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. 2023 Oct;30(48):106660-106670.
doi: 10.1007/s11356-023-29696-y. Epub 2023 Sep 21.

Evaluation of concentration procedures, sample pre-treatment, and storage condition for the detection of SARS-CoV-2 in wastewater

Francesca Cutrupi  1 , Michele Rossi  2 , Maria Cadonna  3 , Elisa Poznanski  4 , Serena Manara  5 , Mattia Postinghel  3 , Giulia Palumbi  6 , Marta Bellisomi  6 , Elena Nicosia  6   7 , Giorgia Allaria  8 , Lorenzo Dondero  8 , Carolina Veneri  9 , Pamela Mancini  9 , Giusy Bonanno Ferraro  9 , Giuseppina Rosa  9 , Elisabetta Suffredini  10 , Paola Foladori  11 , Elena Grasselli  8
Affiliations

Evaluation of concentration procedures, sample pre-treatment, and storage condition for the detection of SARS-CoV-2 in wastewater

Francesca Cutrupi et al. Environ Sci Pollut Res Int. 2023 Oct.

Abstract

Crucial information on the pandemic's spread has been gathered by monitoring the trend of SARS-CoV-2 in wastewater. This surveillance has highlighted that the initial concentration is a critical step of the analytical procedure due to the low viral titer that may be present in this matrix. This paper presents the results of the evaluation of two different wastewater concentration protocols to determine the most efficient and cost-effective. The two methods tested were the following: (a) a biphasic separation system with PEG-dextran and (b) a PEG/NaCl precipitation protocol. Other aspects of the detection method were also investigated including the influence of storage temperature on virus recovery and the heat treatment of pasteurization, which aims to make samples safer for operators and the environment. The PEG/NaCl precipitation method was found to perform better than the biphasic separation system, allowing for more sensitive identification of the presence of the virus and the detection of a higher viral titer than that identified with the biphasic separation in all results. Storage of the samples at 4.3±0.2°C for up to 3 weeks did not adversely affect the virus titer and the pasteurization pre-treatment increases operator safety and maintains the identification of the viral concentration.

Keywords: Concentration methods; Pasteurization; SARS-CoV-2; Storage temperature; Wastewater; Wastewater-based epidemiology.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Flowchart of the compared protocols (biphasic separation system and PEG/NaCl precipitation protocol)
Fig. 2
Fig. 2
Positivity percentage obtained by the three research groups (Trentino, Liguria, and Lazio). The chart displays the sample size and the percentage of samples testing positive for the presence of SARS-CoV-2 using the biphasic separation system or the PEG/NaCl precipitation protocol
Fig. 3
Fig. 3
Comparison of the positive Cq results of the two protocols
Fig. 4
Fig. 4
Comparison of SARS-CoV-2 concentration measured for each method (biphasic separation system and PEG/NaCl precipitation protocol) and groups: A Trentino, B Lazio, C Liguria for the molecular target ORF-1b. The boxplot, median values, and outliers are shown. The grey lines indicate the position of the same sample for the two methods compared. Outliers (highest 5%) were removed to allow better visualization
Fig. 5
Fig. 5
Percentage recovery of Mengovirus (MgV) for the two methods. In these plots, the values distributions of percentage recovery are represented by half violin plots showing the mono-modality of the data
Fig. 6
Fig. 6
Concentration of SARS-CoV-2 in raw influent wastewater stored at 4.3±0.2°C and −23.2±0.3°C from 1 to 22 days. The range 4–8 comprises samples analyzed 4, 5, and 8 days after sampling, the 12–15 samples analyzed 12, 13, and 15 days after, and 19–22 samples from 19, 20, and 22 days after. Error bars represent the standard deviation between grouped samples
Fig. 7
Fig. 7
SARS-CoV-2 concentrations comparing pasteurized and non-pasteurized samples concentrated with A biphasic separation system and B PEG/NaCl precipitation protocol
See this image and copyright information in PMC

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