key: cord-0794296-wm5sdtct
authors: Boehm, A.; Duong, D.; Hughes, B.; Wolfe, M. K.; White, B.; Chan-Herur, V.
title: Regional replacement of SARS-CoV-2 variant BA.1 with BA.2 as observed through wastewater surveillance
date: 2022-04-22
journal: nan
DOI: 10.1101/2022.04.22.22274160
sha: 2655201679b80fb117ba7dab2ce046ad142577c0
doc_id: 794296
cord_uid: wm5sdtct

An understanding of circulating SARS-CoV-2 variants can inform pandemic response, vaccine development, disease epidemiology, and use of monoclonal antibody treatments. We developed custom assays targeting characteristic mutations in SARS-CoV-2 variants Omicron BA.1 and BA.2 and confirmed their sensitivity and specificity in silico and in vitro. We then applied these assays to daily wastewater solids samples from eight publicly owned treatment works in the greater Bay Area of California, USA, over four months to obtain a spatially and temporally intensive data set. We documented regional replacement of BA.1 with BA.2 in agreement with, and ahead of, clinical sequencing data. This study highlights the utility of wastewater surveillance for real time tracking of SARS-CoV-2 variant circulation.

Knowing when a new SARS-CoV-2 variant arrives locally is of utmost importance to pandemic 37 response in order to ensure a rapid response to new features of the virus. In late 2021, the 38 Omicron variant emerged in South Africa and quickly spread to Europe and then North America 39 during a period of time during which Delta was the dominant circulating variant. The Omicron 40 variant resulted in a virus more transmissible and less susceptible to therapeutics and vaccines 41 than previous lineages 1 . Given these implications for public health, timely identification of its 42 introduction into a new community was critical. Variant identification is usually accomplished 43 through sequencing of clinical specimens, but the time between specimen collection, and the 44 return of sequencing information to public health officials is approximately two weeks. For 45 example, outbreak.info 2 , a website that provides analysis and visualization of SARS-CoV-2 46 clinical sequences deposited in GISAID 3,4 , stresses data are unreliable and incomplete during 47 the 1-2 week window preceding the query date. 48 49 Wastewater represents a biological composite sample from the contributing community. It 50 contains feces, urine, sputum, mucus, blood, vomitus, and any other excretion that goes down a 51 drain into the sewer network. Global investigations have illustrated that SARS-CoV-2 RNA 52 concentrations, whether measured by RT-QPCR or digital RT-PCR, or in the liquid or solid 53 phase of wastewater, correlates well with laboratory-confirmed incident COVID-19 cases in the 54 associated sewersheds 5-8 . A number of studies have illustrated that circulating or novel variants 55 can be identified in wastewater using RT-PCR assays that target a characteristic variant 56 mutation 9-11 and by sequencing SARS-CoV-2 12 . We previously developed targeted RT-PCR 57 assays for characteristic mutations in Alpha, Mu, Lambda, Delta, and Omicron BA. 1, and 58 showed that the concentrations of these mutations, normalized by a pan-SARS-CoV-2 N gene 59 target is strongly correlated to the fraction of clinical specimens classified as the associated 60 variant 10,13 at two publicly owned treatment works (POTWs). Here, we extend that previous work 61 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The sensitivity and specificity of the mutation assays were further tested by diluting target 88 variant gRNA (Table S1 ) in no (0 copies), low (100 copies), and high (10, inhibitors were removed 14 (see SI). RNA was processed immediately to measure concentrations 104 of the N gene of SARS-CoV-2, the BA.1* characteristic mutation del143-145 10 , the BA.2* 105 characteristic mutation (LPPA24S), pepper mild mottle virus (PMMoV), and bovine coronavirus 106 (BCoV) recovery using digital droplet RT-PCR methods 14, 15 . The N gene target is present in all 107 variants and is a pan-SARS-CoV-2 gene target. PMMoV is highly abundant in human stool and 108 wastewater globally 16,17 and is used as an internal recovery for the wastewater samples 18 . 109

BCoV was spiked into the samples and used as an additional recovery control; all samples were 110 required to have greater than 10% BCoV recovery. RNA extraction and PCR negative and 111 positive controls were included to ensure no contamination. Extracted RNA samples from one 112 POTW (SJ) were then stored at -80°C between 1 and 7 days before they were analyzed for 113 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (Table S3 ). This date was earliest (26 Feb) at Ocean, located in San Francisco, and latest at Gil 190 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. should be carried out using data on clinical variants in the sewersheds of the eight POTWs, but 213 such data is not readily available, so we had to rely on state-level data. 214 215 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 22, 2022. ; https://doi.org/10.1101/2022.04.22.22274160 doi: medRxiv preprint State-level clinical sample sequencing data on variant occurrence is typically 1-2 weeks delayed 216 owing to the time required for laboratory processing and reporting. In addition, there can be 217 large uncertainties in the proportion estimates preceding the data for which the most recent data 218 are available. This is illustrated in Figure 3 where we highlight the approximate time period for 219 which data were incomplete on 14 April 2022, when wastewater measurements for the last date 220 in our data series (12 or 13 April 2022) were available publicly on our website 221 (wbe.stanford.edu). During this same time period, wastewater data suggest BA.2* is near 100% 222 and BA.1* is near 0%, which a week later (when data were downloaded from GISAID on 21 223

April 2022) was confirmed by clinical sequencing data. This highlights an important advantage 224 of using wastewater surveillance for variant tracking. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted April 22, 2022. ; https://doi.org/10.1101/2022.04.22.22274160 doi: medRxiv preprint

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