As the popularity of wastewater surveillance programs soared during the COVID-19 pandemic, microbiologist Anthony Maresso and microbial geneticist Michael Tisza from Baylor College of Medicine broadened their horizons. Through the wastewater detection initiative of the Texas Epidemic Public Health Institute (TEPHI), Tisza, Maresso, and their team detected over 400 different human and animal viruses through hybrid capture-based sequencing, testing samples predominantly from urban areas throughout Texas. Their goal was to detect infectious disease outbreaks early, and recent upticks in avian influenza (H5N1) cases beyond bird species caught their attention.
In their latest correspondence published in the New England Journal of Medicine, the researchers sounded the alarm on H5N1 present in the wastewater across 10 Texas cities, and championed agnostic viral monitoring as a sentinel surveillance tool in infectious disease preparedness.1
Searching for Viruses in Wastewater
Maresso and his team had been examining wastewater for about a decade prior to the COVID-19 pandemic, looking for microbes. They had built protocols around going to wastewater plants, getting material, and isolating viruses. “We had the idea that maybe SARS-CoV-2 was in the wastewater, and if we were able to detect it, we might be able to tell an entire community what the levels were, rather than relying on individual tests,” said Maresso. That led to a program at Baylor College of Medicine, developed in conjunction with the Houston Health Department, where the researchers were testing for SARS-CoV-2 on a weekly basis and reporting where outbreaks were occurring. As the pandemic went on and the vaccine came into play, wastewater testing started to grow. Maresso and his team then asked themselves, “What is the next big breakthrough in this? Can we look for not just one virus, but all viruses?” And that is where Tisza and his team came in.
Tisza joined Baylor College of Medicine just as the TEPHI wastewater sequencing initiative, Texas Wastewater and Environmental Biomonitoring (TexWEB), was being conceived. “We were figuring out how we could enrich for these viruses in the lab and then sequence them. That has been a big technical undertaking, and it has been a big technical triumph that people in our organization have pioneered,” Tisza said.
Together, the Baylor College of Medicine team built a program where they now detect and report on as many human viruses as possible in wastewater using sequencing technology. One of the features of the program is that they can detect things people are not thinking about, and one of the things they detected was avian flu, which they started to see a signal for in early March 2024.
Detecting H5N1 in Texas Wastewater
When Tisza first detected a signal for H5N1 in Texas wastewater, he was very surprised. There had been early reports that the virus was circulating in cattle, and at the end of March 2024, there was one case in west Texas of a human who presumably was infected from a dairy cow. Still, the researchers did not expect to see a signal across so many urban areas. “At that point, I thought, ‘Well, that is probably just an isolated situation.’ I did not expect that this would be in our urban areas in Texas,” said Tisza.
He organized an emergency sit down with his colleague Blake Hanson, an epidemiologist at the University of Texas Health Science Center at Houston, to figure out from a computational and mathematical perspective how they could confidently know if H5N1 avian influenza was present in their data.
“We started to see H5N1 signal in March 2024 and we were both very shocked,” Tisza said. “We went back into all of our historical data from May of 2022 until current day, and we saw that we had never seen it before March of 2024, which was even more shocking to us, because in 2022 there was a very large H5N1 avian influenza outbreak in wild birds and poultry, yet we never saw it in the wastewater.”
One of the main factors that is different in 2024 is that this virus is circulating in dairy cows and there have been more human cases reported. “Obviously, that this is now in a dairy cattle reservoir is cause for grave concern,” Maresso explained. “It does mean that the virus is adapting in a way that is one step closer toward human-to-human transmission.” Detectable viral sequences in wastewater are largely thought of as being from a human source, but as Maresso describes their ongoing work, it still requires a lot of detective work to determine the exact source of the viral signal in urban wastewater.
Wastewater Surveillance Challenges
According to Tisza, one challenge of wastewater surveillance is that it is a hard sample to deal with because it has liquid and solid components that can clog filters, and it requires careful biohazard considerations. Beyond working with difficult samples, searching for viral sequences in wastewater is what the researchers describe as a needle in a haystack problem. “If you were to just extract RNA and DNA from wastewater to sequence, you would have almost no sequences from human viruses, they are about one in a million,” Tisza explained. “You need to drastically enrich the viral material.”
The Texas team used a technique called hybrid capture to enrich viral sequences for detection. “It is almost like we have a million little magnets, each of which is specific for a different viral sequence,” Tisza said. In this analogy, the magnets are nucleic acids that anneal to each other. There are representative magnets from more than 3000 human viruses, as well as about 15,000 possible variants of these viruses. Those magnets pull down the viral sequences from the wastewater extraction and the researchers wash away the remaining RNA and DNA. They are then left with sequences highly enriched for human viruses.
After the researchers enrich and sequence this genetic material, they are faced with the challenge of making sense of all those As, Ts, Cs, and Gs. “You have to confidently, specifically, and sensitively assign sequencing reads to the appropriate virus from which they originate, and you also have to be able to distinguish very minor variations in these reads,” explained Tisza. This is particularly challenging because different influenza strains or serotypes will rearrange genetic material with each other. For example, the sequences of H5N1 are sometimes very similar to the sequences from other influenza A strains or serotypes. “It is a really big biological, mathematical, and computational problem that we have been working on for the last two and a half years,” Tisza added.
The Future of Viral Detection
Although finding H5N1 across several cities’ wastewater samples has serious implications for public health, the researchers are also encouraged that their virus-agnostic and comprehensive approach allowed them to detect these events early. “From the technological standpoint, I was pleased that we were able to meet the mark,” said Maresso. “I do think that this is going to be a transformative moment in public health with respect to using sequencing rather than PCR to detect these viruses in wastewater.”
The hope is that, if human transmission is beginning at some low level in the population, they will see that signal in the wastewater and be able to pinpoint how the virus has started to adapt. Unlike PCR-based tests that are currently used to detect viruses, sequence information allows the researchers to track if viral evolution is occurring, right when they detect signal in the wastewater. “We can closely match the sequence of the virus in the wastewater to the virus coming from other reservoirs, like bird, cattle, and other animals, and identify possible origins,” Maresso explained.
Looking beyond their current findings, Tisza dreams that there could one day be more of a nationwide wastewater surveillance program that uses agnostic viral sequencing, which would interface with public health and clinical interventions. For example, if sequencing detects H5N1 in wastewater elsewhere, there could then be resources provided for a targeted public health approach such as voluntary testing of anyone who is coming down with cold or flu-like symptoms. “In that way, we may be able to track down if and how this virus is spreading human-to-human, or if there are people getting it from an animal vector,” Tisza said.
- Tisza MJ, et al. Sequencing-based detection of avian influenza A(H5N1) virus in wastewater in ten cities. N Engl J Med. 2024;391(12):1157-1159.
