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-log 10 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-log 10 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.

Wastewater-Based Epidemiology for Community Monitoring of SARS-CoV-2: Progress and Challenges

“…To compare the results between studies, researchers should use a consensus surrogate and a consistent input concentration of the surrogate virus. 43 …”

“…The Canadian Water Network (CWN) also organized an interlaboratory study among eight laboratories. 43 Wastewater samples spiked with low and high levels of gamma-irradiated inactivated SARS-CoV-2 and human coronavirus were provided to the eight laboratories. Overall, all eight laboratories accurately distinguished the high spike (1800 ± 200 gene copies/mL) from the low spike (18 ± 2 gene copies/mL) of inactivated SARS-CoV-2 with a 1.0 log10 range for both low and high spikes excluding nondetects.…”

Monitoring SARS-CoV-2 in sewage: toward sentinels with analytical accuracy

“…They have observed less variation between laboratories when high-spike conditions were compared to the low-spike conditions. The Ct value Chik et al (2021) obtained from analysis of the surrogates in the low-spike samples was not in the linear range of PCR amplification and approached the sensitivity limit. Therefore, it is recommended to: (1) avoid multiple RNA purification steps for reducing loss of the RNA; (2) and run a two-step RT-qPCR analysis, which allow enhancing the resolution.…”

Inferring Numbers of Wild Poliovirus Excretors Using Quantitative Environmental Surveillance

“…When expanding the use of quantitative wastewater-based epidemiology to other viruses such as SARS-CoV-2, it is necessary to determine the analytic uncertainty at each of the following stages: (1) virus shedding into sewers; (2) sample collection; (3) transportation and storage; (4) concentration; (5) quantitative analysis of the virus concentration in wastewater (including determinations of linearity of response, absolute limits of detection (LOD) and quantitation (LOQ), and inter-experiment repeatability over the dynamic range); and (6) normalization and interpretation, including inferring of numbers of infected individuals [ 58 , 59 , 60 , 61 ]. Standardized protocols, laboratory equipment, sample processing strategies, appropriate quality controls, methods for preparing standard curves, and performance limits need to be established for each new pathogen in order to enable inter-laboratory comparisons [ 62 , 63 ]. As for poliovirus surveillance [ 10 ], it is essential to develop explicit performance standards and proficiency testing panels to validate the methods selected to enhance the ability to compare findings between laboratories [ 64 ].…”