As officials turn to wastewater to monitor the bird flu epidemic, questions about testing are bubbling up

As researchers increasingly rely on wastewater testing to monitor the spread of bird flu, some are questioning the reliability of the tests used. Above, the Hyperion water reclamation plant in Playa Del Rey.

(Jason Armond/Los Angeles Times)

As health officials increasingly focus on wastewater testing as a means to track the spread of H5N1 bird flu among U.S. dairy herds, some researchers are raising questions about the effectiveness of the sewage tests.

Although the U.S. Centers for Disease Control and Prevention says current tests are standardized and will detect bird flu, some researchers were skeptical.

“Right now we're using this kind of broad testing” to test for influenza A viruses in wastewater, said epidemiologist Denis Nash, referring to a category of viruses that includes normal human flu and the avian flu that circulates in dairy cattle and wild birds. and domestic poultry.

“It is possible that there are locations in the country where the primers used in these tests … may not work for H5N1,” said Nash, professor of epidemiology and executive director of the Institute for Implementation Science in Population at the City University of New York. Health.

The reason for this is that the most commonly used tests – polymerase chain reaction or PCR tests – are designed to detect genetic material from a specific organism, such as a flu virus.

But in order to identify the virus, they must be “prepared” to know what they are looking for. Depending on which part of the virus researchers are looking for, they may not be able to identify the bird flu subtype.

There are two common human influenza A viruses: H1N1 and H3N2. The “H” stands for hemagglutinin, an identifiable protein in the virus. The “N” stands for neuraminidase.

Bird flu, on the other hand, is also an influenza A virus. But it has the subtype H5N1.

That means that although the human and avian flu viruses share the N1 signal, they do not have H.

If a test is designed to only look for the H1 and H3 as indicators of the influenza A virus, they will miss the bird flu.

Marc Johnson, a professor of molecular microbiology and immunology at the University of Missouri, said he doesn't think that's likely. He said the generic panels most labs use will capture H1, H3 and H5.

While his lab looks specifically for H1 and H3, he said, “I think we might be the only ones doing that.”

It's only in the last few years that health officials have started using wastewater as a sentinel for public health.

Alexandria Boehm, professor of civil and environmental engineering at Stanford University and principal investigator and program director for WastewaterSCAN, said wastewater monitoring really took off during the pandemic. It has become a routine way to look for hundreds, if not thousands, of viruses and other pathogens in municipal wastewater.

“Three or four years ago, no one was doing it,” said Boehm, who works with a network of researchers in labs at Stanford, Emory University and Alphabet Inc.'s life sciences research organization. “It kind of evolved in response to the pandemic and has continued to evolve.”

Since late March, when bird flu was first reported in Texas dairy cattle, researchers and public health officials have been scouring wastewater samples. Most are using the influenza A tests they already had built into their systems – most of which were designed to detect human flu viruses, not bird flu.

The CDC released this on Tuesday your own dashboard with wastewater locations where influenza A has been detected in the past two weeks.

With a network of more than 650 sites across the country, there were only three locations – in Florida, Illinois and Kansas – where levels of influenza A were considered high enough to warrant further investigation by the agency. There were more than 400 cases where the data were insufficient to allow a determination.

Jonathan Yoder, deputy director of the CDC's Division of Infectious Disease Readiness and Innovation, said these sites were considered to have insufficient data because testing was not conducted long enough, or because there may not have been enough positive influenza A samples to test for. to take.

Asked whether some of the tests used could miss bird flu because of the way they are designed, he said: “We don't have any evidence of that. It seems like we are at a level that is so broad that we don't have any evidence that we wouldn't pick up H5.”

He also said testing had been standardized across the network.

“I'm pretty sure it's the same test at all locations,” he said. “They're all based on… what the CDC has published as a clinical test for influenza A, so it's based on clinical testing.”

But there are discrepancies between the CDC's findings and those of others.

Earlier this week, a team of scientists from Baylor College of Medicine, the University of Texas Health Science Center at Houston, the Texas Epidemic Health Institute and the El Paso Water Utility, published a report showing high levels of bird flu from wastewater in nine Texas cities. Their data shows that H5N1 is the dominant form of influenza A swirling in the wastewater of these Texas cities.

But unlike other research teams, including the CDC, they used an “agnostic” approach known as hybrid-capture sequencing.

“So it doesn't just target one virus or one of several viruses,” as is the case with PCR testing, said Eric Boerwinkle, dean of the UTHealth Houston School of Public Health and a member of the Texas team. “We're actually in a very complex mixture, which is wastewater, which is pulling down viruses and sequencing them.”

“What's critical here is that it is very specific to H5N1,” he said, noting that they had been doing this kind of testing for about two years and had never seen H5N1 before mid-March.

Blake Hanson, an assistant professor at the University of Texas Health Science Center at Houston School of Public Health and a member of the Texas wastewater team, agreed, saying PCR-based methods are “excellent” and “very accurate.”

“But we have the ability to look at the representation of the entire genome, and not just a marker component of it. And that has allowed us to look at H5N1 and distinguish it from some of our seasonal fluids like H1N1 and H3N2,” he said. “It gave us great confidence that it is completely H5N1, while the other papers use part of the H5 gene as a marker for H5.”

Boerwinkle and Hanson emphasized that while they could identify H5N1 in the wastewater, they could not say where it came from.

“Texas is actually a confluence of a number of different flyways for migratory birds, and Texas is also an agricultural state, despite having fairly large cities,” Boerwinkle said. “It's probably right that if you had to put your two cents in and guess what happened, it probably won't come from one source, but from multiple sources. We have no reason to think that one source is more likely for any of these things.”

But they're pretty sure it doesn't come from humans.

“Because we're looking at the entire genome, if we look at the single human H5N1 case, the genomic sequence … has a distinctive amino acid change … compared to all cattle from that same time point,” Hanson said. “We don't see that signature amino acid present in our sequence data. And we have looked very closely at that, which gives us confidence that we do not see any human-to-human transmission.”

The team approach from Texas was very exciting, said Devabhaktuni Srikrishna, the CEO and founder of PatientKnowHow.com, noting that it showed a “proof of principle” for using these types of metagenomic testing protocols for wastewater and air.

He said government agencies, private companies and academics are looking for a reliable way to test thousands of microscopic organisms – such as pathogens – quickly, reliably and at a low cost.

“They have shown that it is possible,” he says.

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