The hunt for Omicron in wastewater

Wastewater sector provides expertise in detecting Omicron variant. © iStock / -1300948439

Two years on from the initial COVID-19 outbreak, the virus continues to disrupt lives and create uncertainties, especially because of variants. The Source reviews a recent webinar on how the sector is responding to this evolving picture.

In August 2021, The Source highlighted the technical approaches being developed to track the COVID-19 virus SARS-CoV-2 and emergent variants within wastewater. Experts across the globe continue to learn about the virus, gaining knowledge about its transmission, the effectiveness of vaccines and how the illness can manifest in different ways. And the wastewater community is embracing its surveillance role by helping to monitor the ongoing presence of COVID-19 in communities and tracking the speed and transition of one variant to another.

Wastewater-based Epidemiology

Wastewater-based Epidemiology (WBE) methods are being refined to support public health decision-making. During a recent IWA webinar, chaired by Professor Joan Rose, of Michigan State University, the IWA COVID-19 Task Force convened microbiologists and epidemiologists to discuss the progress that has been made in monitoring COVID-19 and how this could be developed further.

Wastewater has proved to be a valuable source of information in the campaign to manage the pandemic. Wastewater samples enable fragments of genetic code to be tested from thousands of people, providing the opportunity to study and track the combinations of mutations that define each variant.

Variants – unlocking the mutation profile

The primary task in COVID-19 surveillance is to identify which variants are present and establish whether they are emerging, dominant or waning. The main difficulty is that, because variants are defined by their full combination of mutations, detecting a single variant mutation does not prove the variant itself is present.

Professor Gertjan Medema, principal microbiologist at KWR, leads the use of digital droplet-PCR assessment to track SARS-CoV-2 in the Netherlands. During the webinar, he explained how the Alpha variant that emerged in the UK early in 2021 contained the N501Y, E484K and K417 mutations. However, the N501Y mutation was missing from the Delta variant and traces of the N501Y gene began to disappear from wastewater samples as the Delta variant became more dominant.

The mutation profile for Omicron (variant number B.1.1.529) with 50 mutations is the most mutated variant so far, but, interestingly, the N501Y mutant gene has returned. This ‘inserted’ gene makes it easier to discern Omicron from Delta in the wastewater matrix.

Tracking variants in wastewater

Researchers around the world are developing tools and techniques to define definitively when a variant is present. One approach is to develop Targeted RT-PCR assays to hunt for the unique sequence of mutations of a specific variant. Once clinical data has confirmed the genetic code of a variant through sequencing, the target mutations are identified, for which a specific biological assay is then developed and tested.

Targeting the search for variants in this way offers a high degree of accuracy and is relatively cheap with a fast turnaround (compared with clinical data). However, this approach is dependent on the clinical data phase, and the supply chain for diagnostic kit is beset with delays.

An alternative method is sequencing using a digital droplet-PCR. Here, the range of SARS-CoV-2 genes found in the wastewater are examined and the analysis focuses on the presence of ‘inserted’ or ‘deleted’ mutant genes, indicating the likely emergence of a variant compared with the existing dominant. This approach can be used while waiting for variant-specific assay probes to be delivered.

With the appearance of Alpha in early 2020, work to track it in Amsterdam and Utrecht began immediately. The role of wastewater surveillance was recognised by the European Commission as a tool that could deliver results faster, and at a higher resolution, than the clinical data. EU member states were then recommended to undertake wastewater surveillance.

The August edition of The Source highlighted the effectiveness of national surveillance plans. This included an approach in Denmark where mobile wastewater testing units were sent to areas experiencing exponential growth in infection rates to track the speed of transmission. However, according to Jesper Gamst, from Eurofins Environment Testing, the unprecedented speed at which Omicron spread soon ruled out this approach and the authorities recognised the need to build decentralised capacity with shorter supply chains.

Aircraft sanitation – the key to international travel?

A project targeting four of Australia’s major international airports is spearheading progress towards using wastewater for almost real-time COVID-19 monitoring. Warish Ahmed, senior research scientist in the environmental contaminant mitigation and biotechnologies programme at CSIRO, is leading a pioneering project to find out if analysis of aircraft wastewater samples could reduce passenger quarantine times and increase confidence in international travel. The pilot project samples material from the lavatory survey traps of individual airline sanitation systems to add another layer to the clinical data being used.

As part of this project, 37 repatriation flights to Australia from India, the UK and France were sampled, with 6570 passengers testing negative prior to boarding. During 14 days of quarantine, all passengers were screened multiple times using PCR. The clinical data was then compared with the results from the flight sanitation systems.

More than 100 of the passengers tested positive while in quarantine, all of whom had flown on flights where SARS-CoV-2 was found in the wastewater. The vast majority of flights where SARS-CoV-2 had not been found in the wastewater returned no positive cases. However, a very small number of passengers did test positive from flights where SARS-CoV-2 was not detected in the wastewater.

At the time of writing, a further 240 aircraft arriving in Australia had been sampled and analysed. However, the project has identified limitations, including saliva-based inputs to the sanitation systems (from teeth cleaning, for example), and the health and safety risks to personnel of exposure to SARS-CoV-2 and other viruses from this type of decentralised sampling regime. To improve the project’s performance further, the team is seeking rapid onsite analyses in preference to transferring samples to centralised labs, which can take up to five days to process. As the teams work to remove these risks, the results demonstrate the value of using wastewater surveillance alongside clinical data to provide multiple lines of evidence.

Zoning in on settled solids

It is important when assessing the success of Ahmed’s pilot project to recognise that aircraft wastewater is much more concentrated than regular wastewater because of the lack of dilution. A study led by Alexandra Boehm, of the Woods Institute for the Environment at Stanford University, is focusing on the differing capacity of settled solids and diluted wastewater influent for the detection of SARS-CoV-2 (and variants of concern).

To inform this study, 12 wastewater plants in California are generating daily data from analyses on settled solids collected from primary clarifiers or settled influent. Using the targeted RT-PCR assays approach, researchers have found that SARS-CoV-2 RNA is enriched by orders of magnitude in solids compared to influent. Comparisons with local clinical data show strong correlations in concentrations of single mutations (characteristic of a variant) and patterns of variant within the local population. The project team is confident that the process can now detect SARS-CoV-2 variant RNA at the level of 1:100,000 cases in the sewershed.

A virus that continues to surprise

The Californian project has used the specific assays approach to identify the variants found in the settled solids. Through daily monitoring, the team detects the transition of variants served in the sewersheds. In other viruses, variants tend to be completely replaced as the virus evolves, and so SARS-CoV-2 variants should be expected to disappear. SARS-CoV-2, however, continues to surprise, as data from California, Denmark and the Netherlands all show previous variants lingering (in very small proportions, ~0.1%) rather than disappearing completely.

These results continue to strengthen the evidence for the role and value of wastewater surveillance (in combination with clinical sequencing), to identify and track SARS-CoV-2 variants, for the purpose of local, national, and international decision-making. To continue to build on current understanding, the wider wastewater community is being called on to donate data and information about testing methodologies to global data networks, including the Wastewater SARS Public Health Environmental REsponse (W-SPHERE),

More information

View this webinar at:

W Ahmed, et al, Environment International, Wastewater surveillance demonstrates high predictive value for COVID-19 infection on repatriation flights to Australia, 10.1016/j.envint.2021.106938