Photo by Clay Eals
Dr. David Fredricks doesn't wear full-body protective gear or handle suspicious packages. Nor does he work with anthrax. But the techniques the disease detective uses are every bit as sophisticated as the government laboratories called upon to diagnose agents of biological warfare.
His assignment at the Hutchinson Center is to track down elusive microbes suspected to cause everything from virulent pneumonia in transplant patients to unexplained chronic illnesses that plague otherwise healthy people.
Fredricks, who joined the Clinical Research Division in July, represents a new generation of microbiological sleuths: scientists who find and identify infectious agents that defy standard analysis.
Traditionally, tracking down pathogens has meant swabbing bodily fluids onto Petri dishes to see what grows, like the throat cultures taken to diagnose strep infection. But given the extraordinary niches in which some bacteria thrive, including sulfurous deep-sea vents and the acidic human stomach, it's not surprising that scientists can't precisely mimic nature's complex mixture of nutrients.
Fredricks, also an assistant professor of medicine at the University of Washington, said that so-called cultivation-resistant microbes are more common than most people think.
"If you take a drop of water from Lake Union, you could cultivate about 1 percent of the microbes present in that drop, compared to what you see under a microscope," he said. "I use DNA to detect and identify microbes, a technique that doesn't rely on the ability to grow them."
Just as pond water is filled with an almost unimaginable assortment of invisible creatures, so is the human body. While many of these microbes maintain a peaceful co-existence with humans, some cause disease. An inability to grow such pathogens in the laboratory can mean that ailments may escape rigorous diagnosis and precise treatment.
But detecting microbial DNA in living tissue requires no cultivation outside the body, which lets Fredricks identify organisms as they exist in nature.
With the polymerase chain reaction (PCR), which allows minute quantities of DNA to be expanded to many more copies, and subsequent DNA sequencing, small samples of blood or tissues are all that are needed to identify traces of infection.
Once a microbial DNA sequence is obtained, the challenge is to make the actual identification. Just as a telltale fingerprint at a crime scene must be compared to a database of fingerprints, DNA sequences are compared to a vast collection of previously sequenced bits of microbial DNA.
Variable piece of genome
Most useful for this analysis is a variable piece of the genome found in virtually all organisms, Fredricks said. This bit of genetic information, known as the16S ribosomal RNA, contains the code for the protein-synthesis machinery of the cell. Such molecules from thousands of microorganisms have been sequenced and catalogued in a public database.
"This molecule contains a lot of evolutionary information," he said. "Almost all bacteria have portions of the16S ribosomal RNA gene that are identical. But parts of it vary from organism to organism, meaning that every species has a distinct genetic identity. The degree to which organisms differ in this variable region tells us something about their relatedness."
Using this type of criminal identification system, Fredricks hunts for microbes that may not even be disease-causing suspects, an approach he calls novel pathogen discovery.
"Rather than asking, say, 'Is a person infected with bacteria that causes anthrax,' this technique allows us to ask, 'Is a person infected with any kind of microorganism?'" he said.
The distinction has important implications, both for standard clinical settings and for the military.
"If troops out in the field were infected with an unknown pathogen, it would make sense to use a technique that doesn't rely on testing for only a few possible infections," he said. "You'd want to use a method that lets you test for anything."
Even if the procedure turns up bacteria never seen before, the method still provides valuable information, Fredricks said.
"With so many sequences in the database, we usually can tell which species the novel pathogen is related to, which could provide useful information for treatment," he said.
Fredricks plans to use novel pathogen discovery to identify microbes that may be associated with chronic diseases that have no previously demonstrated association with infectious organisms. He will initially investigate lupus, an autoimmune disease whose symptoms include skin rashes, arthritis and kidney dysfunction, and tropical sprue, an intestinal disorder.
"We need to change the way we thing about microbes and disease," he said. "Not all infectious organisms cause rapid death. We already know of some examples where microorganisms cause chronic diseases, like the bacterium Helicobacter pylori that causes stomach ulcers. There are sure to be many others. And there may be cases where the disease is due to a combination of bacteria."
An example, Fredricks pointed out, is gingivitis (gum disease), which is caused by a complex mixture of bacteria that colonize in multiple layers in the oral cavity.
For some infectious diseases with known origins, rapid diagnosis can be critical to effective treatment. An example of such "real-time" detection methods employs the use of a tool called a molecular beacon.
"This involves using fluorescence to make a quick diagnosis," he said. "We use a probe that only lights up if it finds and sticks to the DNA of a suspected pathogen in human tissue. In addition to their use for diagnostics, fluorescent probes can useful for figuring out exactly where in human tissue the organism is living."
Prior to joining the Hutchinson Center, Fredricks completed an infectious-disease fellowship at Stanford University, where he used fluorescence technology to localize the precise location of the organism that causes Whipple's disease, a chronic intestinal ailment. Although the first diagnosis of Whipple's disease was made in 1907, an infectious agent was not discovered until 1992, when DNA methods were used to identify the cultivation-resistant bacterium Tropheryma whippelii.
As a practicing physician, Fredricks treats immunocompromised patients, including bone-marrow transplant recipients, who are highly susceptible to numerous infections that healthy immune systems withstand.
"Many transplant patients suffer from something called idiopathic pneumonia syndrome," he said. "It looks like infectious pneumonia, but no pathogen has been recovered from patients. I'd like to use DNA-based identification methods to look for an infectious agent."
Fredricks also plans to use DNA technology to identify the types of fungi that can also plague transplant recipients.
"Our existing knowledge of fungal diseases isn't very good," he said. "I'm interested in whether I can use these methods to detect fungi faster and more accurately, which may make these infections more responsive to treatment."
Fredricks considers himself lucky to be in a situation where he can impact patients directly.
"I see patients on the infectious disease ward and ask what is infecting them. Then I go back to the laboratory and find out."