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Beyond Clinical Diagnostics

IVD systems in bioterrorism response

A PCR-based system offers a new approach in testing for biowarfare agents.

Kurt Petersen and William McMillan

Perceptions of bioterrorism have changed since the events of September 11 and the anthrax attacks that followed in October 2001. Everyone now knows that there are organized groups in the world with the motive and the means to launch an attack that could kill thousands—and the opportunity can be as close as the corner mailbox. Conscious of this vulnerability, nations throughout the world are recognizing that they must make realistic decisions about preparedness and response.

In the United States, systems for detection of infectious agents have been under development for some time. But now, a great sense of urgency has accelerated schedules for developing and deploying such systems in public places and for clinical use.

Lessons from the Anthrax Attacks

As frightening and tragic as the October anthrax attacks were, a number of important lessons were learned that will help to avert more-damaging attacks in the future. The first lesson is that, in all probability, there will be future attacks. Bioterrorism has been shown to be relatively easy to perpetrate with impunity. The use of the postal system to attack private homes and offices surprised even some bioterrorism experts. People are now justifiably concerned about the vulnerability of the nations's supplies of water and food, including livestock.

Another lesson is that DNA testing is mature and functional, and will likely be the technology of choice in future emergencies. During the October attacks, culture assays, immunoassays, and tests based on polymerase chain reaction (PCR) were all used to test clinical and environmental samples for anthrax. PCR-based DNA testing was shown to deliver extremely accurate results in a matter of hours. The United States Postal Service has budgeted $200 million in fiscal year 2003 for the deployment of PCR-based testing systems.¹

In this crisis, the availability of sensitive and rapid diagnostic methods in this crisis not only saved many lives, it also produced vital new information for the medical management of inhalation anthrax by showing that immediate treatment leads to good outcomes. The survival rate of those individuals who were treated immediately following the emergence of symptoms was 100%.² All the anthrax victims who died received delayed treatment.

Relying on existing laboratory-based methods, however, even with PCR, could lead to disaster. Screening a few thousand Congressional and postal employees in the Washington, DC, area taxed the local laboratory capacity to the point where rapid diagnosis was impossible. A more-aggressive attack could overwhelm existing capacity over a broad area. There are not enough trained microbiologists in the entire country to prepare the samples that would be required if even only one major metropolitan area or large military base were to be more aggressively attacked. The capability to transport and deploy field-ready testing systems would be crucial.

But just as medical diagnostic testing is necessary for preventing deaths and injuries, testing buildings, machinery, and other objects is necessary for damage control, monitoring the effectiveness of environmental decontamination, and forensic investigation. In future attacks, quantitative testing of air, water, soil, food, and potentially contaminated objects and spaces may be important for rapid response and harm reduction. As many people in the agriculture and food-processing industries have recently realized, bioterrorism need not target humans directly. An outbreak, or even a credible threat, of hoof-and-mouth disease could cause irreparable harm to the entire economy.

Terrorists are unlikely to inform in advance which organisms they are using, so testing systems must be capable of identifying a wide range of possible pathogens, including specific genetic variants, and be rapidly adaptable to new threats.

System Requirements

People first think of the military for bioterrorism response. But in fact, numerous agencies, military and civilian, public and private, medical and nonmedical, have already begun to evaluate their needs for bioterrorism detection systems for both diagnostic and environmental testing. The Centers for Disease Control and Prevention (CDC; Atlanta) and their nationwide Laboratory Response Network were especially effective during the recent anthrax crisis. Interestingly, many of the requirements for the ideal system for field testing in the bioterrorism scenario are strikingly similar to those of the ideal clinic-based diagnostic system for routine use:

• Excellent performance in terms of sensitivity and specialty.
  
• Fast turnaround.
  
• Random access.
  
• Versatility.
  
• Usability with a range of sample types (swab, blood, urine).
  
• Friendly interface and streamlined protocols accessible to operators at a range of skill levels.
  
• Low-maintenance hardware and reagents.
  
• Physically both compact and robust.

Currently Deployed Systems

Technologies currently in use in military and security situations are for environmental testing only. Fairly advanced autonomous sensing or "sniffer" systems have been developed that monitor the air in a public environment, such as a subway tunnel or a sports stadium. For example, the Joint Program Office for Biological Defense (JPOBD), which defines all the military programs for biological defense, has developed the Portal Shield, an autonomous system that extracts organisms or toxins from ambient air for testing by onboard immunoassay-based sensors.³

Sniffer systems are obviously part of first-line biodefense. However, they need to be made inexpensive and robust enough to be deployed like security cameras or radar traffic control. This capability will not be ready for many years. Developers of PCR-based sensors to replace the insensitive immunoassays have encountered the familiar problems of automating PCR: complex sample preparation and reagent fragility.

Another military device for environmental biothreat detection, the Biological Integrated Detection system, is a mobile microbiology laboratory, housed in a shell on the back of a Humvee. Its utility is limited to situations of suspected imminent attack, such as in a war zone. The system requires two skilled operators to count and characterize particles collected from the air by immunoassay, flow cytometry, mass spectrometry, and other types of assays.

Technology in Development

Figure 1. The GeneXpert by Cepheid Inc. (Sunnyvale, CA) is a compact system with four independent PCR-reactor modules and disposable single-test cartridges. One or more systems can be linked to a computer for results display and data handling.

Notwithstanding the recent attacks on private and government office buildings, military bases are still considered likely prime targets. Military buildings, equipment, and personnel would certainly be involved in any response to attacks elsewhere. Some medical diagnostic and environmental testing applications for military use were well along in development even before the anthrax attacks of October 2001. Recently, a program was funded to procure the Joint Biological Agent Identification and Diagnostic System (JBAIDS) for biothreat and endemic infectious disease detection and for periodic environmental analysis on military bases.⁴

The primary military facility for field diagnostic testing is the Theater Army Military Laboratory (TAML), a diagnostic laboratory facility that would be transported on a flatbed truck, a plane, or a boat, to accompany troops in the field. Ideally, the TAML would have systems for both medical diagnostic and environmental testing. JPOBD would prefer to use PCR-based testing, if practicable assays can be developed. However, microbiologists trained in sample prep procedures may not be available in most field situations.

Other technologies are being developed for the detection and identification of biothreat organisms, such as mass spectrometry, optical particle characterization and detection, flow cytometry based on binding immunoassays, and detection of various spore surface molecules. However, none of these techniques has yet been shown to be as sensitive and specific as PCR, and none of them provides information on genetic virulence or antibiotic resistance.

Companies in the private sector have also been doing their part to contribute to the effort against bioterrorism. For several years, Cepheid Inc. (Sunnyvale, CA) has been collaborating with the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID; Fort Detrick, MD) and the Soldier Biological and Chemical Command at Aberdeen Proving Ground (Aberdeen, MD) on the development of fully portable, field-ready systems for detection of infectious agents from a wide variety of clinical and environmental sample types. USAMRIID and several other government agencies, such as the CDC, are currently evaluating Cepheid assays for the detection of Bacillus anthracis (anthrax), Yersinia pestis (plague), Francisella tularensis (tularemia), and Clostridium botulinum (botulism) on Cepheid's Smart Cycler systems. In December 2001, Cepheid delivered a prototype of GeneXpert, its next-generation system, to the U.S. Department of Defense. This system integrates fully automated sample preparation into PCR-based testing, using cartridges preloaded with stable test-specific reagents (see Figure 1).

The Cepheid Smart Cycler and GeneXpert systems use PCR-based assays for the simultaneous detection and quantitation of up to four targets (multiplex reactions) in real time. In addition, they combine the advantages of PCR with rapid protocols and the logistical advantages of random access and point-of-need testing. While both systems are suitable for use in a TAML, because it is designed to have a compact, rugged construction and fully automated sample preparation, the GeneXpert will be deployed in other situations that lack even the minimal infrastructure support and skilled personnel available in the TAML.

As is frequently the case, this new technology poses new risks not specifically related to the technology. Some bioterrorism experts fear that the rapid availability of results of biothreat testing may lead to the premature release of information regarding unconfirmed positive results, bringing unnecessary fear and panic, and possibly other adverse social, medical, and economic consequences. A recent false-positive test result at the Salt Lake City Winter Olympic Games made it clear that guidelines regarding confirmatory testing and information control must be developed for what may be difficult and quickly evolving situations.

PCR in the Field?

The problems limiting the use of PCR in bioterrorism response and other field applications are the same ones that have limited its acceptance in the clinical laboratory: sample preparation is technically complex, resource intensive, and subject to error leading to cross-contamination of samples; samples may contain substances that inhibit amplification, leading to false-negative results; and PCR reagents are delicate and unstable outside the freezer.

In the GeneXpert, a self-contained sample-preparation cartridge replaces complex manual procedures and eliminates amplicon contamination and amplification-inhibiting substances. Lyophilized analyte-specific PCR reagents are loaded into the cartridge at the time of manufacture and remain stable under an extremely wide range of ambient conditions for one year. The cartridge integrates with a modular PCR reactor, the I-CORE (integrated cooling/heating optics reaction), and each GeneXpert is comprised of four independent, randomly accessible I-CORE modules.

Automated Sample Preparation

The disposable single-test GeneXpert sample-preparation cartridge measures 4 x 1.5 x 1.5 in. wide and consists of four functional components: the cap, the cartridge body, the valve body assembly, and the microvolume PCR reaction tube (see Figure 2).

Figure 2. The GeneXpert sample-preparation cartridge consists of four functional components: the cap, the cartridge body, the valve body assembly, and the microvolume PCR reaction tube.

The cartridge body is divided internally into a number of chambers of various sizes and functions, some containing the lyophilized reagent beads, and each with a port at the bottom for fluidic inflow and outflow. The chambers are radially arranged around the syringe barrel in the center.

The valve body assembly, located below the cartridge body, is the site of cell lysing and DNA purification. Under software control, a rotary valve on the instrument moves the valve body assembly so that fluids can be aspirated from or dispensed into the appropriate chamber for mixing, dilution, and washing, according to the programmed assay protocol. The reaction tube, which projects from the cartridge, receives the prepared sample and interfaces with the PCR reactor for amplification and detection of the target analyte.

To perform a test on the GeneXpert system, the operator opens the cartridge cap and loads the liquid sample into the sample chamber. When the operator closes the cap, the cartridge is permanently sealed throughout the testing procedure and biohazard disposal, eliminating any risk of cross-contamination of samples. To start the procedure, the operator loads the cartridge onto the system and chooses the test protocol from the menu. From this point, all sample preparation and testing is carried out automatically by the system, with no further operator intervention. At the conclusion of the procedure, the operator disposes of the sealed cartridge.

The flexible interface of the cartridge body assembly and the rotary valve permits the programming of widely varying target-specific protocols. For anthrax, the sample solution is pushed down into the filter capture region inside the valve body. The cells are separated from the sample matrix, washed, and concentrated on the filter. A relatively large volume and wide variety of raw sample types (up to 5 ml of swab eluent, blood, emulsified tissue, emulsified food, or water) can be reduced to 50 µl of clean, concentrated cellular material. This capability extends the lower limit of detection for dilute environmental samples or clinical samples containing a very low concentration of disease organisms.

Figure 3. All components within the I-CORE module function together for rapid thermal cycling and real-time detection and quantitation of signal from fluorescent probes. Any assay developed for the GeneXpert can be run on any module in random-access mode. (click to enlarge)

Other PCR-based technologies use chemicals to lyse the cells and release the DNA, which must then be extracted and purified from the chemical solution. In the GeneXpert, cells are lysed ballistically: tiny glass beads in the valve body assembly are agitated by ultrasound generated directly below the cartridge, breaking open even tough spores. The purified DNA is then mixed with the analyte-specific PCR reagents stored in one of the cartridge body chambers and delivered to the reaction chamber of the PCR reaction tube.

Amplification and Detection

The GeneXpert system is built around the I-CORE module, Cepheid's miniature microprocessor-controlled integrated thermal cycler and fluorimeter within which PCR reactions are controlled and monitored in real time (see Figure 3). All components within the I-CORE module, which measures about 4 x 5 x 1 in., are designed to function together for rapid thermal cycling and real-time detection and quantitation of signal from fluorescent probes. Optical and temperature sensors are calibrated at the time of manufacture, and the calibration coefficients are stored electronically on the microelectronic circuit board in each module. Any assay that is developed for the GeneXpert can be run on any module in random-access mode.

Figure 4. Resolution of a segmental gradient-gel system displaying results for a donor plasma sample in which LDL particles became smaller after incubation. The relative distance of migration from the top of the gel is presented on the x-axis and the absorbance of 405 nm is presented on the y-axis. (click to enlarge)

The PCR reaction tube is manufactured from optically and thermally optimized polypropylene materials and has a flat, diamond-shaped reaction chamber designed for maximum surface-to-volume ratio (see Figure 3). The flat sides of the chamber are in direct contact with two high-thermal-conductivity ceramic heater plates in the I-CORE. The thin edges of the diamond interface with the optical components. The heater plates and a high-efficiency fan reduce thermocycling times to seconds. Integrated temperature sensors on the heater plates interact with the optical system to coordinate the timing of critical events during the PCR reaction.

The I-CORE system uses fluorescent probes, single-stranded DNA molecules labeled with a marker that emits characteristic fluorescence only when bound to the target sequence and illuminated by an incident beam of the correct wavelength. If the target sequence is present in the specimen, the signal from the bound probes doubles following each amplification cycle. Multiplex testing is possible because of the specificity of genetic probes and the availability of a variety of distinctive fluorescent markers. The I-CORE module can detect and identify up to four different targets (molecules or groups of molecules) in a single assay.

Figure 5. Linearity of GeneXpert cycle thresholds versus spore concentration. (click to enlarge)

Each I-CORE module has its own four-channel solid-state fluorimeter for simultaneous excitation and detection of signal from the hybridized probes. Small optical excitation and detection modules interface with the thin edges of the reaction chamber. The excitation block contains four high-intensity LEDs with color spectra keyed to the various fluorescent markers. The detection block has four silicon photodetectors with several filters to capture quantitative signal data in four separate spectral bands.

At the point in the thermal cycle when fluorescence is optimal, each LED flashes on, and light from the fluorescent dyes present in the solution is detected by the four photodetectors. The entire reaction volume is optically interrogated with each flash. In multiplex reactions, the LEDs flash in rapid sequence, each eliciting a characteristic signal from their corresponding probe type. Optical information is passed to the computer for analysis and display. Thus, the reaction is monitored in real time, and the concentration of up to four targets can be derived from the signal growth curves.

System Performance

The performance of the system for anthrax detection was assessed by in-house testing of samples containing purified spores of the Bacillus anthracis Sterne strain; the pXO1 plasmid in this strain contains genes that encode the anthrax toxin. Aliquots (1 ml) with decreasing concentrations of purified spores were processed.

Sample processing and real-time PCR detection of pXO1 was completed approximately 26 minutes after insertion of the cartridge. The detection limit of the system was between 30 and 150 spores per ml. The results of these experiments show that the system achieved good precision at all concentrations and that the cycle threshold declines with increasing concentration of spores in the sample as expected (see Figure 4).

The I-CORE and the GeneXpert sample cartridge were designed to be adaptable to widely varying target-specific protocols. The flexible interface of the valve body assembly and the rotary valve permits the programming of specific multistep protocols for mixing, dilution, and washing.

Conclusion

Directly and through its partners, Cepheid is developing assays to detect bacterial, viral, and fungal pathogens in aqueous solutions and biological specimens from humans and livestock animals. The company is hoping to develop assays targeting specific DNA sequences in organisms known as well as unknown for use in healthcare and industrial settings.

Cepheid is also working to expand the multiplexing capability of future versions of GeneXpert from four detectable targets per assay to six and possibly more. In bioterrorism response applications, the sooner several target sequences can be detected, the sooner an agent can be positively identified as to specific strain and provenance. This knowledge could have relevance for medical treatment decisions, law enforcement, and military action.

References

1. U.S. Postal Service Emergency Preparedness Plan for Protecting Postal Employees and Postal Customers from Exposure to Biohazardous Materials (Washington, DC: U.S. Postal Service, 2002), p. ES-9.

2. Centers for Disease Control and Prevention, "Update: Investigation of Bioterrorism-Related Anthrax and Interim Guidelines for Clinical Evaluation of Persons with Possible Anthrax," Morbidity and Mortality Weekly Report 50, no. 43 (2001): 941–948.

3. LD Kosaryn, "Defending against Invisible Killers—Biological Agents," [on-line] (Washington, DC: Department of Defense, 2002 [cited 19 April 2002]); available from Internet: http://www.defenselink.mil/specials/chembio.

4. "Improved Immunodiagnostic Platform FY Research Program Plan Objective Summary," Chemical/Biological Defense Program Budget Item Justification Sheet, (Washington, DC: Department of Defense, 2001).

Kurt Petersen, PhD, is president and COO and William McMillan is vice president for biotechnology at Cepheid Inc. (Sunnyvale, CA). The authors can be reached via petersen@cepheid.com and mcmillan@cepheid.com, respectively.

Photo courtesy USAMRIDD/Fort Detrick
Photos courtesy Cepheid

Copyright ©2002 IVD Technology