Quantification of multiple respiratory viruses in wastewater in the Kathmandu Valley, Nepal: Potential implications of wastewater-based epidemiology for community disease surveillance in developing countries.

Despite being the major cause of death, clinical surveillance of respiratory viruses at the community level is very passive, especially in developing countries. This study focused on the surveillance of three respiratory viruses [severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (IFV-A), and respiratory syncytial virus (RSV)] in the Kathmandu Valley, Nepal, by implication of wastewater-based epidemiology (WBE). Fifty-one untreated wastewater samples were from two wastewater treatment plants (WWTPs) between April and October 2022. Among eight combinations of the pre-evaluated methods, the combination of concentration by simple centrifugation, pretreatment by DNA/RNA Shield (Zymo Research), and extraction by the QIAamp Viral RNA Mini Kit (QIAGEN) showed the best performance for detecting respiratory viruses. Using this method with a one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR), SARS-CoV-2 RNA was successfully detected from both WWTPs (positive ratio, 100 % and 81 %) at concentrations of 5.6 ± 0.6 log10 copies/L from each WWTP. Forty-six SARS-CoV-2 RNA-positive samples were further tested for three mutation site-specific one-step RT-qPCR (L452R, T478K, and E484A/G339D), where G339D/E484A mutations were frequently detected in both WWTPs (96 %). IFV-A RNA was more frequently detected in WWTP A (84 %) compared to WWTP B (38 %). RSV RNA was also detected in both WWTPs (28 % and 8 %, respectively). This is the first study on detecting IFV-A and RSV in wastewater in Nepal, showing the applicability and importance of WBE for respiratory viruses in developing countries where clinical data are lacking.

Detection of SARS-CoV-2 and Omicron variant RNA in wastewater samples from Manila, Philippines.

Manila, a highly urbanized city, is listed as one of the top cities with the highest recorded number of coronavirus disease 2019 (COVID-19) cases in the Philippines. This study aimed to detect and quantify the RNA of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the Omicron variant in 51 wastewater samples collected from three locations in Manila, namely Estero de Santa Clara, Estero de Pandacan, which are open drainages, and a sewage treatment plant (STP) at De La Salle University-Manila, between July 2022 and February 2023. Using one-step reverse transcription-quantitative polymerase chain reaction, SARS-CoV-2 and Omicron variant RNA were detected in 78 % (40/51; 4.9 ± 0.5 log10 copies/L) and 60 % (24/40; 4.4 ± 0.3 log10 copies/L) of wastewater samples collected from all sampling sites, respectively. SARS-CoV-2 RNA was detected frequently at Estero de Santa Clara (88 %, 15/17); its highest concentration was at the STP (6.3 log10 copies/L). The Omicron variant RNA was present in the samples collected (4.4 ± 0.3 log10 copies/L) from all sampling sites, with the highest concentration at the STP (4.9 log10 copies/L). Regardless of normalization, using concentrations of pepper mild mottle virus RNA, SARS-CoV-2 RNA concentrations exhibited the highest positive correlation with COVID-19 reported cases in Manila 5 days after the clinical report. These findings revealed that wastewater-based epidemiology may aid in identifying and monitoring of the presence of pathogens in open drainages and STPs in the Philippines. This paper provides the first documentation on SARS-CoV-2 and the Omicron variant in wastewater from Manila.

COVID-19 infection risk assessment and management at the Tokyo 2020 Olympic and Paralympic Games: A scoping review.

The Tokyo 2020 Olympic and Paralympic Games was one of the largest international mass-gathering events held after the beginning of coronavirus disease 2019 (COVID-19) pandemic. In this scoping review, we extracted papers discussing COVID-19 risk assessment or management at the Tokyo 2020 Games to determine the nature of studies that were conducted. Among the 75 papers obtained from two search engines (PubMed and ScienceDirect) and four papers collected from hand-searches, 30 papers were extracted. Only eight papers performed both COVID-19 prior risk assessment and quantitative evaluation of effectiveness measures, highlighting the importance of rapid, solution-focused risk assessment. Furthermore, this review revealed that the findings regarding the spread of COVID-19 infection to citizens in the host country were inconsistent depending on the assessment methods and that assessments of the spread of infection outside the host country were lacking.

Detection of enteroviruses related to hand foot and mouth disease in wastewater of Asian communities.

Hand, foot, and mouth disease (HFMD) is contagious and predominantly affects children below the age of five. HFMD-associated serotypes of Enterovirus A (EVA) family include EVA71, Coxsackievirus A type 6 (CVA6), 10 (CVA10), and 16 (CVA16). Although prevalent in numerous Asian countries, studies on HFMD-causing agents in wastewater are scarce. This study aimed to conduct wastewater surveillance in various Asian communities to detect and quantify serotypes of EVA associated with HFMD. In total, 77 wastewater samples were collected from Indonesia, the Philippines, Thailand, and Vietnam from March 2022 to February 2023. The detection ratio for CVA6 RNA in samples from Vietnam was 40 % (8/20). The detection ratio for CVA6 and EVA71 RNA each was 25 % (5/20) for the Indonesian samples, indicating the need for clinical surveillance of CVA6, as clinical reports have been limited. For the Philippines, 12 % (2/17) of the samples were positive for CVA6 and EVA71 RNA each, with only one quantifiable sample each. Samples from Thailand had a lower detection ratio (1/20) for CVA6 RNA, and the concentration was unquantifiable. Conversely, CVA10 and CVA16 RNAs were not detected in any of the samples. The minimum and maximum concentrations of CVA6 RNA were 2.7 and 3.9 log10 copies/L and those for EVA71 RNA were 2.5 and 4.9 log10 copies/L, respectively. This study underscores the importance of wastewater surveillance in understanding the epidemiology of HFMD-associated EVA serotypes in Asian communities. Long-term wastewater surveillance is recommended to monitor changes in dominant serotypes, understand seasonality, and develop effective prevention and control strategies for HFMD.

Pyrofollis japonicus gen. nov. sp. nov., a novel member of the family Pyrodictiaceae isolated from the Iheya North hydrothermal field.

A novel hyperthermophilic, heterotrophic archaeon, strain YC29T, was isolated from a deep-sea hydrothermal vent in the Mid-Okinawa Trough, Japan. Cells of strain YC29T were non-motile, irregular cocci with diameters of 1.2-3.0 µm. The strain was an obligatory fermentative anaerobe capable of growth on complex proteinaceous substrates. Growth was observed between 85 and 100 °C (optimum 90-95 °C), pH 4.9-6.4 (optimum 5.1), and in the presence of 1.4-4.0% (w/v) NaCl (optimum 3.0%). Inorganic carbon was required as a carbon source. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the isolate was a member of the family Pyrodictiaceae. The genome size was 2.02 Mbp with a G+C content of 49.4%. The maximum values for average nucleotide identity (ANI), average amino acid identity (AAI), and in silico DNA-DNA hybridization (dDDH) value of strain YC29T with relatives were 67.9% (with Pyrodictium abyssi strain AV2T), 61.1% (with Pyrodictium occultum strain PL-19T), and 33.8% (with Pyrolobus fumarii strain 1AT), respectively. Based on the phylogenetic, genomic, and phenotypic characteristics, we propose that strain YC29T represents a novel genus and species, Pyrofollis japonicus gen. nov., sp. (type strain YC29T = DSM 113394T = JCM 39171T).

Haliovirga abyssi gen. nov., sp. nov., a mesophilic fermentative bacterium isolated from the Iheya North hydrothermal field, and proposal of Haliovirgaceae fam. nov.

A novel mesophilic, obligately anaerobic, facultatively sulphur-reducing bacterium, designated strain IC12T, was isolated from a deep-sea hydrothermal field in the Mid-Okinawa Trough, Japan. The cells were Gram-negative, motile, short rods with a single polar flagellum. The ranges and optima of the growth temperature, NaCl concentration and pH of strain IC12T were 15-40 °C (optimum, 30-35 °C), 10-60 g l-1 (optimum, 20-30 g l-1) and pH 4.9-6.7 (optimum, pH 5.8), respectively. Yeast extract was utilized as a sole carbon and energy source for fermentative growth. Major fatty acids of strain IC12T were C14 : 0, C16 : 0 and C16 : 1 ω7. Results of phylogenetic analysis based on 16S rRNA gene sequences indicated that strain IC12T was affiliated to the phylum Fusobacteriota and was most closely related to Ilyobacter insuetus VenChi2T (86.5 % sequence similarity). Strain IC12T contained a chromosome of 2.43 Mbp and a large plasmid of 0.30 Mbp. The G+C content of the genomic DNA was 26.4 mol%. The maximum values for average nucleotide identity and in silico DNA-DNA hybridization between strain IC12T and related strains of the phylum Fusobacteriota were 71.4 and 26.4 %, respectively. Phylogenomic, physiological and chemotaxonomic analyses indicate that strain IC12T represents a novel genus and species within the phylum Fusobacteriota, for which the name Haliovirga abyssi gen. nov., sp. nov. is proposed, with strain IC12T (= DSM 112164T=JCM 39166T) as the type strain. We also propose the family Haliovirgaceae fam. nov. to accommodate this novel genus.

Tracking the effects of the COVID-19 pandemic on viral gastroenteritis through wastewater-based retrospective analyses.

The COVID-19 pandemic possibly disrupted the circulation and seasonality of gastroenteritis viruses (e.g., Norovirus (NoV), Sapovirus (SaV), group A rotavirus (ARoV), and Aichivirus (AiV)). Despite the growing application of wastewater-based epidemiology (WBE), there remains a lack of sufficient investigations into the actual impact of the COVID-19 pandemic on the prevalence of gastroenteritis viruses. In this study, we measured NoV GI and GII, SaV, ARoV, and AiV RNA concentrations in 296 influent wastewater samples collected from three wastewater treatment plants (WWTPs) in Sapporo, Japan between October 28, 2018 and January 12, 2023 using the highly sensitive EPISENS™ method. The detection ratios of SaV and ARoV after May 2020 (SaV: 49.8 % (134/269), ARoV: 57.4 % (151/263)) were significantly lower than those before April 2020 (SaV: 93.9 % (31/33), ARoV: 97.0 % (32/33); SaV: p < 3.5×10-7, ARoV: p < 1.5×10-6). Furthermore, despite comparable detection ratios before (88.5 %, 23/26) and during (66.7 %, 80/120) the COVID-19 pandemic (p = 0.032), the concentrations of NoV GII revealed a significant decrease after the onset of the pandemic (p < 1.5×10-7, Cliff's delta = 0.72). NoV GI RNA were sporadically detected (24.7 %, 8/33) before April 2020 and after May 2020 (6.5 %, 17/263), whereas AiV was consistently (100 %, 33/33) detected from wastewater throughout the study period (95.8 %, 252/263). The WBE results demonstrated the significant influence of COVID-19 countermeasures on the circulation of gastroenteritis viruses, with variations observed in the magnitude of their impact across different types of viruses. These epidemiological findings highlight that the hygiene practices implemented to prevent COVID-19 infections may also be effective for controlling the prevalence of gastroenteritis viruses, providing invaluable insights for public health units and the development of effective disease management guidelines.

Association of wastewater SARS-CoV-2 load with confirmed COVID-19 cases at a university hospital in Sapporo, Japan during the period from February 2021 to February 2023.

Wastewater surveillance for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been used to monitor trends in SARS-CoV-2 prevalence in a community without being influenced by clinical testing resources or healthcare-seeking behaviors. Since the rate of mortality from COVID-19 is higher in elderly patients with comorbidities, it is important to protect hospitalized patients from nosocomial infections caused by SARS-CoV-2. SARS-CoV-2 dissemination within a hospital ward was mostly mediated by healthcare workers (HCWs) and patients. HCWs need to understand the occurrence of COVID-19 and reflect this in their infection control measures. The aim of the present study was to determine the potential of SARS-CoV-2 RNA in wastewater as a leading indicator of confirmed COVID-19 cases at a university hospital. The trend of the geometric mean RNA concentrations in wastewater collected in Sapporo corresponded well with that of the number of newly confirmed COVID-19 cases at Hokkaido University Hospital between February 15, 2021 and February 26, 2023 (Pearson's r = 0.8823, p < 0.0001). Our results showed that monitoring SARS-CoV-2 RNA in municipal wastewater was useful for estimating the number of COVID-19 patients in healthcare facilities in the city.

Economic Evaluation of Wastewater Surveillance Combined with Clinical COVID-19 Screening Tests, Japan.

The COVID-19 pandemic has imposed substantial burdens on the global society. To find an optimal combination of wastewater surveillance and clinical testing for tracking COVID-19, we evaluated the economic efficiency of hypothetical screening options at a single facility in Japan. To conduct cost-benefit analyses, we developed standard decision models in which we assumed model parameters from literature and primary data, such as screening policies used at the Tokyo Olympic and Paralympic Village in 2021. We compared hypothetical 2-step screening options that used clinical PCR to diagnose COVID-19 after a positive result from primary screening using antigen tests (option 1) or wastewater surveillance (option 2). Our simulation results indicated that option 2 likely would be economically more justifiable than option 1, particularly at lower incidence levels. Our findings could help justify and promote the use of wastewater surveillance as a primary screening at a facility level for COVID-19 and other infectious diseases.

Moving forward with COVID-19: Future research prospects of wastewater-based epidemiology methodologies and applications.

Wastewater-based epidemiology (WBE) has been demonstrated for its great potential in tracking of coronavirus disease 2019 (COVID-19) transmission among populations despite some inherent methodological limitations. These include non-optimized sampling approaches and analytical methods; stability of viruses in sewer systems; partitioning/retention in biofilms; and the singular and inaccurate back-calculation step to predict the number of infected individuals in the community. Future research is expected to (1) standardize best practices in wastewater sampling, analysis and data reporting protocols for the sensitive and reproducible detection of viruses in wastewater; (2) understand the in-sewer viral stability and partitioning under the impacts of dynamic wastewater flow, properties, chemicals, biofilms and sediments; and (3) achieve smart wastewater surveillance with artificial intelligence and big data models. Further specific research is essential in the monitoring of other viral pathogens with pandemic potential and subcatchment applications to maximize the benefits of WBE beyond COVID-19.

Optimization and performance evaluation of an automated filtration method for the recovery of SARS-CoV-2 and other viruses in wastewater.

A rapid virus concentration method is needed to get high throughput. Reliable results of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) detection in wastewater are necessary for applications in wastewater-based epidemiology. In this study, an automated filtration method using a concentrating pipette (CP Select; Innovaprep) was applied to detect SARS-CoV-2 in wastewater samples with several modifications to increase its sensitivity and throughput. The performance of the CP Select method was compared to other concentration methods (polyethylene glycol precipitation and direct capture using silica column) to evaluate its applicability to SARS-CoV-2 detection in wastewater. SARS-CoV-2 RNA was successfully detected in six of eight wastewater samples using the CP Select method, whereas other methods could detect SARS-CoV-2 RNA in all wastewater samples. Enteric viruses, such as noroviruses of genogroups I (NoVs-GI) and II (NoVs-GII) and enteroviruses, were tested, resulting in 100 % NoVs-GII detection using all concentration methods. As for NoVs-GI and enteroviruses, all methods gave comparable number of detected samples in wastewater samples. This study showed that the optimized CP Select method was less sensitive in SARS-CoV-2 detection in wastewater than other methods, whereas all methods were applicable to detect or recover other viruses in wastewater.

Near full-automation of COPMAN using a LabDroid enables high-throughput and sensitive detection of SARS-CoV-2 RNA in wastewater as a leading indicator.

Wastewater-based epidemiology (WBE) is a promising tool to efficiently monitor COVID-19 prevalence in a community. For WBE community surveillance, automation of the viral RNA detection process is ideal. In the present study, we achieved near full-automation of a previously established method, COPMAN (COagulation and Proteolysis method using MAgnetic beads for detection of Nucleic acids in wastewater), which was then applied to detect SARS-CoV-2 in wastewater for half a year. The automation line employed the Maholo LabDroid and an automated-pipetting device to achieve a high-throughput sample-processing capability of 576 samples per week. SARS-CoV-2 RNA was quantified with the automated COPMAN using samples collected from two wastewater treatment plants in the Sagami River basin in Japan between 1 November 2021 and 24 May 2022, when the numbers of daily reported COVID-19 cases ranged from 0 to 130.3 per 100,000 inhabitants. The automated COPMAN detected SARS-CoV-2 RNA from 81 out of 132 samples at concentrations of up to 2.8 × 105 copies/L. These concentrations showed direct correlations with subsequently reported clinical cases (5-13 days later), as determined by Pearson's and Spearman's cross-correlation analyses. To compare the results, we also conducted testing with the EPISENS-S (Efficient and Practical virus Identification System with ENhanced Sensitivity for Solids, Ando et al., 2022), a previously reported detection method. SARS-CoV-2 RNA detected with EPISENS-S correlated with clinical cases only when using Spearman's method. Our automated COPMAN was shown to be an efficient method for timely and large-scale monitoring of viral RNA, making WBE more feasible for community surveillance.

Identification of SARS-CoV-2 variants in wastewater using targeted amplicon sequencing during a low COVID-19 prevalence period in Japan.

Wastewater-based epidemiology is expected to be able to identify SARS-CoV-2 variants at an early stage via next-generation sequencing. In the present study, we developed a highly sensitive amplicon sequencing method targeting the spike gene of SARS-CoV-2, which allows for sequencing viral genomes from wastewater containing a low amount of virus. Primers were designed to amplify a relatively long region (599 bp) around the receptor-binding domain in the SARS-CoV-2 spike gene, which could distinguish initial major variants of concern. To validate the methodology, we retrospectively analyzed wastewater samples collected from a septic tank installed in a COVID-19 quarantine facility between October and December 2020. The relative abundance of D614G mutant in SARS-CoV-2 genomes in the facility wastewater increased from 47.5 % to 83.1 % during the study period. The N501Y mutant, which is the characteristic mutation of the Alpha-like strain, was detected from wastewater collected on December 24, 2020, which agreed with the fact that a patient infected with the Alpha-like strain was quarantined in the facility on this date. We then analyzed archived municipal wastewater samples collected between November 2020 and January 2021 that contained low SARS-CoV-2 concentrations ranging from 0.23 to 0.43 copies/qPCR reaction (corresponding to 3.30 to 4.15 log10 copies/L). The targeted amplicon sequencing revealed that the Alpha-like variant with D614G and N501Y mutations was present in municipal wastewater collected on December 4, 2020 and later, suggesting that the variant had already spread in the community before its first clinical confirmation in Japan on December 25, 2020. These results demonstrate that targeted amplicon sequencing of wastewater samples is a powerful surveillance tool applicable to low COVID-19 prevalence periods and may contribute to the early detection of emerging variants.

Influence of membrane pore-size on the recovery of endogenous viruses from wastewater using an adsorption-extraction method.

The ongoing COVID-19 pandemic has emphasized the significance of wastewater surveillance in monitoring and tracking the spread of infectious diseases, including SARS-CoV-2. The wastewater surveillance approach detects genetic fragments from viruses in wastewater, which could provide an early warning of outbreaks in communities. In this study, we determined the concentrations of four types of endogenous viruses, including non-enveloped DNA (crAssphage and human adenovirus 40/41), non-enveloped RNA (enterovirus), and enveloped RNA (SARS-CoV-2) viruses, from wastewater samples using the adsorption-extraction (AE) method with electronegative HA membranes of different pore sizes (0.22, 0.45, and 0.80 µm). Our findings showed that the membrane with a pore size of 0.80 µm performed comparably to the membrane with a pore size of 0.45 µm for virus detection/quantitation (repeated measurement one-way ANOVA; p > 0.05). We also determined the recovery efficiencies of indigenous crAssphage and pepper mild mottle virus, which showed recovery efficiencies ranging from 50% to 94% and from 20% to 62%, respectively. Our results suggest that the use of larger pore size membranes may be beneficial for processing larger sample volumes, particularly for environmental waters containing low concentrations of viruses. This study offers valuable insights into the application of the AE method for virus recovery from wastewater, which is essential for monitoring and tracking infectious diseases in communities.

Impact of the COVID-19 pandemic on the prevalence of influenza A and respiratory syncytial viruses elucidated by wastewater-based epidemiology.

Since the COVID-19 pandemic, a decrease in the prevalence of Influenza A virus (IAV) and respiratory syncytial virus (RSV) has been suggested by clinical surveillance. However, there may be potential biases in obtaining an accurate overview of infectious diseases in a community. To elucidate the impact of the COVID-19 on the prevalence of IAV and RSV, we quantified IAV and RSV RNA in wastewater collected from three wastewater treatment plants (WWTPs) in Sapporo, Japan, between October 2018 and January 2023, using highly sensitive EPISENS™ method. From October 2018 to April 2020, the IAV M gene concentrations were positively correlated with the confirmed cases in the corresponding area (Spearman's r = 0.61). Subtype-specific HA genes of IAV were also detected, and their concentrations showed trends that were consistent with clinically reported cases. RSV A and B serotypes were also detected in wastewater, and their concentrations were positively correlated with the confirmed clinical cases (Spearman's r = 0.36-0.52). The detection ratios of IAV and RSV in wastewater decreased from 66.7 % (22/33) and 42.4 % (14/33) to 4.56 % (12/263) and 32.7 % (86/263), respectively in the city after the COVID-19 prevalence. The present study demonstrates the potential usefulness of wastewater-based epidemiology combined with the preservation of wastewater (wastewater banking) as a tool for better management of respiratory viral diseases.

Wastewater-based prediction of COVID-19 cases using a highly sensitive SARS-CoV-2 RNA detection method combined with mathematical modeling.

Wastewater-based epidemiology (WBE) has the potential to predict COVID-19 cases; however, reliable methods for tracking SARS-CoV-2 RNA concentrations (CRNA) in wastewater are lacking. In the present study, we developed a highly sensitive method (EPISENS-M) employing adsorption-extraction, followed by one-step RT-Preamp and qPCR. The EPISENS-M allowed SARS-CoV-2 RNA detection from wastewater at 50 % detection rate when newly reported COVID-19 cases exceed 0.69/100,000 inhabitants in a sewer catchment. Using the EPISENS-M, a longitudinal WBE study was conducted between 28 May 2020 and 16 June 2022 in Sapporo City, Japan, revealing a strong correlation (Pearson's r = 0.94) between CRNA and the newly COVID-19 cases reported by intensive clinical surveillance. Based on this dataset, a mathematical model was developed based on viral shedding dynamics to estimate the newly reported cases using CRNA data and recent clinical data prior to sampling day. This developed model succeeded in predicting the cumulative number of newly reported cases after 5 days of sampling day within a factor of √2 and 2 with a precision of 36 % (16/44) and 64 % (28/44), respectively. By applying this model framework, another estimation mode was developed without the recent clinical data, which successfully predicted the number of COVID-19 cases for the succeeding 5 days within a factor of √2 and 2 with a precision of 39 % (17/44) and 66 % (29/44), respectively. These results demonstrated that the EPISENS-M method combined with the mathematical model can be a powerful tool for predicting COVID-19 cases, especially in the absence of intensive clinical surveillance.

Occurrence of multiple respiratory viruses in wastewater in Queensland, Australia: Potential for community disease surveillance.

The early warning and tracking of COVID-19 prevalence in the community provided by wastewater surveillance has highlighted its potential for much broader viral disease surveillance. In this proof-of-concept study, 46 wastewater samples from four wastewater treatment plants (WWTPs) in Queensland, Australia, were analyzed for the presence and abundance of 13 respiratory viruses, and the results were compared with reported clinical cases. The viruses were concentrated using the adsorption-extraction (AE) method, and extracted nucleic acids were analyzed using qPCR and RT-qPCR. Among the viruses tested, bocavirus (BoV), parechovirus (PeV), rhinovirus A (RhV A) and rhinovirus B (RhV B) were detected in all wastewater samples. All the tested viruses except influenza B virus (IBV) were detected in wastewater sample from at least one WWTP. BoV was detected with the greatest concentration (4.96-7.22 log10 GC/L), followed by Epstein-Barr virus (EBV) (4.08-6.46 log10 GC/L), RhV A (3.95-5.63 log10 GC/L), RhV B (3.74-5.61 log10 GC/L), and PeV (3.17-5.32 log10 GC/L). Influenza viruses and respiratory syncytial virus (RSV) are notifiable conditions in Queensland, allowing the gene copy (GC) concentrations to be compared with reported clinical cases. Significant correlations (ρ = 0.60, p < 0.01 for IAV and ρ = 0.53, p < 0.01 for RSV) were observed when pooled wastewater influenza A virus (IAV) and RSV log10 GC/L concentrations were compared to log10 clinical cases among the four WWTP catchments. The positive predictive value for the presence of IAV and RSV in wastewater was 97 % for both IAV and RSV clinical cases within the four WWTP catchments. The overall accuracy of wastewater analysis for predicting clinical cases of IAV and RSV was 97 and 90 %, respectively. This paper lends credibility to the application of wastewater surveillance to monitor respiratory viruses of various genomic characteristics, with potential uses for increased surveillance capabilities and as a tool in understanding the dynamics of disease circulation in the communities.

Wastewater monitoring in tourist cities as potential sentinel sites for near real-time dynamics of imported SARS-CoV-2 variants.

Wastewater-based epidemiology (WBE) complements the clinical surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants' distribution in populations. Many developed nations have established national and regional WBE systems; however, governance and budget constraints could be obstacles for low- and middle-income countries. An urgent need thus exists to identify hotspots to serve as sentinel sites for WBE. We hypothesized that representative wastewater treatment plants (WWTPs) in two international gateway cities, Bangkok and Phuket, Thailand, could be sentineled for SARS-CoV-2 and its variants to reflect the clinical distribution patterns at city level and serve as early indicators of new variants entering the country. Municipal wastewater samples (n = 132) were collected from eight representative municipal WWTPs in Bangkok and Phuket during 19 sampling events from October 2021 to March 2022, which were tested by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) using the US CDC N1 and N2 multiplex and variant (Alpha, Delta, and Omicron BA.1 and BA.2) singleplex assays. The variant detection ratios from Bangkok and Phuket followed similar trends to the national clinical testing data, and each variant's viral loads agreed with the daily new cases (3-d moving average). Omicron BA.1 was detected in Phuket wastewater prior to Bangkok, possibly due to Phuket's WWTPs serving tourist communities. We found that the Omicron BA.1 and BA.2 viral loads predominantly drove the SARS-CoV-2 resurgence. We also noted a shifting pattern in the Bangkok WBE from a 22-d early warning in early 2021 to a near real-time pattern in late 2021. The potential application of tourist hotspots for WBE to indicate the arrival of new variants and re-emerging or unprecedented infectious agents could support tourism-dependent economies by complementing the reduced clinical regulations while maintaining public health protection via wastewater surveillance.

Correlation between SARS-CoV-2 RNA concentration in wastewater and COVID-19 cases in community: A systematic review and meta-analysis.

Wastewater-based epidemiology (WBE) has been considered as a promising approach for population-wide surveillance of coronavirus disease 2019 (COVID-19). Many studies have successfully quantified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA concentration in wastewater (CRNA). However, the correlation between the CRNA and the COVID-19 clinically confirmed cases in the corresponding wastewater catchments varies and the impacts of environmental and other factors remain unclear. A systematic review and meta-analysis were conducted to identify the correlation between CRNA and various types of clinically confirmed case numbers, including prevalence and incidence rates. The impacts of environmental factors, WBE sampling design, and epidemiological conditions on the correlation were assessed for the same datasets. The systematic review identified 133 correlation coefficients, ranging from -0.38 to 0.99. The correlation between CRNA and new cases (either daily new, weekly new, or future cases) was stronger than that of active cases and cumulative cases. These correlation coefficients were potentially affected by environmental and epidemiological conditions and WBE sampling design. Larger variations of air temperature and clinical testing coverage, and the increase of catchment size showed strong negative impacts on the correlation between CRNA and COVID-19 case numbers. Interestingly, the sampling technique had negligible impact although increasing the sampling frequency improved the correlation. These findings highlight the importance of viral shedding dynamics, in-sewer decay, WBE sampling design and clinical testing on the accurate back-estimation of COVID-19 case numbers through the WBE approach.