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Use of MDx Tests for Animal Health on the Rise, but Cost Concerns Present Significant Hurdle

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In the second part of this two-part story, GenomeWeb spoke with commercial developers, end users, and federal regulators about trends in animal health molecular diagnostics. Part one of the story, which focused on the overlap between animal and human MDx development, can be read here.

NEW YORK (GenomeWeb) – Compared to molecular diagnostics developed for use on humans, the animal health MDx market is uniquely constrained by cost. With agricultural diagnostics in particular, patients are also inventory, and veterinary diagnostics labs must be ever aware of the bottom line.

In recent interviews with GenomeWeb, industry and federal sources suggested that molecular methods remain on the rise in veterinary diagnostics labs. More US Department of Agriculture-licensed kits may be imminent, but test makers are also attempting to incorporate quality controls into assays while remaining on the analyte-specific reagent side of the regulatory fence.

More molecular

Use of molecular techniques, particularly PCR, has been increasing in veterinary diagnostics labs for a number of years, explained Beverly Schmitt, director of the Diagnostic Virology Lab at the US Department of Agriculture's National Veterinary Services Laboratories (NVSL), which is under the umbrella of the Animal and Plant Health Inspection Service (APHIS).

"I would say over the last 10 years the trend has definitely been toward using PCR, maybe even in lieu of virus isolation or culture," she said, noting however that this can depend on the pathogen and the subfield, and that other laboratory methods are still essential.

Although there is increasing use of molecular, "The trend generally speaking in our industry is to drive more value for each sample that's being sent to the lab, and, while interrogating those samples, try to get as much information as possible at the lowest cost possible," said Martin Guillet, global head and general manager of animal health at Thermo Fisher.

Panelization and automation are increasingly used, just as in the human MDx market, but the animal health industry is more fragmented, and each lab's testing volume tends to be smaller, Guillet said.

Jeff Lorch is a research associate at the Department of Pathobiological Sciences in the School of Veterinary Medicine at University of Wisconsin-Madison. He conducts his work in collaboration with the US Geological Survey's National Wildlife Health Center, an agency responsible for assessing wildlife and ecosystem health.

The majority of samples submitted to NWHC are from wildlife die-off events, Lorch explained in an email to GenomeWeb. "We also assist with determining cause of death in endangered species, in certain legal cases, and other instances in which disease has the potential to impact wildlife populations and ecosystems," Lorch said. The NWHC also researches wildlife diseases, and assists with disease surveillance, "including diseases that can spill over from wildlife into humans and domestic animals," he said.

For example, the NWHC recently helped identify the novel fungus responsible for a massive mortality event of hibernating bats, and developed molecular tests to help diagnose White-nose syndrome.

It also was part of a coordinated effort to investigate a recent waterfowl die-off. Along with the Washington State Department of Fish and Wildlife and the USDA, the lab found that the birds had died of an opportunistic fungal infection, but that some of them also had a highly pathogenic H5 strain of avian influenza. Specifically, H5N2 was found in northern pintail ducks and H5N8 in captive gyrfalcons that were fed wild birds killed by hunters. Just last week, an H5N1 reassortant strain was reported by the NWHC in a green-wing teal. That strain had segments identical to the gyrfalcon H5N8 as well as segments from North American low-pathogenic avian influenza viruses of wild bird origin.

Monitoring wild bird flu can help prevent transmission to domesticated birds, and hopefully stave off another bird flu epidemic in the US. To surveil wild animals with the USGS, "We are using more molecular assays than in years past," Lorch said, adding that it is likely "this will continue to increase in the next few years as more assays are developed and as molecular diagnostics continue to become more affordable."

However, he asserted that in some scenarios, molecular assays will likely remain just a part of a more comprehensive molecular biology toolkit. "Particularly with novel wildlife diseases, or diseases about which little is known, other techniques including histopathology, pathogen isolation, and immunohistochemistry will remain essential for understanding pathology and making diagnoses," he said.

Likewise, in many cases, assays for rare or novel pathogens affecting wildlife do not exist, Lorch said, and demand is low because there are few laboratories in the space.  "Thus, we often have to develop assays ourselves or work with other labs that are investigating wildlife disease to design and share assays."

Kits and QC

Commercially available molecular assays are federally regulated. They can be sold as reagents, but can only be packaged into "kits" if they are USDA licensed.

A licensed assay will include full protocols, internal positive controls, and describe the interpretation of the results. The assay is then called a kit. For sale and distribution in the US, assays that are not USDA licensed may not provide all of these elements ― performance data or validation, for example, can't be part of a product insert with assay reagents ― and a set of reagents can not be called a "kit" unless it has a USDA license. Interestingly, this rule does not always apply in international markets.

Thermo Fisher makes four of the six molecular diagnostic kits with USDA licenses. There is currently "a big push" at the company to start validating assays and pursuing USDA licensing for more, Guillet said.

"Kits come with an internal positive control built into those assays, so the lab can run their tests, look for the targets, and if they see the IPC rising in their PCR reaction and the target is not rising, then they can be sure that they're getting good results," he said.

Tetracore, a maker of molecular diagnostics and immunoassays for both the human and animal health markets, has two USDA licensed kits, but has not pursued more. In the first part of this series, Bill Nelson, Tetracore Co-founder and CEO, explained that the cost of pursuing licensure must be made up later by sales. "From an economic standpoint, it is not necessarily a good proposal," he said. "It's a balancing act, and you hope over the long term you'll recoup that cost of getting the licensure versus selling [an assay] as a reagent."

To enhance quality control, Thermo Fisher is also working with an industry association called The American Association of Veterinary Laboratory Diagnosticians ― a body overseeing standardization, quality assurance, and control ― to ensure labs are using methods, workflows, and protocols that they can rely on, so that "the vet has assurance that if he or she is sending samples to that lab that they'll get consistent results," Guillet said.

Quality control is vital to other users as well. "The same attributes that make molecular assays so valuable can also cause issues when they are not used appropriately," Lorch noted. Many real-time PCR assays are so sensitive that they are prone to contamination and can yield false-positive results, he said. "The inclusion of numerous DNA extraction controls and PCR controls is essential to ensure accurate results and to make sure that assays are consistent across different reagents and platforms," he said, noting that in the wildlife testing domain they often rely on lab-developed tests rather than USDA licensed kits.

Specificity of assays, lab-developed or otherwise, is another common issue, Lorch noted, because "standards for what constitutes rigorous screening of an assay, especially those that are not commercially available, are often poorly defined," Lorch said.

Customer cost-cutting

A persistent trend in the animal health industry seems to be strict attention to cost. Makers of molecular diagnostics for pigs or cows must match their pricing to the narrow profit margins of farmers and veterinary labs.

Although clinicians in the human diagnostics space are embracing pooled nucleic acid testing more and more — for HIV testing and assays of nasopharyngeal swabs, for example — pooling samples is even more common in the veterinary diagnostics world.

Pigs can contract a virus called porcine reproductive and respiratory syndrome, or PRRS, and Tetracore makes an assay for PRRS that can detect virus from saliva samples. Nelson explained that end users often pool these samples using a rope.

Essentially, a farmer or veterinarian ties a short length of rope to the side of the pig pen. "Say a pen has got 25 pigs in it, they'll all come over and chew on that rope, it becomes a game for them," Nelson said. Samples of this rope are then used for viral extraction, and, if there is a positive result, more detailed testing can be done.

Likewise, for Tetracore's other USDA licensed test for Johne's disease, diagnosticians often use pooled fecal samples.

"Basically a large portion of [molecular testing] is massive screening for transportation and for security … you're trying to make sure your herd is clear and free of the disease," Nelson said. At least for agricultural diagnostics, "There aren't a lot of things that they're necessarily testing individual animals for," he said.

The future of animal MDx

Still, at some labs cutting-edge molecular biology is sometimes more important than cost-cutting. Like their counterparts in the human MDx universe, veterinary diagnostics labs are also adopting next-generation sequencing and bioinformatics to identify pathogens, particularly in cases of mystery mass die-offs.

At the NVSL, Schmitt says they use Ion Torrent technology and also have MiSeq on campus.

"What I think is going to happen, and what I think everybody else is seeing, is that the veterinary diagnostics labs, just like with [the uptake of] PCR, will move toward doing next-gen sequencing of clinical samples," she said. "They're already doing that and we have done it also … particularly if you suspect a certain family of viruses. … We've [also] used next-gen sequencing to detect unknowns," she said.  

Given this trend, Schmitt suggested that one of the issues labs are going to have to work through in the future is "the IT component of that … having the bioinformatics on site to crunch that information and to look at trends, [and] combining that with epidemiology of a disease," in order to get a better picture of what's going on in the field. Thus, labs may soon need more staff with "some sort of training in bioinformatics to be able to interpret that sequencing information [or] put it into phylogenetic trees," Schmitt said.

Meanwhile, at the USGS, Lorch said he primarily uses PCR assays for molecular work. These vary from conventional and real-time PCR tests for specific pathogens, to 'DNA barcoding' for fungi or bacteria. He said the lab does follow-up DNA sequencing after barcoding to confirm the identity of a microorganism, and also uses NGS for pathogen discovery work.

So-called "penside" molecular testing is also being developed for a few animal pathogens, such as a point-of- care porcine epidemic diarrhea virus test in the works from Lucigen.

Thermo Fisher is also contemplating developing these. "We're looking into it for sure," Guillet said. "There's no doubt that the technology is heading that way. In our case it's more in terms of epidemiological surveillance, for example for migratory birds that we keep an eye on," he said.

But end users seem uncertain about how useful penside molecular tests may ultimately be.

"There's been a lot of development in regards to some foreign animal diseases," particularly for penside immunoassays, Schmitt said, but added, "generally, still, most of the samples are collected on the farm and then they go to a veterinary diagnostic lab."

Schmitt noted that penside molecular testing might be most useful for illnesses called foreign animal diseases. These are pathogens that can wipe out herds, but have either been kept out or controlled within theUS.

For these pathogens, "It would be very useful to know right away so that you can restrict movements and respond to trying to eradicate the disease."

There are also avian influenza molecular tests that can be run in the field ― literally ― but they are still more often used in the lab, where the expertise resides, Schmitt said. In addition, higher test volumes are easier to run on high-throughput systems, and often vets do not need to know "within five seconds" whether a particular disease is present.

At the USDA's CVB, Byron Rippke said he believes that one way or another, there will likely be more molecular tests for animal health soon, and more of those will be USDA licensed.

"I honestly think that with all the funding that [has been] poured into research on rapid diagnostics … that we're at the leading edge of commercialization of a lot of those technologies," Rippke said. "I think there's going to be a fairly significant uptick in molecular-based diagnostic assays in the next few years," he said. 

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