Long-term SARS-CoV-2 surveillance in the wastewater of Stockholm: What lessons can be learned from the Swedish perspective?

Abstract

Wastewater-based epidemiology (WBE) can be used to track the spread of SARS-CoV-2 in a population. This study presents the learning outcomes from over two-year long monitoring of SARS-CoV-2 in Stockholm, Sweden. The three main wastewater treatment plants in Stockholm, with a total of six inlets, were monitored from April 2020 until June 2022 (in total 600 samples). This spans five major SARS-CoV-2 waves, where WBE data provided early warning signals for each wave. Further, the measured SARS-CoV-2 content in the wastewater correlated significantly with the level of positive COVID-19 tests (r = 0.86; p << 0.0001) measured by widespread testing of the population. Moreover, as a proof-of-concept, six SARS-CoV-2 variants of concern were monitored using hpPCR assay, demonstrating that variants can be traced through wastewater monitoring. During this long-term surveillance, two sampling protocols, two RNA concentration/extraction methods, two calculation approaches, and normalization to the RNA virus Pepper mild mottle virus (PMMoV) were evaluated. In addition, a study of storage conditions was performed, demonstrating that the decay of viral RNA was significantly reduced upon the addition of glycerol to the wastewater before storage at -80 °C. Our results provide valuable information that can facilitate the incorporation of WBE as a prediction tool for possible future outbreaks of SARS-CoV-2 and preparations for future pandemics.

Keywords: COVID-19; Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); Sewage surveillance; Storage conditions; Wastewater-based epidemiology (WBE); hpPCR.

Graphical abstract

Fig. 1

Long-term monitoring of SARS-CoV-2 in the Stockholm region. Three major plants in Stockholm were monitored, Käppala WWTP (700,000 inhabitants, purple bar), Bromma WWTP (377,500 inhabitants, pink bar) and Henriskdal WWTP (862,100 inhabitants, light-blue bar). SARS-CoV-2 content was expressed as the Total N-gene copy number per week, which has been normalized with PMMoV content (using PMMoV factor) and flow rate. Two or three biological replicates were analyzed for each data point. For the same monitoring period, the positive case numbers in Stockholm based on laboratory-confirmed PCR (yellow line/red dots) are plotted as well as the estimated case numbers based on SARS-CoV-2 measurements in wastewater (black interrupted line/green dots). Grey areas in the figure indicate the different waves in Stockholm during the monitoring of SARS-CoV-2. (A) SARS-CoV-2 measurements during the whole monitoring period (week 16, April 2020 to week 26, June 2022) (B) Zooming in on waves 1 and 2, reported data from week 16, 2020 until week 3, 2021. Two RNA methods and two sampling protocols were applied. In Fig. 1A, we present in which week the second protocol was applied in each case. In the samples denoted with ** in Fig. 1A, the Sickla inlet was not analyzed. Samples denoted with * in Fig. 1B were kept at −20 °C before analysis.

Fig. 2

Correlation and simple linear regression between the clinical positive Covid-19 cases and estimated positive Covid-19 cases in Stockholm. The statistical analysis was performed using estimated positive cases and clinical positive cases from week 16, 2020 until week 6, 2022. The Pearson correlation coefficient (r) and the r-squared coefficient (r²) of the linear regression are presented in the figure.

Fig. 3

Detection of SARS-CoV-2 variants of concern using hpPCR method. The variants of concern analyzed were Alpha (Al), Beta (Be), Delta (De), Gamma (Ga), Kappa (Ka), and Omicron (Om) BA.1, BA.2, BA.4, BA.5 and BA.2.12.1. Delta (green line), Omicron BA.1 (pink line) and Omicron BA.2 (brown line) were detected. From a total of 104-week samples from Stockholm, 36 representative samples were analyzed, covering the main five waves. The results presented in the figure are the median of three replicates.

Fig. 4

Surveillance of SARS-CoV-2 in Käppala WWTP. (A) SARS-CoV-2 content was expressed as the Total N-gene copy number per week, which has been normalized using the PMMoV-factor approach and flow rate. (B) SARS-CoV-2 content was expressed as N-gene copy number per PMMoV gene copy number x10⁴, which has been normalized using the N3/PMMoV approach and flow rate. For the same monitoring period, the positive case numbers based on laboratory-confirmed PCR (yellow line/red dots) in the Käppala area are plotted. The standard deviation was calculated from two or three biological replicates.

Fig. 5

Surveillance of SARS-CoV-2 in Henriksdal WWTP. SARS-CoV-2 content in Henriksdal WWTP was the sum of the Sickla inlet (blue bar) and Henriksdal inlet (light-blue bar). SARS-CoV-2 content was expressed as the Total N-gene copy number per week, which has been normalized using the PMMoV-factor approach and flow rate. (*) No Sickla sample from week 22 to week 26, 2022. Two or three biological replicates were analyzed for each data point. For the same monitoring period, the positive case numbers based on laboratory-confirmed PCR (yellow line/red dots) in the Sickla and Henriksdal areas are plotted.

Fig. 6

Surveillance of SARS-CoV-2 in Bromma WWTP. SARS-CoV-2 content in Bromma WWTP was the sum of Riksby inlet (pink bar), Järva (dark red, bar) and Hässelby inlet (fuchsia bar). SARS-CoV-2 content was expressed as the Total N-gene copy number per week, which has been normalized using the PMMoV-factor approach and flow rate. Two or three biological replicates were analyzed for each data point. For the same monitoring period, the positive case numbers based on laboratory-confirmed PCR (yellow line/red dots) in the Riksby, Järva and Hässelby areas are plotted.

Fig. 7

N-gene and PMMoV copy numbers under different storage conditions. Samples were analyzed after 1 day (week 0, fresh sample), 1 week, 18 weeks and 24 weeks. Three temperature conditions are shown in the figure: 4 °C, −80 °C without the addition of glycerol (no glycerol) and −80 °C with the addition of glycerol. C1: one freeze-thaw cycle, C2: two freeze-thaw cycles, C3: three freeze-thaw cycles and C4: four freeze-thaw cycles. Three biological replicates were analyzed for each data point.