Medical Device Link.

Originally Published IVD Technology May 2002

A whole new world

Overlooked areas in nonclinical testing are providing opportunities for IVD companies.

Renee DiIulio

The Guardian Reader and BioThreat Alert test strips from Alexeter Technologies (Wheeling, IL) provide lab-quality results to emergency personnel within 15 minutes.

Human clinical diagnostics provide an enormous market for IVD companies. But for those companies willing to look outside this realm at niche markets in nonclinical areas, there are ample opportunities for growth. Areas such as veterinary testing, agricultural monitoring, environmental safety, and biodefense are begging for new products that are faster, simpler, and less expensive.

Faster, Better, Cheaper Than What?

Rapid DNA tests will likely be the next generation of testing in nonclinical areas, according to Robert Bohannon, chief science officer at Osborn Scientific Group (Lakeside, AZ). Amplified DNA tests using polymerase chain reaction (PCR) have already revolutionized the ability to detect disease and causative agents, according to Lynn Joens, a professor of veterinary science and microbiology at the University of Arizona (Tucson). Despite the development of alternative amplification techniques, says Joens, PCR will likely remain the leading technology in nonclinical testing for the next 6–15 years. Using specialized equipment, the PCR process takes less than one hour, and the tests can be performed in the field. Although they are often handled in a laboratory, not all laboratories have the capability to perform those PCR tests which require skilled technicians.

The Rapid Anthrax detection device from Osborn Scientific Group (Lakeside, AZ) displays a positive result.

But other technologies are also being developed for use in nonclinical testing. Test strips are currently being touted as the solution to quite a few nonclinical needs and will continue to play an important role. Lon Crosby, director of research and development at Numedloc (Webster City, IA), suggests that end-users themselves may provide a strong market for test strips.

"There is a large consumer market in veterinary, agricultural, and even industrial areas. If test strips were available at reasonable prices, people could check their pets to determine if a trip to the veterinarian is necessary; they could check to make sure the water at the drinking fountain is free of nitrates and nitrites; they could check the municipal swimming pool for chlorine; they could check their gasoline for water," Crosby says.

The key to this technology, however, is having either a lateral-flow test or a reader that can work with multiple strips. Such reading devices eliminate the subjectivity and increase the reliability of these tests. Industry experts suggest there is room in the market for such a reader that works with multiple strips. "A reader that can handle multiple strips is more cost-effective," says Crosby.

Such a device would also benefit farmers and veterinarians. "There is an enormous demand for diagnostic tests in this area," says Crosby. "Veterinary practice has changed a lot over the past 20 years, moving from farm animals to commercial and companion animals. While laboratories consider veterinary testing to be merely a niche market, there is actually a clear need. One company has based its entire success on a single ferret test. Therefore, the fact that some companies are making significant amounts of money in this market area suggests that it is being overlooked."

Opportunity Knocks

Several areas that have been overlooked for many years until recently are where the biggest opportunities in nonclinical diagnostics lie. The events of September 11 have brought to the forefront the government's concern regarding the need to protect against and be prepared for terrorist threats. During the past few months, the general public has come to realize that biological and chemical warfare can be waged just about anywhere: in public transportation systems, arenas, the general atmosphere, even the food and water supplies.

Due to last year's anthrax incidents, biological warfare have been garnering the lion's share of attention. The Centers for Disease Control and Prevention (CDC; Atlanta) have identified the following six organisms as having the greatest potential to cause harm in a bioterrorism act: Bacillus anthracis (anthrax), Clostridium botulinum toxin (botulism), Yersinia pestis (pneumonic plague), Variola major (smallpox), Francisella tularensis (tularemia), and viral hemorrhagic fevers. CDC also identified 14 other organisms as lesser priorities but still as valid threats.¹

CDC's identification of these organisms as viable and dangerous biowarfare agents is not unfounded, according to experts. Tom O'Brien, vice president at Tetracore Inc. (Gaithersburg, MD), knows firsthand that anthrax and botulism toxin have been developed as weapons in Iraq based on his work there as a United Nations inspector after the Gulf War.

The colostrum Bovine IgG test from Midland BioProducts Corp. (Boone, IA) measures the concentration of IgG in fresh or frozen bovine colostrum. The test device gives a yes or no answer in less than 20 minutes on a concentration of 50 mg/mL of IgG. Colostrum provides the necessary immunity agents for a calf's first months of life, and the test provides a way to ensure the quality of the colostrum. This same type of lateral-flow device is being used for quality control checks in other areas of industry. In the food industry, the numbers of checks of incoming material with a quick-test device are on the rise.

In an article that was published in the Journal of the American Medical Association, Raymond A. Zilinskas, a member of the United Nations Special Commission investigative team in Iraq, warned that Iraq's biological warfare program is still intact. In the article, Zilinskas stated, "Iraqi scientists had investigated the biological warfare potential of five bacterial strains, one fungal strain, five viruses, and four toxins."² Those biological agents that are considered weapons of mass destruction pose the greatest danger when released in an aerosol form, since doing so allows a standoff delivery with a wide area of distribution.

Aerosol distribution is also effective for chemical agents, which are even easier to disperse than biological agents. "Chemical warfare agents are much less sophisticated to produce and more readily available than biological agents," says Jimmie Valentine, a professor of pediatrics and pharmacology at the University of Arkansas College of Medicine (Fayetteville, AR).

Chemicals have been used as military weapons by various nations throughout the 20th century because of the relative ease in delivering them to an enemy. During World War I, both the Allies and their enemies released large cylinders of mustard gas and let the wind carry the gas to enemy forces.

More recently, chemical attacks occurred in two Japanese cities when the Aum Shinrikyo cult released the nerve gas sarin in Matsumoto in 1994 and in Tokyo in 1995. According to Valentine, the Matsumoto attack resulted in 7 deaths, 53 hospitalizations, and 253 outpatient treatments, while in Tokyo, 12 persons died, 1300 were hospitalized, and approximately 5500 persons sought medical aid. Investigators subsequently discovered that the cult leaders had been stockpiling anthrax and botulism toxin, illustrating that protection against and preparation for both agents are necessary.

Biological Agent Detection Technology

While various technologies can detect and identify biological and chemical warfare agents, there is a definite need for more research, particularly in the area of biological agents. According to a report by the National Institute of Justice (Washington, DC), "one reason for the lack of available biological detection equipment is that detection of biological agents requires extremely high sensitivity and an unusually high degree of selectivity. Another reason for the lack of biological detection equipment is that biological agents, compared with chemical agents, are very complex systems of molecules, which makes them much more difficult to identify. The need for high-efficiency collection and concentration of the sample, high sensitivities, and high selectivities make all chemical detectors in their current form unusable for biological agent detection."³

Flow cytometry systems like FACSCalibur Automated Cell Analysis System from BD Biosciences (San Jose, CA) can be used to analyze a variety of sample types for the presence of biologically-active agents, including bacteria and toxins, in approximately 30 minutes. BD Biosciences supplies cell analysis systems and biotechnology tools to clinicians who study genes, proteins, and cells.

So which methods are currently being used? "The standard method of testing for biological agents has been microbiological culture. This is the only test that CDC recommends for confirmation," says Jim Whelan, general manager at Alexeter Technologies LLC (Dublin, CA). This method is accurate, reliable, and available in many diagnostic laboratories, but it takes a long time to turn around results, due to transportation and the length of time to plate. PCR tests are also reliable, but reagents for them are not readily available. According to Whelan, CDC is currently evaluating four PCR tests for anthrax detection, although all of them are in the reagent stage.

Overall, from a diagnostics standpoint, the problems are that most of the suspect biological agents are generally uncommon, clinical laboratories have little experience with these agents, and the diagnostic reagents to detect them are limited.

"There are not a lot of background reagents that have been developed for this use because it has never been a high-volume market. The challenge is convincing manufacturers that this is not a short-term problem. The reagents may sit there but they are necessary as insurance," says Whelan.

The types of tests that many companies are looking to develop right now are immunoassay technologies with disposable matrix devices, such as handheld immunochromatographic assays. Even though such tests are not approved for clinical testing by FDA, they are finding increased use due largely to their adaptability to automated readers as well as manual readers. Primarily designed for military use as rapid field tests, these tests are now wanted for first-response teams as well. The new generation of tests have increased sensitivity, and specificity approaches 100% accuracy, according approches Daniel J. Faubion, CEO of Osborn Scientific Group.

"The need for these tests did not exist before September 11. People were not overly concerned; therefore not many were developed," says Valentine, who adds that the need for these tests will not go away any time soon.

Challenges and Issues

"But the government is not going to buy a billion different tests. It wants an integrated system, one that will also include atmospheric monitors and sniffers," says Whelan.

The applications for such integrated systems have expanded into different areas, including airports, arenas, public transportation, corporate mailrooms, and postal services. "But because we have no idea which chemicals are likely to be released, we need to determine what types of devices could give multiplicity and avoid false-positives. False-positives are dangerous because they lead to complacency," says Valentine.

Government officials also want the ability to identify individuals who are exposed to a biological or chemical agent before they become sick. The Defense Advanced Research Projects Agency (Arlington, VA) has looked into whether detectors can be built which are sensitive enough to identify those persons who have been exposed before they actually get sick. Academic researchers have also begun developing such screening tests. Valentine is developing tests that identify metabolites in urine which are similar to the immunoassay-based tests used for drug testing. Exposure to a nerve gas could then be determined in an emergency room, resulting in quicker treatment.

There is also a need for tests that show which persons are more susceptible to which chemical agents. "Studies have shown a proven ethnic stratification of genes that causes varying responses to these agents," says Valentine. "For example, sarin was a particularly potent gas to use in Japan because Japanese people lack an enzyme that allows the substance to be metabolized quickly. The military might be able to use such a test in forming battalions; industries could identify at-risk employees; and appropriate first-response teams can be formed."

The same is true for biological agents, which are effective in very large doses. Biological agent detection systems, however, require high sensitivity. The complex and rapidly changing environmental background requires that these detection systems need to exhibit a high degree of selectivity. In addition, rapid determination is necessary, posing a significant technical challenge.

Another challenge in creating new tests is validation. In some instances, such as smallpox, no clinical reference material is available, so even if a diagnostic test were developed, there is no reference with which to analyze it. Since current regulations require studies on human subjects, which is not an option for tests detecting exposure to terrorist chemical agents, the acceptability of animal subjects is being considered, according to Whelan.

Field Tests

Another growing need in biowarfare testing is taking diagnostics out of the laboratory and moving them into the field. Doing so eliminates delays that occur due to transporting specimens and produces more-rapid results.

Faster results allow the identification of both antigens and hoaxes sooner, when a couple of hours can make a significant difference. "We need to merge environmental testing with clinical testing to deliver answers within a short time frame. Quicker decisions will affect facility closures, local economies, and victims' emotions. It took eight days to analyze the 1000 samples from the AMI building anthrax attack in Florida, which is too long," says Whelan.

First-response teams are the primary users of such field tests. These teams include those individuals and organizations that initially encounter and deal with biowarfare and possible bioterrorism incidents. There are more than 90,000 units in the United States, including police, fire, Haz-Mat, emergency medical teams, the National Guard, and other specialized organizations (see Table I).⁴

First-Responder Units	Establishments	Employees	Vehicles
State and local law enforcement agencies (U.S.)	18,760	932,780	255,000
Fire departments (U.S.)	54,000	1,065,150	189,000
Military (worldwide)	—	849,380,000	—
Ambulance services (U.S.)	3,524	107,312	17,885
Hospitals (U.S.)	6,964	5,011,337	—
Federal law enforcement agencies (U.S.)	16,660	36,439	—
Total	99,908	856,533,018	461,885

Table I. First-response teams provide the largest market for biowarfare field tests. Source: Osborn Scientific Group

Tests employed by these teams need to be simple, fast, and accurate, and, with a specific substance being tested, specificity is more important than sensitivity. "First-responders are called out in response to a threat and will test the suspect material, not a random building or room," says Tetracore's O'Brien.

Beyond being able to test people, field tests are also needed in other areas, especially those related to food safety issues. According to a program by the Genelex Corp. (Redmond, WA), biological attacks on the agricultural sector may be easier to conduct than attacks on the civilian population, and are likely to produce devastating effects on a nation's food supply and economy. Past incidents from other countries clearly demonstrate this point. One example is the mad cow disease epidemic in the UK. The costs that resulted from the most recent bovine spongiform encephalopathy outbreak exceeded $3 billion.⁵ A single domestic case of mad cow disease would stop all exports of mutton and cattle for at least one year and would halt the movement of such animals within the nation.

Animal-disease surveillance is therefore an important part of any complete security program, and President Bush signed the Defense Appropriations Act, which accordingly provided an additional $328 million to the Department of Agriculture for homeland security protection.⁶

Even absent the threat of terrorism, livestock farmers would benefit from the ability to conduct on-site field tests. Sick animals can affect production and the rest of the herd. The ability to weed out those sick animals sooner means higher revenues for farmers, but not if they must spend a lot of time and money to test their animals. Therefore, these tests need to be not only affordable, but also simple and fast.

Rapid on-site tests would also benefit the monitoring of genetically engineered (GE) food. Greenpeace notes that more than 35 countries have laws that require the labeling of food containing GE ingredients, or restrict the import of some genetically modified organisms.⁷ If simple, fast, inexpensive tests to verify the absence of genetic modifications were available, government monitoring of such food would be made easier.

In addition, other areas that utilize consistent monitoring would benefit from the development of field tests. For instance, there is a large market concerned about environmental safety. The water supply is not at a high risk of being contaminated by terrorists, but pollution is always a threat. "Huge amounts of data are needed to understand a regional system, or to track pollution of a watershed," says Crosby.

Money Generates Interest

With few exceptions, until recently, it has been difficult to fund research and development for nonclinical diagnostics. Much of the investment has come from government organizations, such as the Department of Agriculture and the Defense Department, since there did not appear to be much demand for such diagnostics.

In addition, test strips were not seen as high technology and therefore did not create a lot of excitement in the investment community, according to Crosby. That perception is likely to change, particularly where bioterrorism is concerned.

On January 10, 2002, President Bush signed a $2.9 billion bioterrorism appropriations bill, a tenfold increase in spending from approximately $290 million in 2001.⁸ Some of this money has been earmarked for tests and training for first responders, and many of these units have earmarked future funds to spend on the purchase of testing equipment, says O'Brien. These new funds in fact have already had an effect on some companies' revenues. According to O'Brien, Tetracore's sales were increasing steadily throughout 2001, but saw a marked increase after September 11.

Even so, the money has not yet trickled down to make a real difference. The government seems to be looking for ideas right now, and those companies with the right ideas and timing may receive grants to pursue their research. It is another opportunity that should not be overlooked.

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References

1. Biological Diseases/Agents Listing, [on-line] (Atlanta: Centers for Disease Control and Prevention, 2002 [cited 10 April 2002]); available from Internet: http://www.bt.cdc.gov/Agent/Agentlist.asp.

2. RA Zilinskas, "Iraq's Biological Weapons the Past as Future," Journal of the American Medical Association 278, no. 5 (1997): 418–424.

3. AA Fatah et al., An Introduction to Biological Agent Detection Equipment for Emergency First Responders: NIJ Guide 101-00. (Washington, DC: National Institute of Justice/U.S. Department of Justice, 2001).

4. Biological Weapons and Threat Detection (Biowarfare Agent Detection Devices White Paper)., (Lakeside, AZ: Osborn Scientific Group, 2002.)

5. Bioterrorism Facts for the Biodefense Mobilization Conference, [on-line] (Redmond, WA: Genelex Corp., 2002 [cited 10 April 2002]); available from Internet: http://www.bio-defense.org/bioterrorism.html.

6. "President's Budget to Provide $146 Million Increase in Funding to Protect Agriculture and the Nation's Food Supply," (Washington, DC: United States Department of Agriculture, 2002 [cited 10 April 2002]); available from Internet: http://www.usda.gov/news/releases/2002/01/0026.htm.

7. GE Crops—Increasingly Isolated as Awareness and Rejection Grow, [on-line] (Berlin: Greenpeace/International Genetic Engineering Campaign, 2002 [cited 10 April 2002]); available from Internet: http://www.greenpeace.org/~geneng/highlights/gmo/usda_mar28.pdf.

8. Center for Civilian Biodefense Strategies, "HHS Announces $1.1 Billion for State BY Preparedness" in Biodefense Quarterly [on-line] Winter 2002 [cited 10 April 2002]; available from Internet: http://www.hopkins-biodefense.org/pages/news/quarter.html.

Renee DiIulio is a freelance writer in Los Angeles. She has written for IVD Technology's sister publication MX as well.

Photo Courtesy Alexeter Technologies
Photo Courtesy Osborn Scientific Group
Photo Courtesy Midland Bioproducts Corp.
Photo Courtesy BD Biosciences

Copyright ©2002 IVD Technology