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Central nervous system (CNS) infections cause substantial morbidity and mortality worldwide, with mounting concern about new and emerging neurologic infections. Stratifying etiologies based on initial clinical and laboratory data would facilitate etiology-based treatment rather than relying on empirical treatment. Here, we report the epidemiology and clinical outcomes of patients with CNS infections from a prospective surveillance study that took place between 2013 and 2016 in Singapore. Using multiple correspondence analysis and random forest, we analyzed the link between clinical presentation, laboratory results, outcome and etiology. Of 199 patients, etiology was identified as infectious in 110 (55.3%, 95%-CI 48.3-62.0), immune-mediated in 10 (5.0%, 95%-CI 2.8-9.0), and unknown in 79 patients (39.7%, 95%-CI 33.2-46.6). The initial presenting clinical features were associated with the prognosis at 2 weeks, while laboratory-related parameters were related to the etiology of CNS disease. The parameters measured were helpful to stratify etiologies in broad categories, but were not able to discriminate completely between all the etiologies. Our results suggest that while prognosis of CNS is clearly related to the initial clinical presentation, pinpointing etiology remains challenging. Bio-computational methods which identify patterns in complex datasets may help to supplement CNS infection diagnostic and prognostic decisions.

To understand the genetic diversity and patterns of circulation of rhinoviruses (RV) and enteroviruses (EV) in Singapore, we retrospectively screened 2950 nasal swab samples collected from adults presenting to primary care services with signs of febrile illness in Singapore during 2007-2013 using sequencing and phylogenetic methods. Through sequencing and phylogenetic analysis, our results show the year-round circulation of the three rhinovirus species, A, B, and C. A diverse set of RV/EV serotypes were detected in Singapore with a predominance of RV-A in all years, whereas serotypes EV-C A21 and EV-D68 were only sporadically detected. This study highlights the previously unrecognized diversity and burden in the adult population in Singapore., (© 2019 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.)

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.

Background: Undifferentiated acute febrile illness (AFI) is a common presentation among adults in primary care settings in Singapore but large gaps exist in the understanding of the characteristics of these patients. We studied clinical and epidemiological characteristics of AFI patients and factors associated with delayed recovery from AFI., Methods: We performed a secondary data analysis using data from the Early DENgue infection and outcome (EDEN) study on 2046 adult patients presenting at 5 Singapore polyclinics between December 2007 and February 2013 with a history of fever (≥38 °C) for less than 72 h. We used an accelerated failure time model to investigate factors associated with delayed recovery from AFI., Results: The mean age of patients was 36.6 years, 65 % were male, 51 % were of Chinese ethnicity, and 75 % lived in public housing. Median illness duration was 5 days (interquartile range, 3-7). In multivariable analysis, the unemployed and white collar workers had longer illness duration compared with blue collar workers (time ratio (TR), 1.10; 95 % confidence interval (CI), 1.03-1.17 and TR, 1.08; 95 % CI, 1.02-1.15, respectively). Patients with more symptoms at initial consultation had slower recovery (TR, 1.03 per additional symptom; 95 % CI, 1.02-1.03). Other clinical factors were also associated with longer duration of illness, including use of analgesics (TR, 1.21; 95 % CI, 1.15-1.28); use of cough medicines (TR, 1.14; 95 % CI, 1.08-1.20); use of antibiotics (TR, 1.14; 95 % CI, 1.07-1.21); and hospitalization (TR, 1.59; 95 % CI, 1.39-1.82). Compared to patients with normal WBC count at first consultation, those with low WBC count had slower recovery (TR, 1.14; 95 % CI, 1.07-1.21), while the reverse was observed among patients with high WBC count (TR, 0.94; 95 % CI, 0.88-1.00)., Conclusions: Differences in illness duration among different types of employment may reflect differences in their underlying general health status. Early identification of factors delaying recovery could help triage management in a primary care setting. In-depth characterization of fever etiology in Singapore will improve surveillance and control activities.

Methods: A novel, fully bacterially produced recombinant virus-like particle (VLP) based influenza vaccine (gH1-Qbeta) against A/California/07/2009(H1N1) was tested in a double-blind, randomized phase I clinical trial at two clinical sites in Singapore. The trial evaluated the immunogenicity and safety of gH1-Qbeta in the presence or absence of alhydrogel adjuvant. Healthy adult volunteers with no or low pre-existing immunity against A/California/07/2009 (H1N1) were randomized to receive two intramuscular injections 21 days apart, with 100μg vaccine, containing 42μg hemagglutinin antigen. Antibody responses were measured before and 21 days after each immunization by hemagglutination inhibition (HAI) assays. The primary endpoint was seroconversion on Day 42, defined as percentage of subjects which reach a HAI titer ≥40 or achieve an at least 4-fold rise in HAI titer (with pre-existing immunity). The co-secondary endpoints were safety and seroconversion on Day 21., Results: A total of 84 Asian volunteers were enrolled in this study and randomized to receive the adjuvanted (n=43) or the non-adjuvanted (n=41) vaccine. Of those, 43 and 37 respectively (95%) completed the study. There were no deaths or serious adverse events reported during this trial. A total of 535 adverse events occurred during treatment with 49.5% local solicited symptoms, of mostly (76.4%) mild severity. The most common treatment-related systemic symptom was fatigue. The non-adjuvanted vaccine met all primary and secondary endpoints and showed seroconversion in 62.2% and 70.3% of participants respectively on Day 21 and Day 42. While the adjuvanted vaccine showed an increased seroconversion from 25.5% (Day 21) to 51.2% (Day 42), it did not meet the immunogenicity endpoint., Conclusion: In summary, non-adjuvanted gH1-Qbeta showed similar antibody mediated immunogenicity and a comparable safety profile in healthy humans to commercially available vaccines. These results warrant the consideration of this VLP vaccine platform for the vaccination against influenza infection (HSA CTC1300092)., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Dengue infection is the most common mosquito-borne viral disease worldwide, but no suitable antiviral drugs are available. We tested the α-glucosidase inhibitor celgosivir as a treatment for acute dengue fever., Methods: To establish eligibility for inclusion in a phase 1b, randomised, double-blind, placebo-controlled, proof-of-concept trial, individuals aged 21-65 years who had had a fever (≥38°C) for less than 48 h, met at least two criteria indicating probable dengue infection, and had a positive result on a dengue point-of-care test kit or PCR assay were referred for screening at a centre in Singapore between July 30, 2012, and March 4, 2013. Using a web-based system, we randomly assigned patients who met full inclusion criteria after screening (1:1; random permuted block length four) to celgosivir (initial 400 mg loading dose within 6 h of randomisation, followed by 200 mg every 12 h for a total of nine doses) or matched placebo. Patients and the entire study team were masked to group assignment. The primary endpoints were mean virological log reduction (VLR) from baseline for days 2, 3, and 4, and area under the fever curve (AUC) for a temperature above 37°C from 0 h to 96 h. Efficacy analyses were by intention to treat. This study is registered with ClinicalTrials.gov, number NCT01619969., Findings: We screened 69 patients and randomly assigned 50 (24 to celgosivir, 26 to placebo). Mean VLR was greater in the celgosivir group (-1·86, SD 1·07) than in the placebo group (-1·64, 0·75), but the difference was non-significant (-0·22, 90% CI -0·65 to 0·22; one-sided p=0·203). The mean AUC was also higher in the celgosivir group (54·92, SD 31·04) than in the placebo group (40·72, 18·69), but again the difference was non-significant (14·20, 90% CI 2·16-26·25; one-sided p=0·973). We noted similar incidences of adverse events between groups., Interpretation: Although generally safe and well tolerated, celgosivir does not seem to reduce viral load or fever burden in patients with dengue., Funding: STOP Dengue Translational Clinical Research., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Aims: To characterise carbapenemase-producing Enterobacteriaceae (CRE) isolated in Singapore., Methods: Carbapenemase genes and their flanking regions were amplified and sequenced by PCR. Isolates were typed by pulsed-field gel electrophoresis and multi-locus sequence typing. Plasmids bearing carbapenemase genes were sized by S1 nuclease digestion, Southern blotting, and DNA hybridisation with appropriate probes. Transfer of these plasmids was attempted by conjugation and transformation. Successfully transferred plasmids were characterised by polymerase chain reaction (PCR) replicon typing and restriction digestion., Results: Isolates of Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae and Citrobacter species carried a variety of carbapenemase genes including blaIMP-1, blaIMP-4, blaNDM-1, blaNDM-7, blaOXA-181, blaOXA-48 and blaKPC-2. Apart from K. pneumoniae with blaOXA-181, and some K. pneumoniae with blaNDM-1, the other isolates were not clonal. However there appears to be some spread of plasmids with blaIMP-1, blaNDM-1,blaKPC-2, and blaOXA-48., Conclusions: The number of isolates of CRE has increased in Singapore, especially since 2010. There is a diversity of carbapenemase types that reflects the geographical proximity of other countries with CRE.

Background: In 2001 and 2002, fatal myocarditis resulted in the sudden deaths of four, two adult and two juvenile, orang utans out of a cohort of 26 in the Singapore Zoological Gardens., Methods: Of the four orang utans that underwent post-mortem examination, virus isolation was performed from the tissue homogenates of the heart and lung obtained from the two juvenile orang utans in Vero cell cultures. The tissue culture fluid was examined using electron microscopy. Reverse transcription and polymerase chain reaction with Encephalomyocarditis virus (EMCV)-specific primers targeting the gene regions of VP3/VP1 and 3D polymerase (3Dpol) confirmed the virus genus and species. The two EMCV isolates were sequenced and phylogenetic analyses of the virus genes performed. Serological testing on other animal species in the Singapore Zoological Gardens was also conducted., Results: Electron microscopy of the two EMCV isolates, designated Sing-M100-02 and Sing-M105-02, revealed spherical viral particles of about 20 to 30 nm, consistent with the size and morphology of members belonging to the family Picornaviridae. In addition, infected-Vero cells showed positive immunoflorescence staining with antiserum to EMCV. Sequencing of the viral genome showed that the two EMCV isolates were 99.9% identical at the nucleotide level, indicating a similar source of origin. When compared with existing EMCV sequences in the VP1 and 3Dpol gene regions, the nucleotide divergence were at a maximum of 38.8% and 23.6% respectively, while the amino acid divergence were at a maximum of 33.9% and 11.3% respectively. Phylogenetic analyses of VP1 and 3Dpol genes further grouped the Sing-M100-02 and Sing-M105-02 isolates to themselves, away from existing EMCV lineages. This strongly suggested that Sing-M100-02 and Sing-M105-02 isolates are highly divergent variants of EMCV. Apart from the two deceased orang utans, a serological survey conducted among other zoo animals showed that a number of other animal species had neutralizing antibodies to Sing-M105-02 isolate, indicating that the EMCV variant has a relatively wide host range., Conclusions: The etiological agent responsible for the fatal myocarditis cases among two of the four orang utans in the Singapore Zoological Gardens was a highly divergent variant of EMCV. This is the first report of an EMCV infection in Singapore and South East Asia.

Scrub typhus is a major infectious threat in the Asia-Pacific region. We report an unusual case of scrub typhus in a patient in Singapore who presented with sepsis and acute respiratory distress syndrome but lacked the pathognomonic eschar. The patient recovered after appropriate diagnosis and doxycycline treatment. Rickettsial diseases should be included in the differential diagnosis of febrile illnesses in regions where the diseases are endemic, and absence of eschar should not be the criterion used to rule out scrub typhus.

A growing body of evidence suggests that dengue infection in Singapore predominantly occurs away from the home, but when and where dengue transmission occurs is unclear, confounding control efforts. The authors estimate days of the week in which dengue inpatients in Singapore were infected during the period 2006-2008, based on the day they became febrile and historical data on the incubation period, using Bayesian statistical methods. Among male inpatients, the relative risk of infection is an estimated 57% higher at the weekend, suggesting infections associated with the home or leisure activities. There was no evidence of elevated risk of infection at the weekend for female inpatients. The study motivates further research identifying locales frequented in the week leading up to onset to improve the effective targeting of vector control efforts.

This study was performed to determine the prevalence, distribution of specimen sources, and antimicrobial susceptibility of the Acinetobacter calcoaceticus-Acinetobacter baumannii (Acb) species complex in Singapore. One hundred and ninety-three non-replicate Acb species complex clinical isolates were collected from six hospitals over a 1-month period in 2006. Of these, 152 (78·7%) were identified as A. baumannii, 18 (9·3%) as 'Acinetobacter pittii' [genomic species (gen. sp.) 3], and 23 (11·9%) as 'Acinetobacter nosocomialis' (gen. sp. 13TU). Carbapenem resistance was highest in A. baumannii (72·4%), followed by A. pittii (38·9%), and A. nosocomialis (34·8%). Most carbapenem-resistant A. baumannii and A. nosocomialis possessed the bla(OXA-23-like) gene whereas carbapenem-resistant A. pittii possessed the bla(OXA-58-like) gene. Two imipenem-resistant strains (A. baumannii and A. pittii) had the bla(IMP-like) gene. Representatives of carbapenem-resistant A. baumannii were related to European clones I and II.

Background: Dengue illness causes 50-100 million infections worldwide and threatens 2.5 billion people in the tropical and subtropical regions. Little is known about the disease burden and economic impact of dengue in higher resourced countries or the cost-effectiveness of potential dengue vaccines in such settings., Methods and Findings: We estimate the direct and indirect costs of dengue from hospitalized and ambulatory cases in Singapore. We consider inter alia the impacts of dengue on the economy using the human-capital and the friction cost methods. Disease burden was estimated using disability-adjusted life years (DALYs) and the cost-effectiveness of a potential vaccine program was evaluated. The average economic impact of dengue illness in Singapore from 2000 to 2009 in constant 2010 US$ ranged between $0.85 billion and $1.15 billion, of which control costs constitute 42%-59%. Using empirically derived disability weights, we estimated an annual average disease burden of 9-14 DALYs per 100 000 habitants, making it comparable to diseases such as hepatitis B or syphilis. The proportion of symptomatic dengue cases detected by the national surveillance system was estimated to be low, and to decrease with age. Under population projections by the United Nations, the price per dose threshold for which vaccines stop being more cost-effective than the current vector control program ranged from $50 for mass vaccination requiring 3 doses and only conferring 10 years of immunity to $300 for vaccination requiring 2 doses and conferring lifetime immunity. The thresholds for these vaccine programs to not be cost-effective for Singapore were $100 and $500 per dose respectively., Conclusions: Dengue illness presents a serious economic and disease burden in Singapore. Dengue vaccines are expected to be cost-effective if reasonably low prices are adopted and will help to reduce the economic and disease burden of dengue in Singapore substantially.

Dengue activity depends on fluctuations in Aedes populations which in turn are known to be influenced by climate factors including temperature, humidity and rainfall. It has been hypothesized that haze may reduce dengue transmission. Due to its geographical location Singapore suffers almost every year from hazes caused by wildfires from Indonesia. Such hazes have a significant impact on pollution indexes in Singapore. We set out to study the relationship of dengue activity and haze (measured as pollution standard index) in Singapore, using ARIMA models. We ran different univariate models, each encompassing a different lag period for the effects of haze and temperature (from lag 0 to lag 12 weeks). We analysed the data on a natural logarithmic scale to stabilize the variance and improve the estimation. No association between dengue activity and haze was found. Our findings do not lend support to the hypothesis that haze is associated with reduced dengue activity in Singapore.

Nine imipenem-resistant Pseudomonas aeruginosa isolates were found to contain a variety of metallo-beta-lactamase genes, including bla(IMP-1), bla(IMP-7), bla(VIM-2), bla(VIM-6), and the novel bla(IMP-26). Multilocus sequence typing showed a diversity of sequence types. Comparison with isolates from an earlier study showed that the epidemic clones from 2000 have not become established.

Introduction: Outbreaks of acute respiratory illness occur commonly in long-term care facilities (LTCF), due to the close proximity of residents. Most influenza outbreak reports have been from temperate countries. This study reports an outbreak of influenza B among a highly immunised resident population in a welfare home in tropical Singapore, and discusses vaccine efficacy and the role of acute respiratory illness surveillance for outbreak prevention and control., Materials and Methods: During the period from 16 to 21 March 2007, outbreak investigations and active case finding were carried out among residents and nursing staff at the welfare home. Interviews and medical notes review were conducted to obtain epidemiological and clinical data. Hospitalised patients were tested for respiratory pathogens. Further genetic studies were also carried out on positive respiratory samples., Results: The overall clinical attack rate was 9.4% (17/180) in residents and 6.7% (2/30) in staff. All infected residents and staff had received influenza immunisation. Fifteen residents were hospitalised, with 2 developing severe complications. Genetic sequencing revealed that the outbreak strain had an 8.2% amino acid difference from B/Malaysia/2506/2004, the 2006 southern hemisphere influenza vaccine strain, which the residents and staff had earlier received., Conclusions: A mismatch between the vaccine and circulating influenza virus strains can result in an outbreak in a highly immunised LTCF resident population. Active surveillance for acute respiratory illness in LTCFs could be implemented for rapid detection of antigenic drift. Enhanced infection control and other preventive measures can then be deployed in a timely manner to mitigate the effect of any outbreaks.

Background: All influenza pandemic plans advocate pandemic vaccination. However, few studies have evaluated the cost-effectiveness of different vaccination strategies. This paper compares the economic outcomes of vaccination compared with treatment with antiviral agents alone, in Singapore., Methodology: We analyzed the economic outcomes of pandemic vaccination (immediate vaccination and vaccine stockpiling) compared with treatment-only in Singapore using a decision-based model to perform cost-benefit and cost-effectiveness analyses. We also explored the annual insurance premium (willingness to pay) depending on the perceived risk of the next pandemic occurring., Principal Findings: The treatment-only strategy resulted in 690 deaths, 13,950 hospitalization days, and economic cost of USD$497 million. For immediate vaccination, at vaccine effectiveness of >55%, vaccination was cost-beneficial over treatment-only. Vaccine stockpiling is not cost-effective in most scenarios even with 100% vaccine effectiveness. The annual insurance premium was highest with immediate vaccination, and was lower with increased duration to the next pandemic. The premium was also higher with higher vaccine effectiveness, attack rates, and case-fatality rates. Stockpiling with case-fatality rates of 0.4-0.6% would be cost-beneficial if vaccine effectiveness was >80%; while at case-fatality of >5% stockpiling would be cost-beneficial even if vaccine effectiveness was 20%. High-risk sub-groups warrant higher premiums than low-risk sub-groups., Conclusions: The actual pandemic vaccine effectiveness and lead time is unknown. Vaccine strategy should be based on perception of severity. Immediate vaccination is most cost-effective, but requires vaccines to be available when required. Vaccine stockpiling as insurance against worst-case scenarios is also cost-effective. Research and development is therefore critical to develop and stockpile cheap, readily available effective vaccines.

Chikungunya infections were detected in Singapore among returning travelers who had visited friends and relatives (VFR) in India and Malaysia. These sporadic imported cases occurred over a year before the 2008 chikungunya outbreaks in Singapore, demonstrating the potential for introducing this emerging viral infection into new areas via VFR travel.

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.

A gastroenteritis outbreak occurred in a military camp where a laboratory and epidemiological investigation was carried out. The early onset of symptoms indicated probable food contamination with Clostridium perfringens. Stool samples collected from affected patients were tested within 4 h via real-time polymerase chain reaction (PCR) for the presence of the C. perfringens plc gene. Ten out of the 12 stool samples were positive. Confirmation of the molecular test results was carried out by enumeration of C. perfringens in stool by culture and shown to be in excess of 106 spores/g stool. The isolates obtained from culture were further analysed by PCR for the presence of the chromosomal enterotoxin (cpe) gene. Based on the clinical symptoms, epidemiological and laboratory investigations, C. perfringens was implicated as the aetiological agent. The ability to conduct real-time PCR analysis greatly shortens the time to diagnosis and allows for preventive and control measures to be effected quickly.

We report a case of naturally acquired Plasmodium knowlesi in Singapore, a malaria-free country. Diagnosis was confirmed by PCR with validated species-specific primers. In industrialized countries, free-ranging primates are a potential source of P. knowlesi human infection. P. knowlesi infection is a differential diagnosis of febrile illness acquired in Singapore.

Objective: To reconstruct the historical changes in force of dengue infection in Singapore, and to better understand the relationship between control of Aedes mosquitoes and incidence of classic dengue fever., Methods: Seroprevalence data were abstracted from surveys performed in Singapore from 1982 to 2002. These data were used to develop two mathematical models of age seroprevalence. In the first model, force of infection was allowed to vary independently each year, while in the second it was described by a polynomial function. Model-predicted temporal trends were analysed using linear regression. Time series techniques were employed to investigate periodicity in predicted forces of infection, dengue fever incidence and mosquito breeding., Findings: Force of infection estimates showed a significant downward trend from 1966, when vector control was instigated. Force of infection estimates from both models reproduced significant increases in the percentage and average age of the population susceptible to dengue infections. Importantly, the year-on-year model independently predicted a five to six year periodicity that was also displayed by clinical incidence but absent from the Aedes household index., Conclusion: We propose that the rise in disease incidence was due in part to a vector-control-driven reduction in herd immunity in older age groups that are more susceptible to developing clinical dengue.

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.

An increase in the number of reported melioidosis cases was observed in the first 4 months of 2004. These cases were associated with a significant increase in case-fatality rate compared with the past 5 years. In order to exclude the possibility of a single source, including the possibility of intentional release of Burkholderia pseudomallei, we applied a multiplex PCR-based multilocus variable-number tandem repeat (VNTR) assay to determine the clonality of the clinical isolates. Our investigation indicated that a total of 30 different VNTR types could be distinguished in the 32 clinical isolates of B. pseudomallei obtained during this period, thus indicating that infection was unlikely to have occurred from a single source. Our experience underscores the usefulness of a rapid strain typing method in augmenting an epidemiological investigation into an infectious disease outbreak, particularly at a time where the intentional use of biological agents is a potential threat to public health.

After a 15-year period of low incidence, dengue has reemerged in Singapore in the past decade. We identify potential causes of this resurgence. A combination of lowered herd immunity, virus transmission outside the home, an increase in the age of infection, and the adoption of a case-reactive approach to vector control contribute to the increased dengue incidence. Singapore's experience with dengue indicates that prevention efforts may not be sustainable. For renewed success, Singapore needs to return to a vector control program that is based on carefully collected entomologic and epidemiologic data. Singapore's taking on a leadership role in strengthening disease surveillance and control in Southeast Asia may also be useful in reducing virus importation.

The laboratory diagnosis of dengue has largely relied on serological assays, although many different RT-PCR protocols have been reported. Owing to its limited use, the value of RT-PCR in the clinical laboratory has not been fully evaluated. During the outbreak of severe acute respiratory syndrome (SARS) in Singapore in 2003, RT-PCR to detect dengue viral RNA was used as a rapid diagnostic tool to differentiate dengue from SARS among patients who presented to a hospital designated to manage and quarantine SARS cases. A total of 343 results for RT-PCR and 439 results for serology were analysed and compared with the final discharge diagnosis. Our experience indicates that RT-PCR for dengue can be set up rapidly in a clinical laboratory, with very sensitive and specific results for the diagnosis of dengue, particularly in the first 5 days from onset of symptoms.

Background: An outbreak of dengue occurred among Chinese migrant workers at a construction site in Singapore that was characterized by a number of unusual features., Methods: Clinical and laboratory data were prospectively collected from workers who were hospitalized with fever. We compared the frequency of disease manifestations in these patients with historical data from other cases of dengue admitted to the same centre. A serological survey was performed at the construction site one month after the onset of the outbreak., Results: Thirty-nine patients were admitted from the affected construction site with confirmed acute dengue infection in March and April 2002. Fever, headache and rash were common and occurred at similar frequency in outbreak patients and historical controls. Gastrointestinal manifestations were significantly more common in the outbreak patients compared with controls (nausea 68%, diarrhea 41%, hyponatremia 47% and elevated urea 20%). Of the 274 studied in the serological survey, 27 had serological evidence of acute dengue, of whom 24 (89%) had experienced a febrile illness in the previous month. The virus was cultured and identified as Dengue-2. Few patients had a positive IgG for dengue indicating that this was likely a primary infection., Conclusion: This outbreak of primary dengue infection was characterized by unusually prominent gastrointestinal symptoms and a high proportion of symptomatic transmission. Manifestations of dengue may be altered when outbreaks involve immunologically naïve adults who have migrated to dengue endemic areas.

Legionellosis is endemic in Singapore, with sporadic cases reported throughout the year. The absence of outbreak could be due to the low prevalence of the highly pathogenic Pontiac subtype of Legionella pneumophila in the urban environment. Mandatory maintenance of cooling towers and water fountains has been put in place, and the effectiveness of legislation in minimizing the occurrence and risk of outbreak of legionellosis is being evaluated.

Japanese encephalitis virus (JEV) transmission in Singapore appeared to have ceased after pig farming in Singapore was phased out from the early 1980s. However, the recent detection of neutralizing antibodies to JEV in a population of wild boars in an offshore island, as well as the notification of two human cases of JE in Singapore in 2001, prompted us to reconsider the presence and hence the public health threat of JEV in Singapore. We report here a serological study of animals, birds and humans for neutralizing antibodies to JEV. The results indicate that JEV may still be actively transmitted in the peripheral part of the Singapore island and that regular serological surveys of farm animals and birds, such as chickens, may be useful to further elucidate the activity of JEV in Singapore.

Background: The SARS coronavirus is the etiologic agent for the epidemic of the Severe Acute Respiratory Syndrome. The recent emergence of this new pathogen, the careful tracing of its transmission patterns, and the ability to propagate in culture allows the exploration of the mutational dynamics of the SARS-CoV in human populations., Methods: We sequenced complete SARS-CoV genomes taken from primary human tissues (SIN3408, SIN3725V, SIN3765V), cultured isolates (SIN848, SIN846, SIN842, SIN845, SIN847, SIN849, SIN850, SIN852, SIN3408L), and five consecutive Vero cell passages (SIN2774_P1, SIN2774_P2, SIN2774_P3, SIN2774_P4, SIN2774_P5) arising from SIN2774 isolate. These represented individual patient samples, serial in vitro passages in cell culture, and paired human and cell culture isolates. Employing a refined mutation filtering scheme and constant mutation rate model, the mutation rates were estimated and the possible date of emergence was calculated. Phylogenetic analysis was used to uncover molecular relationships between the isolates., Results: Close examination of whole genome sequence of 54 SARS-CoV isolates identified before 14th October 2003, including 22 from patients in Singapore, revealed the mutations engendered during human-to-Vero and Vero-to-human transmission as well as in multiple Vero cell passages in order to refine our analysis of human-to-human transmission. Though co-infection by different quasipecies in individual tissue samples is observed, the in vitro mutation rate of the SARS-CoV in Vero cell passage is negligible. The in vivo mutation rate, however, is consistent with estimates of other RNA viruses at approximately 5.7 x 10-6 nucleotide substitutions per site per day (0.17 mutations per genome per day), or two mutations per human passage (adjusted R-square = 0.4014). Using the immediate Hotel M contact isolates as roots, we observed that the SARS epidemic has generated four major genetic groups that are geographically associated: two Singapore isolates, one Taiwan isolate, and one North China isolate which appears most closely related to the putative SARS-CoV isolated from a palm civet. Non-synonymous mutations are centered in non-essential ORFs especially in structural and antigenic genes such as the S and M proteins, but these mutations did not distinguish the geographical groupings. However, no non-synonymous mutations were found in the 3CLpro and the polymerase genes., Conclusions: Our results show that the SARS-CoV is well adapted to growth in culture and did not appear to undergo specific selection in human populations. We further assessed that the putative origin of the SARS epidemic was in late October 2002 which is consistent with a recent estimate using cases from China. The greater sequence divergence in the structural and antigenic proteins and consistent deletions in the 3'--most portion of the viral genome suggest that certain selection pressures are interacting with the functional nature of these validated and putative ORFs.

Background: Severe acute respiratory syndrome (SARS) is a newly recognized infectious disease that has recently emerged in East Asia and North America. Although the clinical features of acute infection have been well described, mildly symptomatic or asymptomatic infections have not been well characterized., Objective: To assess the spectrum of illness in health-care workers (HCWs)., Methods: A prospective seroepidemiologic cohort study was conducted on 372 HCWs in a large teaching hospital in Singapore who were both exposed and not exposed to patients with SARS. Participating HCWs completed a questionnaire and provided paired serum samples, which were analyzed by 2 different laboratories blinded to clinical data, by use of an enzyme-linked immunosorbent assay based on a protocol developed by the Centers for Disease Control and Prevention and a dot-blot immunoassay, with confirmation by a viral neutralization assay., Results: A total of 21 patients with SARS were treated at our hospital. They were associated with transmission to 14 staff members, patients, and visitors in our hospital. Of the 372 HCWs participating in the present study, 8 were found to have positive antibodies to the SARS coronavirus in both samples by use of both test methods, and 6 had pneumonia and had been hospitalized for either probable or suspected SARS infection, whereas 2 had fever but did not have changes on chest radiographs. All seropositive HCWs had been exposed either directly or indirectly to patients with SARS. No asymptomatic, nonexposed staff members were found to be seropositive. There was a trend towards protection for HCWs who, while fully protected, had had contact with patients with SARS., Conclusions: Although the majority of cases of SARS are associated with pneumonia, a small number of mildly symptomatic individuals do seroconvert. HCWs who are exposed to patients with SARS can be infected with SARS, regardless of the intensity of exposure. This has implications for surveillance and infection control planning, in the event that SARS returns next winter.

Serum samples were obtained from healthcare workers 5 weeks after exposure to an outbreak of severe acute respiratory syndrome (SARS). A sensitive dot blot enzyme-linked immunosorbent assay, complemented by a specific neutralization test, shows that only persons in whom probable SARS was diagnosed had specific antibodies and suggests that subclinical SARS is not an important feature of the disease.

Malaria is primarily an imported disease in Singapore. Local outbreaks are uncommon. We describe a localised outbreak of three patients with Falciparum malaria, which we believe to be locally acquired. There was one fatality due to severe disease and late presentation. Malaria should be considered as a cause of febrile illness as the likelihood of cure depends on early detection and treatment.

Human enterovirus 71 has caused outbreaks in many parts of the world, especially Southeast Asia, with some fatal cases. The epidemiology of this viral infection is not well understood. We conducted a serologic survey in Singapore children, and the results indicate that infection occurs largely in preschool settings.

A study was conducted to determine the presence of hemagglutination inhibition (HI) and neutralization antibodies against Japanese encephalitis virus (JEV) in wild pigs on an offshore island in Singapore. Blood samples were collected from 28 wild pigs on the island. All the sera tested with HI assay and plaque reduction neutralization tests were found to be positive for antibodies against JEV. Our results indicate that the wild pigs have been infected with JEV on the offshore island and there is JEV transmission.

Nasopharyngeal carcinoma (NPC) is tightly associated with Epstein-Barr virus (EBV) infection and a heavy infiltration of lymphoid cells in the tumor tissue. Although various lines of evidence have shown that the immune systems of NPC patients have the potential to attack the tumor cells, it is not yet understood how this potential is blocked. In this study we determined the circulatory soluble tumor necrosis factor receptors (sTNFRI and sTNFRII), which are proven to be inhibitory to the anti-tumor effects of tumor necrosis factor-alpha (TNF-alpha), in NPC patients. The serum concentration of both sTNFRI and sTNFRII was determined with an ELISA method, and shown to be significantly higher in 28 NPC patients than in matched healthy controls. This elevation was found to be positively correlated with the serum titers of IgA against EBV early antigens and viral capsid antigens in NPC patients, suggesting that the increased serum concentration of sTNFRI and sTNFRII is possibly due to the EBV infection in NPC tumor cells. This is partly supported by FACS analysis of the circulatory T cells. Phenotypical expression of activation markers such as CD25, CD38, CD69 and CD71 in blood T cells was not significantly different between the NPC and control individuals, indicating the elevation of the sTNFRs is indeed derived from the local immune response in the tumor area. Based on these results, it seems that the increased sTNFRs may act as an inhibitor to decrease the host immune response towards tumor cells in NPC patients.