Diagnosing Bioterrorism: Applying New Technologies

Chills, fever, headache, muscle pain, and appetite loss are classic flu symptoms; they are also markers of the biological warfare agents tularemia, Staphylococcus enterotoxin B, and Q-fever. At the moment, the diagnostic methods that would distinguish, within a timely manner, the cause of these symptoms do not exist. But researchers are working towards that end, as well as trying to find the appropriate treatments. Advanced diagnostic methods, ranging from genetic analyses to breath analysis, are among the approaches being examined. Stephen Morse, director of the center for public health preparedness at the Mailman School of Public Health, Columbia University, says progress is being made in developing new, fast techniques that would allow a diagnosis well before conventional signs and symptoms manifest themselves. "You will not be able to give a city of eight million people antibiotics for weeks if you think they've been exposed to anthrax," says Morse. "You'd like to be able to find the ones who really need it and get to them first." Morse, commenting in an interview, is a former program manager in the Defense Advanced Research Projects Agency (DARPA). This federal agency is funding a number of projects to develop those speedy techniques. One approach is to analyze the way the genes of an infected person react. Marti Jett, chief of the molecular pathology department at Walter Reed Army Institute of Research, and her colleagues, are studying how monkeys and piglets genetically respond to infection when exposed to several potential biowarfare weapons -anthrax, plague, Staphylococcus enterotoxin B (SEB), brucella, and botulinum toxi. In one study submitted for publication to the Journal of Biological Chemistry Jett found that monkey exposed to SEB (which can, within hours, induce fatal, massive shock) show genetic changes when genes from blood samples were analyzed by differential display PCR. "Even after 30 minutes we get an inkling that this was a lethal SEB exposure," says Jett. The monkeys didn't show any overt sign of illness for four hours, she says. Death can occur after about two and a half days. Crucially, she found that 120 genes were altered when exposed to SEB. At least six of those genes were unique compared to lipopolysaccharides, which are released by gram negative bacteria that can induce severe shock, indicating the assay's value in providing a differential diagnosis and potentially being able to quickly identify a bioterrorist attack. Jett is working to develop a gene chip, comprised of a few hundred genes, which could be used as a field diagnosis device. Quickly discovering such clues, says Morse, is vital. "If we could read those signals early on, pick up those signals and interpret them, we could have important clues that would tell us who's been exposed [and] to what agents they've been exposed to, and what optimal therapy would be," he says. Taking a somewhat broader approach, Patrick Brown, associate professor of biochemistry and David Relman, associate professor of microbiology and immunology and medicine at Stanford University, are using DNA microarrays to look at genes expressed by lymphocytes and monocytes, "professional reconnaissance agents that are just searching for trouble anywhere in the body," says Brown. These genes, taken from human volunteers, were exposed to pathogens. "We are teaching ourselves to interpret the reports they give to us when we look at their gene expression programs," he says. The researchers are investigating whether the genes orchestrate the body's response to a pathogen or another potential harmful event. In laboratory tests, says Relman, the genes are activated within minutes. He says they are beginning to look at how human cells might respond in vivo to stress, such as that induced by exercise. Analyzing a Pathogen's Genes Other researchers want to develop technologies that will identify pathogens by directly examining the microbial DNA. Doing so is clearly vital, says J. Craig Venter, president and chief scientific officer of Celera Genomics Corp. of Rockville, MD. "If we obtain a complete repertoire of pathogen genome sequences, it will provide the ability to detect and track either an emerging pathogen or synthetic organism. If we can readily detect whether someone has altered an organism or tried to make a synthetic one from simple PCR assays, we would know that and know how to treat such an organism." Venter made his comments at the annual meeting of the American Association for the Advancement of Science (AAAS), held earlier this year. Daniel Branton, a biology professor at Harvard University, is developing a lipid bilayer containing a nanopore that is between 1.5 and 2.5 nanometers wide. A single strand of DNA would be passed through the nanopore. The pathogen's identity would be determined by changes in electrical conductivity, induced by the different bases. "The general notion of using a pore and measuring conductivity to investigate what is going through the pore is completely new," says Branton. Branton is working to develop a silicon nitride chip with such a nanopore. In a test system described last year, he says he was able to identify a molecule passing through a nanopore in a millisecond.2 To identify a potential bioterrorism agent in the field, one might take a swab from a possibly infected individual, extract the DNA and put it in solution. "One could envision an instrument, no bigger than a thumb, which one would dip into a solution, looking for certain patterns, and when that pattern showed up it would ring a bell," he says. The pattern would be the pathogen's DNA sequence. Cepheid, of Sunnyvale, Calif., has made a battery-operated instrument that uses PCR to identify a bioterrorism agent in the field. The company says it also has developed a device to prepare a biological sample that can be analyzed. Readying a biological sample for analysis is a key step in identification, says Allen Northrup, the company's founder and chief technology officer. If the sample is in soil, air, water, "or an envelope that is delivered by the mailman, all of those have varying requirements to extract the organism from the background. So even once you get the spore, like anthrax, from the sample, you have to open those up," he says, and get to the organisms' DNA for analysis. The company has already made portable "spore lysis and a sample preparation module," he says. The next step, he continues, is to integrate a sample preparation module with a PCR analysis. Looking at Breath Not all diagnostic approaches are genetics-based. An industry-university collaboration in Maine has been working to develop diagnostics based on breath. Initially, Robert Lad, professor of physics at the University of Maine, and F. Gregory Grillo, a physician and vice president of Sensor Research and Development in Orono, Maine, wanted to produce a handheld sensor that could detect breath-based increases in nitric oxide (NO) as a marker of disease. NO is a marker of inflammation, explains Grillo. The goal behind DARPA's funding the sensor development would be to triage individuals exposed to a biological weapon. The idea was to determine which members of an exposed group were really at risk a day or six hours after a group was exposed. Those who showed elevated NO levels would then be "pulled out from the rest of the population and treated prophylactically," he says. Though a sensor has been developed,1 the task is more complex than originally thought. While a person's NO level remains relatively consistent from day to day, differences exist among individuals, making it difficult to interpret a given reading in the event of an attack. What's more, notes Grillo, virtually any inflammatory condition leads to a rise in NO. Consequently, he is trying to determine whether he can develop an instrument that can provide a diagnosis using a ratio between NO and another breath-based compound. "We are looking today for more subtle markers that are going to be identifiers of clinical infection," he says. Grillo is working in hospitals, taking gas chromatography mass spectrometer measurements of patients' breath to assess composition, "to see if there are markers that would make this whole idea work." While these new technologies are aimed at dealing with bioterrorism, they clearly could offer help in quick diagnoses in everyday medicine; many researchers say their efforts would have that impact. And of course, there is the question of whether these technologies, no matter how innovative, will actually succeed in their chosen missions. Morse readily acknowledges that these efforts are high risk in terms of success. But, as he commented said at the AAAS meeting, "Creativity is what we need." Harvey Black (73773.2227@compuserve.com) is a freelance science writer in Madison, Wis. References 1. B. Fruhberger, et al, "Detection and quantification of nitric oxide in human breath using a semiconducting oxide based chemiresistive microsensor," Sensors and Actuators B: Chemical, 76(1-3):226-34, 2001. 2. A. Meller et al., "Rapid nanopore discrimination between single polynucleotide molecules," Proceedings of the National Academy of Sciences, 97:1079-84, 2000. Readying the Public Health System As researchers work to develop highly sophisticated, fast-acting devices to diagnose illnesses caused by bioweapons, efforts are also underway to improve the nation's public health system, which would be responsible for helping the afflicted. But in the view of one former health official, the public health system needs help itself. "The United States is ill-prepared to handle epidemic disease," says Margaret Hamburg, former assistant secretary for planning and evaluation at the Department of Health and Human Services. "Our public health infrastructure is woefully inadequate. For many years it has been the poor step-sister of modern medicine. [It] is not well-integrated with the medical system," she told a symposium at the 2001 meeting of the American Association for the Advancement of Science, held earlier this year. The Centers for Disease Control and Prevention is attempting to redress this problem with its Centers for Public Health Preparedness Program. Started last year with a $4.5 million budget for fiscal years 2000 and 2001, the program establishes four schools of public health to "foster individual preparedness at the front line," according to the CDC. In particular, the program is aimed at preparing public health personnel to cope with bioterrorism, says the CDC's Maureen Lichtveld, associate director for workforce development. The program, which funds work at the University of Illinois at Chicago, University of North Carolina, University of Washington, Columbia University, is aimed at getting this knowledge to the country's 450,000 public health workers through Internet schooling. Among the projects: At Columbia's Mailman School of Public Health, the focus will be on training emergency health workers on what to do in a bioterrorist attack, Lichtveld says. The school, along with the New York City Health Department, is also developing a national bioterrorism-preparedness plan. The University of North Carolina is setting up a surveillance model for a particular disease outbreak or health condition in four diverse counties, and will assess how competently health workers handle the outbreak. The school also will train local workers on how to follow the surveillance model. At the University of Illinois, curricula are being developed to help health educators, managers, nurses, community health planners, and others, to assume such responsibilities as talking with the press, explaining what public health is to a general audience, and analyzing a public health problem in a community. The University of Washington School of Public Health and Community Medicine is providing training on injury prevention and public health preparedness in several western states. --Harvey Black

Diagnosing Bioterrorism: Applying New Technologies

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