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. ### Competing Interest Statement U.N. and R.S. are co-inventors of the hpPCR methodology according to patent application WO/2021/206614. U.N. holds shares in Aplex Bio AB. Other authors declare no competing financial interests. ### Funding Statement The data handling was enabled by resources provided by the National Academic Infrastructure for Supercomputing in Sweden (NAISS), partially funded by the Swedish Research Council through grant agreement no. 2022-06725. Ion Torrent sequencing was performed by the SNP&SEQ Technology Platform in Uppsala. The facility is part of the National Genomics Infrastructure (NGI) Sweden and Science for Life Laboratory. The SNP&SEQ Platform is also supported by the Swedish Research Council and the Knut and Alice Wallenberg Foundation. This work was funded by governmental grants provided to the Public Health Agency of Sweden for assignment S2022/04841 ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study used only openly available human data obtained from the open data portals CoV-Spectrum (https://cov-spectrum.org) and GISAID (https://gisaid.org). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors