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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.

Anticoagulant rodenticides are commonly used in rodent control because they are economical and have great deployment versatility. However, rodents with Single Nucleotide Polymorphism (SNP) mutations within the Vkorc1 gene are resistant to the effects of anticoagulant rodenticide use and this influences the effectiveness of control strategies that rely on such rodenticides. This study examined the prevalence of rat SNP mutations in Singapore to inform the effectiveness of anticoagulant rodenticide use. A total of 130 rat tail samples, comprising 83 Rattus norvegicus (63.8%) and 47 Rattus rattus complex (36.2%) were conveniently sampled from November 2016 to December 2019 from urban settings and sequenced at exon 3 of Vkorc1. Sequencing analysis revealed 4 synonymous and 1 non-synonymous mutations in Rattus rattus complex samples. A novel synonymous mutation of L108L was identified and not previously reported in other studies. Non-synonymous SNPs were not detected in the notable codons of 120, 128 and 139 in R. norvegicus, where these regions are internationally recognised to be associated with resistance from prior studies. Our findings suggest that the prevalence of anticoagulant rodenticide resistance in Singapore is low. Continued monitoring of rodenticide resistance is important for informing rodent control strategies aimed at reducing rodent-borne disease transmission.

Rodents living alongside humans increases the probability of encounter and also the transmission of rodent-borne diseases. Singapore’s cosmopolitan urban landscape provides a perfect setting to study the prevalence of four rodent-borne pathogens: Seoul hantavirus (SEOV), Leptospira species, Rickettsia typhi and Yersinia pestis, and identify the potential risk factors which may influence rodent density and transmission of rodent-borne diseases. A total of 1143 rodents were trapped from 10 unique landscape structures throughout Singapore. Real-time quantitative Polymerase Chain Reactions were used to detect pathogenic and intermediate Leptospira spp. and Yersinia pestis, whereas the seroprevalence of SEOV and R. typhi were analysed by Enzyme-Linked Immunosorbent Assay and Immunofluorescence Assay respectively. Multivariable logistic regression analysis was used to evaluate the association between prevalence of infection in rodent reservoirs and risk factors. Most of the rodents were caught in public residential developments (62.2%). Among the tested rodents, 42.4% were infected with Leptospira spp., while 35.5% and 32.2% were seropositive for SEOV and R. typhi respectively, whereas Yersinia pestis was not detected. Furthermore, risk factors including habitat, species, gender, and weight of rodents, influenced prevalence of infection to a varying extent. This study highlights the presence of Leptospira spp., SEOV and R. typhi in Singapore’s rodent population, suggesting the need for effective rodent management and sanitation strategies to prevent further circulation and transmission to humans.

Mosquito-borne flaviviruses are emerging pathogens of an increasing global public health concern because of their rapid increase in geographical range and the impact of climate change. Japanese encephalitis virus (JEV) and West Nile virus (WNV) are of concern because of the risk of reemergence and introduction by migratory birds. In Singapore, human WNV infection has never been reported and human JEV infection is rare. Four sentinel vector surveillance sites were established in Singapore to understand the potential risk posed by these viruses. Surveillance was carried out from August 2011 to December 2012 at Pulau Ubin, from March 2011 to March 2013 at an Avian Sanctuary (AS), from December 2010 from October 2012 at Murai Farmway, and from December 2010 to December 2013 at a nature reserve. The present study revealed active JEV transmission in Singapore through the detection of JEV genotype II in Culex tritaeniorhynchus collected from an Avian Sanctuary. Culex flavivirus (CxFV), similar to the Quang Binh virus isolated from Cx. tritaeniorhynchus in Vietnam and CxFV-LSFlaviV-A20-09 virus isolated in China, was also detected in Culex spp. (vishnui subgroup). No WNV was detected. This study demonstrates the important role that surveillance plays in public health and strongly suggests the circulation of JEV among wildlife in Singapore, despite the absence of reported human cases. A One Health approach involving surveillance, the collaboration between public health and wildlife managers, and control of mosquito populations remains the key measures in risk mitigation of JEV transmission in the enzootic cycle between birds and mosquitoes.

Japanese encephalitis virus (JEV) and West Nile virus (WNV) are arboviruses primarily transmitted by Culex spp. mosquitoes. Birds are the primary hosts for JEV and WNV. Recent WNV outbreaks in Europe and United States and their association with migratory birds highlight the importance of understanding the feeding host preference of potential vectors for outbreak preparedness, especially in nonendemic settings. Singapore is nonendemic to JEV and WNV, but is a stopover site for migratory birds of the East Asian-Australasian Flyway. Therefore, we elucidated the feeding host range of Culex spp. mosquitoes captured in four natural (bird) habitats in Singapore from January 2011 to December 2012. We characterized feeding host DNA in field-caught mosquitoes using a PCR sequencing-based assay targeting the mitochondrial gene regions. Of 22,648 mosquitoes captured, 21,287 belonged to the Culex vishnui subgroup. The host DNA analysis showed that mosquitoes from the Cx. vishnui subgroup are opportunistic biters, feeding on a range of birds and mammals. Cx. vishnui subgroup, Culex sitiens and Culex bitaeniorhynchus, was primarily ornithophagic, although they fed opportunistically on mammals, including humans. Culex gelidus and Culex quinquefasciatus, in contrast, fed mainly on mammals. The presence of ornitho- and anthropophilic mosquito vectors and susceptible avian and mammalian hosts poses a risk spill-over transmission of JEV and WNV among humans, should these viruses be introduced through migratory birds and establish persistent transmission in resident birds and animal hosts in Singapore.

Japanese encephalitis virus (JEV) is an important arbovirus in Asia that can cause serious neurological disease. JEV is transmitted by mosquitoes in an enzootic cycle involving porcine and avian reservoirs, in which humans are accidental, dead-end hosts. JEV is currently not endemic in Singapore, after pig farming was abolished in 1992; the last known human case was reported in 2005. However, due to its location along the East-Asian Australasian Flyway (EAAF), Singapore is vulnerable to JEV re-introduction from the endemic regions. Serological and genetic evidence in the last decade suggests JEV’s presence in the local fauna. In the present study, we report the genetic characterization and the first isolation of JEV from 3214 mosquito pools consisting of 41,843 Culex mosquitoes, which were trapped from April 2014 to May 2021. The findings demonstrated the presence of genotype I of JEV (n = 10), in contrast to the previous reports of the presence of genotype II of JEV in Singapore. The genetic analyses also suggested that JEV has entered Singapore on several occasions and has potentially established an enzootic cycle in the local fauna. These observations have important implications in the risk assessment and the control of Japanese encephalitis in non-endemic countries, such as Singapore, that are at risk for JEV transmission.

Avian pox is a highly contagious avian disease, yet relatively little is known about the epidemiology and transmission of Avipoxviruses. Using a molecular approach, we report evidence for a potential link between birds and field-caught mosquitoes in the transmission of Fowlpox virus (FWPV) in Singapore. Comparison of fpv167 (P4b), fpv126 (VLTF-1), fpv175-176 (A11R-A12L) and fpv140 (H3L) gene sequences revealed close relatedness between FWPV strains obtained from cutaneous lesions of a chicken and four pools of Culex pseudovishnui, Culex spp. (vishnui group) and Coquellitidea crassipes caught in the vicinity of the study site. Chicken-derived viruses characterized during two separate infections two years later were also identical to those detected in the first event, suggesting repeated transmission of closely related FWPV strains in the locality. Since the study location is home to resident and migratory birds, we postulated that wild birds could be the source of FWPV and that bird-biting mosquitoes could act as bridging mechanical vectors. Therefore, we determined whether the FWPV-positive mosquito pools (n=4) were positive for avian DNA using a polymerase chain reaction-sequencing assay. Our findings confirmed the presence of avian host DNA in all mosquito pools, suggesting a role for Cx. pseudovishnui, Culex spp. (vishnui group) and Cq. crassipes mosquitoes in FWPV transmission. Our study exemplifies the utilization of molecular tools to understand transmission networks of pathogens affecting avian populations, which has important implications for the design of effective control measures to minimize disease burden and economic loss.

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

Wastewater-based surveillance has been widely used as a non-intrusive tool to monitor population-level transmission of COVID-19. Although various approaches are available to concentrate viruses from wastewater samples, scalable methods remain limited. Here, we sought to identify and evaluate SARS-CoV-2 virus concentration protocols for high-throughput wastewater testing. A total of twelve protocols for polyethylene glycol (PEG) precipitation and four protocols for ultrafiltration-based approaches were evaluated across two phases. The first phase entailed an initial evaluation using a small sample set, while the second phase further evaluated five protocols using wastewater samples of varying SARS-CoV-2 concentrations. Permutations in the pre-concentration, virus concentration and RNA extraction steps were evaluated. Among PEG-based methods, SARS-CoV-2 virus recovery was optimal with 1) the removal of debris prior to processing, 2) 2 h to 24 h incubation with 8% PEG at 4 °C, 3) 4000 xg or 14,000 xg centrifugation, and 4) a column-based RNA extraction method, yielding virus recovery of 42.4-52.5%. Similarly, the optimal protocol for ultrafiltration included 1) the removal of debris prior to processing, 2) ultrafiltration, and 3) a column-based RNA extraction method, yielding a recovery of 38.2%. This study also revealed that SARS-CoV-2 RNA recovery for samples with higher virus concentration were less sensitive to changes in the PEG method, but permutations in the PEG protocol could significantly impact virus yields when wastewater samples with lower SARS-CoV-2 RNA were used. Although both PEG precipitation and ultrafiltration methods resulted in similar SARS-CoV-2 RNA recoveries, the former method is more cost-effective while the latter method provided operational efficiency as it required a shorter turn-around-time (PEG precipitation, 9-23 h; Ultrafiltration, 5 h). The decision on which method to adopt will thus depend on the use-case for wastewater testing, and the need for cost-effectiveness, sensitivity, operational feasibility and scalability. Published version

Wastewater-based surveillance for SARS-CoV-2 has been used for the early warning of transmission or objective trending of the population-level disease prevalence. Here, we describe a new use-case of conducting targeted wastewater surveillance to complement clinical testing for case identification in a small community at risk of COVID-19 transmission. On 2 July 2020, a cluster of COVID-19 cases in two unrelated households residing on different floors in the same stack of an apartment building was reported in Singapore. After cases were conveyed to healthcare facilities and six healthy household contacts were quarantined in their respective apartments, wastewater surveillance was implemented for the entire residential block. SARS-CoV-2 was subsequently detected in wastewaters in an increasing frequency and concentration, despite the absence of confirmed COVID-19 cases, suggesting the presence of fresh case/s in the building. Phone interviews of six residents in quarantine revealed that no one was symptomatic (fever/ respiratory illness). However, when nasopharyngeal swabs from six quarantined residents were tested by PCR tests, one was positive for SARS-CoV-2. The positive case reported episodes of diarrhea and the case's stool sample was also positive for SARS-CoV-2, explaining the SARS-CoV-2 spikes observed in wastewaters. After the case was conveyed to a healthcare facility, wastewaters continued to yield positive signals for five days, though with a decreasing intensity. This was attributed to the return of recovered cases, who had continued to shed the virus. Our findings demonstrate the utility of wastewater surveillance as a non-intrusive tool to monitor high-risk COVID-19 premises, which is able to trigger individual tests for case detection, highlighting a new use-case for wastewater testing., Graphical abstract Unlabelled Image

Fomite-mediated transmission has been identified as a possible route for the spread of COVID-19 disease caused by SARS-CoV-2. In healthcare settings, environmental contamination by SARS-CoV-2 has been found in patients&rsquo, rooms and toilets. Here, we investigated environmental presence of SARS-CoV-2 in non-healthcare settings and assessed the efficacy of cleaning and disinfection in removing virus contamination. A total of 428 environmental swabs and six air samples was taken from accommodation rooms, toilets and elevators that have been used by COVID-19 cases. By using a reverse transcription polymerase chain reaction assay, we detected two SARS-CoV-2 RNA positive samples in a room where a COVID-19 patient stayed prior to diagnosis. The present study highlights the risk of fomite-mediated transmission in non-healthcare settings and the importance of surface disinfection in spaces occupied by cases. Of note, neither air-borne transmission nor surface contamination of elevators, which were transiently exposed to infected individuals, was evident among samples analyzed.

Mosquito-borne flaviviruses are emerging pathogens of an increasing global public health concern because of their rapid increase in geographical range and the impact of climate change. Japanese encephalitis virus (JEV) and West Nile virus (WNV) are of concern because of the risk of reemergence and introduction by migratory birds. In Singapore, human WNV infection has never been reported and human JEV infection is rare. Four sentinel vector surveillance sites were established in Singapore to understand the potential risk posed by these viruses. Surveillance was carried out from August 2011 to December 2012 at Pulau Ubin, from March 2011 to March 2013 at an Avian Sanctuary (AS), from December 2010 from October 2012 at Murai Farmway, and from December 2010 to December 2013 at a nature reserve. The present study revealed active JEV transmission in Singapore through the detection of JEV genotype II in Culex tritaeniorhynchus collected from an Avian Sanctuary. Culex flavivirus (CxFV), similar to the Quang Binh virus isolated from Cx. tritaeniorhynchus in Vietnam and CxFV-LSFlaviV-A20-09 virus isolated in China, was also detected in Culex spp. (vishnui subgroup). No WNV was detected. This study demonstrates the important role that surveillance plays in public health and strongly suggests the circulation of JEV among wildlife in Singapore, despite the absence of reported human cases. A One Health approach involving surveillance, the collaboration between public health and wildlife managers, and control of mosquito populations remains the key measures in risk mitigation of JEV transmission in the enzootic cycle between birds and mosquitoes.

Background Singapore was previously endemic for JEV but the threat of JE remains. There is continued JEV transmission in animal hosts despite the banning of pig farming in 1992. Surveillance of mosquitoes, ardeid birds and swine gives public health officials useful warning signs for the monitoring of JEV. In this study, we aim to develop a JE Risk Map based on the data gathered by spatial and quantitative survey of the JE-associated vectors and animal hosts. Methods For over a year across the island, the distribution and population of herons were visually surveyed and the distribution of wild boars was captured with wildlife camera traps. Mosquito population data was collected from 16 sites with ecological factors suitable for JE transmission. The animal and vector data were then spatially analysed using open-sourced GIS software. Results Four JE high risk clusters were interpolated from the combined risk factors of data points. A JEV positive Culex tritaeniorynchus pool was subsequently detected in the largest of these JE high risk clusters.

Fomite-mediated transmission has been identified as a possible route for disease spread of the COVID-19 pandemic. In healthcare settings, evidence of environmental contamination by SARS-CoV-2 has been found in patients’ rooms and toilets. Here, we investigate environmental contamination of SARS-CoV-2 in non-healthcare settings and assessed the efficacy of cleaning and disinfection in removing SARS-CoV-2 contamination. A total of 428 environmental swabs and six air samples was taken from accommodation rooms, toilets and elevators that have been used by COVID-19 cases. Through the use of a sensitive nested RT-PCR assay, we found two SARS-CoV-2 RNA positive samples from the room resided by a COVID-19 case, highlighting the risk of fomite-mediated transmission in non-healthcare settings and the importance of surface disinfection of spaces occupied by cases. Of note, we did not find evidence for air-borne transmission, nor of environmental contamination of elevators, which were transiently exposed to infected persons.

Background Acute respiratory infections (ARIs) are among the most common human illnesses globally. Previous studies that examined the associations between climate variability and ARIs or ARI pathogens have reported inconsistent findings. Few studies have been conducted in Southeast Asia to date, and the impact of climatic factors are not well-understood. This study aimed to investigate the short-term associations between climate variability and ARIs in Singapore. Methods We obtained reports of ARIs from all government primary healthcare services from 2005 to 2019 and analysed their dependence on mean ambient temperature, minimum temperature and maximum temperature using the distributed lag non-linear framework. Separate negative binomial regression models were used to estimate the association between each temperature (mean, minimum, maximum temperature) and ARIs, adjusted for seasonality and long-term trend, rainfall, relative humidity, public holidays and autocorrelations. For temperature variables and relative humidity we reported cumulative relative risks (RRs) at 10th and 90th percentiles compared to the reference value (centered at their medians) with corresponding 95% confidence intervals (CIs). For rainfall we reported RRs at 50th and 90th percentiles compared to 0 mm with corresponding 95% CIs. Results Statistically significant inverse S-curve shaped associations were observed between all three temperature variables (mean, minimum, maximum) and ARIs. A decrease of 1.1 °C from the median value of 27.8 °C to 26.7 °C (10th percentile) in the mean temperature was associated with a 6% increase (RR: 1.06, 95% CI: 1.03 to 1.09) in ARIs. ARIs also increased at 23.9 °C (10th percentile) compared to 24.9 °C of minimum temperature (RR: 1.11, 95% CI: 1.07 to 1.16). The effect of maximum temperature for the same comparison (30.5 °C vs 31.7 °C) was non-significant (RR: 1.02, 95% CI: 0.99 to 1.05). An increase in ambient temperature to 28.9 °C (90th percentile) was associated with an 18% decrease (RR: 0.82, 95% CI: 0.80 to 0.83) in ARIs. Similarly, ARIs decreased with the same increase to 90th percentile in minimum (RR: 0.84, 95% CI: 0.80 to 0.87) and maximum (RR: 0.89, 95% CI: 0.86 to 0.93) temperatures. Rainfall was inversely associated with ARIs and displayed similar shape in all three temperature models. Relative humidity, on the other hand, exhibited a U-shaped relationship with ARIs. Conclusion Our findings suggest that lower temperatures increase the risk of ARIs. Anticipated extreme weather events that reduce ambient temperature can be used to inform increased healthcare resource allocation for ARIs.

Plasmodium knowlesi is a simian malaria parasite currently recognized as the fifth causative agent of human malaria. Recently, naturally acquired P. cynomolgi infection in humans was also detected in Southeast Asia. The main reservoir of both parasites is the long-tailed and pig-tailed macaques, which are indigenous in this region. Due to increased urbanization and changes in land use, there has been greater proximity and interaction between the long-tailed macaques and the general population in Singapore. As such, this study aims to determine the prevalence of simian malaria parasites in local macaques to assess the risk of zoonosis to the general human population. Screening for the presence of malaria parasites was conducted on blood samples from 660 peridomestic macaques collected between Jan 2008 and Mar 2017, and 379 wild macaques collected between Mar 2009 and Mar 2017, using a Pan-Plasmodium-genus specific PCR. Positive samples were then screened using a simian Plasmodium species-specific nested PCR assay to identify the species of parasites (P. knowlesi, P. coatneyi, P. fieldi, P. cynomolgi, and P. inui) present. All the peridomestic macaques sampled were tested negative for malaria, while 80.5% of the 379 wild macaques were infected. All five simian Plasmodium species were detected; P. cynomolgi being the most prevalent (71.5%), followed by P. knowlesi (47.5%), P. inui (42.0%), P. fieldi (32.5%), and P. coatneyi (28.5%). Co-infection with multiple species of Plasmodium parasites was also observed. The study revealed that Singapore’s wild long-tailed macaques are natural hosts of the five simian malaria parasite species, while no malaria was detected in all peridomestic macaques tested. Therefore, the risk of simian malaria transmission to the general human population is concluded to be low. However, this can be better demonstrated with the incrimination of the vectors of simian malaria parasites in Singapore., Author summary Malaria is one of the most important vector-borne diseases in the world. In Southeast Asia, the incidence of human malaria infections caused by simian malaria parasites that originate from macaques has been increasing in the last decade, possibly due to closer proximity between the macaque and human population as a result of deforestation. In Singapore, urbanization and change in land use have resulted in closer proximity and interactions between macaques and the general human population. In order to assess the risk of potential simian malaria zoonosis, 1039 macaques’ blood samples were screened in this study for simian malaria parasites. Our results showed that simian malaria parasites were only detected in macaques living deep in the forest within the military protected zone, while macaques living close to human habitations are malaria-free. This study illustrated a low risk of zoonotic transmission of simian malaria parasites to the general population in Singapore.