aegypti (Ooi et al., 2006).
In many countries, health-sector reform poses
new challenges for programme delivery, including
decentralization and issues of selection, purchase,
procurement, and use and monitoring of insecticide application. Moreover, a limited number of
new, cost-effective chemical pesticides suitable for
public-health use have been developed in recent
years. This problem is particularly acute with regard
to larvicides suitable for use in stored water for
domestic consumption (Ooi et al., 2006). For these
reasons, better strategies for programme delivery
are needed.
150
BACKGROUND
Dengue has been successfully prevented via vector control in at least three instances. The first was
the highly successful, vertically-structured paramilitary hemispheric eradication campaign directed by
the Pan American Sanitary Board (Ooi et al., 2006).
Campaigns to eradicate Ae. aegypti were successful
between 1948 and 1972, when complete vector eradication was achieved in 21 countries of the Americas
(Rodrguez Cruz, 2002). The second was also a rigorous, top-down, military-like vector control operation in Cuba that was based on intensive insecticidal
treatment followed by reduction of available larval habitats (source reduction) in 1981 (Kouri et
al., 1989). The third successful programme was in
Singapore. However, none of these programmes
was sustainable, with consequent reinfestation and
a loss of the progress made in previous years (Ooi
et al., 2006).
Since the early 1970s, the World Health Organization
(WHO) has been actively involved in developing
and promoting strategies for the treatment and control of dengue. In resolution WHA46.31, the Fortysixth World Health Assembly in 1993 confirmed that
dengue prevention and control should be among
the priorities of WHO. In 1995, the WHO Global
Strategy for Prevention and Control of Dengue Fever
and Dengue Haemorrhagic Fever was developed
(WHO, 1999). It comprises five major components:
selective integrated vector control, with community and intersectoral participation; active disease
surveillance based on a strong health information
system; emergency preparedness, capacity building and training; and vector control research (WHO,
2000). Global and regional strategies emphasizing
the need for effective prevention, active surveillance
and outbreak preparedness have since been developed in the Regions of the Americas, Western Pacific
and South-East Asia.
The Pan American Health Organization (PAHO)
developed regional guidelines for dengue prevention in 1994 (PAHO, 1994) and, during the meeting
of its Directing Council in 2001, adopted Resolution
CD43.R4, which is a political declaration concerning
the alarming situation and regarding support for a
new generation of dengue programming (PAHO,
2001a). The new generation of programmes for the
prevention and control of dengue aims to strengthen
prevention and control through community participation and health education (PAHO, 2001b). In
2003, the 44th Directing Council of PAHO/WHO
approved Resolution CD44.R9, promoting the adoption of the Integrated Management Strategy for
Dengue Prevention and Control (PAHO, 2003).
The WHO Regional Office for South-East Asia developed a regional strategy for the prevention and control of dengue fever/dengue haemorrhagic fever
in 1995, revising it in July 2001. Different countries
formulated control programmes according to their
own priorities, infrastructure capacity, and resources
(e.g. Thailand, Indonesia, Myanmar, Sri Lanka). The
countries of this region have developed various
models of community-based control programmesbased source reduction, which have met with varying degrees of success (WHO, 2006).
Dengue fever is also a growing problem in the WHO
Region of the Western Pacific; more than 160000
cases of dengue and dengue haemorrhagic fever
were reported in this region in 2004. Despite the significance of dengue, activities for the prevention and
control of dengue are under-funded in many countries of this region (WHO, 2005a).
PROGRAMME DELIVERY: THE

TRADITIONAL MODEL OF
ERADICATION VERSUS CONTROL
Control and eradication are two different strategies, with different methodologies and targets. The
eradication strategy implies universal coverage of
every breeding site of the mosquito in every house
of every locality infested in the entire country, for
the total elimination of the vector and subsequent
permanent surveillance to detect reinfestation. The
up-front cost of this strategy is high, but once the
mosquito is eliminated, the cost of surveillance to
detect reinfestation is much smaller and the transmission of dengue and urban yellow fever is totally
prevented (Rodrguez Cruz, 2002).
The first eradication campaigns had great success
in the 1950s and 1960s primarily because there was
great political will for the implementation of the
strategy, which was reflected in internal and external financing for personnel, insecticides and equipment. Great emphasis was placed on the reduction
of sources of vector breeding; development and
implementation of policies for adequate use of
insecticides, including residual insecticides; and
management through vertical, centralized and wellorganized programmes based on strict discipline.
However, from the 1970s onwards, these results
were not maintained and receded notably; the programme lost political importance and priority in the
majority of the countries that had achieved eradication. Once reinfestation was detected, government
response was very late; high costs were associated
with providing materials, equipment, salaries and
benefits for the workers that were not kept in their

positions, and reinfestation was concomitant with
the appearance in Ae. aegypti of resistance to organochlorated insecticides and the fast and rampant
growth of surban centres. Currently, few countries
in the world maintain a strategy of eradication, for
example, in the WHO Region of the Americas only
Cuba maintains these principles of work (Rodrguez
Cruz, 2002).
A control strategy is based on preventing or reducing dengue epidemics and deaths caused by severe
dengue; a secondary focus is on the prevention of
urban yellow fever. This strategy identifies areas
at greater risk and concentrates efforts on these
areas in order to reduce, but not eradicate, the vector (Rodrguez Cruz, 2002). The cost of the control
strategy is less than the cost of the attack phase of
the eradication strategy, but higher than the maintenance phase of the eradication strategy (surveillance
against vector reinfestation).
An intermediate strategy between control and
eradication, especially when there are insufficient
resources for universal coverage, would be the total
elimination of the vector in limited high-risk areas,
with a progressive expansion of these areas as funds
permit, and with surveillance against reinfestation
(Rodrguez Cruz, 2002).
National programmes, especially in the Americas,
have been predominantly vertically structured;
however, there is a growing trend in recent years
towards decentralization of dengue control programmes. Unfortunately, this decentralization has
often been applied indiscriminately and with little
decentralization of financial and human resources,
with a consequent loss of control capacity.
Current status of vector control programmes

Currently dengue is presented as a health problem
whose magnitude exceeds the borders of the health
sector; the prevention and control of dengue is the
responsibility of not only the health sector but also
of other government sectors.
There are several barriers to addressing the shortcomings of dengue programmes. These obstacles
are very similar to those encountered in the past, but
current working models are not sufficiently comprehensive and participatory to address service delivery
problems in all its magnitude and dimensions. We
highlight some elements that make this relevant:
Macrofactors related to dengueenvironmental,
socioeconomic, political and social factors have
a strong impact on dengue, and are associated
151
with the re-emergence of dengue as a serious

issue. Climate change and ecosystem alterations
have provided ideal conditions for expanding
the geographical distribution of pathogens and
vectors, and increases in migration and international traffic favour the spread of the vector and
the disease.
Unprecedented population growththe worlds
population has tripled in the last 70 yearsis
also contributing to increasing the number of
vector breeding sites. Also, the presence of dengue in large urban centres, and especially in megacities (e.g. Rio de Janeiro, So Paulo, and Caracas),
associated with urbanization that is neither
planned nor controlled and poverty, with the
absence of basic services (electricity, running
water, sewer systems, refuse collection), poses
new challenges and requirements for prevention
activities and control. Such activities are expensive and require great coordination and synchronization, and the incorporation of extrasectorial
actors, such as the tyre industry.
The local health services, now politically and
administratively responsible for disease prevention and control programmes, are generally not
sufficiently prepared for the management of dengue control programmes, and resources are usually insufficient. The lack of human resources to
cover the large number of houses reduces the
quality of work, programme managers do not
know how to prioritize areas of high complexity,
and the work is converted into a routine household inspection with standard container-control
messages offered to homeowners and businesses.
The sustainability and continuity of control
actions are always given a lower priority than
other health demands and policy, with which
they compete.
Elements such as employment instability of the
workers (i.e. vector control inspectors), training methods that continue to employ curricula content that does not lead to participatory
models for vector control, and use of old control/
eradication models in which the vector control
inspector carries out control actions during his
household visit prevent the transfer of responsibilities and creation of abilities to prevent and
control Aedes breeding in the household and surrounding areas.
In general, ministries have very few external partners and little ability to negotiate partnerships.
There exists little communication, collaboration
or integration between key components within
ministries of health, (epidemiology, entomology,
environment, health promotion, laboratory, etc.),
as well as with other ministries, and governmental, nongovernmental and community agencies.
152
Establishment of partnerships, traditional and

non-traditional, may help to address the problem
in all its magnitude and dimensions.
Countries carry out vector control primarily using
insecticides. Frequently, larvicides are applied to
containers that could be destroyed or better managed; there is excessive use of ultra-low-volume application of adulticides in areas where
there is no transmission of dengue. This method
is useful as a support for the suppression of epidemics, but not for routine control (Rodrguez
Cruz, 2002).
Participation of the community in the prevention
and control of dengue the community has transferred the responsibility for Ae. aegypti control
to the health sector as a result of the long-standing traditional vertical model (Toledo-Romani
et al., 2006b). It is limited to response to official
demands and control actions, and is not viewed
as an empowerment process for the community.
The work dynamic of the vector control inspectors and their interactions with families can be
paternalistic; their focus is the destruction of the
containers in which mosquitoes breed, with little ability to motivate residents towards ongoing
environmental management of their premises.
There is an evident need for matching the interests of residents and health-care providers in
order to attain a significant social mobilization
(Toledo-Romani et al., 2006b).
Incorporation of the Communication-forBehavioural-Impact (COMBI) planning methodology is opening new roads; in contrast to
intensified routine control activities, a community-based intervention approach promises to
be sustainable (Mosquera et al., 2006; ToledoRomani et al., 2006a). There is still a need for
monitoring and impact assessment of this planning instrument, and we cannot say that has
been introduced and generalized in all programmes in the Americas.
Water-supply and waste-management systems are
limited in many high-risk areas; this facilitates
vector proliferation and persistence. We point out
that the high presence of plastic containers that
can contain water and that are not biodegradable also facilitates vector persistence, because
these containers remain for long periods in the
environment and must be eliminated properly
by man.
Operational research on new approaches and control strategies has not been sufficient to investigate and monitor its impact.
COMBI: http://www.paho.org/english/ad/dpc/cd/
den-step-by-step.htm
The role of the vector control inspectors:

what do we expect from them?
Any strategy or programme plan that is adopted
may need the presence of field inspectors, employed
either by the public or the community. The household visit is important as a preliminary and basic
prevention activity for health promotion. These visits are a great opportunity (particularly in countries
at risk of dengue, where large groups of the population have a low level of education), to review and
determine the application of control actions.
However, the function of vector control personnel should be analysed seriously; traditional programmes do not have a sufficient impact in disease
control owing to severe and ongoing reductions
in personnel. A significant programmatic change
is needed, and health services must have personnel who are able to interact with residents and who
can assume a greater role as a health promoters and
evaluators, without losing the point of entomological surveillance and vector control. These personnel should be part of epidemiological surveillance
teams and the actions that they recommend or take
should not be routine, but should be based on an
analysis of the situation.
The great challenge is to provide these field staff with
good communication skills, thus training is very
important. Given that residents must also assume
some responsibility and capacity for self-care, it is
hoped that having better relationships with householders will improve the development of practical prevention actions, taking into account that the
residents may need not only increased knowledge
related to health, but also skill-building to carry out
the recommended behaviour. Changing the current passive nature of the house visits by emphasizing communication and interpersonal contact can
help transmit more appropriate messages that may
modify behaviours related to breeding sites of Ae.
aegypti. For this, the system has to provide adequate
tools and materials for the inspectors that respond
to this objective.
Dengue prevention and control programmes need
to work with the community, women, young people and children directly; using organized networks
that exist in the community is one way to achieve
this. This may be a means to create comprehensive
control with co-responsibility that is led jointly by
the residents and municipalities; the programmes
will have to change from the traditional model
toward a participatory model, giving a comprehensive nature to the control measures. To achieve this
end-point, models of mass interactive community
institution communication may need to be developed and tested.
STRATEGIC PARTNERSHIPS
FOR VECTOR CONTROL
Strategic partnerships for dengue prevention
and control have been identified as an important
source of support for vector control programmes.
These partnerships can promote the coordination
of actions among the government, health sector and
other social and economic sectors, volunteer and
nongovernmental organizations, churches, local
authorities, industry and mass media. Furthermore,
the importance of adapting the programmes to the
realities and local needs is recognized, taking into
account social, cultural and economic differences.
Stateindustrycommunity partnership
Environmental management that promotes the elimination of vector breeding sites should be a priority
in control programmes. Programmes that involve
the creation of strategic partnerships should include
intersectoral participation of public and private corporations with a strong component of community
participation, as well as participation of different
ministries and institutions with a greater direct relationship to the various components that lead to continued dengue transmission (e.g. ministry of health,
of protection of the environment, of finance, of construction, of transportation, of sports), universities,
nongovernmental organizations, importers of tyres,
tyre repair shops, municipal government, among
others. There could also be partnerships between the
ministries of health and education, promoting dengue prevention during the teaching process among
elementary-school students.
These partnerships can be promoted by the state,
through the promulgation and implementation of
laws that serve as a framework. For example, Puerto
Rico, the United States of America, Spain, Costa
Rica, Israel and Brazil have established decrees or
laws for the adequate control and management of
used tyresthe habitual breeding site of the vector
in many countries and for which few or no adequate
mechanisms exist for final disposal. Experience
gained in Brazil is a positive example. In Brazil, the
tyre-recycling industry employs more than 20000
people directly, and involves nearly 15 companies and 21 factories. To date, 18 municipalities in
8 states are promoting tyre recycling. Other models of application of this have been observed such as
the creation of artificial reefs (Colombia, Malaysia,
Thailand, the Philippines), use of tyres in the cement
industry (Brazil, Barbados), and use of tyres in
153
c onstruction, lamination and for exportation. Used

tyres also have uses in the construction of athletic
fields, as roofing materials, vibration insulation and
carpets, among others.
Framework for Integrated Vector Management provides a basis for strengthening vector control in a
manner that is compatible with national health systems (WHO, 2000a).
Ecoclubs
The integrated vector management (IVM) process aims to be effective and efficient. It uses indicators of impact on vector populations and disease
transmission, and employs approaches compatible
with local health systems. It is also robust enough to
allow for effective planning and decision-making to
take place at the lowest possible administrative levels (e.g. community level). It encourages a multi-disease and multi-strategy control approach whenever
possible, and efficient integration with other disease control measures as well as the application of a
range of interventions. Such a commitment requires
an approach that effectively integrates the roles of
the various sectors, including health, within a strategic management framework. Finally, IVM can also
strengthen the rational use of insecticides, increasing their efficiency and impact and for the achievement of the Millennium Goals (WHO, 2004b).
Ecoclubs are democratic organizations, with more

than 15000 volunteers distributed in 600 networks around the world (International Network of
Ecoclubs, INE**). These networks link actions to various institutions of the community, visualizing an
improvement of the quality of life. Ecoclubs promote actions in the healthenvironment axis, such
as strategies for the rational use of water, dengue
prevention, and waste management, among other
topics. With sensitization campaigns coordinated
with other institutions and communities, Ecoclubs
involve neighbours via the use of participatory
strategies and actions in the implementation of programmes that are characterized by their sustainability and that can be evaluated practically.
These experiences have demonstrated that large
budgets are not necessarily needed to implement
community programmes for the prevention and
control of dengue; it is this philosophy, including different social actors for a common cause that
Ecoclubs promote. But management guidance is
needed and this is a role that should be played by
health workers. However, there still are large gaps
in information on the overall impact of the work of
these associations.
OTHER PERSPECTIVES AND NEW

TOOLS FOR VECTOR CONTROL
Integrated vector management
Vector control has mainly relied on the use of chemical insecticides and has not been very successful
owing to human, technical, operational, ecological, and economic factors. Problems of insecticide
resistance, costs and environmental concerns have
resulted in a reduced reliance on insecticides, and
an emphasis on the need for other vector control measures involving environmental management, biological control and personal protection. In
addition, the Stockholm Convention on Persistent
Organic Pollutants (POPs) adopted in 2001 (UNEP,
2001) requires a reduced reliance on, with a goal
to eliminate, the use of DDT and other intentionally produced POPs and the promotion of research
and development of safe alternative products, methods and strategies. The WHO Global Strategic
** Ecoclubs International: http://www.ecoclubes.org/DENGUE/
ingles/dengue.asp
154
IVM has been effectively applied in several regions

and steps towards its implementation have been
taken in the WHO South-East Asia, Western Pacific,
Americas, Eastern Mediterranean and African
Regions (WHO, 2005a, 2005b, 2004b). Good examples
of its application have been provided by researchers
in Viet Nam (Kay & Vu, 2005; Vu et al., 2005) and in
Africa (Mukabana et al., 2006). IVM is based on the
premise that effective control is not the sole preserve
of the health sector but requires the collaboration of
various public and private agencies, and community participation. The engagement of communities
is a key factor in assuring sustainability, but further
operational research is required to develop surveillance systems that are practical, affordable, effective
and acceptable so that community-based IVM can
be implemented (Vanek et al., 2006).
Ecohealth approach for dengue control

and prevention
The aim of the Ecosystem Approach to Human
Health (Ecohealth)*** is to improve community
health through a holistic approach to the management of complex socio-ecological ecosystems. The
International Development Research Centre (IDRC)
of Canada has made an emphasis on assessing the
potential of the Ecohealth approach to contribute to
the prevention of vector-borne diseases, and more
specifically with dengue (Lebel, 2003).
*** Ecohealth: http://www.idrc.ca/in_focus_health/
The Ecohealth approach is being supported by the

Tropical Diseases (TDR), an independent collaborative programme financed jointly by the United
Nations Development Programme (UNDP), the
United Nations Childrens Fund (UNICEF), World
Bank and WHO. With support from IDRC, TDR is
applying the Ecohealth approach in two research
programmes in South America. Furthermore, the
Pan American Health Organization (PAHO) provides support for the implementation of this
approach in two projects on dengue in Central
America and the Caribbean. These projects are also
supported by the United Nations Environment
Programme (UNEP). In Guatemala, researchers are
developing a Community strategy for the reduction
of dengue and diarrhoeal diseases in urban ecosystems; on the border of Guatemala and Mexico the
Development and validation of a community strategy for the reduction of the risk of dengue and diarrhoea in urban ecosystems is being carried out; and
in the City of Havana, Cuba, a model for sustainable
development and healthy municipal environments
in an approach to ecosystem in human health for
the prevention of dengue at the local level is being
tested (Lebel, 2003). In these large agglomerations,
many groups with diverse interests interact: the private sector, civil society, municipal authorities, different ethnic groups, castes, and social classes, men
and women. All play a role in the management of
the urban ecosystem.
Integrated Management Strategy for Dengue

Prevention and Control (EGI-Dengue)
The Integrated Management Strategy for Dengue
Prevention and Control in the Americas (EGIDengue) addresses the issue of how to achieve
effective programmatic integration of prevention
and control actions. This introduces a new form of
technical cooperation between PAHO and member
countries through the dengue task force (known by
its Spanish abbreviation GT-Dengue International).
The GT-Dengue task force is a group of technical
experts from across the region who, starting with
a regional analysis, works with the dengue technical teams in each country to develop a national
strategy for integrated operations. From these initial
work plans, efforts are made in consultation with
other countries to change existing programme practices and implement the new integrated strategy for
dengue prevention and control. The new integrated
management strategy is horizontal, intersectoral,
inter-programmatic, and seeks changes in behaviour
at all levels to reduce the risk factors for dengue.
The purpose of this strategy is to achieve a sustainable
national strategy that allows a functional integration of actions among its key components (social
communication, epidemiological surveillance, entomology, patient care, laboratory and environment),
designed by the country with technical cooperation
from the GT-Dengue, using a multisectoral, intersectoral, and interdisciplinary (integrated) approach,
based on new practices that permit the evaluation and continuity of the activities, with national
resources (PAHO, 2003).
The Integrated Management Strategy for Dengue
Prevention and Control demands research on new
indicators that better measure the risk of transmission, and environmental and behaviour indicators
in order to know what the behavioural impact has
been. Indicators are also needed to investigate new
or modified existing practices both for surveillance
(e.g. MosquiTrap, LIRAa), control (e.g. impregnated
curtains, dabbed bleach), and management and integration processes that each country prepares using a
log-frame matrix (EGI-Dengue).
Communication for Behavioural

Impact (COMBI)
COMBI is a novel approach in the design and implementation of behaviourally focused social mobilization and communication actions for the control of
communicable diseases. It is a planning methodology for programme managers to prepare, implement
and evaluate the social mobilization and communication interventions developed as part of the integrated plans (Mosquera et al., 2006).
The general strategy for preventing and controlling
dengue and dengue haemorrhagic fever is based on
promoting behaviour changes that lead to involving the community as a partner in controlling the
disease, particularly the vector. In order to achieve
this, dengue communication programmes should
have two primary aims: converting information into
practice and working with the community to adopt
and maintain appropriate and relevant prevention
and control measures. The new generation of programmes should be designed taking into account
the local sanitation structure (water distribution and
waste disposal) as well as information on community organizations and the roles of different family
members. Furthermore, new vector control models
should incorporate all ten components of an integrated programme (PAHO, 2001b): epidemiological surveillance, intersectoral actions, community
participation, managing the environment and basic
services, patient care, case reporting, education,
rational use of insecticides and vector control, training, and preparing for emergencies. Communication
155
should be aimed at supporting positive mosquitocontrol behaviours among individuals and the community, and their empowerment to identify and
carry out community-relevant prevention and control measures.
Geographic information systems

While investigating the spatial patterning of health
events and disease outcomes has a long history, the
development of geographic information systems
(GIS) has facilitated the inclusion of a spatial component in epidemiological and entomological studies.
GIS is a computer system that allows the collection,
storage, integration, analysis, and display of spatially referenced data. In the field of health, GIS has
been widely used for disease mapping of different
pathologies, in analysis of space and spacetime distributions of disease data, in identifying risk factors,
and in mapping risk areas. In most studies, each
patient or person exposed to a disease is located at
the residential address, and these locations are integrated into GIS for mapping and analysis. Because
GIS allows epidemiologists to map environmental factors associated with disease vectors, it has
become especially relevant for the surveillance of
infectious and vector-borne diseases such as dengue
and malaria (Napier, 2003; Tran et al., 2004).
Examples of the use of this technology include the
geographic analysis conducted for the 20012002 outbreak of dengue fever in the state of Hawaii (Tran et
al., 2004). In another study, a GIS spatial/temporal
analysis depicting the spread of the disease and a spatial dengue threat model (DTM) were created. In addition, GIS case-clustering and mean/median distance
comparison analysis of homes in rural and semi-urban
areas was conducted. This model may be adapted for
use as a predictor in other arbovirus (arthropod-borne
virus) outbreaks in various geographic locals.
Rapid Survey Index for Ae. aegypti for

estimating the Breteau and
house indices (LIRAa)
Simpler methods for sampling have been proposed,
with the objective of facilitating the acquisition of
information that contributes to the evaluation of
health-services programmes through the conduct
of systematic and periodic research. There are simplified methods to estimate entomological indices,
associated with acceptable errors of margin that are
also rapid and economical. Such is the example of
the Rapid Survey Index for Ae. aegypti for estimating Breteau and house indices developed in Brazil
(LIRAa in Portuguese). The implementation of this
system permits the dengue programme manager to
156
target control measures to the areas of highest risk,

thereby permitting better use of human resources
and of available materials not only during routine control activities but also in critical periods
with higher numbers of cases that might indicate
an outbreak. The National Program for the Control
of Dengue (PNCD) of Brazil, launched in July 2002
by the Ministry of Health, uses this methodology
as a component of epidemiological surveillance
(Ministrio Da Sade, 2005).
MosquiTRAP
MosquiTRAP is a novel, simple, easy-to-use, lowcost, and efficient trap developed to catch Aedes
mosquitoes. It relies on visual cues and synthetic
oviposition attractants (AtrAedes), based on volatile substances identified from grass infusions.
Compared with ovitraps, the MosquiTRAP allows
the identification of mosquito species in the field,
thus saving time and avoiding laboratory routines such as counting eggs and larval identification. Trapped mosquitoes can also be used for virus
diagnosis. New entomological indices are: (a) the
positive MosquiTRAP index (PMI), which is the percentage of positive traps; and (b) the adult density
index for Ae. aegypti and Ae. albopictus. Field data
can be collected using hand-held PDAs (personal
digital assistants) and then loaded directly into a
GIS program, for an efficient determination of local
entomological indices. At the moment, a national
monitoring programme in Brazil using this technology is being established (Eiras et al., 2004).
The new technology for the monitoring and generation
of indices for entomological surveillance, composed of
MosquiTRAP, AtrAedes for oviposition, and a system
of computerized monitoring is promising and should
be considered for possible future use as results on efficacy and efficiency are published in the literature.
RESEARCH AND DEVELOPMENT:

OBSERVATIONS
Efforts based solely on chemical vector control have
been insufficient in modern times. Moreover, evidence demonstrates that educational measures do
not modify the behaviours or habits of the population (Texeira et al, 2005). Thus, as a vaccine is not
available, further dengue control depends on potential results from basic interdisciplinary research
and intervention studies, integrating environmental change, community participation and education,
epidemiological and virological surveillance, and
strategic technological innovations aimed at stopping transmission. Some examples of these research
efforts are:
The Innovative Vector Control Consortium

(IVVC) will address the market for new insecticides by developing a portfolio of chemical
and technological tools that will be directly and
immediately accessible to populations in the
developing world (Hemingway et al., 2006).
Searching for new bioactive, environmentally
friendly and biodegradable natural insecticides and repellents, particularly from botanical
sources in Thailand, China, Libya, Burkina Fasso,
India and other countries (Choochote et al., 1999,
2005; Bassole et al., 2003; Tuetun et al., 2005;
Kamsuk et al., 2006; Chaiyasit et al., 2006; Ravi
Kiran et al., 2006; Amer et al., 2006).
Ae. aegypti population replacement: A proposed
strategy to aid in controlling the growing burden
of vector-borne disease is population replacement, in which a natural vector population is
replaced by a population with a reduced capacity for disease transmission. Endosymbiotic
Wolbachia bacteria are potential transgene drivers. Stable infections of wAlbB Wolbachia were
established in Ae. aegypti and caused high rates
of cytoplasmic incompatibility (that is, elimination of egg hatching). Laboratory cage tests demonstrated the ability of wAlbB to spread into an
Ae. aegypti population after seeding of an uninfected population with infected females, reaching
infection fixation within seven generations (Xi et
al., 2005).
A web-based multimedia spatial information system was used to support a study of the re-invasion of Ae. aegypti in the deserts of the south-west
United States/north-west Mexico. The system
was developed by applying open geospatial consortium and worldwide web consortium open
specifications and using open source software.
The system creates a sensory-rich environment,
one that allows users to interact with the system to explore connections among data (maps,
remotely sensed images, text, graphs, 360 degree
panoramas and photos), visualize information, formulate their own interpretations, generate hypotheses and reach their own conclusions.
(Moreno-Sanchez et al., 2006).
Evaluating the practicality of a survey method
based on the rationale that certain water containers are particularly productive of the dengue
vector, Ae. aegypti and whether this can consistently identify and classify particularly productive
classes of container, and so provides guidance
on the development of targeted control strategies. This was done as study involving nine Latin
American, Asian, and African countries (Focks &
Alexander, 2006).
The time has come to restore vector control to its key

position in the prevention of disease transmission,
albeit with an increased emphasis on multiple measures, which may include use of pesticides and environmental modification, and with a strengthened
managerial and operational capacity (Townson et
al., 2005). Today, prevention and control of dengue
require consideration of a wider perspective than
simply tropical disease. Many of the affected countries are also some of the poorest. Approaches that
are realistic for limited infrastructures need to be
urgently developed. A systematic approach and a
clear international research agenda can quickly bring
forward the frontiers of knowledge. Better understanding of the above will not only feed into operational policies for dengue control, but also provide
fertile terrain for vaccine application strategies in the
future. Accelerating the research programme, with
emphasis on mechanisms of transmission dynamics, validation and improvement of existing or new
vector control methods and their application, partnership building, and formulation of guidelines for
research will help in these strategic areas (GuhaSapir et al., 2005).
Based upon and guided by scientific knowledge and
operational research, and subject to routine monitoring and evaluation of control activities, the strategies and interventions need to be adapted to local
vector ecology, epidemiology and resources. Welltargeted operational research is urgently needed to
make progress in dengue prevention and control.
PRIORITY RESEARCH
RECOMMENDATIONS FOR THE
NEXT five YEARS (20072011)
Assessment of the impact of dengue prevention
and control activities that have incorporated the
use of new methodological instruments, strategies, technologies etc.
Investigate potential indicators for risk of transmission with greater sensitivity than the current
entomological indicators.
Development of mathematical prognostic models, geographic or others, which consider different levels of risk of transmission.
Studies of costeffectiveness of the new tools,
strategies, and instruments being developed and
incorporated into programmes.
157
CONCLUSIONS
In this document we have summarized current
approaches and the status of recent ideas and technologies that are being tested, in particular in the
Americas, in response to the broader question of
how dengue prevention and control interventions
are currently being delivered and/or developed.
Nevertheless, some questions still do not have a
conclusive answer:
1. What do we expect from vector control services, particularly from vector control inspectors
during household visits? Should they continue
these visits or does this component need to be
changed? Do we need to seek other associations in order to transfer the responsibilities of
what they currently do to a more appropriate
group locally?
2. Can we change the current control services in other
ways? How can we work with the population to
change attitudes toward control strategies?
3. Which are the most cost-effective strategies, comparing traditional vector control with new tools
and managerial and organizational strategies? If
the new tools are effective, (COMBI, LIRAa, GIS,
among others) can they be generalized? What
operational research is needed to strengthen vector control service delivery?
We look forward to a rich scientific exchange
that will contribute new ideas and knowledge to
these issues.
158
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TDR/SWG/07
Report on Dengue

Report on
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Special Programme for Research & Training

Scientific Working Group

TDR/World Health Organization

Fax: (+41) 22 791-4854

The Special Programme for Research and Training in Tropical Diseases

Special Programme for Research & Training

Report of the Scientific Working Group

LIST OF PARTICIPANTS: Scientific Working Group on Dengue . . . . . . . . . . . . . . . . . . . . . . . . 23

Repor t of the Scientif ic Working Group on Dengue, 2006 TDR/SWG/08

Repor t of the Scientif ic Working Group on Dengue, 2006 TDR/SWG/08

Repor t of the Scientif ic Working Group on Dengue, 2006 TDR/SWG/08

Global dengue research agenda

Stream 1: Research related to reducing disease severity and case fatality

Repor t of the Scientif ic Working Group on Dengue, 2006 TDR/SWG/08

Process and impact evaluation of staff training

Critical issues in dengue pathogenesis

Stream 2: Research related to transmission control through improved vector management

Surveillance and response

Stream 3: Research related to primary and secondary prevention

Repor t of the Scientif ic Working Group on Dengue, 2006 TDR/SWG/08

Stream 4: Health policy research contributing to adequate publichealth response

Repor t of the Scientif ic Working Group on Dengue, 2006 TDR/SWG/08

1. Dengue as a public health problem and efforts

Dengue as an expanding public health problem

International efforts to control dengue

Repor t of the Scientif ic Working Group on Dengue, 2006 TDR/SWG/08

Repor t of the Scientif ic Working Group on Dengue, 2006 TDR/SWG/08

2. Ongoing dengue research

Repor t of the Scientif ic Working Group on Dengue, 2006 TDR/SWG/08

Towards improved case management

Towards improved primary prevention through support

Repor t of the Scientif ic Working Group on Dengue, 2006 TDR/SWG/08

Towards improved epidemiological surveillance

Repor t of the Scientif ic Working Group on Dengue, 2006 TDR/SWG/08

Repor t of the Scientif ic Working Group on Dengue, 2006 TDR/SWG/08

Policy research, implementation research related to

3. Purpose and objectives of the

Repor t of the Scientif ic Working Group on Dengue, 2006 TDR/SWG/08

Stream 1: Research related to reducing disease severity and case fatality

4. Global research agenda recommended

Process and impact evaluation of staff training

It is recommended to analyse the:

Critical issues in dengue pathogenesis

Stream 2: Research related to transmission control through improved vector management

Repor t of the Scientif ic Working Group on Dengue, 2006 TDR/SWG/08

Surveillance and response

It is recommended to analyse the:

Stream 3: Research related to primary and secondary prevention

Repor t of the Scientif ic Working Group on Dengue, 2006 TDR/SWG/08

Correlates of protective immunity for use as an endpoint in vaccine trials.

Stream 4: Health policy research contributing to adequate publichealth response

Repor t of the Scientif ic Working Group on Dengue, 2006 TDR/SWG/08

1. World Health Assembly. Dengue fever

Repor t of the Scientif ic Working Group on Dengue, 2006 TDR/SWG/08

Repor t of the Scientif ic Working Group on Dengue, 2006 TDR/SWG/08

Repor t of the Scientif ic Working Group on Dengue, 2006 TDR/SWG/08

Dr D.L. Heymann, Acting ADG/CDS

Issues and challenges for dengue research

Dengue research needs related to surveillance and emergency