John Leavell bends down, grabs a 50-pound cast-iron manhole cover with a T-hook, and slides it aside. He then attaches one end of a thin hose to a battery-operated pump and drops the other into the darkness below. “Yesterday we were unable to retrieve any samples,” says Leavell, a contractor with the nonprofit Current Water. “Everything just froze. It was not pleasant.”

The manhole, located outside the Baton Show Lounge in Chicago, is his second stop of the day. Once he and his team have pulled, labeled and double-bagged two 50-milliliter bottles of raw sewage here, they’ll drive across town to sample another manhole — then deliver their bounty to a lab of microbial ecology. Rinse and repeat, four days a week.

It is a ritual that takes place throughout the country. In September 2020, the CDC launched its National Wastewater Monitoring System to monitor COVID-19 upsurges using clues that Americans are evacuating. It became the first widespread use of wastewater-based epidemiology since the technique was first used to track polio in the mid-20th century, and it is already filling critical gaps in clinical testing.

Read more: Why scientists don’t want our shit wasted

“We know that people infected with SARS-CoV-2 shed fragments of the virus in their stool whether they have symptoms or not,” says microbiologist Amy Kirby, program manager at CDC. Wastewater surveillance thus detects infections from the entire population, including people who never seek a test or who take a test at home and neglect to report their results to a health service. And since the virus can be identified in stool early in infection, potentially days before noticeable symptoms appear, sewage can even predict future trends in cases.

From the sewers to the laboratory

Rachel Poretsky, an associate professor of biological sciences at the University of Illinois at Chicago, stands next to a chest-high stack of cardboard boxes in her lab. Each contains a sample of downstate wastewater surrounded by blocks of ice and labeled with a QR code by 120 Water, a supplier that quickly moved from shipping Chicago public school water samples for sewage lead testing during the pandemic. Also present are samples of treatment plants and samples taken from city manholes by Current Water and engineering firm CDM Smith.

Microbial ecologist Rachel Poretsky stands next to a fresh delivery of sewage samples. (Credit: Christian Elliott)

The past two years have been a whirlwind, says Poretsky — expanding the lab to receive, organize, process and record data from hundreds of samples in less than a day is hard work. The sewage-based epidemiology project is truly scientific at an unprecedented rate. “Usually when you start a new project, you spend time refining your methods, doing various experiments, and then settling on something,” she says – sometimes it takes decades. In this case, “everyone uses the analogy of building the plane while flying it”.

She and her colleagues load the samples into an instrument that concentrates pieces of virus using magnetic beads in a few microliters of water, then extracts the viral RNA. But labs across the United States are using a variety of methods to try to intensify the treatment, including centrifuges and even skimmed milk to cause the virus to clump together. Clinical tests skip these steps because viral concentrations in nasal swabs are high enough to be detected directly; wastewater, on the other hand, is a “complex matrix” of microorganisms, organic matter and SARS-CoV-2 fragments diluted in varying amounts of water.

Next comes the critical step: a reverse transcription polymerase chain reaction, or RT-PCR, which exponentially copies the target RNA sequences to detectable levels. The latest addition from the Poretsky lab is a digital PCR machine that divides a sample into 26,000 partitions with one piece of RNA per partition, on a tray that looks like a giant cartridge in a high-tech printer. Unlike standard PCR machines that spit out a simple “yes” or “no” to find out if the virus is present, this one tells scientists how many copies of RNA were in the starting sample – or other terms, exactly how much virus was in the sewage.

Poretsky Lab’s digital PCR machine. (Credit: Christian Elliott)

Poretsky then sends the analyzed samples to Argonne National Laboratory in suburban Chicago for sequencing. It’s the job of geneticist Sarah Owens to look for any mutations, like the 40 or more that usually match the omicron variant. “It’s quite a complex problem, to sort out these viral genomes that are very similar to each other to determine the variants of concern,” she says.

It’s even harder to sequence the virus from millions of contributors in a sewage sample, rather than a single person’s nasal swab. On the one hand, RNA can degrade in wastewater. Virus sequencing is a new challenge for Owens, who previously focused on DNA-based bacterial pathogens in samples from urban streams. Yet she recently managed to disambiguate the variants in the samples and calculate the relative abundance of each. By the time the next worrisome variant of COVID-19 emerges, she says, she should be able to track its spread over time in wastewater across the state.

And Poretsky’s lab archives all samples at -112 degrees Fahrenheit. That way, when a new variant inevitably arrives in the United States, she and Owens can go back to samples and sequencing data to find out exactly when it started showing up in the city. “I think a lot of people wish it existed when it all started,” Poretsky says. “We could have gone back and said, ‘Hey, was this here in April 2020? “”

Frozen samples chill to -80 degrees Celsius in Poretsky’s lab. (Credit: Christian Elliott)

From laboratory to public health action

The final challenge is to understand what the data means and how to make it “actionable,” in public health parlance. That’s where Aaron Packman, a professor of civil and environmental engineering at Northwestern University, comes in. Using maps of sewer lines, his team can chase outbreaks from any manhole. . “If you see a spike of SARS-CoV-2 RNA, it’s possible to go further upstream and pinpoint the source,” Packman says. “That’s something you can’t do with a sewage treatment plant, but you can do it once you’re working in the sewer system.”

Some challenges remain. When it rains, for example, sewage sometimes backs up into buildings or overflows into the nearby river and lake. During storms (made more frequent by climate change), sewage is diverted 300 feet underground and out of town to a 6.5 billion gallon reservoir. All of this means scientists have to adjust the volume to prevent diluted samples from skewing the data.

“It’s difficult to directly link a sewage measurement to an actual number of cases,” Packman says. “But we have accumulated a lot of data now and we can make better estimates of the total number of sick people using sewage data plus clinical data than clinical data alone.”

Modou Lamin Jarju, a lab technician in Poretsky’s lab, pipettes samples. (Credit: Christian Elliott)

The Illinois Department of Public Health and the Chicago Department of Public Health meet with the research team every two weeks to discuss trends in wastewater data and plan where to deploy more testing, clinical vaccination and additional hospital staff based on this data. “Everything with COVID is new, including sewage monitoring,” says CDPH medical director Isaac Ghinai. “And so there’s a lot to understand about this data before it can be used in exactly the same way as case-based surveillance when there’s a bit more history.”

With the monitoring system finally expanded and data pouring in, sewage has become commonplace. Even if COVID-19 eventually relents, some public health departments hope to use wastewater to monitor future unknown pathogens, monitor drug-resistant organisms in long-term care facilities, track influenza seasonally, and even find hotspots for opioid use.

“The infectious disease tracking system in this country was put in place 50 years ago,” says Packman. “And it was basically based on people going to hospitals. But now it is absolutely clear that we will be more successful in identifying and responding to public health issues if we combine clinical and environmental surveillance information. This is the new frontier.