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S U P P L E M E N T A R T I C L E
Developing Molecular Amplification Methodsfor Rapid Diagnosis of Respiratory TractInfections Caused by Bacterial Pathogens
Fred C. Tenover
Cepheid, Sunnyvale, California
Current diagnostic methods for bacterial respiratory tract infections are slow and often of marginal value for
patient management if the adequacy of the specimen is not confirmed before culture. Molecular amplification
tests, which are highly sensitive, can provide results in hours rather than days but may not distinguish
colonization from infection unless a quantification step is included. Defining the reference method to be used
for evaluating a novel molecular assay, with input from the US Food and Drug Administration (FDA), is critical
before initiating development of a potential product. Although expectorated sputum may be the clinician’s
specimen of choice for testing because of ease of collection, the poor quality of such specimens may pose
problems for clinical trials of novel amplification tests. There are still many gaps in our understanding of
the interplay between colonization and infection and of the role that amplification tests may play in guiding
anti-infective therapy. Thus, the performance parameters of a new diagnostic method should be closely
matched to a precisely defined intended use statement.
Developing accurate methods for diagnosing bacterial
respiratory tract infections has long been a challenge for
the clinical microbiology laboratory [1, 2]. The semi-
quantitative culture methods used in most clinical
microbiology laboratories today, although adequate for
recovering and identifying a wide variety of bacterial
species from respiratory specimens, cannot differentiate
between colonization and infection, especially when the
majority of specimens submitted for testing either are
contaminated with upper respiratory tract flora or are
simply saliva, as shown by Gram stain [2, 3]. In fact,
a Gram stain of expectorated sputum can be used both
to ascertain the quality of a specimen and to guide
empirical therapy [4], but the closing of satellite labo-
ratories where residents, fellows, and attending physi-
cians could perform and interpret their own Gram
stains to aid in rapid decision making for patient
management has significantly lengthened the time it
takes for physicians to receive results [1, 5]. Recognizing
infection in a ventilated patient is even more of a chal-
lenge, because colonization of the respiratory tree with
a succession of gram-negative bacteria is common
during prolonged hospital stays, and most of the species
recovered from endotracheal aspirates would typically
be considered true pathogens if recovered from the
bloodstream [6]. Thus, many physicians feel compelled
to initiate empirical antimicrobial therapy on the basis
of culture results from endotracheal aspirates [7]. The
key reason for performing bacterial culture is to opti-
mize anti-infective therapy, which hinges not only on
identifying the causative agent of infection in a timely
manner but also on determining its antimicrobial sus-
ceptibility profile as well. In this age of antimicrobial
resistance, the current turn-around times (ranging from
48 to 96 hours to complete testing) are often inadequate
for optimal patient care [6, 8]. With the ever-expanding
array of multidrug-resistant pathogens, including Kleb-
siella pneumoniae, Pseudomonas aeruginosa, and Acine-
tobacter species, getting anti-infective therapy for
bacterial pneumonia correct the first time is critical for
Correspondence: Fred C. Tenover, PhD, Cepheid, 904 Caribbean Dr, Sunnyvale,CA 94089 (fred.tenover@cepheid.com).
Clinical Infectious Diseases 2011;52(S4):S338–S345� The Author 2011. Published by Oxford University Press on behalf of theInfectious Diseases Society of America. All rights reserved. For Permissions,please e-mail: journals.permissions@oup.com.1058-4838/2011/52S4-0009$14.00DOI: 10.1093/cid/cir049
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positive patient outcomes [4, 6, 7]. Thus, to many physicians,
the rapid and accurate identification of bacterial respiratory
pathogens, particularly for hospital-acquired pneumonia (HAP)
and ventilator-associated pneumonia (VAP), has become an
unmet clinical need.
In 2005, the disease management guidelines published by
the European Respiratory Society noted the potential role of
molecular amplification methods in improving the diagnosis of
lower respiratory tract infections [8]. Although many physicians
find the potential for enhanced sensitivity and more rapid
turn-around times attractive, there are both challenges and
barriers that industry will face in developing and marketing
molecular amplification tests for bacterial respiratory pathogens.
This article will review those challenges and barriers.
CURRENT DIAGNOSTIC METHODS FOR
RESPIRATORY TRACT INFECTIONS
The current semiquantitative agar plate-based culture method
used for analyzing expectorated sputum from patients with
suspected community-acquired pneumonia (CAP) or HAP is
slow and results may bemisleading, particularly if a Gram stain is
not performed in parallel to ascertain specimen adequacy [6, 8].
Expectorated sputum samples that contain .10 squamous epi-
thelial cells and ,25 polymorphonuclear leukocytes per low
power field are likely to contain saliva and not lower respiratory
tract material from the site of infection [2]. Unfortunately,
however, only 32% to 76% of sputum samples obtained from
hospitalized patients recently admitted with severe CAP met the
above acceptance criteria, according to a recent meta-analysis of
published CAP studies [8]. Even if the sputum sample is deemed
to be adequate using Gram stain criteria, the culture may still not
yield an obvious pathogen [2, 8]. The highest predictive value for
a culture of expectorated sputum occurs when Gram stain shows
a predominant morphotype (eg, gram-positive lancet-shaped
cocci in pairs) and the culture yields predominant growth of
a single recognized respiratory pathogen of that morphotype
(eg, Streptococcus pneumoniae) [9]. Unfortunately, such con-
cordance decreases rapidly when specimens are collected after
initiation of antimicrobial therapy [9] or when their arrival at the
microbiology laboratory is significantly delayed. On the other
hand, for aspiration pneumonia, which is often caused by a mix
of anaerobic bacterial species, a Gram stain of expectorated
sputum is often the only diagnostic test of value, because sputa
are not cultured anaerobically and aerobic cultures will yield only
mixed normal respiratory flora.
The other issue that impacts patient care is the slow turn-
around time of the test procedure. When antimicrobial sus-
ceptibility testing is included, the culture method requires
a minimum of 48 hours to complete. This is often too late to be
effective in guiding anti-infective therapy, especially when the
bacterial pathogen is multidrug resistant. Urine antigen tests for
pneumococci and Legionella pneumophila can provide rapid
results, but the available commercial tests have variable sensi-
tivity (range 66%–92% [4, 10]) and antigen tests for
L. pneumophila are limited to serogroup 1[10].
CHALLENGES FOR DIAGNOSTIC TEST
MANUFACTURERS
One approach that may improve the diagnosis of respiratory
tract infections and shorten the time necessary to place patients
on appropriate therapy is the use of nucleic acid amplification
methods. Real-time polymerase chain reaction (PCR) assays
[11–13], pyrosequencing [14, 15], and nucleic acid arrays [16]
are all options for improving the speed and accuracy of labo-
ratory results. The key challenge for industry is to develop assays
that are not only rapid but also readily accessible and perceived
by either laboratories or health care systems as cost-effective.
Development of an assay that is rapid but unavailable on evening
or night shifts or on weekends because of its technical complexity
limits the medical value of the test. From the industry perspec-
tive, cost-effectiveness should be determined not only by com-
parison to costs of performing slower conventional methods in
the laboratory but also by consideration of the cost savings
achieved from optimized antimicrobial therapy, decreased use of
additional diagnostic tests, and shorter hospital stays.
To have a positive impact on patient management, molecular
amplification tests will need to provide clear, definitive results
that will give physicians the data necessary to start, or in some
cases withhold, antimicrobial agents [6]. To be successful,
industry must determine (1) which combination of molecular
targets and clinical specimens will produce results that will
effectively guide anti-infective therapy regimens for patients
with pneumonia or other respiratory tract diseases; (2) which
reference method will provide the most meaningful results
for test development and clinical trials; and (3) which assays
companies will be able to afford to develop, given both the costs
of development and clinical trials and the ever-changing
reimbursement schedules from both private insurers and the
federal government. The potential return on investment is a key
factor when industry chooses which assays to develop. Input
from the US Food and Drug Administration (FDA) regarding
each of these determinations can be helpful and should be
sought. The development of novel diagnostic assays, especially
when the reference methods are not obvious, is certainly one of
the major challenges facing the industry.
CHOOSING MOLECULAR TARGETS
Molecular assays may target either a single pathogen, such as
Mycobacterium tuberculosis [17], or multiple respiratory
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pathogens in a single assay. Several multiplex assays for viral
respiratory pathogens have been described [12, 13, 16], a few of
which have been commercialized and have received FDA
clearance. There are merits to both single-pathogen and multi-
plex approaches.
DETECTION OF A SINGLE BACTERIAL SPECIES
Certain bacterial respiratory pathogens cause such distinct
clinical syndromes that assays that target them individually
still have clinical utility. These include organisms such
as M. tuberculosis, L. pneumophila, and Bordetella pertussis.
Molecular assays have been developed for all of these organisms,
although for the sake of brevity, discussion will be limited to the
issues surrounding the development of assays forM. tuberculosis,
where there is little doubt that detection of the organisms is
highly associated with disease.
RAPID DETECTION OF MYCOBACTERIUM
TUBERCULOSIS
The World Health Organization has reported that in 2008 there
were an estimated 9.4 million new cases of tuberculosis world-
wide [18]. Although the overall number of cases of tuberculosis
is decreasing in the United States, the proportion of cases among
foreign persons is steadily increasing [19, 20]. Of particular
importance from a diagnostic point of view is the fact that in
more than 26,000 cases of tuberculosis diagnosed in US-bound
refugees and immigrants, the acid-fast stains of expectorated
sputum samples from the patients yielded negative results. Such
infections are likely to be missed by tuberculosis screening
programs that rely primarily on acid-fast staining of respiratory
secretions instead of chest x-rays and slower culture methods
[19]. In addition, infections among foreign-born persons are
more likely to be caused by multidrug-resistant M. tuberculosis
strains than are cases among US domestic persons [20, 21].
Thus, particularly from the standpoint of immigrant and refugee
screening programs, there is a need for rapid tests that have
adequate sensitivity for detecting M. tuberculosis among smear-
negative samples. Although molecular diagnostics have been
used for direct detection ofM. tuberculosis in clinical samples for
more than a decade, these assays typically show unacceptably
low sensitivity levels for smear-negative specimens [17]. How-
ever, a recently released PCR-based commercial assay (which is
currently available only outside of the United States) showed
sensitivity of.90% on smear-negative culture-positive samples
in a multicountry, multicenter study [22]. Assays that in-
corporate direct detection of resistance markers in addition to
direct detection ofM. tuberculosisDNA targets are important for
guiding anti-tuberculosis therapy, particularly in an era of
multidrug-resistant and extensively drug-resistant strains [11,
23, 24]. The current drawback to developing and marketing tests
for M. tuberculosis in the United States is that tests for this
organism are classified by the FDA as Class III (ie, having high
impact on patient management). Thus, molecular tests to detect
the organism require what is known as a Pre-Market Approval
(PMA) application rather than the less rigorous, but still tech-
nically demanding and costly, Pre-Market Notification (510(k))
submission. In a PMA submission, the company must
independently show that its device is safe and effective in the
context of its intended use. This includes demonstrating
through carefully conducted clinical trials that the results of the
tests are highly accurate compared with a robust reference
method and that the company has designed and implemented
sufficient controls of its manufacturing process (including
product design, production, and quality control) to assure
the FDA that the method or device can be manufactured
reproducibly. An FDA Expert Panel is typically convened to
review the data to further assure the safety and effectiveness of
the device. By contrast, a 510(k) submission requires only that
the company demonstrate through clinical trials that its device
or method is ‘‘substantially equivalent’’ to a ‘‘predicate device or
method’’—that is, one that previously has been cleared by the
FDA for use in the United States. Although early molecular
probe assays for M. tuberculosis did not require a PMA, the
reclassification ofM. tuberculosis tests as Class III under current
regulations poses a significant barrier to test development and
marketing in the United States primarily because of the com-
plexity, breadth, and cost of the clinical trials required. Nucleic
acid amplification tests for M. tuberculosis, including at least 2
that simultaneously identify antimicrobial resistance markers
[11, 23], are widely available outside of the United States.
However, although there are published data describing the
performance of the GenProbe Amplified M. tuberculosis Direct
(MTD) test, the Roche Amplicor MTB test, the Cobas Amplicor
test, the Abbott LCx test, and the BD-ProbeTec Strand Dis-
placement Amplification (SDA) test for detection of M. tuber-
culosis in clinical samples, as noted by Ling et al [25] in their
recent meta-review of nucleic acid amplification testing meth-
ods, the GenProbe MTD is now the only FDA-cleared test
available in the United States. The paucity of molecular di-
agnostic options for M. tuberculosis detection in the United
States may reflect both real and perceived regulatory and mar-
ket-related barriers that are substantial enough to keep many in
the diagnostics industry on the sidelines.
DIRECT DETECTION OF MULTIPLE
ORGANISMS IN RESPIRATORY TRACT
SAMPLES BY SYNDROME
Potential multiplex assays for respiratory tract disease caused by
bacterial pathogens include those for CAP, HAP, VAP, acute
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bronchitis, sinusitis, exacerbation of chronic obstructive pul-
monary disease (COPD), and aspiration pneumonia. In
designing assays, it is critical to understand whether an assay for
a limited number of bacterial pathogens will meet physicians’
needs and provide adequate data for initiating or altering anti-
infective therapy. The targets chosen for molecular assays are
usually the key pathogens likely to be present in respiratory
samples. However, in this age of multidrug resistance, expand-
ing the target selection to include key antimicrobial resistance
genes that would alter existing therapy or guide empirical
therapy should also be considered. Potential groups of targets
for respiratory tract samples obtained from patients with
CAP, HAP, or VAP are presented in Table 1. The addition of
resistance gene targets, particularly those that mediate carba-
penem resistance (ie, resistance to doripenem, ertapenem, imi-
penem, and meropenem), may be very useful, especially because
carbapenems now play a prominent role in empirical therapy of
both HAP and VAP [6, 8]. For example, the emerging Klebsiella
pneumoniae carbapenemase (KPC) b-lactamases (encoded by
blaKPC genes, which mediate resistance to penicillins; cepha-
losporins, including extended-spectrum cephalosporins; and
carbapenems) [26–28] can compromise therapy for infections
caused by K. pneumoniae, Enterobacter aerogenes, and other
species of Enterobacteriaceae [29, 30], whereas the metallo-
b-lactamases, IMP and VIM (encoded by the blaIMP and blaVIMgenes, respectively) that mediate resistance to all b-lactam drugs
including carbapenems (with the exception of the mono-
bactams), can compromise therapy for Pseudomonas aeruginosa
infections [30]. Specific OXA b-lactamases (eg, OXA-40,
which also mediates resistance to extended-spectrum cepha-
losporins and carbapenems) can also compromise therapy for
Acinetobacter infections [31]. For gram-positive pathogens, the
presence of mecA in samples positive for S. aureus suggests that
vancomycin (or perhaps linezolid), either alone or in combi-
nation with other anti-infective agents, is necessary. If physicians
have confidence in the results of molecular assays, then negative
results for a specific pathogen should prompt physicians to
withhold treatment for that infectious agent (eg, to withhold
therapy for suspected methicillin-resistant Staphylococcus aureus
[MRSA] if the molecular assay for MRSA is negative). Of course,
laboratories may still culture respiratory specimens that are
positive by molecular methods to recover the pathogen to per-
form standard antimicrobial susceptibility tests on organisms
obtained in pure culture. Standard susceptibility testing provides
a broader susceptibility profile that extends beyond the few
antimicrobial resistance genes likely to be included in a molec-
ular assay.
RESPIRATORY SAMPLES FOR ANALYSIS
A variety of respiratory samples are amenable to molecular
testing, including expectorated sputum, bronchoalveolar lavages
(BALs), protected bronchial brushes, and endotracheal aspirates.
Of these, expectorated sputum samples are by far the most
common respiratory samples submitted to the clinical micro-
biology laboratory but are also the poorest in overall quality. As
the Infectious Diseases Society of America/American Thoracic
Society CAP guideline notes, one of the problems with di-
agnostic tests for respiratory tract infections ‘‘is driven by the
poor quality of most sputum microbiological samples and the
low yield of positive culture results’’ [4]. Getting specimens from
the site of infection that are not contaminated with upper
respiratory tract flora is a constant problem [3, 6, 32]. Endo-
tracheal aspirates from ventilated patients are often of better
Table 1. Potential Targets for Multiplex or Individual Molecular Amplification Assays by Syndrome [2, 4, 6, 8, 41]
CAP/exacerbations of COPD HAP/VAP Individual organisms
Streptococcus pneumoniae Staphylococcus aureus Mycobacterium tuberculosis
Haemophilus influenzae mecA genea Bordetella pertussis
blaTEM geneb Pseudomonas aeruginosa .
Moraxella catarrhalis blaVIM, blaIMP genesc .
Staphylococcus aureus Acinetobacter spp .
mecA genea blaOXA genesd .
Mycoplasma pneumoniae Enterobacteriaceae .
Chlamydophila pneumoniae blaKPC genec .
Chlamydophila psittaci Stenotrophomonas maltophilia .
Legionella pneumophila . .
NOTE. CAP, community-acquired pneumonia; COPD, chronic obstructive pulmonary disease; HAP, hospital-acquired pneumonia; VAP, ventilator-associated
pneumonia.a Mediates resistance to all b-lactam agents with the exception of the novel anti–methicillin-resistant S. aureus cephalosporins.b Mediates resistance to penicillins and first-generation cephalosporins.c Mediates resistance to cephalosporins and carbapenems; metallo-b-lactamases, such as VIM and IMP, typically do not mediate resistance to monobactams.d Some OXA b-lactamases can mediate resistance to carbapenems.
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quality than that of expectorated sputum from patients with
HAP but may still be contaminated with upper respiratory tract
flora [6, 32]. BALs and protected brush samples are more likely
to yield samples from the site of infection but require signifi-
cantly more effort to obtain and thus offer a much smaller
market for a new molecular test. In fact, there is a significant gap
in our knowledge as to how well molecular tests for bacterial
pathogens would perform on expectorated sputum samples,
compared with performance on BALs or protected brush
samples from the same patient collected within a similar period.
This knowledge gap is also a barrier to test development, because
a molecular test that cannot be performed on expectorated
sputum (given all the problems with specimen quality) may
not have broad enough appeal among physicians to make it
a financially viable product (from the industry perspective).
CHOOSING A REFERENCE METHOD FOR TEST
DEVELOPMENT AND CLINICAL TRIALS
Choosing a reference method for both test development and
clinical trials again highlights the issue of the ability of multiple
bacterial species to colonize and to infect the respiratory tract
[4, 32]. Antimicrobial stewardship programs frequently stress
the need to treat infections but to withhold antimicrobial agents
from patients who are asymptomatically colonized [6, 33]. The
enhanced sensitivity of molecular methods is essentially
negated if physicians perceive the test as ‘‘overly sensitive,’’
detecting colonization in the absence of infection. Balancing
sensitivity and specificity (and by analogy positive and negative
predictive values) for molecular tests is another example of
a critical gap in our understanding of the best manner in which
to apply molecular diagnostics. Is quantification of the targets
the answer for molecular tests? A recent Cochrane report
concludes that the use of quantitative cultures did not improve
the outcomes of patients with VAP, compared with the out-
comes of patients who were managed with qualitative results
[34]. However, the decreased turn-around time ofmolecular test
results may well prove advantageous over the two culture
methods (ie, qualitative and quantitative) that produced results
in similar, and significantly slower, time frames.
To illustrate this point, a brief review of the semiquantitative
culture method for expectorated sputum is appropriate.
Assessments of bacterial growth are performed on a series of
agar plates without an enrichment step. The constellation of
growth media usually includes blood agar, chocolate agar (for
fastidious organisms), MacConkey agar (or a similar selective
medium for gram-negative organisms), and colistin-nalidixic
acid agar (or a similar selective medium for gram-positive
pathogens). Results such as rare (often recorded as ‘‘one plus’’)
growth of pneumococci mixed with other respiratory flora from
an expectorated sputum sample on blood or chocolate agar
would likely be reported as ‘‘mixed respiratory flora’’ (often with
no specific mention of pneumococci on the report), whereas
heavy growth of pneumococci on the same agar plate may be
reported as ‘‘four plus growth, S. pneumoniae.’’ Although both
samples contain S. pneumoniae, in the former example, the or-
ganism is regarded as normal flora and is not reported, whereas
in the latter it gains ‘‘pathogen’’ status and is reported. It is likely
that both samples would yield a positive molecular amplification
test result. Thus, quantification of the amount of target present
with a defined threshold value that correlates with infection
rather than colonization may be necessary to provide confidence
in the molecular results [4, 32]. This adds complexity to the test
development process (because the test result is not simply the
presence or absence of the target organism) but is technically
feasible. High concentrations of target organism sequences
recognized through the amplification process are highly sug-
gestive of infection, but can one trust the absence of amplifi-
cation to be an indication of lack of infection if there was
no initial assessment of the specimen to ensure that it was of
adequate quality? Will it be necessary to include some host cell
marker to indicate that the specimen originated from the site of
infection and is not saliva? Although Gram stains of specimens
are critical for interpretation of culture results, requiring a Gram
stain of a specimen before performing a molecular assay would
defeat the purpose of having a rapid molecular test, especially
one that may ultimately be used outside of the clinical micro-
biology laboratory as a ‘‘point of care’’ test [10]. Once again, we
have identified a gap in our knowledge that is critical to assay
development. It is imperative, particularly for clinical trials of
novel molecular assays, that the end points that represent pos-
itive results for each bacterial species be established, especially
for organisms that can both colonize and infect the respiratory
tract. Quantification would not be required for organisms
such as M. tuberculosis or B. pertussis, because the presence of
these organism is synonymous with disease. The issue of
whether quantitative bacterial cultures would be necessary for
clinical trials must be answered. Appropriate cut-offs for
quantitative culture are debated, but several sources suggest
that the following numbers of organisms of a single bacterial
species indicate infection and not colonization: for expectorated
sputum, .1 x 107 colony forming units (CFU)/mL; for endo-
tracheal aspirates, .1 x 106 CFU/mL; for bronchoalveolar
lavage, .1 x 104 CFU/mL; and for protected brush specimen,
.1 x 103 CFU/mL [6, 32, 35]. It likely will be necessary to
evaluate the semiquantitative analysis of expectorated sputum
and quantitative methods (such as with BAL specimens) in
parallel to indicate whether a molecular amplification assay
could be used successfully on expectorated sputum specimens.
Because molecular assays are more sensitive than culture, one
may ask whether enrichment broths should be required to
maximize detection by culture during clinical trials. However,
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there are no defined enrichment broths for the majority of
bacterial respiratory pathogens; thus, this is not likely to be of
value.
ASSAY DEVELOPMENT—WHAT IS THE GOLD
STANDARD FOR BACTERIAL IDENTIFICATION?
One question that arises when using amplification methods
during both test development and clinical trials is whether the
amplification products truly represent the target organism
sequences. To establish this, it is often necessary to determine
the nucleic acid sequence of a sample of the amplification
products for the targeted pathogens. DNA sequencing is often
assumed to be completely accurate, showing 100% sensitivity
and specificity (particularly by non–molecular biologists).
However, because of a variety of technical factors, including the
quality of the sequencing template (ie, the amount of contam-
inating DNA from other sources), the selection and quality of
the primers used for amplification and sequencing, the robust-
ness of the base-calling software, and the method for compiling
the ‘‘consensus sequence’’ from multiple forward and reverse
reactions, sequencing does not always represent the ultimate
reference standard [36–38]. Nonetheless, companies must now
consider what measures of sequence quality (eg, using the
quality scores obtained by means of the ‘‘Phred’’ and ‘‘Phrap’’
computer programs [37, 39]) would constitute acceptable
standards if sequencing were included in clinical trial design. For
example, for individual sequencing runs, a length of sequence
that has,100 cumulative ‘‘Phred 20’’ quality score bases (where
the Phred software estimates the statistical likelihood of an in-
accurate base determination) is often considered to be un-
acceptable, because it represents a probability of error level of
1% [40]. On the other hand, using another software program,
a ‘‘Phrap’’ quality score of 40, which is the quality score for the
consensus sequence assembled from multiple reads of a DNA
sequence (equivalent to an error rate of�1 base for every 10,000
bases sequenced), is often considered acceptable—that is, of
publication quality (http://www.phrap.com/background.htm).
Such key parameters of quality have yet to be promulgated
by regulatory agencies, although sequencing data have been
included in multiple 510(k) submissions. Such guidelines would
be of value to diagnostic test developers as they contemplate
beginning clinical trials of novel diagnostic products.
SENSITIVITY AND SPECIFICITY, POSITIVE,
AND NEGATIVE PREDICTIVE VALUES
The key parameters that define the accuracy of a novel test,
compared with the accuracy of the reference method, include
sensitivity, specificity, and positive and negative predictive val-
ues. At present, there is no consensus as to which of these
parameters is most critical for potential molecular assays for
respiratory pathogens. Is a highly sensitive test that rarely misses
the presence of a given pathogen in a respiratory sample desir-
able, or is a test with a high negative predictive value indicating
the absence of a given pathogen of greater value? The former
scenario indicates that an antimicrobial agent is likely necessary,
whereas the latter indicates that antimicrobial agents can be
withheld. Although both high sensitivity and high specificity
rates are desirable (and are sometimes achievable in an assay),
the predictive values depend on disease prevalence and cannot
be manipulated by assay design. Thus, it is critical to define the
intended use of the assay before initiation of any clinical trials.
This is best accomplished in consultation with representatives of
the appropriate FDA center. Other issues that arise concerning
the design of clinical trials include the age ranges, ethnicity, and
clinical severity of the populations to be studied. Will trials need
to be conducted in both high prevalence and low prevalence
populations, stratified not only by age but also by gender and
even socioeconomic group? The broader and more diverse the
study population is, the larger the cost of conducting the trial.
Clearly, such clinical trials, especially if conducted in accordance
with the guidelines of a PMA, can become prohibitively
expensive rather quickly. Thus, the intended use of the assay is
perhaps one of the most critical decisions industry needs to
make before any development is initiated.
A POSSIBLE VAP ASSAY
To illustrate the above issues, a possible molecular amplification
assay for VAP is described. The intended use for the test is as
a diagnostic aid where the results of the test are considered in
association with other laboratory and clinical information to
establish a diagnosis of VAP. Whether the intended use includes
the ability of the test to guide anti-infective therapy has to be
considered carefully (eg, can this aspect of the test be proven in
a clinical trial?). The target organisms are S. aureus, P. aerugi-
nosa, Acinetobacter baumannii, a DNA sequence common to all
Enterobacteriaceae, and target sequences that are highly specific
for the following antimicrobial resistance determinants: mecA,
blaKPC, blaIMP, blaVIM, and blaOXA. The target specimens are
protected bronchial brush samples and endotracheal aspirates.
The reference methods include quantitative cultures on blood
chocolate, MacConkey, and CNA agar; a Gram stain of the
specimen; and a clinical diagnosis of VAP (fever, leukocytosis,
and purulent respiratory secretions) supported by radiologic
findings [32]. If a high degree of correlation between culture
results, Gram stain results, molecular test results, and clinical
diagnosis are achieved in alpha trials, beta trials could be con-
ducted to gather information on the potential cost effectiveness
(and physician acceptance) of the assay. If the assay results are
promising, a molecular test that ultimately could be performed
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on sputum samples from patients with HAP (rather than VAP)
may be feasible. Tests for CAP would require a different selec-
tion of targets.
SUMMARY
There are many challenges to developing molecular tests for
bacterial respiratory pathogens, particularly organisms that can
be both asymptomatic colonizers and overt pathogens of the
respiratory tract. Defining the reference method and the in-
tended use of the product before initiating clinical studies is
critical. Input from the FDA also is highly desirable. Although
expectorated sputum may be the clinician’s specimen of choice
because of ease of collection, the poor quality of such specimens
may require development of a unique host cellular target to
ensure specimen adequacy. Molecular amplification assays
will be costly to develop and clinical trials will be expensive, yet
the need is great. There are still many gaps in our understanding
of the interplay between colonization and infection. The key
question industry must answer is: ‘‘Is the juice worth the
squeeze?’’
Acknowledgments
I thank Drs Peter Dailey, Ellen Jo Baron, David Persing, Kerry Flom,
Michele Schoonmaker, AlanWortman, and Russel Enns for their input into
the development of this article.
Financial support. This work was supported by Cepheid.
Supplement sponsorship. This article was published as part of a supple-
ment entitled ‘‘Workshop on Molecular Diagnostics for Respiratory Tract
Infections.’’ The Food and Drug Administration and the Infectious Dis-
eases Society of America sponsored the workshop. AstraZeneca Pharma-
ceuticals, Bio Merieux, Inc., Cepheid, Gilead Sciences, Intelligent MDX,
Inc., Inverness Medical Innovations, and Roche Molecular Systems pro-
vided financial support solely for the purpose of publishing the supplement.
Potential conflicts of interest. F. C. T. is an employee of Cepheid,
a molecular diagnostics company.
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