Summary

From heartening tales of selflessness, compassion and heroism at ground zero to generous donations far from the site, the terrible events of September 11 united this diverse land in a hunger for a sense of community and a need to do something to help. At Mayo Clinic in Rochester, a team of microbiologists fulfilled that need by working around the clock to produce a one-hour diagnostic anthrax test. Team members laid the groundwork for development of the test over a period of years, but completed the final stages in a matter of weeks. With limited resources and personnel, investigators chose carefully their focus of research to maximize clinical relevance. Their story offers a behind-the-scenes glimpse at the way in which Mayo scientists apply the results of basic science research to clinical medicine to produce a new product or procedure that improves patient care. Their story also illustrates how that process can be dramatically altered by events far beyond their laboratories’and far beyond their control.

A Race Against Time: Turning Weeks into Minutes

Since 1997 the microbiology team had been interested in developing ways to shorten the turnaround time for microbiology tests. Culture tests involve isolating the microorganisms, growing them on culture, and identifying them by the way their colonies grow on culture plates. Culture times vary and can be as long as three weeks. "If you can identify that a person has tuberculosis today instead of three weeks from now you can begin the right treatment immediately," explains Franklin Cockerill, M.D., chair of the Division of Microbiology and the microbiologist who led the development team. "And you can isolate the patient and stop the spread of disease."

Such advantages piqued the team's interest in applying and improving polymerase chain reaction (PCR) testing to decrease the turnaround time of many diagnostic tests. PCR-based testing is a three-step process that was developed over the past two decades. First, DNA is extracted from the microorganisms in the sample. "We spend a great deal of our time searching the genomes of micro-organisms," says Dr. Cockerill. "Humans evolved from these organisms and some of their DNA is the same as ours. So we need to find their fingerprints—unique segments of DNA that specifically identify them." The second step incorporates PCR technology—using an enzyme called Taq polymerase to copy the DNA. This chemical reaction is repeated 30 to 40 times by heating and cooling the sample. Because each DNA copy serves as a template for producing yet another copy of DNA with each heating and cooling cycle, the amount of DNA is increased exponentially, so eventually there is enough DNA available to run further tests on it. In the final step, the amplified DNA is probed for evidence of the organism's presence. Historically, each of these steps has required hours to complete.

Speeding Up PCR Technology

The key to producing a rapid PCR test was the development of a new technology, trademarked as the LightCycler, and its application to clinical microbiology diagnostic testing. It was a feat that evolved from international, multidisciplinary collaboration between its inventor, Carl Wittwer, Ph.D., a University of Utah chemist with an engineering background; researchers at Boehringer Mannheim, a Bavarian chemistry plant that later merged with the Swiss company Roche Applied Science; and the Mayo microbiology team. Mayo Clinic was the first institution in the United States to use the LightCycler when, in February 1998, the microbiology team procured it for a 30-day trial. Roche Applied Science was marketing the tool to research laboratories, but Lester Wold, M.D., chair of the Department of Laboratory Medicine and Pathology, credits the team with envisioning the LightCycler as a tool that could be used for clinical assays as well. "That's the beauty of the LightCycler," says Dr. Wold. "We can use it today to identify the presence of anthrax in a research laboratory, but tomorrow it can be used to diagnose strep throat in a physician's office." The LightCycler is a modest-looking instrument. "It looks like a coffee maker and acts like a sophisticated hair dryer," quips Dr. Cockerill. "It contains a heating element and a fan that blows air on the sample to heat and cool it much faster than the old heating blocks were capable of doing. It also incorporates a mechanism to detect if a probe attaches to the DNA, indicating that an infectious agent is present."

Changing the Microbiology Landscape

The team began by prioritizing the list of disease-causing microorganisms they slated for developmental activities using the LightCycler. They focused on common diseases, such as streptococcal sore throat, whooping cough and chicken pox. The anthrax bacillus was well down the list because anthrax cases had not been seen in the United States for decades. For each targeted microorganism, team members reduced the DNA extraction step to a few minutes or less. Then, using the LightCycler technology, they combined the second and third steps—amplification and probing—into a single, 30-minute analysis. The entire test eventually took less than one hour. By September 11, the Mayo laboratory stopped using culture tests for several common infectious diseases. The rapid PCR tests were faster, cut costs in half and were more accurate. "The rapid PCR test can diagnose 219 percent more cases of whooping cough than conventional culture," says Dr. Cockerill. "With increasing emphasis on molecular diagnostic testing, patients can soon expect results from their infectious disease tests on the same day - just as they do now for a blood glucose test."

September 11: The Anthrax Outbreaks and the Final Sprint

"September 11 revealed a fundamentally different level of terrorism that caused us to turn the focus of our development efforts toward biologic agents," says Dr. Wold. Dr. Cockerill was scheduled to teach at Mayo Medical School on the morning of September 11 but canceled classes when he saw the level of distress among the students. "Instead, I met with Tom Smith and Jim Uhl," he explains. "As soon as we saw the second plane hit the World Trade Center, we knew that anthrax attacks would be a possibility." Tom Smith, Ph.D., is Mayo's director of Virology. Jim Uhl, M.Sc., is an associate member of the Division of Microbiology. Other clinical microbiologists who are members of the team include: Jon Rosenblatt, M.D.; Mark Espy and Lynne Sloan; and Maj. Constance Bell, Ph.D., a United States Army officer who completed a clinical microbiology fellowship at Mayo Clinic in June 2001. Maj. Bell laid the groundwork for the anthrax test by developing reagents for it but finalized her fellowship and returned to active duty in the armed forces before the development of the test could be completed. Ironically, four days before the terrorist attacks, she requested additional time to complete the paperwork on the anthrax test. Presenting the project would be difficult, she thought, without an apparent, immediate need. In a case of famous last words, Uhl replied, "Don't worry. There's no urgency on that one." After September 11, the team put other projects aside and worked around the clock to complete the test. "We had already worked with Mayo Medical Ventures to establish a successful collaboration with Roche Applied Science," says Dr. Cockerill. "When the first anthrax case appeared in October, we stepped up our collaboration to produce laboratory test kits for the rapid anthrax test. In a matter of days, we were able to send the formula for the reagents to the Roche Applied Science plant in Penzsberg, Germany. With air travel being limited, we were concerned about the logistics of transporting shipments, but we had tremendous cooperation from the federal government." To speed the preliminary diagnosis, the test kits needed to be available in other LightCycler-equipped laboratories around the country. "Making this test available in a very short time frame is our contribution to the fight against bioterrorism. The work is a direct outcome of the excellent cooperation between Roche Applied Science and Mayo Clinic," says Martin Madaus, president and chief executive officer of Roche Diagnostics Corporation. "Mayo researchers have several years of experience in developing these types of assays. By combining our efforts, we were able to make this test available to qualified laboratories only a few weeks after beginning this initiative." Since Nov. 9, Roche Applied Science made the test widely available to public health agencies, hospital laboratories and reference laboratories in the United States and other countries.

Testing in the Rochester Field

By October, as anthrax outbreaks began to dominate the news, Roche Applied Science sent its commercial-ly prepared reagents back to Mayo for testing. "We didn't want to announce the test until sufficient quantities of testing kits became available - the work called us out of hiding," says Dr. Cockerill. The police brought Dr. Cockerill's team several suspicious packages to examine for anthrax contamination. When indentification came quickly, the research was revealed. The substances were harmless powders. Their quick identification allowed Mayo Clinic to remain open and available to patients without public health risk.

Cooperating with the Federal Government

The team has worked with dignitaries from the United States Department of Defense as well as Tommy Thompson, Secretary of the United States Department of Health and Human Services. The cooperative effort has made the rapid anthrax test formula available to federal and state agencies nationwide. Roche Applied Science is working with the United States Food and Drug Administration (FDA) to determine requirements for expedited regulatory approval. "On Dec. 21, 2001, the FDA approved the rapid anthrax test as an investigational test for testing human samples," says Dr. Cockerill. "Validating the test for human specimens is difficult because there are restrictions on the limited number of human specimens that now exist and are needed to conduct proper clinical testing. However, in cooperation with the federal government, we're in the process of testing human samples." The rapid success of the project was facilitated by collaboration and cooperation. In addition to the contributions by Mayo scientists and researchers from other institutions, Mayo Medical Ventures assisted with the process of developing relationships with companies in other countries. The development of the same-day diagnostic anthrax test in a period of weeks, which under normal circumstances would have taken months or years, illustrates how rapidly and effectively Mayo can react in response to a societal need.