Hyperplex PCR enables the next-generation of wastewater-based surveillance systems: long-term SARS-CoV-2 variant surveillance in Sweden as a case study

Wastewater-based epidemiology aims at measuring pathogens in wastewater as a means of deriving unbiased epidemiological information at a population scale, ranging from buildings and aircrafts to entire cities or countries. After gaining significant mainstream attention during the SARS-CoV-2 pandemic, the field holds significant promise as a continuous monitoring and early warning system tracking emerging viral variants or new pathogens with pandemic potential. To expand the current toolbox of analytical techniques for wastewater analysis, we explored the use of Hyperplex PCR (hpPCR) to analyse SARS-CoV-2 mutations in wastewater samples collected weekly in up to 22 sites across Sweden between October 2022 and December 2023. Approximately 900 samples were tested using a dynamic probe panel with a multiplexity ranging from 10- to 18-plex, continuously adapted within 1-2 weeks to quantify relevant mutations of concern over time. The panel simultaneously covered deletions, single nucleotide substitutions, as well as variable regions resorting to probe degeneracy. By analysing all samples in parallel resorting to gold standard methods including qPCR and two different NGS technologies, the performance of hpPCR is herein shown to bridge the gap between these methods by providing (1) systematic single nucleotide sensitivity with a simple probe design, (2) high multiplexity without panel re-optimization requirements and (3) 4-5-week earlier mutation detection compared to NGS with excellent quantitative linearity and a good correlation for mutation frequency (r=0.88). Based on the demonstrated performance, the authors propose the combined use of NGS and hpPCR for routine discovery and high-frequency monitoring of key pathogens/variants as a potential alternative to the current analysis paradigm.