Texas Team's Wastewater Surveillance Can Pick Up Concerning H5N1 Mutations

— TEPHI's monitoring gets more granular than CDC's influenza A wastewater dashboard

 A computer rendering of H5N1 viruses.

While the CDC's recently launched reports levels of influenza A, a team in Texas is getting far more granular in its wastewater monitoring.

The team at the Texas Epidemic Public Health Institute (TEPHI) in Houston can detect H5N1 specifically -- and it can even pick up potentially concerning genetic changes that could provide an early warning about adaptation to humans.

That kind of surveillance could sound an alarm well before standard symptomatic surveillance like emergency department visits or hospitalizations, which would only pick up an H5N1 outbreak after it's already spreading in people.

"This allows us to monitor over time for the emergence of new mutations or strains" of H5N1, said Eric Boerwinkle, PhD, director of TEPHI and dean of UTHealth Houston School of Public Health. "We can look for mutations that could increase virulence or increase transmission, even transmission to and among humans."

For instance, the team can look for the hallmark E627K mutation in H5N1's polymerase basic protein 2 (PB2) gene, which has been identified in human cases of the disease -- including the dairy cattle worker in Texas whose only symptom of infection was conjunctivitis.

"Going forward, a major concern will be looking at the different alleles and seeing if there's this hallmark mutation or potentially others," said Michael Tisza, PhD, of TEPHI and Baylor College of Medicine in Houston. "This technology would allow us to do that."

The E627K mutation hasn't turned up in their surveillance yet, but the team is keeping an eye out for it, they said.

Detecting an early signal like that would enable TEPHI to work with its public health partners who can then "direct other resources to look at things like syndromic surveillance to better assess what's going on in those locations as far as the sources go," said Blake Hanson, PhD, of TEPHI and UTHealth in Houston.

Hanson described wastewater monitoring as having varying levels of resolution. The widest resolution is detecting influenza A, which covers a wide range of viruses including H2N3, H1N1, and H5N1. That's what the CDC's dashboard monitors.

While the CDC's wastewater monitoring efforts may be helpful right now -- during the late spring and the summer when it would be unusual to have high levels of influenza A circulating -- it won't work as well during the regular flu season in fall and winter.

"When symptoms and our seasonal influenza levels are low, it's a reasonable proxy for other strains of influenza A circulating in wastewater," Hanson said.

The next level would be detecting the H5 gene using polymerase chain reaction (PCR). That's what a team from Verily Life Sciences, Emory University, and Stanford University recently did, as . They detected influenza A at 59 wastewater treatment plants in the U.S. this spring. They then searched for -- and found -- the H5 gene using PCR at three wastewater treatment plants with a positive flu A signal. All three plants were in a state with confirmed outbreaks of H5N1 in dairy cattle, they reported.

"They can only confidently say that's H5, where our method has a third level of resolution where we can represent the entirety of the genome for the H5N1 strains," Hanson said.

TEPHI's virome sequencing would provide the most granular look, detecting H5N1 specifically, along with any potential mutations in its genome.

Tisza said they only need about a 50-mL sample of wastewater to detect sequences from any given viruses, as long as they're present at about 100 or more genome copies per mL.

The team captures viral genetic material from the sample, then runs it through a sequencer -- the Illumina NovaSeq machine in this case -- which generates a readout of those sequences.

They then use bioinformatics software to identify viruses from those genomes and genome fragments, also scanning for interesting mutations. With COVID, for instance, they're able to see new strains as they come to dominate, Tisza said.

The team recently detected H5N1 in wastewater in nine Texas cities during a sampling period of March 4 through April 25, and they . For that paper, Tisza and Hanson both separately confirmed the sequences manually to be sure they were dealing with H5N1.

TEPHI has been conducting wastewater surveillance since May 2022, testing samples from plants across Texas on a weekly basis, and has detected more than 400 human and animal viruses to date.

The group's methodology is public, in October 2023.

"All our methods are published, and we welcome others to adopt these techniques because we think they're incredibly powerful," Hanson said, particularly for H5N1 surveillance.

The team continues to observe H5N1 sequences in their wastewater sampling, Boerwinkle said. The point of the surveillance is to help keep public health prepared for a potential outbreak: "If we're prepared, we can keep the economy strong," he added.

Andrew Pekosz, PhD, of Johns Hopkins University in Baltimore, who isn't involved in TEPHI, said the team's work is "an important part of the 'pyramid' of analysis that we need to do with wastewater."

"First, you need easy rapid tests to identify the small percentage of systems that might have a signal for influenza," Pekosz told app in an email. "You then need to take those samples and do an in-depth analysis by sequencing to clearly identify what kind of influenza ... is driving that positivity. If your sequencing is good enough, you can even identify the proportion of the sample that has important mutations like the PB2 mutation."

"It's all about using the various virus surveillance tools to maximize efficiency and minimize cost," he said.

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    Kristina Fiore leads MedPage’s enterprise & investigative reporting team. She’s been a medical journalist for more than a decade and her work has been recognized by Barlett & Steele, AHCJ, SABEW, and others. Send story tips to