The ongoing COVID-19 pandemic has reminded us once again of our vulnerability, as individuals and as a society, to infection diseases. To mitigate some of the ramifications, an effective pandemic management requires dependable information on the current situation and future trends. This way, ordinances, from mask mandates, up to closure of businesses, restrictions on private gatherings, and the relieve thereof, can be adjusted to the actual necessities. During the last two years, able countries around the world spend considerable resources to mass test their populations to gain a current overview of the pandemic situation in terms of case numbers and genetic make-up of circulating virus variants. To this end, people with and without symptoms, were incentivised to regularly test for SARS-CoV-2. Some positive samples underwent further examinations to determine the present virus haplotype. Beside high expenditures to maintain such elaborated programmes, this approach comes with a critical dependency on an active and steady participation of a representative share of the public. Hence, gaining a comprehensive overview of the entire population might be impeded, since asymptomatic individual are likely to be less compliant to any voluntary testing scheme, public health policies get more and more politicised, and generally, access to the public health system is unequally distributed across the society. Furthermore, economic and logistic constraints are serious obstacles for a global implementation of such individual case-based testing programmes, leaving substantial parts of the worlds in the blind spot of pandemic surveillance.
In recognition of these intricacies, alternative ways to gain insights over the pandemic situations were suggested early on during the pandemic. Notably, wastewater-based epidemiological methods experienced a strong impetus. SARS-CoV-2 infected individuals can shed virus through their saliva, sputum, urine, and stool, often already days before they develop symptoms. These excretions can make their way into the sewer, where they mix with the wastewater from other sources and are transported into a receiving water. On their way there, samples can be drawn, representing the aggregated signal of the catchment population connected upstream. With appropriate molecular techniques, the virus in the wastewater can be quantified and examined despite its high dilution. After concentration and extraction of the virus genomes, its abundance can be determined with real-time polymerase chain reaction (RT-PCR). After normalisation for the number of contributing individuals, trends in the wastewater signal were shown to reflect the epidemiological dynamic in the associated catchment population. Even though the analysis of a single wastewater sample needs slightly more laborious procedures, such a testing scheme comes with several potential advantage over testing individuals. Foremost, a single sample can represent accumulated information of dozens to millions of individuals, driving down operating expenses to register the entire population. Furthermore, individual turnout is maximized since everyone connected to the sewer is automatically participating.
Beside the quantification of virus material, the amalgamation of virus particles can also be used to learn about the genetic composition of the virus population circulating in the catchment. To this end, the genetic material, RNA in the case of SARS-CoV-2, is amplified, sequenced, and analysed for virus mutations and their frequencies within the examined virus population. While virus quantification became a standard practice for many public health authorities around the world, deducing pathogenomic information through sequencing is less established yet. In our contribution we explored the potentialities of variant-resolved wastewater surveillance of SARS-CoV-2 at national-scale in Austria/Europe. Austria is especially suited for such a proof-of-principle study due to several reasons. In Austria 93% of the population is connected to a public sewer network. Its extensive health care system conducted a comprehensive case-based variants surveillance programme, providing an in-depth understanding of the circulating SARS-CoV-2 variants. Beside the globally dominating Alpha, Delta, and Omicron virus variants, Austria experienced local infection clusters of Beta, Gamma, and Mu virus variants. Last, but probably most importantly, through the initiative of academic institutions and with support from public authorities, wastewater-based SARS-CoV-2 surveillance was established as an important tool for pandemic management already early on during the pandemic.
Equipped with these premises we started out to explore the potential of sequencing SARS-CoV-2 genomes from wastewater samples to delineate the pandemic variant dynamics in the Austrian population. To this end, between December 2020 and February 2022, we processed 3,413 wastewater samples from 94 wastewater treatment plants, with varying catchment population sizes, ranging from as small as 1,490 up to 1.9 million persons. Collectively, around 60% of the Austrian population discharge their wastewater into a monitored wastewater treatment plant. We developed a dedicated software tool, names VaQuERo (short for Variant Quantification in Sewerage designed for Robustness), to deduce the relative virus variant abundance from the complex mutation patterns observed in the deep sequenced samples.
Comparison with comprehensive case-based virus variants surveillance records of more than 300 thousand cases, validated that the wastewater signal indeed allowed a robust deduction of the circulating virus variants and their share of the overall rate of new infections. The overall sensitivity of our surveillance set up could be evaluated. The data suggest that a signal of more than 2 cases within a catchment or a relative share of 3.7% can reliably be detected. Given the fact that the population size of the catchment has profound impact on the observed sensitivity, our data allow to optimize the size of surveillance (sub)catchment. Building on the robust virus variant quantification, the relative growth advantage of single variants could be calculated for individual catchments, corroborating previous reports on the advantage of Alpha against prior prevalent variants, and the advantage of Omicron against the Delta variant. We also explored the possibility to deconvolute novel variants from the rather noise afflicted wastewater sequencing data. For that purpose we attempted to detect the rise of the Alpha variant specific genotypes without using prior information on Alpha specific mutation patterns. Instead, we grouped mutations which exhibited a similar frequency dynamic in a collocation of catchments. Indeed, we could show that, even though challenging, wastewater-based epidemiology enables to detect the emergence of unknown genotypes.
Altogether, our national scale, long term surveillance study demonstrate that examining wastewater allows to draw a detailed picture of the epidemiological situation of an infectious disease across a broad population with maintainable efforts. Key parameter, such as prevalent variants and their effective reproduction number can be reliably deduced. This allows a second, independent perspective in situations where individual case-based surveillance is challenged. Especially, it provides insights into populations which are not easily accessible by classic public health programmes. Be it due to their hesitance to actively participate or be it due to logistic and economic constraints. The latter might be of particular value to close surveillance gaps in low resource areas on a global scale.
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