Sunlight photolysis of SARS-CoV-2 N1 gene target in the water environment: considerations for the environmental surveillance of wastewater-impacted surface waters.

Wastewater surveillance of SARS-CoV-2 has been used around the world to supplement clinical testing data for situational awareness of COVID-19 disease trends. Many regions of the world lack centralized wastewater collection and treatment infrastructure, which presents additional considerations for wastewater surveillance of SARS-CoV-2, including environmental decay of the RT-qPCR gene targets used for quantification of SARS-CoV-2 virions. Given the role of sunlight in the environmental decay of RNA, we evaluated sunlight photolysis kinetics of the N1 gene target in heat-inactivated SARS-CoV-2 with a solar simulator under laboratory conditions. Insignificant photolysis of the N1 target was observed in a photosensitizer-free matrix. Conversely, significant decay of the N1 target was observed in wastewater at a shallow depth (<1 cm). Given that sunlight irradiance is affected by several environmental factors, first-order decay rate models were used to evaluate the effect of water column depth, time of the year, and latitude on decay kinetics. Decay rate constants were found to decrease significantly with greater depth of the well-mixed water column, at high latitudes, and in the winter. Therefore, sunlight-mediated decay of the N1 gene target is likely to be minimal, and is unlikely to confound results from wastewater-based epidemiology programs utilizing wastewater-impacted surface waters.

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.

Systematic Review and Meta-Analysis of the Persistence of Enveloped Viruses in Environmental Waters and Wastewater in the Absence of Disinfectants.

Enveloped viruses are characterized by a lipid-containing envelope that encapsulates the virion, and they have been the cause of major outbreaks and pandemics. Some enveloped viruses are excreted in feces and other bodily fluids of infected people and animals, raising the question of their fate in the aquatic environment. Consequently, we conducted a systematic review and meta-analysis of the decay rate constants (k) of enveloped viruses from 12 families (i.e., Coronaviridae, Cystoviridae (specifically Phi6), Filoviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Paramyxoviridae, Pneumoviridae, Poxviridae, Retroviridae, Rhabdoviridae, Togaviridae) in environmental waters and wastewater to evaluate their decay kinetics and identify the environmental and virus characteristics that influence k. A total of 812 k that met inclusion criteria were identified in the literature, with the number of k for each family ranging from 0 to 560, and the virus family averaged values of k ranging from 0.11 d-1 and 1.85 d-1. Virus type (i.e., genus, species, subspecies, or subtype), method of virus enumeration (i.e., culture-based or (RT-)QPCR), and experimental water matrix type, temperature and sterility were found to have significant effects on k. Additionally, enveloped viruses were found to have statistically significantly greater k than nonenveloped viruses. Multiple linear regression models that allow prediction of log10k as a function of virus type, enumeration method, water temperature, and water type are provided for six virus families that had sufficient data available for model fitting (i.e., Coronaviridae, Phi6, Herpesviridae, Orthomyxoviridae, Rhabdoviridae, Togaviridae). Compiled log10k and multiple regression models can be used to inform management of human and animal waste, operation of water and wastewater facilities, and exposure risks to treatment plant workers and communities living in regions that lack treatment facilities. Given limited data available for some enveloped virus families with a potential water-related transmission route, there is need for additional data collection to aid academic researchers, public health agencies, and water and wastewater professionals involved in outbreak response.

Sunlight Photolysis of Extracellular and Intracellular Antibiotic Resistance Genes tetA and sul2 in Photosensitizer-Free Water.

Antibiotic resistance genes (ARGs; the genetic material in bacteria that encode for resistance to antibiotics) have been found in the aquatic environment, raising concerns of an environmental transmission route. In an effort to contribute to models predicting the fate of ARGs in the environment-to design control measures, predict health risks, inform ARG surveillance activities, and prioritize policy interventions-and given the importance of sunlight in damaging DNA, we evaluated the sunlight photolysis kinetics of antibiotic-resistant bacteria (ARB) and ARGs under laboratory conditions, focusing on Escherichia coli SMS-3-5 and its ARGs tetA and sul2. Experiments were conducted in the absence of photosensitizers, and ARG decay rates were quantified by quantitative polymerase chain reaction (qPCR) with short and long amplicon targets. Long amplicon qPCR targets quantified greater photolysis rate constants, due to greater ARG coverage. After a lag phase, intracellular ARG had faster decay rates than extracellular ARG, likely due to the contribution of intracellular indirect photolysis processes. Furthermore, all ARG decay rates were significantly slower than those of E. coli. Decay rate constants and quantum yields are presented as foundational work in the development of models to describe the persistence of ARGs in sunlit, environmental waters.

Systematic review of the relative concentrations of noroviruses and fecal indicator bacteria in wastewater: considerations for use in quantitative microbial risk assessment.

Human noroviruses are a leading cause of food- and water-borne disease, which has led to an interest in quantifying norovirus health risks using quantitative microbial risk assessment (QMRA). Given the limited availability of quantitative norovirus data to input to QMRA models, some studies have applied a conversion factor to estimate norovirus exposure based on measured fecal indicator bacteria (FIB) concentrations. We conducted a review of peer-reviewed publications to identify the concentrations of noroviruses and FIB in raw, secondary-treated, and disinfected wastewater. A meta-analysis was performed to determine the ratios of norovirus-FIB pairs in each wastewater matrix and the variables that significantly impact these ratios. Norovirus-to-FIB ratios were found to be significantly impacted by the norovirus genotype, month of sample collection, geographic location, and the extent of wastewater treatment. Additionally, we evaluated the impact of using a FIB-to-virus conversion factor in QMRA and found that the choice of conversion ratio has a great impact on estimated health risks. For example, the use of a conversion ratio previously used in the World Health Organization Guidelines for the Safe Use of Wastewater, Excreta and Greywater predicted health risks that were significantly lower than those estimated with measured norovirus concentrations used as inputs. This work emphasizes the gold standard of using measured pathogen concentrations directly as inputs to exposure assessment in QMRA. While not encouraged, if one must use a FIB-to-virus conversion ratio to estimate norovirus dose, the ratio should be chosen carefully based on the target microorganisms (i.e., strain, genotype, or class), prevalence of disease, and extent of wastewater treatment.

Global Sensitivity Analysis of Environmental, Water Quality, Photoreactivity, and Engineering Design Parameters in Sunlight Inactivation of Viruses.

Sunlight-mediated inactivation of microorganisms is a low-cost approach to disinfect drinking water and wastewater. The reactions involved are affected by a wide range of factors, and a lack of knowledge about their relative importance makes it challenging to optimize treatment systems. To characterize the relative importance of environmental conditions, photoreactivity, water quality, and engineering design in the sunlight inactivation of viruses, we modeled the inactivation of three-human adenovirus and two bacteriophages-MS2 and phiX174-in surface waters and waste stabilization ponds by integrating solar irradiance and aquatic photochemistry models under uncertainty. Through global sensitivity analyses, we quantitatively apportioned the variability of predicted sunlight inactivation rate constants to different factors. Most variance was associated with the variability in and interactions among time, location, nonpurgeable organic carbon (NPOC) concentration, and pond depth. The photolysis quantum yield of the virus outweighed the seasonal solar motion in the impact on inactivation rates. Further, comparison of simulated sunlight inactivation efficacy in maturation ponds under different design decisions showed that reducing pond depth can increase the log inactivation at the cost of larger land area, but increasing hydraulic retention time by adding ponds in series yielded greater improvements in inactivation.

Modeling the Endogenous Sunlight Inactivation Rates of Laboratory Strain and Wastewater E. coli and Enterococci Using Biological Weighting Functions.

Models that predict sunlight inactivation rates of bacteria are valuable tools for predicting the fate of pathogens in recreational waters and designing natural wastewater treatment systems to meet disinfection goals. We developed biological weighting function (BWF)-based numerical models to estimate the endogenous sunlight inactivation rates of E. coli and enterococci. BWF-based models allow the prediction of inactivation rates under a range of environmental conditions that shift the magnitude or spectral distribution of sunlight irradiance (e.g., different times, latitudes, water absorbances, depth). Separate models were developed for laboratory strain bacteria cultured in the laboratory and indigenous organisms concentrated directly from wastewater. Wastewater bacteria were found to be 5-7 times less susceptible to full-spectrum simulated sunlight than the laboratory bacteria, highlighting the importance of conducting experiments with bacteria sourced directly from wastewater. The inactivation rate models fit experimental data well and were successful in predicting the inactivation rates of wastewater E. coli and enterococci measured in clear marine water by researchers from a different laboratory. Additional research is recommended to develop strategies to account for the effects of elevated water pH on predicted inactivation rates.

Sunlight inactivation of viruses in open-water unit process treatment wetlands: modeling endogenous and exogenous inactivation rates.

Sunlight inactivation is an important mode of disinfection for viruses in surface waters. In constructed wetlands, for example, open-water cells can be used to promote sunlight disinfection and remove pathogenic viruses from wastewater. To aid in the design of these systems, we developed predictive models of virus attenuation that account for endogenous and exogenous sunlight-mediated inactivation mechanisms. Inactivation rate models were developed for two viruses, MS2 and poliovirus type 3; laboratory- and field-scale experiments were conducted to evaluate the models' ability to estimate inactivation rates in a pilot-scale, open-water, unit-process wetland cell. Endogenous inactivation rates were modeled using either photoaction spectra or total, incident UVB irradiance. Exogenous inactivation rates were modeled on the basis of virus susceptibilities to singlet oxygen. Results from both laboratory- and field-scale experiments showed good agreement between measured and modeled inactivation rates. The modeling approach presented here can be applied to any sunlit surface water and utilizes easily measured inputs such as depth, solar irradiance, water matrix absorbance, singlet oxygen concentration, and the virus-specific apparent second-order rate constant with singlet oxygen (k2). Interestingly, the MS2 k2 in the open-water wetland was found to be significantly larger than k2 observed in other waters in previous studies. Examples of how the model can be used to design and optimize natural treatment systems for virus inactivation are provided.

Sunlight inactivation of MS2 coliphage in the absence of photosensitizers: modeling the endogenous inactivation rate using a photoaction spectrum.

The endogenous sunlight inactivation rates of MS2 coliphage in photosensitizer-free water were measured (kobs) under different light conditions and compared to modeled inactivation rates (kmod) computed using a previously published action spectrum. Experiments were conducted under simulated and natural sunlight. There was generally good agreement between modeled and observed MS2 sunlight inactivation rates in the summer and winter, suggesting that the action spectrum can be used to predict changes in the inactivation rate caused by diurnal and seasonal changes in natural sunlight irradiance. However, we show that a major source of uncertainty in the predictions is the ability to accurately measure or model the comparatively weak and highly variable solar irradiance between 280 and 300 nm, a range to which the inactivation rate is very sensitive. The action spectrum was also used to predict the endogenous inactivation rates of MS2 at different depths in a column of strongly humic-colored [i.e., solar ultraviolet (UV)-attenuating] wetland water under simulated sunlight; we observed fairly good agreement between kobs and kmod, suggesting that the action spectrum can be used to estimate the decrease in the endogenous inactivation rate caused by spectrally selective sunlight attenuation in the water column.

Determining wavelength-dependent quantum yields of photodegradation: importance of experimental setup and reference values for actinometers.

Accurate quantum yields are crucial for modeling photochemical reactions in natural and engineered treatment systems. Quantum yields are usually determined using a single representative light source such as xenon lamps to mimic sunlight or UVC light for water treatment. However, photodegradation modeling can be improved by understanding the wavelength dependence of quantum yields and the potential errors introduced by the experimental setup. In this study, we investigated the effects of experimental setup on measured quantum yields using four photoreactor systems and up to 11 different light sources. When using a calibrated spectroradiometer to measure incident irradiance on an open solution surface, apparent quantum yields were up to two times higher if light reflection and light screening were not accounted for in the experimental setup. When the experimental setup was optimized to allow for accurate irradiance measurements, quantum yields were reproducible across photoreactors. The optimized experimental setup was then used to determine quantum yields of uridine, atrazine, p-nitroanisole (PNA), sulfamethoxazole, and diclofenac across the UV spectrum. No significant wavelength dependence of quantum yields was observed for sulfamethoxazole and diclofenac, in contrast to wavelength-dependent quantum yields for uridine, atrazine, and PNA. These reference values can be used for determining wavelength-dependent quantum yields of other compounds of interest. Additionally, more accurate results can be obtained when using (1) an actinometer with similar light absorption and photoreactivity compared to that of the target chemical, (2) optically transparent actinometer solutions that can account for light reflection within reaction vessels, and (3) a quantum yield that corresponds to the spectrum of the selected light source.

Sunlight inactivation of human viruses and bacteriophages in coastal waters containing natural photosensitizers.

Sunlight inactivation of poliovirus type 3 (PV3), adenovirus type 2 (HAdV2), and two bacteriophage (MS2 and PRD1) was investigated in an array of coastal waters to better understand solar inactivation mechanisms and the effect of natural water constituents on observed inactivation rates (k(obs)). Reactor scale inactivation experiments were conducted using a solar simulator, and k(obs) for each virus was measured in a sensitizer-free control and five unfiltered surface water samples collected from different sources. k(obs) values varied between viruses in the same water matrix, and for each virus in different matrices, with PV3 having the fastest and MS2 the slowest k(obs) in all waters. When exposed to full-spectrum sunlight, the presence of photosensitizers increased k(obs) of HAdV2, PRD1 and MS2, but not PV3, which provides evidence that the exogenous sunlight inactivation mechanism, involving damage by exogenously produced reactive intermediates, played a greater role for these viruses. While PV3 inactivation was observed to be dominated by endogenous mechanisms, this may be due to a masking of exogenous k(obs) by significantly faster endogenous k(obs). Results illustrate that differences in water composition can shift absolute and relative inactivation rates of viruses, which has important implications for natural wastewater treatment systems, solar disinfection (SODIS), and the use of indicator organisms for monitoring water quality.

Quantification of human norovirus GII, human adenovirus, and fecal indicator organisms in wastewater used for irrigation in Accra, Ghana.

Quantitative microbial risk assessment (QMRA) is frequently used to estimate health risks associated with wastewater irrigation and requires pathogen concentration estimates as inputs. However, human pathogens, such as viruses, are rarely quantified in water samples, and simple relationships between fecal indicator bacteria and pathogen concentrations are used instead. To provide data that can be used to refine QMRA models of wastewater-fed agriculture in Accra, stream, drain, and waste stabilization pond waters used for irrigation were sampled and analyzed for concentrations of fecal indicator microorganisms (human-specific Bacteroidales, Escherichia coli, enterococci, thermotolerant coliform, and somatic and F+ coliphages) and two human viruses (adenovirus and norovirus genogroup II). E. coli concentrations in all samples exceeded limits suggested by the World Health Organization, and human-specific Bacteroidales was found in all but one sample, suggesting human fecal contamination. Human viruses were detected in 16 out of 20 samples, were quantified in 12, and contained 2-3 orders of magnitude more norovirus than predicted by norovirus to E. coli concentration ratios assumed in recent publications employing indicator-based QMRA. As wastewater irrigation can be beneficial for farmers and municipalities, these results should not discourage water reuse in agriculture, but provide motivation and targets for wastewater treatment before use on farms.

Comparison of enterovirus and adenovirus concentration and enumeration methods in seawater from Southern California, USA and Baja Malibu, Mexico.

Despite being important etiological agents of waterborne illness, the sources, transport and decay of human viruses in recreational waters are not well understood. This study examines enterovirus and adenovirus concentrations in coastal water samples collected from four beaches impacted by microbial pollution: (1) Malibu Lagoon, Malibu; (2) Tijuana River, Imperial Beach; (3) Baja Malibu, Baja California; and (4) Punta Bandera, Baja California. Water samples were concentrated using a flocculation-based skim milk method and dead-end membrane filtration (MF). Viruses were enumerated using cell culture infectivity assays and reverse transcription quantitative polymerase chain reaction (RT-QPCR). Across concentration and quantification methods, enteroviruses were detected more often than adenoviruses. For both viruses, MF followed by (RT)QPCR yielded higher concentrations than skim milk flocculation followed by (RT)QPCR or cell culture assays. Samples concentrated by skim milk flocculation and enumerated by (RT)QPCR agreed more closely with concentrations enumerated by cell culture assays than MF followed by (RT)QPCR. The detection of viruses by MF and (RT)QPCR was positively correlated with the presence of infectious viruses. Further research is needed to determine if detection of viruses by rapid methods such as (RT)QPCR can be a useful water quality monitoring tool to assess health risks in recreational waters.

Effect of pH on endogenous sunlight inactivation rates of laboratory strain and wastewater sourced E. coli and enterococci.

Understanding the influence of environmental factors like pH on solar disinfection in sunlight-dependent wastewater treatment systems can aid in improving their design. Previous research found pH to influence the solar disinfection rates of bacteria in water containing exogenous photosensitizers that facilitate photo-oxidative inactivation. However, limited research has been conducted on the role of external pH on endogenous solar inactivation processes that occur independent of exogenous photosensitizers. As such, we studied the inactivation rates of laboratory-cultured and wastewater-sourced E. coli and enterococci in sensitizer-free matrices with pH ranging from 4 to 10 under full-spectrum and UVB-filtered simulated sunlight. Elevated solar inactivation rates were observed at pH 4 for all bacterial populations evaluated, and at pH 10 for laboratory-cultured and wastewater-sourced E. coli. Dark inactivation was observed at the pH extremes for some bacteria, but did not contribute significantly to the increased inactivation rates observed under simulated sunlight at these pH, except for laboratory-cultured E. coli at pH 10. UVB light was found to play an important role in sunlight inactivation, albeit the contribution of UVB light to solar inactivation observed for Enterococcus spp. diminished at pH 4 and 5, suggesting that indirect endogenous inactivation pathways facilitated by longer wavelength light were enhanced under acidic conditions. Our findings demonstrate that external pH affects the kinetics of endogenous sunlight inactivation processes, and the results have potential to be integrated into models for predicting inactivation kinetics in sunlight-mediated treatment systems that operate over a range of pH conditions.

Assessment of Commonly Measured Wastewater Parameters to Estimate Sewershed Populations for Use in Wastewater-Based Epidemiology: Insights into Population Dynamics in New York City during the COVID-19 Pandemic.

Understanding per capita rates of disease incidence or prevalence from wastewater surveillance data requires an estimate of the population contributing to wastewater samples, given that populations in large urban areas are dynamic, especially if major events, such as the onset of the COVID-19 pandemic, cause large population shifts. To assess whether commonly measured wastewater parameters can be used to estimate sewershed populations, we used wastewater data collected from New York City's (NYC) 14 wastewater treatment facilities to evaluate the relationship between influent loads of four wastewater parameters-ammonia, total Kjeldahl nitrogen, total suspended solids, and five-day carbonaceous biochemical oxygen demand-and census-based population estimates of the corresponding sewersheds during 2019, when populations were assumed to be relatively stable. Ammonia mass load had the most consistent relationship with sewershed population, regardless of wet weather contributions to NYC's predominantly combined sewer system. Changes in ammonia loads due to COVID-19 restrictions enacted in March 2020 generally reflected population shifts in sewersheds serving areas of Manhattan and Brooklyn, for which previous studies report decreased commuter mobility and residential populations. Our findings highlight the utility of ammonia mass load in influent wastewater as a population indicator to normalize wastewater-based epidemiology data and track sewershed population dynamics.

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.

Making waves: Uses of real-time, hyperlocal flood sensor data for emergency management, resiliency planning, and flood impact mitigation.

Flooding is expected to increase due to intensification of extreme precipitation events, sea-level rise, and urbanization. Low-cost water level sensors have the ability to fill a critical data gap on the presence, depth, and duration of street-level floods by measuring flood profiles (i.e., flood stage hydrographs) in real-time with a time interval on the order of minutes. Hyperlocal flood data collected by low-cost sensors have many use cases for a variety of stakeholders including municipal agencies, community members, and researchers. Here we outline examples of potential uses of flood sensor data before, during, and after flood events, based on dialog with stakeholders in New York City. These uses include inputs to predictive flood models, generation of real-time flood alerts for community members and emergency response teams, storm recovery assistance and cataloging of storm impacts, and informing infrastructure design and investment for long-term flood resilience project planning.

Interlaboratory performance and quantitative PCR data acceptance metrics for NIST SRM® 2917.

Surface water quality quantitative polymerase chain reaction (qPCR) technologies are expanding from a subject of research to routine environmental and public health laboratory testing. Readily available, reliable reference material is needed to interpret qPCR measurements, particularly across laboratories. Standard Reference Material® 2917 (NIST SRM® 2917) is a DNA plasmid construct that functions with multiple water quality qPCR assays allowing for estimation of total fecal pollution and identification of key fecal sources. This study investigates SRM 2917 interlaboratory performance based on repeated measures of 12 qPCR assays by 14 laboratories (n = 1008 instrument runs). Using a Bayesian approach, single-instrument run data are combined to generate assay-specific global calibration models allowing for characterization of within- and between-lab variability. Comparable data sets generated by two additional laboratories are used to assess new SRM 2917 data acceptance metrics. SRM 2917 allows for reproducible single-instrument run calibration models across laboratories, regardless of qPCR assay. In addition, global models offer multiple data acceptance metric options that future users can employ to minimize variability, improve comparability of data across laboratories, and increase confidence in qPCR measurements.

Scaling of SARS-CoV-2 RNA in Settled Solids from Multiple Wastewater Treatment Plants to Compare Incidence Rates of Laboratory-Confirmed COVID-19 in Their Sewersheds.

Published and unpublished reports show that SARS-CoV-2 RNA in publicly owned treatment work (POTW) wastewater influent and solids is associated with new COVID-19 cases or incidence in associated sewersheds, but methods for comparing data collected from diverse POTWs to infer information about the relative incidence of laboratory-confirmed COVID-19 cases, and scaling to allow such comparisons, have not been previously established. Here, we show that SARS-CoV-2 N1 and N2 concentrations in solids normalized by concentrations of PMMoV RNA in solids can be used to compare incidence of laboratory confirmed new COVID-19 cases across POTWs. Using data collected at seven POTWs along the United States West Coast, Midwest, and East Coast serving ∼3% of the U.S. population (9 million people), we show that a 1 log change in N gene/PMMoV is associated with a 0.24 (range 0.19 to 0.29) log10 change in incidence of laboratory confirmed COVID-19. Scaling of N1 and N2 by PMMoV is consistent, conceptually, with a mass balance model relating SARS-CoV-2 RNA to the number of infected individuals shedding virus in their stool. This information should support the application of wastewater-based epidemiology to inform the response to the COVID-19 pandemic and potentially future viral pandemics.