Inferring transmission fitness advantage of SARS- CoV-2 variants of concern from wastewater samples using digital PCR, Switzerland, December 2020 through March 2021

Background: Throughout the COVID-19 pandemic, SARS-CoV-2 genetic variants of concern (VOCs) have repeatedly and independently arisen. VOCs are characterised by increased transmissibility, increased virulence or reduced neutralisation by antibodies obtained from prior infection or vaccination. Tracking the introduction and transmission of VOCs relies on sequencing, typically whole genome sequencing of clinical samples. Wastewater surveillance is increasingly used to track the introduction and spread of SARS-CoV-2 variants through sequencing approaches. Aim: Here, we adapt and apply a rapid, high-throughput method for detection and quantification of the relative frequency of two deletions characteristic of the Alpha, Beta, and Gamma VOCs in wastewater. Methods: We developed drop-off RT-dPCR assays and an associated statistical approach implemented in the R package WWdPCR to analyse temporal dynamics of SARS-CoV-2 signature mutations (spike Delta 69-70 and ORF1a Delta 3675-3677) in wastewater and quantify transmission fitness advantage of the Alpha VOC. Results: Based on analysis of Zurich wastewater samples, the estimated transmission fitness advantage of SARS-CoV-2 Alpha based on the spike Delta 69-70 was 0.34 (95% confidence interval (CI): 0.30-0.39) and based on ORF1a Delta 3675-3677 was 0.53 (95% CI: 0.49-0.57), aligning with the transmission fitness advantage of Alpha estimated by clinical sample sequencing in the surrounding canton of 0.49 (95% CI: 0.38-0.61). Conclusion: Digital PCR assays targeting signature mutations in wastewater offer near real-time monitoring of SARS-CoV-2 VOCs and potentially earlier detection and inference on transmission fitness advantage than clinical sequencing.

Automated summary

This study adapts a rapid, high-throughput method to detect and quantify the relative frequency of characteristic deletions of the Alpha, Beta, and Gamma variants of SARS-CoV-2 in wastewater. The results provide insights into the transmission fitness advantage of the Alpha variant and demonstrate the potential of wastewater surveillance for real-time monitoring of VOCs.