Implementing wastewater surveillance for SARS-CoV-2 on a university campus: Lessons learned.

Wastewater surveillance, also known as wastewater-based epidemiology (WBE), has been successfully used to detect SARS-CoV-2 and other viruses in sewage in many locations in the United States and globally. This includes implementation of the surveillance on college and university campuses. A two-phase study was conducted during the 2020-2021 academic year to test the feasibility of a WBE system on campus and to supplement the clinical COVID-19 testing performed for the student, staff, and faculty body. The primary objective during the Fall 2020 semester was to monitor a large portion of the on-campus population and to obtain an understanding of the spreading of the SARS-CoV-2 virus. The Spring 2021 objective was focused on selected residence halls and groups of residents on campus, as this was more efficient and relevant for an effective follow-up response. Logistical problems and planning oversights initially occurred but were corrected with improved communication and experience. Many lessons were learned, including effective mapping, site planning, communication, personnel organization, and equipment management, and obtained along the way, thereby paving an opportune guide for future planning efforts. PRACTITIONER POINTS: WBE was successful in the detection of many SARS-CoV-2 variants incl. Alpha, Beta, Gamma, Delta, Lambda, Mu, and Omicron. Careful planning and contingencies were essential for a successful implementation of a SARS-CoV-2 monitoring program. A surveillance program may be important for detection and monitoring of other public health relevant targets in wastewater incl. bacteria, viruses, fungi and viruses. Diverse lessons were learned incl. effective mapping, site planning, communication, personnel organization, and equipment management, thereby providing a guide for future planning efforts.

Monkeypox outbreak: Wastewater and environmental surveillance perspective.

Monkeypox disease (MPXD), a viral disease caused by the monkeypox virus (MPXV), is an emerging zoonotic disease endemic in some countries of Central and Western Africa but seldom reported outside the affected region. Since May 2022, MPXD has been reported at least in 74 countries globally, prompting the World Health Organization to declare the MPXD outbreak a Public Health Emergency of International Concern. As of July 24, 2022; 92 % (68/74) of the countries with reported MPXD cases had no historical MPXD case reports. From the One Health perspective, the spread of MPXV in the environment poses a risk not only to humans but also to small mammals and may, ultimately, spread to potent novel host populations. Wastewater-based surveillance (WBS) has been extensively utilized to monitor communicable diseases, particularly during the ongoing COVID-19 pandemic. It helped in monitoring infectious disease caseloads as well as specific viral variants circulating in communities. The detection of MPXV DNA in lesion materials (e.g. skin, vesicle fluid, crusts), skin rashes, and various body fluids, including respiratory and nasal secretions, saliva, urine, feces, and semen of infected individuals, supports the possibility of using WBS as an early proxy for the detection of MPXV infections. WBS of MPXV DNA can be used to monitor MPXV activity/trends in sewerage network areas even before detecting laboratory-confirmed clinical cases within a community. However, several factors affect the detection of MPXV in wastewater including, but not limited to, routes and duration time of virus shedding by infected individuals, infection rates in the relevant affected population, environmental persistence, the processes and analytical sensitivity of the used methods. Further research is needed to identify the key factors that impact the detection of MPXV biomarkers in wastewater and improve the utility of WBS of MPXV as an early warning and monitoring tool for safeguarding human health. In this review, we shortly summarize aspects of the MPXV outbreak relevant to wastewater monitoring and discuss the challenges associated with WBS.

Relationship between SARS-CoV-2 in wastewater and clinical data from five wastewater sheds.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has resulted in a global pandemic starting in 2019 with nearly 500 million confirmed cases as of April 2022. Infection with SARS-CoV-2 is accompanied by shedding of virus in stool, and its presence in wastewater samples has been documented globally. Therefore, monitoring of SARS-CoV-2 in wastewater offers a promising approach to assess the pandemic situation covering pre-symptomatic and asymptomatic cases in areas with limited clinical testing. In this study, the presence of SARS-CoV-2 RNA in wastewater from five wastewater resource recovery facilities (WRRFs), located in two adjacent counties, was investigated and compared with the number of clinical COVID-19 cases during a 2020-2021 outbreak in United States. Statistical correlation analyses of SARS-CoV-2 viral abundance in wastewater and COVID-19 daily vs weekly clinical cases was performed. While a weak correlation on a daily basis was observed, this correlation improved when weekly clinical case data were applied. The viral fecal indicator Pepper Mild Mottle Virus (PMMoV) was furthermore used to assess the effects of normalization and the impact of dilution due to infiltration in the wastewater sheds. Normalization did not improve the correlations with clinical data. However, PMMoV provided important information about infiltration and presence of industrial wastewater discharge in the wastewater sheds. This study showed the utility of WBE to assist in public health responses to COVID-19, emphasizing that routine monitoring of large WRRFs could provide sufficient information for large-scale dynamics.

Correlation between Clinical and Wastewater SARS-CoV-2 Genomic Surveillance, Oregon, USA.

SARS-CoV-2 variant proportions in a population can be estimated through genomic sequencing of clinical specimens or wastewater samples. We demonstrate strong pairwise correlation between statewide variant estimates in Oregon, USA, derived from both methods (correlation coefficient 0.97). Our results provide crucial evidence of the effectiveness of community-level genomic surveillance.

Evaluation of a Wastewater-Based Epidemiological Approach to Estimate the Prevalence of SARS-CoV-2 Infections and the Detection of Viral Variants in Disparate Oregon Communities at City and Neighborhood Scales.

BACKGROUND: Positive correlations have been reported between wastewater SARS-CoV-2 concentrations and a community's burden of infection, disease or both. However, previous studies mostly compared wastewater to clinical case counts or nonrepresentative convenience samples, limiting their quantitative potential. OBJECTIVES: This study examined whether wastewater SARS-CoV-2 concentrations could provide better estimations for SARS-CoV-2 community prevalence than reported cases of COVID-19. In addition, this study tested whether wastewater-based epidemiology methods could identify neighborhood-level COVID-19 hotspots and SARS-CoV-2 variants. METHODS: Community SARS-CoV-2 prevalence was estimated from eight randomized door-to-door nasal swab sampling events in six Oregon communities of disparate size, location, and demography over a 10-month period. Simultaneously, wastewater SARS-CoV-2 concentrations were quantified at each community's wastewater treatment plant and from 22 Newport, Oregon, neighborhoods. SARS-CoV-2 RNA was sequenced from all positive wastewater and nasal swab samples. Clinically reported case counts were obtained from the Oregon Health Authority. RESULTS: Estimated community SARS-CoV-2 prevalence ranged from 8 to 1,687/10,000 persons. Community wastewater SARS-CoV-2 concentrations ranged from 2.9 to 5.1 log10 gene copies per liter. Wastewater SARS-CoV-2 concentrations were more highly correlated (Pearson's r=0.96; R2=0.91) with community prevalence than were clinically reported cases of COVID-19 (Pearson's r=0.85; R2=0.73). Monte Carlo simulations indicated that wastewater SARS-CoV-2 concentrations were significantly better than clinically reported cases at estimating prevalence (p<0.05). In addition, wastewater analyses determined neighborhood-level COVID-19 hot spots and identified SARS-CoV-2 variants (B.1 and B.1.399) at the neighborhood and city scales. DISCUSSION: The greater reliability of wastewater SARS-CoV-2 concentrations over clinically reported case counts was likely due to systematic biases that affect reported case counts, including variations in access to testing and underreporting of asymptomatic cases. With these advantages, combined with scalability and low costs, wastewater-based epidemiology can be a key component in public health surveillance of COVID-19 and other communicable infections. https://doi.org/10.1289/EHP10289.

Detection of SARS-CoV-2 B.1.351 (Beta) Variant through Wastewater Surveillance before Case Detection in a Community, Oregon, USA.

Genomic surveillance has emerged as a critical monitoring tool during the SARS-CoV-2 pandemic. Wastewater surveillance has the potential to identify and track SARS-CoV-2 variants in the community, including emerging variants. We demonstrate the novel use of multilocus sequence typing to identify SARS-CoV-2 variants in wastewater. Using this technique, we observed the emergence of the B.1.351 (Beta) variant in Linn County, Oregon, USA, in wastewater 12 days before this variant was identified in individual clinical specimens. During the study period, we identified 42 B.1.351 clinical specimens that clustered into 3 phylogenetic clades. Eighteen of the 19 clinical specimens and all wastewater B.1.351 specimens from Linn County clustered into clade 1. Our results provide further evidence of the reliability of wastewater surveillance to report localized SARS-CoV-2 sequence information.

Passive sampling to scale wastewater surveillance of infectious disease: Lessons learned from COVID-19.

Much of what is known and theorized concerning passive sampling techniques has been developed considering chemical analytes. Yet, historically, biological analytes, such as Salmonella typhi, have been collected from wastewater via passive sampling with Moore swabs. In response to the COVID-19 pandemic, passive sampling is re-emerging as a promising technique to monitor SARS-CoV-2 RNA in wastewater. Method comparisons and disease surveillance using composite, grab, and passive sampling for SARS-CoV-2 RNA detection have found passive sampling with a variety of materials routinely produced qualitative results superior to grab samples and useful for sub-sewershed surveillance of COVID-19. Among individual studies, SARS-CoV-2 RNA concentrations derived from passive samplers demonstrated heterogeneous correlation with concentrations from paired composite samples ranging from weak (R2 = 0.27, 0.31) to moderate (R2 = 0.59) to strong (R2 = 0.76). Among passive sampler materials, electronegative membranes have shown great promise with linear uptake of SARS-CoV-2 RNA observed for exposure durations of 24 to 48 h and in several cases RNA positivity on par with composite samples. Continuing development of passive sampling methods for the surveillance of infectious diseases via diverse forms of fecal waste should focus on optimizing sampler materials for the efficient uptake and recovery of biological analytes, kit-free extraction, and resource-efficient testing methods capable of rapidly producing qualitative or quantitative data. With such refinements passive sampling could prove to be a fundamental tool for scaling wastewater surveillance of infectious disease, especially among the 1.8 billion persons living in low-resource settings served by non-traditional wastewater collection infrastructure.

Impact of Sampling Type, Frequency, and Scale of the Collection System on SARS-CoV-2 Quantification Fidelity

With the rapid onset of the COVID-19 pandemic, wastewater-based epidemiology sampling methodologies for SARS-CoV-2 were often implemented quickly and may not have considered the unique drainage catchment characteristics. This study assessed the impact of grab versus composite sampling on the detection and quantification of SARS-CoV-2 in four different catchment scales with flow rates ranging from high flow (wastewater treatment plant influent) to medium flow (neighborhood scale) to low-flow (city block scale) to ultralow flow (building scale). At the high-flow site, grab samples were comparable to 24 h composite samples with SARS-CoV-2 detected in all samples and differed in concentration from the composite by <1 log 10 unit. However, as the size of the catchment decreased, the percentage of negative grab samples increased despite all respective composites being positive, and the SARS-CoV-2 concentrations of grab samples varied from those of the composites by up to almost 2 log 10 units. At the ultra-low-flow site, increased sampling frequencies generated composite samples with higher fidelity to the 5 min composite, which is the closest estimate of the true SARS-CoV-2 composite concentration that could be measured. Thus, composite sampling is more likely to compensate for temporal signal variability while grab samples do not, especially as the catchment basin size decreases.

Evaluation of multiple analytical methods for SARS-CoV-2 surveillance in wastewater samples.

In this study, 14 virus concentration protocols based on centrifugation, filtration, polyethylene glycol (PEG) precipitation and ultrafiltration were tested for their efficacy for the quantification of SARS-CoV-2 in wastewater samples. These protocols were paired with four RNA extraction procedures resulting in a combination of 50 unique approaches. Bovine respiratory syncytial virus (BRSV) was used as a process control and seeded in each wastewater sample subjected to all 50 protocols. The recovery of BRSV obtained through the application of 50 unique approaches ranged from <0.03 to 64.7% (±1.6%). Combination of centrifugation as the solid removal step, ultrafiltration (Amicon-UF-15; 100 kDa cut-off; protocol 9) as the primary virus concentration method, and Zymo Quick-RNA extraction kit provided the highest BRSV recovery (64.7 ± 1.6%). To determine the impact of prolonged storage of large wastewater sample volume (900 mL) at -20 °C on enveloped virus decay, the BRSV seeded wastewaters samples were stored at -20 °C up to 110 days and analyzed using the most efficient concentration (protocol 9) and extraction (Zymo Quick-RNA kit) methods. BRSV RNA followed a first-order decay rate (k = 0.04/h with r2 = 0.99) in wastewater. Finally, 21 wastewater influent samples from five wastewater treatment plants (WWTPs) in southern Maryland, USA were analyzed between May to August 2020 to determine SARS-CoV-2 RNA concentrations. SARS-CoV-2 RNA was quantifiable in 17/21 (81%) of the influent wastewater samples with concentration ranging from 1.10 (±0.10) × 104 to 2.38 (±0.16) × 106 gene copies/L. Among the RT-qPCR assays tested, US CDC N1 assay was the most sensitive followed by US CDC N2, E_Sarbeco, and RdRp assays. Data presented in this study may enhance our understanding on the effective concentration and extraction of SARS-CoV-2 from wastewater.

Differentiating between the possibility and probability of SARS-CoV-2 transmission associated with wastewater: empirical evidence is needed to substantiate risk

People infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) shed the virus and its genetic material via their sputum, nasopharyngeal secretions, saliva, urine and feces (Cevik et al.2021). Hence, public health and water quality scientists throughout the world have been monitoring untreated and/or primary treated wastewater and sludge for the surveillance of SARS-CoV-2 in communities (https://arcg.is/1aummW). Numerous reviews have discussed the possibility of SARS-CoV-2 transmission to humans from exposure to wastewater or waters receiving untreated or inadequately treated wastewater based on limited empirical evidence (Adelodun et al. 2020;Bilal et al. 2020;Olusola-Makinde and Reuben 2020;Elsamadony et al. 2021;Khorram-Manesh, Goniewicz and Burkle 2021;Shutler et al. 2021). Multiple transmission routes have been suggested, including waterborne transmission, airborne transmission, contact with contaminated surfaces (fomites) and subsequent touching of mucous membranes such as the mouth, nose, or eyes. Herein, we briefly summarize the empirical evidence pertaining to the transmission of SARS-CoV-2 associated with wastewater exposure.

Variability in RT-qPCR assay parameters indicates unreliable SARS-CoV-2 RNA quantification for wastewater surveillance.

Due to the coronavirus disease 2019 (COVID-19) pandemic, wastewater surveillance has become an important tool for monitoring the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within communities. In particular, reverse transcription-quantitative PCR (RT-qPCR) has been used to generate large datasets aimed at detecting and quantifying SARS-CoV-2 RNA in wastewater. Although RT-qPCR is rapid and sensitive, there is no standard method yet, there are no certified quantification standards, and experiments are conducted using different assays, reagents, instruments, and data analysis protocols. These variations can induce errors in quantitative data reports, thereby potentially misleading interpretations, and conclusions. We review the SARS-CoV-2 wastewater surveillance literature focusing on variability of RT-qPCR data as revealed by inconsistent standard curves and associated parameters. We find that variation in these parameters and deviations from best practices, as described in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines suggest a frequent lack of reproducibility and reliability in quantitative measurements of SARS-CoV-2 RNA in wastewater.