Background:Accurate COVID-19 incidence estimates, including undiagnosed cases, are vital for epidemic management but are often unavailable in real time. Participatory surveillance can capture community illness episodes; however, quantifying undiagnosed infections remains difficult. We assessed a Singaporean cohort to estimate medically unattended COVID-19 infections by combining symptom models with proxy epidemic indicators. Objective:This study aims to estimate COVID-19 incidence and medically attended fractions using participatory surveillance data and to evaluate the consistency of these estimates against independently derived serological measures of infection in a community cohort in Singapore. Methods:We analyzed 11 survey waves (September 2021 to November 2022) from the SOCRATEs (Strengthening Our Community's Resilience Against Threats from Emerging Infections) community cohort (n=1899), spanning Delta and Omicron variant waves. Respondents reported recent illness, symptoms, health care use, and COVID-19 diagnoses. Multilevel logistic regression of medically attended episodes estimated the probability of COVID-19 in unattended episodes, incorporating symptoms and external indicators-wastewater viral-load index and health care staff surveillance. The estimates of total infections and medically attended fractions were validated against independent serological survey results. Results:Among 2284 illness episodes, 756 were diagnosed with COVID-19, of which 62.4% (472/756) were medically attended. Health care-seeking declined from 83.9% (26/31) of COVID-19 episodes early in 2022 to 55.3% (47/85) by late 2022. Regression models demonstrated strong associations between COVID-19 infection and key symptoms and epidemic activity indicators. Estimated total infections were substantially higher than notified cases, reaching 1.0 to 2.9 times the reported incidence across successive variant waves. Model-based estimates of medically attended fractions were broadly consistent with serological benchmarks. Incidence estimates closely matched serological estimates in earlier intervals, with slight overestimation in later periods due to reinfection. Conclusions:Participatory surveillance, when combined with probabilistic modeling and external indicators of epidemic activity, can generate robust estimates of infection incidence and health care use. Agreement with serological data supports the validity of this integrated framework, although discrepancies persist in later epidemic phases due to reinfection dynamics. This approach provides a scalable and timely complement to traditional and serological surveillance systems.

Serologic tests to detect specific IgGs to antigens related to viral infections are urgently needed for diagnostics and therapeutics. We present a diagnostic method for serotype-specific IgG identification of dengue infection by a competitive enzyme-linked immunosorbent assay (ELISA), using high-affinity unnatural-base-containing DNA (UB-DNA) aptamers that recognize the four categorized serotypes. Using UB-DNA aptamers specific to each serotype of dengue NS1 proteins (DEN-NS1), we developed our aptamer-antibody sandwich ELISA for dengue diagnostics. Furthermore, IgGs highly specific to DEN-NS1 inhibited the serotype-specific NS1 detection, inspiring us to develop the competitive ELISA format for dengue serotype-specific IgG detection. Blood samples from Singaporean patients with primary or secondary dengue infections confirmed the highly specific IgG detection of this format, and the IgG production initially reflected the serotype of the past infection, rather than the recent infection. Using this dengue competitive ELISA format, cross-reactivity tests of 21 plasma samples from Singaporean Zika virus-infected patients revealed two distinct patterns: 8 lacked cross-reactivity, and 13 were positive with unique dengue serotype specificities, indicating previous dengue infection. This antigen-detection ELISA and antibody-detection competitive ELISA combination using the UB-DNA aptamers identifies both past and current viral infections and will facilitate specific medical care and vaccine development for infectious diseases.

Objective To investigate community and health-care workers' perspectives on the coronavirus disease 2019 (COVID-19) pandemic and on early pandemic responses during the first 2 weeks of national lockdown in Zimbabwe. Methods Rapid qualitative research was carried out between March and April 2020 via phone interviews with one representative from each of four community-based organizations and 16 health-care workers involved in a trial of community-based services for young people. In addition, information on COVID-19 was collected from social media platforms, news outlets and government announcements. Data were analysed thematically. Findings Four themes emerged: (i) individuals were overloaded with information but lacked trusted sources, which resulted in widespread fear and unanswered questions; (ii) communities had limited ability to comply with prevention measures, such as social distancing, because access to long-term food supplies and water at home was limited and because income had to be earned daily; (iii) health-care workers perceived themselves to be vulnerable and undervalued because of a shortage of personal protective equipment and inadequate pay; and (iv) other health conditions were sidelined because resources were redirected, with potentially wide-reaching implications. Conclusion It is important that prevention measures against COVID-19 are appropriate for the local context. In Zimbabwe, communities require support with basic needs and access to reliable information to enable them to follow prevention measures. In addition, health- care workers urgently need personal protective equipment and adequate salaries. Essential health-care services and medications for conditions other than COVID-19 must also continue to be provided to help reduce excess mortality and morbidity.

Carbapenemase-producing Enterobacteriaceae (CPE) are a group of drug-resistant Gram-negative pathogens that are classified as a critical threat by the World Health Organization (WHO). Conventional methods of detecting antibiotic-resistant pathogens do not assess the resistance mechanism and are often time-consuming and laborious. We have developed a magnetic digital microfluidic (MDM) platform, known as MDM Carba, for the identification of CPE by measuring their ability to hydrolyze carbapenem antibiotics. MDM Carba offers the ability to rapidly test CPE and reduce the amount of reagents used compared with conventional phenotypic testing. On the MDM Carba platform, tests are performed in droplets that function as reaction chambers, and fluidic operations are accomplished by manipulating these droplets with magnetic force. The simple droplet-based magnetic fluidic operation allows easy system automation and simplified hands-on operation. Because of the unique "power-free" operation of MDM technology, the MDM Carba platform can also be operated manually, showing great potential for point-of-care testing in resource-limited settings. We tested 27 bacterial isolates on the MDM Carba platform, and the results showed sensitivity and specificity that were comparable to those of the widely used Carba NP test. MDM Carba may shorten the overall turnaround time for CPE identification, thereby enabling more timely clinical decisions for better clinical outcomes. MDM Carba is a technological platform that can be further developed to improve diagnostics for other types of antibiotic resistance with minor modifications.

Prior immunological exposure to dengue virus can be both protective and disease-enhancing during subsequent infections with different dengue virus serotypes. We provide here a systematic, longitudinal analysis of B cell, T cell, and antibody responses in the same patients. Antibody responses as well as T and B cell activation differentiate primary from secondary responses. Hospitalization is associated with lower frequencies of activated, terminally differentiated T cells and higher percentages of effector memory CD4 T cells. Patients with more severe disease tend to have higher percentages of plasmablasts. This does not translate into long-term antibody titers, since neutralizing titers after 6 months correlate with percentages of specific memory B cells, but not with acute plasmablast activation. Overall, our unbiased analysis reveals associations between cellular profiles and disease severity, opening opportunities to study immunopathology in dengue disease and the potential predictive value of these parameters.

Genetic alphabet expansion of DNA by introducing unnatural bases (UBs), as a fifth letter, dramatically augments the affinities of DNA aptamers that bind to target proteins. To determine whether UB-containing DNA (UB-DNA) aptamers obtained by affinity selection could spontaneously achieve high specificity, we have generated a series of UB-DNA aptamers (K-D: 27-182 pM) targeting each of four dengue non-structural protein 1 (DEN-NS1) serotypes. The specificity of each aptamer is remarkably high. and the aptamers can recognize the subtle variants of DEN-NS1 with at least 96.9% amino acid sequence identity, beyond the capability of serotype identification (69-80% sequence identities). Our UB-DNA aptamers specifically identified two major variants of dengue serotype 1 with 10-amino acid differences in the DEN-NS1 protein (352 aa) in Singaporeans' clinical samples. These results suggest that the high-affinity UB-DNA aptamers generated by affinity selection also acquire high target specificity. Intriguingly, one of the aptamers contained two different UBs as fifth and sixth letters, which are essential for the tight binding to the target. These two types of unnatural bases with distinct physicochemical properties profoundly expand the potential of DNA aptamers. Detection methods incorporating the UB-DNA aptamers will facilitate precise diagnoses of viral infections and other diseases.

Dengue virus (DENV) and Zika virus (ZIKV) belong to the Flaviviridae family of viruses spread by Aedes aegypti mosquitoes in tropical and subtropical areas. Accurate diagnostic tests to differentiate the 2 infections are necessary for patient management and disease control. Using characterized ZIKV and DENV patient plasma in a blind manner, we validated an ELISA and a rapid immunochromatographic test for ZIKV detection. We engineered the ZIKV nonstructural protein 1 (NS1) for sensitive serologic detection with low cross reactivity against dengue and developed monoclonal antibodies specific for the ZIKV NS1 antigen. As expected, the serologic assays performed better with convalescent than acute plasma samples; the sensitivity ranged from 71% to 88%, depending on the performance of individual tests (IgM/IgG/NS1). Although serologic tests were generally less sensitive with acute samples, our ZIKV NS1 antibodies were able to complement the serologic tests to achieve greater sensitivity for detecting early infections.

OBJECTIVES:WHO recommends that low burden countries consider systematic screening and treatment of latent tuberculosis infection (LTBI) in migrants from high incidence countries. We aimed to determine LTBI prevalence and risk factors and evaluate cost-effectiveness of screening and treating LTBI in migrants to Singapore from a government payer perspective.DESIGN:Cross-sectional study and cost-effectiveness analysis.SETTING:Migrants in Singapore.PARTICIPANTS:3618 migrants who were between 20 and 50 years old, have not worked in Singapore previously and stayed in Singapore for less than a year were recruited.PRIMARY AND SECONDARY OUTCOME MEASURES:Costs, quality-adjusted life-years (QALYs), threshold length of stay, incremental cost-effectiveness ratios (ICERs), cost per active TB case averted.RESULTS:Of 3584 migrants surveyed, 20.4% had positive interferon-gamma release assay (IGRA) results, with the highest positivity in Filipinos (33.2%). Higher LTBI prevalence was significantly associated with age, marital status and past TB exposure. The cost-effectiveness model projected an ICER of S$57 116 per QALY and S$12 422 per active TB case averted for screening and treating LTBI with 3 months once weekly isoniazid and rifapentine combination regimen treatment compared with no screening over a 50-year time horizon. ICER was most sensitive to the cohort's length of stay in Singapore, yearly disease progression rates from LTBI to active TB, followed by the cost of IGRA testing.CONCLUSIONS:For LTBI screening and treatment of migrants to be cost-effective, migrants from high burden countries would have to stay in Singapore for ~50 years. Risk-stratified approaches based on projected length of stay and country of origin and/or age group can be considered.

Current methods for dengue virus (DENV) genome amplification, amplify parts of the genome in at least 5 overlapping segments and then combine the output to characterize a full genome. This process is laborious, costly and requires at least 10 primers per serotype, thus increasing the likelihood of PCR bias. We introduce an assay to amplify near full-length dengue virus genomes as intact molecules, sequence these amplicons with third generation "nanopore" technology without fragmenting and use the sequence data to differentiate within-host viral variants with a bioinformatics tool (Nano-Q). The new assay successfully generated near full-length amplicons from DENV serotypes 1, 2 and 3 samples which were sequenced with nanopore technology. Consensus DENV sequences generated by nanopore sequencing had over 99.5% pairwise sequence similarity to Illumina generated counterparts provided the coverage was>100 with both platforms. Maximum likelihood phylogenetic trees generated from nanopore consensus sequences were able to reproduce the exact trees made from Illumina sequencing with a conservative 99% bootstrapping threshold (after 1000 replicates and 10% burn-in). Pairwise genetic distances of within host variants identified from the Nano-Q tool were less than that of between host variants, thus enabling the phylogenetic segregation of variants from the same host.

[This corrects the article DOI: 10.1002/cti2.1066.].

Background A range of immunomodulatory therapies have been proposed as adjuncts to conventional antivirals to suppress harmful inflammation during severe influenza infection. We conducted a systematic review to assess available data of the effect of adjunctive non-corticosteroid immunomodulatory therapy and potential adverse effects. Method We searched MEDLINE, Embase, Web of Science and clinical trial databases for published and unpublished studies, and screened the references of included articles. We included RCTs, quasi-RCTs and observational studies of virologically confirmed influenza infections in hospitalised patients. We did not restrict studies by language of publication, influenza type/subtype or age of participants. Where possible, we pooled estimates of effect using random-effects meta-analysis models. Results We identified 11 eligible studies for inclusion: five studies (4 RCTs and 1 observational; 693 individuals) of passive immune therapy; four studies (3 RCTs and 1 observational; 1120 individuals) of macrolides and/or non-steroidal anti-inflammatory drugs (NSAIDs), one RCT of mTOR inhibitors (38 individuals), and one RCT of statin therapy (116 individuals). Meta-analysis of RCTs of passive immune therapy indicated no significant reduction in mortality (OR 0.84, 0.37-1.90), but better clinical outcomes at Day 7 (OR 1.42, 1.05-1.92). There was a significant reduction in mortality associated with macrolides and/or NSAIDs (OR 0.28; 0.10-0.77). Conclusions Passive immune therapy is unlikely to offer substantial mortality benefit in treatment of severe seasonal influenza, but may improve clinical outcomes. The effect of other immunomodulatory agents is uncertain, but promising. There is a need for high-quality RCTs with sufficient statistical power to address this evidence gap.

Abstract Objective To evaluate how public perceptions and trust in government communications affected the adoption of protective behaviour in Singapore during the coronavirus disease 2019 (COVID-19) pandemic. Methods We launched our community-based cohort to assess public perceptions of infectious disease outbreaks in mid-2019. After the first case of COVID-19 was reported in Singapore on 23 January, we launched a series of seven COVID-19 surveys to both existing and regularly enrolled new participants every 2 weeks. As well as sociodemographic properties of the participants, we recorded changing responses to judge awareness of the situation, trust in various information sources and perceived risk. We used multivariable logistic regression models to evaluate associations with perceptions of risk and self-reported adopted frequencies of protective behaviour. Findings Our cohort of 633 participants provided 2857 unique responses during the seven COVID-19 surveys. Most agreed or strongly agreed that information from official government sources (99.1%; 528/533) and Singapore-based news agencies (97.9%; 522/533) was trustworthy. Trust in government communication was significantly associated with higher perceived threat (odds ratio, OR: 2.2; 95% confidence interval, CI: 1.6–3.0), but inversely associated with perceived risk of infection (OR: 0.6; 95% CI: 0.4–0.8) or risk of death if infected (OR: 0.6; 95% CI: 0.4–0.9). Trust in government communication was also associated with a greater likelihood of adopting protective behaviour. Conclusion Our findings show that trust is a vital commodity when managing an evolving outbreak. Our repeated surveys provided real-time feedback, allowing an improved understanding of the interplay between perceptions, trust and behaviour.

Background: To demonstrate the use of a cohort for assessing knowledge, perceptions and behaviours of the Singapore population towards infectious disease outbre

Objectives Recent Zika virus (ZIKV) outbreaks challenged existing laboratory diagnostic standards, especially for serology-based methods. Because of the genetic and structural similarity of ZIKV with other flaviviruses, this results in cross-reactive antibodies, which confounds serological interpretations. Methods Plasma from Singapore ZIKV patients was screened longitudinally for antibody responses and neutralising capacities against ZIKV. Samples from healthy controls, ZIKV patients and DENV patients were further assessed using ZIKV and DENV peptides of precursor membrane (prM), envelope (E) or non-structural 1 (NS1) viral proteins in a peptide-based ELISA for epitope identification. Identified epitopes were re-validated and diagnostically evaluated using sera of patients with DENV, bacteria or unknown infections from Thailand. Results Long-lasting ZIKV-neutralising antibodies were elicited during ZIKV infection. Thirteen potential linear B-cell epitopes were identified, and of these, four common flavivirus, three ZIKV-specific and one DENV-specific differential epitopes had more than 50% sensitivity and specificity. Notably, ZIKV-specific peptide 26 on domain I/II of E protein (amino acid residues 271-288) presented 80% sensitivity and 85.7% specificity. Importantly, the differential epitopes also showed significance in differentiating non-flavivirus patient samples. Conclusion Linear B-cell epitope candidates to differentiate between ZIKV and DENV infections were identified, providing the first step towards the design of a much-needed serology-based assay.

Targeted proteomic mass spectrometry is emerging as a salient clinical diagnostic tool to track protein biomarkers. However, its strong analytical properties have not been exploited in the diagnosis and typing of flaviviruses. Here, we report the development of a sensitive and specific single-shot robust assay for flavivirus typing and diagnosis using targeted mass spectrometry technology. Our flavivirus parallel reaction monitoring assay (fvPRM) has the ability to track secreted flaviviral nonstructural protein 1 (NS1) over a broad diagnostic and typing window with high sensitivity, specificity, extendibility, and multiplexing capability. These features, pivotal and pertinent to efficient response toward flavivirus outbreaks, including newly emerging flavivirus strains, circumvent the limitations of current diagnostic assays. fvPRM thus carries high potential in positioning itself as a forerunner in delivering early and accurate diagnosis for disease management.

Background. Since its unexpected reemergence, Zika virus (ZIKV) has caused numerous outbreaks globally. This study characterized the host immune responses during ZIKV infection. Methods. Patient samples were collected longitudinally during the acute, convalescence and recovery phases of ZIKV infection over 6 months during the Singapore outbreak in late 2016. Plasma immune mediators were profiled via multiplex microbead assay, while changes in blood cell numbers were determined with immunophenotyping. Results. Data showed the involvement of various immune mediators during acute ZIKV infection accompanied by a general reduction in blood cell numbers for all immune subsets except CD14(+) monocytes. Importantly, viremic patients experiencing moderate symptoms had significantly higher quantities of interferon gamma-induced protein 10, monocyte chemotactic protein 1, interleukin 1 receptor antagonist, interleukin 8, and placental growth factor 1, accompanied by reduced numbers of peripheral CD8(+) T cells, CD4(+) T cells, and double-negative T cells. Levels of T-cell associated mediators, including interferon gamma-induced protein 10, interferon gamma, and interleukin 10, were high in recovery phases of ZIKV infection, suggesting a functional role for T cells. The identification of different markers at specific disease phases emphasizes the dynamics of a balanced cytokine environment in disease progression. Conclusions. This is the first comprehensive study that highlights specific cellular changes and immune signatures during ZIKV disease progression, and it provides valuable insights into ZIKV immunopathogenesis.

Zika virus (ZIKV) has re-emerged in the population and caused unprecedented global outbreaks. Here, the transcriptomic consequences of ZIKV infection were studied systematically firstly in human peripheral blood CD14+ monocytes and monocyte-derived macrophages with high density RNA-sequencing. Analyses of the ZIKV genome revealed that the virus underwent genetic diversification and differential mRNA abundance was found in host cells during infection. Notably, there was a significant change in the cellular response with crosstalk between monocytes and natural killer (NK) cells as one of the highly identified pathway. Immune-phenotyping of peripheral blood from ZIKV-infected patients further confirmed the activation of NK cells during acute infection. ZIKV infection in peripheral blood cells isolated from healthy donors led to the induction of IFNγ and CD107a — two key markers of NK-cell function. Depletion of CD14+ monocytes from peripheral blood resulted in a reduction of these markers and reduced priming of NK cells during infection. This was complemented by the immunoproteomic changes observed. Mechanistically, ZIKV infection preferentially counterbalances monocyte and/or NK-cell activity, with implications for targeted cytokine immunotherapies.

BACKGROUND:Methicillin-resistant Staphylococcus aureus (MRSA) is the most common healthcare-associated multidrug-resistant organism. Despite the interconnectedness between acute care hospitals (ACHs) and intermediate- and long-term care facilities (ILTCFs), the transmission dynamics of MRSA between healthcare settings is not well understood. METHODS:We conducted a cross-sectional study in a network comprising an ACH and 5 closely affiliated ILTCFs in Singapore. A total of 1700 inpatients were screened for MRSA over a 6-week period in 2014. MRSA isolates underwent whole-genome sequencing, with a pairwise single-nucleotide polymorphism (Hamming distance) cutoff of 60 core genome single-nucleotide polymorphisms used to define recent transmission clusters (clades) for the 3 major clones. RESULTS:MRSA prevalence was significantly higher in intermediate-term (29.9%) and long-term (20.4%) care facilities than in the ACH (11.8%) (P < .001). The predominant clones were sequence type [ST] 22 (n = 183; 47.8%), ST45 (n = 129; 33.7%), and ST239 (n = 26; 6.8%), with greater diversity of STs in ILTCFs relative to the ACH. A large proportion of the clades in ST22 (14 of 21 clades; 67%) and ST45 (7 of 13; 54%) included inpatients from the ACH and ILTCFs. The most frequent source of the interfacility transmissions was the ACH (n = 28 transmission events; 36.4%). CONCLUSIONS:MRSA transmission dynamics between the ACH and ILTCFs were complex. The greater diversity of STs in ILTCFs suggests that the ecosystem in such settings might be more conducive for intrafacility transmission events. ST22 and ST45 have successfully established themselves in ILTCFs. The importance of interconnected infection prevention and control measures and strategies cannot be overemphasized.