A process for identifying infectious pathogens by DNA analysis instead of laboratory culture won U.S. patent No. 5,849,492. Dated Dec. 15, 1998, it is headed "A method of rapid identification of prokaryotic and eukaryotic organisms."
The sole inventor is molecular geneticist Peter Rogan, president of Phylogenetix Laboratories Inc. (PLI), of Pittsburgh. His company acquired all rights to the patent from Penn State University, in State College, Pa., where Rogan did much of the research for the technology. It was funded by the Commonwealth of Pennsylvania. PLI is located in the university's campus-based Applied Research Center.
A retrospective trial, pitting the invention's process against conventional microbiology culturing, is halfway to completion, Rogan told BioWorld Today. "It involves testing 200 varied clinical specimens of infectious bacteria," he said, "and so far we've been able to confirm 90 percent of the diagnoses made in conventional laboratories - except that we've been able to do it much faster, in six hours instead of days or weeks. Once we have determined the sequence, 15 seconds to a minute later, we have the identification.
"The purpose of this study," he explained, "is to prove the accuracy and sensitivity of our technology when applied to actual clinical samples, without culturing the infectious agents. To date, we have identified the pathogen in more than 125 fluid specimens, including pleural, cerebrospinal, peritoneal, synovial and blood.
Test Patented, Dubbed OmniAmp
"Our now-patented test," he said, "which we call OmniAmp [standing for "omnibus amplification"], should be particularly helpful in high-risk patients who are acutely ill from a bacterial infection, or in whose cases the cultures fail. Also [in] those who are septicemic or develop infections after surgery, or while in chemotherapy."
As the patent text summarizes, the method involves "retrieval of phylogenetically informative sequences, which comprise searching for a highly divergent segment of genomic DNA surrounded by two highly conserved segments, designing universal primers for PCR amplification of the highly divergent region and amplifying the genomic DNA by PCR technique using universal primers."
Rogan recounted one actual clinical situation last year in which his OmniAmp system scored:
"I had a phone call from an orthopedist at a local hospital, concerning one of his patients who had an artificial knee joint. This lady was in a great deal of pain, and he felt that she had a bacterial infection, because he saw pus in the wound when he looked inside the knee joint.
"He had taken samples on a couple of occasions," Rogan said, "but because it was an artificial knee, it was not very well endowed with a blood supply. So, they were unable to culture anything in the microbiology lab. I suggested that he inject some saline, then withdraw it, and send me that specimen.
"He did so and we demonstrated that the patient had a Pseudomonas cepacia infection. They had no evidence for an infection prior to that at all. But, where twice he had sent this out for culture, and had failed to get any results, we were able to make an identification. So, with this information, he treated her infection, and she did better.
"This was before we had a full lab here," Rogan recalled. "We were doing everything by hand working directly from the saline wash of the wound. And then, we had a result back to him in 24 hours."
Rogan made an additional point: "The interesting thing about Pseudomonas is that there's a lot of antibiotic resistance to it, as indeed to many other bacteria. So, even if you know what the organism is, as in that case, sometimes it's not very helpful, because you still have to determine antibiotic sensitivity. And, to be honest, we aren't able to do that yet. Well, the genome sequences of these organisms are going to provide us with new classes of drugs that will be impenetrable to mechanisms bacteria use for resisting antibiotics.
"A lot of people," Rogan said, "have been doing sequencing of bacterial ribosomal DNA genes, but their general approach has been a research approach. So they sequenced the entire gene. What we came to realize a number of years ago, based on information theory, was that we could get equivalent information in just a short segment of the gene. PLI's technology applies information theory to molecular biology. That is really what made our OmniAmp process a clinical test."
Conceptually, this means tracking a cellular agent's unique DNA sequence back to the point in evolutionary time when an individual microorganism diverged from its common ancestor on the phylogenetic family tree, thus betraying its taxonomic identity.
Next: A Pivotal Prospective Clinical Study
Now, with patent in hand, Rogan said, his objective is a prospective human trial. "We want to compare the time it takes for us to get a result with that of the microbiology laboratory," he said. "For example, we would definitely expect to get identity much faster than the conventional lab for a patient with tuberculosis. Because that organism grows very slowly.
"Besides these comparisons," he continued, "we also want to try to quantify these clinical effects in dollars. If we can work with large enough clinical partners who have hospital information systems, we can go back to their records and find patients who were diagnosed at day one instead of day five, and ask how much less time they spent in the hospital, how much less it cost to take care of them - whether in fact that early diagnosis in those cases resulted in a change in treatment, better outcome, and a decrease in the cost of their antibiotics.
"So, we want to run this prospective clinical trial at more than one institution," Rogan said. "We see our initial partners in these studies as being our first customers. Because, obviously, the data that comes out of this would give us rather compelling arguments to sell them not only on the basis of a better standard of care but also a much more economic way of delivering care." He and his associates "hope to start marketing our diagnostic service to hospitals at the beginning of the millennium - perhaps the first or second quarter of the year 2000," Rogan concluded. *