High-resolution and real-time wastewater viral surveillance by Nanopore sequencing.

Wastewater genomic sequencing stands as a pivotal complementary tool for viral surveillance in populations. While long-read Nanopore sequencing is a promising platform to provide real-time genomic data, concerns over the sequencing accuracy of the earlier Nanopore versions have somewhat restrained its widespread application in wastewater analysis. Here, we evaluate the latest improved version of Nanopore sequencing (R10.4.1), using SARS-CoV-2 as the model infectious virus, to demonstrate its effectiveness in wastewater viral monitoring. By comparing amplicon lengths of 400 bp and 1200 bp, we revealed that shorter PCR amplification is more suitable for wastewater samples due to viral genome fragmentation. Utilizing mock wastewater samples, we validated the reliability of Nanopore sequencing for variant identification by comparing it with Illumina sequencing results. The strength of Nanopore sequencing in generating real-time genomic data for providing early warning signals was also showcased, indicating that as little as 0.001 Gb of data can provide accurate results for variant prevalence. Our evaluation also identified optimal alteration frequency cutoffs (>50 %) for precise mutation profiling, achieving >99 % precision in detecting single nucleotide variants (SNVs) and insertions/deletions (indels). Monitoring two major wastewater treatment plants in Hong Kong from September 2022 to April 2023, covering over 4.5 million population, we observed a transition in dominant variants from BA.5 to XBB lineages, with XBB.1.5 being the most prevalent variants. Mutation detection also highlighted the potential of wastewater Nanopore sequencing in uncovering novel mutations and revealed links between signature mutations and specific variants. This study not only reveals the environmental implications of Nanopore sequencing in SARS-CoV-2 surveillance but also underscores its potential in broader applications including environmental health monitoring of other epidemic viruses, which could significantly enhance the field of wastewater-based epidemiology.

Tracking diarrhea viruses and mpox virus using the wastewater surveillance network in Hong Kong.

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.

Comparison of RT-ddPCR and RT-qPCR platforms for SARS-CoV-2 detection: Implications for future outbreaks of infectious diseases.

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.

Development and application of influenza virus wastewater surveillance in Hong Kong.

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.

Wastewater genomic sequencing for SARS-CoV-2 variants surveillance in wastewater-based epidemiology applications.

Wastewater-based epidemiology (WBE) has been widely used as a complementary approach to SARS-CoV-2 clinical surveillance. Wastewater genomic sequencing could provide valuable information on the genomic diversity of SARS-CoV-2 in the surveyed population. However, reliable detection and quantification of variants or mutations remain challenging. In this study, we used mock wastewater samples created by spiking SARS-CoV-2 variant standard RNA into wastewater RNA to evaluate the impacts of sequencing throughput on various aspects such as genome coverage, mutation detection, and SARS-CoV-2 variant deconvolution. We found that wastewater datasets with sequencing throughput greater than 0.5 Gb yielded reliable results in genomic analysis. In addition, using in silico mock datasets, we evaluated the performance of the adopted pipeline for variant deconvolution. By sequencing 86 wastewater samples covering more than 6 million people over 7 months, we presented two use cases of wastewater genomic sequencing for surveying COVID-19 in Hong Kong in WBE applications, including the replacement of Delta variants by Omicron variants, and the prevalence and development trends of three Omicron sublineages. Importantly, the wastewater genomic sequencing data were able to reveal the variant trends 16 days before the clinical data did. By investigating mutations of the spike (S) gene of the SARS-CoV-2 virus, we also showed the potential of wastewater genomic sequencing in identifying novel mutations and unique alleles. Overall, our study demonstrated the crucial role of wastewater genomic surveillance in providing valuable insights into the emergence and monitoring of new SARS-CoV-2 variants and laid a solid foundation for the development of genomic analysis methodologies for WBE of other novel emerging viruses in the future.

Refining detection methods for emerging SARS-CoV-2 mutants in wastewater: A case study on the Omicron variants.

COVID-19 is an ongoing public health threat worldwide driven by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Wastewater surveillance has emerged as a complementary tool to clinical surveillance to control the COVID-19 pandemic. With the emergence of new variants of SARS-CoV-2, accumulated mutations that occurred in the SARS-CoV-2 genome raise new challenges for RT-qPCR diagnosis used in wastewater surveillance. There is a pressing need to develop refined methods for modifying primer/probes to better detect these emerging variants in wastewater. Here, we exemplified this process by focusing on the Omicron variants, for which we have developed and validated a modified detection method. We first modified the primers/probe mismatches of three assays commonly used in wastewater surveillance according to in silico analysis results for the mutations of 882 sequences collected during the fifth-wave outbreak in Hong Kong, and then evaluated them alongside the seven original assays. The results showed that five of seven original assays had better sensitivity for detecting Omicron variants, with the limits of detection (LoDs) ranging from 1.53 to 2.76 copies/µL. UCDC-N1 and Charité-E sets had poor performances, having LoDs higher than 10 copies/µL and false-positive/false-negative results in wastewater testing, probably due to the mismatch and demonstrating the need for modification of primer/probe sequences. The modified assays exhibited higher sensitivity and specificity, along with better reproducibility in detecting 81 wastewater samples. In addition, the sequencing results of six wastewater samples by Illumina also validated the presence of mismatches in the primer/probe binding sites of the three assays. This study highlights the importance of re-configuration of the primer-probe sets and refinements for the sequences to ensure the diagnostic effectiveness of RT-qPCR detection.

Circulation of SARS-CoV-2 Omicron sub-lineages revealed by multiplex genotyping RT-qPCR assays for sewage surveillance.

Sewage surveillance has proven to be an essential complementary tool to clinical diagnosis in combating the COVID-19 pandemic by tracking the spread of the SARS-CoV-2 virus and evaluating infection levels in populations. With the striking spreading and continuous evolution of SARS-CoV-2 Omicron VOC that characterized with higher transmissibility and potential immune evasion, there is an urgent need for the rapid surveillance of this prevalent strain and its sub-lineages in sewage. In this study, based on three multiplex allele-specific (AS) RT-qPCR assays, we established a rapid and high-throughput detection workflow for the simultaneous discrimination of Omicron sub-lineages BA.2.2, BA.2.12.1, BA.4 and BA.5 (hereafter referred to as BA.4/BA.5) to track their community circulation in Hong Kong. All primer-probe sets in the multiplex assays could correctly discriminate and quantitate their target genotypes with high sensitivity and specificity, even when multiple variants co-existed in the sewage samples. Using the established multiplex assays, the trends of SARS-CoV-2 total viral load and variant dynamics in influent samples collected from 11 wastewater treatment plants (WWTPs) during June 2022 and September 2022, aligned with the clinical data, successfully unveiling the swift emergence and predominance of Omicron BA.4/BA.5 in Hong Kong. The study highlights the feasibility and applicability of multiplex RT-qPCR assays for monitoring epidemic trends and tracking variant displacement dynamics in sewage samples, providing a more rapid, high-throughput and cost-effective alternative to enhance the current sewage surveillance system.

The city-wide full-scale interactive application of sewage surveillance programme for assisting real-time COVID-19 pandemic control - A case study in Hong Kong.

The paper discusses the implementation of Hong Kong's tailor-made sewage surveillance programme led by the Government, which has demonstrated how an efficient and well-organized sewage surveillance system can complement conventional epidemiological surveillance to facilitate the planning of intervention strategies and actions for combating COVID-19 pandemic in real-time. This included the setting up of a comprehensive sewerage network-based SARS-CoV-2 virus surveillance programme with 154 stationary sites covering 6 million people (or 80 % of the total population), and employing an intensive monitoring programme to take samples from each stationary site every 2 days. From 1 January to 22 May 2022, the daily confirmed case count started with 17 cases per day on 1 January to a maximum of 76,991 cases on 3 March and dropped to 237 cases on 22 May. During this period, a total of 270 "Restriction-Testing Declaration" (RTD) operations at high-risk residential areas were conducted based on the sewage virus testing results, where over 26,500 confirmed cases were detected with a majority being asymptomatic. In addition, Compulsory Testing Notices (CTN) were issued to residents, and the distribution of Rapid Antigen Test kits was adopted as alternatives to RTD operations in areas of moderate risk. These measures formulated a tiered and cost-effective approach to combat the disease in the local setting. Some ongoing and future enhancement efforts to improve efficacy are discussed from the perspective of wastewater-based epidemiology. Forecast models on case counts based on sewage virus testing results were also developed with R2 of 0.9669-0.9775, which estimated that up to 22 May 2022, around 2,000,000 people (~67 % higher than the total number of 1,200,000 reported to the health authority, due to various constraints or limitations) had potentially contracted the disease, which is believed to be reflecting the real situation occurring in a highly urbanized metropolis like Hong Kong.

A rapid, high-throughput, and sensitive PEG-precipitation method for SARS-CoV-2 wastewater surveillance.

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.

Quantification of SARS-CoV-2 RNA in wastewater treatment plants mirrors the pandemic trend in Hong Kong.

Wastewater-based epidemiology (WBE) for the SARS-CoV-2 virus in wastewater treatment plants (WWTPs) has emerged as a cost-effective and unbiased tool for population-level testing in the community. In the present study, we conducted a 6-month wastewater monitoring campaign from three WWTPs of different flow rates and catchment area characteristics, which serve 28 % (2.1 million people) of Hong Kong residents in total. Wastewater samples collected daily or every other day were concentrated using ultracentrifugation and the SARS-CoV-2 virus RNA in the supernatant was detected using the N1 and E primer sets. The results showed significant correlations between the virus concentration and the number of daily new cases in corresponding catchment areas of the three WWTPs when using 7-day moving average values (Kendall's tau-b value: 0.227-0.608, p < 0.001). SARS-CoV-2 virus concentration was normalized to a fecal indicator using PMMoV concentration and daily flow rates, but the normalization did not enhance the correlation. The key factors contributing to the correlation were also evaluated, including the sampling frequency, testing methods, and smoothing days. This study demonstrates the applicability of wastewater surveillance to monitor overall SARS-CoV-2 pandemic dynamics in a densely populated city like Hong Kong, and provides a large-scale longitudinal reference for the establishment of the long-term sentinel surveillance in WWTPs for WBE of pathogens which could be combined into a city-wide public health observatory.

Real-time allelic assays of SARS-CoV-2 variants to enhance sewage surveillance.

To effectively control the ongoing outbreaks of fast-spreading SARS-CoV-2 variants, there is an urgent need to add rapid variant detection and discrimination methods to the existing sewage surveillance systems established worldwide. We designed eight assays based on allele-specific RT-qPCR for real-time allelic discrimination of eight SARS-CoV-2 variants (Alpha, Beta, Gamma, Delta, Omicron, Lambda, Mu, and Kappa) in sewage. In silico analysis of the designed assays for identifying SARS-CoV-2 variants using more than four million SARS-CoV-2 variant sequences yielded ∼100% specificity and >90% sensitivity. All assays could sensitively discriminate and quantify target variants at levels as low as 10 viral RNA copy/µL with minimal cross-reactivity to the corresponding nontarget genotypes, even for sewage samples containing mixtures of SARS-CoV-2 variants with differential abundances. Integration of this method into the routine sewage surveillance in Hong Kong successfully identified the Beta variant in a community sewage. Complete concordance was observed between the results of viral whole-genome sequencing and those of our novel assays in sewage samples that contained exclusively the Delta variant discharged by a clinically diagnosed COVID-19 patient living in a quarantine hotel. Our assays in this method also provided real-time discrimination of the newly emerging Omicron variant in sewage two days prior to clinical test results in another quarantine hotel in Hong Kong. These novel allelic discrimination assays offer a rapid, sensitive, and specific way for detecting multiple SARS-CoV-2 variants in sewage and can be directly integrated into the existing sewage surveillance systems.

Evaluation of RT-qPCR Primer-Probe Sets to Inform Public Health Interventions Based on COVID-19 Sewage Tests.

Sewage surveillance is increasingly employed as a supplementary tool for COVID-19 control. Experiences learnt from large-scale trials could guide better interpretation of the sewage data for public health interventions. Here, we compared the performance of seven commonly used primer-probe sets in RT-qPCR and evaluated the usefulness in the sewage surveillance program in Hong Kong. All selected primer-probe sets reliably detected SARS-CoV-2 in pure water at 7 copies per µL. Sewage matrix did not influence RT-qPCR determination of SARS-CoV-2 concentrated from a small-volume sewage (30 mL) but introduced inhibitory impacts on a large-volume sewage (920 mL) with a ΔCt of 0.2-10.8. Diagnostic performance evaluation in finding COVID-19 cases showed that N1 was the best single primer-probe set, while the ORF1ab set is not recommended. Sewage surveillance using the N1 set for over 3200 samples effectively caught the outbreak trend and, importantly, had a 56% sensitivity and a 96% specificity in uncovering the signal sources from new cases and/or convalescent patients in the community. Our study paves the way for selecting detection primer-probe sets in wider applications in responding to the COVID-19 pandemic.

Use of Sewage Surveillance for COVID-19: A Large-Scale Evidence-Based Program in Hong Kong.

BACKGROUND: Sewage surveillance, by detecting SARS-CoV-2 virus circulation at the community level, has the potential to supplement individual surveillance for COVID-19. However, to date, there have been no reports about the large-scale implementation and validation of sewage surveillance for public health action. OBJECTIVE: Here, we developed a standardized approach for SARS-CoV-2 detection in sewage and applied it prospectively to supplement public health interventions. METHODS: We analyzed 1,169 sewage samples collected at 492 sites from December 2020 to March 2021. Forty-seven of 492 sites tested positive, 44 (94%) of them had traceable sources of viral signals in the corresponding sewershed, either from previously unsuspected but subsequently confirmed patients or recently convalescent patients or from both patient groups. RESULTS: Sewage surveillance had a sensitivity of 54%, a specificity of 95%, a positive predictive value of 53%, and a negative predictive value of 95% for identifying a previously unsuspected patient within a sewershed. Sewage surveillance in Hong Kong provided a basis for the statutory public health action to detect silent COVID-19 transmission. DISCUSSION: Considering the epidemiological data together with the sewage testing results, compulsory testing was conducted for individual residents at 27 positive sewage sites and uncovered total of 62 previously unsuspected patients, demonstrating the value of sewage surveillance in uncovering previously unsuspected patients in the community. Our study suggests that sewage surveillance could be a powerful management tool for the control of COVID-19. https://doi.org/10.1289/EHP9966.

Comparison of virus concentration methods and RNA extraction methods for SARS-CoV-2 wastewater surveillance.

Wastewater surveillance is a promising tool for population-level monitoring of the spread of infectious diseases, such as the coronavirus disease 2019 (COVID-19). Different from clinical specimens, viruses in community-scale wastewater samples need to be concentrated before detection because viral RNA is highly diluted. The present study evaluated eleven different virus concentration methods for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wastewater. First, eight concentration methods of different principles were compared using spiked wastewater at a starting volume of 30 mL. Ultracentrifugation was the most effective method with a viral recovery efficiency of 25 ± 6%. The second-best option, AlCl3 precipitation method, yielded a lower recovery efficiency, only approximately half that of the ultracentrifugation method. Second, the potential of increasing method sensitivity was explored using three concentration methods starting with a larger volume of 1000 mL. Although ultracentrifugation using a large volume outperformed the other two large-volume methods, it only yielded a comparable method sensitivity as the ultracentrifugation using a small volume (30 mL). Thus, ultracentrifugation using less volume of wastewater is more preferable considering the sample processing throughput. Third, a comparison of two viral RNA extraction methods showed that the lysis-buffer-based extraction method resulted in higher viral recovery efficiencies, with cycle threshold (Ct) values 0.9-4.2 lower than those obtained for the acid-guanidinium-phenol-based method using spiked samples. These results were further confirmed by using positive wastewater samples concentrated by ultracentrifugation and extracted separately by the two viral RNA extraction methods. In summary, concentration using ultracentrifugation followed by the lysis buffer-based extraction method enables sensitive and robust detection of SARS-CoV-2 for wastewater surveillance.

Use of sewage surveillance for COVID-19 to guide public health response: A case study in Hong Kong.

Sewage surveillance could help develop proactive response to the Coronavirus Disease 2019 (COVID-19) pandemic, but currently there are limited reports about examples in practical exercises. Here, we report a use case of intensified sewage surveillance to initiate public health action to thwart a looming Delta variant outbreak in Hong Kong. On 21 June 2021, albeit under basically contained COVID-19 situation in Hong Kong, routine sewage surveillance identified a high viral load of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a sewage sample from one site covering over 33,000 population, suggesting infected cases living in the respective sewershed. The use of a newly developed method based on allele-specific real-time quantitative polymerase chain reaction (AS RT-qPCR) served to alert the first documentation of the Delta variant in local community sewage three days before the case was confirmed to be a Delta variant carrier. Intensified sewage surveillance was triggered. Targeted upstream sampling at sub-sewershed areas pinpointed the source of positive viral signal across spatial scales from sewershed to building level, and assisted in determining the specific area for issuing a compulsory testing order for individuals on 23 June 2021. A person who lived in a building with the positive result of sewage testing was confirmed to be infected with COVID-19 on 24 June 2021. Viral genome sequences determined from the sewage sample were compared to those from the clinic specimens of the matched patient, and confirmed that the person was the source of the positive SARS-CoV-2 signal in the sewage sample. This study could help build confidences for public health agencies in using the sewage surveillance in their own communities.

The first case study of wastewater-based epidemiology of COVID-19 in Hong Kong.

Early detection and surveillance of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) virus are key pre-requisites for the effective control of coronavirus disease (COVID-19). So far, sewage testing has been increasingly employed as an alternative surveillance tool for this disease. However, sampling site characteristics impact the testing results and should be addressed in the early use stage of this emerging tool. In this study, we implemented the sewage testing for SARS-CoV-2 virus across sampling sites with different sewage system characteristics. We first validated a testing method using "positive" samples from a hospital treating COVID-19 patients. This method was used to test 107 sewage samples collected during the third wave of the COVID-19 outbreak in Hong Kong (from June 8 to September 29, 2020), covering sampling sites associated with a COVID-19 hospital, public housing estates, and conventional sewage treatment facilities. The highest viral titer of 1975 copy/mL in sewage was observed in a sample collected from the isolation ward of the COVID-19 hospital. Sewage sampling at individual buildings detected the virus 2 days before the first cases were identified. Sequencing of the detected viral fragment confirmed an identical nucleotide sequence to that of the SARS-CoV-2 isolated from human samples. The virus was also detected in sewage treatment facilities, which serve populations of approximately 40,000 to more than one million people.