Wild type and variants of SARS-COV-2 in Parisian sewage: presence in raw water and through processes in wastewater treatment plants.

The presence of SARS-CoV-2 RNA has been extensively reported at the influent of wastewater treatment plants (WWTPs) worldwide, and its monitoring has been proposed as a potential surveillance tool to early alert of epidemic outbreaks. However, the fate of the SARS-CoV-2 RNA in the treatment process of WWTP has not been widely studied yet; therefore, in this study, we aimed to evaluate the efficiency of treatment processes in reducing SARS-CoV-2 RNA levels in wastewater. The treatment process of three WWTPs of the Parisian area in France was monitored on six different weeks over a period of 2 months (from April 14 to June 9, 2021). SARS-CoV-2 RNA copies were detected using digital polymerase chain reaction (dPCR). Investigation on the presence of variants of concern (Del69-70, E484K, and L452R) was also performed. Additionally, SARS-CoV-2 RNA loads in the WWTPs influents were expressed as the viral concentration in per population equivalent (PE) and showed a good correlation with French public health indicators (incidence rate). SARS-CoV-2 RNA loads were notably reduced along the water treatment lines of the three WWTPs studied (2.5-3.4 log reduction). Finally, very low SARS-CoV-2 RNA loads were detected in effluents (non-detected in over half of the samples) which indicated that the potential risk of the release of wastewater effluents to the environment is probably insignificant, in the case of WWTPs enabling an efficient biological removal of nitrogen.

Fate of SARS-CoV-2 coronavirus in wastewater treatment sludge during storage and thermophilic anaerobic digestion.

Since the COVID-19 outbreak has started in late 2019, SARS-CoV-2 has been widely detected in human stools and in urban wastewater. No infectious SARS-CoV-2 particles have been detected in raw wastewater until now, but it has been reported occasionally in human stools. This has raised questions on the fate of SARS-CoV-2 during wastewater treatment and notably in its end-product, wastewater treatment sludge, which is classically valorized by land spreading for agricultural amendment. In the present work, we focused on SARS-CoV-2 stability in wastewater treatment sludge, either during storage (4 °C, room temperature) or thermophilic anaerobic digestion (50 °C). Anaerobic digestion is one of the possible processes for sludge valorization. Experiments were conducted in laboratory pilots; SARS-CoV-2 detection was based on RT-quantitative PCR or RT-digital droplet PCR. In addition to SARS-CoV-2, Bovine Coronavirus (BCoV) particles were used as surrogate virus. The RNA from SARS-CoV-2 particles, inactivated or not, was close to the detection limit but stable in wastewater treatment sludge, over the whole duration of the assays at 4 °C (55 days) and at ambient temperature (∼20 °C, 25 days). By contrast, the RNA levels of BCoV and inactivated SARS-CoV-2 particles decreased rapidly during the thermophilic anaerobic digestion of wastewater treatment sludge lasting for 5 days, with final levels that were close to the detection limit. Although the particles' infectivity was not assessed, these results suggest that thermophilic anaerobic digestion is a suitable process for sludge sanitation, consistent with previous knowledge on other coronaviruses.

From Alpha to Omicron BA.2: New digital RT-PCR approach and challenges for SARS-CoV-2 VOC monitoring and normalization of variant dynamics in wastewater.

Throughout the COVID-19 pandemic, new variants have continuously emerged and spread in populations. Among these, variants of concern (VOC) have been the main culprits of successive epidemic waves, due to their transmissibility, pathogenicity or ability to escape the immune response. Quantification of the SARS-CoV-2 genomes in raw wastewater is a reliable approach well-described and widely deployed worldwide to monitor the spread of SARS-CoV-2 in human populations connected to sewage systems. Discrimination of VOCs in wastewater is also a major issue and can be achieved by genome sequencing or by detection of specific mutations suggesting the presence of VOCs. This study aimed to date the emergence of these VOCs (from Alpha to Omicron BA.2) by monitoring wastewater from the greater Paris area, France, but also to model the propagation dynamics of these VOCs and to characterize the replacement kinetics of the prevalent populations. These dynamics were compared to various individual-centered public health data, such as regional incidence and the proportions of VOCs identified by sequencing of strains isolated from patient. The viral dynamics in wastewater highlighted the impact of the vaccination strategy on the viral circulation within human populations but also suggested its potential effect on the selection of variants most likely to be propagated in immunized populations. Normalization of concentrations to capture population movements appeared statistically more reliable using variations in local drinking water consumption rather than using PMMoV concentrations because PMMoV fecal shedding was subject to variability and was not sufficiently relevant in this study. The dynamics of viral spread was observed earlier (about 13 days on the wave related to Omicron VOC) in raw wastewater than the regional incidence alerting to a possible risk of decorrelation between incidence and actual virus circulation probably resulting from a lower severity of infection in vaccinated populations.