Wastewater: On the trail of the Evolving COVID-19 Variants


Author: Katharina Schwaiger, acib GmbH

Even as we approach the end of 2023, the COVID-19 pandemic, which once seemed like a narrative out of a science fiction tale, continues to evolve. Austria, having lifted all COVID-19 restrictions, now faces the challenge of monitoring the virus’s ever-changing landscape. The persistence of COVID-19 is evident in the ongoing circulation of Omicron subvariants like EG.5 and BA.2.86, which are currently prevalent across Europe1.
In light of these developments, a July 2022 publication by Acib scientists Petra Heidinger, Fabian Amman, Andreas Bergthaler, Heribert Insam and Norbert Kreuzinger alongside other noted scientists, for example Peter Klimek and Niki Popper, who gained special media attention in their expert role during the Corona pandemic in Austria, gains renewed attention. Their research on wastewater-based epidemiology (WBE), featured in Nature Biotechnology, highlights the potential of this approach. The methodologies developed, particularly the VaQuERo system remains highly relevant as it provides critical insights into tracking the evolving strains of COVID-19.
This blog revisits the July 2022 publication to underscore the ongoing importance of WBE in our current health landscape. WBE not only aids in tracking the shifts in viral strains but also plays a crucial role in shaping public health responses in Austria’s post-pandemic era.

What’s the benefit of wastewater-based epidemiology?

Wastewater surveillance is not new in epidemiology. Detection and monitoring of the spread of viruses and pathogens, such as cholera, polioviruses, noroviruses and influenza have been used for decades. The emergence of COVID-19 has reaffirmed its role as a cost-effective tool for disease surveillance and early detection of infections in the population. It is difficult to test all individulas who have COVID-19. This is especially true for infections with a long incubation period, as was the case with early SARS-CoV-2 variants. In addition, asymptomatic infections are sometimes not even noticed by the affected individuals themselves. These populations, as well as individuals with limited access to health care, may be underrepresented in surveillance strategies based on screening and sequencing of individual positive cases. Neglecting such groups potentially introduces bias in the detection of variants with altered clinical manifestations. WBE can overcome this potential selection bias, as well as some economic limitations associated with whole-population testing. SARS-CoV-2 RNA is excreted in faeces, urine, and saliva from infected individuals, and what enters the sewage system can potentially be found in wastewater.

How to detect virus variants in wastewater?

In this study, scientists analyzed 3,413 wastewater samples from 94 municipal catchments, covering more than 59% of the Austrian population between December 2020 and February 2022. They used quantitative PCR (qPCR)-based approache, in which viral RNA was first transcribed into the complementary DNA sequence (reverse transcription). This DNA was amplified in a second step. The amplification step is important to obtain sufficient material for sequencing. The sequencing method must be very accurate to detect mutations in the DNA that are specific to certain virus variants. This is called deep sequencing, and means that a genomic region is sequenced multiple times, sometimes hundreds or even thousands of times. The challenge is to analyze this sequencing data for the regional occurrence of specific variants (a total of 33 variants were included in the analysis). They began by grouping the detected mutations into mutations for specific variants and created a comprehensive set of marker mutations. This allowed them to develop a method, which they named VaQuERo (variant quantification in sewage designed for robustness), to detect and quantify the variant composition in the wastewater over time. Moreover, to visualize the regional patterns of expansion and decline of single variants, they reduced Austria to its main axis of mobility (west-east and east-south direction) and were thus able to generate a space-time coordinate system. This was demonstrated by the steady retreat of B.1.160 and B.1.258 and their replacement by Alpha, which began in January 2021 in the eastern provinces of Austria and was completed only about three months later in the western provinces. The displacements of Alpha by the Delta variant in early summer 2021 and subsequently of Delta by Omicron in winter 2021/22 began almost simultaneously throughout Austria and were completed within one month.

Detection of one single case of infection

All of these data were validated through a comprehensive integration with available surveillance data from individual cases in the catchment areas (>310,000). Between January 1st 2021 and February 10th 2022 50.6% of all positive cases in Austria were tested for variants. Comparison of these two data sets (wastewater sequencing and individual case testing) indicated a high concordance. In some cases, even time points with a single confirmed infection case in the catchment area were reflected by a proportionate signal in the wastewater analysis. However, the number of individuals connected to the sewerage system influenced the sensitivity in detecting individual infection cases. The authors found that a variant can be reliably detected if it is linked to more than 2 cases and more than 3.75% of all cases in the catchment area. In addition, the data can further provide useful information about the spreading benefit of certain variants, thus, their reproduction number. Overall, the comparative analysis revealed that wastewater monitoring provides a detailed reflection of the epidemiological dynamics at regional resolution across Austria.

Initial recognition of mutation constellations

The detection of newly emerging variants is as important as the detection and quantification of already known variants. However, it is a difficult task to decipher information about individual variants of viruses from the totality of genetic information found in wastewater. Moreover, sequenced DNA fragments are rather short (~400 bases) and linkage of mutations across these fragments is limited. To overcome these obstacles, the scientists clustered mutations according to their corresponding frequency pattern in time and space. They identified a mutation constellation in Carinthia, which they again used as input for VaQuERo. VaQuERo immediately indicated that the observed constellation was indeed new, once again impressively demonstrating the accuracy and wealth of information of sequence-based wastewater monitoring.


WBE offers a high-resolution surveillance of epidemiological trends while requiring fewer samples and less logistical effort compared to traditional methods. In the current context, where individual case-based surveillance faces challenges due to the reduced frequency of testing and the presence of multiple variants, WBE stands out as a crucial tool. It provides a broad overview of the pandemic situation, supplementing and informing traditional epidemiology, and aiding in the adjustment of testing strategies at a regional level. As we continue to navigate the pandemic, comprehensive wastewater monitoring emerges not just as a scientific tool, but as a strategic asset in understanding and responding to the dynamic nature of COVID-19.

Link to online publication:

Picture credits: Pixabay