Discussion
Our results show that the HV69–70 and Del156–157/R158G mutation assays as used for wastewater settled solids were sensitive and specific. By using these PCR mutation assays, we found strong correlation between wastewater estimates and case isolate sequencing–derived estimates of circulating Alpha and Delta in 2 large metropolitan communities in California, USA. Mutations were detected in wastewater samples collected 1–3 weeks earlier than when Alpha and Delta variant estimates generated by case-isolate sequencing were available and reliable. Targeted mutation assays applied to SARS-CoV-2 RNA extracted from wastewater solids can be a rapid, efficient, and reliable way to monitor VOCs introduced to and circulating in a community. Monitoring for VOCs using wastewater may provide earlier complementary surveillance data than from case isolate sequencing data, if mutation assays are or can be developed for new and existing VOCs and put into use in a timely manner.
Use of PCRs targeting characteristic mutations thought to be particular to a SARS-CoV-2 variant may concurrently detect other SARS-CoV-2 strains that carry the same mutations. Targeting a single mutation in wastewater, as was done in our study for Alpha, carries an increased potential risk for mischaracterization. For example, on September 8, 2021, according to GISAID (https://www.gisaid.org), a global repository of case isolate sequence data, 1,043,561 (97%) of the 1,077,360 Alpha (B.1.1.7 and Q sublineages) sequences contained the HV69–70 mutation. However, HV69–70 was also present in other variants, such as B.1.258.19, where it was present in all 141 B.1.258.19 sequences in GISAID, and B.1.617.2, where it was present in 647 (0.2%) of 402,038 sequences. Targeting multiple mutations, as was done in our study with Delta, can increase specificity. Of the 937,570 sequences in GISAID classified as Delta (B.1.617.2 and AY sublineages), 842,354 (90%) have the Del156–157/R158G mutations (referred to as E156G/del157–158 in GISAID). Although this combination of mutations can be present in other variants, it is rarer; the non-Delta variant with the highest percentage of sequences with these mutations is B.1.617.3, for which there were 266 isolates in the global GISAID database and only 77 (29%) possessing these mutations. The non-Delta variant with these mutations for which there are the largest number of isolates in GISAID is B.1.1.7, for which 6 (0.0006%) of the >1,053,637 million sequences have these mutations.
Our findings show that use of mutation assays (HV69–70 for Alpha, Del156–157/R158G for Delta) to estimate circulating variants in wastewater correlated well with estimates from case isolate sequencing data. Wastewater estimates for Alpha, based on a single deletion assay, were robust over time in 2 large municipalities over 8 months (rp 0.82, p<10–5 in San Jose; rp 0.88, p<10–7 in Sacramento), including periods of high (tail of 2020 winter, 2021 summer) and low (2021 spring) community SARS-CoV-2 transmission. Similarly, estimates for Delta, based on multiple mutations, correlated highly with estimates from sequenced case isolates (rp 0.97, p<10–15 for both San Jose and Sacramento). Concurrent monitoring of VOCs in both wastewater and case isolates can confirm whether targeted mutation assays used are correlated with the VOCs being monitored and mitigate risks for misinterpreting wastewater results. Discrepant or divergent estimates between the 2 datasets should be noticeable within weeks and would suggest another variant with the same mutations circulating at abundance, prompting investigation if unexpected.
Emergence of the Omicron VOC in November 2021[3] provides an excellent example of the importance of interpreting wastewater mutation assay data in the context of case isolate sequencing data. Omicron also includes the HV69–70 mutation. At the time this study was conducted, the HV69–70 mutation, as noted previously, was rarely circulating in non-Alpha variants, suggesting that positive detections likely represented Alpha. However, Alpha disappeared from California circulation by end of summer 2021 and by December 2021, public health concern was for Omicron. With zero Alpha case isolates detected in either sewershed during September 1–December 1, the HV69–70 mutation assay was deployed on wastewater to screen for presence of Omicron, a more likely VOC to emerge in California than Alpha, while a more specific assay was developed.[22]
For validated assays deployed in established wastewater sites, wastewater surveillance for VOCs could be an important adjunctive estimate of variant circulation. Because cost and limited genomic testing capacity make sequencing all COVID-19 isolates impractical, especially during times of high case incidence, health departments and decision-makers extrapolate information from relatively small numbers or proportions of sequenced isolates, which may be biased and unrepresentative. For our case dataset, 14-day VOC estimates were derived from as few as 2–20 total case isolates and <1% of total cases sequenced.
Wastewater variant monitoring can overcome biases and delays seen with case isolate sequencing. Because everyone living in a sewershed contributes waste to the system, wastewater monitoring is independent of testing and care accessibility biases and results are more representative of cases in that sewershed. In addition, wastewater mutation assay results are available in a shorter time than VOC estimates from sequencing of case isolates. In our monitored sewersheds, the total average turnaround time from wastewater collection to testing results was <8 hours. In contrast, for our 2 sewersheds, it took 2–3 weeks after sample collection date for 75% of case isolate sequence results to be received and 3 weeks for most 14-day VOC estimates to be within 10% of their final estimate. These delays do not include the additional delay between case symptom onset and test taking that could further accentuate time advantages of wastewater variant monitoring.
Several limitations exist for using wastewater (solids or liquids) for SARS-CoV-2 variant monitoring. Laboratory limits of detection for SARS-CoV-2 RNA in wastewater and for targeted mutations may result in no detection, especially at times with lower community COVID-19 case counts and consequent lower overall concentrations of SARS-COV-2 RNA in wastewater. However, even in mid-May 2021, when case counts in these 2 sewersheds were as low as 1–2 cases/100,000 population, both SARS-CoV-2 RNA levels and variant abundance could still be measured and accurately estimated. Estimates of circulating Alpha and Delta mutations were also able to be consistently detected even at levels <5% of total SARS-CoV-2 RNA. Limits of detection, both for SARS-CoV-2 and for different mutations associated with variants, are likely to vary depending on laboratory methods used and which mutation is targeted; delineating these limits for each laboratory, sewershed, and assay is important for interpreting what a nondetect result implies about variant circulation.
Because newly identified variants to be monitored require new mutation assays to be designed, the time needed to design, test, and begin using assays is a crucial consideration (Appendix Figure 3). Although the time to design an assay in silico (<1 day) and test its sensitivity and specificity in vitro (3–5 days) is short, the time to receive reagents, including synthesized oligos and positive control RNA, from vendors can take 4–6 weeks because of supply chain issues and increased demand during the pandemic. In addition, before an assay can be designed, variant sequences and mutations must be accurately characterized, which can delay the assay design process. Efforts to develop assays before variants become VOCs and proactively order reagents can help ensure assays are available when needed for public health response.
Monitoring for VOCs will continue to be an important public health function and a need that will become more salient if SARS-CoV-2 testing of cases and sequencing resources or utilization decrease over time. Difficulty in surveillance based on case isolate sequencing, including difficulties attributable to nonrepresentative sampling and delayed results, mean that complementary variant surveillance methods are needed. Detection and monitoring of variants in wastewater has been proposed as an adjunct methodology, and our experiences monitoring for 2 VOCs in 2 large California municipalities support the use of targeted PCR mutation assays as a useful method to estimate abundance of circulating VOCs and inform public health. In conjunction with continued COVID-19 case isolate sequencing, wastewater variant monitoring can be strategically deployed as an adjunct public health surveillance tool.
Acknowledgments
We thanks the many people who contributed to wastewater sample collection, including Srividhya Ramamoorthy, Michael Cook, Ursula Bigler, James Noss, Lisa C. Thompson, Payak Sarkar, Noel un Enoki, and Amy Wong. We also thank the many groups involved in the sequencing of case isolates, including California, Santa Clara, and Sacramento County Public Health departments and the many public, private, and academic laboratories who have dedicated substantial resources towards the understanding of SARS-CoV-2 variants. In addition, we thank the California Department of Public Health COVID-19 Epidemiology and Data teams for their help with COVID-19 data.
Parts of this study were performed at Stanford University, which sits on the ancestral and unceded lands of the Muwekma Ohlone people. We pay our respects to them and their Elders, past and present, and are grateful for the opportunity to live and work here.
This work is supported in part by a gift from the CDC Foundation. This study was supported in part by the Epidemiology and Laboratory Capacity for Infectious Diseases Cooperative Agreement (no. 6NU50CK000539-03-02) from CDC. B.W., D.D., and B.H. are employees of Verily Life Sciences.
Emerging Infectious Diseases. 2022;28(5):940-947. © 2022 Centers for Disease Control and Prevention (CDC)
