Wastewater-based epidemiology (WBE) is a useful tool that has the potential to act as a complementary approach to monitor the presence of SARS-CoV-2 in the community and as an early alarm system for COVID-19 outbreak. Many studies reported low concentrations of SARS-CoV-2 in sewage and also revealed the need for methodological validation for enveloped viruses concentration in wastewater. The aim of this study was to evaluate different methodologies for the concentration of viruses in wastewaters and to select and improve an option that maximizes the recovery of SARS-CoV-2. A total of 11 concentration techniques based on different principles were evaluated: adsorption-elution protocols with negatively charged membranes followed by polyethylene glycol (PEG) precipitation (Methods 1-2), PEG precipitation (Methods 3-7), aluminum polychloride (PAC) flocculation (Method 8), ultrafiltration (Method 9), skim milk flocculation (Method 10) and adsorption-elution with negatively charged membrane followed by ultrafiltration (Method 11). To evaluate the performance of these concentration techniques, feline calicivirus (FCV) was used as a process control in order to avoid the risk associated with handling SARS-CoV-2. Two protocols, one based on PEG precipitation and the other on PAC flocculation, showed high efficiency for FCV recovery from wastewater (62.2 % and 45.0 %, respectively). These two methods were then tested for the specific recovery of SARS-CoV-2. Both techniques could recover SARS-CoV-2 from wastewater, PAC flocculation showed a lower limit of detection (4.3 × 10 2 GC/mL) than PEG precipitation (4.3 × 10 3 GC/mL). This work provides a critical overview of current methods used for virus concentration in wastewaters and the analysis of sensitivity for the specific recovery of SARS-CoV-2 in sewage. The data obtained here highlights the viability of WBE for the surveillance of COVID-19 infections in the community.

The effect of yeast concentration on ultraviolet (UV) inactivation of five strains of Escherichia coli O157: H7 from different sources, inoculated both individually and simultaneously in orange juice, was analyzed and mathematically modeled. The presence of yeast cells in orange juice decreases the performance of UV radiation on E. coli inactivation. UV absorption coefficients in the juice increased with increasing yeast concentration, and higher UV doses were necessary to inactivate bacterial strains. UV intensities of I=3.00±0.3 mW/cm 2 and exposure times (t) between 0 and 10 min were applied; radiation doses (energy, E=I×t) ranging between 0 and 2 J/cm 2 were measured using a UV digital radiometer. All the tested individual strains showed higher resistance to the treatment when UV radiation was applied at 4°C in comparison to 20°C. UV inactivation of E. coli O157:H7 individual strain was satisfactory fitted with a first order kinetic model. A linear relationship was found between UV absorptivities and D values (radiation doses required to decrease microbial population by 90%) for each strain. The dose required to reach 5-log reduction for the most unfavorable conditions that is the most UV resistant strain, and maximum background yeast concentration was 2.19 J/cm 2 at 4°C (corresponding to 11 min of UV treatment) and 2.09 J/cm 2 at 20°C (corresponding to 10.55 min of UV treatment). When a cocktail of strains was inoculated in orange juice, the logistic equation was the best model that fits the experimental results due to the deviation from the log-linear kinetics. The UV resistance between strain cocktail and single strain were mathematically compared. Slopes of the decline curves for strain cocktail at high UV doses were lower than the slopes of the log-linear equation calculated for the individual strains, even for the most resistant one. Therefore, microbial inactivation tests using a cocktail of strains are particularly important to determine the performance of the UV inactivation treatment.

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