Stopping rogue infectious agents in the nick of time
July 2003 Paul Karr
In most hospitals, using the best available technology, it could
take from two days to a week to recognize an outbreak
of bacterial infection, positively identify the strain of pathogen
and its history of resistance to antimicrobial agents, and then
begin fighting back with the correct antibiotics.
But a new technique
may be able to cut response time substantially if it proves to be
reliable. And that’s just what pathologists and technologists
at Johns Hopkins Hospital in Baltimore are hoping to find out.
The technique
works by searching for special repeated chromosomal
DNA sequences in locations unique to pathogens and pathogenic strains,
then typing and cataloging the distinctive pattern of each bacteria’s
DNA fingerprint for comparison with close relatives and historically
archived infections.
At Johns Hopkins,
a medical technologist loads DNA extracted from organisms recovered
from infected patients’ blood, respiratory, or stool specimens
into a small tray of computer chips, then inserts the tray into
an analyzing device. Within hours, reactions will have been performed
to amplify certain segments of the DNA, and computer software will
have typed the DNA of the segments, compared them with each other
and a library of archived data, and picked them out of a database
lineup as a specific strain of, for example, methicillin-resistant
Staphylococcus aureus. The infection might be contained before it
becomes a full-blown outbreak.
“The advantage
to real-time molecular typing,” says Karen Carroll, MD, associate
professor of pathology at Johns Hopkins University School of Medicine
and principal investigator in the pilot program to test the typing
product in Johns Hopkins Hospital, “is that you may have an
answer [about strain type] the same day an organism is identified.
Notification of the hospital’s infection control staff would
ideally lead to interventions such as improvements in hand hygiene
and isolation of patients, among other control strategies, 24 to
48 hours sooner. And sooner is better.”
Although clinical
studies have not yet been published, the system Johns Hopkins is
testing—unveiled by Houston-based Bacterial Barcodes in 1999
and improved in 2002 and again in 2003—may type pathogenic
strains of DNA more quickly, compactly, and cost-effectively than
other existing methods. It was developed by medical researchers
at Baylor College of Medicine, at Texas biotechnology company Bacterial
Barcodes, and at a California molecular microtechnology firm.
The typing with
Bacterial Barcodes’ technology takes “hours instead
of days, in some cases,” says Dr. Carroll, director of the
hospital’s Division of Medical Microbiology. “In this
situation, you’re trying to determine whether you have a transmission
problem in the hospital and what you can do to intervene. Isolate
the patients? Get health care workers to change their hand-washing
procedures? Diagnostic labs can have a real impact on this intervention
process by doing real-time typing, and Bacterial Barcodes’
kits may be able to help hospitals achieve that goal.”
Hospital-acquired
infections are a serious and growing problem
in American hospitals.
A Feb. 13 article
in the New England Journal of Medicine (Burke JP. 2003;348:651-656)
said incidents of nosocomial infection afflict five to 10 percent
of all patients admitted to acute-care hospitals, causing 90,000
deaths and generating $4.5 to $5.7 billion in health care costs
annually in the United States alone. The incidence of hospital-acquired
infections appears to be steadily increasing, and for dangerous
bloodstream infections and antibiotic-resistant Staphylococcus aureus
infections, it has risen at alarming rates during the past two decades.
“Although
rare, laboratories may recover organisms that are resistant to every
antibiotic that is tested,” Dr. Carroll says. “We certainly
don’t want those organisms to be disseminated throughout our
hospital environments.”
The key to arresting
such infections and outbreaks is to rapidly detect drug-resistant
or variant strains of a pathogen. In a 1999 study conducted at Chicago’s
Northwestern Memorial Hospital and reported in the American
Journal of Clinical Pathology, the use of DNA typing cut the
nosocomial infection rate at the hospital by 23 percent in two years,
reducing patient infections by an estimated 270 cases per year and
saving nearly $4.4 million in estimated health care costs during
that period (Hacek DM, et al. 1999;111:647-654).
“Such
an approach for managing nosocomial infections is technically possible,
medically useful, and economically justified,” the authors
of the report concluded.
“They
analyzed outbreaks in a timely fashion, and that was the key difference,”
explains James Versalovic, MD, PhD, director of microbiology laboratories
at Texas Children’s Hospital in Houston and assistant professor
of pathology at Baylor College of Medicine—and a co-founder
of Bacterial Barcodes. “With DNA typing, they could perform
the tracking much more rapidly and accurately.”
Traditionally,
pulsed-field gel electrophoresis has been used to track rogue infectious
agents. PFGE is a useful, if tedious, method of separating out long
fragments of pathogenic DNA by administering staggered bursts of
electrical currents to the gel from two different angles—a
process that keeps the large molecules cohesive and intact enough
to be readily identified. But manually preparing and obtaining results
from gels means
it can take many hours to get reactions and up to a week to get
complete results.
Bacterial Barcodes’
proprietary technique appears to be more efficient than PFGE and
similar processes, thanks to several innovations. First among them
was the repetitive sequence-based polymerase chain reaction technique,
or rep-PCR, invented by Dr. Versalovic and co-founder James R. Lupski,
MD, PhD, during the early 1990s. Dr. Lupski, professor of molecular
and human genetics at Baylor College of Medicine, was examining
bacterial and mammalian DNA in his Houston laboratory when he and
Dr. Versalovic noticed that certain relatively short intergenic
sequences of bases seemed to repeat themselves regularly, in high
copy numbers, throughout the chromosomes of pathogens.
Using oligonucleotide
primers with nicknames like Eric and Box, the polymerase chain reaction
process could be applied to separate, mark, and amplify these unique
repeated fragments of DNA; analyze them; and produce bar-code-like
banding patterns in gels. The resulting graphs could then be compared
with those of existing profiles of pathogenic strains.
“To our
surprise, we found many copies of these conserved, interspersed,
repetitive sequences in bacteria,” says Dr. Versalovic, who
was then working in Dr. Lupski’s laboratory. “We had
a sense that we had hit on something that might be useful in DNA
typing of pathogens, and we
were very excited about that—although it did take some time
to convince ourselves that what we had found was truly going to
prove robust and useful.”
It did indeed.
A 1999 literature review by the Wisconsin research firm Millennium
Strategies reported that rep-PCR is not only the quickest but also
the most cost-effective of the six most commonly used methods of
typing pathogenic DNA.
Drs.
Lupski and Versalovic’s microbial typing process was a time
improvement over PFGE, but still it demanded that the samples be
prepared manually and results came 16 hours later. Seeking to automate
and further quicken its process, Bacterial Barcodes began searching
for a technological alliance—and found it in California’s
Silicon Valley. In December 2001 the company incorporated a second
innovation: Mountain View-based Caliper Technologies’ “lab-on-a-chip.”
The two-inch-by-two-inch
chips play a critical role in speeding the analysis and making it
more uniform. After the rep-PCR process is performed on the patient
samples in a thermal cycler—a device that accelerates the
heating and cooling cycles necessary to separate and amplify segments
of pathogenic DNA—the PCR products, along with molecular weight
and internal “markers,” are loaded into each lab-on-a-chip.
The chips are placed into a proprietary analyzer and activated by
electrical currents; the samples and fluorescent chemical reagents
flow through tiny channels (each about the width of a human hair)
in a sieving process that separates multiple fragments of the pathogenic
DNA on the basis of their size.
A focused laser
beam detects the results of these reactions within minutes and converts
them into peak-profile graphs. Those data are then swiftly uploaded
in digital format to a central database via the Internet, where
the company’s specially designed software analyzes correlations
and draws gel-like images, graphs, dendrograms, and scatter plots
that can be used to visualize clonal relationships between the genetic
fingerprints of the problem strain and those of other known strains.
The system implementing
the lab chips cuts the total time needed for fragmentation and analysis
of pathogenic DNA an additional 75 percent from that previously
required by the manual rep-PCR process; its analysis equipment now
takes up no more desk space than, say, a personal computer’s
hard drive mini-tower.
Mimi Healy,
PhD, who joined Bacterial Barcodes as chief scientific officer in
February 2002, has been instrumental in the effort to develop analysis
software. Thanks to her streamlining of the test protocols, results
can now be obtained within three or four hours of sampling.
“PFGE
is technically challenging and requires experienced operators,”
says Dr. Healy. The agarose plugs and enzymatic reactions required
for gel methods and the original, manual rep-PCR technique tend
to cause greater variations in results, she adds. Bacterial Barcodes’
DiversiLab System, as the platform is known, has standardized kit
reagents and protocols. “Detection is automated, simple, and
reproducible, and the analysis is by computational methods—less
subject to interpretation errors,” she says.
The system is
also less expensive than other methods because labor costs are lower
when using the automated laboratory chips.
The company
is still working, however, to develop the rigorous interpretive
standards that will enable clinical laboratory scientists to definitively
say whether a problem strain is a clone of, related to, or unrelated
to a known strain in an outbreak. PFGE technicians already use a
hard-and-fast standard: If the gel band pattern is identical to
that of a known organism, it is considered identical to the organism
causing the outbreak; if there are fewer than three “band
differences” between the mystery strain and a known strain,
it is considered related and part of the outbreak; and if there
are more than three differences in band size, it is considered unrelated
to the known organism and the outbreak.
As clinical
studies—most of them comparing data from the latest beta version
of the automated rep-PCR technique with data derived from PFGE typing
results—filter in from Johns Hopkins and other hospitals and
reference labs, Bacterial Barcodes will develop stronger standards
and a larger library of pathogen strain types, says Dr. Carroll
of Johns Hopkins.
The company
shipped its first test kit—which can be ordered for small
or large laboratory setups—for Staphylococcus bacteria last
January. Soon afterward came test kits for Enterococcus, Clostridium,
Salmonella, Acinetobacter, and Listeria bacteria using the same
principles and procedures; then, in May, kits for Streptococcus,
Pseudomonas, Campylobacter, Shigella, Escherichia, Klebsiella, Serratia,
and Stenotrophomonas bacteria as well as tests for two major fungal
pathogens, Candida and Aspergillus.
Future versions
of the DiversiLab kits may make it possible to load blood, urine,
or other specimens directly into an integrated lab-on-a-chip, which
would save additional time by automating the extraction, amplification,
and DNA fragment separation steps in the process. Future releases
of the software will speed analysis even more by going beyond the
typing of pathogenic DNA to specific identification of the pathogenic
strains involved in an infection.
The rep-PCR
technique and kits may also be useful in such areas as food safety,
air quality (to detect the presence of deleterious fungi), viticulture
(to identify strains of bacteria or yeasts involved in beer and
wine manufacturing), veterinary medicine, parasitology, and even
bioterrorism. For example, the company has tested a kit that could
be used to quickly type the DNA of mysterious spores found in an
envelope as being—or not being—a strain of Bacillus
anthracis.
“I believe this technology,” says Dr. Carroll, “when completely
developed, has the potential to allow us to identify an organism to the strain
level while simultaneously allowing the lab to tell whether the organism in
question is part of the hospital clone—or a new strain introduced into
that environment.”
Paul Karr is a writer in North Bergen, NJ.
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