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

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 2 L 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 2 L 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 min. 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.