Wastewater SARS-CoV-2 RNA Concentration and Loading Variability from Grab and 24-Hour Composite Samples

The ongoing COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires a significant, coordinated public health response. Assessing case density and spread of infection is critical and relies largely on clinical testing data. However, clinical testing suffers from known limitations, including test availability and a bias towards enumerating only symptomatic individuals. Wastewater-based epidemiology (WBE) has gained widespread support as a potential complement to clinical testing for assessing COVID-19 infections at the community scale. The efficacy of WBE hinges on the ability to accurately characterize SARS-CoV-2 RNA concentrations in wastewater. To date, a variety of sampling schemes have been used without consensus around the appropriateness of grab or composite sampling. Here we address a key WBE knowledge gap by examining the variability of SARS-CoV-2 RNA concentrations in wastewater grab samples collected every 2 hours for 72 hours compared with three corresponding 24-hour flow-weighted composite samples collected over the same period. Results show relatively low variability (respective means for N1, N2, N3 assays = 608, 847.9, 768.4 copies 100 mL^-1, standard deviations = 501.4, 500.3, 505.8 copies 100 mL^-1) for grab sample concentrations, and good agreement between most grab samples and their respective composite (mean deviation from composite = 159 copies 100 mL^-1). When SARS-CoV-2 RNA concentrations are used to calculate viral load (RNA concentration * total influent flow the sample day), the discrepancy between grabs (log₁₀range for all grabs = 11.9) or a grab and its associated 24-hour composite (log₁₀difference = 11.6) are amplified. A similar effect is seen when estimating carrier prevalence in a catchment population with median estimates based on grabs ranging 63-1885 carriers. Findings suggest that grab samples may be sufficient to characterize SARS-CoV-2 RNA concentrations, but additional calculations using these data may be sensitive to grab sample variability and warrant the use of flow-weighted composite sampling. These data inform future WBE work by helping determine the most appropriate sampling scheme and facilitate sharing of datasets between studies via consistent methodology.