Medical Device Link.

Originally Published IVD Technology June 2001

Taking IVD test technology beyond human clinical diagnostics

Alan Reder

Market opportunities in food safety, veterinary, and environmental testing hold promise for large and small IVD companies.

Alan Reder is a freelance writer in Rogue River, OR. He has written for IVD Technology's sister publication, Medical Device & Diagnostic Industry as well.

Some of these markets promise the kinds of returns that attract major corporations now involved in human IVD testing. These companies hope to realize added value from intellectual property they originally developed for human use. Other nonhuman-clinical IVD markets are modest and slow-growing. They offer niche opportunities for companies too small to compete successfully against the majors in human testing.

Three extraclinical areas in which both established and entrepreneurial IVD companies are gaining footholds are food safety, veterinary testing, and environmental monitoring.

Food Safety

The Envirologix QuickStix strip can detect Cry9C, the unique protein expressed in the bioengineered StarLink corn. The strips can be used to test bulk grain as well as partially processed corn products.

Bob Minarchi, president of OEM Concepts (Toms River, NJ), a manufacturer of clinical testing reagents, estimates the market for tests mandated by the U.S. Food Safety Inspection Service (FSIS) to be some 1.45 million tests per year. FSIS is the Department of Agriculture (USDA) agency responsible for meat inspection. It oversees 6000 meat-processing plants nationwide. Its jurisdiction covers most meat-safety testing, because such testing takes place primarily at the plant level rather than at the farm or sale barn.

With the public concern about safe meat increasing and the meat-processing industry desperate to reassure its customers, the food safety–testing market could grow to $1.5 billion annually, suggests Minarchi. It would encompass both instrument-based, high-throughput testing for large processing companies such as Tyson Foods and Durkee and rapid, dipstick-type testing suitable for smaller plants.

Food safety testing relies to a large extent on immunoassays that were developed for human clinical use, but Minarchi believes that this separate market is "much, much bigger potentially than the clinical marketplace for these same products."

BSE has grabbed many of the recent headlines, but most food-safety testing involves the "big four" pathogens:

• Listeria, a microorganism found in meats that can cause spontaneous abortions or stillbirths in humans and make young children, the elderly, and the immune-compromised severely ill.
• Escherichia coli O157:H7, a meat contaminant from fecal matter that, like Listeria, can be life-threatening to the very young, the very old, and the immune-depressed.
• Salmonella, a bacteria found in meats and eggs that can cause mild to severe gastrointestinal symptoms and, occasionally, more-severe damage or death.
• Campylobacter, the least-serious type of bacterial food poisoning, and the most common.

These food contaminants have been of long-standing interest. In addition to testing for them, the demand for testing for newer food-borne dangers is increasing. For instance, more global travel means that contaminated foods are being smuggled past checkpoints in luggage and purses. This phenomenon introduces alien pathogens into the food supply and thus creates a need for new tests to weed them out.

Genetically modified content is another area in which testing opportunities abound. The U.S. public has not seemed to worry much about genetically engineered foods, but customers in Europe and other foreign markets have indicated that they want no part of them. This means that U.S. companies exporting food products to many countries need to test for genetically modified content.

In addition, U.S. biotechnology companies are working hard to develop new genetically modified attributes—for example, lower fat or higher vitamin content—that will attract consumers to engineered food products. As these products are readied for market, they must be tested for the expression of the inserted gene, points out Dean Layton, vice president of marketing and sales for Envirologix (Portland, ME), which produces both food safety and environmental monitoring tests. "Plus, the market itself will demand a certain amount of testing in terms of the segregation required for a company's different customers and market segments," Layton adds.

Microbiological contamination of fresh fruits and vegetables is another area of growing concern. With global trade, more produce is entering the United States from countries whose water-quality, hygiene, and other pertinent standards may be lax.

Weschler notes also that consumers are buying more packaged produce, such as salad mixes, which they expect to be clean in the bag, despite packagers' cautions to the contrary. These consumer expectations present a daunting challenge to both produce packagers and those who regulate them, Weschler says, because unlike the case of meats, there is no "kill step" with these foods. Even though meat and dairy testing cannot possibly catch all dangerous pathogens, organisms that escape detection at the testing stage are usually eliminated at some later point—when milk is pasteurized, for example, or meat is cooked at high heat. For produce served raw, no such safety net exists.

IVD methods are the best way to tackle the problem, suggests Mark Rasmussen, a research scientist at the National Animal Disease Center (NADC; Ames, IA) of the USDA Agricultural Research Service (ARS).

Some animal-health issues have crossover impact on the food safety–testing marketplace. For instance, dairy farmers worry about paratuberculosis (also called Johne's disease) and bovine viral diarrhea (BVD), both of which are debilitating to cows, affecting milk production. These diseases, which can spread through a herd, generate indirect food safety concerns. When unproductive cows are identified, they are culled from the herd, sent to slaughter, and end up as meat. Most hamburger meat comes from culled dairy cattle.

Then, of course, the BSE crisis in Europe presents, along with the staggering health challenge, an enormous business opportunity for test companies. Currently, only three tests for BSE have been approved by the European Commission. Five more are awaiting approval.

Figure 1. The Bio-Rad Platelia BSE screening test detects the presence of a resistant form of protein prion, using monoclonal antibodies in a standard ELISA format.

Schmerr points out another significant disadvantage of postmortem tests: if testing turns up an infected animal at a slaughterhouse, that means the plant's equipment could be contaminated and thus must be thoroughly disinfected. Were a live-animal screen available, slaughterhouses could demand that animals be tested at the farm so as to minimize the expense and danger associated with this problem.

The first companies to develop effective, easy-to-administer screening tests stand to take over a major share of the BSE-testing market. Two tests currently in development, including a blood test that Schmerr herself is working on, show promise. However, validation of the tests as predictors may still be years away because BSE can take two to five years to reach clinical proportions in animals.

But even without an effective screening test for live cattle, the market for BSE test kits is considerable. On January 1, 2001, member countries of the European Union (EU) commenced mandatory testing of all beef cattle more than 30 months old. This comprises some 7 million head this year. At roughly $14 per kit, that amounts to a market of close to $100 million. Although no cases of BSE have been reported in the United States, testing in this country would multiply the market more than 14 times. About 100 million cattle live on American soil, according to the National Cattlemen's Beef Association.

American companies prominent in food-safety testing today, besides those already mentioned, include Becton Dickinson (Sparks, MD), Millipore Corp. (Bedford, MA), Neogen Corp. (Lansing, MI), Pall Corp. (East Hills, NY), Remel Inc. (Lenexa, KS), and 3M (St. Paul, MN). Overseas food-safety companies include Becton Dickinson in Belgium, Foss in Denmark, bioMérieux in France, and Oxoid in the UK.

The IVD technologies employed in food-safety testing often involve antibody-based or polymerase chain reaction processes. Minarchi of OEM Concepts notes that human clinical IVD methods that successfully identify whole organisms are particularly well suited for applications in food-safety testing.

New approaches may begin to crowd out traditional methods, however, at least in certain applications. Consider, for instance, E. coli. This pathogen inevitably contaminates carcasses when animals are disassembled in processing plants. The time-to-result of tests for microbes is long because of the need to first draw samples and then culture the organisms and perform other process steps. To overcome that problem, Mark Rasmussen, his NADC colleague Tom Casey, and Iowa State University photochemist Jacob Petrich have together developed a prototype of an optical device that detects luminescence in metabolites of gastrointestinal contents, the source of the contamination. The device theoretically can identify every contaminated carcass in real time as it passes by on a conveyor belt or other transport.

The optical device is not a magic bullet. It does not work with hamburger, for instance, because contaminants are often mixed into the interior portion of the meat. But it could be tremendously useful for initial screening. And even if the optical technology is effective only at the carcass stage, other new approaches may be up to the hamburger challenge. One innovative method involving immunomagnetic-electrochemiluminescent detection has proven effective in finding E. coli O157:H7 in ground meat. Gerald C. Crawford, technology transfer coordinator of the USDA ARS and a developer of the technique through a Cooperative Research and Development Agreement with Igen International Inc. (Gaithersburg, MD), says that while this approach does not offer real-time detection, it seems to be much more sensitive than some traditional methods. Igen International Inc. has since commercialized the test (see Figure 2). Igen is just starting to penetrate the U.S. market after debuting in the UK.

Figure 2. The PathIgen E. coli O157:H7 test uses immunomagnetic separation and electrochemiluminescent technologies in a no-wash immunoassay format.

Of course, a technologically innovative method designed to meet such technical challenges can face problems in getting approved. The federal government has in recent years allowed industry groups to perform the actual approval of testing methods. There is a significant investment of resources and time needed to gain approvals and certification, says Weschler. Nevertheless, new thinking is clearly needed if the considerable challenges of food-safety testing are to be overcome.

Veterinary Testing

The veterinary testing marketplace comprises two primary subsectors: commercial production animals (mainly dairy cattle) and companion animals—that is, pets—such as dogs, cats, horses, and parrots. The subsectors bear little relation to each other in terms of the types of tests required, the types of companies attracted to each sector, and the types of issues that testing companies face.

In the commercial production area, animal health and human food-safety issues often overlap, as already noted. For discussion purposes in this section, however, consider veterinary issues pertaining to commercial animals to be those that affect primarily an animal's productive value.

For instance, mastitis does not seriously threaten a dairy cow's health—according to Beth Claypoole, executive director of Cornell University's Wayne County Cooperative Extension (Newark, NY). The condition affects the cow like a persistent cold affects us—but the animal gives less milk when she is not feeling well. Therefore, dairy farm performance can be improved by identifying the illness and then either treating it or culling the sick animal from the herd.

Take, for example, a cow that is producing 80 lb of milk per day, which is a perfectly acceptable output in the judgment of many farmers. A test might show the cow to be suffering from a mild case of mastitis, the treatment of which could enable the animal to produce as much as 90 lb of milk daily. But that is a tricky case to make to a cash-conscious farmer, in Claypoole's experience.

Concentration of the dairy industry should increase opportunities for diagnostic companies. As farms grow larger and are more centrally managed, farmers have less knowledge of each cow's productivity and health; thus, testing can play a vital role in improving herd management.

A test developed by Midland Bioproducts, for instance, determines whether a calf received enough colostrum in its first weeks. Without sufficient colostrum, the calf is likely to be illness-prone, to be contagious to the rest of the herd, to grow more slowly, and to be less productive over its lifetime. Testing could identify it as a prime candidate for culling.

The issue, again, is how to convince the farmer to spend the money. McVicker cautions companies eyeing this potential customer base to consider the substantial marketing effort they will have to invest. He thinks that one reason not many large companies see the farm marketplace as a profitable arena is the education necessary to generate big sales.

Companies also must understand the special needs of this market. Ideally, the time to results should not exceed 2 hours for any test, advises Claypoole. (Tests for Johne's disease and BVD now require 6 weeks.) Also important are ease of use, to enable nontechnical farmhands to administer the test, and durability, so the kit can stand up to rugged handling in a highly septic environment. Interpretation of results should be straightforward (i.e., positive or negative, without gray areas), and the chemistry should be self-contained (e.g., no external mixing of reagents). Low cost, of course, is paramount.

The veterinary market for companion animals may not have nearly the potential that McVicker sees in dairy cattle, but it offers a more natural fit for established human clinical companies. The reason is simple: sick animals require many of the same tests that sick people do, or slight variations of them. In fact, veterinary testing is sometimes done in the very labs that test human samples. Although they rarely advertise it, many human hospitals have increased their billables by taking in veterinary work.

Veterinary testing is also performed at veterinary colleges, of which there are 27 in the United States. Private referral labs do considerable veterinary testing, too; the majority of these have been bought up by two companies, Idexx and Antech Diagnostics (Irvine, CA). In addition, more and more testing is being performed in individual veterinary practices—even small one-doctor businesses—as instruments have become more affordable. Test menus typically can handle a variety of species, both mammalian and nonmammalian.

The veterinary IVD marketplace is a fraction of the size of the human clinical one. The American Veterinary Medical Association (AVMA) has verified that at least 45,200 veterinarians were in private practice in the United States as of January 1, 1999. They had a mean 1997 pretax income of $65,208. (The statistics here are the most recent available.) These veterinarians worked in a verified total of 22,400 practices. Another 10,180 veterinarians were in public or corporate practice as of the beginning of 1999, according to AVMA. AVMA estimates that 31.2 million U.S. households owned dogs and spent an average of $186.80 on veterinary care in 1996. Some 27 million households owned cats and spent $147.19 on vet care that year; 4.6 million owned birds and spent $10.95; and 1.5 million owned horses and spent $226.26. With rare exceptions, such as insurance for high-value horses, there is no third-party payer system in place for companion-animal health. Almost all owner expenditures are out of pocket.

Environmental Monitoring

Small IVD companies seeking a place to play should take a careful look at the environmental monitoring area. The overall environmental market comprises some 35 million monitoring tests annually worldwide, according to The Industrial Microbiology Market Review, a report published by Strategic Consulting in 1998 (see sidebar, page 39). That figure does not measure up to the market for food safety testing, of course, but the same modest numbers that keep big players away also reduce competition overall.

The environmental monitoring marketplace takes in the subsectors of drinking water, recreational water, wastewater, and soil remediation. Unlike the situation in the food industry, environmental testing methods must be approved by the regulator in the United States, specifically, the Environmental Protection Agency (EPA). That can be challenging to IVD companies for several reasons. For one thing, EPA does not embrace novel methods quickly. Layton of Envirologix notes that it took five years for the agency to approve his company's new water test for E. coli, though he believes approvals can now be completed in one or two years.

A second and more formidable challenge is that, for many applications, EPA is more familiar with gas chromatography (GC) than IVD methods, says Brian Skoczenski, president of Beacon Analytical Systems (Portland, ME). Consequently, it is hard to get IVD methods approved, whether novel or not. Beacon has waited more than four years for approval of an immunoassay that tests levels of the pesticide atrazine in drinking water.

But companies like Envirologix and Beacon can still find customers for their products, even without approvals. For instance, in the U.S. corn belt where atrazine is heavily used, spring rains wash the water-soluble pesticide into the water table. Water companies there install activated-charcoal filtration systems during the rainy season in order to meet government requirements for safe levels of the chemical. That is an expensive process. Use of a test like Beacon's immunoassay allows the utilities to forestall implementation of the filtration system until it is absolutely necessary (see Figure 3).

Figure 3. The Beacon atrazine tube kit is capable of quantitating atrazine residues between 0.05 and 5.0 parts per billion in 30 minutes.

Not all global markets are so promising. The EU in particular has much more stringent drinking-water standards than the United States; not all U.S. IVD tests have the sensitivity necessary to fulfill their requirements. U.S. standards are widely accepted in nations outside the EU, however, so a test approved or certified in this country should have excellent potential global utility.

Conclusion

Hot-button issues like BSE and hoof-and-mouth disease promise to create extensive new testing markets for IVD companies and to intensify public demand for better and more comprehensive food-safety testing in general.

In addition, Americans seem to be growing more skeptical about government food standards. Although they have long trusted that any foods the authorities allowed to be sold must be safe, 30% annual growth in organic-food sales suggests that public confidence is not what it used to be. This trend could lead to more testing.

And while biotechnology companies hope to seduce U.S. consumers with engineered food products like low-fat corn, resistance to genetically modified content of any kind remains strong in the EU and other places. Testing can be expected to continue to play a vital role in the food export business.

Skoczenski cautions that the miniaturization of GC instruments probably will cut into the IVD share of the environmental monitoring market. When GC instruments can be backpacked into the field, one of the main advantages of IVD kits—their portability—disappears. Nevertheless, even in the environmental area, IVD penetration should grow as regulators become more familiar with IVD methods.

Globalization, Third World industrialization, biotechnology, and other such contemporary trends are making the world a more contaminated place—or at least causing it to be seen as such. The only way that public safety can be maintained is if IVD and other testing technologies play an ever greater role in guaranteeing it.

The Transmission of Prion Disease

Prions are pathogenic variants of proteins that are naturally produced in nerve cells and certain other cells. They have been implicated in a variety of transmissible spongiform encephalopathies, including sheep scrapie, mad cow disease, and human new-variant Creutzfeldt-Jakob disease. Normal, healthy prions are referred to as prion protein cellular (PrPc). In the illustration, the production of PrPc is shown from the nucleus (a). RNA that codes for PrPc is produced in the nucleus and exits via the nuclear pore. The RNA then passes along ribosomes attached to the rough endoplastic reticulum (rER). PrPc is formed in the rER and then progresses up through the Golgi complex. At the upper face of the Golgi, vesicles containing PrPc bud off and travel to the cell surface (b). There, they fuse with the cell membrane and so discharge their cargo (c). By this means, the cellular proteins come to sit on the exterior of the cell. In this illustration, PrPc particles encounter rogue prions, shown in purple (d). These are termed prion protein scrapie (PrPsc), for the prion disease of sheep. Such rogue prions seem to force normal proteins to change shape. Both types of protein—PrPc and its corresponding prions—are the same chemical, but in different shapes. Equivalent to the transmission of infection, such conformational shifting may take place at the cell surface or in caveolae (one is shown as a small invagination in the cell membrane). In such vesicles, residual PrPc may continue to be flipped by contact with rogue conformations for some time. Prions polymerize, finally appearing as purple fibrils (e). PrPsc is resistant to degradation by the enzymes contained in lysosomes (shown here floating nearby). Consequently, PrPsc accumulates in the cell. PrPsc vesicles may also travel to the Golgi and intercept PrPc that is being processed there. In this way, PrPc particles can be switched to the rogue form before they reach the surface of the cell. By such mechanisms, PrPc may be switched to PrPsc at various points in and on the cell. Prions may enter the brain along the axons of neurons (f). This probably happens by a retrograde flow of prion-filled vesicles (shown here as purple spheres ascending the axon). Another route of entry could be the blood, probably in immune cells. A lymphocyte is shown exiting the capillary at bottom left (g), where it could then contact the astrocyte (h). Astrocytes and other glial cells also support the production of prions. SOURCE: Russell Kightley, courtesy Russell Kightley Media Scientific Illustrations and Medical and Health Education Videos (http://www.rkm.com.au).

Photo Courtesy Envirologix Corp.

Photo Courtesy Bio-Rad Laboratories

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