Comparison of viral concentration techniques for native fecal indicators and pathogens from wastewater.

Viral pathogens are typically dilute in environmental waters, necessitating a concentration step prior to subsequent quantification or analysis. Historically, studies on viral concentration efficiency have been done by spiking known viruses into the sample; however, spike-in controls may not have the same behavior as "native" viruses exposed to environmental conditions. In this study, four concentration methods, including polyethylene glycol precipitation (PEG), skimmed milk flocculation (SMF), pH drop followed by filtration through a 0.45 µm filter (pH), and centrifugation using an Amicon filter (Amicon), were evaluated to concentrate native viral targets in wastewater. Viral targets included both indicators (crAssphage and pepper mild mottle virus) and pathogens (adenovirus, norovirus GII, human polyomavirus, and SARS-CoV-2) in addition to a bacterial marker (HF183). A non-native spike-in control was also added to compare native and spike-in recoveries. Recovery varied widely across targets and methods, ranging from 0.1 to 39.3 %. The Amicon method was the most broadly effective concentration for recovery efficiency. For the lowest-titer target, the PEG method resulted in the lowest number of non-detections, with 96.7 % positive detections for SARS-CoV-2, compared to 66.7 %, 80 %, and 76.7 % positive detections for SMF, pH, and Amicon, respectively. The non-native spike-ins chosen were only representative of a few native recovery trends, varying by both target and concentration method, and consistently under or over-estimated recovery. Overall, this study suggests the utility of including native targets in viral concentration evaluation and determining the efficiency of concentration methods for a specific target of interest.

Comparison of RT-dPCR and RT-qPCR and the effects of freeze-thaw cycle and glycine release buffer for wastewater SARS-CoV-2 analysis.

Public health efforts to control the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic rely on accurate information on the spread of the disease in the community. Acute and surveillance testing has been primarily used to characterize the extent of the disease. However, obtaining a representative sample of the human population is challenging because of limited testing capacity and incomplete testing compliance. Wastewater-based epidemiology is an agnostic alternative to surveillance testing that provides an average sample from the population served by the treatment facility. We compare the performance of reverse transcription quantitative PCR (RT-qPCR) and reverse transcription digital droplet PCR (RT-dPCR) for analysis of SARS-CoV-2 RNA in a regional wastewater treatment facility in northern Indiana, USA from the earliest stages of the pandemic. 1-L grab samples of wastewater were clarified and concentrated. Nucleic acids were extracted from aliquots and analyzed in parallel using the two methods. Synthetic viral nucleic acids were used for method development and generation of add-in standard-curves. Both methods were highly sensitive in detecting SARS-CoV-2 in wastewater, with detection limits as low as 1 copy per 500 mL wastewater. RT-qPCR and RT-dPCR provided essentially identical coefficients of variation (s/[Formula: see text] = 0.15) for triplicate measurements made on wastewater samples taken on 16 days. We also observed a sevenfold decrease in viral load from a grab sample that was frozen at - 80 °C for 92 days compared to results obtained without freezing. Freezing samples before analysis should be discouraged. Finally, we found that treatment with a glycine release buffer resulted in a fourfold inhibition in RT-qPCR signal; treatment with a glycine release buffer also should be discouraged. Despite their prevalence and convenience in wastewater analysis, glycine release and freezing samples severely and additively (~ tenfold) degraded recovery and detection of SARS-CoV-2.

Inferring SARS-CoV-2 RNA shedding into wastewater relative to the time of infection.

Since the start of the coronavirus disease-2019 (COVID-19) pandemic, there has been interest in using wastewater monitoring as an approach for disease surveillance. A significant uncertainty that would improve the interpretation of wastewater monitoring data is the intensity and timing with which individuals shed RNA from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into wastewater. By combining wastewater and case surveillance data sets from a university campus during a period of heightened surveillance, we inferred that individual shedding of RNA into wastewater peaks on average 6 days (50% uncertainty interval (UI): 6-7; 95% UI: 4-8) following infection, and that wastewater measurements are highly overdispersed [negative binomial dispersion parameter, k = 0.39 (95% credible interval: 0.32-0.48)]. This limits the utility of wastewater surveillance as a leading indicator of secular trends in SARS-CoV-2 transmission during an epidemic, and implies that it could be most useful as an early warning of rising transmission in areas where transmission is low or clinical testing is delayed or of limited capacity.

Making waves: Plausible lead time for wastewater based epidemiology as an early warning system for COVID-19.

Wastewater-based epidemiology (WBE) has emerged as a useful tool in the fight to track and contain COVID-19 spread within communities. One of the motives behind COVID-19 WBE efforts is the potential for 'early warning' of either the onset of disease in a new setting or changes in trends in communities where disease is endemic. Many initial reports of the early warning potential of WBE have relied upon retrospective sample analysis, and delays in WBE analysis and reporting should be considered when evaluating the early warning potential of WBE that enable public health action. Our purpose in this manuscript is to establish a framework to critique the potential of WBE to serve as an early warning system, with special attention to the onset of viral shedding and the differential between results reporting for WBE and clinical testing. While many uncertainties remain regarding both COVID-19 clinical presentation and technical factors influencing WBE results, our analysis suggests at most a modest lead time interval ranging from six days for clinical testing to four days for WBE during community-level wastewater surveillance where clinical testing is accessible on-demand with a rapid time to results. This potential lead time for WBE subsequently increases in settings with limited clinical testing capacity or utilization. Care should be taken when reporting 'early detection' of COVID-19 disease trends via WBE to consider underlying causes (e.g., clinical testing lag or delayed result reporting) to avoid misrepresenting WBE potential.

CrAssphage abundance and correlation with molecular viral markers in Italian wastewater.

Current fecal indicators for environmental health monitoring are primarily based on fecal indicator bacteria (FIB) which do not accurately represent viral pathogens. There is a need for highly abundant, human-associated viral fecal indicators to represent viral pathogens in sewage-contaminated water. In the present study, we evaluate the abundance of the emerging viral fecal indicator crAssphage in 156 Italian wastewater samples collected between 2014 and 2018. Samples were collected using two separate viral concentration methods, glycine-CF and PEG-dextran and qPCR assays were run for crAssphage (CPQ56) and Human Polyomavirus (HPyV) and endpoint PCR assays were run for Human Bocavirus (HBoc) and Hepatitis E Virus (HepE). CrAssphage was detected in 96% of samples and no statistically significant difference was observed in crAssphage abundance between concentration methods (p = 0.39). CrAssphage concentrations also did not correlate with location (latitude) or size (load and capacity) of the wastewater treatment plant. HPyV detection rates with the glycine-CF and PEG-dextran methods were 64% and 100%, respectively, and the concentrations of HPyV were statistically significantly influenced by the concentration method (p < 0.0001). CrAssphage was measured at significantly higher concentrations than HPyV for both concentration methods (p < 0.0001). The observed concentration ranges were 3.84-7.29 log10GC/100 mL for crAssphage and 3.45-5.17 log10GC/100 mL for HPyV. There was a strong positive correlation between crAssphage and HPyV abundance for both concentration methods; however, the slope of the correlation depended on the concentration method. CrAssphage presence correlated with the presence of HBoc in samples concentrated with glycine-CF, but did not correlate with the presence of HBoc concentrated with the PEG-dextran method or with the presence of HepE. Overall, these results demonstrate that crAssphage is an abundant viral fecal indicator in wastewater with statistically significant correlation with human viral pathogens (e.g., HPyV) and viral concentration methods influence the interpretation of fecal viral indicator detection.