Effect of storage conditions on SARS-CoV-2 RNA quantification in wastewater solids

Abstract

SARS-CoV-2 RNA in wastewater settled solids is associated with COVID-19 incidence in sewersheds and therefore, there is a strong interest in using these measurements to augment traditional disease surveillance methods. A wastewater surveillance program should provide rapid turn around for sample measurements (ideally within 24 hours), but storage of samples is necessary for a variety of reasons including biobanking. Here we investigate how storage of wastewater solids at 4 °C, −20 °C, and −80 °C affects measured concentrations of SARS-CoV-2 RNA. We find that short term (7 or 8 d) storage of raw solids at 4 °C has little effect on measured concentrations of SARS-CoV-2 RNA, whereas longer term storage at 4 °C (35–122 d) or freezing reduces measurements by 60%, on average. We show that normalizing SARS-CoV-2 RNA concentrations by concentrations of pepper mild mottle virus (PMMoV) RNA, an endogenous wastewater virus, can correct for changes during storage as storage can have a similar effect on PMMoV RNA as on SARS-CoV-2 RNA. The reductions in SARS-CoV-2 RNA in solids during freeze thaws is less than those reported for the same target in liquid influent by several authors.

High-Frequency, High-Throughput Quantification of SARS-CoV-2 RNA in Wastewater Settled Solids at Eight Publicly Owned Treatment Works in Northern California Shows Strong Association with COVID-19 Incidence

“…As such, normalizing by PMMoV likely served to adjust for variable recovery. Additionally, recent work by Simpson et al ( 18 ) suggests that normalizing by PMMoV can serve to correct for degradation of SARS-CoV-2 RNA during sample storage.…”

SARS-CoV-2 RNA and N Antigen Quantification via Wastewater at the Campus Level, Building Cluster Level, and Individual-Building Level

“… 6 Wolfe et al 7 showed that normalizing by PMMoV allows for comparison of measurements made using diverse methods as it likely corrects for changes in recovery across the methods. Simpson et al 29 show that normalizing by PMMoV may also aid in controlling for RNA degradation in samples stored for different lengths of time. Therefore, normalizing by PMMoV may be the most useful when comparing measurements across studies or methods and serve as an internal process control.…”

Making waves: Wastewater surveillance of SARS-CoV-2 in an endemic future

“…Furthermore, the lack of clinical viral shedding data from vaccinated (single or multiple doses) and unvaccinated individuals, patients with varied severity of symptoms, as well as across SARS-CoV-2 variants also hampers a wide range of WBE endeavours, including calculating the incidence of infection in sewersheds, estimating the effective reproduction number of SARS-CoV-2 in a population ( Huisman et al, 2021 ) and inferring transmission fitness of VOCs from wastewater data ( Caduff et al, 2021 ). On the other hand, viral concentrations in wastewater are impacted by the transient flow rates, water pH, viral degradation and detention in wastewater lines ( Amoah et al, 2022 ; Khan et al, 2021 ; Simpson et al, 2021 ), further undermining WBS data applications.…”