Concentration of SARS-CoV-2 from large volumes of raw wastewater is enhanced with the inuvai R180 system (preprint)

Wastewater-based epidemiology (WBE) for severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) is a powerful tool to complement syndromic surveillance: first, as an early-warning system for the spread of the virus in the community, second, to find hotspots of infection, and third, to aid in the early detection and follow-up of circulating virus variants. Although detection of SARS-CoV-2 in raw wastewater may be prompted with good recoveries during periods of high community prevalence, in the early stages of population outbreaks concentration procedures are required to overcome low viral concentrations. Several methods have become available for the recovery of SARS-CoV-2 from raw wastewater, generally involving filtration. However, these methods are limited to small sample volumes, possibly missing the early stages of virus circulation, and restrained applicability across different water matrices. The aim of this study was thus to evaluate the performance of three methods enabling the concentration of SARS-CoV-2 from large volumes of wastewater: i) hollow fiber filtration using the inuvai R180, with an enhanced elution protocol and polyethylene glycol (PEG) precipitation; ii) PEG precipitation; and iii) skimmed milk flocculation. The performance of the three approaches was evaluated in wastewater from multiple wastewater treatment plants (WWTP) with distinct singularities, according to: i) effective volume; ii) percentage of recovery; iii) extraction efficiency; iv) inhibitory effect; and v) the limits of detection and quantification (The inuvai R180 system had the best performance, with detection of spiked controls across all samples, average recovery percentages of 64% for SARS-CoV-2 control and 68% for porcine epidemic diarrhea virus (PEDV), with low variability. The inuvai R180 enables the scalability of volumes without negative impact on the costs, time for analysis, and recovery/inhibition. Moreover, hollow fiber filters favor the concentration of different microbial taxonomic groups. Such combined features make this technology attractive for usage in environmental waters monitoring.

A Wastewater-based Epidemiology tool for COVID-19 Surveillance in Portugal (preprint)

Shedding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in the feces and urine of infected patients and subsequent presence in wastewater has produced interest on the use of this matrix for sentinel surveillance at a community level and as a complementary approach to syndromic surveillance. With this work, we set the foundations for wastewater-based epidemiology (WBE) in Portugal by monitoring the trends of SARS-CoV-2 RNA circulation in the community, on a nationwide perspective during different epidemiological phases of the pandemic. The Charite assays (E_Sarbecco, RdRP, and N_Sarbecco) were applied to monitor, over 32-weeks (April to December 2020), the dynamics of SARS-CoV-2 RNA at the inlet of five wastewater treatment plants (WWTP), which together serve more than two million people in Portugal. Raw wastewater from three COVID-19 reference hospitals was also analyzed during this period. In total, more than 600 samples were tested. Sampling started late April 2020, during lockdown, and, for the first weeks, detection of SARS-CoV-2 RNA was sporadic, with concentrations varying from 103 to 105 genome copies per liter (GC/L). Prevalence of SARS-CoV-2 RNA increased steeply by the end of May into late June, mainly in Lisboa e Vale do Tejo region (LVT), during the reopening phase. After the summer, with the reopening of schools in mid-September and return to partial face-to-face work, a pronounced increase of SARS-CoV-2 RNA in wastewater was detected. In the LVT area, SARS-CoV-2 RNA load agreed with reported trends in hotspots of infection. Synchrony between trends of SARS-CoV-2 RNA in raw wastewater and daily new COVID-19 cases highlights the value of WBE as a surveillance tool for this virus, particularly after the phasing out of the epidemiological curve and when hotspots of disease re-emerge in the population which might be difficult to spot based solely on syndromic surveillance and contact tracing.