key: cord-0891874-leba21s1
authors: La Rosa, G.; Iaconelli, M.; Veneri, C.; Mancini, P.; Ferraro, G. Bonanno; Brandtner, D.; Lucentini, L.; Bonadonna, L.; Rossi, M.; Grigioni, M.; Suffredini, E.
title: The rapid spread of SARS-COV-2 Omicron variant in Italy reflected early through wastewater surveillance
date: 2022-05-06
journal: Sci Total Environ
DOI: 10.1016/j.scitotenv.2022.155767
sha: 55150e55104c9f3e0e87b4464f48943251532e3a
doc_id: 891874
cord_uid: leba21s1

The SARS-CoV-2 Omicron variant emerged in South Africa in November 2021, and has later been identified worldwide, raising serious concerns. A real-time RT-PCR assay was designed for the rapid screening of the Omicron variant, targeting characteristic mutations of the spike gene. The assay was used to test 737 sewage samples collected throughout Italy (19/21 Regions) between 11 November and 25 December 2021, with the aim of assessing the spread of the Omicron variant in the country. Positive samples were also tested with a real-time RT-PCR developed by the European Commission, Joint Research Centre (JRC), and through nested RT-PCR followed by Sanger sequencing. Overall, 115 samples tested positive for Omicron SARS-CoV-2 variant. The first occurrence was detected on 7 December, in Veneto, North Italy. Later on, the variant spread extremely fast in three weeks, with prevalence of positive wastewater samples rising from 1.0% (1/104 samples) in the week 5–11 December, to 17.5% (25/143 samples) in the week 12–18, to 65.9% (89/135 samples) in the week 19–25, in line with the increase in cases of infection with the Omicron variant observed during December in Italy. Similarly, the number of Regions/Autonomous Provinces in which the variant was detected increased from one in the first week, to 11 in the second, and to 17 in the last one. The presence of the Omicron variant was confirmed by the JRC real-time RT-PCR in 79.1% (91/115) of the positive samples, and by Sanger sequencing in 66% (64/97) of PCR amplicons. In conclusion, we designed an RT-qPCR assay capable to detect the Omicron variant, which can be successfully used for the purpose of wastewater-based epidemiology. We also described the history of the introduction and diffusion of the Omicron variant in the Italian population and territory, confirming the effectiveness of sewage monitoring as a powerful surveillance tool.

The SARS-COV-2 Omicron variant emerged in South Africa on 24 November 2021 and has later been identified in numerous countries worldwide. On 26 November 2021, WHO designated B.1.1.529 as a Variant of Concern, named Omicron, asking to enhance surveillance and sequencing efforts to better understand circulation of SARS-CoV-2 variants.

As of 20 January 2022, Omicron has been identified in all EU/EEA countries, and as on 30 January it was the dominant variant (accounting for >50% of sequenced viruses) in 19 of the 22 EU/EEA countries with adequate sequencing volume, with 268.835 Omicron cases reported (https://www.ecdc.europa.eu/en/covid-19/country-overviews).

In Italy, the first Omicron clinical case was described on 22 November in Milan, in an Italian man who had returned on 11 November from Mozambique (first Omicron sequence submitted in (10.5281/zenodo.5985196) and 28 November and 3 December 2021 (10.5281/zenodo.5985276) showed, as expected, the predominance of the Delta variant associated to significant variability, and no presence of the Omicron variant by analysis of raw data of Next Generation amplicon sequencing.

As soon as the detection of the SARS-CoV-2 Omicron variant was first reported in Italy, we designed a real-time RT-PCR assay to enable rapid identification of the novel VoC, targeting characteristic mutations in the spike gene, and evaluated its specificity and sensitivity against other SAR-CoV-2 variants. In parallel, we tested an assay developed by the European Commission, Joint

Research Centre (JRC) designed in silico for the detection of Omicron targeting a spike region with a unique cluster of mutations (Petrillo et al., 2021) . The JRC invited control laboratories worldwide to validate it in vivo on clinical samples (https://ec.europa.eu/jrc/en/news/efficient-tracing-omicronnew-pcr-test). Subsequently, both the assays were used to test sewages collected throughout Italy in the period between 11 November (date of entry in Italy of the first Omicron case in Italy) and 25

December 2021, to investigate how the Omicron variant spread over time and geographically.

Moreover, Sanger sequencing was performed on positive samples, to validate results and study sequence variability.

A new real-time RT-PCR assay targeting the spike region of the Omicron variant was designed using the Primer3Plus software (Primer3Plus (bioinformatics.nl). The spike region of Omicron harbours a number of mutations, some of which are in common with other variants (e.g. the deletion 69/70 shared with Alpha, T95I and G142D shared with Delta, N501Y shared with Alpha, Beta and Gamma), while other are unique for Omicron, and therefore suitable for the design of specific assays. We selected the region harbouring mutations H655Y, N679K and P681H, which are each present in >98.9% of the Omicron variant sequences (Supplementary materials, Figure S1 ) as on 8 January 2022. Only mutation P681H is in common with the Alpha variant which was deescalated by the European Centre for Diseases Control on 23 September 2021 due its drastically reduced circulation in Europe.

For the optimization of the assay different reaction conditions were evaluated: primer/probe concentrations, annealing temperatures (58°C and 60°C), and real time RT-PCR reagents (UltraSense One-Step qRT-PCR System by Invitrogen and AgPath-ID One-Step RT-PCR Reagents J o u r n a l P r e -p r o o f Journal Pre-proof by Applied Biosystems). The newly designed assay was tested on a RNA sample extracted from a nasopharyngeal swab of a patient resident in the region of Apulia, collected on 7 December 2021, and kindly provided by Dr. Maria Chironna, Department of Biomedical Sciences and Human Oncology, School of Medicine and Surgery, University of Bari Aldo Moro. This sample had been previously characterized as Omicron variant by whole genome sequencing and submitted to GISAID with ID EPI_ISL_7565149. Viral RNA was quantified using a previously described realtime RT-qPCR (La Rosa et al., 2021c; Pierri et al., 2021) and was standardized at 6×10 2 genome copies (g.c.)/l for the use as a positive control in PCR runs and for sensitivity assessment. For the latter, the standardized RNA was used to prepare serial dilutions which were analysed in eight replicates to calculate the limit of detection (LOD 50 ) of the assay on pure target. The same dilutions were also used to spike nucleic acids extracted from wastewater samples collected in July 2021 (i.e. before the emergence of the Omicron variant) to assess the real-time assay LOD 50 on environmental samples. Calculation were performed according to Wilrich and Wilrich (2009) November -3 December 2021, had already been analysed in the monthly "flash surveys" on SARS-CoV-2 variants by Sanger and NGS (report available at ac78131e-73eb-695e-4c64-65ad2379cc45 (iss.it). Samples were collected, registered, processed for virus concentration and subjected to RNA extraction by the members of the of SARS-CoV-2 environmental surveillance network in Italy (SARI network) in the framework of the surveillance program established according to EU Rec. 2021/472. The same reference analytical protocol (SARI protocol rev. 3, 10.5281/zenodo.5758725) was adopted by all network members. Briefly, 24 h composite sewage sample was concentrated by PEG precipitation followed by centrifugation following the method described by Wu and collaborators (Wu et al., 2020) with minor modifications (sample pasteurization at 56°C per 30 min and removal of solid debris before precipitation through centrifugation at 4500 × g per 30 min).

RNA was extracted by magnetic silica beads and subsequently purified by the OneStep PCR Inhibitor Removal Kit (Zymo Research, CA, USA). RT-qPCR testing for SARS-CoV-2 was conducted according to previously published protocols (La Rosa et al., 2021c) and quality insurance controls (process control virus, inhibition control) were included in the process to assess viral recovery and PCR inhibition. Purified RNAs were then shipped in dry ice to the Department of Environment and Health at ISS. Detection of the Omicron variant by real-time PCR was carried out with two different protocols: a) Newly designed assay (PCR ID_999 assay) Following optimization of conditions, the RT-PCR mix (25μL total volume) was prepared using the AgPath-ID One-Step RT-PCR Reagents (Applied Biosystems, MA, USA), and 5 μL of sample RNA were added to reactions containing 1× buffer, 1 μL of RT-PCR enzyme mix, 1.67 μL of detection enhancer. Primer/probe (Table 1) As no PCR conditions were specifically provided in the reference publication, the reaction mix was prepared as for the PCR ID_999 and the same thermal profile was adopted. Amplification with the JRC assay was performed for confirmation purpose on samples testing positive by the real-time PCR assay ID_999.

J o u r n a l P r e -p r o o f Journal Pre-proof All real-time reactions were run on a QuantStudio 12K Flex (Applied Biosystems). Samples with Ct values <40 were considered positive; samples with Ct values >40 were considered uncertain but were retained for nested RT-PCR testing.

A summary of the characteristics of the samples testing positive by the two real-time PCRs is shown in Table 3 .

A newly designed nested RT-PCR assay targeting the spike protein and designed PCR ID_995/996 (first cycle/nested reaction), was used for further confirmation and to study viral genetic diversity though sequencing. The Omicron's Spike protein has several amino acid mutations (substitutions and deletions) in the selected region that are distinct compared to other VoCs (Kannar et al., 2022) .

Specifically, the nested assay amplifies a region of 577 bps, covering the amino acids 220 to 412 of the spike protein.

First-strand cDNA was synthesized using Super Script IV Reverse Transcriptase (ThermoFisher Scientific) according to the manufacturer's instructions, using reverse primer 2347. First PCR reaction was performed with 4 μL of cDNA and 1 μL of each primer (10 μM) in a final volume of 25 μL (Kit Platinum SuperFi Green PCR Master Mix, Thermo). The PCR conditions were as follows: 98 °C for 2 min, followed by 35 cycles at 98 °C for 10 s, 62 °C for 30 s, and 72 °C for 1 min and a final extension at 72 °C for 5 min. Nested PCR was performed using 2 μL of the first PCR product under the same conditions, with the exception of the annealing temperature raised at 63°C. To avoid false-positive results, standard laboratory precautions were taken.

The PCR products were visualised by gel electrophoresis purified using a Montage PCRm96

Microwell Filter Plate (Millipore, Billerica, MA, USA), and were then sequenced on both strands (BioFab Research, Rome, Italy). Mutations were detected using the CoVsurver mutation Analysis of hCoV-19 implemented in GISAID (GISAID -CoVsurver mutations App).

Sequences were submitted to GenBank under the accession numbers ON196927-ON197019.

The optimization of PCR conditions showed that the RT-PCR reagents used had a significant impact on the performance of both real-time PCR assays, with Ct values between the results provided by the different amplification systems equal or above 5 cycles (see Supplementary J o u r n a l P r e -p r o o f Journal Pre-proof Material, Figure S3 ). Following optimization, the standardized Omicron RNA (6×10 2 g.c./l) was successfully amplified by both the newly designed PCR ID_999 (ISS assay) and the OmMet PCR (JRC assay), although higher Ct values were obtained for the latter (30.15±0.40 vs. 27.38±0.66, Table S1 ). The ISS real-time RT-PCR provided a LOD 50 of 0.28 g.c./l of tested nucleic acids (1.4 g.c./reaction) on pure Omicron RNA, and of 2.67 g.c./l (13.3 g.c./reaction) on nucleic acids extracted from sewage samples and spiked with the Omicron variant.

As for the specificity of the assays, no amplification was obtained for the six Coronavirus RNAs included in the EVAg Coronavirus panel, but a partial cross-reactivity was observed with Alpha and Gamma variants which were however amplified at a lower efficiency (Ct of 5.02 and 3.78, respectively, compared to Omicron; Supplementary Material, Figure S4 ). 

Overall, 97 of the 115 Omicron-positive wastewater samples (84.3 %) were successfully amplified with the nested RT-PCR assay targeting the S protein, and subsequently sequenced by Sanger J o u r n a l P r e -p r o o f Journal Pre-proof sequencing. Of these, 64 samples showed mutations associated with the Omicron variant, three samples showed amino acid substitutions not discriminatory for variant assignment, and 4 were unreadable due to mixed electropherograms (Table 3) . Moreover, 26 sequences showed no mutations.

Sequence analysis showed a high degree of sequence variability within the Omicron variant sequences. In total, 10 amino acid substitutions were detected in the 577-bps fragment, with 12 different combinations (Table 3 ). The most frequent combination of mutations included the panel "G339D, S371L, S373P, S375F" (29 samples) followed by the panels "G339D, S371F, S373P, S375F" and "G339D, R346K, S371L, S373P, S375F" (11 samples each). This last combination of mutation is associated with sublineage BA1.1.

The SARS-CoV-2 Omicron variant has rapidly replaced SARS-CoV-2 Delta in most European Union/European Economic Area (EU/EEA) countries (Assessment of the further spread and potential impact of the SARS-CoV-2 Omicron variant of concern in the EU/EEA, 19th update (europa.eu)). Immunity acquired through previous infection seems to be less effective against Omicron than against other variants, but the risk of severe COVID-19 remains low (Mallapaty 2022 moreover, it is more adept at infecting people who are vaccinated and even boosted (Lyngse et al., 2022) . Early studies suggest that the BA.2 lineage might prolong the Omicron wave, but won't necessarily cause a fresh surge of COVID-19 infections (Callaway 2022 Positive samples by the newly designed ISS real-time RT-PCR were also tested with another assay (JRC OmMet) to further confirm the results, and compare the performances of the two assays.

Overall, 79.1% of positive samples were also confirmed by the second assay, discrepancies being mostly associated with samples with Ct values higher than the average and, presumably, lower concentration of the target. Indeed, considering the results of the optimization tests and of the analysis on wastewater samples, the JRC assay seems to provide higher amplification stringency (no cross-reactivity with other variants), but lower sensitivity (Ct values on average higher than the ISS assay). Since a protocol for the OmMet assay was not given in the published article (Petrillo et al., 2021) , the OmMet assay was run at the same condition of the newly designed assay, as specific optimization of the assay was outside the scope of the work. Increase of the sensitivity of the assay could be incremented by further optimize reaction conditions.

It should be noted that only qualitative real-time RT-PCR assays were used in this study, aiming at evaluating presence/absence of the target; we therefore did not exactly measure the Omicron template by comparison of the Ct values to a standard curve. Indeed the aim of the present study was not comparing Omicron SARS-CoV-2 concentrations, but describing the history of the introduction and diffusion of the Omicron variant in the Italian population. However, by comparing

absolute Ct values, we were able to show that they (and consequently the variant concentration) varied considerably. It is known that many factors impact the absolute value of Ct besides the concentration of the target, however a comparison can be done for data obtained in experiments using the same reaction conditions (instruments, reagents and assays). Therefore, the observation that the Ct value from one sample is higher than that of the other, could be valuable in concluding that the amount of template is lower in the first sample. Ct values ranged from 31,5 up to >40, that J o u r n a l P r e -p r o o f 

☒ 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.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: J o u r n a l P r e -p r o o f 

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