Challenges in Measuring the Recovery of SARS-CoV-2 from Wastewater

Abstract

Wastewater-based epidemiology is an emerging tool for tracking the spread of SARS-CoV-2 through populations. However, many factors influence recovery and quantification of SARS-CoV-2 from wastewater, complicating data interpretation. Specifically, these factors may differentially affect the measured virus concentration, depending on the laboratory methods used to perform the test. Many laboratories add a proxy virus to wastewater samples to determine losses associated with concentration and extraction of viral RNA. While measuring recovery of a proxy virus is an important process control, in this piece, we describe the caveats and limitations to the interpretation of this control, including that it typically does not account for losses during RNA extraction. We recommend reporting the directly measured concentration data alongside the measured recovery efficiency, rather than attempting to correct the concentration for recovery efficiency. Even though the ability to directly compare SARS-CoV-2 concentrations from different sampling locations determined using different methods is limited, concentration data (uncorrected for recovery) can be useful for public health response.

Tools for interpretation of wastewater SARS-CoV-2 temporal and spatial trends demonstrated with data collected in the San Francisco Bay Area

“…For example, crAssphage is not expected to be a good surrogate for SARS-CoV-2 stability, partitioning, and extraction ( Ye et al, 2016 ), and as a DNA virus, crAssphage may be incompatible with some extraction methods used for SARS-CoV-2 RNA. Other controls (e.g., endogenous biomarkers, recovery controls) and modeling may be applied to improve measurement accuracy and translate results across labs and methods, although there are challenges associated with these corrections ( Kantor et al, 2021b ; Wolfe et al, 2021). Degradation modeling with target-specific decay constants ( Ahmed et al, 2020a ) and sewershed-specific parameters could assist in correcting for degradation or determining sample integrity, but no comprehensive approach for this correction exists, and more work is needed developing these methods.…”

“…BCoV and SOC concentrations were significantly and strongly correlated (τ=0.57, p<0.05). Because it is not possible to independently quantify the surrogate spike without the influence of extraction efficiency ( Kantor et al, 2021b ), extraction controls were used to assess consistency of extractions rather than recovery. Outlier analysis (alpha=0.05) was conducted for BCoV and SOC Cq values using Grubbs test.…”

Wastewater-based estimation of the effective reproductive number of SARS-CoV-2

“…People's behaviours, such as defecation timing outside of a daily routine (39) and/or movement into or out of the catchment (40) can influence Rww estimates, particularly when the number of infected individuals is low. RNA signals may also be impacted during sewer transport, with persistence influenced by environmental conditions (i.e., temperature) and/or sewage composition (i.e., solids content) (41)(42)(43)(44). Furthermore, sample processing required to quantify SARS-CoV-2 RNA may introduce variation, as suggested by substantial day-to-day variation in measurements (13,15,38,45 Briefly, 24-hour flow-proportional composite samples were collected in 500 mL polystyrene or polypropylene plastic bottles, shipped on ice, and stored at 4°C for up to 8 days before processing.…”

Assessing multiplex tiling PCR sequencing approaches for detecting genomic variants of SARS-CoV-2 in municipal wastewater

“…One possible explanation for this finding could be that the sludge matrix was inhibitory to RT-PCR (11); however, no inhibition of RT-qPCR on sludge RNA extracts was detected using internal controls (Text S1, Table S2). Another potential reason for the lower genome coverage in sludge is that SARS-CoV-2 was more nonintact or its RNA more degraded with the direct sludge extraction compared to ultrafiltration of influent wastewater, as has been previously hypothesized (22). A third potential cause of discrepancies in genome coverage between sludge and influent wastewater samples could be higher off-target amplification in sludge extracts.…”