As researchers increasingly rely on wastewater testing to monitor the spread of bird flu, some question the reliability of the tests being used. Above, the Hyperion Water Recovery Plant in Playa Del Rey.
(Jason Armond / Los Angeles Times)
As health officials increasingly turn to wastewater testing as a means to track the spread of H5N1 bird flu among U.S. dairy herds, some researchers raise questions about the effectiveness of water testing. residuals.
Although the U.S. Centers for Disease Control and Prevention says current tests are standardized and will detect bird flu, some researchers expressed skepticism.
“Right now we are using this type of broad testing” to detect influenza A viruses in wastewater, said epidemiologist Denis Nash, referring to a category of viruses that includes normal human flu and bird flu circulating in dairy cattle and wild birds. and domestic poultry.
“There may be some places around the country where the primers that are used in these tests … don't work for H5N1,” said Nash, distinguished professor of epidemiology and executive director of the Institute for Implementation of Population Sciences. 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 the flu virus.
But in order for them to identify the virus, they must be “prepared” to know what they are looking for. Depending on what part of the virus researchers are looking for, they may not identify the bird flu subtype.
There are two common human influenza A viruses: H1N1 and H3N2. The “H” stands for hemagglutinin, which is an identifiable protein on the virus. The “N” stands for neuraminidase.
Avian flu, on the other hand, is also an influenza A virus. But it has the H5N1 subtype.
That means that while the human and bird flu viruses share the N1 signal, they do not share an H signal.
If a test is designed to look only for H1 and H3 as indicators of the influenza A virus, it will not detect bird flu.
Marc Johnson, a professor of molecular microbiology and immunology at the University of Missouri, said he doesn't think that's very likely. He said the generic panels most labs use will capture H1, H3 and H5.
He said that while his lab specifically looks for H1 and H3, “I think we may be the only ones doing that.”
Only in recent years have health officials begun using wastewater as a sentinel for community health.
Alexandria Boehm, a professor of civil and environmental engineering at Stanford University and principal investigator and program director for WastewaterSCAN, said wastewater surveillance really kicked into high gear during the pandemic. It has become routine 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 collaborates with a network of researchers in labs at Stanford, Emory University and Verily, 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 analyzing 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.
On Tuesday, the CDC released its own dashboard showing the wastewater sites where it detected influenza A in the past two weeks.
Showing a network of more than 650 sites across the country, there were only three sites (in Florida, Illinois and Kansas) where influenza A levels were considered high enough to warrant further investigation by the agency. There were more than 400 for which the data were insufficient to allow a determination.
Jonathan Yoder, deputy director of the CDC's Division of Infectious Disease Preparedness and Innovation, said those sites were considered to have insufficient data because testing has not been done long enough, or there may not have been enough positive samples of influenza A to include.
Asked if some of the tests used might not detect bird flu because of the way they were designed, he said: “We don't have any evidence of that. “It seems like we're at a broad enough level that we don't have any evidence that we wouldn't pick up H5.”
He also said the tests were standardized across the network.
“I'm pretty sure the same assay is used at all sites,” he said. “They're all based on…what the CDC has published as a clinical trial for influenza A, so it's based on clinical evidence.”
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 Public Health Institute and the El Paso Water Utility released a report showing High levels of bird flu in 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's not just one virus or multiple viruses,” as is done with PCR tests, said Eric Boerwinkle, dean of the UTHealth Houston School of Public Health and a member of the Texas team. “In reality, we are in a very complex mixture, which is wastewater, which eliminates viruses and sequences them.”
“What's critical here is that it's very specific to H5N1,” he said, noting that they had been doing this type 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 the College of Public Health in Houston and a member of the Texas wastewater team, agreed, saying that PCR-based methods are “ exquisite” and “highly precise”.
“But we have the ability to look at the representation of the entire genome, not just a marker component of it. And that has allowed us to look at H5N1 and differentiate it from some of our seasonal fluids like H1N1 and H3N2,” he said. “That's what gave us great confidence that this is exclusively H5N1, while the other papers use a part of the H5 gene as a marker for H5.”
Boerwinkle and Hanson stressed that while they were able to identify H5N1 in the wastewater, they can't say where it came from.
“Texas is really a confluence of a couple 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 penny down and bet what's happening, it's probably coming from not just one source but multiple sources. “We have no reason to think that any of those things are more likely than any other source.”
But they're pretty sure it's not coming from people.
“Because we're looking at the entire genome, when we look at a single human case of H5N1, the genomic sequence… has a characteristic amino acid change… compared to all cattle at that same time.” Hanson said. “We don't see that characteristic amino acid present in any of our sequencing data. “And we've looked at it very carefully, which gives us some confidence that we're not seeing human-to-human transmission.”
The Texas team's approach was really exciting, said Devabhaktuni Srikrishna, CEO and founder of PatientKnowHow.com, noting that it exhibited “proof of principle” for employing this type of metagenomic testing protocol for wastewater and air.
He said government agencies, private companies and academics have been looking for a reliable way to test for thousands of microscopic organisms, such as pathogens, quickly, reliably and at low cost.
“They showed it can be done,” he said.
