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.

Extractionless nucleic acid detection: a high capacity solution to COVID-19 testing.

We describe an extractionless real-time reverse transcriptase-PCR (rRT-PCR) protocol for SARS-CoV-2 nucleic acid detection using heat as an accurate cost-effective high-capacity solution to COVID-19 testing. We present the effect of temperature, transport media, rRT-PCR mastermixes and gene assays on SARS-CoV-2 gene amplification and limits of detection. Utilizing our heated methodology, our limits of detection were 12.5 and 1 genome copy/reaction for singleplex E- and N1-gene assays, respectively, and 1 genome copy/reaction by utilizing an E/N1 or Orf1ab/N1 multiplex assay combination. Using this approach, we detected up to 98% of COVID-19 positive patient samples analyzed in our various cohorts including a significant percentage of weak positives. Importantly, this extractionless approach will allow for >2-fold increase in testing capacity with existing instruments, circumvent the additional need for expensive extraction devices, provide the sensitivity needed for COVID-19 detection and significantly reduce the turn-around time of reporting COVID-19 test results.