The detection and stability of the SARS-CoV-2 RNA biomarkers in wastewater influent in Helsinki, Finland

Hokajarvi, A.-M.; Rytkonen, A.; Tiwari, A.; Kauppinen, A.; Oikarinen, S.; Lehto, K.-M.; Kankaanpaa, A.; Gunnar, T.; Al-Hello, H.; Blomqvist, S.; Miettinen, I. T.; Savolainen-Kopra, C.; Pitkanen, T.

Wastewater-based surveillance of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is used to monitor the population-level prevalence of the COVID-19 disease. In many cases, due to lockdowns or analytical delays, the analysis of wastewater samples might only be possible after prolonged storage. In this study, the effect of storage conditions on the RNA copy numbers of the SARS-CoV-2 virus in wastewater influent was studied over time at cold storage temperatures using the reverse-transcription quantitative PCR (RT-qPCR) assays N2 and E-Sarbeco. For the first time in Finland, quantifiable numbers of SARS-CoV-2 RNA copies were detected in 24 h composite incoming wastewater samples collected from Viikinmaki wastewater treatment plant, Helsinki, Finland. The SARS-CoV-2 RNA counts of the wastewater sample aliquots taken during 19-20 April 2020 and stored for 29, 64, and 84 days remained surprisingly stable at 4{degrees}C, -20{degrees}C, and -75{degrees}C. The SARS-CoV-2 copy numbers in the stored samples seemed slightly higher when analyzed from the pre-centrifugation pellet-that is, the particulate matter of the influent-as compared with the supernatant (i.e., water fraction) used for ultrafiltration, although the difference was not statistically significant. With an N2 assay, on average the sample aliquots contained 3.3 {+/-} 0.18 and 2.9 {+/-} 0.06 SARS-CoV-2 RNA copies (log10 {+/-} SE 100 ml-1) in a pre-centrifugation pellet and supernatant ultrafiltrated, respectively. The corresponding numbers with an E-Sarbeco assay were 3.2 {+/-} 0.24 in the pellet and 2.8 {+/-} 0.12 in supernatant. Furthermore, when wastewater was spiked with SARS-CoV-2, linear decay at 4{degrees}C was observed on the first 28 days, while no decay was visible within 58 days at -20{degrees}C or -75{degrees}C. In conclusion, freezing temperatures should be used for storage when immediate SARS-CoV-2 analysis from the wastewater influent is not possible. Analysis of the particulate matter of the sample, in addition to the water fraction, can improve the detection frequency.