Abstract
BACKGROUND: During the COVID-19 pandemic, the spread and diversity of SARS-CoV-2 was monitored non-invasively in more than 99% of Dutch households by a comprehensive wastewater-based epidemiology (WBE) programme which analysed viral RNA in wastewater samples taken at every household wastewater-treatment plant (WWTP) in the Netherlands. In this study, we analyse next-generation sequencing data generated from these wastewater samples for tracking SARS-CoV-2 lineages. We aimed to determine how well patterns of lineage abundances correspond to findings from individual surveillance, including community testing and testing at hospitals, and how WBE can be employed efficiently for future surveillance of SARS-CoV-2. METHODS: Whole-genome short-read sequencing was performed on 16,631 wastewater samples collected between Apr 9, 2021, and Dec 31, 2023, at 311 WWTPs. Observed lineages were grouped and week-aggregate estimates were compared to estimates from 138,374 individual surveillance sequences generated during the same period. Furthermore, we used resampling to examine the effect of sample size and catchment type on observed lineage dynamics. RESULTS: WBE enabled monitoring of SARS-CoV-2 lineages even during periods of reduced circulation in the Dutch population. Wastewater lineage proportions consistently mirrored those of individual surveillance. During periods of low turnover of dominant lineages, nationally representative lineage estimates can be obtained by sequencing only 24 samples per week, whereby population density or total population size of catchment areas would have a negligible effect on country-wide aggregate lineage estimates. CONCLUSIONS: WBE at household WWTPs is a valuable tool for SARS-CoV-2 surveillance, whereby comparatively little sequence data can be generated to identify lineage dynamics trends in large populations, and which can be scaled up and down in response to emerging lineages of concern. As such, it provides a useful complement to existing surveillance tools, to ensure optimal insight into pathogen diversity and spread.