Acute infectious diarrhea imposes a substantial socioeconomic burden globally. In Singapore, information on etiological agents of diarrhea remains limited. Through analysis of data from public hospitals, this study presents key insights into the epidemiology of diarrheagenic pathogens. Two datasets comprising gastrointestinal multiplex polymerase chain reaction (PCR) panel results from adult and pediatric populations, drawn from five hospitals across several years, were collated. Stool samples included in the study were screened for 22 pathogens using a commercial PCR assay. Overall sample positivity, pathogen relative abundances, and co-detection rate were determined. Potential associations between co-detected pathogens were assessed via pairwise correlations. Additionally, exploratory analysis was undertaken to investigate seasonal patterns and climatic influence on pathogen detection. Sample positivity was 42.6% (3,212/7,543) in the adult data set and 52.1% (1,572/3,015) in the pediatric data set. Despite differences in ranking, the most frequently detected organisms in both groups were pathogenic Escherichia coli, Clostridioides difficile, Salmonella, and Norovirus. Differences in pathogen proportions between data sets reveal distinct epidemiological profiles in adults and children. Geographic variations in the prevalence of pathogens such as Plesiomonas shigelloides and Vibrio underscore the influence of climate change on infectious disease transmission. Co-detection of two or more organisms occurred in 14.7% of adult samples. Pathogen detections peaked in the warmer, drier mid-year months. This study highlights the significant contribution of non-notifiable pathogens to acute diarrhea burden, contextualizing local data within global epidemiological trends. Our findings carry important implications for clinical decision-making, public health policy, One Health disease prioritization, and optimization of surveillance strategies.IMPORTANCEAcute infectious diarrhea remains a major global health issue, yet in many cases, specific etiologies are often undiagnosed or underreported. This study uses multiplex PCR panel testing to identify a broad spectrum of diarrheal pathogens from thousands of patients across public hospitals, offering one of the most comprehensive assessments in the Southeast Asian region to date. It highlights the potentially overlooked role of non-notifiable pathogens and reveals distinct epidemiological patterns in adult and pediatric populations. Furthermore, it uncovers seasonal trends and potential links between climate and disease transmission. These findings fill critical knowledge gaps, improving clinical decision-making and strengthening public health responses. Our study supports the integration of environmental and epidemiological data to optimize health planning and offers useful insights for enhancing national surveillance strategies under a One Health approach.

Background: COVID-19 pandemic response measures caused a broad decline of other respiratory pathogens, but these did not return immediately when measures were lifted. We assessed the dynamics of pathogen re-emergence in Singapore.

INTRODUCTION:Influenza is an important cause of paediatric illness across the globe. However, information about the relationships between air pollution, meteorological variability and paediatric influenza A and B infections in tropical settings is limited.METHODS:We analysed all daily reports of influenza A and B infections in children <5 years old obtained from the largest specialist women and children's hospital in Singapore. In separate negative binomial regression models, we assessed the dependence of paediatric influenza A and B infections on air quality and meteorological variability, using multivariable fractional polynomial modelling and adjusting for time-varying confounders.RESULTS:Approximately 80% of 7329 laboratory-confirmed reports were caused by influenza A. We observed positive associations between sulphur dioxide (SO2) exposure and the subsequent risk of infection with both influenza types. We observed evidence of a harvesting effect of SO2 on Influenza A but not Influenza B. Ambient temperature was associated with a decline in influenza A reports (Relative Risk at lag 5 [RRlag5]: 0.949, 95% CI: 0.916-0.983). Rainfall was positively associated with a subsequent increase in influenza A reports (RRlag3: 1.044, 95% CI: 1.017-1.071). Nitrogen dioxide (NO2) concentration was positively associated with influenza B reports (RRlag5: 1.015, 95% CI: 1.005-1.025). There was a non-linear association between CO and influenza B reports. Absolute humidity increased the ensuing risk of influenza B (RRlag5: 4.799, 95% CI: 2.277-10.118). Influenza A and B infections displayed dissimilar but predictable within-year seasonal patterns.CONCLUSIONS:We observed different independent associations between air quality and meteorological variability with paediatric influenza A and B infections. Anticipated seasonal infection peaks and variations in air quality and meteorological parameters can inform the timing of community measures aimed at reducing influenza infection risk.

Respiratory syncytial virus (RSV) is an important cause of respiratory illness among children. While studies have focused on the air-quality and climate dependence of RSV infections, few have been undertaken in South-East Asia where the burden of respiratory illness is among the highest across the globe. This study aimed to determine the relationships between climatic factors and air quality with RSV infections among children in Singapore. We obtained all laboratory-confirmed reports of RSV infections in children below 5 years old from the largest public hospital specializing in pediatric healthcare in Singapore. We assessed the independent cumulative effects of air quality and meteorological factors on RSV infection risk using the Distributed Lag Non-Linear Model (DLNM) framework in negative binomial models adjusted for long-term trend, seasonality and changes in the diagnostic systems. We included 15,715 laboratory-confirmed RSV reports from 2009 to 2019. Daily maximum temperature exhibited a complex, non-linear association with RSV infections. Absolute humidity (Relative Risk, 90th percentile [RR90th percentile]: 1.170, 95% CI: [1.102, 1.242]) was positively associated with RSV risk. Higher levels of particulate matter of aerodynamic diameter of less than (i) 2.5 µm (PM2.5), (ii) 10 µm (PM10), carbon monoxide (CO) and sulfur dioxide (SO2) were associated with lower RSV infection risk. RSV infections exhibited both annual and within-year seasonality. Our findings suggest that falls in ambient temperature and rises in absolute humidity exacerbated pediatric RSV infection risk while increases in air pollutant concentrations were associated with lowered infection risk. These meteorological factors, together with the predictable seasonality of RSV infections, can inform the timing of mitigation measures aimed at reducing transmission.

Objectives: Evidence of the relationship between climate variability, air pollution and human parainfluenza virus (HPIV) infections has been inconsistent. We assessed this in a paediatric population from a highly urbanized tropical city-state. Methods: We analysed all reports of HPIV infections in children <5 years old obtained from a major specialist women and children's hospital in Singapore. Assuming a negative binomial distribution and using multivariable fractional polynomial modelling, we examined the relations between climate variability, air quality and the risk of HPIV infections, adjusting for time-varying confounders. Results: We identified 6393 laboratory-confirmed HPIV infections from 2009 to 2019. Every 1 C decline in temperature was associated with a 5.8% increase (RR: 0.943, 95% Confidence Interval [95% CI]: 0.903-0.984) in HPIV infection risk 6 days later. Every 10% decrease in relative humidity was associated with a 15.8% cumulative increase in HPIV risk over the next 6 days (cumulative RR: 0.842, 95% CI: 0.771-0.919). Rainfall was positively associated with HPIV risk 2 days later (RR: 1.021, 95% CI: 1.000-1.043). A within-year seasonal rise of HPIV was driven by HPIV-3 and HPIV-1 and preceded by a seasonal decline in temperature. Gender was an effect modifier of the climate-HPIV relationship. Air quality was not associated with HPIV risk. Conclusions: This study demonstrates a close association between HPIV infection risk and tropical climate variability. The climate dependence and seasonal predictability of HPIV can inform the timing of community campaigns aimed at reducing infection risk and the development of hospital resources and climate adaption plans.

Dear Editor, Mycoplasma pneumoniae has become the leading cause of paediatric community-acquired pneumonia in countries where pneumococcal vaccination is included in the national immunisation programme, including Singapore. 1 M. pneumoniae is intrinsically resistant to beta-lactams due to the absence of cell walls.Macrolides, tetracyclines, and fluoroquinolones are used to treat M. pneumoniae infections, and macrolides are recommended in children, due to the potential adverse effects of tetracyclines and fluoroquinolones.Macrolideresistant M. pneumoniae (MRMP) isolates were first observed in 2001. 2 The prevalence of MRMP worldwide varies from 0.2% in Europe to 90% in East Asia. 3In Singapore, the prevalence of MRMP in hospitalised children was 13% in 2017. 4Studies in Asia have reported increased disease severity in persons infected with MRMP, 5 while others have not detected differences in the clinical course in persons with MRMP versus those with macrolide-susceptible M. pneumoniae (MSMP) infections. 6We compare differences between hospitalised children with MSMP and those with MRMP infections.From July 2019 to February 2020, patients younger than 16 years in KK Women's and Children's Hospital, Singapore, who tested positive for M. pneumoniae by polymerase chain reaction on nasopharyngeal or throat swab samples, were screened for inclusion.Assessment of genotypic macrolide resistance was done as previously described. 4Sequencing was performed retrospectively; thus, the results of genotypic macrolide resistance were not known at the time of treatment.Electronic medical records of patients with M. pneumoniae infection were retrospectively reviewed after informed consent was obtained.Demographic and clinical data were collected and matched to the results of genotypic macrolideresistance testing.Comparisons between binary groups were analysed using chi-square or Fisher's Exact test for categorical variables, and Mann-Whitney U test for continuous variables.All P values were two-tailed and differences were considered statistically significant at <0.05.

BACKGROUND:Clinical utility of universal antigen rapid test (ART) in the pediatric setting is unknown. We aimed to assess the performance and utility of universal ART in hospitalized children (≥5-year-old) to prevent nosocomial COVID-19 transmission. METHODS:Cross-sectional study involving all hospitalized pediatric patients aged ≥5-year-old from 2 periods during Omicron wave. Clinical data, ART and polymerase chain reaction test results were collected. RESULTS:A total of 444 patients were included from the 2 study periods, and 416 patients (93.7%) had concordant results between ART and polymerase chain reaction. The overall sensitivity and specificity of ART were 83.3% (95% CI: 75.2-89.3) and 97.5% (95% CI: 95.0-98.8), respectively. Negative predictive values of ART between the Omicron emergence and Omicron peak periods for a probable case group were 71.4% and 66.7%, respectively, and for a suspect case group 91.4% and 75.0%, respectively. Negative predictive values for an unlikely case group was >95% in both periods. Positive predictive value of ART was >85% for probable and suspect case groups in both periods. Seventy-five percent of patients (n = 15) who were incorrectly classified as SARS-CoV-2 negative by ART had potentially viable virus. No large nosocomial transmission clusters were detected. CONCLUSIONS:Universal ART screening may limit nosocomial outbreaks in hospitalized children. The performance can be optimized by considering clinical symptoms, exposure and periods within COVID waves.

This cross-sectional study assessed the changes in respiratory virus prevalence in 2020 vs 2019 associated with the COVID-19 pandemic.

It is important to understand the temporal trend of the paediatric severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load to estimate the transmission potential of children in schools and communities. We determined the differences in SARS-CoV-2 viral load dynamics between nasopharyngeal samples of infected asymptomatic and symptomatic children. Serial cycle threshold values of SARS-CoV-2 from the nasopharynx of a cohort of infected children were collected for analysis. Among 17 infected children, 10 (58.8%) were symptomatic. Symptomatic children, when compared to asymptomatic children, had higher viral loads (mean cycle threshold on day 7 of illness 28.6 vs. 36.7, P = 0.02). Peak SARS-CoV-2 viral loads occurred around day 2 of illness in infected children. Although we were unable to directly demonstrate infectivity, the detection of significant amount of virus in the upper airway of asymptomatic children suggest that they have the potential to shed and transmit SARS-CoV-2. Our study highlights the importance of contact tracing and screening for SARS-CoV-2 in children with epidemiological risk factors regardless of their symptom status, in order to improve containment of the virus in the community, including educational settings.

AbstractImportanceCOVID-19 pandemic control measures affect the prevalence of other respiratory viruses. Effects on some viruses have been described; however, the broader impact and temporal relationship of control measures on virus decline and subsequent re-emergence have not been thoroughly documented. Understanding these phenomena may influence health policies.ObjectiveTo examine the prevalence of unrelated respiratory viruses in relation to population-wide pandemic response measures and phases in 2020 in Singapore.Design, Setting, and ParticipantsData from respiratory multiplex PCRs from 3 major hospitals (total 3700 beds) in Singapore were collated. The full dataset consisted of 42,558 test results, 19,898 from 2019 and 22,660 from 2020.Main Outcomes and MeasuresWeekly virus prevalence data were mapped onto prevailing pandemic response measures, in order to elucidate temporal relationships and differential virus responses. Pre-pandemic data from 2019 were compared with data from 2020.ResultsEarly response measures, even before national lockdown, were followed by a dramatic reduction of influenza viruses and a more gradual decline of other respiratory viruses, including respiratory syncytial virus, parainfluenza viruses, endemic coronaviruses and metapneumovirus. Marked decline of enterovirus/rhinovirus and adenovirus, however, was only observed during lockdown. About 13 weeks into phased reopening, enterovirus/rhinovirus re-emerged, followed by adenovirus, the latter mainly in the pediatric population. All other viruses remained at low levels until the end of 2020.Conclusions and RelevanceCOVID-19 control measures in Singapore had a significant impact on a broad range of respiratory viruses. Effects of various control measures varied between phases and different viruses. Influenza viruses declined earliest and most dramatically; relaxation of measures was followed by re-emergence of enterovirus/rhinovirus and adenovirus. These patterns are presumably a result of different propensities for contact versus droplet and overall ease of transmission, and different virus reservoirs. Further studies into these phenomena are a matter of public health importance.Key PointsQuestionWhat were the effects of COVID-19 pandemic control measures in Singapore on the prevalence of other respiratory viruses?FindingsViruses responded differently to control measures. Influenza viruses declined rapidly after early control measures and remained near-absent during reopening after lockdown. Enterovirus/rhinovirus and adenovirus declined later and re-emerged earlier than other viruses during phased reopening.MeaningPopulation-wide interventions resulted in a broad decline and subsequent differential re-emergence of non-targeted respiratory viruses, corresponding to different patterns of virus response to control measures.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was detected from at least 1 buccal specimen in 9 of 11 coronavirus disease 2019 (COVID-19)-infected children (81.8%). Viral loads in buccal specimens were substantially lower than those in nasopharyngeal specimens. Buccal swabs are not good as COVID-19 screening specimens in children.

Background It is often impractical for each laboratory to establish its own paediatric reference intervals. This is particularly true for specimen types collected using invasive procedures, for example, cerebrospinal fluid (CSF). Methods Published CSF reference intervals for white cell count, and concentrations of total protein and glucose were reviewed by stakeholders in a paediatric hospital. Consensus reference intervals for the three CSF parameters were then subjected to verification using guidelines from the Clinical Laboratory Standards Institute and residual CSF specimens. Results Consensus paediatric reference intervals adapted from published studies with minor modifications were locally verified as follows. White cell count (x10(6) cells/L): 0-20 (<1 month); 0-10 (1-2 months); 0-5 (>2 months). Total protein (g/L): 0.3-1.2 (<1 month); 0.2-0.6 (1-3 months); 0.1-0.4 (>3 months). Glucose (mmol/L): 2.0-5.6 (<6 months); 2.4-4.3 (6 months or older).

Aims & Objectives: Elizabethkingia anophelis (E. anophelis) is an emergent pathogen first described from mid-gut specimens of the Anopheles gambiae mosquito. We report the first confirmed E. anophelis case cluster in a paediatric critical care unit (CCU). Methods A clinical and molecular epidemiological investigation to decipher the clinical manifestation, effective outbreak interventions as well as transmission route of E.anophelis in CCU. All samples were tested using 16S rDNA PCR to identify E.anophelis. Results The cluster was composed of 3 patients positive for Elizabethkingia species from blind bronchial samples detected within a 13 day period. The patients’ age ranged from 2.8 months-4.8 years old and all had significant comorbidity. All case patients’ rooms or cubicles were confirmed positive for E. anophelis from their respective tap outlets. Removal of tap aerators was effective in eliminating E. anophelis from tap water sources. Of the two healthcare staff included in the transmission study, one acquired E. anophelis on her hands following hand washing with contaminated water. After hand hygiene using alcohol based hand rub (ABHR), there was no detectable E. anophelis growth on her hands. Phylogenetic data confirmed the association between tap outlet and patient sample. Conclusions E. anophelis is a nosocomial pathogen with outbreak potential in paediatric CCUs. Hand washing, despite using chlorhexidine soap, is a possible vehicle of transmission for E. anophelis. Effective outbreak control measures should include removal of aerators from tap outlets and the use of ABHR as the primary hand hygiene method of choice.

We report an Elizabethkingia anophelis case cluster associated with contaminated aerators and tap water in a children's intensive care unit in Singapore in 2017. We demonstrate a likely transmission route for E. anophelis to patients through acquisition of the bacteria on hands of healthcare workers via handwashing.

Dear Editor, Mycoplasma pneumoniae belongs to the class Mollicutes and is a small bacterium without a cell wall that is a common cause of community-acquired pneumonia in adults and children. Due to its unique physical structure, M. pneumoniae is resistant to cell wall-targeting agents, such as beta-lactam antibiotics. Therefore, the antibiotics of choice for the treatment of suspected M. pneumoniae infections, particularly for children, are the macrolides, with their main class representative being erythromycin. Alternatives for treatment include the tetracyclines and fluoroquinolones. Reduced susceptibility or resistance of M. pneumoniae to macrolide antibiotics has been described; this is the consequence of point mutations in domain V (peptidyltransferase region) of the 23S rRNA gene.1,2 The most common mutation is the substitution of adenine with guanine (A2063G) at base position 2063 (M. pneumoniae numbering, equivalent to base position 2058 in Escherichia coli).3 This mutation has been found associated with a 103-fold to >105-fold elevation of the minimum inhibitory concentration (MIC) of erythromycin as compared to susceptible strains.4 Less common mutations occur at positions 2063 (A→T, A→C), 2064 (A→G, A→C), 2067 (A→G) and 2617 (the latter not studied in this work). These mutations reduce the affinity of macrolides for the ribosome,4,5 thereby reducing their efficacy. Macrolide resistance due to erm genes or enzymes that inactivate macrolides have not been detected thus far in M. pneumoniae.2 Studies conducted from 2011 onwards in Japan, South Korea and China have demonstrated alarmingly high levels of macrolide resistance (ranging from 63% to 98%) in M. pneumoniae strains.2 On the other hand, macrolide resistance in M. pneumoniae from Europe, North America and Australia has been less common (ranging from 0% to 26%).2 The prevalence of macrolide resistance in M. pneumoniae in Singapore or its neighbouring countries in South-east Asia is currently unknown. Thus, we sought to determine the prevalence of genotypic macrolide resistance in M. pneumoniae among clinical specimens submitted for diagnostic purposes at a paediatric hospital in Singapore. We used PCR primers published by Wolff et al6 to amplify a region that encompasses the mutations at positions 2063, 2064 and 2067 that account for more than 99% of macrolide resistance in published works.2

BACKGOUND:Due to difficulties of culturing Human metapneumovirus (HMPV) much of the current understanding of HMPV replication can be inferred from other closely related viruses. The slow rates of virus replication prevent many biochemical analyses of HMPV particles. In this study imaging was used to examine the process of HMPV morphogenesis in individually infected LLC-MK2 cells, and to better characterise the sites of HMPV assembly. This strategy has circumvented the problems associated with slow replication rates and allowed us to characterise both the HMPV particles and the sites of HMPV morphogenesis.METHODS:HMPV-infected LLC-MK2 cells were stained with antibodies that recognised the HMPV fusion protein (F protein), attachment protein (G protein) and matrix protein (M protein), and fluorescent probes that detect GM1 within lipid-raft membranes (CTX-B-AF488) and F-actin (Phalloidin-FITC). The stained cells were examined by confocal microscopy, which allowed imaging of F-actin, GM1 and virus particles in HMPV-infected cells. Cells co-expressing recombinant HMPV G and F proteins formed virus-like particles and were co-stained with antibodies that recognise the recombinant G and F proteins and phalloidin-FITC and CTX-B-AF594, and the distribution of the G and F proteins, GM1 and F-actin determined.RESULTS:HMPV-infected cells stained with anti-F, anti-G or anti-M revealed a filamentous staining pattern, indicating that the HMPV particles have a filamentous morphology. Staining of HMPV-infected cells with anti-G and either phalloidin-FITC or CTX-B-AF488 exhibited extensive co-localisation of these cellular probes within the HMPV filaments. This suggested that lipid-raft membrane domains and F-actin structures are present at the site of the virus morphogenesis, and are subsequently incorporated into the HMPV filaments. Furthermore, the filamentous virus-like particles that form in cells expressing the G protein formed in cellular structures containing GM1 and F-actin, suggesting the G protein contains intrinsic targeting signals to the sites of virus assembly.CONCLUSIONS:These data suggest that HMPV matures as filamentous particles and that virus morphogenesis occurs within lipid-raft microdomains containing localized concentrations of F-actin. The similarity between HMPV morphogenesis and the closely related human respiratory syncytial virus suggests that involvement of F-actin and lipid-raft microdomains in virus morphogenesis may be a common feature of the Pneumovirinae.

Background: Human metapneumovirus (HMPV) is now a major cause of lower respiratory infection in children. Although primary isolation of HMPV has been achieved in several different cell lines, the low level of virus replication and the subsequent recovery of low levels of infectious HMPV have hampered biochemical studies on the virus. These experimental methodologies usually require higher levels of biological material that can be achieved following HMPV infection. In this study we demonstrate that expression of the HMPV F, G and M proteins in mammalian cells leads to HMPV virus-like particles (VLP) formation. This experimental strategy will serve as a model system to allow the process of HMPV virus assembly to be examined.Methods: The HMPV F, G and M proteins were expressed in mammalian cell lines. Protein cross-linking studies, sucrose gradient centrifugation and in situ imaging was used to examine interactions between the virus proteins. VLP formation was examined using sucrose density gradient centrifugation and electron microscopy analysis.Results: Analysis of cells co-expressing the F, G and M proteins demonstrated that these proteins interacted. Furthermore, in cells co-expression the three HMPV proteins the formation VLPs was observed. Image analysis revealed the VLPs had a similar morphology to the filamentous virus morphology that we observed on HMPV-infected cells. The capacity of each protein to initiate VLP formation was examined using a VLP formation assay. Individual expression of each virus protein showed that the G protein was able to form VLPs in the absence of the other virus proteins. Furthermore, co-expression of the G protein with either the M or F proteins facilitated their incorporation into the VLP fraction.Conclusion: Co-expression of the F, G and M proteins leads to the formation of VLPs, and that incorporation of the F and M proteins into VLPs is facilitated by their interaction with the G protein. Our data suggests that the G protein plays a central role in VLP formation, and further suggests that the G protein may also play a role in the recruitment of the F and M proteins to sites of virus particle formation during HMPV infection.

UNLABELLED:Since the introduction of the pertussis vaccine into the standard immunization program, very few cases of pertussis have been detected. In 2007, it was felt that the number of cases being admitted for pertussis had increased and this was verified on a retrospective review done from 2004 to 2007 of children diagnosed with pertussis in KK Women's and Children's Hospital. AIM:To review the cases diagnosed with pertussis, the demographic profile and the outcome of these patients. METHODS:A retrospective review was done of patients diagnosed with pertussis from 2004 to 2007. The patients were identified from records of the positive results obtained from the microbiology laboratory. RESULTS:In the preceding years, only 1-2 cases/year were reported with pertussis but this increased to 33 cases in 2007. 45 confirmed cases were analysed. Most infections were in infants below 6 months old (mean age 4.1 months) and almost all were not vaccinated. The average length of stay was 4.96 days (Range 2-14 days, SD 2.55). Children under 6 months had more severe disease in terms of ICU admissions (6% vs. 0%, p=0.70) and average length of stay (5.1 vs. 3.5 days, p=0.25) as compared to those above 6 months of age. Exposure to a symptomatic adult was documented in 64%, mainly parents (45%), older siblings (29%). Healthcare workers may also be a source of infection as one child had symptoms as early as the first week of life and none of the family members were coughing. CONCLUSION:There is a resurgence of pertussis in recent years with high morbidity in children who have not been vaccinated. A booster with Tdap vaccine should be considered for young adults and healthcare workers looking after children.