Detection and quantification of SARS-CoV-2 RNA in wastewater influent in relation to reported COVID-19 incidence in Finland

Abstract

Wastewater-based surveillance is a cost-effective concept for monitoring COVID-19 pandemics at a population level. Here, SARS-CoV-2 RNA was monitored from a total of 693 wastewater (WW) influent samples from 28 wastewater treatment plants (WWTP, N = 21-42 samples per WWTP) in Finland from August 2020 to May 2021, covering WW of ca. 3.3 million inhabitants (∼ 60% of the Finnish population). Quantity of SARS-CoV-2 RNA fragments in 24 h-composite samples was determined by using the ultrafiltration method followed by nucleic acid extraction and CDC N2 RT-qPCR assay. SARS-CoV-2 RNA signals at each WWTP were compared over time to the numbers of confirmed COVID-19 cases (14-day case incidence rate) in the sewer network area. Over the 10-month surveillance period with an extensive total number of samples, the detection rate of SARS-CoV-2 RNA in WW was 79% (including 6% uncertain results, i.e., amplified only in one out of four, two original and two ten-fold diluted replicates), while only 24% of all samples exhibited gene copy numbers above the quantification limit. The range of the SARS-CoV-2 detection rate in WW varied from 33% (including 10% uncertain results) in Pietarsaari to 100% in Espoo. Only six out of 693 WW samples were positive with SARS-COV-2 RNA when the reported COVID-19 case number from the preceding 14 days was zero. Overall, the 14-day COVID-19 incidence was 7.0, 18, and 36 cases per 100 000 persons within the sewer network area when the probability to detect SARS-CoV-2 RNA in wastewater samples was 50%, 75% and 95%, respectively. The quantification of SARS-CoV-2 RNA required significantly more COVID-19 cases: the quantification rate was 50%, 75%, and 95% when the 14-day incidence was 110, 152, and 223 COVID-19 cases, respectively, per 100 000 persons. Multiple linear regression confirmed the relationship between the COVID-19 incidence and the SARS-CoV-2 RNA quantified in WW at 15 out of 28 WWTPs (overall R² = 0.36, p < 0.001). At four of the 13 WWTPs where a significant relationship was not found, the SARS-CoV-2 RNA remained below the quantification limit during the whole study period. In the five other WWTPs, the sewer coverage was less than 80% of the total population in the area and thus the COVID-19 cases may have been inhabitants from the areas not covered. Based on the results obtained, WW-based surveillance of SARS-CoV-2 could be used as an indicator for local and national COVID-19 incidence trends. Importantly, the determination of SARS-CoV-2 RNA fragments from WW is a powerful and non-invasive public health surveillance measure, independent of possible changes in the clinical testing strategies or in the willingness of individuals to be tested for COVID-19.

Keywords: Community sewage; Coronavirus; National surveillance; RT-qPCR assay; Wastewater-based epidemiology.

Graphical abstract

Fig. 1

Total daily reported COVID-19 cases in the municipalities covered in the national wastewater sample collection (for the list of municipalities, see Table S1).

Fig. 2

Relationship between COVID-19 incidence and the detection and quantification rates of SARS-CoV-2 RNA determined using N2-assay in wastewater (the data points presented in Tables 2 and S6).

Fig. 3

Fourteen-day COVID-19 incidence rate thresholds in the sewer network area covered by national surveillance in Finland required for wastewater-based SARS-CoV-2 detection and quantification estimated with logistic regression (the equations presented in Table S5).

Fig. 4

The trend of SARS-CoV-2 RNA (GC/day/person) in WW samples and COVID-19 incidence (per 100 000 persons). Sample dots with (-) denote SARS-CoV-2 was not detected, (+) denotes that the detection was uncertain, (*) denotes detected, but below quantification limit (LOQ). (A) City areas (Helsinki, Espoo, Turku, Hämeenlinna) with quantification rate (QR) 48–79%. (B) City areas (Jyväskylä, Tampere, Lahti II, Vihti) with QR 30–38%. (C) City areas (Mikkeli, Lahti I, Salo, Vaasa) with QR 18–29%. (D) City areas (Kuopio, Oulu, Rauma, Maarianhamina) with QR 14–17%. (E) City areas (Kokkola, Kouvola, Pietarsaari, Savonlinna) with QR 9–10%. (F) City areas (Kajaani, Kotka, Pori, Lappeenranta) with QR 0–5%. (G) City areas (Rovaniemi, Joensuu, Kemi, Seinäjoki) with QR 0%. Caution: the Y-axis of each graph is different, and the x-axis is the same. Samples with arrival temperature higher than 15 °C (C) and transportation delay more than or equal to three days (D) are shown.