Medical Device Link.

Beyond Clinical Diagnostics

The responsibility of quickly ascertaining the validity of bioterrorist attacks and protecting the public from such potential attacks has fallen to first responders (i.e., police, military, public health authorities). They have come to rely on IVD technologies that have been adapted from clinical settings into various field arenas and can rapidly determine the degree of hazard in prospective biological incidents. The primary questions that such technologies need to address are: “Is this threat valid?” and “How can this threat be dealt with so that it has the least possible impact?”

CDC has prepared a list of biological agents that could be used in a harmful manner to pose a risk to national security (see Table I). They are divided into three categories based on their potential levels of harm. Public health officials and first responders must be able to identify quickly in the field such agents in order to minimize their danger to individuals and prevent any panic situations.1

Requirements and Processes

Biodefense IVDs are deployed in environments that are very different from a clinical laboratory. Such IVDs used in the field are lightweight, portable, rugged, not sensitive to light, and easy to operate by inexperienced users. They are self-contained or turnkey systems, with few extraneous accessories required. Most importantly, they must be designed for users wearing personal protective equipment, which can constrict manual dexterity and limit vision.

Biological detection systems for examining suspected biological situations are used in the field for many of the same reasons as clinical settings. They primarily provide rapid, on-site testing of biological samples to support effective decision making. Each suspected biological incident must be treated at the onset as a legitimate threat, which often results in hours of inconvenience and anxiety for both the public and safety personnel. Those responsible for handling the threat must patiently wait for the eventual negative or positive result and be prepared for its ramifications. Therefore, while speed and timeliness of results are important, the information must also be accurate in order to avoid an inappropriate and possibly disastrous response.

The use of field-based systems and the rapid results that are obtained provide first responders with various options to deal with bioterrorist situations. In many cases, sending a sample to an off-site laboratory for confirmatory testing can take 48 hours or more, which is not a viable solution from both an economic and human standpoint. The cost of quarantining a building and its occupants for such a time period is not feasible. Moreover, the victims and first responders suffer if they are made to wait for confirmatory results. Rapid on-site determination provides responders with options to contain the threat immediately, if any, and mitigate any potential fallout. A recent report noted that 24 states and the District of Columbia lack the capacity to deliver and receive lab specimens, such as bioterrorism agents or new disease outbreak samples, 24 hours a day, seven days a week.2

If the authorities have determined that a bioterrorist situation is credible and warrants further investigation, the initial step is to use rudimentary IVD technologies to obtain a rapid initial assessment. This process involves basic pH or protein test strips or kits to find out if a sample indeed has biological origins.

If pH were determined to be within a range in which a potential biological agent could exist, most emergency-response protocols would likely call for the incident to be escalated. A positive reading on a simple protein test would also generate such an escalation. Such protein tests do not distinguish between harmful or benign proteins. Rather, the tests indicate their presence and that the substance in question has biological origins. During this phase, additional testing with specialized equipment would also evaluate the substance’s chemical, explosive, or radiation hazard properties. Numerous portable chemical and radiation samplers of varying complexities are used for such purposes.

Protein kits currently deployed in the field include the BioChek Powder Screening Kit by 20/20 GeneSystems Inc. (Rockville, MD). An evaluation by Edgewood Chemical Biological Center (Aberdeen Proving Ground, MD) on inexpensive field tests for pH and protein showed that they still had 7% false-positive and 11% false-negative rates in laboratory settings, and the findings were the same in field settings.3 They concluded that such strips provide valuable information but are not infallible, and the information alone cannot be used to make an action decision.

Technologies and Devices

If an initial evaluation indicates a sample could have biological origins and could be within physiological harm range, the next step is to identify exactly what it is. During this phase, first responders encounter the largest variety of technologies that could provide additional information for the decision-making process.

Such technologies are usually immunoassay or polymerase chain reaction (PCR), and can take the form of the simplest handheld assay (HHA) devices that are similar to home pregnancy tests and can determine a positive or negative result depending on a color change of a visible indicator line on a test strip. While such devices are inexpensive and easy to use, they require large and pure samples to produce accurate test results due to the sensitivity limitations of the technology. They are also subject to an individual user’s interpretation of the degree of color change, leading to the risk of a misinterpretation of results. In addition, the tests require manual timing, which can be difficult during a busy incident. The strips are single-use only, with a single-labeled antibody/antigen interaction for producing results.

Lately, some interest has emerged in the ability to test for multiple biological agents using multiple antibodies on a single test strip. Some strips identify the agent in the sample, with visual results in an individual window on the test cartridge. Others provide results indicating the sample possibly has biological origins, but without identifying the agent. Examples of such immunoassay strips include the BioThreat Alert by Tetracore Inc. (Rockville, MD) and the BADD devices by ADVNT Biotechnologies (Phoenix). The limitation is sensitivity, with a reflective or human-eye read line, and there is approximately a 20–25% test-to-test variability. Sensitivity is also not not achievable by these simple tests at the 10,000-spore level for anthrax which, according to CDC, is the lethal-exposure dose.

Electronic readers can assist in interpreting the visible indicator line on a test strip. If the degree of intensity of the visible line on the test cartridge or strip exceeds a programmed amount, the reader will provide a positive result. Such readers eliminate the subjectivity in the interpretation of a test result. They also provide a means of data collection and retention which is sometimes necessary for forensic or evidentiary purposes. However, they too are limited in sensitivity and do not produce results for the lethal dose of anthrax. One such reader system is the Guardian Reader by Alexeter Technologies LLC (Chicago), which has been on the market since 2001.

During the past few years, reader-cartridge combinations have been developed, which eliminate both manual and equipment interpretation of visible test results. In such systems, a test sample is tagged or labeled with a fluorescent or luminescent dye, and placed onto or in a cartridge-like device, which is inserted into a reader. The reader scans the cartridge, measures the emitted signal, and generates the test results. The tests are usually single-use cartridge/reader systems, with the results available in about 15 minutes or less, and they require a smaller amount of sample and can have high sensitivity due to proprietary fluorescent chemistry. An example of such a system is the RAMP System by Response Biomedical Corp. (Vancouver) (see Figure 1). Such systems also offer data retention for later analysis, and due to the use of fluorescence and a proprietary test calibration, they can get below the 10,000 spore level for anthrax.

All the above HHAs provide a positive or negative test result only by visible indication or a digital readout. Most users gain little additional information value in a quantitative result since these tests are only presumptive.

Originally intended for military and laboratory use, PCR devices are being adapted for field use by first responders. They are attractive because they theoretically can detect as little as a single copy of DNA or a single anthrax spore. Such portable devices use freeze-dried reagents and specialized sampling kits, and can provide test results in approximately 30 minutes. An example of such a device is the RAZOR or RAPID system by Idaho Technology Inc. (Salt Lake City). Both the devices and reagents for PCR systems are more expensive ($35,000 and more) than the handheld readers, which are $8000–15,000.

Nonetheless, the test results with PCR can be more sensitive than an immunoassay-based system, although they can be more complex to operate. IVD manufacturers are addressing the ease-of-use issues. For example, Idaho Technology is introducing an automated sample device to ease the sampling step. Additionally, a new PCR system, the EvoCycler HD12, developed by Evogen Inc., (Kansas City, MO), is a small, portable, and easy-to-use market entrant that costs $10,000 (see Figure 2). Like Idaho Technology, Evogen will be introducing an automated sampling preparation system for field use to simplify the previously complex processes.

Users and Funding

One of the biggest users of field-based IVD technologies is the U.S. Postal Service (USPS). Most of its larger facilities use a biohazard detection system (BDS) that combines an aerosol collection device, automated sample preparation on a cartridge, and automated PCR analysis for detecting anthrax. One such system is the SmartCycler by Cepheid (Sunnyvale, CA). BDS can be incorporated into existing mail-processing systems and is designed to send an alarm automatically when it detects a positive test result. The intent of BDS is to provide mail-facility operators with rapid response decision-making capabilities and to assist in the appropriate escalation, containment, decontamination, and notification responses.

Equipment based on gas chromatography mass spectrometry (GCMS) or Fourier transform infrared spectroscopy (FTIR) technology is also available but not often used. These technologies are normally fixed or vehicle-carried (including boats) systems, but their increased complexity comes with a trade-off of decreased portability and ease of use. Most of these technologies also implement aerosol collection systems to collect samples and send an alarm if a biological hazard is present.

A significant amount of the funding for U.S. field-based IVD tests used by first responders is available from a limited number of federal grant programs, such as the Urban Area Security Initiative (UASI) and the Commercial Equipment Direct Assistance Program (CEDAP). However, since many of these programs require matching funds from local agencies, field-based IVD biological detection capabilities are sometimes overlooked. In some cases, it is necessary for the limited funds at the local level to be spent on more basic gear, so biological detection equipment ends up lower on each agency’s priority list.

The problem of funding is magnified since currently all the technologies discussed above involve consumables (e.g., test strips, cartridges, reagents, sampling materials). For example, in 2008, USPS used approximately 2 million test cartridges. In addition, such consumables are perennial charges that must be considered with any purchase. Many local agencies are reluctant to commit to technologies that necessitate continual funding and expenditures.

Following the Continuum

Most first responder protocols for bioterrorist situations operate as a continuum. If during an incident, responders obtained a positive result from a protein test, they would next perform a rapid strip test. If that test also turned out positive, they might then conduct an immunoassay, a PCR test, or both for additional presumptive analysis. Most first responders’ protocols recommend using multiple devices to identify a sample and not relying on only a single tool at their disposal. As the continuum escalates, the need to definitively determine the biological agent becomes more important. Most protocols require any presumptive positive samples to be sent to a laboratory for confirmatory testing, which is still considered the gold standard.

As the biodefense technologies become more complex, the information value obtained also increases, along with the user competency required to perform any field evaluation. Training in sampling methods, handling, and specific equipment operation technique is necessary for any first responder assigned to perform field testing. As biological detectors are not used as frequently as chemical or explosive gas detectors, such skills are perishable and must be continually renewed in order for users to remain competent in their operations.

Due to the proliferation of multiple test equipment among first responder groups, some confusion has emerged regarding the use of detectors as chemical identification devices, which involves comparing a sample against an onboard library of chemical signatures. Such identifiers do not name actual biological agents and can be used as a rule-in/rule-out only for biological agents. Such tests are also an essential part of the continuum since knowing if a suspect substance does not have biological origins is just as important.

Standards and Studies

During a suspected biological incident, the priority is to preserve as much sample as possible to allow confirmation of its identity. Even though an incident may be a hoax, determining its origin for possible legal prosecution is imperative. Despite the advantages of rapid on-site decision making, federal authorities stress the need for preserving a sample so that it is not consumed during testing.

In November 2004, the FBI, the Department of Homeland Security (DHS), the Department of Health and Human Services (HHS), and CDC jointly released a document that stated, “In situations where biological threat agents are suspected, the item(s) should be field safety screened and immediately transported in law enforcement custody to a laboratory response network (LRN) laboratory. Field safety screening should be limited to ruling out explosive devices, radiological materials, and volatile organic compounds. Currently, there are no definitive field tests for identifying biological agents. Additional field testing can mislead response efforts by providing incorrect or incomplete results, and destroy limited materials critical for definitive laboratory testing required to facilitate any appropriate public health and law enforcement response.”

Following this mandate, many states, counties, and first responder groups have prohibited the use of IVDs in the field. However, most large cities still use some sort of IVD testing; and in most cases, they use multiple tests to obtain a presumptive identification of a biological agent.

Recognizing the need for a practical approach to field testing, ASTM International (West Conshohocken, PA) formed a committee to develop a standard that would address the needs of first responders, federal authorities, and LRN. In 2006, ASTM published the “Standard Practices for Bulk Sample Collection and Swab Sample Collection of Visible Powders Suspected of Being Biological Agents from Nonporous Surfaces (ASTM Designation E 2458).”

This standard recommends that the bulk of any sample should be collected and retained strictly for laboratory analysis. The tiny residual amount can then be used for field sampling and testing. This procedure necessitates using IVD technologies sensitive enough to identify minute amounts of sample. However, implementing the standard has been less than universal, with most groups continuing to use their own protocols.

In 2002, first responders and public health organizations recognized that no standards for IVD field equipment performance were available and that the significantly important information provided could come from unqualified equipment. With such knowledge and a high level of concern, DHS funded a collaborative study for rapid anthrax detection systems, which was conducted by AOAC International (Gaithersburg, MD).4

This study involved a comprehensive evaluation of five commercially available field tests at 12 separate laboratories led by the U.S. Army Dugway Proving Ground. The task force responsible for this evaluation included 50 experts on anthrax, assay development, validation study design, and statistics from 45 federal government and military agencies (e.g., DHS, the Department of Defense, FDA, FBI) as well as representatives from state and municipal agencies, academia, and first responders. Only one anthrax test, the RAMP anthrax test by Response Biomedical, received AOAC certification. AOAC is now considering performance tests for ricin test kits and tests for portable PCR devices.

Conclusion

The quest for the ideal biological detection device continues. The nature of biological agent identification that involves a specific antigen/antibody interaction or PCR analysis precludes developing an all-inclusive lab-on-a-chip type device. Unlike today’s chemical identification devices that can quickly identify numerous hazardous chemicals, no equivalent device is available for biological agents. Development work is being done in the use of microarray, microfluidics, mass spectrometry, and nanotechnology in the quest for a field-based confirmatory test. However, any such devices developed must be rugged and portable, a challenge for many technologies.