Flush. We all do it. Out of sight, out of mind. Or is it?
Believe it or not, the human-deposited contents that disappear down a toilet bowl contain valuable health data. Researchers can harvest insights from wastewater pertaining to the health of an entire city, or they can zero in on a neighborhood or even a specific building. This is why studying wastewater may be the public health wave of the future.
With the right tools and methods, researchers can use wastewater to learn about infectious disease and drug use trends in real-time. The COVID-19 pandemic upgraded and accelerated the adoption of this decades-old technology as public health officials sought non-invasive tools to assess daily and weekly infection trends in communities and metropolitan areas.
University of Florida investigators in the College of Public Health and Health Professions (PHHP), the College of Veterinary Medicine, and the Emerging Pathogens Institute—have spent the past two years building testing capacity and refining wastewater analysis techniques to better detect viruses, bacteria and even chemical markers of health—such as pesticides and illegal drugs.
Their efforts started early in the pandemic when PHHP and EPI researchers brainstormed how to adapt their skills to help protect the health of Floridians and those who work and study on UF’s campus. Tara Sabo-Attwood, an associate dean of PHHP, and Joseph Bisesi, an assistant professor in the UF Department of Environmental and Global Health (EGH) in PHHP, had experience surveying for toxins in environmental samples.
They began talking with two microbiologists in their department about how their skills could be combined for detecting the novel coronavirus, SARS-CoV-2, and its genetic variants in wastewater. Tony Maurelli is a professor and associate chair of the EGH department, and an expert in infectious disease surveillance. John Lednicky is a research professor who specializes in detecting and isolating viruses.
“Tara and Joe bought to the table years of experience in how to detect substances from the environment,” Maurelli said. “And John and I were essentially the microbiologists on the team.”Sabo-Attwood said the initial push to analyze wastewater for COVID-19 infection trends came in the form of a request for help from a small island community at the beginning of the pandemic. Next, it was UF officials asking for assistance monitoring infection trends on campus. “We are a group of environmental toxicologists and environmental biologists, and we’re really good at finding stuff in air, water and soil,” said Sabo-Attwood, who is chair of the EGH department. “Working with wastewater is not too much of an oddity for us. The main change when the pandemic began was that we started tracking pathogens rather than chemicals.”
A short history of wastewater-based epidemiology at UF
Monitoring wastewater to protect public health is not new. Its origins can be traced to the 1940s when U.S. epidemiologists used cell-culture methods to monitor communities for poliovirus—an effort that is still in use internationally. It’s also been used to monitor other infectious public health threats globally such as epidemics of cholera, hepatitis A virus, and norovirus—and exposures to chemicals ranging from pesticides to illegal drugs. Some researchers have even used bacteria found in wastewater and known to occur in the human gut microbiome to accurately predict a community’s obesity trends.
But the COVID-19 pandemic sparked an urgent interest in monitoring wastewater as a tool for public health, to know in real-time when the virus was surging or ebbing locally.
The efforts to survey wastewater got underway at UF not long after the novel coronavirus arrived in Florida. A team of microbiologists, environmental toxicologists, and public health experts built a high-throughput laboratory in the EPI in March 2020 to test and research symptomless cases of COVID-19 via the now all-too-familiar nasal swab collection system. Soon after, the City of Cedar Key, Fla. reached out and asked for help with the pandemic in their community. The UF researchers realized that in addition to testing nasal swabs, they could use the same lab to analyze wastewater samples for SARS-CoV-2.
Analyzing wastewater allows researchers to formulate a snapshot of community health using far less resources than testing individuals.
“Since we had already set the lab up, and as wastewater surveillance for SARS-CoV-2 was gaining some traction around the world, we were really poised to act,” said Bisesi. “One, because the testing lab was operational, but two because we are environmental toxicologists, so we are very used to testing environmental samples and the procedures that it takes to extract a biological entity from those really complex matrices.”
While the high-capacity lab contained state-of-the-art equipment and capabilities for handling infectious agents, it lacked the certification to offer clinically diagnostic testing. As more diagnostic labs became available, the research on symptomless carriers of SARS-CoV-2 came to a close, and the team’s work with analyzing wastewater for SARS-CoV-2 accelerated.
“Wastewater is composed of a lot of organic matter, detergents, and everything else that we pour down the drain. It’s a complex matrix, not the easiest thing to pull viruses out of,” said Bisesi. “We spent a few weeks in April 2020 optimizing our extraction protocols. We tried a few different things to see what worked best for us. Once that was optimized, we were set up to run wastewater surveillance in communities.”
Figuring out how to retrieve viruses from wastewater, and estimating the efficiency of the process—were they getting most or all of the virus hiding in the wastewater?—took a few weeks. The team used a known number of viral copies from a coronavirus that causes the common cold, strain OC43, and mixed it with wastewater in a laboratory setting. Next, they tested three methods to extract the viral copies and verify their retrieval rate, eventually landing on electronegative membrane filtration as their go to method.
And voila: the team was able to filter virus from the gunky wastewater and be confident that they were capturing most of the virus present.
A matter of scale: cities, neighborhoods, buildings
Bisesi, an expert in molecular toxicology, spearheaded the field work and sample collection in Cedar Key. Wastewater testing began in April 2020, and by June a few samples were turning up with positive test results.
City officials used the results to communicate infection trends and risks to their citizens in almost real-time, and to encourage mitigation measures such as physical distancing and wearing face masks. (At the time, vaccines were not yet available.)
Further study by the UF team linked infection burdens over time with tourism to the island—a locally beloved community for its fishing, art, and seafood restaurants. The lessons learned in this island community were brought back to UF.
“We next moved into monitoring wastewater for the City of Gainesville,” Bisesi said. Soon the team was also testing wastewater on UF’s campus.
Whereas the work in Cedar Key was at the community level—there is only one water reclamation facility from which to collect samples, the work in Gainesville got more granular. Gainesville has two water reclamation facilities, fed by different areas of the city; sampling from each facility gave a rough comparative picture of how much virus was in the different areas.
“At about the same time, in the summer of 2020, we started thinking about how we could expand this tool to help the University of Florida,” said Bisesi. “And we proposed to the university that we conduct surveillance but instead of collecting from the water reclamation facility, that we target individual buildings and residence halls.”
They trademarked their strategy and methods as GatorWATCH™ which became part of the now-retired UF Health Screen, Test, and Protect program (WATCH stands for Wastewater Analysis Tracking for Community Health).
Where it was possible to do so, the team set up wastewater sampling stations on campus that were positioned to capture outflow directly from specific residential buildings. This required close collaboration with UF Facilities, which provided maps of the sewer lines to the team.
“I remember those very hot days in June, July and August, when we were basically mapping out where and how to collect samples. That was a lot of fieldwork,” Maurelli recalled. “But once we identified the manholes which serviced individual buildings, it meant that we could sample from manholes we knew were coming from just one building or a small set of buildings.”
At first, the team collected samples by hand dipping collection bottles into open manhole covers. But soon autosamplers arrived that could be left in the manhole to collect samples automatically once every hour for 24 hours, to yield a composite sample.
“Those autosamplers were a great advance for us,” Maurelli said.
When GatorWATCH detected a rise in the virus, UF STP was alerted, and they used a messaging system to advise those living in the building that they should consider getting tested.
“The automatic samplers gave us the precision that the STP program needed to be able to send out the alerts,” Maurelli said. “And STP guided us on the optimal number of people to include in a wastewater sample, less than 500, to facilitate messaging about positive samples. They didn’t want to send the alerts to thousands of people every time we found a hotspot. It needed to be more focused. And I think that was the big, innovative advance we made was being able to home in on residential buildings, and UF Facilities was key to us being able to do that.”
As the pandemic ground on and their funding to monitor wastewater on campus ran out, Sabo-Atwood and Bisesi’s team came full circle.
“We had a lot of blood and sweat equity invested in this high-capacity lab,” Sabo-Attwood explained. “So we began thinking about what else we could test wastewater for. What other interesting things could be measured related to community health?”
That discussion brought them back to their roots: detecting chemicals. Sabo-Attwood said they were fortunate to team up with Linda Cottler, the associate dean of research in the College of PHHP and professor in the Department of Epidemiology, who is also the lead investigator for the National Drug Early Warning System coordinating center at UF. Known as NDEWS, the program collects national epidemiological data on drug use.
The team proposed adding a wastewater analysis component to the program and validating wastewater trends with clinical overdose data. Their work detecting fentanyl in wastewater is now funded and underway as a pilot program in four different cities nationally: Chicago, Detroit, Philadelphia and Gainesville. The team plans to use what they learn from the pilot study and apply it to more than 70 other chemical health signals in future work.
The team uses high-powered chemical analyses to isolate and measure chemical compounds present in the wastewater. First, they filter and concentrate the wastewater samples before performing liquid chromatography to trap fractions of the samples that contain the chemicals of interest. Next, portions of these concentrated samples are fed in a stratified fashion into a mass spectrometer which can identify and count the molecules specific to each chemical by their mass, allowing the team to distinguish fentanyl from other drugs.
Epidemiologists would usually analyze blood and urine samples to determine illicit drug use in individuals, but wastewater offers nearly real-time community-wide results.
“It’s pretty powerful because we get instant results for that day, that week,” said Sabo-Attwood. “Whereas the health department has to wait months for fentanyl overdose data before they can start seeing patterns.”
GatorWATCH is working directly with epidemiologists at the departments of health so that they produce data that is of high value to public health intervention. Through this lens, the team is also developing plans to test wastewater for hepatitis A and the sexually transmitted disease agents of gonorrhea and chlamydia.
“The bacteria that cause these diseases are found in the genital tract,” Maurelli said. “In both men and women, the infectious agent is washed out in the urine, which ends up in the wastewater, and we should be able to detect those organisms in wastewater.”
From a public health perspective, officials need to know which diseases are in a community, who is getting them, and if they are increasing or decreasing.
“Wastewater gives us another tool to get answers to those questions,” Maurelli said. “It’s about using these new tools and applying them to a discipline that can really benefit—public health.”
For a very long time, disease surveillance has depended upon tracking diseases as people get sick. In other words, clinical cases documented in a health care setting and reported to public health officials. But the beauty of using wastewater for tracking markers of specific diseases, is that researchers gain insight on the presence of the disease in a community even if people are not experiencing symptoms. For example, women can have silent infections of chlamydia—just like silent COVID infections.
In a related project, their work to understand population-level exposure to pesticides is getting started in Immokalee, Fla.
While analyzing wastewater does not pinpoint who is infected, who is taking drugs or who is exposed to pesticides, it offers real-time insight into community-wide health signals. But the key to making wastewater-based epidemiological data useful for community health lies in turning it into messaging or interventions that protect or improve health outcomes.
“That’s where we need to push for more research,” Sabo-Attwood said. “The dissemination and operationalization piece needs to be worked out.”
The team is working with a consulting firm that designs dashboards for public health officials to overlay data onto, for example. In theory, results of wastewater analyses could be fed into a dashboard to show trends in fentanyl use or COVID-19 infections. This firm can also help visualize their data by placing boundaries within a map around the areas of a city that are represented by a specific wastewater reclamation facility.
“People could look at it and say, Oh, there’s a COVID-19 hotspot at this part of the city right now,” said Sabo-Attwood. “Or city officials could use it internally to see where fentanyl levels are rising and do a rapid response in that area.”
Time will tell how far these research programs will go. But who would have ever thought such rich insight might be at the other end of a simple flush?
By: DeLene Beeland