Development of a large volume concentration method for recovery of coronavirus from wastewater

Abstract

Levels of severe acute respiratory coronavirus type 2 (SARS CoV 2) RNA in wastewater could act as an effective means to monitor coronavirus disease 2019 (COVID-19) within communities. However, current methods used to detect SARS CoV 2 RNA in wastewater are limited in their ability to process sufficient volumes of source material, inhibiting our ability to assess viral load. Typically, viruses are concentrated from large liquid volumes using two stage concentration, primary and secondary. Here, we evaluated a dead-end hollow fiber ultrafilter (D-HFUF) for primary concentration, followed by the CP Select™ for secondary concentration from 2L volumes of primary treated wastewater. Various amendments to each concentration procedure were investigated to optimally recover seeded OC43 (betacoronavirus) from wastewater. During primary concentration, the D-HFUF recovered 69 ± 18% ( n = 29) of spiked OC43 from 2L of wastewater. For secondary concentration, the CP Select™ system using the Wastewater Application settings was capable of processing 100 mL volumes of primary filter eluates in <25 minutes. A hand-driven syringe elution proved to be significantly superior ( p = 0.0299) to the CP Select™ elution for recovering OC43 from filter eluates, 48 ± 2% compared to 31 ± 3%, respectively. For the complete method (primary and secondary concentration combined), the D-HFUF and CP select/syringe elution achieved overall 22 ± 4% recovery of spiked OC43 through ( n = 8) replicate filters. Given the lack of available standardized methodology confounded by the inherent limitations of relying on viral RNA for wastewater surveillance of SARS CoV 2, it is important to acknowledge these challenges when interpreting this data to estimate community infection rates. However, the development of methods that can substantially increase sample volumes will likely allow for reporting of quantifiable viral data for wastewater surveillance, equipping public health officials with information necessary to better estimate community infection rates.

Performance evaluation of virus concentration methods for implementing SARS-CoV-2 wastewater based epidemiology emphasizing quick data turnaround

“…The Cp Select method yielded an average of 29.29% BCoV recovery, which is similar to results reported in other studies using MS2 and OC43 for recovery ( (Forés et al, 2021;McMinn et al, 2021). When comparing the BCoV recovery between the two methods under consideration, the Cp Select method outperformed EMF in terms of both median value and average recovery value, as shown in Figure 1.…”

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

“…Additionally, the filter used in our study has a larger membrane area (1.8 m 2 for inuvai R180 vs 1.5 m 2 for Rexeed S15) and larger fiber inner diameters (220 μm for inuvai R180 vs 185 μm for Rexeed S15). In addition to the optimized elution and secondary concentration protocols, such features might help justify the differences registered in the recovery efficiencies of our study and McMinn et al (2021).…”

“…Gonzalez et al (2020), testing the CDC assay (N1, N2, and N3), reported different theoretical limits of detection depending on the RT-qPCR assay used but the LoD were similar to those obtained in our study. A comparison between the performance of our method (evaluated through LoD and LoQ) and the method reported by McMinn et al (2021) would have been useful, given that the authors have also used hollow-fiber filters for primary concentration, but such parameter information is missing on the former report. In fact, information on LoQ is missing from most publications with very few exceptions, such as LaTurner et al…”

“…In our study, recovery values for PEG precipitation were higher than those reported by Pérez-Cataluña et al (2021) when using similar nucleic acid extraction method (spin column). McMinn et al (2021) developed a method for the recovery of coronavirus from raw wastewater also using hollow fibers as a primary concentration approach, followed by Concentrating Pipette Select TM (CP Select TM ), reporting overall recovery values for human coronavirus OC43 of 22%. Differences in recovery between our study and that of McMinn et al (2021) may be attributed to the filter that used in our study (inuvai R180 vs Rexeed), coupled with an enhanced elution strategy with three steps that we adopted, and/or to the secondary concentration protocol.…”

“…McMinn et al (2021) developed a method for the recovery of coronavirus from raw wastewater also using hollow fibers as a primary concentration approach, followed by Concentrating Pipette Select TM (CP Select TM ), reporting overall recovery values for human coronavirus OC43 of 22%. Differences in recovery between our study and that of McMinn et al (2021) may be attributed to the filter that used in our study (inuvai R180 vs Rexeed), coupled with an enhanced elution strategy with three steps that we adopted, and/or to the secondary concentration protocol. The inuvai R180 filter has a reduced nominal pore size (≤ 5.5 nm with a correspondent cut-off ≤ 18.8 Kda) compared to the Rexeed 15S, which has a more open pore structure.…”