Though PCR clinical laboratory testing is widely used, some scientists are concerned its specificity may limit the ability to identify all variants of bird flu in wastewater
Wastewater testing of infectious agents appears to be here to stay. At the same time, there are differences of opinion about which methodologies and clinical laboratory tests are best suited to screen for specific contagions in wastewater. One such contagion is avian influenza, the virus that causes bird flu.
Wastewater testing by public health officials became a valuable tool during the COVID-19 pandemic and has now become a common method for detecting other diseases as well. For example, earlier this year, scientists used wastewater testing to learn how the H5N1 variant of the bird flu virus was advancing among dairy herds across the country.
In late March, the bird flu was first detected in dairy cattle in Texas, prompting scientists to begin examining wastewater samples to track the virus. Some researchers, however, expressed concerns about the ability of sewage test assays to detect all variants of certain diseases.
“Right now we are using these sort of broad tests to test for influenza A viruses,” Denis Nash, PhD, Distinguished Professor of Epidemiology at City University of New York (CUNY) and Executive Director of CUNY’s Institute for Implementation Science in Population Health (SPH), told the Los Angeles Times. “It’s possible there are some locations around the country where the primers being used in these tests might not work for H5N1.” Clinical laboratory PCR genetic testing is most commonly used to screen for viruses in wastewater. (Photo copyright: CUNY SPH.)
Effectiveness of PCR Wastewater Testing
Polymerase chain reaction (PCR) tests are most commonly used to distinguish genetic material related to a specific illness such as the flu virus. For PCR tests to correctly identify a virus, the tests must be designed to look for a specific subtype. The two most prevalent human influenza A viruses are known as H1N1 (swine flu) and H3N2, which was responsible for the 1968 pandemic that killed a million people worldwide. The “H” stands for hemagglutinin and the “N” for neuraminidase.
Hemagglutinin is a glycoprotein that assists the virus to attach to and infect host cells. Neuraminidase is an enzyme found in many pathogenic or symbiotic microorganisms that separates the links between neuraminic acids in various molecules.
Avian flu is also an influenza A virus, but it has the subtype H5N1. Although human and bird flu viruses both contain the N1 signal, they do not share an H. Some scientists fear that—in cases where a PCR test only looks for H1 and H3 in wastewater—that test could miss the bird flu altogether.
“We don’t have any evidence of that. It does seem like we’re at a broad enough level that we don’t have any evidence that we would not pick up H5,” Jonathan Yoder, Deputy Director, Infectious Disease Readiness and Innovation at the US Centers for Disease Control and Prevention (CDC) told the Los Angeles Times.
The CDC asserts current genetic testing methods are standardized and will detect the bird flu. Yoder also affirmed the tests being used at all the testing sites are the same assay, based on information the CDC has published regarding testing for influenza A viruses.
Genetic Sequencing Finds H5N1 in Texas Wastewater
In an article published on the preprint server medRxiv titled, “Virome Sequencing Identifies H5N1 Avian Influenza in Wastewater from Nine Cities,” the authors wrote, “using an agnostic, hybrid-capture sequencing approach, we report the detection of H5N1 in wastewater in nine Texas cities, with a total catchment area population in the millions, over a two-month period from March 4th to April 25th, 2024.”
The authors added, “Although human to human transmission is rare, infection has been fatal in nearly half of patients who have contracted the virus in past outbreaks. The increasing presence of the virus in domesticated animals raises substantial concerns that viral adaptation to immunologically naïve humans may result in the next flu pandemic.”
“So, it’s not just targeting one virus—or one of several viruses—as one does with PCR testing,” Eric Boerwinkle, PhD, Dean of the UTHealth Houston School of Public Health told the LA Times. “We’re actually in a very complex mixture, which is wastewater, pulling down viruses and sequencing them. What’s critical here is it’s very specific to H5N1.”
Epidemiologist Blake Hanson, PhD, Assistant Professor, Department of Epidemiology, Human Genetics, and Environmental Sciences at the UT Health Houston Graduate School of Biomedical Science, agreed with Boerwinkle that though the PCR-based methodology is highly effective at detecting avian flu in wastewater samples, the testing can do more.
“We have the ability to look at the representation of the entire genome, not just a marker component of it. And so that has allowed us to look at H5N1, differentiate it from some of our seasonal fluids like H1N1 and H3N2,” Hanson told the LA Times. “It’s what gave us high confidence that it is entirely H5N1, whereas the other papers are using a part of the H5 gene as a marker for H5.”
Human or Animal Sources
Both Boerwinkle and Hanson are epidemiologists in the team studying wastewater samples for H5N1 in Texas. They are not sure where the virus originated but are fairly certain it did not come from humans.
“Texas is really a confluence of a couple of different flyways for migratory birds, and Texas is also an agricultural state, despite having quite large cities,” Boerwinkle noted. “It’s probably correct that if you had to put your dime and gamble what was happening, it’s probably coming from not just one source but from multiple sources. We have no reason to think that one source is more likely any one of those things.”
“Because we are looking at the entirety of the genome, when we look at the single human H5N1 case, the genomic sequence has a hallmark amino acid change, compared to all of the cattle from that same time point,” Hanson said. “We do not see that hallmark amino acid present in any of our sequencing data. And we’ve looked very carefully for that, which gives us some confidence that we’re not seeing human-human transmission.”
CDC Updates on Bird Flu
In its weekly updates on the bird flu situation, the CDC reported that 48 states have outbreaks in poultry and 14 states have avian flu outbreaks in dairy cows. More than 238 dairy herds have been affected and, as of September 20, over 100 million poultry have been affected by the disease.
In addition, the CDC monitored more than 4,900 people who came into contact with an infected animal. Though about 230 of those individuals have been tested for the disease, there have only been a total of 14 reported human cases in the US.
The CDC posts information specifically for laboratory workers, healthcare providers, and veterinarians on its website.
The CDC also states that the threat from avian flu to the general public is low. Individuals at an increased risk for infection include people who work around infected animals and those who consume products containing raw, unpasteurized cow’s milk.
Symptoms of H5N1 in humans may include fever or chills, cough, headaches, muscle or body aches, runny or stuffy nose, tiredness and shortness of breath. Symptoms typically surface two to eight days after exposure.
Scientists and researchers have been seeking a reliable clinical laboratory test for disease organisms in a fast, accurate, and cost-effective manner. Wastewater testing of infectious agents could fulfill those goals and appears to be a technology that will continue to be used for tracking disease.
—JP Schlingman
Related Information:
As National Wastewater Testing Expands, Texas Researchers Identify Bird Flu in Nine Cities
Experts Blast CDC over Failure to Test Sewage for Signs of H5N1 Bird Flu Virus
The Bird Flu Outbreak Has Spread to Humans: Are We Too Late to Prevent the Next Pandemic?
Virome Sequencing Identifies H5N1 Avian Influenza in Wastewater from Nine Cities