Molecular epidemiology of dengue in Southeast Asia (SEA): Protocol of systematic review and meta-analysis

Abstract

Introduction

Dengue fever is a major global public health challenge caused by the Arbovirus and transmitted by Aedes mosquitoes. The increasing incidence of dengue, particularly in the Southeast Asia (SEA) region, including Malaysia, highlights the urgent need for a comprehensive understanding of dengue molecular epidemiology. This study aims to systematically review various aspects of dengue molecular epidemiology to gain insights into the disease’s dynamics, transmission and circulation. Providing evidence-based insights is crucial for the prevention and control of dengue.

Methods and analysis

A systematic review and meta-analysis will be conducted following the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols guidelines. Eligible studies will include observational designs from the inception of time to 31 December 2024, in the SEA region. The review will encompass various molecular epidemiology domains as the exposures and assess the outcomes, such as confirmed dengue cases and severity. Descriptive and meta-analytical methods will determine prevalence, genetic changes and associations. Grading of Recommendations Assessment, Development, and Evaluation methodology will evaluate the quality of evidence, and reporting biases will be addressed. This review aims to bridge the gap in dengue molecular epidemiology in the SEA region by providing comprehensive insights crucial for effective dengue prevention and control.

Ethics and dissemination

No primary data will be collected; thus, the ethical exemption was obtained from Medical Research Ethics Committee with reference number 23-03212-AE6 and ethics approval from the IMU University Joint Committee. The results will be disseminated through a peer-reviewed publication and conference presentation.

PROSPERO registration number

CRD42023480417.

STRENGTHS AND LIMITATIONS OF THIS STUDY.

• This review adopts a comprehensive framework of molecular epidemiology which includes the following domains, namely causation, pathogenesis, sources of the infectious agent, reservoirs, circulation patterns, transmission, drug therapy and vaccine development, which will bridge the gap in knowledge of molecular epidemiology of dengue in Southeast Asia (SEA).

• The various domains relating to the molecular epidemiology of dengue will be systematically retrieved and synthesised.

• Meta-analysis will provide validated evidence-based pooled quantitative estimates of serotype distribution and other molecular characterisation of dengue across SEA.

• Differences in study quality and molecular methods may affect the robustness of meta-analyses or pooled outcomes.

Introduction

Background

Dengue fever, caused by the Arbovirus (DENV-1 to DENV-4) and transmitted through Aedes aegypti and Aedes albopictus mosquitoes, remains a critical global public health concern.¹ Most infections are asymptomatic or marked by intense flu-like symptoms that last up to 10 days.² Globally, the WHO has reported a 10-fold increase in dengue cases over the last two decades, where cases have increased from 505 430 in 2000 to more than 5.0 million in 2023.¹³,⁵ Currently, 40% of the world’s population is living in dengue endemic areas, where a staggering 3.9 billion people are at risk of dengue infection.⁶ Furthermore, studies have reported that 60% of the world’s population will be exposed to dengue infection by 2080.⁷ In addition, one modelling study in 2010 estimated around 390 million dengue infections per year, with 96 million manifesting clinically.³

Dengue has been endemic in more than 100 countries in Asia, Africa, the Americas, the Eastern Mediterranean and the Western Pacific. Across these regions, Asia accounts for 70% of the global dengue burden, with the highest dengue burden reported in Southeast Asia (SEA).³

Since early 2023 (post COVID-19 pandemic), there has been an upsurge of dengue cases and deaths reported globally, which is characterised by a significant increase in the number, scale and simultaneous occurrence of multiple outbreaks, spreading into regions previously unaffected by dengue.⁵ In addition, increasing dengue infection is projected in the coming years within the SEA region, which has been more rampant 30-fold over the last 50 years.⁵

Since SEA contributes to a significant burden of dengue globally, a systematic review of dengue molecular epidemiology must explore dengue dynamics, transmission and circulation; as no studies are currently available within SEA. Also, focusing on this region allows for a detailed examination of region-specific viral genotypes, mutations and the emergence of new lineages that may not be observed elsewhere. Such a review would be able to do so in the following ways.

In order to control and prevent dengue infections effectively, it is crucial to have a comprehensive understanding of the dengue virus (DENV), including its sources and reservoirs, pathogenesis, circulation patterns, transmission, drug therapy and vaccine development. Dengue molecular epidemiology is a field that can provide us with the necessary knowledge to achieve this understanding.⁸ In addition, understanding the dengue causation factors and pathogenesis is essential in establishing disease aetiology and determining host susceptibility, severity and dengue outcomes. This knowledge is pivotal in developing targeted dengue prevention and control measures. Different levels of disease severity caused by a pathogen can be due to the difference in the human host response to the infection, individuals’ genetic susceptibility to, or inherent immunity against the pathogens.

Furthermore, molecular epidemiology would also allow for a better understanding of sources and reservoirs for dengue. Molecular epidemiology enhances the ability to detect trace amounts of infectious genetic materials in the environment that can vary depending on the specific pathogen and its transmission mode. Hence, understanding the sources or reservoirs of dengue is crucial for implementing effective dengue prevention and control measures. Also, molecular epidemiology provides detailed information on the circulating DENV, which enables the identification of predominantly circulating serotypes and serotype shifts that would influence the transmission of dengue. Transmission of dengue in molecular epidemiology provides deep information about the spread of dengue within the population, which is determined by molecular and genetic characterisation of the DENV. Therefore, this allows for tracking of dengue transmission patterns, identification of sources of infection and determining the relatedness of DENV in different individuals or populations, which will enables constructing a transmission network and identifying the transmission rate, transmission clusters and hotspots. Despite several studies exploring various aspects of dengue at a molecular level, systematic analyses of findings on the subject collectively, especially in the SEA region, are unavailable. Hence, conducting a systematic review of these studies would be essential as dengue research is a rapidly evolving field, and with the utilisation of molecular techniques, information on genetic diversity, the evolution of infectious agents and their hosts can be determined. Furthermore, in the context of infectious diseases, molecular epidemiology has proven to be a powerful tool for disease surveillance, outbreak investigations and the formulation of targeted control measures. By deciphering the genetic makeup of viral strains and tracing their spread, molecular epidemiology offers crucial insights into disease dynamics, aiding in developing effective prevention and control strategies.

Therefore, this study aims to bridge these gaps by exploring and synthesising evidence on the various domains of dengue molecular epidemiology, to provide comprehensive insights into the dynamics of dengue disease, mainly focusing on transmission and circulation. The findings are pivotal in informing evidence-based decision-making for health and contributing to the prevention, control and management of dengue.

Why is it important to do this review?

The reasons and mechanisms that lead to dengue severity and pathogenicity still need to be fully understood. The present knowledge indicates that several factors involved in virology and the host immune system are correlated with dengue haemorrhagic fever (DHF)/dengue shock syndrome (DSS) occurrence. In addition, climate change also plays an important role in Aedes mosquito distribution, subsequently having an impact on DENV transmission. Combining this information indicates that dengue infections and the development of severe dengue syndromes are complex.

In addition to the above, the dengue early warning systems are essential tools to predict outbreak occurrences and disease severity, facilitating early dengue response measures.⁹ Using molecular epidemiology of dengue would improve existing dengue early warning systems by first predicting dengue outbreaks through seroprevalence and detection of serotype change within a specific population that is immune naïve to the new dengue strain.⁹,¹¹ Second, examining the past dengue molecular transmission patterns of different viral strains would give an understanding of the current and future tracking of dengue transmission patterns over different locations. Although cross-border transmission from neighbouring regions may influence dengue dynamics, focusing on SEA will provide insight into intra-regional transmission patterns, which are key to understanding and controlling the disease locally, and are directly applicable to regional health authorities, enabling more effective disease surveillance, vector control and management.

To date, there are no universally accepted vaccines or antiviral therapies for dengue available worldwide because of challenges and limited knowledge of the DENV genetic variations and its subsequent immunopathogenesis. Certain countries in Latin America and the SEA region are currently using dengue vaccines such as (ie, Dengvaxia, TAK003) for the control and prevention of dengue. This study would help to (1) assist vaccine deployment by understanding the molecular epidemiology of dengue which includes the past and current virus genetic profile, circulating strains and the disease seroprevalence and (2) provide information on molecular epidemiology that would serve as a reference to the changes following the dengue vaccination and the effects of future dengue vaccinations. These findings would improve the deployment and understanding of the vaccine-disease dynamics.¹²,¹⁴

To our knowledge, no documented reviews explicitly discuss or synthesise current evidence on the molecular epidemiology of dengue; therefore, a comprehensive systematic review could provide the best available evidence on the molecular epidemiology of dengue. This review is essential to identify an effective strategy to reduce dengue cases. Information from this review will provide potential benefits to the service providers, policymakers, researchers, and other institutions on dengue control.

Objectives

This study aims to systematically (a) identify the prevalence of dengue cases and evaluate the effect of molecular epidemiology (causation, pathogenesis, sources of the infectious agent, reservoirs, circulation patterns, transmission, drug therapy and vaccine development) in dengue control and management; (b) determine the trend of predominating circulating serotypes/genotypes; and (c) determine the association between dengue serotypes/genotypes with dengue severity in the SEA region.

Review questions

We will retrieve any studies on dengue molecular epidemiology that report on causation, pathogenesis, infectious agents' sources, reservoirs, circulation patterns, transmission, drug therapy and vaccine development.

1. What is the current knowledge in the field of dengue molecular epidemiology, specifically regarding causation, pathogenesis, infectious agents’ sources, reservoirs, circulation patterns, transmission, drug therapy, and vaccine development?

2. What is the distribution and prevalence of different dengue serotypes, genotypes, and lineages across different periods in the SEA region, and how has genetic diversity evolved?

3. Is there an association between dengue serotypes and the severity of dengue infection?

4. Is there an association between DENV genetic change and disease transmission rates?

Methods

We will conduct a review in accordance with the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols guidelines (online supplementary appendix 1).¹⁵ This protocol has been registered on the Prospective Register of Systematic Reviews (PROSPERO) with registration number CRD42023480417. Any changes to the protocol will be recorded on PROSPERO. Our review will rigorously examine observational study designs with meta-analysis as an outcome. We will integrate our reporting with the Meta-Analysis of Observational Studies in Epidemiology guidelines.¹⁶

Patient and public involvement

This review does not require patient and/or public involvement.

Eligibility criteria

Types of studies

This review will include peer-reviewed published articles restricted to observational study designs that include case-control studies, cohort studies, cross-sectional studies, case reports and case series on the molecular epidemiology of dengue, from the inception of time to 31 December 2024, in the SEA region. We will exclude reviews, editorials, letters to editors and randomised controlled trial studies in this review.

Population

This review will include infected individuals or those at risk of dengue infection in the SEA region (Malaysia, Singapore, Indonesia, Brunei, Cambodia, East Timor (Timor-Leste), Laos, Myanmar (Burma), the Philippines, Thailand and Vietnam).

Types of exposure

We will include all the domains of molecular epidemiology of dengue (ie, causation, pathogenesis, infectious agent’s sources, reservoirs, circulation patterns, transmission, drug therapy and vaccine development) as exposures to understand dengue disease dynamics in the population.^{17 18} These exposures are retrieved from a publication by Sana Eybpoosh in 2017.⁸

The exposures are defined as domains of molecular epidemiology:

• Causation of dengue is defined as the pathogens responsible for dengue infection.

• Pathogenesis of dengue is defined as the role of pathogenic genes and host genetic factors affecting the disease initiation and progression.

• Sources and reservoirs of dengue are defined as:

  • Humans infected with the dengue virus serve as a source.

  • Aedes mosquitoes act as a reservoir for the dengue virus.

• Circulation of dengue is defined as strain-specific incidence and prevalence.

• Transmission of dengue encompasses routes, patterns, probabilities and dynamics of disease transmission.

• Drug therapy for dengue encompasses pharmacogenetics, drug resistance markers and relevant biomarkers.

• Dengue vaccine encompasses molecular characteristics, influences on development and effectiveness, and considerations for safety and impact within the context of molecular epidemiology.

Types of outcome measures

The types of outcomes of this study include:

1. Number of confirmed dengue cases of any severity involving all individuals as defined by the standard case definitions by WHO and Centres for Disease Control and Prevention guidelines.

2. Dengue severity will be categorised according to the WHO Dengue Case Classification 1997 and 2009 based on the authors’ classification.

The WHO 1997 classification includes Dengue Fever (DF), Dengue Haemorrhagic Fever (DHF), and Dengue Shock Syndrome (DSS), while the WHO 2009 classification comprises Dengue Without Warning Signs (DWS), Dengue With Warning Signs (DWWS) and Severe Dengue (SD). The dengue severity will be classified based on the original categorisation used in each study. Hence, the dengue severity in the included studies will be classified based on the WHO criteria corresponding to the respective years.

Information sources

Electronic searches

Electronic searches

The search will be conducted in five electronic databases, including PubMed, Wiley, Scopus, Embase and Cochrane Library, to identify potential studies. We will retrieve eligible published articles from the inception of time to 31 December 2024. A Boolean search strategy will be used, combining the following concepts: molecular epidemiology (concept 1), dengue (concept 2) and the SEA region (concept 3). We will adapt the search strategy to the key elements of the research question: population, intervention and outcome. Two main search strategy approaches include using the MeSH and free-text terms in the title and abstract on databases (online supplementary appendix 2). These MeSH terms and keywords were derived from the search strategies used in previous systematic reviews and meta-analyses on molecular epidemiology of dengue.^{19 20} We will also screen the reference lists of all included articles to identify additional eligible studies.

Selection of studies

All citations identified in the search will be imported into the reference management software Mendeley, where duplicates will be identified and removed using the software.²¹ Two independent reviewers will then screen all articles to exclude those that do not meet the eligibility criteria.

Data extraction

Two researchers will be involved in data extraction. A third researcher will be consulted in the event of any disagreement. A standardised data extraction form will be developed to aid in the extraction of the following (online supplementary appendix 3):

• Study characteristics, including publication year, study setting, sample size and study design.

• Molecular epidemiology domains that include (a) causation of infectious disease; (b) pathogenesis of infectious disease; (c) sources/reservoirs; (d) circulation patterns; (e) transmission; (f) vaccine development; and (g) drug therapy.

• Number of confirmed dengue cases.

• Dengue severity.

Quality assessment

To evaluate the risk of bias in the included studies, the Newcastle-Ottawa Scale (NOS) will be used.²² The NOS assigns stars to each study based on three domains: selection of participants, comparability of study groups and outcome assessment. The scale ranges from 0 to 10 stars, with higher scores indicating a lower risk of bias. The NOS has been widely used in systematic reviews and meta-analyses to evaluate the quality of evidence and inform conclusions about the effectiveness of interventions. It is a valuable tool for assessing the risk of bias in observational studies and can help ensure that study findings are reliable and valid. NOS scores ≤2 or ≥7 indicate poor and good quality, respectively.²³

Summary of findings table

Summary tables will be tabulated for the respective studies’ objectives as follows:

• Dengue molecular epidemiology domains: (a) causation of infectious disease; (b) pathogenesis of infectious disease; (c) sources/reservoirs; (d) circulation patterns; (e) transmission; (f) vaccine development; and (g) drug therapy.

• The distribution and prevalence of different dengue serotypes, genotypes and lineages across different time periods in the SEA region, and the evolution of genetic diversity.

• The association between rates of DENV genetic change and disease transmission

• The association between dengue serotypes and the severity of dengue infection

We will assess the quality of evidence for all outcomes in the systematic review and meta-analysis using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) methodology. The GRADE elements consist of study limitations, consistency of effect, indirectness, imprecision and publication bias.²⁴ The application of the GRADE framework into the current review will be conducted as follows:

• Risk of bias and imprecision: all studies will be assessed methodologically for risk of bias in the quality assessment stage using the NOS and Joanna Briggs Institute checklist, and subsequently categorised into poor, moderate and high quality.

• Inconsistency: variability in findings (eg, genetic markers, serotype distribution) will be assessed by comparing trends across studies and, where applicable, using statistical measures such as I² and Cochran’s Q test for heterogeneity.

• Indirectness: we will assess whether the study populations, lab methods, or molecular targets are relevant to this review.

• Publication bias: we will assess using funnel plot analyses where applicable.

Data analysis and statistical analysis

Molecular epidemiology data for DENV serotypes will be categorised based on the seven specified domains. Geographic classification will be conducted at the country level within SEA, while temporal trends will be analysed according to each study’s timeframe, extending up to 31 December 2024. DENV serotypes will be categorised in the following ways: (a) overall SEA region by the respective years and (b) country-specific by respective years. Following this, dengue virus serotypes will be analysed spatiotemporally, focusing on within and between countries analysis.

We will conduct descriptive data analysis using the statistical software STATA (V.18.0) and R Software (V.4.4.3).

The data on prevalence/frequency/mutation rate/seroprevalence/Bayesian Coalesce/genetic distance/dengue severity/associations will be tabulated. In addition, the distribution of dengue serotypes/genotypes/lineages across the SEA region will be spatially mapped.

Meta-analysis will be performed on measures presented in prevalence, frequency, mutation rate, genetic distance, OR, and RR (risk ratio) or prevalence OR by calculating the pooled estimates of these measures using the random effects model. We will conduct prespecified subgroup analyses by country, time period and methodological characteristics, complemented by sensitivity analyses and meta-regression to explore whether study-level variables explain observed variations. To address heterogeneity between studies from different Southeast Asian countries, we will implement a comprehensive analytical approach combining quantitative and qualitative methods. We will also measure the extent of interstudy variability; we will statistically assess heterogeneity using the I² statistic and Cochran’s Q test. To account for the between-study variance that may arise from differences in healthcare systems, laboratory capabilities, diagnostic methodologies and surveillance reporting practices, we will be using a random-effects meta-analysis model.

Assessment of reporting biases

Publication bias will be assessed using Begg’s rank correlation and Egger’s weighted regression methods, and funnel plots will be created and examined for publication bias.²⁵

Reaching conclusions

This review will assess the risk of publication bias by identifying and determining the distribution of published articles relating to the molecular epidemiology of dengue across countries within the SEA region. Therefore, since the review does not include unpublished data, we will acknowledge it as a limitation. We will base our conclusions only on findings from the quantitative or narrative synthesis of included studies for this review. The conclusions can be used as guidelines by health authorities for the prevention and control of dengue. Our implications for research will suggest priorities for future research and outline the remaining uncertainties in the area.

Ethics and dissemination

This review has been registered with the National Medical Research Register, Ministry of Health Malaysia (NMRR ID-23-03212-AE6) and obtained Medical Research Ethics Committee (MREC) exemption (reference number 23-03212-AE6), as well as ethics approval from IMU University Joint-Committee. This protocol has been registered with PROSPERO (ID number CRD42023480417). The results will be disseminated through peer-reviewed publications and conference presentations.

The funders played no role in study design, data collection or analysis, preparation of the manuscript, or publication decision.