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May 2005
Cover Story
William Check, PhD
Ready for a pop quiz? When testing for viral respiratory pathogens, the
best method to use is which of the following: rapid culture, rapid immunoassay
antigen detection kits, immunofluorescent antibody methods such as direct
fluorescent antibody, or nucleic acid amplification-based molecular tests?
If you’re thinking its all of the above, give yourself a pat on the
back.
Certainly there is a prevalent perception that molecular methods based
on nucleic acid amplification, such as PCR and NASBA, are the ideal technologies
for viral diagnosis. Speaking at a session on rapid detection of respiratory
pathogens at last year’s meeting of the Association for Molecular Pathology,
Kelly Henrickson, MD, associate professor of pediatrics and microbiology
and director of the respiratory virus molecular diagnostics laboratory
at the Medical College of Wisconsin, Milwaukee, was explicit about this
position: "I am going to try to convince you that molecular methods are
the new gold standard for sensitivity and specificity."
But does that mean molecular methods are best in all situations? Angela
Caliendo, MD, PhD, vice chair of pathology and laboratory medicine at
Emory University School of Medicine, offered a different perspective in
her talk at the same AMP session: When choosing rapid tests for respiratory
viruses, "there is no answer that is perfect for everyone." Later she
told CAP TODAY, "I would recommend that you ask what test best fits your
clinical situation based on your patient population—inpatient versus
outpatient, pediatric versus adult, immunocompromised versus immunocompetent—your
desired turnaround time, and your resources and financial situation. If
a particular test does not allow you to impact clinical outcome or clinical
management, maybe that test is not for you."
"Dr. Caliendo and I mostly agree," Dr. Henrickson told CAP TODAY. To
say that molecular tests may not be the best option in every clinical
setting, he says, "doesn’t contradict that molecular is the gold standard
for sensitivity and specificity. There are many places where more rapid,
cheaper, and less sensitive and specific tests are preferable, such as
in the outpatient setting." However, he maintains that molecular methods
are best for all hospitalized patients and for all seriously ill patients,
such as those with cancer, where it is critical to identify viral pathogens
accurately.
Marie Louise Landry, MD, professor of laboratory medicine at Yale University
School of Medicine and director of the clinical virology laboratory at
Yale-New Haven Hospital, has evaluated many methods for detecting viral
respiratory pathogens. "Certainly the greatest increase in interest right
now is in molecular testing," she says. Whether you choose a molecular
test for detecting viral respiratory pathogens, however, depends on your
clinical setting and capabilities. "I know that some virology laboratories
are starting to replace conventional culture with molecular tests for
respiratory viruses in hospitalized patients. That approach has a lot
to say for it," says Dr. Landry, who does not use molecular methods now
as her primary testing modality.
"If a molecular assay is as sensitive as culture or better, and if it
gives you an answer in one day or less, there can be a benefit." But,
she adds, "there is a cost to be paid, both in development time and for
equipment." She notes also that molecular testing can be difficult to
troubleshoot because these tests are generally in-house assays or analyte-specific
reagents.
In viral diagnostics generally, molecular testing has moved quickly to
replace traditional testing, says David Hillyard, MD, associate professor
of pathology at the University of Utah and medical director for molecular
infectious disease testing at ARUP Laboratories, who organized and chaired
the AMP session. "These tests have demonstrated increased speed and sensitivity,
and for some viral pathogens, such as HIV and hepatitis C virus, molecular
is essentially the only practical method. Many people have assumed that
what has been true of other viral scenarios—replacement of traditional
methods by molecular—would also be true automatically for viral
respiratory pathogens," Dr. Hillyard says. "But testing for viral respiratory
pathogens is more complicated."
The field is very nuanced and rapidly changing, Dr. Hillyard says, and
realizing there are many different clinical scenarios is important. "What
is indicated for RSV [respiratory syncytial virus] testing may not be
true for influenza or human metapneumovirus. Different approaches are
taken for pediatric versus adult populations, inpatient versus clinic
patients, and the demands on small versus large laboratories vary considerably."
Analyzing the value of molecular testing in all these settings is complex.
For this reason, Dr. Hillyard chose two individuals—Drs. Caliendo
and Henrickson—experienced in viral testing "to showcase two contrasting
perspectives" at the AMP session.
One undisputed fact is that viral respiratory infections are a major
source of illness, with an estimated 1.5 billion upper respiratory infections
(colds, conjunctivitis, pharyngitis, otitis media, sinusitis) and 16 million
lower respiratory infections (pneumonia, bronchiolitis, bronchitis, croup)
in the U.S. annually. Each year 430,000 children and 208,000 adults are
hospitalized with viral lower respiratory infections. At higher risk for
severe viral LRI are elderly, immunocompromised people, infants, and children,
but the majority of those who get serious LRI have no known risk factor.
Influenza causes about 36,000 deaths in adults each year and RSV an additional
17,000 deaths. (Corresponding figures for children are about 150 and 500.)
The most common viral respiratory pathogens (in decreasing order of hospitalizations)
are RSV A and B, influenza A and B, human parainfluenza virus (HPIV)1-3,
human metapneumovirus, adenovirus, and rhinovirus. Because these viruses
cause similar-appearing clinical illness, Dr. Henrickson says, "You can’t
diagnose which virus is causing a patient’s illness by the clinical presentation."
And though several viruses show clear seasonality, it’s not possible to
make a viral identification by the time of year.
Detection of viral respiratory pathogens can be used for cohorting (to
guide accurate isolation procedures), for surveillance (to determine which
viruses are circulating in a community at a given time), and to affect
patient management—to reduce unnecessary antibiotic use, promote
appropriate antiviral use, and reduce unnecessary diagnostic studies.
The clinical benefits of rapid diagnosis of respiratory viruses have been
demonstrated (Woo PC, et al. J
Clin Microbiol. 1997;35:1579-1581; Barenfanger J, et al. J
Clin Microbiol. 2000;38:2824-2828).
Dr. Caliendo reviewed relevant data about traditional tests. (She told
CAP TODAY, "I was in a role with which I am less familiar—not advocating
for molecular testing.") Children shed much higher titers of many viruses,
so a test may have much lower sensitivity in adults than in children.
Sampling methods are not all equal. Using 14-day cultures for influenza,
one group found an 80 percent positivity rate with nasal aspirates, 90
percent with sputum, 65 percent with nasopharyngeal swabs, and only 52
percent with throat swabs (Covalciuc KA, et al. J
Clin Microbiol. 1999;37:3971-3974). For this reason, some viral
laboratories will not accept throat swabs for testing.
Major testing methods include immunofluorescent antibody (IFA) methods,
rapid immunoassay kits, and conventional and rapid culture.
Dr. Landry tested a multiplex (seven-virus) direct fluorescent anti body
(DFA) pool against culture. Using cytospin-prepared slides, she found
the respiratory screen reagent equivalent to or better than culture for
detecting all viruses except adenovirus (Landry ML, Ferguson D. J
Clin Microbiol. 2000;38:708-711). In separate studies, she found
that DFA and rapid immunoassay kits were closer in sensitivity for influenza
detection in children, especially when nasopharyngeal aspirates were tested,
but that DFA was much superior to ELISA for swab samples collected from
adults (Landry ML, et al. J
Clin Microbiol. 2000;38:429-430). She also found rapid immunoassay
kits for influenza A and B to have low sensitivity, just over 50 percent,
and to be inferior to DFA (Landry ML, Ferguson D.
J Clin Microbiol. 2003;41:3407-3409; Landry ML, et al. J
Clin Virol. 2004;31:113-115).
Though they have low sensitivity, immunoassay kits are rapid, Dr. Caliendonoted,
yielding results in less than an hour, and are extremely simple to use.
They may be appropriate for use in outpatient clinics and as point-of-care
devices.
Dr. Caliendo called IFA "a workhorse in many labs." Its turnaround time
can be almost stat, and it is possible to run several batches per day.
Since the specimen is scored under a microscope, this is the only method
in which the quality of the sample can be assessed—at least 25 ciliated
columnar epithelial cells are required for an adequate sample. However,
use of the fluorescence microscope also makes IFA a high-complexity test
demanding trained personnel.
Conventional culture has a turnaround time of two to seven days or longer,
so it can’t affect management decisions. Rapid culture using the shell
vial method and commercial mixed cell cultures such as R-Mix is much faster.
Dr. Caliendo finds that more than 95 percent of positives are detected
at day one. "We set it up in the late afternoon and report out the next
afternoon," she says.
Dr. Henrickson spoke for molecular testing, focusing on the Hexaplex
assay, which he called "the oldest and most widely used" kit for detecting
respiratory viruses. (Dr. Henrickson co-invented and co-developed Hexaplex,
which is marketed by Prodesse, a company he founded but with which he
currently has no management role.) Hexaplex is an RT-PCR assay using strepavidin
plate technology; it detects RSV A and B, influenza A and B, and HPIV
1-3. A newer Hexaplex Plus kit also detects human metapneumovirus. In
unpublished work by Dr. Henrickson and pediatrician John DeVincenzo, MD,
of the University of Tennessee, Memphis, results from Hexaplex for RSV
A were tightly correlated with a viral plaque assay.
Pooled results for several studies in which Hexaplex was compared with
culture, EIA, or DFA showed sensitivity and specificity for Hexaplex from
97 percent to 100 percent. True specificity for the molecular test is
probably close to 100 percent, Dr. Henrickson said, an anomaly that arises
from the extremely high sensitivity of molecular tests. "If the molecular
test is positive and the old method is negative, by definition we have
to say that is a false-positive for molecular," he explained. However,
when discrepant results are adjudicated with a second RT-PCR assay using
primers specific for a different genomic region than that used in the
Hexaplex assay, molecular "false-positives" almost always turn out to
be true positives, according to Dr. Henrickson.
Working with Sue Kehl, PhD, of Children’s Hospital of Wisconsin, Dr.
Henrickson evaluated Hexaplex on clinical laboratory samples. Of 456 samples
that tested negative by EIAs for influenza A and RSV, 209 were positive
by Hexaplex. Similar results were obtained with samples that were negative
by DFA—133 of 502 were positive by Hexaplex (Kehl SC, et al. J
Clin Microbiol. 2001;39:1696-1701).
Overall, RT-PCR assays are more sensitive and specific than traditional
methods and are rapid and clinically useful, with a turnaround time of
six hours, Dr. Henrickson said. (He summarized the advantages of this
technology in Henrickson KJ. Pediatr
Ann. 2005;34:24-31.) Remaining issues in molecular diagnostics
are cost, reimbursement, reliable reagents, the need for open platforms
so that new pathogens can be added easily, and the need for proficiency
panels.
Dr. Caliendo has several thoughts about whether and when molecular tests
are the best choice. "How will you be using the test?" she asks. If it
is to cohort patients, current molecular methods are not yet rapid enough,
though she predicts they will eventually meet that standard. "We still
have to rely on EIA or DFA," she says. "If you are interested in surveillance,
then any test will do, since the turnaround time is not so urgent."
Guiding clinical management of individual patients is a separate context.
Testing affects clinical management when it is "as accurate as possible
as quickly as possible," Dr. Caliendo says. In outpatient settings, molecular
is not rapid enough and the faster turnaround time of EIA or DFA offsets
their lower sensitivities.
For inpatients, laboratories must ask themselves how quickly they need
a result to influence clinical care. "From my perspective," Dr. Caliendo
says, "I would like the result that day or the next day at the most. We
have found that rapid cultures are very successful. Molecular can also
have a place here. If you do choose molecular, make sure you are in a
situation in which you are going to get a result in the time frame you
need it."
Molecular testing’s higher sensitivity has also raised new questions
and new issues of which labs need to be aware, Dr. Caliendo notes. A positive
result on a molecular test may be analytically true but of unclear clinical
importance. For example, molecular tests can detect both viable and nonviable
virus—do these have similar clinical significance? Also, people
can shed adenovirus for days or weeks after they recover from the clinical
illness—will detection of adenovirus always correlate with disease?
Finally, following administration of live attenuated vaccine, recipients
may shed the virus for up to three weeks, which may give positive test
results.
Evaluating the role of molecular testing for respiratory pathogens, Dr.
Hillyard notes that it can be rapid, but that there are rapid immunological
methods "that can be done in 20 minutes to an hour, much more quickly
than molecular."
"Molecular assays, especially for respiratory viruses, can be much more
difficult to perform than some immunological assays," he says, since you
are often looking for multiple pathogens. Real-time PCR assays are starting
to emerge, with the potential for increased speed, but they must be run
once or even twice a day to take advantage of their speed. Not all hospital
laboratories can do this.
An important question, Dr. Hillyard says, "is how much the adoption of
molecular testing is driven by a clear medical necessity." In patients
with encephalitis, testing for enterovirus and herpes simplex should be
done only by molecular methods. Though a case can be made for molecular
testing for respiratory viruses, Dr. Hillyard says, "it is probably safe
to say it is a work in progress."
The development of specific therapies could tip the balance in favor
of molecular. "It’s not as though once you identify a respiratory virus
you have a clear unique therapy, a drug that will be used only with that
virus," Dr. Hillyard points out. "That could change dramatically as work
proceeds on therapeutics for viral respiratory infections. A specific
therapeutic ups the ante, and rapid molecular testing may naturally fall
out of that equation."
In Dr. Landry’s weighing of molecular testing, cost is a major consideration.
"You will need automated extractors," she says. "Doing a large volume
of testing with manual extraction using conventional PCR is very labor-intensive."
As real-time instruments enter the laboratory, they make things faster
and simpler but increase the investment in equipment even further.
In addition, Dr. Landry says, "All the time in development and validation
of molecular methods gives people pause in switching over. And in terms
of charges, a single PCR charge is often $150 to $200 or more." If you
test for all important respiratory viruses, charges multiply, unless they
are multiplexed. "Is the cost justified by the benefit?" Dr. Landry asks.
Like Dr. Hillyard, she says, "If we had clearly effective treatments for
these viruses, a better case could be made."
Molecular tests have a place in reference laboratories, in Dr. Landry’s
view. Samples are shipped long distances, with a decline in infectivity,
so higher sensitivity is more important. And a reference laboratory is
more likely to be able to afford the up-front investment in equipment.
But why would a respiratory virus sample be sent to a reference laboratory?
A tertiary care hospital would have a virology laboratory on site that
could do rapid culture or DFA. For a hospital without a virology laboratory,
sending a sample to a reference laboratory would not help with cohorting
or patient management because of the time factor. "In some situations
you may need to document a very serious infection," Dr. Landry says. "But
the cost of a send-out PCR would be steep and the hospital has to ask,
Does this impact patient care? What is the benefit to justify this cost?"
Drs. Caliendo, Henrickson, Landry, and Hillyard all work in different
settings and use different strategies for diagnosing respiratory viruses.
"We have in recent years changed our approach to respiratory virus testing,"
Dr. Caliendo says. She now primarily uses rapid culture with R-Mix, which
meets the need for a broad diagnostic tool. Emory University Hospital
does not have many pediatric patients; it serves a large population of
transplant patients. "When respiratory viruses are in the differential,
we recommend rapid culture of nasopharyngeal aspirate or swab or a BAL
[bronchoalveolar lavage]," Dr. Caliendo says. She also does DFA testing
for RSV.
They are considering molecular. "It would really only be practical for
us to run it once per day. Does that improve our turnaround time that
much? And we would have to assess the cost and labor," Dr. Caliendo says.
If she went to molecular, Dr. Caliendo would want a real-time assay. "Would
one multiplex assay be adequate?" she asks. "Or would we need to run two
or three assays to cover all pathogens?" Real-time PCR has a limited ability
to multiplex because of the technical limitation on the number of dyes.
Running multiple assays would increase the expense.
Dr. Hillyard has used Hexaplex for several years. "The current version
of the assay shows good sensitivity," he says. His laboratory can get
a result out the same day, with a turnaround time of about eight to 10
hours from receipt of the sample. They run it many times during the week.
Still, since ARUP is a reference laboratory and samples come from a distance,
would the sending physician get the result in time to influence patient
care? "Even with electronic reporting, we are looking at a turnaround
time of a couple of days," Dr. Hillyard says.
Dr. Landry uses different tests in different clinical circumstances.
Her primary test, for patients who come to the emergency department when
the virology laboratory is open, is "a very labor-intensive" multiplex
cytospin-enhanced DFA test for RSV, influenza A and B, HPIV 1-3, and adenovirus
that meets the sensitivity of culture. Negative specimens are cultured.
"It requires a lot of effort on the part of the laboratory to turn around
results in 1.5 to two hours," she says. The laboratory does the test continuously
throughout the operating hours. Under these circumstances, DFA is a useful
test for the ER. "They are trying to move patients through," Dr. Landry
says. "To wait several hours for results is a long time for them. We have
stayed with DFA because of the benefits. But we wonder: People get backed
up, since the ER physicians don’t make decisions until they get the result,
whereas rapid tests for influenza and RSV give results in 15 to 20 minutes."
The problem is that rapid tests are much less sensitive than DFA done
well, and Dr. Landry found some false-positive results in early evaluations
of rapid tests.
It requires a major effort to do the DFA test well, Dr. Landry says.
"A lot of laboratories do not do it well so they get poor results. We
put in a lot of effort training our people. It is something you have to
make a major commitment to."
To make this test available as much of the time as possible, virology
is open seven days a week year round, from 7 AM to 8:30 PM Monday to Friday
and for eight hours on Saturday and Sunday. Beginning in November, depending
on virus activity, weekend hours increase to 12 hours a day. For two to
three months in peak respiratory season, they are open 18 hours a day,
seven days a week.
During peak season when the virology laboratory is closed, a rapid EIA
influenza test is done in the chemistry laboratory. "We train and oversee
that," Dr. Landry says.
Her laboratory does human metapneumovirus by real-time PCR. "We may bring
other respiratory viruses on in PCR format," Dr. Landry says, "those that
are difficult to culture, such as new coronaviruses." She may also add
a PCR assay for rhinovirus, for which there is no rapid or DFA assay and
which may be involved in more lower respiratory disease than previously
thought. A molecular test for the dangerous avian influenza virus would
also be helpful.
In a comparison study, Dr. Landry found a real-time PCR assay for influenza
A to be almost as sensitive as her laboratory’s DFA method (Habib-Bein
NF, et al. J
Clin Microbiol. 2003;41:3597-3601). She chose to stay with DFA
because she would be able to do the molecular assay only once per day,
greatly extending turnaround time and making the test irrelevant for much
of what rapid testing is used for—bed assignments, moving patients
out of the ER, and deciding on the type of infection control. "We would
lose the rapid turnaround times that we are providing now," she says.
"We are not in a position to run molecular tests several times during
the day. It would be too expensive and is not an option for us."
For 10 years Dr. Henrickson ran a virology service laboratory. Until
last year he also ran a reference laboratory. (Currently he does only
research.) During that time he recommended Hexaplex testing on all hospitalized
children with moderate to severe lower respiratory infection of unknown
etiology, all immunocompromised children or with chronic medical conditions
with any LRI, and many adults in the same categories. He performed the
assay once daily, six days per week. He collected specimens until 1 PM,
then set up the assay and (depending on whether he had one or two shifts)
ran it overnight and reported results in the morning or ran it straight
through (about six hours) and reported results at 7 to 8 PM. Between 1996
and 2004, his lab tested about 1,500 respiratory samples per year from
Children’s Hospital of Wisconsin, one of the busiest children’s hospitals
in the country. The lab also tested many hundreds per year from the adult
university hospital.
During his talk at the AMP meeting, Dr. Henrickson presented cost and
reimbursement figures for standard format and real-time PCR in singleplex
and multiplex mode. His conclusion: "You can make money doing molecular
diagnostics, despite what people say."
But Dr. Caliendo showed different figures and draws a different conclusion,
and says, "The issue of reimbursement is complex and different for reference
labs compared with hospital-based clinical laboratories." For the latter,
she says, "making money on molecular testing on inpatients, most of whom
are covered by DRGs, can be difficult. For outpatients, whether the laboratory
makes money depends on its payer mix, cost, and contracting agreements."
Even as laboratories struggle with PCR, yet more innovative formats are
being developed. Dr. Henrickson previewed an 87-probe microarray from
Metrigenix, a multiplex gene chip assay with a detection threshold of
12 copies from Nanogen, a microbead method to detect multiplex PCR products
from Luminex, and a combination RT-PCR and capillary electrophoresis approach.
Only for this last technology is there clinical data so far (Erdman DD,
et al. J
Clin Microbiol. 2003;41:4298-4303). Whatever methods come into
clinical use, Dr. Henrickson said, "Multiplexing is how we must go."
What could tip the scales in favor of broader molecular testing? Again,
the introduction of specific anti virals. Evidence that using existing
molecular tools leads to better patient management or is cost-effective
relative to traditional methods might also do it, in Dr. Hillyard’s view.
Still another factor would be a technological breakthrough. "We need a
rapid, inexpensive, robust technology that creates a test that is as simple
to perform as an immunological test and is price competitive," Dr. Hillyard
says. "There are candidate assays that people are taking a hard look at."
These include rapid isothermal techniques, technology that minimizes or
eliminates sample preparation, and major improvements in real-time technology.
In the meantime, laboratory directors have to make decisions about existing
technology. Despite Dr. Landry’s reservations about current versions of
molecular tests, she says it is important to become familiar with them.
"Where does that technology belong," she asks, "in the molecular lab
or the virology lab? I think virology labs need to start getting molecular
expertise because many tests are going in that direction."
William Check is a medical writer in Wilmette, Ill.
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