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

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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.

Introduction

During the COVID-19 pandemic, Wastewater-Based Epidemiology (WBE) has emerged as an important source of additional information to predict and understand the spreading trend of SARS-CoV-2 within the population [1]. The first pan-European Sewage Sentinel System Surveillance was set up by the European Commission to assess the technical and economic feasibility of monitoring the spread of the SARS-CoV-2 virus in wastewater [2]. Since the beginning of the pandemic, several Member States and many countries outside the EU established their own wastewater national surveillance schemes, anticipating the invitation formalised in the European Commission Recommendation of March 2021, which asked Member States (MS) to systematically roll out sewage surveillance and collaborate with the Commission in the building of a so-called EU Sewage Sentinel System for SARS-CoV-2 (EU4S) [3]. Furthermore, the Recommendation declared that:“…putting in place the recommended surveillance system and procedures will also have an added value beyond SARS-CoV-2 surveillance [.]”, since “[.] it will provide an early warning for future possible outbreaks of other pathogens of concern or threats from other pollutants of emerging concern [.]”.

Such outcomes assert the global-scale recognition that WBE can be implemented as an additional support tool to classical epidemiological parameters in monitoring the state of viral outbreaks, in the detection of variants of concern, and in the evaluation of the spread of viral infections within a population [4], [5], [6].

With this outlook, methods’ harmonization and comparability are fundamental to guarantee absence of systematic bias and validity of data obtained from detection of SARS-CoV-2 in wastewater, if those are to be used as a support tool for decision making by water and health authorities. The need for definition of common procedures and mechanisms of collection and handling, processing and analysis of wastewater samples has been widely touched upon in the literature of the past two years [7], [8].

Among the wastewater sample handling procedures for screening for SARS-CoV-2, lack of consistency has been identified regarding the storing temperature of wastewater samples, which can have consequences on detection results of SARS-CoV-2 viral loads [9]. While studies on the influence of temperature on other enveloped viruses in wastewater have identified storage at + 4 °C as appropriate for a short-period of time (1–5 days) [10], SARS-CoV-2 wastewater samples have been kept at + 4 °C, − 20 °C and − 80 °C before analysis [9], [11] and transported at + 4 °C [12], with little information on their stability. The results of a survey on the methodologies applied to wastewater samples for quantification of SARS-CoV-2 by participants of the Pan-European Sewage Sentinel System confirmed variability of storing conditions (+4 °C, <10 °C and −20 °C) that were applied by the respondents [2].

A study by Ahmed et al. (2020) quantified the decay rate of SARS-CoV-2 RNA and of murine hepatitis virus (MHV) in different water matrices, including untreated wastewater, at storing temperatures + 4 °C, + 15 °C, + 25 °C, + 37 °C [10]. The results confirmed 1-log decay in 27.8 ± 4.5 at + 4 °C, while storage at higher temperatures (+25 °C and +37 °C) was discouraged. Markt et al., although using another surrogate (BRSV), partly supported such findings, by comparing composite samples of raw influent wastewater of two different waste water treatment plants (WWTPs) in Austria, stored at + 4 °C for up to 9 days, and − 20 °C for up to 3 days [13]. Besides variability of detected N gene values due to differences in sampled wastewater characteristics, no significant degradation was reported at + 4 °C, while freezing lead to a decreased signal [13]. While these studies are important to support the choice of proper storage temperature, more investigation is required to assess the impacts of varied transport conditions on SARS-CoV-2 concentrations in the sampled wastewater, to harmonize the WBE sampling process. Indeed, the EU-wide WBE exercise involved various sampling locations around Europe, from which wastewater samples were transported to a central analytical facility. Logistics and transport conditions, rather than storage, thus become objective of investigation. As recognized by Gawlik et al. (2012), in case of EU-wide monitoring exercises, it becomes essential to provide information on the short-term transport stability of samples, not only qualitatively, as indicative stability over time, but also quantitatively, as a defined expiry date or shelf life [14].

The isochronous stability exercise (wastewater samples storage at different temperatures for different times) described in this paper thus aims at deepening the understanding of the effect of temperature on SARS-CoV-2 concentration in wastewater during handling and to support the development of homogeneous and comparable operative procedures for national and international wastewater surveillance schemes. The exercise, following Gawlik et al. (2012) study design and statistics, involved three research laboratories, namely the National Institute of Biology, Slovenia, Nireas-International Water Research Centre of the University of Cyprus, Cyprus, and the University of Tartu, Estonia, referred to as Laboratory 1, 2 and 3, respectively, throughout the paper. This study allowed the assessment of short-term stability results relative to the handling of different wastewater samples under specific reproducibility conditions, in an experimental design where concentration methods and targeted genes varied.

Section snippets

Statistics

The stability assessment was done according to an isochronous stability study scheme in collected influent wastewater samples, in which measurements are performed at the same time at the end of the experiment [15]. The testing scheme was designed according to the research developed previously by Gawlik at et al. (2012) and Linsinger et al. (2001).

In an isochronous stability study, the stability and related uncertainty of quantification are based on regression analysis of concentration

Results

The results obtained in the isochronous stability study are presented in Figs. 2–6, for each of the participating laboratories. The graphs show, for the whole duration of the experiment (0–8 days), the trend of variation of the SARS-CoV-2 gene concentrations (Y), normalised by the gene concentration of the samples kept for 0 days at ± 20 °C (i.e.: reference samples kept at +4 °C for the entire duration of the experiment). The study of the trend of the ratio (Y/Y0) allows to eliminate variations

Discussion

The scenario regarding available data on stability of SARS-CoV-2 in wastewater samples reported in previous literature is complex, because of a limited number of studies, different experimental approaches used and genes measured. Even in this study, each participating laboratory has utilized different wastewater processing approaches and investigated different gene markers, depending on their SARS-CoV-2 wastewater surveillance method in place. Moreover, the application of different

Conclusions

This isochronous stability study aimed at identifying proper storage and transport conditions that allow, under controlled uncertainty, the execution of wastewater-based surveillance programmes on national and international scale. The chosen reference temperature of + 4 °C seems to be the more cautious and practical suggestion for handling wastewater samples for SARS-CoV-2 detection, provided that the analysis could be performed within a limited time frame of few days from collection. Indeed,

Declaration of Competing Interest

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.

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