key: cord-1050054-6c1pdhzo
authors: Cutrupi, Francesca; Cadonna, Maria; Manara, Serena; Postinghel, Mattia; La Rosa, Giuseppina; Suffredini, Elisabetta; Foladori, Paola
title: The wave of the SARS-CoV-2 Omicron variant resulted in a rapid spike and decline as highlighted by municipal wastewater surveillance
date: 2022-05-21
journal: Environ Technol Innov
DOI: 10.1016/j.eti.2022.102667
sha: 3ecabd93d7f9908628c482e480f18d1642c86d73
doc_id: 1050054
cord_uid: 6c1pdhzo

This paper highlights the extraordinarily rapid spread of SARS-CoV-2 loads in wastewater that during the Omicron wave in December 2021-February 2022, compared with the profiles acquired in 2020–21 with 410 samples from two wastewater treatment plants (Trento+suburbs, 132,500 inhabitants). Monitoring of SARS-CoV-2 in wastewater focused on: (i) 3 samplings/week and analysis, (ii) normalization to calculate genomic units (GU) inh(−1) d(−1); (iii) calculation of a 7-day moving average to smooth daily fluctuations; (iv) comparison with the ‘current active cases’/100,000 inh progressively affected by the mass vaccination. The time profiles of SARS-CoV-2 in wastewater matched the waves of active cases. In February–April 2021, a viral load of 1.0E＋07 GU inh(−1) d [Formula: see text] corresponded to 700 active cases/100,000 inh. In July–September 2021, although the low current active cases, sewage revealed an appreciable SARS-CoV-2 circulation (in this period 2.2E＋07 GU inh(−1) d(−1) corresponded to 90 active cases/100,000 inh). Omicron was not detected in wastewater until mid-December 2021. The Omicron spread caused a 5–6 fold increase of the viral load in two weeks, reaching the highest peak (2.0–2.2E＋08 GU inh(−1) d(−1) and 4500 active cases/100,000 inh) during the pandemic. In this period, wastewater surveillance anticipated epidemiological data by about 6 days. In winter 2021–22, despite the 4–7 times higher viral loads in wastewater, hospitalisations were 4 times lower than in winter 2020–21 due to the vaccination coverage >80%. The Omicron wave demonstrated that SARS-CoV-2 monitoring of wastewater anticipated epidemiological data, confirming its importance in long-term surveillance.

in Supplementary Material.

Raw wastewater samples were collected and analysed for 17 months, from December 1, 2020, for WWTPSouth. The first two months, December 2020 and January 2021, were a run-in period with changes in the analytical methods, and wastewater was collected once or twice a week.

From February 2021 to April 2022 (more than one year), three samplings were performed per week, resulting in more data, a more accurate calculation of the 7-day moving average, and a 158 better understanding of trends. Then, each composite sample was carefully mixed and collected in 250 mL aliquots before its refrigerated transport to the lab and storage at 4°C until further analysis. Samples were not frozen at any time, except for 7 samples (6 from WWTPNorth and 1 from WWTPSouth) due to unforeseen logistical problems. From October 1, 2021, in accordance with the European in that period, the analysis of 66% of the samples began within three hours from the time of 178 delivery to the laboratory.

For each sampling day, the daily flow rate (expressed as m 3 /d) was recorded.

Initially, from December 2020 to January 2021, the analysis of SARS-CoV-2 was based on the adopted in agreement with the National Institute of Health (Italy) and shared with the Italian 188 network involved in the national programme for wastewater surveillance (SARI project; La Rosa et al., 2021a) . This latter method enables detection of SARS-CoV-2 even at low Institute of Health (Italy).

All samples were analyzed in duplicate in qPCR. Analyses were considered acceptable if the 223 standard curves for the quantification of SARS-CoV-2 provided a slope close to -3.32 224 (minimum -3.1, maximum -3.6) and a regression coefficient equal to or above 0.98 (R 2 ≥0.98),

and if the other quality controls were within acceptability limits (recovery ≥1% and PCR 226 inhibition ≤50%).

In the absence of a general agreement on the acceptable recovery rate for SARS-CoV-2 in 228 sewage, we used the criterion of recovery rate >1% according to ISO 15216-1:2017, which 229 concerns the quantification of viruses in complex food categories. Other protocols indicate that 230 a recovery of 1% or higher may be considered acceptable; i.e., the recovery process will not be 231 repeated or the data will not be rejected (inter alia Omura et al., 2022) . Instead, when the 232 recovery rate is below 1% the results are discarded and the samples can be re-extracted and 233 analyzed, thus providing an overall improvement in data quality. The recovery of the procedure 234 is indeed higher on average. Here, recovery rate of the process control virus was not used to where Qd is the daily volume of wastewater entering the plant (daily flow rate, in m 3 /d) and P is the population (inhabitants) in the served sewershed. 

The calculation of is performed for 'n' municipalities or suburbs served by the sewer, obtaining 1 , 2 , …, . If a municipality is served by two WWTPs (as in our case), is that discharge their wastewater (and positive faeces) in a WWTP can be calculated by summing 294 all the of the municipalities and suburbs served by the WWTP:

In particular, in the present case n= 7, which includes two parts of Trento city and 7 suburbs as 297 indicated in Figure S1 in Supplementary Material. 

The profiles of the normalized SARS-CoV-2 RNA loads over time (calculated according to the 316 procedure described in section 2.4) obtained in WWTPNorth and WWTPSouth are shown in Figure   317 1. The original daily values acquired by RT-qPCR are represented as discrete points in Figure   318 1; data are affected by a 'natural' noise that can be associated with the 'nugget effect' (Wurtzer 

Therefore ̅ at day 't' was calculated considering the 'n' data acquired within the previous last In short, the viral loads observed between December 2021 and January 2022 were the highest during the period of the study, and the slope was the steepest ever seen since monitoring began 431 in the middle of 2020.

At the end of January 2022, the viral loads in wastewater began to decline rapidly, along with 433 COVID-19 active cases (Figure 2 ). On February 15, 2022, the viral load dropped by more than

77% from the peak of the Omicron wave observed in January 2022.

The relationship between SARS-CoV-2 loads in wastewater and current active cases cannot be 

The rapid increase of the SARS-CoV-2 load in wastewater during the Omicron wave and the 

The results also highlighted that wastewater surveillance allows to appreciate the circulation of 507 the virus even with an underestimation of positive cases due to mass vaccination.

Understanding the evolution of the diffusion of emerging VOCs is essential for adopting WWTPNorth and WWTPSouth, respectively. These maximum loads were 4 to 7 times higher than 557 those in winter 2020-21. During the Omicron wave, the number of active cases at the peak was 558 4700 cases/100,000 inh that is 4.7 times higher than the previous winter. 

The steep spike of positive cases has raised concerns that the healthcare system will once again 570 come under pressure, due to the extremely high rate of the spread of the Omicron variant. 

-the viral loads in wastewater during the Omicron wave were 4-20 times higher than in 603 the Alpha and Delta waves;

-the Omicron wave lasted two months from December 2021 to February 2022.

-a marked concordance was observed between viral loads in wastewater and 'current 606 active cases';

-wastewater is able to show the increase in viral loads with an anticipation of 6 days.

In the rapidly changing and challenging context of emerging VOCs, wastewater surveillance 609 has proven to be a valuable tool, independent of diagnostic testing and important in the long-610 term monitoring of the pandemic.

Declaration of competing interest

The authors declare that they have no competing interests that could have influenced the work 615 reported in this manuscript.

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