Nucleic Acid-Based Tests Move Slowly
into Clinical Labs
Sample preparation problems, contamination, and
entrenched attitudes slow but cannot stop adoption of analytically
Diagnostic methods based on hybridizing and amplifying
nucleic acids are gradually making inroads into clinical microbiology
laboratories. And more recently, sequencing the DNA of pathogenic
organisms is looking as if it, too, will eventually gain clinical
So far, however, the practical diagnostic usefulness of
such molecular methods has consistently lagged behind their promisein
part, because these methods are time-consuming, expensive, and demand a
high level of expertise from experienced technologists, who tend to be
in short supply and whose services are expensive. But because molecular
assays often offer means to measure or detect pathogens for which
existing tests fare badly or no tests are available, the newer methods
have gained a foothold in some larger and research-oriented clinical
laboratories. In these laboratories, the ensuing familiarity, in turn,
makes it easier to adopt additional molecular methods for diagnostic
Molecular Methods for Diagnosing Pathogens Still Face
"With the introduction of the Roche Amplicor
Monitor for HIV viral load, molecular testing has become a fairly
routine assay," says Tim Alcorn, Director of Infectious Diseases at
the LabCorp Center for Molecular Biology and Pathology in Research
Triangle Park, N.C. Availability of procedures for quantifying HIV
levels in patients was followed by amplification assays for detecting
the hepatitis C virus (HCV). Neither virus can be readily cultured from
patients. Alcorn sees molecular assays for cytomegalovirus (CMV), herpes
simplex virus (HSV), and hepatitis B as being next in line for
widespread adoption at clinical laboratories. Molecular tests for CMV
and HSV exemplify replacement of existing methods. "As more places
develop molecular testing menus, this methodology is expanding in all
markets from big reference laboratories to small hospital
laboratories," Alcorn says.
But there are still hurdles to overcome before nucleic
acid-based methods achieve the goals cited by their advocates. "I
remain very optimistic about the potential of molecular methods,"
says David Relman, associate professor of microbiology and immunology
and of medicine at Stanford University School of Medicine in Stanford,
Calif. "But I am the first to admit that these putative promises
are still largely unmet or unfulfilled. I think part of the problem is
that in some cases the technology in a pure sense has advanced beyond
the peripheral technologies that will be necessary to bring these
methods into clinical laboratory practice."
In addition, there is a need to distinguish sensitivity
and specificity in the analytical sense from the clinical sense.
"Most often the wonderful data you hear at meetings are about
analytical performance rather than clinical data," Relman points
out. "At this point we need both."
Patrick Murray, Director of Clinical Microbiology at the
University of Maryland Medical System in Baltimore, compares the current
status of nucleic acid-based diagnostic methods to the first wave of
enzyme-linked immunosorbent assay (ELISA) tests that became available
15-20 years ago. Initially, immunoassays were very cumbersome and took
4-5 hours to do. "I think that many laboratories had a difficult
time justifying doing many of these immunoassays or replacing what we
were using with immunoassays on the market," he recalls. Now,
rapid, reliable immunoassays abound in clinical laboratories and even in
Similarly, nucleic acid-based molecular diagnostics are
now a first-generation technology with major problems. "Not to say
that it won't be adopted in the future," Murray says. "I am
convinced it will be." But improvements will be needed.
Specimen Handling, Other Challenges Need Facing
before New Testing Is Widely Adopted
Murray points out that the ability to isolate target
nucleic acids, to amplify those targets, and to use sophisticated probes
to identify them is far ahead of the preanalytical phasewhich entails
processing specimens to make them suitable for input into the assays.
Inhibitors in clinical specimens can interfere with critical analytic
reactions. Moreover, it can be difficult to concentrate organisms that
are present in very small numbers, and the possibility of
cross-contamination is still real.
Even improved molecular technology will have inherent
limits. According to Ellen Jo Baron, Director of the Clinical
Microbiology and Virology Laboratory and associate professor of
pathology at Stanford University Medical Systems, Calif.,
"Molecular technology is not going to be able to answer every
question about etiology of infections." Pneumonia is one example:
many agents are carried in the respiratory tract of asymptomatic
individuals. A sensitive amplification assay can be overloaded with
organisms that are not relevant to a disease process. "It is wrong
to believe that we will do everything by amplification in the
future," she says.
Nucleic acid sequencing, the most futuristic method for
analyzing pathogenic microorganisms in clinical settings, is just now
emerging from the research laboratory. Instruments are beginning to be
designed specifically for clinical laboratory diagnostics and kit-based
assays are being developed that have greater reliability than in-house
assays, says Steve Day, Director of Medical Affairs for Visible Genetics
in Atlanta, Ga. There is still a pressing need for standardization of
methods, reference sequences, and interpretation. But, he adds,
"Sequencing- and pharmacogenomics-based assays are revolutionizing
infectious disease diagnostics and disease management, and will continue
to do so."
In addition to the commercially developed applications
of molecular technology to diagnostics, some clinical microbiologists
are developing their own in-house amplification assays for less-common
but clinically important pathogens, including Epstein-Barr virus,
enterovirus, and Ehrlichia, Rickettsia, and Toxoplasma species.
Having this capability is one advantage of having a well-trained and
experienced microbiologist as laboratory director.
One Lab's Approach toward Adopting Nucleic Acid-based
A representative menu of molecular methods might be the
one put into place over the last few years by Lizzie Harrell, Associate
Director of the Clinical Microbiology Laboratory and associate research
professor of microbiology and pathology at Duke University Medical
Center in Raleigh, N.C. "Our main molecular test right now is HIV
viral load testing," Harrell says. The laboratory offers both the
standard assay, usually ordered by physicians for a baseline value at
the initiation of therapy and the ultrasensitive assay, typically
ordered after a period of therapy, when viral load is expected to
To detect CMV DNA, Harrell has instituted Digene's
hybrid capture DNA hybridization (nonamplified) test, which is performed
directly on patient specimens. Since Harrell is staying with Food and
Drug Administration (FDA)-approved tests, she uses the qualitative
assay. "A few laboratories are doing the quantitative assay, but
that raises reimbursement issues," she says. CMV testing is used
mostly in transplant recipients who have developed symptoms to help the
clinician rule out organ rejection as a source of a patient's problems.
Harrell has installed the nucleic acid-based test in lieu of antigenemia.
Quantitative assays for CMV provide an indication of the risk of
infection. So, as they are approved, they will increasingly replace
antigenemia tests and will be used for detecting early signs of
infection to allow for preemptive therapy.
Harrell also offers pulsed-field gel electrophoresis (PFGE)
testing for molecular epidemiology at the request of infection control
officers, typically to analyze a suspected nosocomial outbreak. PFGE
displays characteristic fragments of the entire chromosome of a
microorganism after restriction enzymes are used to digest its DNA at
infrequent intervals. Patterns of identity are easy to spot and indicate
a common-source outbreak.
Harrell is looking at both qualitative and quantitative
methods for diagnosing and monitoring patients with HCV infection and,
eventually, to determine whether amplification testing can be brought
in-house. Amplification assays are useful to confirm HCV infection in
patients who have abnormal liver enzymes and who are HCV-antibody
positive by ELISA. Clinicians who manage liver transplant patients and
those who treat HIV-positive patients will be the primary users of these
Harrell's laboratory also is using a (nonamplified)
GenProbe assay on request to identify mycobacterial species, including Mycobacterium
tuberculosis, Mycobacterium avium complex, and Mycobacterium
gordonae, on growth in culture. In the United States, the most
common clinically significant mycobacterium is M. avium complex
(among individuals with HIV), an organism that is relatively inert.
Using traditional biochemical tests, identification can take several
weeks or even months, while probes do it in a couple of hours, once
growth has occurred. Harrell is also evaluating the use of an amplified
test to detect M. tuberculosis directly in clinical specimens.
One of the first diagnostic applications of nucleic
acid-based technology to be approved was for Chlamydia trachomatis and
Neisseria gonorrhoeae in patient specimens. Nucleic acid
amplification assays quickly proved more sensitive than culture and are
now considered standard for characterizing the pathogens responsible for
these sexually transmitted diseases. At Duke, Harrell says, this test is
being done in two sites outside the microbiology laboratory. "We
firmly believe that this kind of testing should be done in the
microbiology laboratory," Harrell says. "Tests should be
grouped by discipline, not by technique."
An exception might be the amplification assay for human
papillomavirus (HPV) in cervical specimens, which has been incorporated
into cervical screening by many institutions to triage indeterminate Pap
smears. At Duke, this testing is being done in the pathology lab to
better correlate results with histology findings.
Nucleic Acid-Based Tests Sometimes Prove Misleading
While amplification assays are contributing to
infectious disease diagnostics, they sometimes cause problems. Before
moving to Maryland, Murray was at Washington University, St. Louis, Mo.,
where one such incident occurred, he recalls. In that case, use of
Abbott's ligase chain reaction to detect C. trachomatis and N.
gonorrhoeae yielded a cluster of results right at the limit of
sensitivity, the so-called "gray zone," a situation that
Murray calls "very unsettling" since it led to false-positive
results. Further exploration revealed that this problem occurred
throughout the assay's range. False-negative results were also found,
particularly with urine specimens, since urine inhibits the ligase chain
reaction. Murray explains that these errors were due to a technical
problem with the then-current assay. After repeating every positive they
had done, they shifted to the Becton Dickinson assay, which has an
internal amplification control that shows whether an inhibitor is
present in the sample.
But their experience with the Becton Dickinson assay
provides a second example of another pitfallcontamination from the
amplification control. The vendor was very helpful, but eventually they
had to replace the instrument and move amplification testing into
another room. "We were unable to clean the laboratory," Murray
says. "These problems have been reported from a number of
laboratories with both systems." He is optimistic that systems will
improve, but believes that such problems are not appreciated by many
Besides detecting antibiotic resistance markers in C.
trachomatis and N. gonorrhoeae, other nucleic acid-based
testing to detect resistance markers such as mecA in methicillin-resistant
Staphylococcus aureus and van genes in vancomycin-resistance
enterococci also sometimes proves highly valuable over conventional
approaches. "Amplification of resistance genes is the most
sensitive technique for detecting resistance in those organisms,"
Murray says. Indeed, detecting oxacillin and vancomycin resistance in
these organisms by standard meansdisks and breakpointsoften proves
difficult if not impossible, due to their "heteroresistant"
property: not all organisms that carry the gene express it.
As Baron notes, amplification assays can be misleading
when they are used for diagnosing pneumonia cases. "A lot of people
would think pneumonia is a situation where molecular technology might
fill a big void in cases where no etiologic diagnosis is made,"
agrees John Bartlett, chief of infectious diseases at Johns Hopkins
University, Baltimore, Md. However, amplification methods need to be
restricted to those organisms that are not present as normal flora, such
as Legionella, Mycoplasma pneumoniae, and Chlamydia
pneumoniae. Organisms commonly causing pneumoniaHaemophilus
influenzae and the pneumococcuswill be present as contaminants
too often for amplification testing ever to be definitive.
Sample Preparation, Other Technical Problems Also
Fundamental obstacles will have to be overcome before
amplification assays become routine in clinical laboratories. Important
needs include learning how to prepare clinical specimens to obtain the
target nucleic acid while avoiding inhibitory activities and how to
concentrate nucleic acids without concentrating inhibitors. Choosing
specimens also requires some thought. "We think we know where to
find microorganisms," says Relman. "But I am not sure we have
a full understanding of what kinds of samples might be useful and which
might be nonproductive."
Baron points out that there are still no satisfactory
automated extraction methods with which to prepare specimens for
amplification assays. Her laboratory is testing two commercial methods.
"They are not as sensitive as manual methods, but manual methods
have ergonomic problems," she says, referring to the repetitive
pipetting that sometimes causes problems among laboratorians.
Relman also cites the broad problem of background signal
contamination, which he believes is more aptly called the endogenous
microbial molecular background of the human body. "With molecular
methods, we have even less idea what we should find in people who are
healthy than with standard cultivatable organisms," he says.
"Sequence diversity is much greater than cultivar diversity."
Understanding the endogenous microbial "molecular flora" is
crucial if clinical microbiologists are to interpret correctly results
of molecular tests on samples taken from diverse patients.
Shortages of Trained Personnel Further Complicate
And then there is the appropriately skilled technologist
shortage. Baron cites projections that 9,300 laboratory personnel will
be needed annually for the next 10 years, but only about 5,000 persons
will be entering the profession each year. Eventually, automation will
lessen the impact of this shortage, but not completely. And what can be
done during the next 5 to 10 years, while new technology is slowly
filtering into the laboratory? "We will still have to depend on
people reading Gram stains, performing susceptibility testing by
standard methods, and reading culture plates or CPE in virus
culture," Baron says. "We haven't done a very good job of
making clinical laboratory science attractive. We don't pay well."
But that is part of a larger issue. "The health
care industry has had a great problem attracting people in support
areas, not just microbiology but pharmacy and nursing, too,"
Bartlett says. "There is a real crisis in support personnel in the
health care industry and I don't see that anybody has a solution."
Over the next several years, until amplification assays
are fully automated, the introduction of this technology could aggravate
this shortage in trained personnel. "I would regard it as a major
problem," says Harrell. "The reason is that there is a
different mindset when you are doing molecular testing." In
traditional clinical microbiology, technologists are taught aseptic
technique and how to avoid contamination. But when it comes to amplicons,
which can be present in millions and billions of copies, the usual
contamination problem escalates to a whole different order of magnitude.
Even with kits that incorporate biochemical safeguards to reduce the
risk of carryover contamination, Harrell says, "You still want to
make sure you are ultra careful or you won't know whether you are
getting a false positive." Avoiding false positives is especially
important with HCV assays, which actually diagnose infection, as opposed
to HIV tests, which quantitate viral load in infected individuals.
Maintaining separate areas for setup and amplification
is part of the solution, as is adequate disposal so that incoming
specimens do not become contaminated. But a high level of vigilance is
crucial, and that requires as much as possible having certified medical
technologists who have bachelor of science degrees or the equivalent and
have passed a national certifying examination. Since so many
already-employed medical technologists went through programs with little
or no training in molecular methods, they require on-the-job training
for amplification assays. To meet this need, Harrell uses a combination
of one-on-one training in the laboratory and both off-site and on-site
training by test and instrument vendors.
Despite Difficulties, Nucleic Acid Sequencing Usage
While nucleic acid sequencing remains even more
challenging than amplification assays, it has come a long way from its
days as a research tool that used reagents that included hazardous
chemicals and radionuclides. Now, it is largely automated and also
equipped with fluorescent dyes instead of radionuclides with which to
visualize results. However, the newer sequencing-based tests still use
polyacrylamide gel electrophoresis (PAGE), depend on research-designed
instruments with large "footprints" on the lab bench, and
entail awkward gel-casting techniques with long polymerization times.
Nonetheless, sequencing is entering at least larger laboratories where
it is used primarily to detect HIV resistance mutations and to determine
Day says that Visible Genetics has developed the first
DNA sequencing system designed explicitly for clinical use, the OpenGene
system, which allows rapid, convenient gel casting and has
interpretative software. The system was approved by the FDA on 27
September as an HIV-1 genotyping kit. "We have taken a process that
used to take two and half hours and you can do it in less than five
minutes," Day says of the system's preparation time.
Sequencers using capillary electrophoresis separation
are also available for research use. Capillary electrophoresis
sequencers are more readily automated; the primary maintenance task is
replacing capillaries after about 100 runs.
"I see sequencing as being a growing market,"
says Alcorn. "When it gets put into kit format, it will break the
ice as viral load testing kits did for amplification." Either
sequencing or sequence detection using probes has the potential to
detect antibiotic resistance genes and to identify bacterial species
that are difficult to grow.
Looking even further into the future, Alcorn believes
that microarrays and gene expression microarrays "have enormous
potential to come into routine clinical use." As sequencing is used
more in clinical laboratory settings, its users will demand ways to do
it faster, a need that microarray platforms can meet, he says. Cost and
technical hurdles will have to be overcome first. But eventually, in
perhaps five years, he predicts, "I think they will become fairly
Molecular methods can also provide basic understandings
that will take clinical microbiology to a new level. "I am a big
proponent that we need soon to undertake a major effort to define and
characterize the normal microbial biome within the human body,"
Relman says. Molecular methods are ideal for obtaining an idea of the
diversity of microbial signatures, such as microbial DNA sequences found
normally in the bodies of healthy people in frequently infected sitesmouth,
intestinal tract, vagina, skinbut also in sites typically thought of
as sterile, such as blood, liver, and spleen. "I think we will find
sequences there that will be helpful when we are looking for disease
agents," Relman says.