William Check, PhD
Amid the rapid pace of technological innovation in laboratory
medicine, one could do worse than to follow the advice of the 18th-century English
poet Alexander Pope:
"Be not the first by whom the new are tried Nor yet the last to lay the old
Prudent advice indeed. And pertinent to today's clinical microbiology laboratory,
where conventional culture and immunoassay are giving way to molecular methods
for detecting bacterial and viral pathogens. But, as is often true of good advice,
there is a catch-converting it into practice. Just when should a laboratory
adopt newer methods? That is, for a given pathogen, how does an individual laboratory
decide which method is optimal for it right now?
Anyone looking for a simple, one-size-fits-all answer shouldn't ask Angela
Caliendo, MD, PhD, medical director of microbiology/ molecular diagnostics in
the Department of Pathology and Laboratory Medicine, Emory University School
of Medicine, Atlanta. "I would say that there is no perfect test for every laboratory,"
says Dr. Caliendo. Although she is speaking specifically about tests for respiratory
viruses, her comments apply more generally.
"Depending on your patient population and whether you are an on-site laboratory
or servicing multiple hospitals as an off-site laboratory, how you choose to
do testing will vary," she says.
What doesn't vary, of course, is the need to provide results rapidly, so the
results can influence clinical care. "You can provide that in a variety of ways,"
Dr. Caliendo says. "You need to decide which is best for your laboratory."
Christine Ginocchio, PhD, lists seven criteria for test selection: availability
and expertise of technical staff, space limits (more pertinent to molecular
methods), test volumes, required turnaround time, clinician demand, equipment
costs (a real-time PCR instrument can cost $100,000), and the extent of the
requirements for assay verification and validation. To complicate matters, "Even
in the hospital setting, laboratory testing is becoming, in part, a business,"
says Dr. Ginocchio, director of microbiology/virology and molecular diagnostics,
North Shore-Long Island Jewish Health System Laboratories, New York. Low reimbursement
and rising health care costs drive financial concerns.
"Years ago we would look at a new laboratory test and if it was exciting, technically
good, and clinically relevant, we didn't think so much about how much it cost
to run," Dr. Ginocchio says. "We would just use it. Now budgets are tight, and
every test we bring in not only must benefit the patient but should be profitable
for the hospital as well."
Every laboratory has money-losing tests that are necessary for patient care,
but those losses have to be made up in tests that are profitable.
Decisions will become only more difficult as tests with improved characteristics
enter clinical practice. Michel G. Bergeron, MD, professor and chair of the
Division of Microbiology and Infectious Disease Research Center at Laval University,
Quebec City, says, "Due to the fact that microbiological identification still
takes around 48 hours, infectious disease management is empirical today. Moreover,
clinicians do not consult microbiological results unless patients do not respond
to initial therapy." Dr. Bergeron has developed a faster molecular method. His
goal is to provide tests that detect and simultaneously identify, in less than
one hour and directly from clinical samples, multiple microbes responsible for
infections and many antibiotic resistance genes. The first commercial test from
his work is for group B streptococcus, or GBS (Bergeron MG, et al. N Engl
J Med. 2000; 343: 175-179).
Methods may be novel, but the traditional ways of evaluating them remain the
best, says Michael Loeffelholz, PhD, director of Public Health Laboratories
in the Arkansas Department of Health, Little Rock. "I would say that if someone
is considering implementing a molecular test, a new analyte or a new test they
haven't done, they really need to look at the peer-reviewed literature," he
says. "Particularly if it is a commercially developed assay, you need an objective
source to get a good idea of test performance and workflow." Don't adopt a new
molecular test just because it is molecular and new. "A particular molecular
test is not always as necessary in some settings as in others," Dr. Loeffelholz
Molecular methods are useful for detecting several clinically
important pathogens: respiratory viruses, Bordetella, enterovirus and
herpes simplex virus in the central nervous system, and GBS in pregnant women.
"In 10 years we have moved eons in turnaround time for detection of respiratory
viruses and with more sensitive assays," says Dr. Caliendo, discussing the details
of selecting a rapid test for laboratory diagnosis of viral respiratory infections.
Early diagnosis can affect patient management by leading to focused care, infection
control, and appropriate use of antivirals. "We finally have some antivirals
for respiratory viruses," she notes. Rapid tests include antigen identification,
rapid culture, and molecular methods.
Patient population and specimen type affect test sensitivity. Children shed
higher titers of virus, 105 to 107 pfu/ mL, within the
range of sensitivity for rapid enzyme immunoassays, or EIA. "As you get away
from the pediatric population," Dr. Caliendo notes, "you lose sensitivity."
To illustrate the interaction of age with collection method, Dr. Caliendo cites
data for detecting influenza A virus (flu A) using an EIA compared with a 14-day
culture as the gold standard. In children, sensitivity was 90 percent with nasopharyngeal
washes; in a geriatric population, sensitivity was 50 to 60 percent with nasopharyngeal
or throat swabs (Landry ML, et al. J Clin Microbiol. 2000; 38: 429-430).
Adding method as a variable, both a fluorescent antibody (FA) method and an
EIA detected all 15 positive nasopharyngeal aspirates in children; for nasopharyngeal
swabs in adults, FA's sensitivity was 90 percent, much higher than the 66 percent
Based on these and other data, Dr. Caliendo says, "you really don't want to
use throat swabs in these rapid assays." Her laboratory refuses them.
Newer, very rapid EIA tests (15 to 45 minutes) are available for flu and respiratory
syncytial virus from Becton Dickinson, Biostar, Binax, ZymeTx (which offers
a waived test), and others. In general, very rapid tests are less sensitive,
with reported figures from 50 to 90 percent, varying by brand. One brand has
reported sensitivity of 85 percent; Dr. Caliendo says this occurred under "ideal"
conditions in a pediatric hospital. Specificity ranges from 80 to 90 percent.
A second category is fluorescent antibody methods, sometimes differentiated
into indirect (IFA) and direct (DFA) fluorescent antibody tests. These assays
use pooled monoclonal antibodies to detect flu A/B, major parainfluenza virus
serotypes, RSV, and adenovirus. FA tests are of high complexity, require more
highly trained personnel, and are run in batches. Their turnaround time is hours
to one day.
Figures show higher sensitivity for FA tests than for very rapid tests: flu
A, 70 to 95 percent; flu B, 60 to 85 percent; RSV, 70 to 95 percent; PIV, 65
to 85 percent. Adenovirus, at 50 percent sensitivity, may be an exception. One
study compared a very rapid EIA, the BD Directigen flu A+B kit, with cytospin
DFA in adults. Of all the DFA-positive specimens, 52 percent were Directigen-positive.
Positivity rates by number of cells were: <10 cells, 10 percent; 11-100, 44
percent; >100, 92 percent; >1,000, 100 percent.
Another advantage of FA assays is that they can be used to detect multiple
pathogens with one test. In a transplant center, for instance, one test can
look for both flu and parainfluenza virus.
Rapid culture is done by the shell vial method, in which cultures of multiple
cell lines are stained at 24, 48, and 72 hours. Because of the need for multiple
cell lines, shell vial cultures are "pretty cumbersome," in Dr. Caliendo's words.
A recent innovation is the use of a mixture of mink lung cells and A549 cells
(R-Mix, Diagnostic Hybrids), which allows all respiratory viruses to be detected
on one cell culture. Cells are stained at one and two days. Dr. Caliendo calls
the use of mixed cell lines "a major improvement in rapid culture methods for
respiratory viruses." Sensitivity remains about the same as with conventional
cell culture and is superior to DFA, with the possible exception of respiratory
syncytial virus, which is efficiently detected by DFA.
The real advantage of mixed cell culture is more rapid results. One analysis
showed positive results being reported in an average of 1.4 days using R-Mix
compared with 5.2 days for conventional culture. On average, 60 to 100 percent
of positives turned positive at day one using mixed cell culture. Dr. Caliendo
says in addition to getting positive results earlier, the negative cultures
are reported earlier, at 48 hours.
Summing up, EIA assays are very rapid but less sensitive and limited to flu
and respiratory syncytial virus; FA testing is rapid and useful for a wider
range of respiratory viruses; and rapid culture with mixtures of cell lines
is "a major improvement over conventional culture methods," Dr. Caliendo says,
though it still has a slower turnaround than FA and EIA tests. "We use very
rapid tests in the ER in the winter respiratory virus season," she says. For
inpatients, she uses FA and rapid culture.
Turning to molecular testing, there are two major options
for RNA amplification, nucleic acid sequence-based amplification (NASBA) or
reverse-transcriptase PCR (RT-PCR).
BioMérieux's NASBA system, called NucliSens, with detection by electrochemiluminescence
or molecular beacons, is available for flu A/B, parainfluenza virus 1-4, and
respiratory syncytial virus. It takes several hours to complete a batch. Since
these are open kits and include only the basic reagents for nucleic acid isolation,
amplification, and detection, laboratories need to provide their own primers
and probes. Analytical sensitivity is <1 TCID50 or 1-<100 RNA copies;
there is no cross-reactivity with other respiratory pathogens. What is lacking
for NASBA, Dr. Caliendo says, is extensive clinical evaluation.
The second molecular option is RT-PCR. Prodesse markets a multiplex RT-PCR
kit (Hexaplex) for RSV, flu A/B, and parainfluenza virus 1-3, as well as one
that simultaneously detects 51 strains of adenovirus (Adenoplex). Hexaplex is
more sensitive than culture for RSV and flu A and equally sensitive for PIV.
It is slightly less specific than culture-for example, 97.4 percent for RSV.
However, Dr. Caliendo cautions that such numbers may be misleading because culture
is less sensitive than molecular methods. How does one adjudicate when culture
is negative and PCR is positive? In-laboratory-developed assays for real-time
RT-PCR have been reported. One such method was as sensitive for respiratory
syncytial virus detection as DFA and culture-augmented DFA in nasopharyngeal
aspirates from children (Whiley DM, et al. J Clin Microbiol. 2002;
Dr. Caliendo concludes that molecular methods are rapid, with a two- to six-hour
turnaround time, and sensitive and specific. Drawbacks are that multiplex assays
can be difficult to design and that it is probably too expensive to do molecular
testing as frequently as DFA. Molecular methods also require technologist expertise
and can be labor-intensive, particularly for the initial extraction step.
"I don't want [laboratory directors] to think they all have to do molecular
testing for respiratory viruses," Dr. Caliendo says. "There is no perfect test."
She advises laboratorians to think critically when reading the literature. "Look
very closely at the methods," she says, noting patient population, specimen
type, culture technique, and assay technique will influence the testing sensitivity
and specificity. Finally, ask yourself, "Is this realistic? Would I really do
it in my laboratory?"
Dr. Ginocchio emphasizes the external challenges that laboratory
directors face when deciding which tests to implement: At the same time that
costs, test volume, medical staff demands, and regulations are increasing, budgets,
technical staff, reimbursement, and approved tests are decreasing. Personnel
shortages are particularly vexing, Dr. Ginocchio says: "We have positions open
that we can't fill."
As Dr. Caliendo enumerates, test options include DFA, direct antigen tests,
culture, and molecular-based methods. Optimal choices might vary by setting,
Dr. Ginocchio suggests. A small community hospital might prefer FDA-approved
tests with rapid turnaround time, such as off-the-shelf DFA and antigen detection
assays, plus limited culture. To this menu, a large community hospital or university
hospital could add molecular assays, both FDA-approved and commercial non-FDA-approved,
and in-laboratory-developed molecular assays. A core reference laboratory, considering
the need for rapid turnaround times and a competitive edge, would use off-the-shelf
assays and more in-laboratory-developed molecular assays for a broad range of
test analytes and low-volume niche testing.
Addressing molecular tests specifically, Dr. Ginocchio notes the virtual alphabet
soup of amplification methods-PCR, RT-PCR, TMA, SDA, and NASBA. For any amplification
method, nucleic acid extraction is "the most labor-intensive step," she says.
Semiautomated or automated extraction robots are available from Qiagen, Gentra,
Abbott, BioMérieux, Roche, and others.
Detection can be done by a traditional endpoint method (colorimetric, luminescent,
or electrochemiluminescent) or by real-time technologies, including dyes such
as Syber Green, FRET probes, TaqMan probes, and molecular beacons. "What we
are moving to in the laboratory is real-time," Dr. Ginocchio says. Simultaneous
amplification and detection favorably affects turnaround time and thus patient
care. Real-time PCR entails a major initial outlay for the instrument, "but
you save a tremendous amount of technologist time and reduce TAT greatly," Dr.
Ginocchio says. Equipment for real-time non-PCR-based applications such as NASBA
and TMA is significantly less expensive since the reactions are isothermal and
don't require a thermocycler. "And you reduce the risk of contaminating your
laboratory with amplicons," she says. Also, it is easy to develop applications
for real-time assays. Many real-time PCR instruments are available, such as
the Corbett RotorGene, Cepheid Smart Cycler, Bio-Rad iCycler, Roche LightCycler,
Applied Biosystems 7000, and Stratagene Mx4000. The BioMérieux EasyQ system
is a real-time instrument for NASBA applications.
To detect RNA viruses, Dr. Ginocchio uses NASBA and molecular beacons; for
DNA viruses, she does real-time PCR. Molecular methods are ideal for laboratories
without classical virology services, those that don't have the ability to grow
viruses in tissue culture, Dr. Ginocchio says. (Even in laboratories that do
perform viral culture, molecular methods often provide a safer alternative to
culture-for identifying the coronavirus that causes SARS, for example.) And
molecular methods have been demonstrated to be more sensitive than traditional
methods in "dozens of journal articles, if we're just talking about respiratory
viruses here," she says.
For instance, one group looked for respiratory viruses in bronchoalveolar lavage
fluids from 43 patients with hematological cancer and pneumonia. By culture
or antigen detection, nine pathogens were found in eight samples (19 percent
detection rate); PCR found 17 pathogens in 15 samples (35 percent), a significant
increase. Other investigators verified that PCR and multiplex RT-PCR are more
sensitive for RSV, parainfluenza virus, rhinovirus, and adenovirus (Freymuth
F, et al. Clin Diagn Virol. 1997; 8:31-40; Osiowy C. J Clin Microbiol.
On balance, Dr. Ginocchio says, although DFA and rapid antigen tests are generally
fast (less than two hours), their sensitivity and specificity are typically
lower than those of amplified tests. Rapid antigen tests are fastest (less than
30 minutes) but least sensitive. Real-time molecular assays, on the other hand,
are much faster than conventional amplification assays and can provide results
from start to finish in as little as 1.5 hours, which is comparable to DFA.
A major consideration in selecting test method is cost. Dr. Ginocchio lists
these representative setup costs for various methods: antigen tests, $0 to $200
(pipettes); DFA, $15,000 to $25,000 (biohazard facility, cytospin, microscope);
viral culture, $30,000 to $40,000 (add centrifuge); molecular, $30,000 to $250,000
(extraction and amplification instruments). Test costs range from $20 to $50,
with molecular tests on the high end. Using R-Mix for viral cultures adds acquisition
cost but saves money overall.
Molecular tests often require greater technical expertise depending on the
complexity of the assay and on whether the assay is commercially available or
developed in-house. Dr. Ginocchio had two positions open for a year in the molecular
laboratory. Her approach was to develop more defined, standardized protocols
for in-laboratory assays she developed so that less-experienced technologists
could readily perform them. Real-time molecular assays are coming out with more
well-developed protocols; however, all laboratories need to do in-house verification
and validation studies.
A second challenge is the lack of FDA-approved tests. "Often, manufacturers
don't want to go through the expensive FDA approval process, particularly for
assays without significant test volumes," she says, "so we must verify and validate
these assays and run numerous appropriate controls to ensure the validity of
our test results and the technical competency of our staff." Her solution: "We
need to put pressure on manufacturers to bring more assays to the FDA." This
is particularly important for smaller labs that don't have the ability to develop
in-house assays or perform the more in-depth verification and validation studies
required for non-FDA-approved assays.
Overall, Dr. Ginocchio says, molecular methods cost more initially but offer
greater sensitivity with equal or better specificity, are faster, and save money
in technologist time.
One place where molecular methods are valuable is in diagnosing
pertussis, especially in light of the atypical presentation seen in adolescents
and adults who now contract this infection due to waning immunity. Newer, more
sensitive tests are contributing to the recognition of pertussis as a re-emerging
infection, Dr. Loeffelholz says. Molecular methods are valuable also for their
specificity: While most pertussis is due to Bordetella pertussis, one
percent to 30 percent of cases are caused by B. parapertussis.
The tools used to diagnose Bordetella infections are culture, DFA,
serology, and molecular. Dr. Loeffelholz last July surveyed participants on
the ClinMicroNet e-mail discussion list, most of whom are laboratory directors,
about Bordetella testing capabilities; 35 laboratories responded. Of
the 24 laboratories using nonmolecular methods, 13 used culture and DFA, eight
used culture alone, one used DFA, and two sent samples out. None depended on
serology. Eleven laboratories, or 35 percent, offered molecular methods, chiefly
PCR. Four laboratories were using conventional PCR and seven had adopted real-time
PCR. "The real-time bandwagon is rolling on," Dr. Loeffelholz says.
(Dr. Loeffelholz was surprised to find that four of five public health laboratories
were using PCR, compared with only seven of 25 hospital laboratories. "Historically,
one thinks of public health laboratories as entrenched in more traditional methods,
in part due to restricted funding," he says. The reality of bioterrorism, he
adds, "has made a huge impact" on money available to public health labs. This
funding has enhanced testing capabilities outside of bioterrorism.) Culture
provides high specificity and variable sensitivity, but is affected by specimen
transport, since Bordetella is labile.
Culture provides isolates for antibody susceptibility testing, erythromycin
resistance (which occurs at a very low rate), and strain typing in outbreaks.
DFA is more rapid, but has poor sensitivity and variable specificity (positive
predictive value <50 percent has been reported). The Centers for Disease Control
and Prevention does not recommend DFA as a primary diagnostic test for Bordetella.
Serology may be the most sensitive method, but optimal specificity requires
paired specimens, to detect seroconversion or a fourfold rise in the antibody
titer. However, it is not always possible to get a patient to return in two
to four weeks. There is no FDA-cleared serology test in the United States and
no standardized interpretation criteria.
Among molecular methods, PCR predominates. Primary amplification targets are
insertion sequences (IS), pertussis toxin, porin, and RecA. One insertion sequence,
IS481, is present at 80-100 copies/ cell (which increases sensitivity) but is
not specific for B. pertussis; it is also found in B. holmesii. IS1001 is present
at 20 copies/ cell and is almost specific for B. pertussis. Conventional and
real-time PCR multiplex assays are available for B. pertussis and B. parapertussis
(Sloan LM, et al. J Clin Microbiol. 2002; 40: 96-100). Of the 11 laboratories
in the survey offering molecular methods, only three included B. parapertussis.
Dr. Loeffelholz delineated four possible solutions to the nonspecificity problem.
First, use a target specific for B. pertussis, such as RecA, porin, or pertussis
toxin. Second, use real-time PCR and do melting curve analysis. Third, use both
IS481 and IS1001, with this scoring: +/-, B. pertussis; -/+, B. parapertussis;
+/+, B. holmesii (Templeton KE, et al. J Clin Microbiol. 2003; 41: 4121-4126).
Fourth, use IS481 and report positive results with a disclaimer. Macrolides
are effective against B. holmesii.
Dr. Loeffelholz reviewed 14 studies comparing PCR to culture. PCR ranged from
1.1- to 9.6-fold more sensitive than culture. However, it is difficult to pinpoint
a true multiple, he says, since studies from the 1990s did not always resolve
discrepant results. Lower multiples typically occurred in studies done in pediatric
populations or when specimens were transported to the laboratory in less than
24 hours. These conditions can influence which test a laboratory adopts.
Asymptomatic people who are PCR-positive for Bordetella present another conundrum.
We can assume that B. pertussis bacteria or DNA are present in the specimen
but that the person was transiently colonized and didn't develop disease because
of a preexisting immunity. What is the clinical relevance of a positive PCR
result in this person? We might call it a clinical false-positive. Whether such
a person can transmit disease is not known. Clinical false-positives are particularly
common in children: 92 percent of PCR-positive children one to six years old
and 33 percent of those seven to 12 years old never develop symptoms. Should
they be treated? Are they Bordetella reservoirs? No one knows.
The problem of possible PCR false-positives also arises during therapy. In
infants and neonates culture positivity decreases to zero after seven days,
while 56 percent of specimens remain PCR-positive at this time (Edelman K, et
al. Pediatr Infect Dis J. 1996;15:54-57). Dr. Loeffelholz says these
figures illustrate the challenge of "interpreting positive results from sensitive
tests that don't require the presence of viable organisms and that don't always
agree with clinical response."
He concludes that molecular diagnostics offers high sensitivity and detection
of more Bordetella infections while helping clarify the organism's epidemiology.
Remaining challenges include standardization and quality assessment.
Speaking more generally about whether to adopt molecular tests, Dr. Loeffelholz
advises, "Consider your patient population. Traditional methods might perform
adequately." Consider, too, how you will implement real-time molecular tests.
"If a method has a two-hour turnaround time, does that mean a technologist is
going to be standing there with open arms 24/7 to receive specimens as they
come in and get a result out in two hours?" Dr. Loeffelholz asks. "Probably
Think about volume and specimen batching. Ultimately, adopting molecular testing
will require a comprehensive approach. "You need several tests to justify setting
up a molecular capability," Dr. Loeffelholz says. One or two is not enough.
Amidst all the talk of technology, Dr. Caliendo brings the discussion back
to its critical core. “The most valuable resource that we have in our labs is
our technical staff,” she reminds us. “We need to focus on them.”
William Check is a medical writer in Wilmette, Ill.