Longitudinal SARS-CoV-2 RNA wastewater monitoring across a range of scales correlates with total and regional COVID-19 burden in a well-defined urban population

Abstract

Wastewater-based epidemiology (WBE) is an emerging surveillance tool that has been used to monitor the ongoing COVID-19 pandemic by tracking SARS-CoV-2 RNA shed into wastewater. WBE was performed to monitor the occurrence and spread of SARS-CoV-2 from three wastewater treatment plants (WWTP) and six neighborhoods in the city of Calgary, Canada (population 1.44 million). A total of 222 WWTP and 192 neighborhood samples were collected from June 2020 to May 2021, encompassing the end of the first-wave (June 2020), the second-wave (November end to December 2020) and the third-wave of the COVID-19 pandemic (mid-April to May 2021). Flow-weighted 24-hour composite samples were processed to extract RNA that was then analyzed for two SARS-CoV-2-specific regions of the nucleocapsid gene, N1 and N2, using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Using this approach SARS-CoV-2 RNA was detected in 98.06% (406/414) of wastewater samples. SARS-CoV-2 RNA abundance was compared to clinically diagnosed COVID-19 cases organized by the three-digit postal code of affected individuals' primary residences, enabling correlation analysis at neighborhood, WWTP and city-wide scales. Strong correlations were observed between N1 & N2 gene signals in wastewater and new daily cases for WWTPs and neighborhoods. Similarly, when flow rates at Calgary's three WWTPs were used to normalize observed concentrations of SARS-CoV-2 RNA and combine them into a city-wide signal, this was strongly correlated with regionally diagnosed COVID-19 cases and clinical test percent positivity rate. Linked census data demonstrated disproportionate SARS-CoV-2 in wastewater from areas of the city with lower socioeconomic status and more racialized communities. WBE across a range of urban scales was demonstrated to be an effective mechanism of COVID-19 surveillance.

Keywords: COVID-19; Census data; Epidemiological monitoring; Neighborhoods; RT-qPCR; Wastewater-based epidemiology.

Graphical abstract

Fig. 1

Map of Calgary showing the catchment of the three WWTPs and the six targeted neighborhoods included in the study. Catchment area for the three WWTPs (Table 1) are shaded on the map as follows: WWTP-1 is green, WWTP-2 and WWTP-3 are in orange owing to their shared catchment area. Six neighborhoods are shown by the smaller shaded regions, and are designated by quadrant: NE1, NE2, NE3, SE1, SE2 and SW1.

Fig. 2

Time series of SARS-CoV-2 RNA concentration for three different wastewater treatment plants. SARS-CoV-2 RNA signal plotted together with daily number of COVID-19 new cases (rolling 5-day average) categorized by three-digit postal codes of diagnosed individuals’ home addresses (orange line). Data were collected from June 29, 2020 to May 31, 2021 from all three WWTPs. SARS-CoV-2 RNA signals are shown for both N1 (black line) and N2 (red line) assays performed on wastewater samples collected weekly from Calgary's three WWTPs: (A) WWTP-1, (B) WWTP-2 and (C) WWTP-3. Error bars correspond to the standard deviation of the three technical replicates. Calgary's second and third waves of COVID-19 are represented as the vertical dotted lines. Asterisks (*) denote the single sample that was not included in the analysis (i.e., WWTP-1 (15/02/2021)) due to the presence of inhibitors.

Fig. 3

Total Calgary mass flux of SARS-CoV-2 RNA compared to COVID-19 clinically diagnosed cases. (A) Daily dynamics of the mass flux of SARS-CoV-2 RNA signal in entire city of Calgary from June 29, 2020 to May 31, 2021 using the N1 (black dots) and N2 (red dots) assays. COVID-19 new cases (5-day average) and clinical test percent positivity rate are represented as orange and green lines. Calgary's second and third waves of COVID-19 are represented as the blue and gray shaded areas. Vertical dotted lines mark the introduction of new restrictions (NR) on November 24 and December 8, 2020 (stricter restrictions). Some of the restrictions that were implemented during the second wave were partially lifted on February 8, 2021. Further relaxation took place on March 8, 2021. On May 7, 2021, new stricter restrictions (NR*) were once again put in place. Decreases in both the SARS-CoV-2 wastewater signal and clinically diagnosed cases are represented as the purple-shaded areas. Arrow denotes the lowest detectable signal obtained when the daily new clinical cases exceeded 1.14 per 100,000 people. Correlations between flow corrected data of the SARS-CoV-2 N1 (copies/day) and new cases (5 days average) (B), COVID-19 total active (C) and the clinical test percent positivity rate (D). Pearson correlations and 95% prediction intervals (dashed line) on the linear regressions (solid line) are shown in each figure. WWTP: wastewater treatment plant, NR: new restrictions, NR*: stricter new restrictions.

Fig. 4

SARS-CoV-2 RNA concentration in six targeted neighborhoods in Calgary. Daily dynamics of COVID-19 new cases (rolling 5-day average) defined to the three-digit postal code level (orange line) and abundance of SARS-CoV-2 RNA signal based on N1 (black line) and N2 (red line) assays in wastewater samples. Neighborhood names correspond to the geographic quadrant of the city (northeast = NE, etc.): A. NE1, B. NE2 and C. NE3. South side of the city: D. SE1, E. SE2 and F. SW1. The number of new cases for each neighborhood were accounted by postal code data corresponding to clinically identified cases. The sampling period varied between the different neighborhoods, as shown on the x axes: NE1, NE2 and NE3 sites were sampled twice weekly from December 1, 2020 to May 31, 2021; SE1 and SE2 sites were sampled twice weekly from March 8, 2021 to May 31, 2021. SW1 was sampled twice weekly from April 14, 2021 to May 31, 2021. Error bars correspond to the standard deviations of the three technical replicates. Asterisks (*) denotes dates that were not included in the analysis (i.e., NE2 (18/01/2021), NE3 (22/03/2021) and NE3 (19/04/2021)) due to the presence of inhibitors.

Fig. 5

Ethno-demographic and socioeconomic status (SES) indicators for neighborhoods monitored for SARS-CoV-2 in wastewater. Ethno-demographic and SES data for each neighborhood were obtained from 2016 Canadian Census data (Statistics Canada, 2016) using postal codes that corresponded for each neighborhood-level sewershed catchment that was sampled. Family-unit (% multiple-census family households), education-level (% university certificate, diploma or degree at bachelor level or above), immigration status (% immigrant to Canada) and member of a visible minority (% visible minority), B. Income (measured as; average household income) and C. Average household size.

Fig. 6

Correlations between data pairs of new COVID-19 cases organized by three-digit postal codes (5-day average) and SARS-CoV-2 N1 copies/ml in (A) all WWTPs or (B) in all neighborhoods sites. Pearson correlations and 95% prediction intervals (dashed line) on the linear regressions (solid line) are shown in each figure. WWTP: wastewater treatment plant.