Rapid Diagnostic Tests for the Detection of the Four Dengue Virus Serotypes in Clinically Relevant Matrices.

The efficient and accurate diagnosis of dengue, a major mosquito-borne disease, is of primary importance for clinical care, surveillance, and outbreak control. The identification of specific dengue virus serotype 1 (DENV-1) to DENV-4 can help in understanding the transmission dynamics and spread of dengue disease. The four rapid low-resource serotype-specific dengue tests use a simple sample preparation reagent followed by reverse transcription-isothermal recombinase polymerase amplification (RT-RPA) combined with lateral flow detection (LFD) technology. Results are obtained directly from clinical sample matrices in 35 min, requiring only a heating block and pipettes for liquid handling. In addition, we demonstrate that the rapid sample preparation step inactivates DENV, improving laboratory safety. Human plasma and serum were spiked with DENV, and DENV was detected with analytical sensitivities of 333 to 22,500 median tissue culture infectious doses (TCID50)/mL. The analytical sensitivities in blood were 94,000 to 333,000 TCID50/mL. Analytical specificity testing confirmed that each test could detect multiple serotype-specific strains but did not respond to strains of other serotypes, closely related flaviviruses, or chikungunya virus. Clinical testing on 80 human serum samples demonstrated test specificities of between 94 and 100%, with a DENV-2 test sensitivity of 100%, detecting down to 0.004 PFU/µL, similar to the sensitivity of the PCR test; the other DENV tests detected down to 0.03 to 10.9 PFU/µL. Collectively, our data suggest that some of our rapid dengue serotyping tests provide a potential alternative to conventional labor-intensive RT-quantitative PCR (RT-qPCR) detection, which requires expensive thermal cycling instrumentation, technical expertise, and prolonged testing times. Our tests provide performance and speed without compromising specificity in human plasma and serum and could become promising tools for the detection of high DENV loads in resource-limited settings. IMPORTANCE The efficient and accurate diagnosis of dengue, a major mosquito-borne disease, is of primary importance for clinical care, surveillance, and outbreak control. This study describes the evaluation of four rapid low-resource serotype-specific dengue tests for the detection of specific DENV serotypes in clinical sample matrices. The tests use a simple sample preparation reagent followed by reverse transcription-isothermal recombinase polymerase amplification (RT-RPA) combined with lateral flow detection (LFD) technology. These tests have several advantages compared to RT-qPCR detection, such as a simple workflow, rapid sample processing and turnaround times (35 min from sample preparation to detection), minimal equipment needs, and improved laboratory safety through the inactivation of the virus during the sample preparation step. The low-resource formats of these rapid dengue serotyping tests have the potential to support effective dengue disease surveillance and enhance the diagnostic testing capacity in resource-limited countries with both endemic dengue and intense coronavirus disease 2019 (COVID-19) transmission.

Assessing the efficacy of male Wolbachia-infected mosquito deployments to reduce dengue incidence in Singapore: study protocol for a cluster-randomized controlled trial.

BACKGROUND: Dengue is a severe environmental public health challenge in tropical and subtropical regions. In Singapore, decreasing seroprevalence and herd immunity due to successful vector control has paradoxically led to increased transmission potential of the dengue virus. We have previously demonstrated that incompatible insect technique coupled with sterile insect technique (IIT-SIT), which involves the release of X-ray-irradiated male Wolbachia-infected mosquitoes, reduced the Aedes aegypti population by 98% and dengue incidence by 88%. This novel vector control tool is expected to be able to complement current vector control to mitigate the increasing threat of dengue on a larger scale. We propose a multi-site protocol to study the efficacy of IIT-SIT at reducing dengue incidence. METHODS/DESIGN: The study is designed as a parallel, two-arm, non-blinded cluster-randomized (CR) controlled trial to be conducted in high-rise public housing estates in Singapore, an equatorial city-state. The aim is to determine whether large-scale deployment of male Wolbachia-infected Ae. aegypti mosquitoes can significantly reduce dengue incidence in intervention clusters. We will use the CR design, with the study area comprising 15 clusters with a total area of 10.9 km2, covering approximately 722,204 residents in 1713 apartment blocks. Eight clusters will be randomly selected to receive the intervention, while the other seven will serve as non-intervention clusters. Intervention efficacy will be estimated through two primary endpoints: (1) odds ratio of Wolbachia exposure distribution (i.e., probability of living in an intervention cluster) among laboratory-confirmed reported dengue cases compared to test-negative controls and (2) laboratory-confirmed reported dengue counts normalized by population size in intervention versus non-intervention clusters. DISCUSSION: This study will provide evidence from a multi-site, randomized controlled trial for the efficacy of IIT-SIT in reducing dengue incidence. The trial will provide valuable information to estimate intervention efficacy for this novel vector control approach and guide plans for integration into national vector control programs in dengue-endemic settings. TRIAL REGISTRATION: ClinicalTrials.gov, identifier: NCT05505682 . Registered on 16 August 2022. Retrospectively registered.

Rapid diagnostic tests for the detection of recent dengue infections: An evaluation of six kits on clinical specimens.

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.

Evaluation of eight commercial Zika virus IgM and IgG serology assays for diagnostics and research.

Several commercial Zika virus (ZIKV) serology assays have been developed since the recognition of ZIKV outbreaks as a Public Health Emergency of International Concern in 2016. However, test interpretation for ZIKV serology can be challenging due to antibody cross-reactivity with other flaviviruses like dengue virus (DENV). Therefore, we sought to evaluate the performance of eight commercially available ZIKV IgM and IgG assays across three testing platforms, namely, immunochromatographic tests (ICT), ELISAs and immunofluorescence tests (IIFT). The test panel comprised of 278 samples, including acute and convalescent sera or plasma from ZIKV-confirmed, DENV-confirmed, non-ZIKV and non-DENV patients, and residual sera from healthy blood donors. The ZIKV IgM and IgG serology assays yielded higher test sensitivities of 23.5% - 97.1% among ZIKV convalescent samples as compared to 5.6% - 27.8% among ZIKV acute samples; the test specificities were 63.3% - 100% among acute and convalescent DENV, non-DENV samples. Among the ELISAs and IIFTs, the Diapro ZIKV IgM ELISA demonstrated high test sensitivity (96%) and specificity (80%) when tested on early convalescent samples, while the Euroimmun ZIKV IgG ELISA yielded the highest test specificity of 97% - 100% on samples from non-ZIKV patients and healthy blood donors. For rapid ICTs, the LumiQuick IgM rapid ICT yielded low test sensitivity, suggesting its limited utility. We showed that commercial ZIKV IgM and IgG serology assays have differing test performances, with some having moderate to high test sensitivities and specificities when used in a dengue endemic setting, although there were limitations in IgG serology.

Serological evidence of continued Japanese encephalitis virus transmission in Singapore nearly three decades after end of pig farming.

BACKGROUND: Singapore used to report an annual average of 14 cases of Japanese encephalitis, but ever since the abolishment of pig farms in the early 1990s, the local incidence rate for Japanese encephalitis virus (JEV) infections has reduced drastically. Studies done in the early 2000s demonstrated the presence of JEV-specific antibodies in animals such as wild boars, dogs, chickens and goats on the offshore island and peripheral parts of the Singapore, indicative of prior JEV exposure. A JEV wildlife and sentinel chicken surveillance system was initiated in 2010 through to 2017 to study the animal host seroprofiles. RESULTS: A total of 12/371 (3.23%) of resident bird samples, 24/254 (9.45%) of migratory bird samples and 10/66 (15.16%) of wild boar samples were positive for the presence of JEV antibodies. Seroconversions in sentinel chickens were observed at two time points. Through this study, two sites with active transmission of JEV amongst avian or porcine hosts were identified. CONCLUSIONS: JEV transmission in animal hosts has continued despite the phasing out of pig farming nearly thirty years ago; however, the public health risk of transmission remains low. Environmental management for mosquito population remains key to keeping this risk low.

Highly Selective Transmission Success of Dengue Virus Type 1 Lineages in a Dynamic Virus Population: An Evolutionary and Fitness Perspective.

Arbovirus transmission is modulated by host, vector, virus, and environmental factors. Even though viral fitness plays a salient role in host and vector adaptation, the transmission success of individual strains in a heterogeneous population may be stochastic. Our large-scale molecular epidemiological analyses of a dengue virus type 1 population revealed that only a subset of strains (16.7%; n = 6) were able to sustain transmission, despite the population being widely dispersed, dynamic, and heterogeneous. The overall dominance was variable even among the "established" lineages, albeit sharing comparable evolutionary characteristics and replication profiles. These findings indicated that virological parameters alone were unlikely to have a profound effect on the survival of viral lineages, suggesting an important role for non-viral factors in the transmission success of lineages. Our observations, therefore, emphasize the strategic importance of a holistic understanding of vector, human host, and viral factors in the control of vector-borne diseases.

Dengue in Singapore from 2004 to 2016: Cyclical Epidemic Patterns Dominated by Serotypes 1 and 2.

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.

Viral and Antibody Kinetics, and Mosquito Infectivity of an Imported Case of Zika Fever Due to Asian Genotype (American Strain) in Singapore.

We report a case of a Singaporean who acquired Zika virus (ZIKV) during a visit to Cuba. The infection was confirmed using molecular and serological methods. This report highlights potential drawbacks of using IgG serology for diagnosis of flavivirus infections in endemic regions. The low viremia detected during the early phase of this case resulted in low mosquito infectivity rates, suggesting the possibility of ZIKV transmission prior to clinical onset. The report also emphasizes the challenges of public health interventions for Zika fever and the importance of sustaining a low vector population to reduce the risk of arbovirus transmission in vulnerable regions.

Epidemic resurgence of dengue fever in Singapore in 2013-2014: A virological and entomological perspective.

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.

Genetic Variability in Probe Binding Regions Explains False Negative Results of a Molecular Assay for the Detection of Dengue Virus.

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.

Intra-epidemic evolutionary dynamics of a Dengue virus type 1 population reveal mutant spectra that correlate with disease transmission.

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.

Development and Evaluation of a SYBR Green-Based Real-Time Multiplex RT-PCR Assay for Simultaneous Detection and Serotyping of Dengue and Chikungunya Viruses.

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.

High rates of inapparent dengue in older adults in Singapore.

Although the dengue iceberg phenomenon is well known, there is a paucity of data on inapparent dengue. Results from a seroepidemiological study conducted during a dengue epidemic in 2007 in Singapore showed a seroprevalence of 65.9% and an inapparent dengue rate of 78%. Older adults (> 45 years old) had significantly higher rates of inapparent dengue infections (P < 0.05).

Clinico-genetic characterisation of an encephalitic Dengue virus 4 associated with multi-organ involvement.

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.

Entomologic and molecular investigation into Plasmodium vivax transmission in Singapore, 2009.

BACKGROUND: Singapore has been certified malaria free since November 1982 by the World Health Organization and despite occasional local transmission, the country has maintained the standing. In 2009, three clusters of malaria cases were reported in Singapore. METHODS: Epidemiological, entomological and molecular studies were carried out to investigate the three clusters, namely Mandai-Sungei Kadut, Jurong Island and Sembawang. RESULTS: A total of 29 malaria patients, with no recent travel history, were reported in the three clusters. Molecular analysis based on the msp3α and msp1 genes showed two independent local transmissions: one in Mandai-Sungei Kadut and another in Sembawang. Almost all cases within each cluster were epidemiologically linked. In Jurong Island cluster, epidemiological link remains uncertain, as almost all cases had a unique genetic profile. Only two cases shared a common profile and were found to be linked to the Mandai-Sungei Kadut cluster. Entomological investigation found Anopheles sinensis to be the predominant Anopheline in the two areas where local transmission of P. vivax was confirmed. Anopheles sinensis was found to be attracted to human bait and bites as early as 19:45 hrs. However, all Anopheles mosquitoes caught were negative for sporozoites and oocysts by dissection. CONCLUSION: Investigation of P. vivax cases from the three cluster areas confirmed the occurrence of local transmission in two areas. Although An. sinensis was the predominant Anopheline found in areas with confirmed transmission, the vector/s responsible for the outbreaks still remains cryptic.

Evaluation of Chikungunya diagnostic assays: differences in sensitivity of serology assays in two independent outbreaks.

BACKGROUND: The sensitivity and specificity of two in-house MAC-ELISA assays were tested and compared with the performance of commercially-available CTK lateral flow rapid test and EUROIMMUN IFA assays for the detection of anti-Chikungunya virus (CHIKV) IgM. Each MAC-ELISA assay used a whole virus-based antigen derived from genetically distinct CHIKV strains involved in two chikungunya disease outbreaks in Singapore (2008); a January outbreak strain with alanine at amino acid residue 226 of the E1 glycoprotein (CHIKV-A226) and a May-to-September outbreak strain that possessed valine at the same residue (CHIKV-226V). We report differences in IgM detection efficacy of different assays between the two outbreaks. The sensitivities of two PCR protocols were also tested. METHODS AND FINDINGS: For sera from January outbreak, the average detection threshold of CTK lateral flow test, MAC-ELISAs and EUROIMMUN IFA assays was 3.75, 4.38 and 4.88 days post fever onset respectively. In contrast, IgM detection using CTK lateral flow test was delayed to more than 7 days after fever onset in the second outbreak sera. However, MAC-ELISA using CHIKV-226V detected IgM in the second outbreak sera 3.96 days after fever onset, which was approximately one day earlier compared to the same assay using CHIKV-A226 (4.86 days). Specificity was 100% for both commercial assays, and 95.6% for the in-house MAC-ELISAs. For sensitivity determination of the PCR protocols, the probe-based real time RT-PCR method was found to be 10 times more sensitive than one based on SYBR Green. CONCLUSION: Our findings suggested that the two strains of CHIKV using variants A226 and 226V resulted in variation in sensitivities of the assays evaluated. We postulated that the observed difference in antigen efficacy could be due to the amino acid substitution differences in viral E1 and E2 envelope proteins, especially the E1-A226V substitution. This evaluation demonstrates the importance of appraisal of different diagnostic assays before their application in clinical and operational settings.

Evaluation of nonstructural 1 antigen assays for the diagnosis and surveillance of dengue in Singapore.

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.

Dengue virus surveillance for early warning, 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.

Re-emergence of Chikungunya virus in South-east Asia: virological evidence from Sri Lanka and Singapore.

Chikungunya fever swept across many South and South-east Asian countries, following extensive outbreaks in the Indian Ocean Islands in 2005. However, molecular epidemiological data to explain the recent spread and evolution of Chikungunya virus (CHIKV) in the Asian region are still limited. This study describes the genetic Characteristics and evolutionary relationships of CHIKV strains that emerged in Sri Lanka and Singapore during 2006-2008. The viruses isolated in Singapore also included those imported from the Maldives (n=1), India (n=2) and Malaysia (n=31). All analysed strains belonged to the East, Central and South African (ECSA) lineage and were evolutionarily more related to Indian than to Indian Ocean Islands strains. Unique genetic characteristics revealed five genetically distinct subpopulations of CHIKV in Sri Lanka and Singapore, which were likely to have emerged through multiple, independent introductions. The evolutionary network based on E1 gene sequences indicated the acquisition of an alanine to valine 226 substitution (E1-A226V) by virus strains of the Indian sublineage as a key evolutionary event that contributed to the transmission and spatial distribution of CHIKV in the region. The E1-A226V substitution was found in 95.7 % (133/139) of analysed isolates in 2008, highlighting the widespread establishment of mutated CHIKV strains in Sri Lanka, Singapore and Malaysia. As the E1-A226V substitution is known to enhance the transmissibility of CHIKV by Aedes albopictus mosquitoes, this observation has important implications for the design of vector control strategies to fight the virus in regions at risk of chikungunya fever.

Entomologic and virologic investigation of Chikungunya, Singapore.

Local transmission of chikungunya, a debilitating mosquito-borne viral disease, was first reported in Singapore in January 2008. After 3 months of absence, locally acquired Chikungunya cases resurfaced in May 2008, causing an outbreak that resulted in a total of 231 cases by September 2008. The circulating viruses were related to East, Central, and South African genotypes that emerged in the Indian Ocean region in 2005. The first local outbreak was due to a wild-type virus (alanine at codon 226 of the envelope 1 gene) and occurred in an area where Aedes aegypti mosquitoes were the primary vector. Strains isolated during subsequent outbreaks showed alanine to valine substitution (A226V) and largely spread in areas predominated by Ae. albopictus mosquitoes. These findings led to a revision of the current vector control strategy in Singapore. This report highlights the use of entomologic and virologic data to assist in the control of chikungunya in disease-endemic areas.