Comparison of approaches to quantify SARS-CoV-2 in wastewater using RT-qPCR: Results and implications from a collaborative inter-laboratory study in Canada

Abstract

Detection of SARS-CoV-2 RNA in wastewater is a promising tool for informing public health decisions during the COVID-19 pandemic. However, approaches for its analysis by use of reverse transcription quantitative polymerase chain reaction (RT-qPCR) are still far from standardized globally. To characterize inter- and intra-laboratory variability among results when using various methods deployed across Canada, aliquots from a real wastewater sample were spiked with surrogates of SARS-CoV-2 (gamma-radiation inactivated SARS-CoV-2 and human coronavirus strain 229E [HCoV-229E]) at low and high levels then provided "blind" to eight laboratories. Concentration estimates reported by individual laboratories were consistently within a 1.0-log₁₀ range for aliquots of the same spiked condition. All laboratories distinguished between low- and high-spikes for both surrogates. As expected, greater variability was observed in the results amongst laboratories than within individual laboratories, but SARS-CoV-2 RNA concentration estimates for each spiked condition remained mostly within 1.0-log₁₀ ranges. The no-spike wastewater aliquots provided yielded non-detects or trace levels (<20 gene copies/mL) of SARS-CoV-2 RNA. Detections appear linked to methods that included or focused on the solids fraction of the wastewater matrix and might represent in-situ SARS-CoV-2 to the wastewater sample. HCoV-229E RNA was not detected in the no-spike aliquots. Overall, all methods yielded comparable results at the conditions tested. Partitioning behavior of SARS-CoV-2 and spiked surrogates in wastewater should be considered to evaluate method effectiveness. A consistent method and laboratory to explore wastewater SARS-CoV-2 temporal trends for a given system, with appropriate quality control protocols and documented in adequate detail should succeed.

Keywords: COVID-19; Public health; Quality assurance; Quality control; Wastewater surveillance.

Graphical abstract

Fig. 1

Estimates of concentrations of SARS-CoV-2 RNA in three wastewater samples with no-spike (WW-N), low-spike (WW-A, 18 ± 2 gene copies/mL) and high-spike (WW-B, 1800 ± 200 gene copies/mL). Concentrations of spikes are denoted by the dashed lines. Non-detects (o), detected but not quantifiable (+), as well as samples not analyzed (◊) are also shown. Laboratories have been anonymized; laboratory A was the coordinating laboratory that was responsible for the preparation and dissemination of the spiked wastewater samples.

Fig. 2

Ct values of SARS-CoV-2 RNA reported for three wastewater samples with no-spike (WW-N), low-spike (WW-A, 18±2 gene copies/mL) and high-spike (WW-B, 1800±200 gene copies/mL). Non-detects (o), detected but not quantifiable (+), as well as samples not analyzed (◊) are also shown.

Fig. 3

Ct values reported for HCoV-229E RNA in three wastewater samples with no-spike (WW-N), low-spike (WW-A, 10 infectious units/mL) and high-spike (WW-B, 1000 infectious units/mL). Non-detects (o), detected, but not quantifiable (+), as well as samples not analyzed (◊) are also shown.

Fig. 4

Differences between Ct values for low-spike and high-spike (i.e. ΔCt) observed for SARS-CoV-2 and HCoV-229E by each laboratory. Error bars represent the standard deviation of the mean ΔCt; the absence of error bars denote cases where no replicates were available. The ΔCt values represent those observed in the supernatant fraction or from the processing of both fractions unless otherwise indicated.

Fig. 5

An example of standard curves generated from a plasmid DNA standard and an RNA standard. In this extreme case, SARS-CoV-2 RNA concentration estimates derived from the use of plasmid DNA standards yielded results that were two orders of magnitude higher than those generated using RNA standards.

Fig. 6

Pepper Mild Mottle Virus RNA concentration estimates observed in the Winnipeg wastewater samples, across all aliquots by three laboratories. Statistics shown are based on log₁₀-concentration estimates. The concentration estimates reflect those observed in the supernatant fraction or from the processing of both fractions unless otherwise indicated.