Alternate: Comparison of two rapid virus concentration methods for implementing SARS-CoV-2 Wastewater-based epidemiology

Wastewater based epidemiology (WBE) has drawn significant attention as an early warning tool to detect and predict the trajectory of COVID-19 cases in a community as it can provide evidence of both symptomatic and asymptomatic COVID-19 cases. However, precise and accurate viral copies quantification in wastewater is a prerequisite for making that tool successful. This ultimately is dependent on the effective and reliable virus concentration method prior to RNA extraction and quantification. Virus concentration is crucial in the wastewater especially when viral titers are very low, as is seen in building-based surveillance (Corchis-Scott et al., 2021; Gibas et al., 2021). Polyethylene glycol (PEG)-based precipitation, Electronegative Membrane Filtration (EMF), and Ultrafiltration are the popular methods that are being used to concentrate virus with successful signal detection (La Rosa et al., 2020; Wu et al., 2020; Kumar et al., 2020). However, in the context of congregate living facilities such as university residence halls, school rapid virus concentration method is necessary for faster data reporting. We previously reported outcomes of SARS-CoV-2 building level surveillance for a large urban college campus during Fall 2020 using EMF as the method of concentration (Gibas et al., 2021). However, to shorten the timeline from sample collection to reporting, we have tested an alternative concentration method using the InnovaPrep CP Select concentrator (https://www.innovaprep.com). We aimed to determine how the optimized CP Select protocol performs compared to the established EMF method in terms of filtration time, external control recovery, and sensitivity of SARS-CoV-2 detection and quantification. Method Wastewater samples was processed using the Innovaprep Cp Select method and EMF method side by side. Bovine Coronavirus or BCoV (BOVILIS® Coronavirus, Merck Animal Health, NE, USA), a surrogate of human coronavirus, was spiked into the wastewater as a process control prior to sample concentration. For sample processing with the CP Select concentrator, wastewater samples were centrifuged for 10 mins at 10000xg to remove solid debris. 10% Tween-20 was added to the supernatant in a ratio of 1:100 before concentration. 40 to 150 mL samples were then filtered through a single use 0.05 PS Hollow Fiber Filter Tips (InnovaPrep) using the automatic CP Select (InnovaPrep). Viral particles attached to the filter tips were recovered by eluting with 0.075% Tween-20/Tris elution fluid using Wet Foam Elution technology (InnovaPrep) into a final volume ranging from 250 uL to 500 uL. Conventional EMF or HA method was followed as previously described in Gibas et al., (2021). Following the EMF or CP Select concentration step, we then used the QIAamp viral mini kit (Qiagen, Valencia, CA, USA) for RNA extraction from 200 uL of concentrated sample. To determine whether a significant amount of virus remained in the pellets following centrifugation step, we quantified recovery of BCoV and natural SARS-CoV-2 from both the pellet and the supernatant of centrifuged samples. As part of the optimization of the Cp Select protocol we tested the sonication step addition prior to the centrifugation step and AVL lysis buffer (Qiagen) addition to the eluted concentrated sample to see the virus recovery performance. Quantitative reverse transcription PCR (RT-qPCR) was used to detect and quantify SARS-CoV-2 using CDC recommended N1 (Nucleocapsid) primer and probe set (Corman et al., 2020) and Bovine Coronavirus a primer/probe set published by Decaro et al., (2008). The detail of the RT-qPCR protocol was discussed in Gibas et al., (2021). Result The CP Select method resulted in a BCoV recovery rate of approximately 37%, which is higher than BCoV recovery from samples processed using an EMF protocol. The CP Select is capable of processing up to 150 mL of wastewater within 30 min, while the EMF method fails at larger volumes and operates optimally with 40 mL input. This allows for a higher effective volume of wastewater to be assayed with the CP Select relative to EMF, which in turn results in increased sensitivity for detection and quantification of SARS-CoV-2 from wastewater. About 25% of samples that tested negative when concentrated with the EMF method produced a positive signal with the CP Select protocol. Virus partitioning result indicated that a significantly smaller fraction of BCoV was recovered from the pellet than from the supernatant, with a P-value of 0.015 (P < 0.05). However, SARS-CoV-2 behaved differently from BCoV in centrifugation, with similar recovery fractions in the supernatant and the pellet (P value of 0.857). This difference may be due to the viral structure itself; the structure of the spike protein may result in SARS-CoV-2 attaching more strongly to a solid surface compared to BCoV. Ai et al. (2021). The optimization of the CP Select protocol by adding AVL lysis buffer significantly improved the virus recovery performance. Sonication step increased Bovine Coronavirus (BCoV) recovery by 19%, which seems to compensate for viral loss during centrifugation. Inhibition to RT-qPCR was not found for both of the method. Filtration time decreases by approximately 30% when using the CP Select protocol, making this an optimal choice for building surveillance applications where quick turnaround time is necessary. In general, the CP Select concentrator is advantageous for concentrating low viral titer wastewater samples, especially when rapid data reporting is necessary, and use of this protocol can also improve recovery and detection sensitivity.