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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.

Although Singapore has an intensive dengue control program, dengue remains endemic with regular outbreaks. We report development and use of a novel adult oviposition trap, the Gravitrap, in managing dengue cluster areas. The Gravitrap is a simple, hay infusion-filled cylindrical trap with a sticky inner surface to serve as an oviposition site for gravid female Aedes mosquitoes. Wire gauze fitted above the water level minimizes the risk of it being an unwanted breeding habitat. The Gravitrap was deployed in 11 dengue cluster areas throughout Singapore. Aedes aegypti was the predominant mosquito caught in the trap and some (5.73%) were found to be positive for dengue virus.

Objective: To conduct an external quality assessment (EQA) of dengue and chikungunya diagnostics among national-level public health laboratories in the Asia Pacific region following the first round of EQA for dengue diagnostics in 2013. Methods: Twenty-four national-level public health laboratories performed routine diagnostic assays on a proficiency testing panel consisting of two modules. Module A contained serum samples spiked with cultured dengue virus (DENV) or chikungunya virus (CHIKV) for the detection of nucleic acid and DENV non-structural protein 1 (NS1) antigen. Module B contained human serum samples for the detection of anti-DENV antibodies. Results: Among 20 laboratories testing Module A, 17 (85%) correctly detected DENV RNA by reverse transcription polymerase chain reaction (RT-PCR), 18 (90%) correctly determined serotype and 19 (95%) correctly identified CHIKV by RT-PCR. Ten of 15 (66.7%) laboratories performing NS1 antigen assays obtained the correct results. In Module B, 18/23 (78.3%) and 20/20 (100%) of laboratories correctly detected anti-DENV IgM and IgG, respectively. Detection of acute/recent DENV infection by both molecular (RT-PCR) and serological methods (IgM) was available in 19/24 (79.2%) participating laboratories. Discussion: Accurate laboratory testing is a critical component of dengue and chikungunya surveillance and control. This second round of EQA reveals good proficiency in molecular and serological diagnostics of these diseases in the Asia Pacific region. Further comprehensive diagnostic testing, including testing for Zika virus, should comprise future iterations of the EQA.

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.

Data from longitudinal analyses can be useful in the design and implementation of control strategies., 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.

Population-based serosurveillance studies provide critical estimates on community-level immunity and the potential for future outbreaks. Currently, serological assays, such as IgG enzyme-linked immunosorbent assays (ELISAs) and indirect immunofluorescence tests (IIFT) based on the inactivated whole virus are used to determine past Chikungunya virus (CHIKV) infection. However, these commercially available tests have variable sensitivities. To develop and evaluate recombinant based CHIKV-specific IgG antibody capture ELISAs (GAC-ELISAs), baculoviruses carrying wild-type (E1-A226, named WT) or mutant (E1-A226V, named MUT) E1 envelope protein genes of CHIKV were generated. The seroreactivity of recombinant CHIKV WT and MUT envelope proteins were determined using residual blood, collected from CHIKV-confirmed patients. The sensitivities of both recombinant CHIKV envelope proteins were 83.0% as measured by GAC-ELISAs. The specificities of both recombinant proteins were 87.8%. These GAC-ELISAs were also able to detect the persistence of anti-CHIKV IgG antibodies up to 6 months after the disease onset, together with rise in sensitivities with increasing time. These results suggest that the baculovirus purified recombinant CHIKV envelope proteins react with anti-CHIKV IgG antibodies and may be useful in population-based seroprevalence surveys. In addition, these GAC-ELISAs offer good diagnostic value to determine the recent/past CHIKV infection status in non-endemic populations.

Background The recommendation from the 2009 World Health Organization guidelines for managing dengue suggests that patients with any warning sign can be hospitalized for observation and management. We evaluated the utility of using warning signs to guide hospital admission and predict disease progression in adults. Methods We conducted a prospective cohort study from January 2010 to September 2012. Daily demographic, clinical and laboratory data were collected from adult dengue patients. Warning signs were recorded. The proportion of admitted patients using current admission criteria and warning signs was compared. The sensitivity, specificity, positive and negative predictive values of warning signs in predicting disease progression were also evaluated. Results Four hundred and ninety-nine patients with confirmed dengue were analyzed. Using warning signs instead of the current admission criteria will lead to a 44% and 31% increase in admission for DHF II-IV and SD cases respectively. The proportion of non-severe dengue cases which were admitted also increased by 32% for non DHF II-IV and 33% for non-SD cases. Absence of any warning signs had a NPV of 91%, 100% and 100% for DHF I-IV, DHF II-IV and SD. Of those who progressed to severe illness, 16.3% had warning signs on the same day while 51.3% had warning signs the day before developing severe illness, respectively. Conclusions Our findings demonstrated that patients without any warning signs can be managed safely with ambulatory care to reduce hospital resource burden. No single warning sign can independently predict disease progression. The window from onset of warning sign to severe illness in most cases was within one day.

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., Author Summary Chikungunya is a mounting public health concern in many parts of the world. Definitive diagnosis is critical in differentiating the diseases, especially in dengue endemic areas. There are some commercial chikungunya kits and published molecular protocols available, but no comprehensive comparative evaluation of them was performed. Using sera collected in outbreaks caused by two variants of Chikungunya virus (A226 and 226V), we tested 2 commercial IgM tests (CTK lateral flow rapid test and EUROIMMUN IFA) alongside our in-house IgM assays (using both variants of the virus). Sensitivities of 2 published PCR protocols were also evaluated based on RNA standards derived from cell-cultured viruses. The commercial assays had different performances in each outbreak, with CTK's lateral flow test having the best performance in the first outbreak and EUROIMMUN IFA being more sensitive in the second outbreak. Use of the current circulating virus in a test assay improves sensitivity of the MAC-ELISAs. For PCR, a probe-based real time RT-PCR method was found to be 10 times more sensitive than the SYBR Green method. Despite this, the latter protocol is found to be more suitable and cost-effective for our diagnostic laboratory. This evaluation demonstrates the importance of appraisal of commercial kits and published protocols before application of a diagnostic tool in the clinical and operational setting.

Chikungunya virus (CHIKV) and clinically-related arboviruses cause large epidemics with serious economic and social impact. As clinical symptoms of CHIKV infections are similar to several flavivirus infections, good detection methods to identify CHIKV infection are desired for improved treatment and clinical management. The strength of anti-E2EP3 antibody responses was explored in a longitudinal study on 38 CHIKV-infected patients. We compared their anti-E2EP3 responses with those of patients infected with non-CHIKV alphaviruses, or flaviviruses. E2EP3 cross-reactive samples from patients infected with non-CHIKV viruses were further analyzed with an in vitro CHIKV neutralization assay. CHIKV-specific anti-E2EP3 antibody responses were detected in 72% to 100% of patients. Serum samples from patients infected with other non-CHIKV alphaviruses were cross-reactive to E2EP3. Interestingly, some of these antibodies demonstrated clearly in vitro CHIKV neutralizing activity. Contrastingly, serum samples from flaviviruses-infected patients showed a low level of cross-reactivity against E2EP3. Using CHIKV E2EP3 as a serology marker not only allows early detection of CHIKV specific antibodies, but would also allow the differentiation between CHIKV infections and flavivirus infections with 93% accuracy, thereby allowing precise acute febrile diagnosis and improving clinical management in regions newly suffering from CHIKV outbreaks including the Americas., Author Summary Chikungunya virus (CHIKV) causes Chikungunya fever in humans. The symptoms, particularly joint pain, can be severe and long lasting, and outbreaks can have serious socioeconomic impact. CHIKV is a mosquito-borne alphavirus that co-exists geographically with other mosquito-borne flaviviruses such as dengue virus (DENV). This causes difficulties in diagnosis because the symptoms are similar between CHIKV and DENV infections. It is important to differentiate between CHIKV and DENV infections, with good diagnostic methods. In this paper, we found that 72%–100% of CHIKV-infected patients had antibodies that recognized E2EP3, a part of a CHIKV protein. In contrast, a low percentage of flavivirus-infected patients had antibodies that recognized E2EP3. This suggests that testing patients for the presence of E2EP3-recognizing antibodies will aid in diagnostic differentiation between CHIKV and DENV infections. Interestingly, patients infected with non-chikungunya alphaviruses had moderate levels of antibodies that recognized E2EP3. While it was generally known that the alphaviruses have fairly conserved amino acid sequences, it was unknown until now, to what extent the antibodies against non-chikungunya viruses would also recognize E2EP3 from CHIKV. This paper provides insights about the E2EP3-recognizing antibodies from patients with different mosquito-borne viral infections and these insights will inform approaches to diagnostics and vaccination.

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. Published version