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Wastewater surveillance for pathogens using reverse transcription-polymerase chain reaction (RT-PCR) is an effective and resource-efficient tool for gathering additional community-level public health information, including the incidence of coronavirus disease-19 (COVID-19). Surveillance of SARS-CoV-2 in wastewater can 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, culminating in recommended strategies that can be implemented to identify and mitigate these errors. Recommendations include stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, amplification 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.

The immense global burden of infectious disease outbreaks and the need to establish prediction and prevention systems have been recognized by the World Health Organization (WHO), the National Institutes of Health (NIH), the United States Agency of International Development (USAID), the Bill and Melinda Gates Foundation, and the international scientific community. Despite multiple efforts, this infectious burden is still increasing. For example, it has been reported that between 1.5 and 12 million people die each year from waterborne diseases and diarrheal diseases are listed within the top 15 leading causes of death worldwide. Rapid population growth, climate change, natural disasters, immigration, globalization, and the corresponding sanitation and waste management challenges are expected to intensify the problem in the years to come.

Enteric viruses are important pathogens found in contaminated surface waters and have previously been detected in waters of the Great Lakes. Human adenoviruses were monitored because of their high prevalence and persistence in aquatic environments. In this study, we quantified adenoviruses in wastewater, surface water, and combined sewer overflows (CSOs) by real-time PCR. Between August 2005 and August 2006, adenovirus concentrations in raw sewage, primary-treated effluent, secondary-treated effluent, and chlorinated effluent from a wastewater treatment plant in Michigan were examined. CSO samples (n ‫؍‬ 6) were collected from a CSO retention basin in Grand Rapids, MI. Adenoviruses were detected in 100% of wastewater and CSO discharge samples. Average adenovirus DNA concentrations in sewage and CSOs were 1.15 ؋ 10 6 viruses/liter and 5.35 ؋ 10 5 viruses/liter, respectively. Adenovirus removal was <2 log 10 (99%) at the wastewater treatment plant. Adenovirus type 41 (60% of clones), type 12 (29%), type 40 (3%), type 2 (3%), and type 3 (3%) were isolated from raw sewage and primary effluents (n ‫؍‬ 28). Six of 20 surface water samples from recreational parks at the lower Grand River showed virus concentrations above the real-time PCR detection limit (average, 7.8 ؋ 10 3 viruses/liter). This research demonstrates that wastewater effluents and wastewater-impacted surface waters in the lower Grand River in Michigan contain high levels of viruses and may not be suitable for full-body recreational activities. High concentrations of adenovirus in these waters may be due to inefficient removal during wastewater treatment and to the high persistence of these viruses in the environment.

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Increasing antibiotic resistance genes in the environment may pose a threat to public health. In this study, tetracycline and sulfonamide resistance genes (Tet-W, Tet-O, and Sul-I) were quantified in 24 manure samples from three farms and 18 biosolids samples from seven different wastewater treatment plants using quantitative polymerase chain reaction methods. Concentrations of Tet-W and Tet-O genes were observed to be significantly higher (p < 0.05) in manure than in biosolids samples. The background soil samples showed significantly lower concentration of the above genes compared with manure and biosolids. Lime-stabilized biosolids showed significantly (p < 0.05) lower concentration of antibiotic resistance genes compared with other biosolids treatment methods. Elevated levels of antibiotic resistance genes (Tet-W, Tet-O, and Sul-I) were observed in the amended soil samples after the land application of manure or biosolids (Site A) monitored for a period of about 4 mo. However, at another site (Site B), no significant increase (p > 0.05) in concentration of antibiotic resistance genes was observed after biosolids application on soil. Even though the concentration of antibiotic resistance genes in manure was statistically higher than that in biosolids, when they were applied on land, the contribution to the soil depended on the background soil concentration and the soil characteristics. Further study of multiple soil samples in various locations is needed.

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