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Dengue has been one of the major public health concerns in the Philippines for more than a century. The annual dengue case burden has been increasing in recent years, exceeding 200,000 in 2015 and 2019. However, there is limited information on the molecular epidemiology of dengue in the Philippines. We, therefore, conducted a study to understand the genetic composition and dispersal of DENV in the Philippines from 2015 to 2017 under UNITEDengue. Our analyses included 377 envelope ( E ) gene sequences of all 4 serotypes obtained from infections in 3 main island groups (Luzon, Visayas, and Mindanao) of the Philippines. The findings showed that the overall diversity of DENV was generally low. DENV-1 was relatively more diverse than the other serotypes. Virus dispersal was evident among the three main island groups, but each island group demonstrated a distinct genotype composition. These observations suggested that the intensity of virus dispersal was not substantive enough to maintain a uniform heterogeneity among island groups so that each island group behaved as an independent epidemiological unit. The analyses suggested Luzon as one of the major sources of DENV emergence and CAR, Calabarzon, and CARAGA as important hubs of virus dispersal in the Philippines. Our findings highlight the importance of virus surveillance and molecular epidemiological analyses to gain deep insights into virus diversity, lineage dominance, and dispersal patterns that could assist in understanding the epidemiology and transmission risk of dengue in endemic regions.

Over 105 million dengue infections are estimated to occur annually. Understanding the disease dynamics of dengue is often difficult due to multiple strains circulating within a population. Interactions between dengue serotype dynamics may result in complex cross-immunity dynamics at the population level and create difficulties in terms of formulating intervention strategies for the disease. In this study, a nationally representative 16-year time series with over 43 000 serotyped dengue infections was used to infer the long-run effects of between and within strain interactions and their impacts on past outbreaks. We used a novel identification strategy incorporating sign-identified Bayesian vector autoregressions, using structural impulse responses, historical decompositions and counterfactual analysis to conduct inference on dengue dynamics post-estimation. We found that on the population level: (i) across-serotype interactions on the population level were highly persistent, with a one time increase in any other serotype associated with long run decreases in the serotype of interest (range: 0.5-2.5 years) and (ii) over 38.7% of dengue cases of any serotype were associated with across-serotype interactions. The findings in this paper will substantially impact public health policy interventions with respect to dengue.

Dengue virus type 2 (DENV-2) contributes substantially to the dengue burden and dengue-related mortality in the tropics and sub-tropics. DENV-2 includes six genotypes, among which cosmopolitan genotype is the most widespread. The present study investigated the evolution, intra-genotype heterogeneity and dispersal of cosmopolitan genotype to understand unique genetic characteristics that have shaped the molecular epidemiology and distribution of cosmopolitan lineages. The spatial analysis demonstrated a wide geo-distribution of cosmopolitan genotype through an extensive inter-continental network, anchored in Southeast Asia and Indian sub-continent. Intra-genotype analyses using 3367 envelope gene sequences revealed six distinct lineages within the cosmopolitan genotype, namely the Indian sub-continent lineage and five other lineages. Indian sub-continent lineage was the most diverged among six lineages and has almost reached the nucleotide divergence threshold of 6% within E gene to qualify as a separate genotype. Genome wide amino acid signatures and selection pressure analyses further suggested differences in evolutionary characteristics between the Indian sub-continent lineage and other lineages. The present study narrates a comprehensive genomic analysis of cosmopolitan genotype and presents notable genetic characteristics that occurred during its evolution and global expansion. Whether those characteristics conferred a fitness advantage to cosmopolitan genotype in different geographies warrant further investigations.

Introduction: Early and rapid confirmation of dengue infections strengthens disease surveillance program and are critical to the success of vector control measures. Rapid diagnostics tests (RDTs) are increasingly used to confirm recent dengue infections due to their ease of use and short turnaround time for results. Several studies undertaken in dengue-endemic Southeast Asia have reported the performance of RDTs against enzyme-linked immunosorbent assay (ELISA), reverse transcriptase polymerase chain reaction (RT-PCR) and virus isolation methods. However, few studies have compared multiple RDTs for the detection of dengue NS1 antigen and IgM antibody in a single combo cassette. We evaluated six RDTs in Singapore for their utility in routine clinical testing to detect recent dengue infections., Methods: The evaluation comprised two phases. The first phase sought to determine each RDT's specificity to dengue NS1 and IgM using zika and chikungunya virus supernatant and zika convalescent samples. RDTs that cross-reacted with zika or chikungunya were not further tested in phase 2. The second phase sought to determine the sensitivity and specificity of the remaining RDTs to dengue NS1 and IgM using pre-characterised dengue specimens and non-dengue/chikungunya febrile clinical specimens., Results: None of the RDTs cross-reacted with zika IgM in Phase 1. Truquick and Quickprofile cross reacted with zika and chikungunya viruses and were not evaluated thereafter. Standard Q had the highest dengue NS1 and IgM sensitivity at 87.0% and 84.3% respectively whereas Bioline (68.5%) and Multisure (58.3%) had the lowest dengue NS1 and IgM sensitivity respectively. Combining dengue NS1/IgM detection results greatly improved the RDT ability to detect recent dengue infection; Standard Q had the highest sensitivity at 99.1% while Multisure had the lowest at 92.6%. All the RDTs were highly specific for dengue NS1 and IgM (96.7% to 100%). All the RDTs had high positive predictive values (98.4% to 100%) for NS1, IgM and combined NS1/IgM parameters whereas Standard Q had the highest negative predictive values at 68.2% (NS1), 63.8% (IgM) and 96.8% (NS1/IgM). For the RDTs, detection of NS1 declined from acute to convalescent phase of illness whereas IgM detection rate gradually increased over time., Conclusion: In our study, several RDTs were evaluated for their diagnostic accuracy and capability in detecting recent dengue infection. Standard Q demonstrated a high degree of diagnostic accuracy and capability in the detection of NS1 and IgM biomarkers. RDTs can provide rapid and accurate confirmation of recent dengue infections and augment dengue surveillance and control programmes. Further studies are required to assess the usefulness of these RDTs in other epidemiology settings., Competing Interests: The authors have declared that no competing interests exist.

Dengue is a major public health concern in the tropical and subtropical world, with no effective treatment. The controversial live attenuated virus vaccine Dengvaxia has boosted the pursuit of subunit vaccine approaches, and nonstructural protein 1 (NS1) has recently emerged as a promising candidate. However, we found that NS1 immunization or passive transfer of NS1 antibodies failed to confer protection in symptomatic dengue mouse models using two non-mouse-adapted DENV2 strains that are highly virulent. Exogenous administration of purified NS1 also failed to worsen in vivo vascular leakage in sublethally infected mice. Neither method of NS1 immune neutralization changed the disease outcome of a chimeric strain expressing a vascular leak-potent NS1. Instead, virus chimerization involving the prME structural region indicated that these proteins play a critical role in driving in vivo fitness and virulence of the virus, through induction of key proinflammatory cytokines. This work highlights that the pathogenic role of NS1 is DENV strain dependent, which warrants reevaluation of NS1 as a universal dengue vaccine candidate., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2020 Lee et al.)

Background: Dengue is a mosquito-borne viral disease caused by one of four serotypes (DENV1-4). Infection provides long-term homologous immunity against reinfection with the same serotype. Plaque reduction neutralization test (PRNT) is the gold standard to assess serotype-specific antibody levels. We analysed serotype-specific antibody levels obtained by PRNT in two serological surveys conducted in Singapore in 2009 and 2013 using cluster analysis, a machine learning technique that was used to identify the most common histories of DENV exposure., Methods: We explored the use of five distinct clustering methods (i.e. agglomerative hierarchical, divisive hierarchical, K-means, K-medoids and model-based clustering) with varying number (from 4 to 10) of clusters for each method. Weighted rank aggregation, an evaluating technique for a set of internal validity metrics, was adopted to determine the optimal algorithm, comprising the optimal clustering method and the optimal number of clusters., Results: The K-means algorithm with six clusters was selected as the algorithm with the highest weighted rank aggregation. The six clusters were characterised by (i) dominant DENV2 PRNT titres; (ii) co-dominant DENV1 and DENV2 titres with average DENV2 titre > average DENV1 titre; (iii) co-dominant DENV1 and DENV2 titres with average DENV1 titre > average DENV2 titre; (iv) low PRNT titres against DENV1-4; (v) intermediate PRNT titres against DENV1-4; and (vi) dominant DENV1-3 titres. Analyses of the relative size and age-stratification of the clusters by year of sample collection and the application of cluster analysis to the 2009 and 2013 datasets considered separately revealed the epidemic circulation of DENV2 and DENV3 between 2009 and 2013., Conclusion: Cluster analysis is an unsupervised machine learning technique that can be applied to analyse PRNT antibody titres (without pre-established cut-off thresholds to indicate protection) to explore common patterns of DENV infection and infer the likely history of dengue exposure in a population.

The frequency of epidemics caused by Dengue viruses 1-4, Zika virus and Chikungunya viruses have been on an upward trend in recent years driven primarily by uncontrolled urbanization, mobility of human populations and geographical spread of their shared vectors, Aedes aegypti and Aedes albopictus. Infections by these viruses present with similar clinical manifestations making them challenging to diagnose; this is especially difficult in regions of the world hyperendemic for these viruses. In this study, we present a targeted-enrichment methodology to simultaneously sequence the complete viral genomes for each of these viruses directly from clinical samples. Additionally, we have also developed a customized computational tool (BaitMaker) to design these enrichment baits. This methodology is robust in its ability to capture diverse sequences and is amenable to large-scale epidemiological studies. We have applied this methodology to two large cohorts: a febrile study based in Colombo, Sri Lanka taken during the 2009-2015 dengue epidemic (n = 170) and another taken during the 2016 outbreak of Zika virus in Singapore (n = 162). Results from these studies indicate that we were able to cover an average of 97.04% ± 0.67% of the full viral genome from samples in these cohorts. We also show detection of one DENV3/ZIKV co-infected patient where we recovered full genomes for both viruses., Competing Interests: The authors have declared that no competing interests exist.

Targeted proteomic mass spectrometry is emerging as a salient clinical diagnostic tool to track protein biomarkers. However, its strong analytical properties have not been exploited in the diagnosis and typing of flaviviruses. Here, we report the development of a sensitive and specific single-shot robust assay for flavivirus typing and diagnosis using targeted mass spectrometry technology. Our flavivirus parallel reaction monitoring assay ( fv PRM) has the ability to track secreted flaviviral nonstructural protein 1 (NS1) over a broad diagnostic and typing window with high sensitivity, specificity, extendibility, and multiplexing capability. These features, pivotal and pertinent to efficient response toward flavivirus outbreaks, including newly emerging flavivirus strains, circumvent the limitations of current diagnostic assays. fv PRM thus carries high potential in positioning itself as a forerunner in delivering early and accurate diagnosis for disease management., Competing Interests: The authors declare no conflict of interest.

The Strategic Advisory Group of Experts (SAGE) on Immunization advises WHO on global policies for vaccines. In April, 2016, SAGE issued recommendations on the use of the first licenced dengue vaccine, CYD-TDV. In November, 2017, a retrospective analysis of clinical trial data, stratifying participants according to their dengue serostatus before the first vaccine dose, showed that although in high seroprevalence settings the vaccine provides overall population benefit, there was an excess risk of severe dengue in seronegative vaccinees. SAGE's working group on dengue vaccines met to discuss the new data and mainly considered two vaccination strategies: vaccination of populations with dengue seroprevalence rates above 80% or screening of individuals before vaccination, and vaccinating only seropositive individuals. We report on the deliberations that informed the recommendation of the pre-vaccination screening strategy, in April, 2018. Important research and implementation questions remain for CYD-TDV, including the development of a highly sensitive and specific rapid diagnostic test to determine serostatus, simplified immunisation schedules, and assessment of the need for booster doses., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: In 2013 and 2014, Singapore experienced its worst dengue outbreak known-to-date. Mosquito breeding in construction sites stood out as a probable risk factor due to its association with major dengue clusters in both years. We, therefore, investigated the contribution of construction sites to dengue transmission in Singapore, highlighting three case studies of large construction site-associated dengue clusters recorded during 2013-16., Methods: The study included two components; a statistical analysis of cluster records from 2013 to 2016, and case studies of three biggest construction site-associated clusters. We explored the odds of construction site-associated clusters growing into major clusters and determined whether clusters seeded in construction sites demonstrated a higher tendency to expand into major clusters. DENV strains obtained from dengue patients residing in three major clusters were genotyped to determine whether the same strains expanded into the surroundings of construction sites., Results: Despite less than 5% of total recorded clusters being construction site-associated, the odds of such clusters expanding into major clusters were 17.4 (2013), 9.2 (2014), 3.3 (2015) and 4.3 (2016) times higher than non-construction site clusters. Aedes premise index and average larvae count per habitat were also higher in construction sites than residential premises during the study period. The majority of cases in clusters associated with construction sites were residents living in the surroundings. Virus genotype data from three case study sites revealed a transmission link between the construction sites and the surrounding residential areas., Conclusions: Significantly high case burden and the probability of cluster expansion due to virus spill-over into surrounding areas suggested that construction sites play an important role as a driver of sustained dengue transmission. Our results emphasise that the management of construction-site associated dengue clusters should not be limited to the implicated construction sites, but be extended to the surrounding premises to prevent further transmission.

Singapore has experienced periodic dengue epidemics despite maintaining a low Aedes house index. Each epidemic was associated with a switch in the predominant serotype. We investigated the temporal dynamics of dengue fever and dengue virus (DENV) and analyzed the epidemiological and entomological patterns of dengue in Singapore from 2004 to 2016. The case surveillance is based on a mandatory notification system that requires all medical practitioners to report clinically suspected and laboratory-confirmed cases. Circulating (DENV) serotypes are monitored through a virus surveillance program. Entomological surveillance involves inspections for larval breeding and monitoring of adults using gravitraps. Singapore experienced a similar epidemic pattern during 2004-2007 and 2013-2016. The pattern involved a 2-year DENV-1 epidemic occurring after a switch in the predominant serotype from DENV-2 to DENV-1, followed by a "lull" year. Thereafter, the predominant serotype switched back to DENV-2, tailed by a small-scale epidemic. Across the years, the highest incidence group was in the 25-44 years age group. The incidence rate of those aged ≥ 55 years was about half of that of the 15-24 years age group during DENV-1 predominant years. However, it was almost equal to the younger age group in DENV-2 predominant years. Types of Aedes aegypti breeding habitats remained similar. Dengue incidence was significantly higher in areas with high breeding percentage (BP) than areas with low BP ( P < 0.05). In conclusion, the oscillation of DENV-1 and DENV-2, throughout the 13-year period, led to a cyclical epidemic pattern and older adults were more affected by DENV-2 than DENV-1.

Objectives: We determined the seroprevalence of chikungunya virus (CHIKV) infection in the adult resident population in Singapore following local outbreaks of chikungunya fever (CHIKF) in 2008-2009., Methods: Our cross-sectional study involved residual sera from 3,293 adults aged 18-79 years who had participated in the National Health Survey in 2010. Sera were tested for IgG antibodies against CHIKV and dengue virus (DENV) and neutralizing antibodies against CHIKV., Results: The prevalence of CHIKV-neutralizing antibodies among Singapore residents aged 18-79 years was 1.9% (95% confidence interval: 1.4%- 2.3%). The CHIKV seroprevalence was highest in the elderly aged 70-79 years at 11.5%, followed by those aged 30-39 years at 3.1%. Men had significantly higher CHIKV seroprevalence than women (2.5% versus 1.3%, p = 0.01). Among the three main ethnic groups, Indians had the highest seroprevalence (3.5%) compared to Chinese (1.6%) and Malays (0.7%) (p = 0.02 and p = 0.01, respectively). Multivariable logistic regression identified adults aged 30-39 years and 70-79 years, men, those of Indian ethnicity and ethnic minority groups, and residence on ground floor of public and private housing apartments as factors that were significantly associated with a higher likelihood of exposure to CHIKV. The overall prevalence of anti-DENV IgG antibodies was 56.8% (95% CI: 55.1%- 58.5%), while 1.5% (95% CI: 1.1%- 2.0%) of adults possessed both neutralizing antibodies against CHIKV and IgG antibodies against DENV., Conclusions: Singapore remains highly susceptible to CHIKV infection. There is a need to maintain a high degree of vigilance through disease surveillance and vector control. Findings from such serological study, when conducted on a regular periodic basis, could supplement surveillance to provide insights on CHIKV circulation in at-risk population.

Dengue fever is currently the most prevalent disease caused by mosquito-borne flaviviruses. Despite being potentially fatal, there are no specific antiviral therapies for Dengue virus (DENV) infections. Therefore, early, accurate, and rapid diagnosis plays an important role in proper patient management. In this study, we evaluated the performance of a probe-based real-time RT-PCR (rRT-PCR) assay against that of a conventional RT-PCR assay in three sample cohorts from Pakistan (n = 94) and Singapore (first cohort; n = 559, second cohort; n = 123). The Pakistan cohort also included a comparison with virus isolation. The rRT-PCR assay showed relatively lower overall sensitivity (20.2%) in the Pakistan cohort than that in first (90.8%) and second (80.5%) Singapore cohorts. Surprisingly, the overall sensitivity of rRT-PCR assay was lower compared with the virus isolation (26.6%) among Pakistan samples, indicating a high percentage (79.8%) of false negatives due to rRT-PCR assay. The analysis of sequences of failed and successful DENV isolates indicated mismatches in probe binding regions as the likely cause of rRT-PCR assay failure. Our observations testify the importance of utilizing a combination of methods for dengue diagnostics and surveillance. We emphasize that a thorough understanding of the genetic composition of local DENV populations as well as regular monitoring of the performance and reviewing of probe/primer sequences are essential to maintain a consistently high diagnostic accuracy of PCR-based assays.

Background: Dengue resurged in Singapore during 2013-14, causing an outbreak with unprecedented number of cases in the country. In the present study, we summarise the epidemiological, virological and entomological findings gathered through the dengue surveillance programme and highlight the drivers of the epidemic. We also describe how the surveillance system facilitated the preparedness to moderate epidemic transmission of dengue in the country., Methods: The case surveillance was based on a mandatory notification system that requires all medical practitioners to report clinically-suspected and laboratory-confirmed cases within 24 hours. The circulating Dengue virus (DENV) populations were monitored through an island wide virus surveillance programme aimed at determining the serotypes and genotypes of circulating virus strains. Entomological surveillance included adult Aedes surveillance as well as premise checks for larval breeding., Results: A switch in the dominant serotype from DENV-2 to DENV-1 in March 2013 signalled a potential spike in cases, and the alert was corroborated by an increase in average Aedes house index. The alert triggered preparedness and early response to moderate the impending outbreak. The two-year outbreak led to 22,170 cases in 2013 and 18,338 in 2014, corresponding to an incidence rate of 410.6 and 335.0 per 100,000 population, respectively. DENV-1 was the dominant serotype in 2013 (61.7 %, n = 5,071) and 2014 (79.2 %, n = 5,226), contributed largely by a newly-introduced DENV-1 genotype III strain. The percentage of houses with Ae. aegypti breeding increased significantly (p < 0.001) from 2012 (annual average of 0.07 %) to 2013 (annual average of 0.14 %), followed by a drop in 2014 (annual average of 0.10 %). Aedes breeding data further showed a wide spread distribution of Ae. aegypti in the country that corresponded with the dengue case distribution pattern in 2013 and 2014. The adult Aedes data from 34 gravitrap sentinel sites revealed that approximately 1/3 of the monitored sites remained at high risk of DENV transmission in 2013., Conclusions: The culmination of the latest epidemic is likely to be due to a number of demographic, social, virological, entomological, immunological, climatic and ecological factors that contribute to DENV transmission. A multi-pronged approach backed by the epidemiological, virological and entomological understanding paved way to moderate the case burden through an integrated vector management approach.

Background: Understanding the interaction between Aedes vectors and dengue viruses (DENV) has significant implications in determining the transmission dynamics of dengue. The absence of an animal model and ethical concerns regarding direct feeding of mosquitoes on patients has resulted in most infection studies using blood meals spiked with laboratory-cultured DENV. Data obtained from such studies may not reflect the natural human-mosquito transmission scenario. This study explored the potential of using membrane feeding of dengue patient's blood as a substitute for direct skin feeding., Methods: Four to six-day old female Ae. aegypti were provided the opportunity to feed via direct exposure to a patient's forearm for 15 min or via exposure to EDTA-treated blood from the same patient through an artificial membrane for 30 min. Mosquitoes from both feeding methods were incubated inside environmental chambers. Mosquitoes were sampled at day 13 post-feeding. Midgut and salivary glands of each mosquito were dissected to determine DENV infection by RT-qPCR and viral titration, respectively., Results: Feeding rates: Direct skin feeding assay (DSFA) consistently showed higher mosquito feeding rates (93.3-100%) when compared with the membrane feeding assay (MFA) (48-98.2%). Midgut infection: Pair-wise comparison between methods showed no significant difference in midgut infection rates between mosquitoes exposed via each method and a strong correlation was observed in midgut infection rates for both feeding methods (r = 0.89, P < 0.0001). Overall midgut viral titers (n = 20) obtained by both methods were comparable (P ≥ 0.06). Salivary gland infection: Pair-wise comparison between both methods revealed no significant difference in salivary gland infection rate. Strong correlation in salivary gland infection was observed between DSFA and MFA (r = 0.81, P < 0.0001). In general, mosquitoes fed directly on dengue patients and those on patients' blood (n = 11) had comparable virus titer (P ≥ 0.09)., Conclusion: DENV midgut and salivary gland infection rates showed good concordance between DSFA and MFA blood meal exposure methods. Freshly-obtained venous blood in EDTA from dengue patients for MFA can be used as a substitute to DSFA, especially in circumstances where bioethics approval or patient recruitment is difficult to obtain for vector competence studies. Nevertheless, mosquito numbers will need to be increased to compensate for lower feeding rate in MFA.

Dengue virus (DENV) is currently the most prevalent mosquito-borne viral pathogen. DENVs naturally exist as highly heterogeneous populations. Even though the descriptions on DENV diversity are plentiful, only a few studies have narrated the dynamics of intra-epidemic virus diversity at a fine scale. Such accounts are important to decipher the reciprocal relationship between viral evolutionary dynamics and disease transmission that shape dengue epidemiology. In the current study, we present a micro-scale genetic analysis of a monophyletic lineage of DENV-1 genotype III (epidemic lineage) detected from November 2012 to May 2014. The lineage was involved in an unprecedented dengue epidemic in Singapore during 2013-2014. Our findings showed that the epidemic lineage was an ensemble of mutants (variants) originated from an initial mixed viral population. The composition of mutant spectrum was dynamic and positively correlated with case load. The close interaction between viral evolution and transmission intensity indicated that tracking genetic diversity through time is potentially a useful tool to infer DENV transmission dynamics and thereby, to assess the epidemic risk in a disease control perspective. Moreover, such information is salient to understand the viral basis of clinical outcome and immune response variations that is imperative to effective vaccine design.

Chikungunya virus (CHIKV) and dengue virus (DENV) have emerged as the two most important arbovirus diseases of global health significance. Similar clinical manifestations, transmission vectors, geographical distribution, and seasonal correlation often result in misdiagnosis of chikungunya infections as dengue cases and vice versa. In this study, we developed a rapid and accurate laboratory confirmative method to simultaneously detect, quantify, and differentiate DENV serotypes 1, 2, 3, and 4 and CHIKV. This SYBR Green I-based one-step multiplex real-time RT-PCR assay is highly sensitive and specific for CHIKV and DENV. Melting temperature analysis of PCR amplicons was used to serotype DENV and to differentiate from CHIKV. The detection limit of the assay was 20, 10, 50, 5, and 10 RNA copies/reaction for DENV-1, DENV-2, DENV-3, DENV-4, and CHIKV, respectively. Our assay did not cross-react with a panel of viruses that included other flaviviruses, alphaviruses, influenza viruses, human enteroviruses, and human coronaviruses. The feasibility of using this assay for clinical diagnosis was evaluated in DENV- and CHIKV-positive patient sera. Accordingly, the assay sensitivity for DENV-1, DENV-2, DENV-3, DENV-4, and CHIKV was 89.66%, 96.67%, 96.67%, 94.12%, and 95.74%, respectively, with 100% specificity. These findings confirmed the potential of our assay to be used as a rapid test for simultaneous detection and serotyping of DENV and CHIKV in clinical samples., (Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Routine national notifications of dengue cases typically do not reflect the true dengue situation due to large proportion of unreported cases. Serosurveys, when conducted periodically, could shed light on the true dengue infections in the population. To determine the magnitude of dengue infections of the adult population in Singapore following the outbreak in 2007, we performed a cross-sectional study on blood donor samples from December 2009 to February 2010. The residual blood of 3,995 donors (aged 16-60 years) was screened for the presence of dengue-specific immunoglobulin G (IgG) and IgM using enzyme-linked immunosorbent assay (ELISA) kits. The age-weighted IgG prevalence of residents was 50.8% (N = 3,627, 95% confidence interval [CI] = 49.4-52.3%). Dengue IgG prevalence increased with age, with the lowest in 16-20 years age group (16.1%) and the highest in 56-60 years age group (86.6%). Plaque reduction neutralization test (PRNT) on samples of young resident adults (aged 16-30 years) revealed lower prevalence of neutralizing antibodies to each serotype, ranging from 5.4% to 20.3% compared with the older age groups. The level of exposure to dengue among the young adults is relatively low despite the endemicity of the disease in Singapore. It partially explains the population's susceptibility to explosive outbreaks and the high incidence rate among young adults., (© The American Society of Tropical Medicine and Hygiene.)

Objective: Accurate laboratory testing is a critical component of dengue surveillance and control. The objective of this programme was to assess dengue diagnostic proficiency among national-level public health laboratories in the World Health Organization (WHO) Western Pacific Region., Methods: Nineteen national-level public health laboratories performed routine dengue diagnostic assays on a proficiency testing panel consisting of two modules: one containing commercial serum samples spiked with cultured dengue viruses for the detection of nucleic acid and non-structural protein 1 (NS1) (Module A) and one containing human serum samples for the detection of anti-dengue virus antibodies (Module B). A review of logistics arrangements was also conducted., Results: All 16 laboratories testing Module A performed reverse transcriptase polymerase chain reaction (RT-PCR) for both RNA and serotype detection. Of these, 15 had correct results for RNA detection and all 16 correctly serotyped the viruses. All nine laboratories performing NS1 antigen detection obtained the correct results. Sixteen of the 18 laboratories using IgM assays in Module B obtained the correct results as did the 13 laboratories that performed IgG assays. Detection of ongoing/recent dengue virus infection by both molecular (RT-PCR) and serological methods (IgM) was available in 15/19 participating laboratories., Discussion: This first round of external quality assessment of dengue diagnostics was successfully conducted in national-level public health laboratories in the WHO Western Pacific Region, revealing good proficiency in both molecular and serological testing. Further comprehensive diagnostic testing for dengue virus and other priority pathogens in the Region will be assessed during future rounds.

Characterization of 14,079 circulating dengue viruses in a cross-border surveillance program, UNITEDengue, revealed that the 2013 outbreaks in Singapore and Malaysia were associated with replacement of predominant serotype. While the predominant virus in Singapore switched from DENV2 to DENV1, DENV2 became predominant in neighboring Malaysia. Dominance of DENV2 was most evident on the southern states where higher fatality rates were observed., (© The American Society of Tropical Medicine and Hygiene.)

Studies on serotype-specific features of dengue and disease severity on adults are limited. We prospectively recruited adult febrile patients without alternate diagnosis to dengue from April 2005 to December 2011. Outcomes were defined using both the World Health Organization (WHO) 1997 and 2009 criteria; Dengue hemorrhagic fever (DHF) and severe dengue (SD). Infecting serotype was identified in 469 dengue-confirmed patients comprising 22.0% dengue virus serotype 1 (DENV-1), 57.1% DENV-2, 17.1% DENV-3, and 3.8% DENV-4. Cases infected with DENV-1 were more likely to present with red eyes whereas presence of joint pain and lower platelet count was associated with DENV-2 cases. After adjusting for potential confounders, DENV-1 was associated with both DHF (adjusted Relative Risk [aRR] = 1.74) and SD (aRR = 2.1) whereas DENV-2 had a lower risk of DHF (aRR = 0.5). DENV-1 genotype 1 and DENV-2 cosmopolitan were the predominant genotypes identified. Infecting dengue serotype and possibly genotype may play an important role in disease severity among adult dengue patients in Singapore., (© The American Society of Tropical Medicine and Hygiene.)

The exact mechanisms of interplay between host and viral factors leading to severe dengue are yet to be fully understood. Even though previous studies have implicated specific genetic differences of Dengue virus (DENV) in clinical severity and virus attenuation, similar studies with large-scale, whole genome screening of monophyletic virus populations are limited. Therefore, in the present study, we compared 89 whole genomes of DENV-2 cosmopolitan clade III isolates obtained from patients diagnosed with dengue fever (DF, n = 58), dengue hemorrhagic fever (DHF, n = 30) and dengue shock syndrome (DSS, n = 1) in Singapore between July 2010 and January 2013, in order to determine the correlation of observed viral genetic differences with clinical outcomes. Our findings showed no significant difference between the number of primary and secondary infections that progressed to DHF and DSS (p>0.05) in our study cohort. Despite being highly homogenous, study isolates possessed 39 amino acid substitutions of which 10 substitutions were fixed in three main groups of virus isolates. None of those substitutions were specifically associated with DHF and DSS. Notably, two evolutionarily unique virus groups possessing C-P43T+NS1-S103T+NS2A-V83I+NS3-R337K+ NS3-I600T+ NS5-P136S and NS2A-T119N mutations were exclusively found in patients with DF, the benign form of DENV infections. Those mutants were significantly associated with mild disease outcome. These observations indicated that disease progression into DHF and DSS within our patient population was more likely to be due to host than virus factors. We hypothesize that selection for potentially less virulent groups of DENV-2 in our study cohort may be an evolutionary adaptation of viral strains to extend their survival in the human-mosquito transmission cycle.

WHO recommendations for dengue diagnosis require laboratory facilities. Antibody-based rapid diagnostic tests (RDTs) have performed poorly, and clinical diagnosis remains the mainstay in dengue-endemic countries. We evaluated a combination antigen-antibody RDT for point-of-care testing in a high-prevalence setting. In this prospective cohort study, adults were enrolled from a tertiary infectious disease centre for evaluation of undifferentiated febrile illness from October 2011 to May 2012. SD Bioline Dengue Duo was evaluated at point-of-care against a WHO-based reference standard of viral isolation, RT-PCR, NS1-, IgM-, and IgG-ELISA. 246 adults were enrolled (median age 34 years, range 18-69), of which 197 could be confirmed definitively as either dengue or non-dengue. DENV-2 was the predominant serotype (79.5%) and the ratio of primary to secondary cases was 1∶1.1. There were no test failures and minimal interobserver variation with a Fleiss' kappa of 0.983 (95% CI 0.827-1.00). Overall sensitivity and specificity were 93.9% (95% CI 88.8-96.8%) and 92.0% (95% CI 81.2-96.9%) respectively. Using WHO clinical criteria alone for diagnosis had similar sensitivities (95.9%, 95% CI 91.4-98.1%) and lower specificities (20.0%, 95% CI 11.2-33.0%). No significant difference in performance was found when testing early versus late presenters, primary versus secondary cases, or DENV-1 versus DENV-2 infections. The use of a combination RDT fulfills WHO ASSURED criteria for point-of-care testing and can enhance dengue diagnosis in an endemic setting. This has the potential to markedly improve clinical management of dengue in the field.

Background: Even though dengue has been recognized as one of the major public health threats in Pakistan, the understanding of its molecular epidemiology is still limited. The genotypic diversity of Dengue virus (DENV) serotypes involved in dengue outbreaks since 2005 in Pakistan is not well studied. Here, we investigated the origin, diversity, genetic relationships and geographic distribution of DENV to understand virus evolution during the recent expansion of dengue in Pakistan., Methods: The study included 200 sera obtained from dengue-suspected patients from 2006 to 2011. DENV infection was confirmed in 94 (47%) sera by a polymerase chain reaction assay. These included 36 (38.3%) DENV-2, 57 DENV-3 (60.6%) and 1 DENV-4 (1.1%) cases. Sequences of 13 whole genomes (6 DENV-2, 6 DENV-3 and 1 DENV-4) and 49 envelope genes (26 DENV-2, 22 DENV-3 and 1 DENV-4) were analysed to determine the origin, phylogeny, diversity and selection pressure during virus evolution., Results: DENV-2, DENV-3 and DENV-4 in Pakistan from 2006 to 2011 shared 98.5-99.6% nucleotide and 99.3-99.9% amino acid similarity with those circulated in the Indian subcontinent during the last decade. Nevertheless, Pakistan DENV-2 and DENV-3 strains formed distinct clades characterized by amino acid signatures of NS2A-I116T + NS5-K861R and NS3-K590R + NS5-S895L respectively. Each clade consisted of a heterogenous virus population that circulated in Southern (2006-2009) and Northern Pakistan (2011)., Conclusions: DENV-2, DENV-3 and DENV-4 that circulated during 2006-2011 are likely to have first introduced via the southern route of Pakistan. Both DENV-2 and DENV-3 have undergone in-situ evolution to generate heterogenous populations, possibly driven by sustained local DENV transmission during 2006-2011 periods. While both DENV-2 and DENV-3 continued to circulate in Southern Pakistan until 2009, DENV-2 has spread in a Northern direction to establish in Punjab Province, which experienced a massive dengue outbreak in 2011.

Neurological manifestations due to Dengue virus (DENV) infection are atypical and uncommon. Genomic information of clinically characterised, neurotrophic DENV in humans is extremely limited albeit their importance in deciphering the pathogenicity is substantial. Here, we report a rare case of fatal DENV-4 infection complicated with encephalitis and multi-organ failure. The clinical presentation was unusual due to its rapid onset of encephalitis despite a very low virus titre. Full genomes of serum and CSF-derived viruses shared 99.99% similarity, indicating the virus dissemination across blood-brain barrier. Even though virus genomes did not reveal any of the neurotrophic substitutions of DENV documented so far, case isolates possessed a combination of 8 novel amino acid alterations, predominantly distributed in non-structural genes of DENV-4., (Copyright © 2013. Published by Elsevier B.V.)

A sensitive membrane-based electrochemical nanobiosensor is developed for the detection of dengue type 2 virus (DENV-2) using nanoporous alumina-modified platinum electrode. Its sensing mechanism relies on the monitoring of electrode's Faradaic current response toward redox probe, ferrocenemethanol, which is sensitive toward the formation of immune complexes within the alumina nanochannels. Anti-DENV-2 monoclonal antibody (clone 3H5, isotype IgG) is used as the biorecognition element in this work. The stepwise additions of antibody, bovine serum albumin (BSA) and DENV-2 are characterized by differential pulse voltammetry (DPV). A low detection limit of 1 pfu mL(-1) with linear range from 1 to 10(3) pfu mL(-1) (R(2)=0.98) can be achieved by the nanobiosensor. The nanobiosensor is selective toward DENV-2 with insignificant cross reaction with non-specific viruses, Chikungunya virus, West Nile virus and dengue type 3 virus (DENV-3). Relative standard deviation (RSD) for triplicate analysis of 5.9% indicates an acceptable level of reproducibility. The first direct quantitation of DENV-2 concentration in whole mosquito vector is demonstrated using this electrochemical nanobiosensor., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Dengue fever, a vector-borne disease, has caused tremendous burden to countries in the tropics and sub tropics. Over the past 20 years, dengue epidemics have become more widespread, severe and frequent. This study aims to understand the dynamics of dengue viruses in cosmopolitan Singapore. Envelope protein gene sequences of all four dengue serotypes (DENV-1-DENV-4) obtained from human sera in Singapore (2008-2010) revealed that constant viral introductions and in situ evolution contribute to viral diversity in Singapore and play important roles in shaping the epidemiology of dengue in the island state. The diversity of dengue viruses reported here could be a reflection of the on-going dengue situation in the region given Singapore's location in a dengue hyperendemic region and its role as the regional hub for travels and trade. Though cosmopolitan genotype of DENV-2 has remained as the predominant strain circulating in Singapore, we uncovered evidence of in situ evolution which could possibly result in viruses with improved fitness. While we have previously shown that a switch in the predominant dengue serotype could serve as a warning for an impending outbreak, our current data shows that a replacement of a predominant viral clade, even in the absence of a switch in predominant serotype, could signal a possible increase in dengue transmission. The circulating dengue viruses in Singapore are highly diverse, a situation which could offer ample opportunities for selection of strains of higher fitness, thus increasing the risk of outbreaks despite a low Aedes population., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A nanoporous alumina membrane-based ultrasensitive DNA biosensor is constructed using 5'-aminated DNA probes immobilized onto the alumina channel walls. Alumina nanoporous membrane-like structure is carved over platinum wire electrode of 76 µm diameter dimension by electrochemical anodization. The hybridization of complementary target DNA with probe DNA molecules attached inside the pores influences the pore size and ionic conductivity. The biosensor demonstrates linear range over 6 order of magnitude with ultrasensitive detection limit of 9.55×10(-12) M for the quantification of ss-31 mer DNA sequence. Its applicability is challenged against real time cDNA PCR sample of dengue virus serotype1 derived from asymmetric PCR. Excellent specificity down to one nucleotide mismatch in target DNA sample of DENV3 is also demonstrated.

Dengue fever is currently one of the most important mosquito-borne diseases that affect humans. With neither vaccines nor treatment available, prevention of the disease relies heavily on surveillance and control of mosquito vectors. In the present study, we have evaluated and showed the potential use of the Dengue NS1 Ag Strip(®) for the detection of dengue virus (DENV) in Aedes aegypti. Initial results showed that the sensitivity of the test kit in detecting DENV in wild-caught mosquitoes is comparable to that of real-time reverse transcriptase-polymerase chain reaction. The detection of naturally infected Ae. aegypti with the NS1 rapid test kit in our dengue cluster investigation further illustrates its potential use for surveillance of DENV in wild mosquito populations. The kit can easily be used in a simple field station, and minimal training is required. The results can be obtained in less than an hour. Employment of the kit in the field could help guide mosquito control operations in the prioritization of resources in controlling the transmission of DENV. In this study the potential of the kit for field surveillance of infected dengue vectors, which are epidemiologically important, has been demonstrated.

Background: The necessity of a venous blood collection in all dengue diagnostic assays and the high cost of tests that are available for testing during the viraemic period hinder early detection of dengue cases and thus could delay cluster management. This study reports the utility of saliva in an assay that detects dengue virus (DENV)-specific immunoglobulin A (Ig A) early in the phase of a dengue infection., Methods and Findings: Using an antigen capture anti-DENV IgA (ACA) ELISA technique, we tested saliva samples collected from dengue-confirmed patients. The sensitivity within 3 days from fever onset was over 36% in primary dengue infections. The performance is markedly better in secondary infections, with 100% sensitivity reported in saliva samples from day 1 after fever onset. Serum and salivary IgA levels showed good correlation (Pearson's r = 0.69, p<0.001). Specificity was found to be 97%., Conclusion: Our findings suggest that this technique would be very useful in dengue endemic regions, where the majority of dengue cases are secondary. The ACA-ELISA is easy to perform, cost effective, and especially useful in laboratories without sophisticated equipment. Our findings established the usefulness and reliability of saliva for early dengue diagnosis.

Early and accurate diagnosis of dengue is imperative for disease surveillance, which helps in the control of dengue in endemic countries. In this study, we evaluated the performance of three commercially available dengue nonstructural 1 (NS1) antigen assays (Bio-Rad Platelia™ Dengue NS1 Antigen ELISA, PanBio Dengue Early ELISA, and Bio-Rad Dengue NS1 Antigen Strip test) and compared them with reverse-transcription polymerase chain reaction (RT-PCR) and other commercially available serological assays for the diagnosis of dengue. The analysis showed RT-PCR to be the most sensitive and specific (100%) diagnostic method during the first 3 days of fever. The overall sensitivity of dengue NS1 antigen assays within the same period was 81.7%, indicating their potential role as a cost-effective and convenient alternative method to RT-PCR for the diagnosis of dengue fever in a primary healthcare setting. However, reduced sensitivity in detecting secondary dengue infections was one of the drawbacks of dengue NS1 antigen assays. Nonetheless, it remains a useful assay for the early detection of dengue and hence could play an important role in routine surveillance efforts to control dengue outbreaks in Singapore.

In Singapore, after a major outbreak of dengue in 2005, another outbreak occurred in 2007. Laboratory-based surveillance detected a switch from dengue virus serotype 1 (DENV-1) to DENV-2. Phylogenetic analysis showed a clade replacement within DENV-2 cosmopolitan genotype, which accompanied the predominant serotype switch, and cocirculation of multiple genotypes of DENV-3.

Introduction: To determine the seroepidemiology of dengue virus infection in a representative sample of the adult resident population aged 18 years old to 74 years old in Singapore and to estimate the proportion of asymptomatic dengue infection during the 2004 epidemic., Materials and Methods: The study was based on 4152 stored blood samples collected between September and December 2004 from participants aged 18 years old to 74 years old during the 2004 National Health Survey. Sera were tested for IgG and IgM antibodies using a commercial test kit (PanBio Capture/Indirect ELISA)., Results: Of the study population, 59.0% and 2.6% tested positive for dengue IgG (past infection) and IgM/high-titre IgG (recent infection), respectively. Only 17.2% of young adults aged 18 years old to 24 years old were dengue IgG positive. Multivariate analyses showed that older age, Indian ethnicity and male gender were significantly associated with past infection, whereas only age was significantly associated with recent dengue infection. Based on the dengue cases notified during the period of survey, it was estimated that for every 23 individuals recently infected with dengue, only 1 was reported to the health authority as a clinical case., Conclusion: The Singapore population is highly susceptible to dengue epidemics despite its aggressive Aedes prevention and control programme. The finding of a high proportion of unreported cases due to asymptomatic and subclinical infection poses a challenge for dengue control.

Dengue is one of the most important emerging diseases of humans, with no preventative vaccines or antiviral cures available at present. Although one-third of the world's population live at risk of infection, little is known about the pattern and dynamics of dengue virus (DENV) within outbreak situations. By exploiting genomic data from an intensively studied major outbreak, we are able to describe the molecular epidemiology of DENV at a uniquely fine-scaled temporal and spatial resolution. Two DENV serotypes (DENV-1 and DENV-3), and multiple component genotypes, spread concurrently and with similar epidemiological and evolutionary profiles during the initial outbreak phase of a major dengue epidemic that took place in Singapore during 2005. Although DENV-1 and DENV-3 differed in viremia and clinical outcome, there was no evidence for adaptive evolution before, during, or after the outbreak, indicating that ecological or immunological rather than virological factors were the key determinants of epidemic dynamics.

Introduction: We investigated the 2005 outbreak of dengue fever (DF)/dengue haemorrhagic fever (DHF) to determine its epidemiological, virological and entomological features to further understand the unprecedented resurgence., Materials and Methods: All physician-diagnosed, laboratory-confirmed cases of DF/DHF notified to the Ministry of Health, Singapore during the outbreak as well as entomological and virological data were analysed retrospectively., Results: A total of 14,006 cases of DF/DHF comprising 13,625 cases of DF and 381 cases of DHF, including 27 deaths were reported, giving an incidence rate of 322.6 per 100,000 and a case-fatality rate of 0.19%. The median age of the cases and deaths were 32 and 59.5 years, respectively. The incidence rate of those living in compound houses was more than twice that of residents living in public and private apartments. The distribution of DF/DHF cases was more closely associated with Aedes aegypti compared to Aedes albopictus breeding sites and the overall Aedes premises index was 1.15% (2.28% in compound houses and 0.33% to 0.8% in public and private apartments). The predominant dengue serotype was DEN-1. A significant correlation between weekly mean temperature and cases was noted. The correlation was strongest when the increase in temperature preceded rise in cases by a period of 18 weeks., Conclusion: The resurgence occurred in a highly densely populated city-state in the presence of low Aedes mosquito population. Factors contributing to this resurgence included lower herd immunity and change in dominant dengue serotype from DEN-2 to DEN-1. There was no evidence from gene sequencing of the dengue viruses that the epidemic was precipitated by the introduction of a new virulent strain. The current epidemiological situation is highly conducive to periodic dengue recurrences. A high degree of vigilance and active community participation in source reduction should be maintained.

Virus detection methodology provides detection of dengue virus in the early phase of the disease. PCR, targeting cDNA derived from viral RNA, has been used as a laboratory-based molecular tool for the detection of Dengue virus. We report the development and use of three real-time one-step reverse transcriptase PCR (RT-PCR) assays to detect dengue cases and serotype the virus involved. The first RT-PCR assay uses SYBR green I as the reporting dye for the purpose of cost-effective screening for dengue virus. The detection limit of the SYBR green I assay was 10 PFU/ml (0.01 equivalent PFU per assay) for all four dengue virus serotypes. The second RT-PCR assay is a duplex fluorogenic probe-based real-time RT-PCR for serotyping clinical samples for dengue viruses. The detection threshold of the probe-based RT-PCR format was 0.1 PFU for serotypes Dengue-1 and Dengue-2, 1 PFU for serotype Dengue-3, and 0.01 PFU for serotype Dengue-4. The third is a fourplex assay that detects any of the four serotypes in a single closed tube with comparable sensitivity. Validation of the assays with local clinical samples collected from 2004 to 2006 revealed that there was an 88% positive correlation between virus isolation and RT-PCR with regard to dengue virus detection and a 100% correlation with seroconversion in subsequent samples. The serotyping results derived from duplex and fourplex assays agree fully with each other and with that derived from immunofluorescence assays.

Introduction: Dengue is a major public health problem in Singapore. Age-specific dengue morbidity rates are highest in the young adult population, unlike in many other Southeast Asian countries where dengue is mainly a paediatric disease. Hence, the World Health Organization (WHO) guidelines on dengue diagnosis and management which were developed using the paediatric experiences, may not be suitable for the management of adult dengue infections., Materials and Methods: The Early DENgue (EDEN) infection and outcome study is a collaborative longitudinal study to investigate epidemiological, clinical, viral and host-specific features of early dengue-infected adults, in an effort to identify new early markers for prognostication. Patients presenting with early undifferentiated fever were included in the study. We carried out an interim analysis to look for early indicators of severe disease., Results: During the period of this interim study analysis, 455 febrile patients were recruited. Of these, 133 were confirmed as acute dengue cases based on dengue-specific polymerase chain reaction (PCR) results. There were significant clinical and epidemiological differences between dengue and febrile non-dengue cases. Nine per cent of the dengue cases experienced persistent tiredness, drowsiness and loss of appetite beyond 3 weeks of illness. Quantitation of viral loads using the crossover (Ct) value of real-time RT-PCR correlated with the duration of symptoms. More than half of both primary and secondary dengue cases were hospitalised. There was no dengue-related mortality in this study., Conclusion: The duration of illness and prolonged symptom duration in 9% of the subjects indicate that the burden of dengue illness is substantially different from other non-dengue febrile illness in our study cohort. Our study also highlights the paucity of early prognostic markers for dengue fever in adults.