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• Testing protocols for DNA (mpox) and RNA (norovirus, rotavirus) viruses in wastewater were developed and validated. • Wastewater surveillance effectively tracked the diarrhea viruses' activity in the community. • The mpox virus signal was detectable in wastewater at a low prevalence area. • Four key components were provided to increase the values of wastewater surveillance network. The wastewater surveillance network successfully established for COVID-19 showed great potential to monitor other infectious viruses, such as norovirus, rotavirus and mpox virus. In this study, we established and validated detection methods for these viruses in wastewater. We developed a supernatant-based method to detect RNA viruses from wastewater samples and applied it to the monthly diarrhea viruses (norovirus genogroup I & II, and rotavirus) surveillance in wastewater treatment plants (WWTPs) at a city-wide level for 16 months. Significant correlations were observed between the diarrhea viruses concentrations in wastewater and detection rates in faecal specimens by clinical surveillance. The highest norovirus concentration in wastewater was obtained in winter, consistent with the seasonal pattern of norovirus outbreak in Hong Kong. Additionally, we established a pellet-based method to monitor DNA viruses in wastewater and detected weak signals for mpox virus in wastewater from a WWTP serving approximately 16,700 people, when the first mpox patient in Hong Kong was admitted to the hospital within the catchment area. Genomic sequencing provided confirmatory evidence for the validity of the results. Our findings emphasized the efficacy of the wastewater surveillance network in WWTPs as a cost-effective tool to track the transmission trend of diarrhea viruses and to provide sensitive detection of novel emerging viruses such as mpox virus in low-prevalence areas. The developed methods and surveillance results provide confidence for establishing robust wastewater surveillance programs to control infectious diseases in the post-pandemic era. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] • RT-ddPCR demonstrated superior quantification accuracy for SARS-CoV-2 detection compared to RT-qPCR and was particularly advantageous at low viral levels with better precision and sensitivity. • RT-ddPCR outperformed RT-qPCR for viral RNA detection in the complex wastewater matrix and yielded a significantly higher detection rate in large-volume SARS-CoV-2 positive wastewater samples. • RT-ddPCR exhibited more robust SARS-CoV-2 detection with the presence of a mismatch in the probe binding region when consensus assay was used. • RT-ddPCR showed superiority over RT-qPCR for virus genotyping with higher accuracy and detection rate, especially at low levels of allele frequencies and concentrations. • By taking SARS-CoV-2 as a model system, this study suggested RT-ddPCR as a promising tool in wastewater surveillance scenarios and other relevant applications for future infectious disease outbreaks. The increased frequency of human infectious disease outbreaks caused by RNA viruses worldwide in recent years calls for enhanced public health surveillance for better future preparedness. Wastewater-based epidemiology (WBE) is emerging as a valuable epidemiological tool for providing timely population-wide surveillance for disease prevention and response complementary to the current clinical surveillance system. Here, we compared the analytical performance and practical applications between predominant molecular detection methods of RT-qPCR and RT-ddPCR on SARS-CoV-2 detection in wastewater surveillance. When pure viral RNA was tested, RT-ddPCR exhibited superior quantification accuracy at higher concentration levels and achieved more sensitive detection with reduced variation at low concentration levels. Furthermore, RT-ddPCR consistently demonstrated more robust and accurate measurement either in the background of the wastewater matrix or with the presence of mismatches in the target regions of the consensus assay. Additionally, by detecting mock variant RNA samples, we found that RT-ddPCR outperformed RT-qPCR in virus genotyping by targeting specific loci with signature mutations in allele-specific (AS) assays, especially at low levels of allele frequencies and concentrations, which increased the possibility for sensitive low-prevalence variant detection in the population. Our study provides insights for detection method selection in the WBE applications for future infectious disease outbreaks. [ABSTRACT FROM AUTHOR]

• Two preanalytical methods (supernatant-based and pellet-based) were established for influenza A/B wastewater surveillance. • Sanger sequencing confirmed the presence of influenza A/B RNA in wastewater. • Wastewater influenza A virus concentration effectively reflected influenza activity in the community. • H1pdm virus was the dominant subtype in the community at the time of this study. • Wastewater surveillance can provide virus types and subtypes information for public health actions in the future. Wastewater surveillance is considered as a powerful tool in providing cost-effective, population-wide and near real-time surveillance results for controlling infectious diseases (i.e., SARS-CoV-2, influenza virus), complementary to clinical surveillance. To facilitate the utility of this emerging tool, we developed two preanalytical protocols (supernatant-based and pellet-based) for influenza A/B virus (IAV/IBV) wastewater surveillance and applied them to the established wastewater surveillance network for large-scale longitudinal monitoring in Hong Kong. We tested 724 wastewater samples from 24 stationary sites for weekly surveillance for 8 months and 458 wastewater samples from 11 wastewater treatment plants (WWTPs) for more frequent (three times per week) city-wide surveillance for 4 months when influenza season commenced. We found the city-wide IAV virus concentration in wastewater were associated with the detection rate and influenza-like illness plus rates (ILI+) of clinical respiratory specimens and increased significantly after the cancelling of mask mandate that was in place for COVID-19. IBV was at low detection rates and low virus concentration levels, consistent with the low detection rates observed by clinical surveillance. In addition, we conducted virus subtype identification in selected wastewater samples, and observed the H1pdm was the major circulation subtype. Moreover, the obtained virus signals were confirmed by Sanger sequencing of PCR products, suggesting the feasibility and applicability of established methods for rapid detection of influenza virus types and subtypes in wastewater surveillance. This study demonstrates the applicability of IAV/IBV wastewater surveillance to current wastewater infrastructures and it could be used as a rapid and cost-effective surveillance strategy to track virus transmission patterns in the community for timely public health actions in the future. [ABSTRACT FROM AUTHOR]

• A PEG-precipitation method was developed for high-throughput wastewater surveillance. • The PEG-precipitation method obtained a process limit of detection of 3286 copies/L. • The PEG-precipitation method could process over 100 samples by two persons per day. • The turnaround time was optimized by reducing the centrifugation and incubation time. The effective application of wastewater surveillance is dependent on testing capacity and sensitivity to obtain high spatial resolution testing results for a timely targeted public health response. To achieve this purpose, the development of rapid, high-throughput, and sensitive virus concentration methods is urgently needed. Various protocols have been developed and implemented in wastewater surveillance networks so far, however, most of them lack the ability to scale up testing capacity or cannot achieve sufficient sensitivity for detecting SARS-CoV-2 RNA at low prevalence. In the present study, using positive raw wastewater in Hong Kong, a PEG precipitation-based three-step centrifugation method was developed, including low-speed centrifugation for large particles removal and the recovery of viral nucleic acid, and medium-speed centrifugation for the concentration of viral nucleic acid. This method could process over 100 samples by two persons per day to reach the process limit of detection (PLoD) of 3286 copies/L wastewater. Additionally, it was found that the testing capacity could be further increased by decreasing incubation and centrifugation time without significantly influencing the method sensitivity. The entire procedure uses ubiquitous reagents and instruments found in most laboratories to obtain robust testing results. This high-throughput, cost-effective, and sensitive tool will promote the establishment of nearly real-time wastewater surveillance networks for valuable public health information. [Display omitted] [ABSTRACT FROM AUTHOR]