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. 2023 Apr;11(2):109623.
doi: 10.1016/j.jece.2023.109623. Epub 2023 Mar 2.

Short-term stability of wastewater samples for storage and shipment in the context of the EU Sewage Sentinel System for SARS-CoV-2

S Tavazzi  1 , C Cacciatori  1 , S Comero  1 , D Fatta-Kassinos  2   3 , P Karaolia  2 , I C Iakovides  2   3 , P Loutsiou  2 , I Gutierrez-Aguirre  4 , Z Lengar  4 , I Bajde  4 , T Tenson  5 , V Kisand  5 , P Laas  5 , K Panksep  5 , H Tammert  5 , G Mariani  1 , H Skejo  1 , B M Gawlik  1
Affiliations

Short-term stability of wastewater samples for storage and shipment in the context of the EU Sewage Sentinel System for SARS-CoV-2

S Tavazzi et al. J Environ Chem Eng. 2023 Apr.

Abstract

In the context of an EU-wide surveillance system for SARS-CoV-2 in wastewater, recommended by the European Commission, this study aims to provide scientific support to the adequacy of transport and storage conditions of samples both in terms of duration and samples temperature. Three laboratories in Slovenia, Cyprus and Estonia investigated the short-term, one-week, isochronous stability of wastewater samples by RT-qPCR based detection of SARS-CoV-2 genes. The results were tested for statistical significance to determine uncertainty of quantification and shelf-life, at testing temperatures of + 20 °C and - 20 °C, relative to reference at + 4 °C. Samples were collected from three urban wastewater treatment plant influents and analysed respectively for SARS-CoV-2 genes N1, N2 (Laboratory 1), N2, E (Laboratory 2) and N3 (Laboratory 3), with various analytical methods. For a period of 7/8 days at + 20 °C, decreasing trends of measured concentrations were observed for all genes resulting in instability according to the statistical analysis, while at - 20 °C the trend of variation was stable only for N1, N2 (Laboratory 1) and N3 (Laboratory 3). Trends for gene E concentrations at - 20 °C (Laboratory 2) could not be tested statistically for stability because of lack of data. Over a period of just 3 days at + 20 °C, the variation was statistically non-significant indicating stability for genes N1, E and N3 for laboratories 1, 2 and 3, respectively. Nonetheless, the outcome of the study presents evidence to support the choice of the selected temperature at which samples shall be preserved during storage before analysis or transport to the laboratory. The conditions (+4 °C, ∼ few days) chosen for EU wastewater surveillance are in accordance with these results, highlighting the importance of stability testing of environmental samples to determine the short-term analytical uncertainty.

Keywords: Isochronous stability testing; SARS-CoV-2; Wastewater samples; Wastewater-based epidemiology.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

ga1
Graphical abstract
Fig. 1
Fig. 1
Isochronous short-term stability designs executed simultaneously at two different testing temperatures. The analysis of all samples was performed at the end of the testing period, as indicated by the arrow.
Fig. 2
Fig. 2
Stability curves of SARS-CoV-2 N1 gene (Laboratory 1) at + 20 °C and − 20 °C.
Fig. 3
Fig. 3
Stability curves of SARS-CoV-2 N2 gene (Laboratory 1) at + 20 °C and − 20 °C.
Fig. 4
Fig. 4
Stability curves of SARS-CoV-2 E gene (Laboratory 2) at + 20 °C and − 20 °C.
Fig. 5
Fig. 5
Stability curves of SARS-CoV-2 N2 gene (Laboratory 2) at + 20 °C and − 20 °C.
Fig. 6
Fig. 6
Stability curves of SARS-CoV-2 N3 gene (Laboratory 3) at + 20 °C and − 20 °C.
See this image and copyright information in PMC

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