Evaluating the impact of sample storage, handling, and technical ability on the decay and recovery of SARS-CoV-2 in wastewater

Abstract

Wastewater based epidemiology (WBE) is useful for tracking and monitoring the level of disease prevalence in a community and has been used extensively to complement clinical testing during the current COVID-19 pandemic. Despite the numerous benefits, sources of variability in sample storage, handling, and processing methods can make WBE data difficult to generalize. We performed an experiment to determine sources of variability in WBE data including the impact of storage time, handling, and processing techniques on the concentration of SARS-CoV-2 in wastewater influent from three wastewater treatment plants (WWTP) in North Carolina over 19 days. The SARS-CoV-2 concentration in influent samples held at 4°C did not degrade significantly over the 19-day experiment. Heat pasteurization did not significantly impact the concentration of SARS-CoV-2 at two of the three WWTP but did reduce viral recovery at the WWTP with the smallest population size served. On each processing date, one filter from each sample was processed immediately while a replicate filter was frozen at -80°C. Once processed, filters previously frozen were found to contain slightly higher concentrations (<0.2 log copies/L) than their immediately processed counterparts, indicating freezing filters is a viable method for delayed quantification and may even improve recovery at WWTP with low viral concentrations. Investigation of factors contributing to variability during sample processing indicated that analyst experience level contributed significantly (p<0.001) to accepted droplet generation while extraction efficiency and reverse transcription efficiency contributed significantly (p<0.05) to day-to-day SARS-CoV-2 variability. This study provides valuable practical information for minimizing decay and/or loss of SARS CoV-2 in wastewater influent while adhering to safety procedures, promoting efficient laboratory workflows, and accounting for sources of variability.

Retrospective Analysis of Wastewater-Based Epidemiology of SARS-CoV-2 in Residences on a Large College Campus: Relationships between Wastewater Outcomes and COVID-19 Cases across Two Semesters with Different COVID-19 Mitigation Policies

“…Some studies have reported significant reductions in the SARS-CoV-2 genetic signal after being frozen from as little as 1 week. 56 , 57 However, other studies reported no significant decrease in the SARS-CoV-2 wastewater signal after being frozen. One such study from Hokajärvi et al reported no visible decal in the genetic signal of SARS-CoV-2 after being stored at −20 or −75 °C after 58 days of storage.…”

“…The effects of storage temperatures and time on the SARS-CoV-2 genetic signal in wastewater have been reported throughout many studies. Some studies have reported significant reductions in the SARS-CoV-2 genetic signal after being frozen from as little as 1 week. , However, other studies reported no significant decrease in the SARS-CoV-2 wastewater signal after being frozen. One such study from Hokajärvi et al.…”

Separating Signal from Noise in Wastewater Data: An Algorithm to Identify Community-Level COVID-19 Surges

“…Wastewater samples collected through NCWMN, based on twice-weekly 24-hour time- or flow-weighted composite sampling at centralized WWTPs, were sent to a UNC laboratory for digital PCR analysis. Detailed UNC wastewater testing methods have been previously published (25).…”

“…The Spearman rank correlation coefficient comparing the average of the N1 and N2 concentrations between the two labs was 0.76, and the ratio of the log-transformed average of the N1 and N2 wastewater viral concentrations ranged from 0.91 to 1. 25…”

Comprative evaluation of Advanced Oxidation Processes (AOPs) for reducing SARS-CoV-2 viral load from campus sewage water

“…The SARS-CoV-2 virus is mainly transmitted through tiny respiratory droplets (Nasir et al, 2021). COVID-19-infected patients, both symptomatic as well as asymptomatic excrete the SARS-CoV-2 virus through faeces and other body secretion (sputum, saliva, urine) that are released via the restroom or lavatory and is introduced into the sewage treatment plant (STP) (Foladori et al, 2022; Gwenzi, 2021; Jalali Milani and Nabi Bidhendi, 2022; Serra-Compte et al, 2021) SARS-CoV-2 RNA can survive in wastewater for around eight days, and it can remain inactive at 4° C for approximately 19 days (Beattie et al, 2022; Wang et al, 2005). Therefore, sewage water from the inlet of STPs can be used to extract SARS-CoV-2 viral RNA, and its presence and quantification can be determined using RT-qPCR.…”