Normalization of viral loads in Wastewater-Based Epidemiology using routine parameters: One year monitoring of SARS-CoV-2 in urban and tourist sewersheds.

In wastewater-based epidemiology, normalization of experimental data is a crucial aspect, as emerged in the recent surveillance of COVID-19. Normalization facilitates the comparison between different areas or periods, and it helps in evaluating the differences due to the fluctuation of the population due to seasonal employment or tourism. Analysis of biomarkers in wastewater (i.e. drugs, beverage and food compounds, microorganisms such as PMMoV or crAssphage, etc.) is complex to perform, and it is not routinely monitored. This study compares the results of alternative normalization approaches applied to SARS-CoV-2 loads in wastewater using population size calculated with conventional hydraulic and/or chemical parameters (i.e. total suspended solids, chemical oxygen demand, nitrogen forms, etc.) commonly used in the routine monitoring of water quality. A total of 12 wastewater treatment plants were monitored, and 1068 samples of influent wastewater were collected in urban areas and in highly touristic areas (summer and/or winter). The results indicated that both census and population estimated with ammonium are effective and reliable parameters with which to normalize SARS-CoV-2 loads in wastewater from urban sewersheds with negligible fluctuating populations. However, this study reveals that, in the case of tourist locations, the population calculated using NH4-N loads can provide a better normalization of the specific viral load per inhabitant.

Evaluation of concentration procedures, sample pre-treatment, and storage condition for the detection of SARS-CoV-2 in wastewater.

Crucial information on the pandemic's spread has been gathered by monitoring the trend of SARS-CoV-2 in wastewater. This surveillance has highlighted that the initial concentration is a critical step of the analytical procedure due to the low viral titer that may be present in this matrix. This paper presents the results of the evaluation of two different wastewater concentration protocols to determine the most efficient and cost-effective. The two methods tested were the following: (a) a biphasic separation system with PEG-dextran and (b) a PEG/NaCl precipitation protocol. Other aspects of the detection method were also investigated including the influence of storage temperature on virus recovery and the heat treatment of pasteurization, which aims to make samples safer for operators and the environment. The PEG/NaCl precipitation method was found to perform better than the biphasic separation system, allowing for more sensitive identification of the presence of the virus and the detection of a higher viral titer than that identified with the biphasic separation in all results. Storage of the samples at 4.3±0.2°C for up to 3 weeks did not adversely affect the virus titer and the pasteurization pre-treatment increases operator safety and maintains the identification of the viral concentration.

The wave of the SARS-CoV-2 Omicron variant resulted in a rapid spike and decline as highlighted by municipal wastewater surveillance.

This paper highlights the extraordinarily rapid spread of SARS-CoV-2 loads in wastewater that during the Omicron wave in December 2021-February 2022, compared with the profiles acquired in 2020-21 with 410 samples from two wastewater treatment plants (Trento+suburbs, 132,500 inhabitants). Monitoring of SARS-CoV-2 in wastewater focused on: (i) 3 samplings/week and analysis, (ii) normalization to calculate genomic units (GU) inh-1 d-1; (iii) calculation of a 7-day moving average to smooth daily fluctuations; (iv) comparison with the 'current active cases'/100,000 inh progressively affected by the mass vaccination. The time profiles of SARS-CoV-2 in wastewater matched the waves of active cases. In February-April 2021, a viral load of 1.0E＋07 GU inh-1 d - 1 corresponded to 700 active cases/100,000 inh. In July-September 2021, although the low current active cases, sewage revealed an appreciable SARS-CoV-2 circulation (in this period 2.2E＋07 GU inh-1 d-1 corresponded to 90 active cases/100,000 inh). Omicron was not detected in wastewater until mid-December 2021. The Omicron spread caused a 5-6 fold increase of the viral load in two weeks, reaching the highest peak (2.0-2.2E＋08 GU inh-1 d-1 and 4500 active cases/100,000 inh) during the pandemic. In this period, wastewater surveillance anticipated epidemiological data by about 6 days. In winter 2021-22, despite the 4-7 times higher viral loads in wastewater, hospitalizations were 4 times lower than in winter 2020-21 due to the vaccination coverage >80%. The Omicron wave demonstrated that SARS-CoV-2 monitoring of wastewater anticipated epidemiological data, confirming its importance in long-term surveillance.

Coronaviruses and SARS-CoV-2 in sewerage and their removal: Step by step in wastewater treatment plants.

The fate of Coronaviruses (CoVs) and in particular SARS-CoV-2 in wastewater treatment plants (WWTPs) has not been completely understood yet, but an adequate knowledge on the removal performances in WWTPs could help to prevent waterborne transmission of the virus that is still under debate. CoVs and SARS-CoV-2 are discharged from faeces into the sewer network and reach WWTPs within a few hours. This review presents the fate of SARS-CoV-2 and other CoVs in the primary, secondary and tertiary treatments of WWTPs as well as in sludge treatments. The viral loads decrease progressively along with the treatments from 20 to 3.0E+06 GU/L (Genomic Units/L) in the influent wastewater to concentrations below 2.50E+05 GU/L after secondary biological treatments and finally to negative concentrations (below detection limit) in disinfected effluents. Reduction of CoVs is due to (i) natural decay under unfavourable conditions (solids, microorganisms, temperature) for relatively long hydraulic retention times and (ii) processes of sedimentation, filtration, predation, adsorption, disinfection. In primary and secondary settling, due to the hydrophobic properties, a partial accumulation of CoVs may occur in the separated sludge. In secondary treatment (i.e. activated sludge) CoVs and SARS-CoV-2 loads can be reduced only by about one logarithm (∼90%). To enhance this removal, tertiary treatment with ultrafiltration (Membrane Bioreactors) and chemical disinfection or UV light is needed. CoVs and SARS-CoV-2 in the sludge (1.2E+04-4.6E+08 GU/L) can be inactivated significantly in the thermophilic digestion (55 °C), while mesophilic temperatures (33-37 °C) are not efficient. Additional studies are required to investigate the infectivity of SARS-CoV-2 in WWTPs, especially in view of increasing interest in wastewater reclamation and reuse.

SARS-CoV-2 from faeces to wastewater treatment: What do we know? A review.

SARS-CoV-2, the virus that causes COVID-19, has been found in the faeces of infected patients in numerous studies. Stool may remain positive for SARS-CoV-2, even when the respiratory tract becomes negative, and the interaction with the gastrointestinal tract poses a series of questions about wastewater and its treatments. This review aims to understand the viral load of SARS-CoV-2 in faeces and sewage and its fate in wastewater treatment plants (WWTPs). The viral load in the faeces of persons testing positive for SARS-CoV-2 was estimated at between 5·103 to 107.6 copies/mL, depending on the infection course. In the sewerage, faeces undergo dilution and viral load decreases considerably in the wastewater entering a WWTP with a range from 2 copies/100 mL to 3·103 copies/mL, depending on the level of the epidemic. Monitoring of SARS-CoV-2 in sewage, although no evidence of COVID-19 transmission has been found via this route, could be advantageously exploited as an early warning of outbreaks. Preliminary studies on WBE seem promising; but high uncertainty of viral loads in wastewater and faeces remains, and further research is needed. The detection of SARS-CoV-2 in sewage, based on RNA sequences and RT-PCR, requires a shared approach on sample pre-treatment and on-site collection to ensure comparable results. The finding of viral RNA in stools does not imply that the virus is viable and infectious. Viability of CoVs such as SARS-CoV-2 decreases in wastewater - due to temperature, pH, solids, micropollutants - but high inactivation in WWTPs can be obtained only by using disinfection (free chlorine, UVC light). A reduction in the quantity of disinfectants can be obtained by implementing Membrane-Bioreactors with ultrafiltration to separate SARS-CoV-2 virions with a size of 60-140 nm. In sludge treatment, thermophilic digestion is effective, based on the general consensus that CoVs are highly sensitive to increased temperatures.