How oysters led to
COVID-19 testing

Situated on the French Mediterranean near Montpelier, the Thau lagoon has a thriving shellfish industry that accounts for 10% of France’s annual oyster production. Oysters are well known for their ability to clean waters as they feed on phytoplankton by filtering it through their gills, and in effect, they also trap bacteria, nitrogen, phosphorus, sediments and harmful contaminants. This is great for water quality, but the stored contaminants could potentially be harmful for human consumption. It’s for these reasons that the impact of industrial and residential wastewater overflow has long been a concern to oyster farmers in the Thau lagoon. And then 2020 introduced a new threat: SARS-CoV-2.

That’s when the local authorities asked I.A.G.E., a French startup founded in 2017 that specializes in environmental biological analyses and digital PCR, to assess water samples from the lagoon. “They asked us if the virus is in the lagoon and if it would infect the oysters,” says Dr.Olivier Couillerot, co-founder and head of business development at I.A.G.E., headquartered in nearby Grabels.

Sampling wastewater for answers

In the past, monitoring sewage in communities’ collection or treatment system had provided early surveillance of polio prevalence at a population-wide level. Today, scientists sample wastewaters in the hope that this approach may be similarly beneficial for the current COVID-19 pandemic. “Eventually, somewhat like David against Goliath, we decided to exploit the digital PCR,” says Dr. Franz Durandet, president and head of R&D.

“Currently, the detection of SARS-CoV-2 in sewage is based on DNA sequencing and RT-PCR. These technologies, though, are not very reliable due to inherent technical issues. For this reason, we decided to develop a new, extremely reliable method using dPCR to overcome these limits,” Couillerot explains. “We were initially really surprised to detect huge loads of SARS-CoV-2 in the waste waters. Based on this and from the scientific literature, we could correlate the amount of SARS-CoV-2 that we could detect in the waste waters to the amount of the population infected. And we’d already analyzed wastewaters contaminated with SARS-CoV-2, so we had samples to validate our method with. As soon as the viral sequences of SARS-CoV-2 and its variants became publicly available, we designed specific probes to target mutations of interest and discriminate them.”

Digital PCR reveals mutating COVID strains

The team uses QIAGEN´s QIAcuity, for precise and multiplexed quantification results for mutation detection and gene expression, and can process more than 1000 samples per day. “A small company like ours must work in a very cost-efficient manner; for example, by screening large numbers of samples automatically. Moreover, our instruments must be reliable to ensure robust and sensitive results,” Couillerot says.

Their approach quantifies the virus in wastewaters, correlates viral loads in sewage with diagnostic testing, and traces three mutations identified in the 3 most known variants of SARS-CoV-2 (British, Japanese and South African). “Our analytical approach relies on the digital PCR,” Couillerot says, “because it’s a very sensitive and reproducible tool to measure the amount of DNA and RNA molecules in a sample. The main advantage of digital PCR in this aspect is the specificity. With digital methods, we are able to detect one mutation within the sequence we are targeting, which is very low.”

Preventing future outbreaks

A population vulnerable to COVID is elderly people with comorbidities. In France, many people in this category live in établissements d’hébergement pour personnes âgées dépendantes (EHPAD), which are long-term residential facilities for senior citizens who require daily care. “We are already proposing and testing methods in EHPADs,” Couillerot says. By testing the wastewater from a specific building, they could identify if there are any infections, and if so, which strain is responsible.

We have developed specific automated collector equipment that can be deployed at the wastewater outputs of a building, getting a sample representative of 24 hours. After we have worked on the extraction and concentration process, and then analyzed by digital PCR, we’ll be able to deliver results about the whole population located in this area in those 24 hours. We developed this for EHPADs specifically, but it can be used for ministries and other public buildings. This is really additive information. It’s really important to be able to manage the epidemic as variants spread faster and might be able to resist the vaccine.”

The I.A.G.E team believes this technology could help detect viruses in wastewater responsible for other conditions, such as gastroenteritis and influenza. Couillerot says, “We are really convinced that we can change the way that environmental diagnostics are done now.” And in the end, it did turn out that the oysters tested negative for the disease.