key: cord-0295345-0zhtaeq6
authors: Wolfe, M.; Hughes, B.; Duong, D.; Chan-Herur, V.; Wigginton, K. R.; White, B.; Boehm, A. B.
title: Detection of SARS-CoV-2 variant Mu, Beta, Gamma, Lambda, Delta, Alpha, and Omicron in wastewater settled solids using mutation-specific assays is associated with regional detection of variants in clinical samples
date: 2022-01-18
journal: nan
DOI: 10.1101/2022.01.17.22269439
sha: 1eb8115d94a6c22a0a5208846a930c80a19363cb
doc_id: 295345
cord_uid: 0zhtaeq6

Changes in the circulation of SARS-CoV-2 variants of concern (VOCs) may require changes in public health response to the COVID-19 pandemic, as they have the potential to evade vaccines and pharmaceutical interventions and may be more transmissive relative to other SARS-CoV-2 variants. As such, it is essential to track and prevent their spread in susceptible communities. We developed digital RT-PCR assays for mutations characteristic of VOCs and used them to quantify those mutations in wastewater settled solids samples collected from a publicly owned treatment works (POTW) during different phases of the COVID-19 pandemic. Wastewater concentrations of single mutations characteristic to each VOC, normalized by the concentration of a conserved SARS-CoV-2 N gene, correlate to regional estimates of the proportion of clinical infections caused by each VOC. These results suggest targeted RT-PCR assays can be used to detect variants circulating in communities and inform public health response to the pandemic.

Changes in the circulation of SARS-CoV-2 variants of concern (VOCs) may require changes in 23 public health response to the COVID-19 pandemic, as they have the potential to evade vaccines 24 and pharmaceutical interventions and may be more transmissive relative to other SARS-CoV-2 25 variants. As such, it is essential to track and prevent their spread in susceptible communities. 26 We developed digital RT-PCR assays for mutations characteristic of VOCs and used them to COVID-19 (6) but also for other respiratory viruses such as respiratory syncytial virus (RSV) (7). 56 Using wastewater to track community health has the advantage of providing information on an 57 entire community without relying on individual clinical testing, which may be expensive or 58 unavailable and requires individuals to alter their behavior to seek testing. Wastewater may be 59 a leading indicator of community health when shedding by infectious individuals precedes 60 symptom onset.

The COVID-19 pandemic has seen SARS-CoV-2 acquire mutations that have given rise to 63 variants with distinguishing characteristics. Variants of concern (VOCs) or interest (VOIs) are 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 January 18, 2022. Omicron and then measure these in wastewater solids from a publicly owned treatment work 102 (POTW) located in the Bay Area of California, USA. We measure concentrations in wastewater 103 settled solids as concentrations of SARS-CoV-2 RNA are enriched several orders of magnitude in 104 solids relative to liquid wastewater (20, 21) . We subsequently compare the measurements to 105 data on occurrence of those variants in clinical specimens, aggregated at the state-level. 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 January 18, 2022. Table 1) .

These characteristic mutations were chosen because they are present in high percentages of 113 the associated variant sequences in GISAID (Table 1 , information accessed through 114 outbreak.info), and they represent deletions or multiple single nucleotide polymorphisms 115 (SNPs) in close proximity and thus are likely to be more specific than assays targeting a SNP. Table S1 . Primers and probe sequences are provided in Table 2 . 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 January 18, 2022. The sensitivity and specificity of the mutation assays were further tested by diluting target 142 variant gRNA (Table 1) for the mutations in no (0 copies), low (100 copies), and high (10,000 Santa Clara County, California, USA (San José-Santa Clara Regional Wastewater Facility) was 152 . 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 January 18, 2022. Samples were collected daily for a larger COVID-19 wastewater surveillance effort starting in 161 November 2020 (1) , and a subset of these samples are used in the present study and were 162 chosen to span the period prior to and including presumed emergence of different variants. 163 Generally, sampling was about once per week or month prior to presumed emergence and then 164 3-7 times per week during and after the period of emergence. Details on sampling frequency 165 are provided in Table 3 . A previous study (19) reported Alpha mutation data for the POTW and 166 those data are included in our analysis for completeness. That same study reported some Delta 167 mutation data (N = 48, data until 1 Aug 2021) for the POTW and that data are included here.

The methods below describe those used for the new measurements including those for Mu, 169 Lambda, Beta/Gamma, Delta (measured daily between 1 Aug 2021 and 2 Jan 2022), and 170 Omicron mutations. 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 January 18, 2022. ; https://doi.org/10.1101/2022.01.17.22269439 doi: medRxiv preprint Irvine, CA) as a means to alleviate inhibition (24) . RNA was subsequently processed immediately 175 (within 24 h of sample collection) to measure concentrations of the N gene of SARS-CoV-2, 176 pepper mild mottle virus (PMMoV), and bovine coronavirus (BCoV) recovery using digital 177 droplet RT-PCR methods described in detail elsewhere (1, 25) . The N gene assay targets a 178 region of the N gene that is conserved across these variants. PMMoV is highly abundant in 179 human stool and wastewater globally (26, 27) and is used as an internal recovery and fecal Table   201 3). Droplets were generated using the AutoDG Automated Droplet Generator (Bio-Rad). PCR 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 January 18, 2022. ; https://doi.org/10.1101/2022.01.17.22269439 doi: medRxiv preprint recoveries were higher than 10% and PMMoV concentrations were within the expected range 263 for the POTW suggesting an efficient and acceptable recovery of RNA during RNA extraction 264 ( Figure S1 ). 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 January 18, 2022. ; https://doi.org/10.1101/2022.01.17.22269439 doi: medRxiv preprint

The mutation present in Beta and Gamma was rarely detected in wastewater solids (Figure 2 ).

It was not detected in wastewater until late May 2021 when it was detected at a very low 286 relative concentration. It was detected a total of 3 times between late May 2021 and the end of Gamma (tau = 0.14, p = 0.5), but the association was not statistically significant. This may be 306 . 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 January 18, 2022. ; https://doi.org/10.1101/2022.01.17.22269439 doi: medRxiv preprint due to the relatively low cadence of measurements as we only measured the mutation once 307 per week; this is low compared to frequency of variability typically observed in wastewater 308 measurements (1). There was no reported case of Lambda in the state from November 2021, 309 and our lack of detection of the Lambda mutation in that month is consistent with this. The These findings suggest that for variants of concern, valuable insights are available on the 363 circulation of the variants through the use of wastewater, and these insights are attainable 364 using assays that target a single characteristic variant mutation. Development of assays for 365 SARS-CoV-2 variants requires in silico assay design, procurement of primers, probes and 366 positive control material, and specificity and sensitivity testing. The rate limiting step in this 367 process, we have found, is the procurement process. Targeted ddRT-PCR assays can be applied 368 to samples with a turnaround time for results of less than 24 hours, and new targeted assays 369 can be quickly developed and applied to wastewater when new variants are identified and 370 expected to spread into communities to gain insight into their local emergence. We were able 371 . 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 January 18, 2022. ; https://doi.org/10.1101/2022.01.17.22269439 doi: medRxiv preprint to implement this process in real-time for development and implementation of the Omicron 372 mutation assay, which we were able to apply to daily samples at this POTW starting 6 Dec 2021 373 to capture the emergence of the variant at high resolution. 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 January 18, 2022. ; https://doi.org/10.1101/2022.01.17.22269439 doi: medRxiv preprint 503 504 505 Figure 1 . Copies (cp) of mutations measured when RNA containing the mutation was diluted 506 into no, low, and high background of WT-gRNA. Low background is 100 copies/well and high 507 background is 10,000 copies/well where "copies" refers to copies of genomes of WT-gRNA. 508

Markers show average cross three replicate wells and error bars represent standard deviations. 509

In some cases, the error bar is not visible because it is smaller than the marker. 510 511 . 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.

The copyright holder for this preprint this version posted January 18, 2022. ; https://doi.org/10.1101/2022.01.17.22269439 doi: medRxiv preprint Table 1 . Variants included in this study (column 1), the characteristic mutations that ddRT-PCR 528 assays were developed for (column 2), the percent of variant genomes with the mutation(s) in 529 column 2 (column 3), the positive control used in the sensitivity testing experiments (column 4), 530 and the SARS-CoV-2 genomes that were used, along with the respiratory panel, in the 531 specificity testing conducted in vitro (column 4). Table 2 . Primer and probe sequences used in this study to target characteristic mutations in 537

variants. The variant containing the characteristic mutation is shown below the name of the 538 targeted mutation. 539 540 . 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 January 18, 2022. Table 3 . Frequency of sample collection for different assay applications, number of samples 544 included in this study, whether any of the data have been published, and the time range that 545 RNA samples were stored between extraction of RNA and running the PCR assays. RNA 546 extraction occurred on the day of sample collection, as explained in the methods. 547 548 549

. 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 January 18, 2022. ; https://doi.org/10.1101/2022.01.17.22269439 doi: medRxiv preprint

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