Looking Forward: The Role of Academic Researchers in Building Sustainable Wastewater Surveillance Programs.

BACKGROUND: In just over 2 years, tracking the COVID-19 pandemic through wastewater surveillance advanced from early reports of successful SARS-CoV-2 RNA detection in untreated wastewater to implementation of programs in at least 60 countries. Early wastewater monitoring efforts primarily originated in research laboratories and are now transitioning into more formal surveillance programs run in commercial and public health laboratories. A major challenge in this progression has been to simultaneously optimize methods and build scientific consensus while implementing surveillance programs, particularly during the rapidly changing landscape of the pandemic. Translating wastewater surveillance results for effective use by public health agencies also remains a key objective for the field. OBJECTIVES: We examined the evolution of wastewater surveillance to identify model collaborations and effective partnerships that have created rapid and sustained success. We propose needed areas of research and key roles academic researchers can play in the framework of wastewater surveillance to aid in the transition from early monitoring efforts to more formalized programs within the public health system. DISCUSSION: Although wastewater surveillance has rapidly developed as a useful public health tool for tracking COVID-19, there remain technical challenges and open scientific questions that academic researchers are equipped to address. This includes validating methodology and backfilling important knowledge gaps, such as fate and transport of surveillance targets and epidemiological links to wastewater concentrations. Our experience in initiating and implementing wastewater surveillance programs in the United States has allowed us to reflect on key barriers and draw useful lessons on how to promote synergy between different areas of expertise. As wastewater surveillance programs are formalized, the working relationships developed between academic researchers, commercial and public health laboratories, and data users should promote knowledge co-development. We believe active involvement of academic researchers will contribute to building robust surveillance programs that will ultimately provide new insights into population health. https://doi.org/10.1289/EHP11519.

Wastewater surveillance using ddPCR accurately tracked Omicron emergence due to altered N1 probe binding efficiency

The emergence of Omicron in routine wastewater surveillance was accurately reflected using droplet digital (dd)PCR due to a mutation in the N1 probe binding region that caused diminished fluorescence within individual droplets. Wastewater from five cities provided a higher resolution of Omicron emergence when comparing data from sequenced clinical samples.

Effect of Time and Temperature on SARS-CoV-2 in Municipal Wastewater Conveyance Systems

Wastewater surveillance for SARS-CoV-2 is becoming a widespread public health metric, but little is known about pre-analytical influences on these measurements. We examined SARS-CoV-2 loads from two sewer service areas with different travel times that were within the same metropolitan area. Throughout the one-year study, case rates were nearly identical between the two service areas allowing us to compare differences in empirical concentrations relative to conveyance system characteristics and wastewater treatment plant parameters. We found time did not have a significant effect on degradation of SARS-CoV-2 when using average transit times (22 vs. 7.5 h) (p = 0.08), or under low flow conditions when transit times are greater (p = 0.14). Flow increased rather than decreased SARS-CoV-2 case-adjusted concentrations, but this increase was only significant in one service area. Warmer temperatures (16.8–19.8 °C) compared with colder (8.4–12.3 °C) reduced SARS-CoV-2 case-adjusted loads by ~50% in both plants (p < 0.05). Decreased concentrations in warmer temperatures may be an important factor to consider when comparing seasonal dynamics. Oxygen demand and suspended solids had no significant effect on SARS-CoV-2 case-adjusted loads overall. Understanding wastewater conveyance system influences prior to sample collection will improve comparisons of regional or national data for SARS-CoV-2 community infections.

SARS-CoV-2 RNA is enriched by orders of magnitude in primary settled solids relative to liquid wastewater at publicly owned treatment works.

Wastewater-based epidemiology has gained attention throughout the world for detection of SARS-CoV-2 RNA in wastewater to supplement clinical testing. Raw wastewater consists of small particles, or solids, suspended in liquid. Methods have been developed to measure SARS-CoV-2 RNA in the liquid and the solid fraction of wastewater, with some studies reporting higher concentrations in the solid fraction. To investigate this relationship further, six laboratories collaborated to conduct a study across five publicly owned treatment works (POTWs) where both primary settled solids obtained from primary clarifiers and raw wastewater influent samples were collected and quantified for SARS-CoV-2 RNA. Settled solids and influent samples were processed by participating laboratories using their respective methods and retrospectively paired based on date of collection. SARS-CoV-2 RNA concentrations, on a mass equivalent basis, were higher in settled solids than in influent by approximately three orders of magnitude. Concentrations in matched settled solids and influent were positively and significantly correlated at all five POTWs. RNA concentrations in both settled solids and influent were correlated to COVID-19 incidence rates in the sewersheds and thus representative of disease occurrence; the settled solids methods appeared to produce a comparable relationship between SARS-CoV-2 RNA concentration measurements and incidence rates across all POTWs. Settled solids and influent methods showed comparable sensitivity, N gene detection frequency, and calculated empirical incidence rate lower limits. Analysis of settled solids for SARS-CoV-2 RNA has the advantage of using less sample volume to achieve similar sensitivity to influent methods.

Standardizing data reporting in the research community to enhance the utility of open data for SARS-CoV-2 wastewater surveillance.

SARS-CoV-2 RNA detection in wastewater is being rapidly developed and adopted as a public health monitoring tool worldwide. With wastewater surveillance programs being implemented across many different scales and by many different stakeholders, it is critical that data collected and shared are accompanied by an appropriate minimal amount of metainformation to enable meaningful interpretation and use of this new information source and intercomparison across datasets. While some databases are being developed for specific surveillance programs locally, regionally, nationally, and internationally, common globally-adopted data standards have not yet been established within the research community. Establishing such standards will require national and international consensus on what metainformation should accompany SARS-CoV-2 wastewater measurements. To establish a recommendation on minimum information to accompany reporting of SARS-CoV-2 occurrence in wastewater for the research community, the United States National Science Foundation (NSF) Research Coordination Network on Wastewater Surveillance for SARS-CoV-2 hosted a workshop in February 2021 with participants from academia, government agencies, private companies, wastewater utilities, public health laboratories, and research institutes. This report presents the primary two outcomes of the workshop: (i) a recommendation on the set of minimum meta-information that is needed to confidently interpret wastewater SARS-CoV-2 data, and (ii) insights from workshop discussions on how to improve standardization of data reporting.

Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance.

Wastewater surveillance for pathogens using reverse transcription-polymerase chain reaction (RT-PCR) is an effective and resource-efficient tool for gathering community-level public health information, including the incidence of coronavirus disease-19 (COVID-19). Surveillance of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in wastewater can potentially provide an early warning signal of COVID-19 infections in a community. The capacity of the world's environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is increasing rapidly. However, there are no standardized protocols or harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can cause false-positive and false-negative errors in the surveillance of SARS-CoV-2 RNA in wastewater, culminating in recommended strategies that can be implemented to identify and mitigate some of these errors. Recommendations include stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, PCR inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly when the incidence of SARS-CoV-2 in wastewater is low. Corrective and confirmatory actions must be in place for inconclusive results or results diverging from current trends (e.g., initial onset or reemergence of COVID-19 in a community). It is also prudent to perform interlaboratory comparisons to ensure results' reliability and interpretability for prospective and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization and detection for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance continues to be demonstrated during this global crisis. In the future, wastewater should also play an important role in the surveillance of a range of other communicable diseases.

SARS-CoV-2 Wastewater Surveillance for Public Health Action.

Wastewater surveillance for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has garnered extensive public attention during the coronavirus disease pandemic as a proposed complement to existing disease surveillance systems. Over the past year, methods for detection and quantification of SARS-CoV-2 viral RNA in untreated sewage have advanced, and concentrations in wastewater have been shown to correlate with trends in reported cases. Despite the promise of wastewater surveillance, for these measurements to translate into useful public health tools, bridging the communication and knowledge gaps between researchers and public health responders is needed. We describe the key uses, barriers, and applicability of SARS-CoV-2 wastewater surveillance for supporting public health decisions and actions, including establishing ethics consideration for monitoring. Although wastewater surveillance to assess community infections is not a new idea, the coronavirus disease pandemic might be the initiating event to make this emerging public health tool a sustainable nationwide surveillance system, provided that these barriers are addressed.