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

Abstract

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 Δ69–70 and ORF1a Δ3675–3677) 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 Δ69–70 was 0.34 (95% confidence interval (CI): 0.30–0.39) and based on ORF1a Δ3675–3677 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.

Monitoring of SARS-CoV-2 variant dynamics in wastewater by digital RT-PCR : from Alpha to Omicron BA.2 VOC

“…The correlation of these results with the regional incidence curve was also the best fitted. The overall dynamic revealed a temporal advance in agreement with other results (Caduff et al, 2022; Cluzel et al, 2022; Wurtzer et al, 2022). Nevertheless, this temporal advance on the wave related to the Omicron VOC appeared to be greater than the advance reported for previous VOCs.…”

“…This study also highlighted the limitations of sequencing in wastewater with respect to analytical sensitivity that may result from the low concentration of SARS-CoV-2 genomes, the very low concentration of mutations in minority variant populations, insufficient sequencing depth, and non-homogeneous coverage along the entire viral genome. Similar limitations have been already reported (Caduff et al, 2022). The accumulation of mutations identified by sequencing is also challenged by the reconstruction of the genomes from which these mutations originate.…”

“…Beta and Gamma VOCs represented at most 13.5% of the sequenced strains (April 26, 2021) and their detection in wastewater was not investigated in this study. The maximum VOC replacement rate for Alpha VOC (2,8%/day) was very close to the growth rate (4%/day) estimated in another study (Caduff et al, 2022). It was quite remarkable that the maximum replacement rate has progressively increased as new VOCs have emerged, suggesting increasingly contagious variants.…”

“…Variant identification results from sequencing, but such approach faces limitations for application in wastewater with respect to RNA quality and quantity (Lou et al, 2022), but also mathematical tools to deconvolute isolated mutations to reconstruct the original genomes in a mixture of variants (Agrawal et al, 2022b; Jahn et al, 2021; Smyth et al, 2022). Different studies have shown that it is also possible to monitor specific mutations suggestive of VOC to identify the emergence of VOC in a territory and to explain the predominance of certain VOC in epidemic waves (Caduff et al, 2022; Carcereny et al, 2022; Erster et al, 2022; Wurtzer et al, 2022). RT-qPCR approaches require calibration tools for absolute quantification or allelic discrimination (wild-type vs mutated) for relative quantification.…”

“…To overcome these limitations, targeted and specific RT-qPCR approaches have been proposed to detect certain mutations suggestive of variant presence or functional mutations whose evolution could reveal the spread of variants designated as VOC (Wurtzer et al, 2022). Different RT-PCR methods have been proposed based on either relative quantification of mutations to the total SARS-CoV-2 genomes (Erster et al, 2022; Wurtzer et al, 2022; Yaniv et al, 2021), either on wild-type and mutated allelic discrimination (Graber et al, 2021), quantification of mutations and total genomes by digital PCR (Boogaerts et al, 2022; Caduff et al, 2022; Heijnen et al, 2021; Lou et al, 2022). These approaches have been developed and applied to the emergence of Alpha and Delta VOC.…”