A longitudinal environmental surveillance study for SARS-CoV-2 from the emergency department of a teaching hospital in Hong Kong.

BACKGROUND: Understanding factors associated with SARS-CoV-2 exposure risk in the hospital setting may help improve infection control measures for prevention. AIM: To monitor SARS-CoV-2 exposure risk among healthcare workers and to identify risk factors associated with SARS-CoV-2 detection. METHODS: Surface and air samples were collected longitudinally over 14 months spanning 2020-2022 at the Emergency Department (ED) of a teaching hospital in Hong Kong. SARS-CoV-2 viral RNA was detected by real-time reverse-transcription polymerase chain reaction. Ecological factors associated with SARS-CoV-2 detection were analysed by logistic regression. A sero-epidemiological study was conducted in January-April 2021 to monitor SARS-CoV-2 seroprevalence. A questionnaire was used to collect information on job nature and use of personal protective equipment (PPE) of the participants. FINDINGS: SARS-CoV-2 RNA was detected at low frequencies from surfaces (0.7%, N = 2562) and air samples (1.6%, N = 128). Crowding was identified as the main risk factor, as weekly ED attendance (OR = 1.002, P=0.04) and sampling after peak-hours of ED attendance (OR = 5.216, P=0.03) were associated with the detection of SARS-CoV-2 viral RNA from surfaces. The low exposure risk was corroborated by the zero seropositive rate among 281 participants by April 2021. CONCLUSION: Crowding may introduce SARS-CoV-2 into the ED through increased attendances. Multiple factors may have contributed to the low contamination of SARS-CoV-2 in the ED, including hospital infection control measures for screening ED attendees, high PPE compliance among healthcare workers, and various public health and social measures implemented to reduce community transmission in Hong Kong where a dynamic zero COVID-19 policy was adopted.

How has the COVID-19 pandemic impacted wastewater-based epidemiology?

Wastewater-based epidemiology (WBE) was one of the areas of scientific knowledge that developed significantly with the COVID-19 pandemic, with robust worldwide application to monitor the circulation of the SARS-CoV-2 virus in urban communities at different scales and levels. This mini-review assesses how the COVID-19 pandemic may have influenced the WBE based on the investigation of 1305 scientific reports published (research, review, and conference papers) up to the end of 2022, considering the research objects, funding sources, actors, and countries involved. As a result, 71 % of all WBE-based publications occurred since the beginning of the pandemic, with 62 % addressing SARS-CoV-2, demonstrating the migration of WBE's relative importance in studies on drug abuse, pharmaceuticals consumption, and other disease-causing organisms to the constitution of a tool to support the monitoring of the coronavirus. Before the pandemic, WBE was a tool used for epidemiological surveillance of several diseases (54 % of studies), drug abuse (30 %), and pharmaceutical consumption (9 %). With the pandemic, these research topics lost to space, constituting only 37 % of the area's studies, and SARS-CoV-2 became the central object of studies. In addition, there has been a 4.7 % expansion of developing country participation in sewage surveillance publications and greater diversification of collaborators and funders, especially from government, businesses, and the water industry. International research partnerships had a reduction of 8 %, consequently, there was an increase in local and regional partnerships. With the COVID-19 pandemic, funding for research in WBE became approximately 6.5 % less dependent on traditional research funds. The future of WBE involves different approaches, including different focuses of research and technological advancements to improve the sensitivity, precision, and applicability of these investigations. The new WBE research arrangements are promising, although the post-pandemic challenges are likely to be in maintaining them and overcoming the trend toward a lack of diversity in study subjects.

Metavirome in Sewage and Fecally-Tainted River Waters in the Philippines: Highlighting the Role of Environmental Surveillance in the Covid-19 Pandemic

Intro: The COVID-19 pandemic has triggered global collaborative efforts on response and research to detect SARS-CoV-2 particles not just in the human population but also in wastewater. While the examination of clinical samples from COVID-19 patients links SARS-CoV-2 to specific individuals, the analysis of an amalgam of human feces through environmental surveillance (ES) links SARSCoV-2 to populations and communities served by the wastewater system. Studies on SARS-CoV-2 in the environment were already done in high-resource countries. However, its epidemiology in wastewater bodies in the Philippines is limited. In this study, we used the National ES for Polio and Other Pathogens Network to investigate the molecular epidemiology and transmission dynamics of SARS-CoV-2 at the outset of the pandemic. Method(s): This is a retrospective study of 250 wastewater samples collected from May 2020 to July 2021. Samples were processed using the two-phase concentration technique. Pepper mild mottle virus RNAs were quantified as the internal control. Real-time PCR was used to detect the N-gene of the SARS-CoV-2. Whole genomes were sequenced using the COVID-19 ARTIC v4.0. Phylogenetic and mutation analysis were done and lineage assignments were established using the PANGOLIN software. Finding(s): Forty-two percent (107/250) of the environmental samples detected SARS-CoV-2 particles. Fifty-nine samples with Ct values <=38 were sequenced and the whole genome analysis revealed B.1.1 and B.6. lineages of SARS-CoV-2. When viral load were plotted with the weekly cases in the respective site, we observed that SARS-CoV2 can be detected in wastewater weeks before the spike of cases in the community. Conclusion(s): This is the first report on the detection of B.1.1 and B.6 SARS-CoV-2 particles in waste/surface waters in the Philippines. With the declining incidence of COVID-19 cases, this study provided data regarding the feasibility of establishing environmental surveillance for SARS-CoV-2 as a supplemental tool for human or case monitoring especially in resource-limited settings.Copyright © 2023

Detection of SARS-CoV-2 RNA in stool and urine specimens of COVID-19 positive patients by Droplet Digital RT-PCR (dd RT- PCR)

SARS-CoV-2 infected cases diagnosis is based on the count of realtime reverse transcription-polymerase chain reaction (RT-PCR). The widely used reverse transcription-polymerase chain reaction (RTPCR) method has some limitations for clinical diagnosis and treatment. However, there are only few reports on the detection of the viral load in the stool and urine samples. While information about other modes of transmission is relatively less, some published literature supporting the possibility of a faecal-oral mode of transmission has been accumulating. Objective(s): The current study's objective was to assess the performance of real-time RT-qPCR assay and a droplet digital RT-PCR (dd RT-PCR) for detecting SARS-CoV-2 in stool and urine specimens. Methodology: One hundred and seven paired samples from 107 COVID-19-confirmed patients were analysed by dd RT-PCR and RTPCR based target gene (N1 and N2). Stool and urine were collected from COVID Care Centers of Pune Region. RNA was isolated using MagMax magnetic beads base procedure for further analysis. Real Time RT-PCR and DD PCR was performed from all the patients. Result(s): In 107 patients, all the stool samples showed 100% positive concordance by both methods, the average of 28.88 cycle threshold (Ct) of RT-PCR was highly correlated with the average copy number of 327.10 copies/mul analyzed in ddPCR. Whereas 27.1% urine samples were tested positive in ddPCR & 1.86% were positive with the average of 36.41 cycle threshold (Ct) in RT-PCR. Using Pangolin COVID-19 Lineage Assigner variants were analyzed and found to be delta prevalent. Conclusion(s): In the context of the COVID-19 pandemic, environmental surveillance for the detection of SARS-CoV-2 has become increasingly important. The findings of this study not only show that SARS-CoV-2 is present in urine and faeces, but they also raise the possibility that low concentrations of the viral target may make it easier to identify positive samples and help resolve situations of inconclusive diagnosis.

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.

Monitoring Enteroviruses and SARS-CoV-2 in Wastewater Using the Polio Environmental Surveillance System in Japan.

In the global strategy for polio eradication, environmental surveillance (ES) has been established worldwide to monitor polioviruses. In addition, nonpolio enteroviruses are simultaneously isolated from wastewater under this ES program. Hence, ES can be used to monitor enteroviruses in sewage to supplement clinical surveillance. In response to the coronavirus disease 2019 (COVID-19) pandemic, we also monitored severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in sewage using the polio ES system in Japan. Enterovirus and SARS-CoV-2 were detected in sewage from January 2019 to December 2021 and from August 2020 to November 2021, respectively. Enterovirus species such as echoviruses and coxsackieviruses were frequently detected by ES in 2019, indicating the circulation of these viruses. After the onset of the COVID-19 pandemic, sewage enterovirus detection and related patient reports were notably reduced in 2020 to 2021, suggesting changes in the hygiene behaviors of the human population in response to the pandemic. Our comparative experiment with a total of 520 reverse transcription-quantitative PCR (RT-qPCR) assays for SARS-CoV-2 detection demonstrated that the solid-based method had a significantly higher detection rate than that of the liquid-based method (24.6% and 15.9%, respectively). Moreover, the resulting RNA concentrations were correlated with the number of new COVID-19 cases (Spearman's r = 0.61). These findings indicate that the existing polio ES system can be effectively used for enterovirus and SARS-CoV-2 sewage monitoring using different procedures such as virus isolation and molecular-based detection. IMPORTANCE Long-term efforts are required to implement surveillance programs for the ongoing COVID-19 pandemic, and they will be required even in the postpandemic era. We adopted the existing polio environmental surveillance (ES) system for SARS-CoV-2 sewage monitoring in Japan as a practical and cost-effective approach. Moreover, the ES system routinely detects enteroviruses from wastewater and, therefore, can be used for enterovirus monitoring. The liquid fraction of the sewage sample is used for poliovirus and enterovirus detection, and the solid fraction can be used for SARS-CoV-2 RNA detection. The present study demonstrates how the existing ES system can be used for monitoring enteroviruses and SARS-CoV-2 in sewage.

Building health system resilience and pandemic preparedness using wastewater-based epidemiology from SARS-CoV-2 monitoring in Bengaluru, India.

The COVID-19 pandemic was a watershed event for wastewater-based epidemiology (WBE). It highlighted the inability of existing disease surveillance systems to provide sufficient forewarning to governments on the existing stage and scale of disease spread and underscored the need for an effective early warning signaling system. Recognizing the potentiality of environmental surveillance (ES), in May 2021, COVIDActionCollaborative launched the Precision Health platform. The idea was to leverage ES for equitable mapping of the disease spread in Bengaluru, India and provide early information regarding any inflection in the epidemiological curve of COVID-19. By sampling both networked and non-networked sewage systems in the city, the platform used ES for both equitable and comprehensive surveillance of the population to derive precise information on the existing stage of disease maturity across communities and estimate the scale of the approaching threat. This was in contrast to clinical surveillance, which during the peak of the COVID-19 pandemic in Bengaluru excluded a significant proportion of poor and vulnerable communities from its ambit of representation. The article presents the findings of a sense-making tool which the platform developed for interpreting emerging signals from wastewater data to map disease progression and identifying the inflection points in the epidemiological curve. Thus, the platform accurately generated early warning signals on disease escalation and disseminated it to the government and the general public. This information enabled concerned audiences to implement preventive measures in advance and effectively plan their next steps for improved disease management.

Retrospective Analysis of Wastewater-Based Epidemiology of SARS-CoV-2 in Residences on a Large College Campus: Relationships between Wastewater Outcomes and COVID-19 Cases across Two Semesters with Different COVID-19 Mitigation Policies

Wastewater-based epidemiology (WBE) has been utilized for outbreak monitoring and response efforts in university settings during the coronavirus disease 2019 (COVID-19) pandemic. However, few studies examined the impact of university policies on the effectiveness of WBE to identify cases and mitigate transmission. The objective of this study was to retrospectively assess relationships between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) wastewater outcomes and COVID-19 cases in residential buildings of a large university campus across two academic semesters (August 2020-May 2021) under different COVID-19 mitigation policies. Clinical case surveillance data of student residents were obtained from the university COVID-19 response program. We collected and processed building-level wastewater for detection and quantification of SARS-CoV-2 RNA by RT-qPCR. The odds of obtaining a positive wastewater sample increased with COVID-19 clinical cases in the fall semester (OR = 1.50, P value = 0.02), with higher odds in the spring semester (OR = 2.63, P value < 0.0001). We observed linear associations between SARS-CoV-2 wastewater concentrations and COVID-19 clinical cases (parameter estimate = 1.2, P value = 0.006). Our study demonstrated the effectiveness of WBE in the university setting, though it may be limited under different COVID-19 mitigation policies. As a complementary surveillance tool, WBE should be accompanied by robust administrative and clinical testing efforts for the COVID-19 pandemic response. © 2022 American Chemical Society.

Wastewater Surveillance for Infectious Disease: A Systematic Review.

Wastewater surveillance of SARS-CoV-2 has been shown to be a valuable source of information regarding SARS-CoV-2 transmission and COVID-19 cases. Though the method has been used for several decades to track other infectious diseases, there has not been a comprehensive review outlining all of the pathogens that have been surveilled through wastewater. Herein we identify what infectious diseases have been previously studied via wastewater surveillance prior to the COVID-19 pandemic. Infectious diseases and pathogens were identified in 100 studies of wastewater surveillance across 38 countries, as well as themes of how wastewater surveillance and other measures of disease transmission were linked. Twenty-five separate pathogen families were identified in the included studies, with the majority of studies examining pathogens from the family Picornaviridae, including polio and non-polio enteroviruses. Most studies of wastewater surveillance did not link what was found in the wastewater to other measures of disease transmission. Among those studies that did, the value reported varied by study. Wastewater surveillance should be considered as a potential tool for many infectious diseases. Wastewater surveillance studies can be improved by incorporating other measures of disease transmission at the population-level including disease incidence and hospitalizations.

Wastewater-based epidemiology approach: The learning lessons from COVID-19 pandemic and the development of novel guidelines for future pandemics.

Wastewater-based epidemiology (WBE) provides a comprehensive real-time framework of population attitude and health status. This approach is attracting the interest of medical community and health authorities to monitor the prevalence of a virus (such as the severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) among a community. Indeed, WBE is currently fine-tuning as environmental surveillance tool for coronavirus disease 2019 (COVID-19) pandemic. After a bibliometric analysis conducted to discover the research trends in WBE field, this work aimed to side-by-side compare the conventional method based on clinical testing with WBE approach. Furthermore, novel guidelines were developed to apply the WBE approach to a pandemic. The growing interest on WBE approach for COVID-19 pandemic is demonstrated by looking at the sharp increase in scientific papers published in the last years and at the ongoing studies on viral quantification methods and analytical procedures. The side-by-side comparison highlighted the ability of WBE to identify the hot-spot areas faster than the conventional approach, reducing the costs (e.g., rational use of available resources) and the gatherings at medical centers. Contrary to clinical testing, WBE has the surveillance capacity for preventing the virus resurgence, including asymptomatic contribution, and ensuring the preservation of medical staff health by avoiding the exposure to the virus infection during clinical testing. As extensively reported, the time in collecting epidemiological data is crucial for establishing the prevention and mitigation measures that are essential for curbing a pandemic. The developed guidelines can help to build a WBE system useful to control any future pandemic.

Retrospective Analysis of Wastewater-Based Epidemiology of SARS- CoV-2 in Residences on a Large College Campus: Relationships between Wastewater Outcomes and COVID-19 Cases across Two Semesters with Different COVID-19 Mitigation Policies

Wastewater-based epidemiology (WBE) has been utilized for outbreak monitoring and response efforts in university settings during the coronavirus disease 2019 (COVID-19) pandemic. However, few studies examined the impact of university policies on the effectiveness of WBE to identify cases and mitigate transmission. The objective of this study was to retrospectively assess relationships between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) wastewater outcomes and COVID-19 cases in residential buildings of a large university campus across two academic semesters (August 2020-May 2021) under different COVID-19 mitigation policies. Clinical case surveillance data of student residents were obtained from the university COVID-19 response program. We collected and processed building-level wastewater for detection and quantification of SARS-CoV-2 RNA by RT-qPCR. The odds of obtaining a positive wastewater sample increased with COVID-19 clinical cases in the fall semester (OR = 1.50, P value = 0.02), with higher odds in the spring semester (OR = 2.63, P value < 0.0001). We observed linear associations between SARS-CoV-2 wastewater concentrations and COVID-19 clinical cases (parameter estimate = 1.2, P value = 0.006). Our study demonstrated the effectiveness of WBE in the university setting, though it may be limited under different COVID-19 mitigation policies. As a complementary surveillance tool, WBE should be accompanied by robust administrative and clinical testing efforts for the COVID-19 pandemic response.

Hands off the Mink! Using Environmental Sampling for SARS-CoV-2 Surveillance in American Mink.

Throughout the COVID-19 pandemic, numerous non-human species were shown to be susceptible to natural infection by SARS-CoV-2, including farmed American mink. Once infected, American mink can transfer the virus from mink to human and mink to mink, resulting in a high rate of viral mutation. Therefore, outbreak surveillance on American mink farms is imperative for both mink and human health. Historically, disease surveillance on mink farms has consisted of a combination of mortality and live animal sampling; however, these methodologies have significant limitations. This study compared PCR testing of both deceased and live animal samples to environmental samples on an active outbreak premise, to determine the utility of environmental sampling. Environmental sampling mirrored trends in both deceased and live animal sampling in terms of percent positivity and appeared more sensitive in some low-prevalence instances. PCR CT values of environmental samples were significantly different from live animal samples' CT values and were consistently high (mean CT = 36.2), likely indicating a low amount of viral RNA in the samples. There is compelling evidence in favour of environmental sampling for the purpose of disease surveillance, specifically as an early warning tool for SARS-CoV-2; however, further work is needed to ultimately determine whether environmental samples are viable sources for molecular epidemiology investigations.

Wastewater-based epidemiology for preventing outbreaks and epidemics in Latin America - Lessons from the past and a look to the future.

Wastewater-based epidemiology (WBE) is an approach with the potential to complement clinical surveillance systems. Using WBE, it is possible to carry out an early warning of a possible outbreak, monitor spatial and temporal trends of infectious diseases, produce real-time results and generate representative epidemiological information in a territory, especially in areas of social vulnerability. Despite the historical uses of this approach, particularly in the Global Polio Eradication Initiative, and for other pathogens, it was during the COVID-19 pandemic that occurred an exponential increase in environmental surveillance programs for SARS-CoV-2 in wastewater, with many experiences and developments in the field of public health using data for decision making and prioritizing actions to control the pandemic. In Latin America, WBE was applied in heterogeneous contexts and with emphasis on populations that present many socio-environmental inequalities, a condition shared by all Latin American countries. This manuscript addresses the concepts and applications of WBE in public health actions, as well as different experiences in Latin American countries, and discusses a model to implement this surveillance system at the local or national level. We emphasize the need to implement this sentinel surveillance system in countries that want to detect the early entry and spread of new pathogens and monitor outbreaks or epidemics of infectious agents in their territories as a complement of public health surveillance systems.

Predicting COVID-19 cases using SARS-CoV-2 RNA in air, surface swab and wastewater samples.

Genomic footprints of pathogens shed by infected individuals can be traced in environmental samples, which can serve as a noninvasive method of infectious disease surveillance. The research evaluates the efficacy of environmental monitoring of SARS-CoV-2 RNA in air, surface swabs and wastewater to predict COVID-19 cases. Using a prospective experimental design, air, surface swabs, and wastewater samples were collected from a college dormitory housing roughly 500 students from March to May 2021 at the University of Miami, Coral Gables, FL. Students were randomly screened for COVID-19 during the study period. SARS-CoV-2 concentration in environmental samples was quantified using Volcano 2nd Generation-qPCR. Descriptive analyses were conducted to examine the associations between time-lagged SARS-CoV-2 in environmental samples and COVID-19 cases. SARS-CoV-2 was detected in air, surface swab and wastewater samples on 52 (63.4 %), 40 (50.0 %) and 57 (68.6 %) days, respectively. On 19 (24 %) of 78 days SARS-CoV-2 was detected in all three sample types. COVID-19 cases were reported on 11 days during the study period and SARS-CoV-2 was also detected two days before the case diagnosis on all 11 (100 %), 9 (81.8 %) and 8 (72.7 %) days in air, surface swab and wastewater samples, respectively. SARS-CoV-2 detection in environmental samples was an indicator of the presence of local COVID-19 cases and a 3-day lead indicator for a potential outbreak at the dormitory building scale. Proactive environmental surveillance of SARS-CoV-2 or other pathogens in multiple environmental media has potential to guide targeted measures to contain and/or mitigate infectious disease outbreaks within communities.

Spectrum of environmental surveillance of SARS-CoV-2 fragments: Questions, quests, and conquest.

This works examines the entire spectrum of 'Environmental Surveillance (EnvSurv)' of SARS-CoV-2 fragments i.e. the questions, quests, and conquests of the technology since early year 2020. The prime focus of the present work to document the journey with achieved objectives and remaining ambitions associated with the technology. Despite the EnvSurv may be regarded as the techniques, which rather achieved more than expected, will it win the struggle for its existence or lose its way once the pandemic and fear associated with it completely fades. Pertaining to this discussions, major researched topics were investigated, followed by enlisting of ten bullets of the past experiences along with corresponding challenges, and finally key targets for the techniques are enlisted. The article targets to be a simple guide of the journey of EnvSur in terms of its effectiveness for treatment, infectivity, monitoring & estimation (TIME) till date.

Surveillance of SARS-CoV-2 in Urban Wastewater in Italy

The presence of SARS-CoV-2 RNA in wastewaters was demonstrated early into the COVID-19 pandemic. Data on the presence of SARS-CoV-2 in urban wastewater can be exploited for different aims, including: i) description of outbreaks trends, ii) early warning system for new COVID-19 outbreaks or for the spread of the virus in new territories, iii) study of SARS-Co V-2 genetic diversity and detection of its variants, and iv) estimating the prevalence of COVID-19 infections. Therefore, wastewater surveillance (known as Wastewater Based Epidemiology, WBE) can be a powerful tool to support the decision-making process on public health measures. Italy was among the first EU countries investigating the occurrence and concentration of SARS-Co V-2 RNA in urban wastewaters, virus detection being accomplished at an early phase of the epidemic, between February and May 2020 in north and central Italy. The present study reports on the methodological issues, related to sample data collection and management, encountered in establishing the systematic, wastewater-based SARS-CoV-2 surveillance, and describes the results of the first six months of surveillance. © 2022 IEEE.

COPMAN: A novel high-throughput and highly sensitive method to detect viral nucleic acids including SARS-CoV-2 RNA in wastewater.

During the coronavirus disease 2019 (COVID-19) pandemic, wastewater-based epidemiology (WBE) attracted attention as an objective and comprehensive indicator of community infection that does not require individual inspection. Although several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection methods from wastewater have been developed, there are obstacles to their social implementation. In this study, we developed the COPMAN (Coagulation and Proteolysis method using Magnetic beads for detection of Nucleic acids in wastewater), an automatable method that can concentrate and detect multiple types of viruses from a limited volume (∼10 mL) of wastewater. The COPMAN consists of a high basicity polyaluminum chloride (PAC) coagulation process, magnetic bead-based RNA purification, and RT-preamplification, followed by qPCR. A series of enzymes exhibiting a high tolerance to PCR inhibitors derived from wastewater was identified and employed in the molecular detection steps in the COPMAN. We compared the detectability of viral RNA from 10-mL samples of virus-spiked (heat-inactivated SARS-CoV-2 and intact RSV) or unspiked wastewater by the COPMAN and other methods (PEG-qPCR, UF-qPCR, and EPISENS-S). The COPMAN was the most efficient for detecting spiked viruses from wastewater, detecting the highest level of pepper mild mottle virus (PMMoV), a typical intrinsic virus in human stool, from wastewater samples. The COPMAN also successfully detected indigenous SARS-CoV-2 RNA from 12 samples of wastewater at concentrations of 2.2 × 104 to 5.4 × 105 copies/L, during initial stages of an infection wave in the right and the left bank of the Sagami River in Japan (0.65 to 11.45 daily reported cases per 100,000 people). These results indicate that the COPMAN is suitable for detection of multiple pathogens from small volume of wastewater in automated stations.

Establishing a national SARS-CoV-2 surveillance network across South Africa to support wastewaterbased genomic and epidemiological monitoring of the COVID pandemic, 2020-2021

Introduction/ Background: Findings from wastewater-based epidemiology (WBE) surveillance of SARS-CoV-2 are increasingly used to monitor the epidemiology of SARS-CoV-2. We report on the findings from the South African Collaboration COVID-19 Environmental Surveillance System (SACCESS) laboratories and compare these with clinically obtained data. Methods: Wastewater grab or passive samples and underwent concentration, RNA extraction, reverse-transcriptasepolymerase chain reaction (RT-PCR) detection and quantification of SARS-CoV-2. Following whole genome amplicon-based sequencing, nonsynonymous mutations in the spike protein specific to variants of concern (VOCs) were identified. Quantitative RNA concentrations in genome copies/mL and the read-frequencies of lineagespecific single nucleotide polymorphisms (SNPs) in the spike protein of SARS-CoV-2 were plotted alongside official case load and SARS-Cov-2 lineage distribution by epidemiological week (provided by NICD SARS-CoV-2 epidemiology team and the Centre for Respiratory Diseases and Meningitis of the NICD). Results: Results from 95 wastewater treatment plants (WWTPs) across 9 provinces and all eight metropolitan areas of South Africa were collected weekly, biweekly or monthly from June 2020-October 2021 illustrate that increases and decreases in SARSCoV- 2 concentrations at all sites corresponded to metro-specific timing of the peak and post-peak decline in clinical cases. Increases in SARS-CoV-2 concentrations consistently preceded increases in clinical cases. In 18 WWTPs genomic analysis detected SNPs corresponding to prevalent VOCs and documented the change from beta to delta variant over the third SARS-CoV-2 wave. Impact: Without sufficient evidence that WBE findings correspond with clinical epidemiology and genomic results, public health authorities are often reluctant to use these data to guide decision making. Our findings illustrated that WBE monitoring of SARS-CoV-2 and detection of SNPs specific to SARS-CoV-2 variants correspond with clinical epidemiology and genomic findings. Conclusion: Wastewater based epidemiology including genotyping should be integrated into SARS-CoV-2 surveillance networks to support decision-making regarding public health interventions to contain SARS-CoV-2. Additional analyses to support interpretation of quantitative and genomic results should be done.

SARS-CoV-2 RNA in Wastewater Was Highly Correlated With the Number of COVID-19 Cases During the Fourth and Fifth Pandemic Wave in Kobe City, Japan.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the current coronavirus disease 2019 (COVID-19) pandemic and associated respiratory infections, has been detected in the feces of patients. Therefore, determining SARS-CoV-2 RNA levels in sewage may help to predict the number of infected people within the area. In this study, we quantified SARS-CoV-2 RNA copy number using reverse transcription quantitative real-time PCR with primers and probes targeting the N gene, which allows the detection of both wild-type and variant strain of SARS-CoV-2 in sewage samples from two wastewater treatment plants (WWTPs) in Kobe City, Japan, during the fourth and fifth pandemic waves of COVID-19 between February 2021 and October 2021. The wastewater samples were concentrated via centrifugation, yielding a pelleted solid fraction and a supernatant, which was subjected to polyethylene glycol (PEG) precipitation. The SARS-CoV-2 RNA was significantly and frequently detected in the solid fraction than in the PEG-precipitated fraction. In addition, the copy number in the solid fraction was highly correlated with the number of COVID-19 cases in the WWTP basin (WWTP-A: r = 0.8205, p < 0.001; WWTP-B: r = 0.8482, p < 0.001). The limit of capturing COVID-19 cases per 100,000 people was 0.75 cases in WWTP-A and 1.20 cases in WWTP-B, respectively. Quantitative studies of RNA in sewage can be useful for administrative purposes related to public health, including issuing warnings and implementing preventive measures within sewage basins.

The role of time-varying viral shedding in modelling environmental surveillance for public health: revisiting the 2013 poliovirus outbreak in Israel.

Environmental pathogen surveillance is a sensitive tool that can detect early-stage outbreaks, and it is being used to track poliovirus and other pathogens. However, interpretation of longitudinal environmental surveillance signals is difficult because the relationship between infection incidence and viral load in wastewater depends on time-varying shedding intensity. We developed a mathematical model of time-varying poliovirus shedding intensity consistent with expert opinion across a range of immunization states. Incorporating this shedding model into an infectious disease transmission model, we analysed quantitative, polymerase chain reaction data from seven sites during the 2013 Israeli poliovirus outbreak. Compared to a constant shedding model, our time-varying shedding model estimated a slower peak (four weeks later), with more of the population reached by a vaccination campaign before infection and a lower cumulative incidence. We also estimated the population shed virus for an average of 29 days (95% CI 28-31), longer than expert opinion had suggested for a population that was purported to have received three or more inactivated polio vaccine (IPV) doses. One explanation is that IPV may not substantially affect shedding duration. Using realistic models of time-varying shedding coupled with longitudinal environmental surveillance may improve our understanding of outbreak dynamics of poliovirus, SARS-CoV-2, or other pathogens.