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New technologies help laboratories fight bioterrorism

From the July-August ACP-ASIM Observer, copyright © 2002 by the American College of Physicians-American Society of Internal Medicine.

By Phyllis Maguire

Last fall's bioterrorism attack began with an index case of inhalational anthrax in Florida. As the patient lay unconscious in a Palm Beach County hospital, the Florida state laboratory rushed to test not only biological samples, but also several dozen environmental samples from the patient's home and office.

The state lab was able to detect the presence of anthrax nucleic acid within three hours, thanks to its new rapid-testing capability. Before announcing those results, officials had to wait 24 hours for the CDC to confirm the diagnosis through conventional culturing and its own rapid-testing assays. But the state lab was able to put the time it gained through rapid detection to good use.

"While waiting for CDC confirmation, we found a site to implement an intervention," said Segaran P. Pillai, PhD, director of the State Public Health Laboratory in Miami. "We also collected nasal swabs from the patient's co-workers to determine the exposure rate, and notified local physicians and lab technicians."

New rapid testing methods—which allow labs to identify pathogens in hours or even minutes, instead of days—are the result of two recent and timely developments: new technology, and increased government funding to improve labs' response to bioterrorism. That rapid detection capability is proving to be a boon not only for security, but also for the field of public health.

New technology

For years, microbiologists have relied on two conventional diagnostic tests to identify pathogens: gammaphage and direct fluorescent antibody staining. Today lab directors have two new rapid- detection methods in their first-line assay arsenal.

One is time resolved fluorescent (TRF) immunoassay testing, which detects antigens or proteins specific to a particular biological agent. The other is real-time polymerase chain reaction (PCR) testing, in which an organism's nucleic acid is analyzed to identify its DNA or RNA.

"The most critical part of real-time PCR is developing the right DNA 'primers' to ensure high specificity and sensitivity, so they won't miss their target," said Dr. Pillai. Genome sequencing technology has made it possible to develop these DNA enzyme probes.

Both methods also rely on innovations in computer software. Dedicated computers—not lab personnel—now comb through sample data for quick answers.

The FDA still considers both procedures "screening" assays, and the CDC requires labs to run at least two assays before a sample can be confirmed as positive.

During last fall's anthrax attacks, for instance, Dr. Pillai primarily relied on real-time PCR and gammaphage. Real-time PCR testing quickly told him whether or not a sampled strain was virulent versus a Pasteur or stern strain, non-pathogenic strains unable to cause disease. The gammaphage lysis assay also provided viability results and served as a confirmation assay.

While the TRF immunoassay can't tell if a sample contains a virulent strain, Dr. Pillai said that the test did come in handy. When lab personnel used real-time PCR and gammaphage to test samples from a second patient who was very ill with anthrax-like symptoms, they were puzzled by the negative results.

A TRF test, however, was positive, indicating the presence of anthrax antigens in the patient's pleural fluid. So why couldn't the lab grow a culture from the sample?

Dr. Pillai's staff solved that mystery by scrutinizing the patient's hospital records: Before samples were taken, the patient had received prophylactic zithromax, which killed the viable anthrax bacteria. Because TRF testing had positively identified anthrax antigens, the lab was able to make the diagnosis.

CDC's key role

State labs would not have been able to implement the new technology if the CDC hadn't taken charge of an effort to get it to local facilities and provide protocols and know-how.

In 1998, HHS charged the CDC with coordinating the country's public health response to bioterrorism. As part of that effort, the CDC established its own core lab for investigating bioterrorist-related activities, the Bioterrorism Rapid Response and Advanced Technology Laboratory.

It also created the Laboratory Response Network, giving financial and technical support to some 130 state, county, military and overseas labs. Three-fourths of these facilities already have rapid-testing capability, according to Richard F. Myer, PhD, director of the CDC's Bioterrorism Rapid Response and Advanced Technology Laboratory. The remainder should have the equipment by the end of this year.

The CDC also developed protocols for using the new rapid detection methods, as well as the specific reagents needed to test for anthrax, plague, tularemia, ricin toxin and staphylococcal Enterotoxin B.

In helping regional facilities buy and use rapid detection technology, the CDC has also developed much closer working relationships with them.

"We now work with the CDC much more closely and frequently, swapping samples and teleconferencing," said David Warshauer, PhD, chief of bacteriology at the Wisconsin State Laboratory of Hygiene in Madison. He also pointed out that state labs' new rapid identification capability has public health applications beyond fighting bioterrorism.

"This equipment can be used for a host of agents outside the bioterrorism realm," Dr. Warshauer said. His lab has used rapid molecular assays to test for pertussis, encephalitis, legionella and West Nile virus. Miami's Dr. Pillai has used real-time PCR to rapidly identify food-borne pathogens like E. coli and salmonella.

New funding and visibility

State labs are planning further upgrades with their share of this year's $1.1 billion federal bioterrorism appropriation. In Florida, for example, lab officials plan to use their new funds to expand their rapid-detection capacity.

"After last fall's event, we realized we had the capability but not the capacity to run the 24-hour, seven-day-a-week operation that was required," Dr. Pillai said. (Florida's state lab normally tests one or two samples for anthrax every few months, but in October 2001 alone, it processed 4,000 samples.) He intends to convert another lab to handle high-volume samples, using funds for new staff and equipment.

All of these developments are helping to mend the nation's frayed public health system—and highlight labs' important diagnostic role. In years past, Dr. Pillai said, much of the attention paid to public health went to epidemiologists who briefed the public and officials on trends in HIV, hepatitis and food-borne outbreaks.

"Epidemiologists have always been at the forefront with data that labs came up with," he said. "The actual testing was always in the background, but we're in the spotlight now. Without lab diagnostics and rapid technology, it would have been impossible to respond as well as we did to last fall's attacks."

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