Mariana Matus has spent years studying what comes out of human bodies in order to better understand what is happening inside us. The computational biologist helped develop heavy-duty devices that are about the size of a milk crate and can be lowered into manholes to dangle over wastewater—steadily sucking up a stream of urine and feces through a strawlike tube.

Matus and her colleagues originally set out to scour sewers for chemical signatures of drug use. But in February reports emerged that it was possible to detect the genetic signature of the novel coronavirus in human stool. So Matus and a company she co-founded, Biobot Analytics, turned their attention to tracking the COVID-19 pandemic. They are now sampling sewage collected from 400 waste treatment plants, which, Matus says, cover an estimated 10 percent of the U.S. population.

It is one of dozens of similar efforts being deployed around the world in an attempt to help countries better focus their containment measures by painting a fuller picture of where and how widespread the virus is. “The big unknown at the moment, because there is still very limited testing going on in [the U.S.], is: No one has a clue how many people are infected and asymptomatic. Not a clue,” says Ian Pepper, an environmental microbiologist at the University of Arizona, who studies pathogens in municipal sewage. Conducting existing diagnostic tests on an entire population is impractical, and such tests are not equitably distributed. Everyone uses the bathroom, however. Thus, wastewater sampling allows for testing for the novel coronavirus’s presence in millions of people all at once, regardless of their socioeconomic status or access to health care. “Our data is naturally aggregated and anonymized at its rawest form,” Matus says.

Still, there are limits to how the approach could be applied in the current pandemic. The technology cannot definitively tell epidemiologists whether an entire population is clear of the virus. But it could be a faster way to detect future flare-ups as regions emerge from lockdown. The approach may also boost the small but growing field of wastewater-based epidemiology—and expand it to detect other diseases.

An Early Warning

Like other viruses, the pathogen causing COVID-19 cannot copy itself. Instead it infiltrates living cells—including those lining the human gut—and enlists them to actively replicate its RNA. Infected patients then shed viral RNA particles in bodily fluids, including saliva, mucus and feces. These end up down the drain in wastewater, where they can be found at levels high enough to be detectable.

Sampling RNA particles from wastewater can help researchers do a few things: For one, they can sequence the coronavirus’s genes to see how various strains mutate and change over time. Such analysis also allows researchers to track the pathogen’s spread, covering all the strains in circulation and peering into their “viral ancestry.” In a recent preprint study, which has not yet been peer-reviewed, microbiologists determined that the dominant strain plucked out of sewage in one city in Montana likely descended from a viral lineage circulating in Europe.

Testing wastewater using the same diagnostic method for processing swab samples (usually taken from individuals’ nose) also answers the simple question of whether or not the coronavirus is present in a given community. “The potential for looking at yes/no—whether it’s there or not—is already here and can be applied on a broad scale pretty quickly,” says Kevin Thomas, a professor at the Queensland Alliance for Environmental Health Sciences in Australia. He published a study demonstrating this technique in Science of the Total Environment in April. A group in Spain, publishing in Water Research, detected positive wastewater samples in an area more than two weeks prior to any COVID-19 cases being diagnosed there—even in places with low prevalence. That early warning could give policy makers a guide for when to lift or reinstate strict shelter-in-place measures. “If it starts spiking up again in certain locations, as we start opening cities, that feedback is going to be really important,” Matus says.

The work has progressed to the point where there are now serious discussions in some countries about expanding existing monitoring. And in the U.S., there are talks about building a new national surveillance program. Vincent Hill, a researcher at the Centers for Disease Control and Prevention’s Waterborne Disease Prevention Branch, says the agency is currently working with researchers to check the reliability of testing with the methods and to standardize them. Wastewater testing will never replace regular individual testing, he says. Yet it is a valuable, quickly obtained source of data about a whole population. Angela Rasmussen, a virologist at Columbia University, agrees. “I see wastewater testing as a potential surveillance tool that could be particularly helpful in terms of managing limited resources like PPE [personal protective equipment] and testing supplies,” she says. “But by no means is it an alternative to diagnostic testing and epidemiological work.”

False Negatives and Unknowns

There are limitations to the usefulness of wastewater detection. One issue is that the consolidated nature of municipal waste means researchers cannot pick out specific high-risk microcommunities such as nursing homes, hospitals, prisons or meatpacking plants. Another is the uncertainty around exactly when RNA from SARS-CoV-2 (the official name of the virus that causes COVID-19) becomes detectable in feces, relative to the onset of symptoms. And scientists have yet to fully clarify how long people continue to shed virus particles after they have recovered. Thomas says it is also unclear how many copies of viral RNA must be present in a sample before it can be detected. According to a model published in April in Science of the Total Environment, a given positive wastewater sample might reveal one infection in as few as 114 people—or as many as two million. But without knowing the minimum number of copies needed to produce a positive result, there is the possibility that a test could generate a false negative, erroneously suggesting a particular area is free of COVID-19. So testing wastewater can indicate that the virus is present but not definitively confirm that it is absent. To work around this problem, many researchers are tracking how the relative number of RNA particles detectable in a specific location changes over time.

Tracking that concentration of particles is the most important step for population-level monitoring, says David Jones, a soil and environmental science researcher at Bangor University in Wales. The reason is that doing so helps chart the course of the pandemic and monitor the effectiveness of lockdowns. Several groups, including Matus and her colleagues, are going a step further: they are attempting to estimate how the concentration of viral particles in wastewater corresponds to the total prevalence of disease in a given population. These calculations attempt to account for many variables, such as the diluting effect of rainfall and storm runoff. In a preprint paper still undergoing peer review, Matus’s group estimated that in one area of Massachusetts, the percentage of people likely infected with COVID-19 was at least five times the number who had officially tested positive—suggesting there were more undiagnosed cases in the community. A more recent preprint by a group in Connecticut found that in a metropolitan area, the concentration of RNA particles peaked about a week before the arrival of new diagnosed COVID-19 cases. But Jordan Peccia, an environmental engineer at Yale University, points out that it remains difficult to pinpoint an exact total of cases just from sewage, precisely “because [regular] testing is limited to only those that have symptoms and go get tested.”

To longtime observers, the renewed interest in the technique reflects a broader maturation of wastewater-based epidemiology. Christian Daughton, a retired Environmental Protection Agency researcher who co-wrote an influential 1999 review paper, recently suggested that the pandemic has already eroded some traditional concerns, such as those about privacy rights. The urgency of dealing with COVID-19 could help establish such monitoring systems, which could help not only with the response to this pandemic but also in tracking other diseases—possibly even new ones that emerge in the future. “I suspect that sewage surveillance will be much more in vogue following this experience with the pandemic,” Pepper says.

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