Charna Albert
November 2024—When it comes to what diseases are predicted to circulate and when, expect a respiratory virus season much like last year’s, and the year before that.
As for the severity? “That’s anybody’s guess,” says Alesia McKeown, PhD, scientific partner, medical and scientific affairs at Roche Diagnostics, speaking in late September. Compared with last season, the Centers for Disease Control and Prevention expects a similar or perhaps lower peak combined hospitalization burden (about 20 per 100,000) from the three major viruses, if all holds steady. In response, laboratories are readying themselves for a SARS-CoV-2, seasonal influenza, and RSV balancing act—and in the background, a push to build testing capabilities for H5N1.
“Flexibility is the name of the game when it comes to respiratory virus season,” Dr. McKeown says. “It’s a big challenge, given how many variables need to be juggled, but it’s also an excellent opportunity because there are so many different solutions that can plug and play to meet the needs of the institution.”
The Cleveland Clinic has made one major change this year to its respiratory testing algorithm. “We’ve discontinued the standalone SARS-CoV-2 [assay],” says Daniel Rhoads, MD, section head of microbiology and associate professor at Case Western Reserve University. “COVID is endemic now and screening is not as essential as it used to be. And the FDA is pushing manufacturers from their EUA labeling to 510(k) labeling, and typically asymptomatic screening is being removed from indications for use with that transition.”
“Our hope is that this simplifies things,” adds Dr. Rhoads, who is vice chair of the CAP Microbiology Committee.
In August, there was a surge of SARS-CoV-2. “So everything stays the same, in some ways,” he says. “One of the challenges with COVID is that it has not yet settled into a single seasonal surge,” so although by mid-September in Northeast Ohio the summer wave was waning and rhinovirus was on the uptick, “I’m not sure we can predict that it’s going to be a long time before the next wave happens.”
RSV and influenza follow a more typical seasonal pattern, albeit one that can still be difficult to predict. “I don’t watch Australia closely,” Dr. Rhoads says. “I don’t know that it’s a great predictor. I do keep an eye on Florida because things tend to move from South to North with RSV and flu. When I see RSV emerging in Florida, I know we should be on watch because it’s coming our way next.”
Last year, the Cleveland Clinic encouraged simultaneous testing for flu A/B, SARS-CoV-2, and RSV, and it will continue to do so this year. “This is an area of active evolution, this testing approach,” he says. “Before COVID, we essentially encouraged not to test for RSV in adults,” but with RSV vaccines now available, “there’s renewed enthusiasm about how we can take better care of patients who might encounter RSV.”
Roche, too, is continuing to promote the value of multiplex testing, Dr. McKeown says. “Targeted multiplex assays, typically a four-plex but really any test detecting between three and five pathogens, are becoming the standard of care.” At the same time, she says, laboratories are strategizing ways to streamline their testing. “With the end of the public health emergency, there were more restrictions on reimbursement and who should be getting what test,” she says. “So those are our main focuses—the value of targeted multiplex as a standard of care and helping to navigate algorithm development with tenets of diagnostic stewardship.”
Upcoming for Roche is FDA submission of its Cobas Respiratory Flex test, which can detect up to 12 of the most common respiratory viruses, including influenza A/B, RSV, and SARS-CoV-2. “In line with the idea of customization,” Dr. McKeown says, the test allows the end user to specify which pathogens to look for using preselected targets from the list of detected targets to analyze and report. The assay, which runs on Roche’s high-throughput Cobas 5800/6800/8800 systems, is designed to be the first to use a new proprietary technology known as TAGS—temperature-activated generation of signal—which expands upon Roche’s current real-time PCR technology by allowing for three targets to be read at unique temperatures within each of the five existing optical channels. It also uses digital reflex, enabling additional results to be analyzed and reported without having to collect another sample or rerun the existing sample. “For instance, you can program the instrument to first test for the four-plex, and if those are negative, also release results for human metapneumovirus and the common seasonal coronaviruses,” she explains.
Some laboratories last year began to consolidate the extra platforms brought on for the pandemic. Others hesitated. “We still weren’t totally sure what the new normal was. We were waiting on a second year of normalcy to confirm what our expectations were. Now, I think we’re there,” she says, noting that discussions about platform consolidation are widespread, though competing priorities may push it to the back burner. Still, quite a few laboratories express concerns about supply shortages. “If we’re all consolidating into one vendor or platform, what happens if we can’t get those materials? That’s one concern that’s driving this hesitancy with platform consolidation.” But many manufacturers, Roche included, have increased their manufacturing capabilities, she says. “That’s not an issue with us.”
The same is true of Hologic, says Jennifer Schneiders, PhD, president of the Division of Diagnostic Solutions. “We have had the opportunity to strengthen our supply chain over the course of the past several years, and we are in a position where we are flexible depending on our customer needs and able to serve those no matter what the season brings.”
Hologic received in August FDA clearance for universal and viral transport media collection for its multiplex flu A/B, SARS-CoV-2, and RSV assay; the multiplex test was 510(k) cleared in 2023. Also new for Hologic is a differentiated collection device called the RespDirect. “That offers flexibility in terms of collection—you don’t have to remove the cap once it comes to the lab; it’s fully contained, with no risk of cross-contamination.” All Hologic respiratory assays—which also include parainfluenzas 1–4, adenovirus, human metapneumovirus, and rhinovirus—are run on the Panther Fusion system. “That offers labs of varying size the flexibility to run whatever volume is required within their laboratory,” she says, as well as a fully automated solution—critical at a time of continuing labor shortages.
“It’s an ongoing issue,” Dr. Rhoads says of the staffing difficulty. “The excitement of the pandemic and rising to the occasion has largely worn off. The adrenaline is gone and we’re now settling into the routine. So it’s hard to ask people to lean into and fix a crisis now, because now is when things should be relatively stable compared to the middle of a pandemic.”
Meanwhile, looking to the next disease with pandemic potential, a growing number of players within and outside the laboratory have set their sights on avoiding a recurrence of the delays that beset early SARS-CoV-2 testing.
During the summer, in response to the H5N1 outbreak in poultry and dairy cattle and the associated human cases, the CDC and state, tribal, local, and territorial public health agencies conducted an enhanced surveillance strategy to monitor for novel influenza A infections. In mid-September, the CDC tapped ARUP, Quest Diagnostics, Labcorp, and other commercial laboratories to develop a high-throughput test for H5N1 clade 2.3.4.4b, the pathogen responsible for the outbreak.
Reference laboratories have the expertise needed to develop high-volume testing for H5N1 or other diseases with pandemic potential, says Benjamin Bradley, MD, PhD, medical director of high consequence pathogen response, virology, and molecular infectious diseases at ARUP. Dr. Bradley credits the CDC for seeking out clinical partners. “All of this stems from learning lessons of the past,” he says. “When COVID started to spike and the CDC and public health labs got put in the position of having to act more like a clinical lab or a reference lab, we saw how that crumbled.”
The CDC already has a 510(k)-cleared H5 assay. “But that was developed several years ago as an epidemiological public health tool for detecting all circulating H5 types,” says Dr. Bradley, who is also assistant clinical professor at the University of Utah School of Medicine and a member of the CAP Microbiology Committee. “We know this H5 strain, clade 2.3.4.4b, that’s been circulating in dairy cattle, has caused these human infections,” he says. “So when we think about building a clinical assay, the focus will be on designing it to detect the pathogen that’s causing disease now.” The differences from the existing assay will be minor, he notes—a one- or two-base change. “We’re tweaking it to make it what we need right now, not trying to use something we’ve made in the past and shoehorning it to fit the current situation.”
“It makes sense to bring on partners who have that expertise,” he says. “Instead of the CDC trying to spin up these super high-volume labs, let’s work with the folks who have been doing this for 40 years, who have the preanalytics and logistics in place for it.”
Dr. Bradley doesn’t see this work as limited to H5N1; rather, it could be applied to any emerging disease. “What we realized from SARS-CoV-2 was that we had the testing volume we needed two months too late,” he says. “So how do we address that for pathogen X that comes out in the future?” The goal is to complete the development work before an outbreak reaches pandemic-like proportions. “So if there is a pathogen like bird flu that starts showing human-to-human transmission . . . the CDC can go to laboratories that have that infrastructure in place to support clinical testing needs, not just public health testing needs.”
Dr. Bradley and Benjamin Pinsky, MD, PhD, of the Stanford University School of Medicine Department of Pathology, in a recent review discussed the limitations associated with the CDC’s assay (Pinsky BA, et al. J Clin Virol. 2024;174:105723). For example, the CDC assay requires four reactions per sample, with a single target in each reaction. “If a laboratory were to run the test as it’s written by the CDC for a 96-well plate, you can do 19 patients,” Dr. Bradley says. “Whereas if you optimized all four targets for the patient into a single well, you could do 93 patients.”
Another challenge they address: navigating the FDA final rule on laboratory-developed tests. “What could be modified from the CDC assay that would allow for staying within the acceptable clearance and being able to do just a verification study, and what would be considered significant alterations requiring premarket review?” Dr. Bradley asks. “My concern is that because this has still been poorly defined, it could potentially discourage some laboratories from bringing on testing earlier,” as they might have pre-FDA final rule. “That is a risk we have with the FDA final rule.”
A declaration of public health emergency would open new pathways for test development under emergency use authorization. “But that still takes time, it relies on the government to issue these declarations, and that puts us at risk of being behind the curve again,” Dr. Bradley says. “What I’m hoping for from these collaborations with the CDC is they’ll see the value clinical laboratories can have in developing these assays, and that we might have effective assays that can be made available without having to go through a full FDA approval process.” It could also make those in government more aware of certain nuances, he says, “that a test for a hemoglobin A1c is different from a test for prenatal screening of aneuploidies is different from a test for H5N1, and the context in which these are applied have different risks associated with them. And certainly, in public health, having some leeway for established laboratories to provide this testing is important.”
At the same time, he adds, “I’m not saying everyone should be given carte blanche for developing these assays. We saw with COVID that there were a lot of fly-by-night operations—people who were trying to make money off a serious event—and we want to avoid that.”
Dr. Bradley points to additional steps that could be taken to ease the process of H5 assay development. “The first thing that comes to mind is resources for validation,” he says. When ARUP began work on its H5 assay in recent months, “we didn’t have positive control material in the form of H5N1 virus from a patient, because there weren’t that many. And that was a problem we saw in COVID as well. We had to make artificial targets to validate this assay, and when you don’t have true patient specimens that’s the best you can do.” He would like to see H5N1 isolates made available and a standardized approach for validation.
Other barriers arise when pathogens are labeled as select agents, he says, requiring a license for import or use. H5N1 fell into this category until recently, when the United States Department of Agriculture exempted it. But select agent regulations are still in place for other pathogens, such as mpox clade 1, the mpox isolate now causing disease. While clade 2 is exempt, if a laboratory wants to develop a clade 1 target, “then they have to deal with select agent licenses and shipping requirements.” No one wants to put laboratory staff at risk, he says, “but if we are getting into a pandemic-type situation where this testing would be indicated, we need a rapid response” from the CDC and others.
What role can existing influenza A diagnostic assays play?
No commercially available assays support H5 subtyping. But the existing testing infrastructure should identify the current H5N1 virus as influenza A. What is important, Dr. Bradley says, is knowing how the laboratory’s influenza assay performs—that is, “making sure your test does have cross-reactivity with the emerging H5 strain, whether the manufacturer has done in silico analysis or another type of analysis, and then sharing that information with the clinical community and hospitals.” For instance: “Our test can detect influenza H5N1, but it’s going to report it out as flu A. If you have someone from a rural county who has exposure to cattle or infected birds and they come back with a flu result, that requires more investigation. You need to get in touch with the laboratory to let them know this specimen should be sent to their state or local public health lab for further typing.”
At the Cleveland Clinic laboratory, Dr. Rhoads and his colleagues have confirmed in vitro that the assays they use can detect H5N1. “Some assays have multiple targets and report those targets discretely for subtyping,” Dr. Rhoads says. “We’ve put some guardrails around it so if we get an unusual pattern of PCR results that could potentially be congruent with a novel type of influenza, then it should bring it to medical director attention for review and potentially sending that sample to public health for further characterization.”
Dr. Schneiders, of Hologic, says the company is monitoring the situation and is in contact with the CDC on intel related to H5N1. The Panther Fusion system, she says, has open access capability. “So that is an opportunity for laboratories to develop their own test.”
With assays that do report subtyping for certain targets, Dr. Bradley says, such as H1N1 or H3N2, the situation to watch out for is when the test reports a result as influenza A without one of those two targets. “That could potentially signal that you have H5N1 or some other type of unsubtypeable influenza,” he says. “Now, you can have target failures because of normal mutations in an H1 or H3. Or if it’s near the limit of detection, the pan-influenza A target may detect and the other two may not.” Having policies and procedures in place to flag such results is important, he says.
Only a quarter of participants in a recent CAP Survey (2023-ID3-C) reported a subtype for influenza when using nucleic acid amplification tests, as he and Dr. Pinsky report in their review, indicating that within the community, the capacity for identifying H5N1 is primarily limited to identification as influenza A.
Hence the push to send samples to the public health laboratory. “Encouraging clinical laboratories to participate in the public health response to the extent they’re able to is a great help,” Dr. Bradley says. “And in my personal opinion, doing subtyping for those influenza A tests may have higher clinical yield than we’re suspecting.” The assays that perform influenza A subtyping tend to be broad respiratory panels, he says, and those aren’t typically ordered on outpatients. “In the community, most of what we have doesn’t provide subtyping. And if we look at where bird flu cases have occurred so far, it’s been in the community. It’s been in rural farmworkers,” he says. “So in addition to testing samples from hospitalized patients, we need to be doing a good job of surveilling flu-positive results from outpatient clinics, community clinics, and point-of-care testing services.”
After the CDC released its enhanced summer influenza surveillance strategy, the Tennessee Public Health Laboratory, led by director Kara Levinson, PhD, MPH, D(ABMM), sent a letter statewide to clinical and commercial laboratories requesting influenza A-positive samples.
“We’ve received over 100 samples since May, which is notable because it’s not our respiratory season—certainly numbers are higher then,” Dr. Levinson says.
Even during respiratory virus season, obtaining samples from clinical laboratories can be challenging. “It takes time and people to save those samples, pull them, and then ship them to the public health lab. I’ve worked in public and clinical labs so I’ve seen both sides and I do get that resources are involved and it’s not always easy. But it does make a difference. We can’t do surveillance without our clinical partners,” she says.
Storage and transport are difficult for laboratories in Tennessee. The CDC’s H5 assay has strict criteria for acceptance, she says. “We have to receive it within 72 hours of collection, which is a challenge if it goes through a clinical laboratory first. Or it can be frozen, but it has to be frozen at minus 70 centigrade and then shipped frozen on dry ice, and that also presents a challenge.” She and her colleagues have discussed ways to reduce the burden, such as allowing batching of samples or providing ice packs and shipping containers. “Anything we can do to make it easier to get those samples to us.”
With avian influenza in the news, her hope is the added publicity will bolster her laboratory’s routine influenza surveillance numbers. If a clinical laboratory gets samples that can’t be subtyped, she notes, “those should come to us at the public health laboratory anyway, no matter what time of year or what year it is.” But this year, given the H5N1 outbreak, “it’s particularly important that it gets to us and gets to us quickly. So we may have more awareness going into this respiratory season.”
Dr. Levinson sounds another hopeful note: “Avian influenza is a perfect example of an opportunity for clinical and public health laboratories to collaborate. Early detection of human-to-human transmission behooves us all, and that’s likely to start with the clinical lab doing that initial testing and then moving it to the public health lab to do the subtyping.”
As with all partnerships, communication is key. “We found that with SARS-CoV-2, and it’s going to be critical to success with avian influenza as well.” In Tennessee, “we’ve got longstanding relationships with our larger clinical partners, our bigger hospital systems. But we’ve also set up a quarterly call with all the clinical labs in our state so we can present updated guidance and answer questions,” she says. “We found coming out of the pandemic that there is turnover in staffing across the board in both clinical and public health laboratories.” Reestablishing those relationships and points of contact has been critical, she says.
The lines of communication are solid in Iowa, where Michael Pentella, PhD, D(ABMM), is director of the State Hygienic Laboratory and clinical professor at the Iowa College of Public Health. “Eleven herds in Iowa were infected with H5,” Dr. Pentella says. “And we were called upon to test patients who had signs and symptoms of influenza-like illness.” Those test requests were treated as an emergency, he says. As the national statistics show, however, “there were no positive patients from Iowa.”
Dr. Pentella similarly promotes collaboration. “It’s important for clinical laboratories to be well connected with the public health labs in their jurisdiction,” he says. “Know the clinical lab’s role in public health surveillance and do the best to contribute samples when requested to do so.” It takes time and money, he acknowledges, “but it’s so important for the public good.”
As for the public health laboratory’s response? It’s firm, Dr. Pentella says. “We stand prepared.”
Charna Albert is CAP TODAY associate contributing editor.