Virus sleuths find Covid-19 clues after toilet flush

John Reilly (right), supervisor plumber at Elmhurst, and Dylan Stephens, a plumber, next to a sewage pipe in the basement at Elmhurst Hospital in Queens. PHOTO: NYTIMES

NEW YORK (NYTIMES) - The Covid-19 pandemic has turned sewage into gold.

People who are infected with the coronavirus shed the germ in their stool. By measuring and sequencing the viral material present in sewage, scientists can determine whether cases are rising in a particular area and which variants are circulating.

People excrete the virus even if they never seek testing or treatment. So wastewater surveillance has become a critical tool for keeping tabs on the virus, especially as Covid-19 testing has increasingly shifted to the home.

The institutions and localities that invested in wastewater surveillance during the past two years are discovering that it can track other health threats, too. The Sewer Coronavirus Alert Network has already begun tracking the monkeypox virus in wastewater. And New York City officials recently announced that polio had been detected in the city's sewage.

Six months ago, NYC Health + Hospitals, a large, local health care system, began piloting its own wastewater surveillance system to track the coronavirus and the flu. Monkeypox and polio monitoring will start soon. There are various approaches to wastewater surveillance.

Here is a description of how coronavirus tracking works at one New York hospital.

In the hospital basement

Where the toilets are flushed, sewage flows through a pipe and two scientists collect it.

New York City was the epicentre of the nation's first Covid-19 wave, and its hospitals were hit hard by several surges in the pandemic. In late 2021, Health + Hospitals decided to build a sustainable, long-term virus surveillance system to get ahead of future outbreaks, said Leopolda Silvera, global health deputy at Elmhurst Hospital Centre in Queens, which is part of the health care network.

The wastewater surveillance initiative is now running at 10 hospitals, but it began, in February, at Elmhurst.

Setting the Contraption

Coronavirus fragments deposited into hospital toilets travel through the plumbing system and enter a sewage pipe in the basement.

"This is our baby," John Reilly, supervisor plumber at Elmhurst, said, banging on the outside of the pipe. Every Monday, a member of the wastewater team drops a collection device, which the team calls the Contraption, into an opening in the pipe.

Over the next 24 hours, the wastewater will rush over, around and through the device.

Elmhurst Hospital in Queens. The wastewater surveillance initiative is now running at 10 hospitals, but it began, in February, at Elmhurst. PHOTO: NYTIMES

The gross part

The next day, two researchers arrive to check on the Contraption. "I must warn you that it's going to be gross," said Sherin Kannoly, who was on collection duty with Justin Silbiger.

Wearing masks and gloves, they carefully remove the device from the pipe and then use tweezers to extract a tampon - yes, a tampon - from the mesh tube.

The researchers have experimented with different designs for the Contraption; one day this spring they were using a porous metal cylinder that contained a tampon to absorb the wastewater. Their current design uses charcoal water filters instead.

Ziploc and chill

The technicians double-bag the waterlogged tampon to ensure it does not leak on the 15-minute drive across Queens.

Then they put the sample on ice and click the cooler shut. The dirty work is done.


On the road

The technicians exit the hospital basement, carry the cooler to the car and drive the sample to Queens College.

In the Queens college lab

Where levels of the virus are measured.

Before the pandemic hit, John Dennehy spent his time studying bacteriophages, or viruses that infect bacteria, often isolating them from wastewater. "When the pandemic came, I felt like I had an obligation as a virologist to contribute my skills," he said.

In 2020, Dennehy, with colleagues including Monica Trujillo, a microbiologist at Queensborough Community College, began testing samples of the city's wastewater for the coronavirus. When they heard that the hospitals wanted to create their own surveillance system, they were eager to help. Dennehy's lab at Queens College is the first stop for the hospital samples.

A researcher from the City University of New York (right) takes a wastewater sample from the plumbing pipes under Elmhurst Hospital in Queens. PHOTO: NYTIMES

First, a hot bath

The sample is pasteurised in a hot-water bath, making it safe for scientists to handle. Then, the water is filtered to remove solids and debris.

The scientists add two compounds, polyethylene glycol and sodium chloride, to help the virus form a solid precipitate.

Next, a cold night

The sample incubates in the fridge overnight and then spins in a centrifuge. When the process is complete, the researchers are left with a tiny pellet of virus.

Researchers at Queens College in New York process wastewater samples collected from a sewage pipe at Elmhurst Hospital. PHOTO: NYTIMES

Finally, the genes

They add a bright pink chemical, TRIzol, to extract the RNA from the viral pellet. In real life, science rarely looks the way it does in the movies - the shockingly pink concoction is an exception, the researchers noted with enthusiasm.

To determine how much virus is present in the sample, the researchers use a polymerase chain reaction, the same method used to test people for the virus. They put the RNA they have extracted into the tiny wells of a PCR plate and then slide the plate into a machine known as a thermal cycler.

The machine will amplify - make copies of - the viral RNA and measure how much is present. The more RNA there is, the more virus presumably is present in the wastewater and, by extension, in the hospital community.

Show and tell

The researchers share the results with hospital officials. The program has already proven promising.

Dennehy, Trujillo and their colleagues have found that the amount of coronavirus and influenza in the hospital's wastewater often began rising 10 to 14 days before the hospital saw an increase in Covid-19 and flu patients.

"When you are testing everything and everybody, the wastewater doesn't give you such a big lead," Trujillo said.

But once coronavirus testing in the city dropped off, the wastewater data became especially valuable. "It's really something that we are hoping that will be incorporated as another tool for public health," she said.

Wastewater samples are processed at the Pandemic Response Lab, a commercial laboratory, in Queens. PHOTO: NYTIMES

Next stop: Sequencing

Silvera, the global health deputy at Elmhurst, ferries the Queens College samples, and some additional bottles of wastewater, to a commercial laboratory and deposits them in a refrigerator to keep them cool until they are ready to be processed.

In the pandemic response lab

Where variants are identified.

Opentrons Labworks, a laboratory robotics company, created the Pandemic Response Lab in 2020 to provide high-volume, high-speed coronavirus testing and, later, coronavirus sequencing of patient samples. The search for viral variants in wastewater involves essentially the same process.

"It just so happens that that sample is not coming from a person but from wastewater, which, you know, has some elements that came from people," said Jonathan Brennan-Badal, CEO of Opentrons.

Wastewater samples are loaded into a sequencer, which determines the genetic sequence of each coronavirus fragment. PHOTO: NYTIMES

Moving in reverse

The Queens College laboratory isolated the virus's RNA. To sequence the genetic material, the Pandemic Response Lab first converts the RNA into DNA, a process known as reverse transcription.

A pipetting robot adds the necessary chemicals and enzymes to a plate containing small amounts of the viral RNA. The plate is then placed into a thermal cycler - each one emblazoned with a name inspired by "Snow White and the Seven Dwarfs" - and the enzymes convert the RNA into DNA.

Bring in the bots

The scientists shuttle the sample back and forth between a small army of laboratory robots.

The robots add chemicals and enzymes, and the samples are manipulated in a variety of ways. The viral DNA is amplified and then chopped into fragments that are short enough to be read by the sequencer.

These fragments are then amplified and marked with molecular bar codes, which allow the scientists to later distinguish individual samples from a pool of them. Finally, the samples are cleaned and then combined, sometimes by hand.

John Dennehy, a virologist, and Monica Trujillo, a microbiologist, at Queens College in New York on July 30, 2022. PHOTO: NYTIMES

Completing the sequence

After all the humans and robots have completed their respective tasks, the pooled samples are loaded into the sequencer, which determines the genetic sequence of each fragment, allowing scientists to determine what mutations and variants are present.

The results are automatically uploaded to a server and processed. The findings are reported to the hospitals weekly.

The sequencing results "reflect what has been seen with clinical data," Silvera said. As the BA.4 and BA.5 variants of the coronavirus spread, for instance, they began to "dominate" the wastewater samples, she added.

Rinse and repeat

The hospital project is just one of many springing up across the country and around the world. New York City has its own citywide wastewater surveillance system, which involves collecting sewage samples from municipal wastewater facilities, including the Newtown Creek Wastewater Treatment Plant.

And the hospital team is already looking toward the future, considering how the same system might be harnessed to monitor a variety of potential health threats. "The information is invaluable, honestly," Silvera said.

And all it takes is a flush.

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