Download Molecular Diagnostics for the Detection and Characterization of

SUPPLEMENT ARTICLE
Molecular Diagnostics for the Detection
and Characterization of Microbial Pathogens
Gary W. Procop
Department of Pathology, Jackson Memorial Hospital and University of Miami Miller School of Medicine, Miami, Florida
New and advanced methods of molecular diagnostics are changing the way we practice clinical microbiology,
which affects the practice of medicine. Signal amplification and real-time nucleic acid amplification technologies
offer a sensitive and specific result with a more rapid turnaround time than has ever before been possible.
Numerous methods of postamplification analysis afford the simultaneous detection and differentiation of
numerous microbial pathogens, their mechanisms of resistance, and the construction of disease-specific assays.
The technical feasibility of these assays has already been demonstrated. How these new, often more expensive
tests will be incorporated into routine practice and the impact they will have on patient care remain to be
determined. One of the most attractive uses for such techniques is to achieve a more rapid characterization
of the infectious agent so that a narrower-spectrum antimicrobial agent may be used, which should have an
impact on resistance patterns.
It has been 12 decades since the inception of the PCR
[1, 2]. Although this technique was almost immediately
implemented in research laboratories to study a variety
of infectious diseases, among other processes, it has
been within only the past 5–7 years that these assays
have been made so user-friendly that they may be implemented in any microbiology laboratory [3]. In addition to PCR, there are numerous competitive technologies that have been developed and shown to be
useful for the detection and characterization of microorganisms. Many of these more recently developed assays may be performed by medical technologists who,
although highly skilled, do not have advanced training
in molecular biology. The simplification of this type of
testing means that it may be performed after hours or
in adverse conditions (i.e., in the field). Several of these
assays are US Food and Drug Administration approved
or cleared, and more are in this process. Today, in the
modern microbiology laboratory, there truly is a “mo-
Reprints or correspondence: Dr. Gary W. Procop, Dept. of Pathology, University
of Miami Miller School of Medicine, Jackson Memorial Hospital (R-5), 1611 NW
12th Ave., Holtz Bldg., Rm. 2090, Miami, FL 33136 ([email protected]).
Clinical Infectious Diseases 2007; 45:S99–111
2007 by the Infectious Diseases Society of America. All rights reserved.
1058-4838/2007/4505S2-0002$15.00
DOI: 10.1086/519259
lecular revolution” under way. Although this is exciting,
the responsibility of the laboratorian and the ordering
physician is to ensure that these tests are used appropriately. The abuse of this technology has serious implications for cost as well as patient care, because, as
with any test, false-positive and false-negative reactions
may occur.
The clinical microbiologist is occasionally asked to
exclude the presence of a particular microorganism in
a clinical specimen. For example, CSF may be submitted
for the exclusion of herpes simplex virus, or a blood
smear may be submitted for the exclusion of Plasmodium species. However, in many instances, several tests
are ordered per clinical specimen, and the questions
asked are of a broader nature. For example, when a
blood or wound culture is ordered, the question really
is “Are there any bacteria or fungi present in this clinical
specimen, and to what antimicrobial agents are they
susceptible?” The clinician rarely draws a blood sample
for culture for the detection or exclusion of a single
type of microorganism (i.e., blood is not drawn for
culture to “rule out [r/o] Pseudomonas” but, rather, to
“r/o bacteremia and fungemia”). The task of the clinical
microbiologist is complicated, because he or she must
be aware of all of the potential pathogens that may be
present in a clinical specimen and must devise or utilize
Molecular Diagnostics • CID 2007:45 (Suppl 2) • S99
means by which to detect and characterize these organisms.
The molecular diagnostic techniques used for the detection
and characterization of microorganisms may be separated into
several broad categories. These are direct hybridization, nucleic
acid amplification, and a variety of methods for postamplification analysis. These methods may be used to either detect or
exclude particular pathogens or, more usefully, to assess a specimen or positive culture sample for a wide variety of microorganisms. The value of such applications must be rigorously
assessed, because these tests are usually added to the battery of
routinely performed assays. The benefits of such assays are less
likely to be found in the laboratory and more likely to be seen
by the clinician or the health care administrator. For example,
the more rapid detection and characterization of an infecting
pathogen afford the clinician the opportunity to tailor antimicrobial therapy. In addition to benefiting the patients, this
may save health care dollars through the pharmacy by identifying situations wherein less expensive antimicrobial agents
may be used [4, 5]. For example, Forrest et al. [5] demonstrated
that $1729 in pharmacy costs per patient could be saved by
switching the patient’s treatment from a more expensive echinocandin to a less expensive azole. The use of narrower-spectrum agents, in contrast to the use of broad-spectrum agents,
should also slow the selection for and spread of antimicrobialresistant microorganisms. Other benefits that have been noted
include better use of ancillary diagnostic tests (e.g., radiology
services) and of hospital beds (i.e., decreasing lengths of stay
or the number of unnecessary admissions) [6].
DIRECT HYBRIDIZATION
There are many direct-hybridization techniques used in clinical
microbiology. Some of these have been used for the rapid identification and characterization of bacteria and fungi in blood
culture samples, whereas others have been used for different
applications (e.g., human papillomavirus [HPV] detection) but
could also potentially be used in this manner. In situ hybridization will predominate this discussion, because it is the newest
signal amplification technique to be used in the clinical microbiology laboratory. The detection process used in in situ
hybridization may be fluorescent (FISH) or chromogenic
(CISH). These tools, which were once used solely in the domains of researchers and anatomic pathologists, have been
demonstrated to be useful for the detection of pathogens in
blood culture samples as well as in other clinical specimens [7–
16]. A chemiluminescence-generating chemical reaction used
after the direct hybridization of oligonucleotide probes to microorganism ribosomal RNA (rRNA) is another technology that
may be used for the rapid characterization of microorganisms.
This technology has been used for more than a decade for the
identification of microorganisms, such as commonly occurring
Mycobacterium species and the dimorphic fungi, in positive
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culture samples [17–21]. Another technology, target-capture
technology, used predominantly for the detection of viruses
(HPV, cytomegalovirus, and hepatitis B virus), could also be
used for the detection of bacteria and fungi in positive blood
culture samples [22–25].
In situ hybridization. In situ hybridization has been introduced into the clinical microbiology laboratory and should
prove to be a useful technology for the rapid characterization
of bacteria and fungi in positive blood culture samples, which
is an area of particular interest to many [5, 7, 9–11, 13, 15,
26–30]. In situ hybridization has also been used successfully in
direct clinical specimens and in histologic sections for the detection of microorganisms [8, 12, 14]. Regardless of the type
of direct hybridization technology used, identifications using
signal hybridizations are most accurately obtained after microorganism growth or biological amplification of the organism.
This technology is often used to differentiate microorganisms
of similar morphotypes in positive culture samples (e.g., grampositive cocci in clusters or acid-fast bacilli). One of the advantages of this technology is that it may be used in conjunction
with traditional methods, such as Gram staining. The information derived from Gram staining may be used to select the
appropriate probes from a battery to more specifically address
select questions. However, the use of the full battery of probes
on all blood culture bottles that indicate positivity without the
Gram stain–derived information would be cost-prohibitive and
an inefficient use of resources. The information derived from
Gram staining is used to select the one or few probes necessary
for bacterial or fungal characterization. For example, if grampositive cocci in clusters are demonstrated in the Gram stain,
then one would select a probe that would detect Staphylococcus
aureus and differentiate it from coagulase-negative staphylococci. Similarly, a battery of probes for the most commonly
occurring yeast species would be used when yeasts are seen in
the Gram stain, whereas probes for Pseudomonas and members
of the Enterobacteriaceae would be used when gram-negative
bacilli are seen in the Gram stain smear. This is an excellent
example of optimizing the use of new technology through the
use of established methods. A variety of oligonucleotide probe
sequences have already been described that generate family-,
genus-, and species-specific information [13, 15].
Two types of oligonucleotide probes are most frequently used
for in situ hybridization. These are traditional nucleic acid oligonucleotide probes and peptide nucleic acid (PNA) probes.
Traditional oligonucleotides used for in situ hybridization need
no further explanation for most in the field of molecular diagnostics, but a limited description of PNA probes is warranted
[31, 32]. Peptide nucleic acid probes are synthetic molecules
that do not naturally occur. The most important difference
between DNA and PNA resides in the “backbone” of the molecule. The backbone of the DNA polymer consists of repeating
deoxyribose sugar and phosphate molecules, the latter of which
have a net negative charge. In contrast, the backbone of the
PNA molecule, used in the studies described here, consists of
repeating glycine molecules. Glycine, importantly, is a sterically
small and neutrally charged amino acid. The similarity between
DNA and PNA oligonucleotide probes is the 4 common nucleotide bases, which participate in Watson and Crick base
pairing. These unique properties afford the construction of a
probe molecule that can penetrate through the cell wall and
cell membrane (i.e., a hydrophobic lipid bilayer) of microorganisms while retaining the specificity achieved with Watson
and Crick base pairing [32]. The charge of the in situ probe is
particularly important in microbiology, wherein the microorganism undergoing FISH analysis is intact, and in distinct contrast to many applications in molecular pathology (e.g., FISH
for Her-2 amplification), wherein the nucleus, which contains
the chromosomal targets for the FISH assay, has been sectioned,
and the probes are applied directly onto their nucleic acid targets (i.e., penetration through a lipid bilayer is not necessary).
Although there is limited commercial availability of FISH
probes in microbiology, some are available, and more are likely
on the horizon. The PNA FISH assays that are currently available in North America detect and differentiate S. aureus from
other gram-positive cocci in clusters and Candida albicans from
other yeasts. The assay may be performed as soon as the blood
culture bottle indicates positivity and Gram staining has been
performed, or the slides to be tested may be tested in batches.
Although batch testing clearly increases the time to result that
is possible with immediate testing, it is a viable alternative for
laboratories with limited personnel and still has a better time
to result than traditional methods. It is now possible, with this
technology, to provide a definitive species-level identification
of microorganisms, such as S. aureus and C. albicans, on the
same day that the blood culture bottle indicates positivity and
demonstrates gram-positive cocci in clusters or yeast, respectively [7, 9–11, 26, 30]. In addition to the commercially available applications, several studies have demonstrated the feasibility of definitively identifying other medically important
bacteria and yeast species [13, 15, 28, 29]. Importantly, these
assays are simple to perform and represent a molecular diagnostic tool that may be introduced into laboratories without
previous experience in molecular microbiology. They have already been adopted and are performed routinely in the several
clinical microbiology laboratories in the United States. From
an economic standpoint, the use of the C. albicans PNA FISH
probe has been shown to be cost-effective, particularly when
linked to limiting the use of more expensive antifungal drugs,
such as an echinocandin, when a less expensive but equally
efficacious drug like fluconazole is a viable alternative [4, 5].
In addition to its use in the differentiation of the causes of
bacteremia and fungemia, in situ hybridization has been used
to identify the most common causes of infection in patients
with cystic fibrosis, to differentiate mycobacteria in culture and
in histologic sections, and to detect trypanosomes in patients
with African sleeping sickness [8, 14, 27].
The same hybridization probes used in the clinical microbiology laboratory could be used in in situ hybridization reactions in histologic sections. Hayden et al. have demonstrated,
in a series of articles [12, 33–35], that chromogenic in situ
hybridization may be used successfully to differentiate morphologically similar microorganisms. They showed the feasibility of this technology to detect Legionella pneumophila and
differentiate filamentous bacteria (i.e., aerobic actinomyces),
filamentous fungi, and yeast and yeastlike fungi. Importantly,
they showed the ability to separate the systemic dimorphic
fungi, which appear as yeast (e.g., Histoplasma capsulatum and
Blastomyces dermatitidis) or spherules (e.g., Coccidioides immitis), from Candida and Cryptococcus species. Although it may
seem a morphologically simple task to differentiate a well-developed spherule of C. immitis from budding yeast, it is considerably more complicated when intact spherules are not seen
and, rather, only immature spherules and endospores are present. In situ hybridization has also been used to identify Pneumocystis jiroveci [36]. Finally, one can imagine an economic
benefit when the same probe sets are used for both microbiology and infectious disease pathology applications. For example, PNA FISH probes have also been used successfully to
identify mycobacteria in histologic sections and from positive
culture samples [27].
Another exciting application that may have practical uses in
the clinical microbiology laboratory is the combined use of in
situ hybridization and flow cytometry. Small-scale flow cytometers that can detect microorganisms have been developed
and are routinely used in the food and pharmaceutical industries. These applications often use nonspecific dyes that bind
to the external aspect of the bacteria. Although such assays are
useful for determining overall bacterial counts in a product,
the results generated do not yield genus- or species-level information. However, it has been demonstrated that this technology can be used to detect microorganisms to which in situ
probes are hybridized [37, 38]. This combination of technologies affords the replacement of human interpretation, which
is subjective and requires advanced training and experience to
perfect, with objective, quantitative measures that are performed by an instrument.
The precise role that in situ hybridization will play in the
clinical microbiology laboratory and its impact on patient care
remain to be seen. Several other competitive technologies, some
of which are described below, offer similar advantages with
regard to rapid identification. Regardless, it has been demonstrated that the identification of the most common microorMolecular Diagnostics • CID 2007:45 (Suppl 2) • S101
ganisms that cause bacteremia and fungemia may be rapidly
determined by use of this technology.
Other methods of signal amplification. There are several
other signal-amplification methods that are currently used in
many clinical microbiology laboratories, the applications of
which could be expanded. The chemiluminescent rRNA probe
hybridization technology that has been available from GenProbe for years could be expanded/promoted to detect many
of these pathogens. This technology uses probes that target
rRNA, which is present in numerous copies in each cell. The
hybridized probe is then detected using a proprietary chemical
reaction that results in the generation of light. Products that
utilize this technology have been used for years in many laboratories for the rapid identification of the most commonly
occurring mycobacteria and for isolates suspected to represent
systemic dimorphic fungi [18–21, 26, 39–41]. Although products have been developed for rapid detection of many of the
more commonly occurring bacteria, such as S. aureus, these
have not gained widespread adoption for the rapid identification of the common causes of bacteremia and fungemia [26,
42, 43]. There have, however, been some hints that this company might be interested in developing panels useful for the
identification of the commonly encountered bacteria and yeast
species, although a commercial product has not become
available.
The hybrid capture (Digene) technology, which has been
used most successfully for the detection of high-risk HPV subtypes, is also a signal amplification technology [44]. This technology has also been used for the detection of cytomegalovirus,
hepatitis B virus, Neisseria gonorrhoeae, and Chlamydia trachomatis [45–47]. Although signal amplification assays that do
not employ direct microscopy are often not as sensitive as
nucleic acid amplification, some have shown the contrary [44,
48]. These assays have adequate sensitivity for many clinical
applications and afford high specificity, depending on the specificity of the probes used. In addition to the types of applications
described, this technology could also be used to characterize
bacteria or fungi that grow in culture and require further
identification.
NUCLEIC ACID AMPLIFICATION
Nucleic acid amplification includes not only PCR but also alternate technologies, such as strand-displacement amplification
and transcription-mediated amplification (i.e., nucleic acid sequence–based amplification), which have also proven useful in
clinically important assays. In addition to the various chemical
reactions that may be used for amplification, the design of these
assays varies widely. Monoplex assays are amplification reactions that detect a single target and provide a “present or absent”–type result. For example, a Legionella pneumophila–specific PCR could be used to rapidly detect this pathogen. If used
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in conjunction with quantitative standards, these could be used
to provide quantitative information (e.g., viral loads). The combination of ⭓2 nucleic acid amplification reactions into 1 assay
is a multiplex reaction. These assays have been used to detect
multiple pathogens simultaneously but require advanced design
and critical assessments, because intramolecular interactions
between the various primers and probes are potential limitations of such assays. Several multiplex (and monoplex) assays
are commercially available as “analyte-specific reagents.” These
are commercial products that contain all the necessary components to conduct a particular amplification reaction, which
are manufactured under Good Manufacturing Practice conditions, but the responsibility of validating such assays resides
with the testing laboratories. One of the more successful of the
multiplex analyte-specific reagents that has been released is a
real-time RT-PCR that simultaneously detects and differentiations the most important respiratory viral pathogens—influenza A, influenza B, and respiratory syncytial virus. The realtime version currently available is a truncated version of
another multiplex assay produced by the same company that
detected and differentiated 6 respiratory viruses [49, 50].
Broad-range nucleic acid amplification is another approach
that could be used to detect any of a wide variety of microorganisms are in a taxonomically related group [51–54]. The
results of broad-range PCR simply indicate that one of the
members of a particular group is present. Quantitative data
(i.e., organism load) may also be obtained if quantitative standards are included in the amplification run and a standard
curve has been produced. Although quantitative data may be
of use, much of the strength of broad-range nucleic acid amplification assays comes from the information that may be derived from the amplified product (i.e., the amplicon) through
postamplification analysis. There are many methods of postamplification analysis, but only a few will be briefly discussed
here, given the scope of the present article. These are postamplification melt curve analysis, reverse hybridization, DNA sequencing (traditional and pyrosequencing), and solid- and liquid-phase microarray analysis.
It seems that advances in laboratory diagnostics more commonly occur quite rapidly in conjunction with technological
advances (i.e., as in Stephen J. Gould’s theory of punctuated
equilibrium), rather than occurring though the accumulation
of small advances over a long period (i.e., a more Darwiniantype change) [55, 56]. The history of the use of PCR in clinical
laboratories supports this notion. Although PCR and other
nucleic acid amplification technologies (note: hereafter, “PCR”
will be used generically to encompass all methods of nucleic
acid amplification) have penetrated into the larger diagnostic
and university laboratories for the detection of microorganisms
that are usually difficult to culture, they have not until recently
become readily available in small, perhaps less molecularly
savvy laboratories or for the detection of more common pathogens. This is because traditional PCR is cumbersome and slow,
especially in light of the time and process necessary to specifically determine the nature of the amplified product by Southern blot analysis. However, all this changed with the advent of
real-time PCR, also known as “homogeneous, rapid cycle nucleic acid amplification.”
The advent of real-time PCR awaited complementary advances in engineering and chemistry. Engineers devised ways
of decreasing the PCR cycling time. This has been accomplished
through the use of heated and cooled air to achieve more rapid
heating and cooling, respectively. This is in contrast to traditional block cycle heating, wherein, traditionally, a metal or
solid block was heated and cooled to change the temperatures
of the reaction vessels contained in the block to achieve those
temperatures necessary for the PCR. This latter method, although effective, is naturally slower and reflects the physical
differences between conduction and convection heating. Next,
the engineers needed to develop sufficient optical systems for
these instruments, to both introduce a beam of light of a particular wavelength and detect minute changes in light emitted
from the solution within the reaction vessel. Fortunately, many
of these technical issues had been addressed by scientists who
used laser and fluorescent dye technology for various applications, such as flow cytometry. This afforded the adaptation
of such technology to real-time PCR platforms.
The advances in chemistry that made real-time PCR possible
were also significant, and modifications of these chemical reactions continue today. The challenge was to devise a way that
amplification and detection reactions could occur within the
same reaction vessel without necessitating the opening of the
vessel and the manipulation of the amplified product. Maintaining a closed system is important in a clinical microbiology
laboratory, because chemical contamination of the environment with amplicon could lead to false-positive reactions [57].
This was achieved through the discovery and construction of
molecules that were nonfluorescent in a nonhybridized state
but were able to generate a fluorescent signal when both bound
to their DNA target and excited by the appropriate electromagnetic energy. The signal generated may be nonspecific,
demonstrating only the presence of double-stranded DNA (i.e.,
evidence that the PCR has occurred), or specific, which is
achieved through the hybridization of specific, fluorescently
labeled oligonucleotide probes. An example of a nonspecific
probe is SYBR Green dye I, which binds to the minor groove
of double-stranded DNA. The signal derived from this molecule, while PCR is under way (thus, in real time), demonstrates
to the user that amplification is occurring. Whether this amplification is specific cannot be determined at this point in the
reaction (see the melt curve analysis below). Conversely, if one
uses a specific, fluorescently labeled oligonucleotide probe, then
the signal that is detected during the PCR is evidence that the
target DNA molecule is present, because the oligonucleotide
probe hybridizes to its target through specific Watson and Crick
base-pairing rules. There are many different types of oligonucleotide probes available. The 3 most commonly used types
are the hydrolysis or “TaqMan” probes, molecular beacons, and
fluorescence resonance energy transfer (FRET) probes. A positive real-time PCR result for any of these probes is demonstrated by an exponential increase in fluorescence with respect
to amplification time or cycle number (figure 1). Specimens
that do not contain the target molecule (i.e., that are negative)
will demonstrate minimal or no change in fluorescence. The
qualitative information derived from such reactions is useful
for demonstrating the presence or absence of a microorganism
of interest. For example, assays have been devised to address
the presence or absence of Legionella pneumophila by use of
real-time PCR. Such an assay may offer times to results that
are similar to those of direct immunofluorescence antigen detection, but the real-time PCR has been shown to be more
sensitive than direct immunofluorescence antigen detection;
additionally, the same assay was described as being more rapid
and possibly more sensitive than culture while retaining a specificity equivalent to that of culture [58].
The specificity of a PCR assay is determined by the target
DNA sequence under evaluation, the sequence of the oligonucleotide probe, similar sequences that may exist elsewhere
in nature, and the intentions of the assay designer. Many assays
are species specific, but genus-generic assays (i.e., a more broadrange PCR) may also be designed. These would be useful when
it is important to detect all members of a group that may be
divergent in nature. For example, one may use a species-specific
PCR for Salmonella serotype Typhi, to detect that particular
serotype, or a broad-range Salmonella assay, to detect all of the
members of that genus [59].
In addition to the detection of microbial pathogens, these
applications may be used to detect genetic elements that encode
for mechanisms of antimicrobial resistance (e.g., the mecA
gene), those that determine subtypes of such genes for epidemiologic purposes (e.g., the mecA cassette IV associated with
community-acquired Staphylococcus aureus infection), and the
detection of virulence factors (e.g., Panton-Valentine leukocidin). Although useful for the detection of certain mechanisms
of resistance, nucleic acid amplification assays for more complicated and genetically diverse mechanisms of antimicrobial
resistance (e.g., the extended-spectrum b-lactamases) have remained elusive and are not likely to replace traditional phenotypic susceptibility testing in the near future.
POSTAMPLIFICATION ANALYSIS
Melt curve analysis. Another feature of some of the chemical
reactions used with real-time PCR is the ability to perform a
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Figure 1. Examples of real-time PCR amplification reactions. The negative control specimen has produced a flat line, whereas the positive control
and other template-containing samples demonstrate an exponential increase in fluorescence. The reactions that become positive earlier (i.e., that have
a lower crossing point, or cycle number threshold [Ct]) contain a greater amount of target template than do those that became positive later.
postamplification melt curve analysis. This provides different
information, depending on the type of probe used. If SYBR
Green dye I or another nonspecific DNA binding dye is used,
then the information derived is similar to that derived from
gel electrophoresis. Larger amplicons are held together by more
hydrogen bonds, whereas smaller amplicons, such as primer
dimers, are held together by fewer hydrogen bonds. The postamplification melt curve derived using these types of DNA
binding molecules demonstrates high melting temperatures
(95C–98C) for large amplicons and lower temperatures for
smaller amplicons and primer dimers. Some researchers have
demonstrated the detection and differentiation of single nucleotide polymorphisms (SNPs) by use of high-resolution melt
curves and nonspecific DNA binding dyes, but, for the most
part, these uses remain experimental [60].
The FRET probes and another oligonucleotide probe called
an “eclipse probe” are the types of specific oligonucleotide
probes that provide the best postamplification melt curves.
Foremost, a melt curve analysis after amplification provides
information regarding the specificity of the reaction. A particular melting temperature is expected for the hybridization between the oligonucleotide probe and its target. The presence
of the expected melt curve after amplification helps to assure
the user that the results are correct. If, for example, the primers
were degenerate and also amplified a related microorganism,
the melt curve generated would have a lower temperature than
expected, if nucleotide polymorphisms existed at the probe
hybridization site. This molecular misidentification would not
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be detected in the instrument’s “quantification” mode, and, if
such a scenario occurred, the specimen would erroneously be
deemed positive. However, if a postamplification melt curve
analysis was performed, then the nucleotide mismatches between the probe and target sequences would be detected, because the probe could not hybridize to its target sequence with
the optimal affinity and would “melt off” at a lower temperature. This technology is so sensitive that it is commonly used
to determine SNPs in human genetic pathology (e.g., the SNP
responsible for factor V Leiden) [61]. Thus, postamplification
melt curve analysis is a method whereby one may obtain additional data concerning the specific nature of the amplified
DNA product after real-time PCR.
This difference in melt curves created by imperfect complementarity between probes and their target hybridization sites
may be exploited to differentiate taxonomically related microorganisms to the species level. Assays have been designed to
differentiate HSV type 1 and 2, the BK and JC polyoma viruses,
human herpesvirus 6 types A and B, the commonly occurring
Bartonella species, and M. tuberculosis from nontuberculous
mycobacteria (figure 2), among others [53, 62]. This application is quite useful, but there is some evidence that there may
be diminished sensitivity for the target that has the lower melting temperature (i.e., the imperfect complementarity) [63].
Therefore, it would be prudent to not use this type of application if the target that had this profile caused serious disease
(i.e., if there would be a significant sequela associated with a
false-negative reaction) or if such a target organism is present
Figure 2. Postamplification melt curves generated after a broad-range PCR for mycobacteria. DNA extracts from Mycobacterium tuberculosis (TB)
and nontuberculous mycobacteria (NTM) are used as controls. The melt curve derived from a specimen that contained mycobacteria is clearly categorized
here as NTM, which means that the patient does not require respiratory isolation or antituberculous therapy.
in small quantities and may be missed because of the limited
sensitivity. The approach we have taken with the mycobacteria
has been to utilize this technology to differentiate mycobacteria
that are detected either in the acid-fast smear or in histologic
sections stained by the Ziehl-Neelsen method. In such an instance, it is not a question of “if” mycobacteria are present but,
rather, a question of “which type of” mycobacteria is present,
with the primary goal being the separation of M. tuberculosis
from the nontuberculous mycobacteria (figure 2). The hybridization probes have 100% homology to M. tuberculosis, with
the nontuberculous mycobacteria having a ⭓1-nt mismatch at
the probe hybridization site. This design makes the assay most
sensitive for the detection of the most important mycobacteria
that we encounter in North American microbiology, M. tuberculosis. If acid-fast bacilli are seen in the smear or stained
tissue section, but amplified product is not detected, then one
may categorize the result as “quantity of mycobacteria below
the limit of detection of this assay,” which is a more useful
result than a false-negative result.
There are a variety of other methods of postamplification
amplicon analysis that require the opening and manipulation
of amplified PCR product. These are acceptable methods, but
care must be taken to avoid the introduction of amplified product into the areas of master mix preparation or specimen preparation, to avoid false-positive reactions. The types of methods
are too numerous to describe in an exhaustive manner in this
article, so I have selected a few to highlight that either have or
will likely have an impact in clinical medicine; these are reverse
hybridization, DNA sequencing—including pyrosequencing—
and microarray analysis, with a focus on limited microarrays
of both the solid and liquid formats.
There is often a need to assess for or identify 11 microbial
pathogen. It is more time- and cost-effective if these assessments can be performed simultaneously or in a single reaction
than if they are performed in numerous monoplex amplification reactions. For example, it is necessary to assay for numerous high-risk HPVs to appropriately diagnose or exclude
a high-risk HPV infection in a woman. It is preferable to perform a single assay to detect and/or differentiate these viruses
rather than to perform 110 individual PCRs for the various
high-risk HPV subtypes. Postamplification analyses are often
employed when the number of results obtained exceeds the
technical differentiation capabilities of multiplex reactions
wherein each reaction contains a different fluorescent label, or
when the number of results achieved may exceed the differentiation capabilities of a melt curve analysis.
Reverse hybridization. One of the most simple-to-perform
methods of detecting a variety of pathogens, after either a multiplex or a broad-range amplification reaction, is through reverse hybridization. This technique is the opposite of Southern
blot analysis in many aspects and is therefore termed “reverse.”
Herein, a variety of probes are placed on a nitrocellulose
membrane strip, and the amplicon rather than the probe is
labeled. The amplicon is applied to the strip under appropriate
hybridization conditions, and then wash steps are performed.
If the amplicon hybridizes to one of the immobilized probes,
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it may be visualized through the application of a color development reagent. The position of the product on the strip reveals
the nature of the pathogen. This technology has been used to
detect and differentiate the commonly occurring mycobacteria
(figure 3), clinically important fungi, HPV subtypes, hepatitis
C virus genotypes, and certain resistance-associated mutations
in HIV [64–67]. This technology is simple to use, but some
applications have been limited because of the expense of the
product. Occasionally, light bands of nonspecific hybridization
are seen (i.e., “ghost bands”) that may confuse interpretation
or suggest dual infections.
DNA sequencing: traditional (Sanger) and pyrosequencing.
The sequencing of the amplicon, through traditional Sanger
sequencing, is the ultimate in postamplification analysis. The
entire composition of the amplified product is thereby determined. Although initially difficult to perform, this technology
is now readily available to any molecular diagnostic laboratory.
Advances in capillary and gel electrophoresis, as well as computer-assisted sequence analysis, afford the expanded use of
this technology to many more laboratories. Traditionally, sequencing after RT-PCR is the method of choice for determining
resistance-associated mutations in HIV, and 2 Food and Drug
Administration–approved tests have been developed for this
application. This technology is also commonly used to identify
bacteria, fungi, and mycobacteria on the basis of sequences
within the ribosomal gene complex [68–71]. Once the sequence
of an unknown microorganism is determined, it may be queried
against a genetic database that contains thousands of sequences.
The results of a query are returned as a percentage match, which
assists the molecular microbiologist with the identification of
the microorganisms. There is clearly great promise for this
approach, and commercial products (e.g., MicroSeq [Applied
Biosystems]) and nonpublic databases (e.g., SmartGene) have
been established. Full-length sequencing is particularly powerful when several nucleotide polymorphisms are needed for
the characterization of a microbe and these are distant from
one another within the amplicon (e.g., some of the mutations
associated with HIV resistance). The drawbacks associated with
this technology would include the cost of the equipment, reagents, and access to private databases; the need to analyze long
lengths of sequenced DNA, which is time and labor consuming;
and the inability to generate high-quality sequence just downstream of the sequencing primer hybridization site.
Another type of DNA sequencing that has been more recently
described is sequencing by synthesis, or pyrosequencing [72,
73]. This technology uses proprietary chemical reactions including several enzymes that generate light when a nucleotide
is incorporated into the strand of DNA that is being synthesized. The amount of this light is in proportion to the pyrophosphate released during nucleotide incorporation. For example, if 2 of the same nucleotides (e.g., …GG) are
S106 • CID 2007:45 (Suppl 2) • Procop
Figure 3. Reverse-hybridization test demonstrating a single postamplification assay that could be used to differentiate the majority of mycobacteria that are clinically encountered. Here, all of the hybridization
sites are developed. The only lines that would be present if a clinical
specimen or a positive culture sample were tested that contained a single
species of mycobacteria would be the lines that correspond to the species,
the Mycobacterium genus, and the conjugate control position. The abbreviations on the left side of the reverse hybridization strip correlate
with the species or groups on the right side of the strip. MAIS,
Mycobacterium avium–M. intracellulare–M. scrofulaceum; MTB, M.
tuberculosis.
incorporated, then the light generated will be twice that generated by the incorporation of a single nucleotide (e.g., …G).
The instrument records the dispensation of the nucleotides and
the light generated. Nucleotides that are dispensed but not
incorporated are hydrolyzed by apyrase, one of the enzymes in
the mixture. Hereby, the DNA sequence is determined as it is
being synthesized. The strengths of this technology are that it
is user friendly, the reactions occur in the commonly used 96well plate, and the sequences immediately downstream of the
sequencing primer are readily determined, which is ideal for
SNP analysis. The limitations of this approach include the cost
of the instrumentation and reagents, its limited ability to appropriately determine the sequence of homopolymers (i.e., numerous nucleotides of the same type occurring one after another), and the length of high-quality DNA sequence that may
be generated, which is !100 nt and frequently is !50 nt. As
noted, this technology is particularly useful for SNP analysis,
because the assessment of only a limited number of nucleotides
is necessary. In addition, this technology, although perhaps not
as discriminating as Sanger sequencing, may be used in the
taxonomic categorization of clinically relevant microorganisms.
For this application, it is necessary to determine the sequence
of variable regions that contain unique or “signature” sequences
for microorganisms within a group. This technology has been
used to appropriately categorize mycobacteria and nocardiae
into clinically important groups (e.g., M. tuberculosis complex)
[74]. In our experience at the Cleveland Clinic, this technique
of identifying mycobacteria was less expensive than our traditional approach that utilized genetic probes and biochemical
reactions (data not shown). It has been used to identify yeasts
(figure 4), filamentous fungi, and a variety of bacteria [75, 76].
It has also been used to differentiate the BK and JC polyoma
viruses, as well as HPV subtypes [77–79].
Regardless of the type of sequencing technology used, both
the laboratorian performing the sequencing and the ordering
clinician must be aware of the strengths and limitations of these
assays. In addition, the identifications achieved using these
methods are only as sound as the entries in the genetic databases
that are being queried. Genetic database entries should be based
on isolates that have been soundly identified using traditional
methods (if feasible) and should be of good sequence quality.
Unfortunately, this is not always the case. Finally, an adequate
number of well-identified isolates from each species should be
examined, so that normal genetic variation may be understood
and incorporated into identification schema (i.e., some variation may reflect only strain variation, whereas other variation
is taxonomically meaningful for the differentiation of species).
The bottom line is that the sequence-based identification of
microorganisms is only as good as the sequences in the genetic
library used and the characterization of the strains from which
these sequences were derived.
Microarray technology. Microarray technology has proven
an important tool for discovery [80–82]. These platforms may
be used to assay for numerous (i.e., hundreds) of targets simultaneously and have been a critical means of studying the
expression of multiple genes simultaneously. This technology,
regardless of its technical format, consists of numerous individual probe-target hybridization reactions that are assayed for
simultaneously. Not surprisingly, this technology has been used
to differentiate numerous microbial pathogens, study their expression profiles, and detect the genetic determinants of drug
resistance [83–86]. For example, soon after its introduction, it
was shown to be capable of differentiating medically important
mycobacteria and detecting the most common causes of resistance to antimycobacterial drugs [87, 88]. Unfortunately, the
limitations of this technology, such as cost, inconsistent reactions in some systems, and data management and interpretation, have surpassed its practical uses. From a practical standpoint, simply more data are generated from a single, large array
than are necessary in clinical medicine, and the cost of the array
is prohibitive for routine use. Fortunately, this is changing. One
Figure 4. Pyrogram (i.e., tracing derived from pyrosequencing) of a 40-bp region that has been used for the identification of the most commonly
occurring yeast species. The sizes of the peaks are measurements of the amount of light generated, which correlates to the amount of pyrophosphate
generated during nucleotide incorporation in the process of DNA synthesis. C. parapsilosis, Candida parapsilosis.
Molecular Diagnostics • CID 2007:45 (Suppl 2) • S107
Figure 5. Limited or small-scale bioelectric microarray demonstrating the feasibility of this type of technology to differentiate most of the clinically
important mycobacteria. A Mycobacterium genus site is located on the far side of the microarray, whereas the remainder is occupied by speciesspecific or complex-specific (e.g., Mycobacterium tuberculosis complex) hybridization sites. ITS, internal transcribed spacer region.
of the most promising areas in molecular diagnostics is the use
of limited microarrays for the assessment of numerous genetic
targets after either a multiplex reaction or a broad-range PCR.
A number of different microarray platforms have been described, including bioelectric arrays and liquid microarrays. As
with any technology, there are strengths and limitations with
each system. These systems used today have improved hybridization and reporting for the individual probe/target combinations (i.e., they have been made more reliable), and, perhaps
most importantly, some have been scaled down from large
platforms to smaller-sized versions that address clinically important issues that require multiple results. In clinical microbiology, a microarray with 20–30 hybridization sites would be
sufficient to address the most common causes of bacteremia
and fungemia (i.e., the most common causes of positive blood
culture results), and similarly sized arrays could be used to
S108 • CID 2007:45 (Suppl 2) • Procop
identify the typical causes of invasive fungal and mycobacterial
infections (figure 5). This approach is optimized with the inclusion of broad family or kingdom probes. For example, a
Raoutella species is an uncommon cause of bacteremia, and
genus- or species-level probe sites are not likely to be included
in a “bacteremia” microarray. The presence of an uncommon
bacterium such as this in the blood culture sample could be
detected through the inclusion of a broad-range bacterial probe
site on the microarray. Hybridization at this site, with the absence of hybridization at a species-determining site, would
demonstrate the presence of a bacterium other than any of the
more common causes of bacteremia that are included in the
panel.
The possibility of producing disease-specific microassays also
exists, because these formats may be used after broad-range
PCR, multiplex PCR, or a combination of such assays. For
example, one could envision a microarray that could distinguish between the various causes of meningoencephalitis. Such
an assay may include a complex multiplex reaction that consisted of a broad-range bacterial PCR, a species-specific PCR
for Cryptococcus neoformans, a broad-range RT-PCR for the
enteroviruses, and a specific PCR for HSV. Although not exhaustive, such an array would, in a single postamplification
assay, address all the most common etiologic agents causing
meningoencephalitis. Furthermore, additional probe sites could
be added, as needed. For example, a West Nile virus site could
be added, if desired. Limited microarrays, from a business perspective, represent a potentially disruptive technology, because
the widespread use of these could change the way that we
practice microbiology. However, their use will depend on numerous factors, such as the development of useful products,
cost, Food and Drug Administration approval, reimbursement,
and, most importantly, their performance in the clinical laboratory and their impact on patient care.
In summary, molecular methods are changing the way that
we practice laboratory medicine and thereby are affecting the
practice of clinical medicine. New methods of direct hybridization, rapid-cycle homogeneous PCR, and a variety of postamplification methods of analysis are making the genetic-based
identification and characterization of pathogenic microorganisms more rapid and, in many instances, more accurate than
ever before. However, for each of these technologies, the additions to health care costs must be weighed against the potential advantages of more rapid diagnostics. The techniques
and the technologies are here. Well-controlled outcome studies are now needed to demonstrate the efficacy of these
technologies.
Acknowledgments
Supplement sponsorship. This article was published as part of a supplement entitled “Annual Conference on Antimicrobial Resistance,” sponsored by the National Foundation for Infectious Diseases.
Potential conflicts of interest. G.W.P. is a scientific advisor for Luminex, AdvanDX, Roche, Cepheid, bioMerieux, and BD GeneOhm and
receives royalties for licensing from Biotage and BD GeneOhm.
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