SUPPLEMENT ARTICLE

Molecular Diagnostics for the Detection

and Characterization of Microbial Pathogens
Gary W. Procop

Downloaded from https://academic.oup.com/cid/article/45/Supplement_2/S99/285931 by guest on 03 August 2021

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 im-
plemented in any microbiology laboratory [3]. In ad-
dition to PCR, there are numerous competitive tech-
nologies that have been developed and shown to be
useful for the detection and characterization of micro-
organisms. Many of these more recently developed as-
says 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 (gprocop@med.miami.edu).
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 appro-
priately. The abuse of this technology has serious im-
plications 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 Plasmo-
dium 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 postamplifi-
cation analysis. These methods may be used to either detect or
exclude particular pathogens or, more usefully, to assess a spec-
imen or positive culture sample for a wide variety of micro-
organisms. 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 anti-
microbial therapy. In addition to benefiting the patients, this
may save health care dollars through the pharmacy by iden-
tifying 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 echi-
nocandin to a less expensive azole. The use of narrower-spec-
trum agents, in contrast to the use of broad-spectrum agents,
should also slow the selection for and spread of antimicrobial-
resistant 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 iden-
tification 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 hybrid-
ization will predominate this discussion, because it is the newest
signal amplification technique to be used in the clinical mi-
crobiology 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 do-
mains 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 mi-
croorganism 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
S100 • CID 2007:45 (Suppl 2) • Procop
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 in-
troduced 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
Downloaded from https://academic.oup.com/cid/article/45/Supplement_2/S99/285931 by guest on 03 August 2021
direct clinical specimens and in histologic sections for the de-
tection of microorganisms [8, 12, 14]. Regardless of the type
of direct hybridization technology used, identifications using
signal hybridizations are most accurately obtained after micro-
organism growth or biological amplification of the organism.
This technology is often used to differentiate microorganisms
of similar morphotypes in positive culture samples (e.g., gram-
positive cocci in clusters or acid-fast bacilli). One of the ad-
vantages of this technology is that it may be used in conjunction
with traditional methods, such as Gram staining. The infor-
mation 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 gram-
positive 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 staphylo-
cocci. 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 ol-
igonucleotide 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 di-
agnostics, 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 mol-
ecule. 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 nu-
cleotide 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 microor-
ganisms 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 microor-
ganism undergoing FISH analysis is intact, and in distinct con-
trast 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 tar-
gets (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 avail-
able 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, respec-
tively [7, 9–11, 26, 30]. In addition to the commercially avail-
able applications, several studies have demonstrated the feasi-
bility 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 diag-
nostic tool that may be introduced into laboratories without
previous experience in molecular microbiology. They have al-
ready 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 micro-
biology laboratory could be used in in situ hybridization re-
actions 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 mor-
phologically similar microorganisms. They showed the feasi-
bility of this technology to detect Legionella pneumophila and
Downloaded from https://academic.oup.com/cid/article/45/Supplement_2/S99/285931 by guest on 03 August 2021
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 im-
mitis), from Candida and Cryptococcus species. Although it may
seem a morphologically simple task to differentiate a well-de-
veloped spherule of C. immitis from budding yeast, it is con-
siderably more complicated when intact spherules are not seen
and, rather, only immature spherules and endospores are pres-
ent. In situ hybridization has also been used to identify Pneu-
mocystis jiroveci [36]. Finally, one can imagine an economic
benefit when the same probe sets are used for both microbi-
ology and infectious disease pathology applications. For ex-
ample, 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 cyto-
meters that can detect microorganisms have been developed
and are routinely used in the food and pharmaceutical indus-
tries. 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 in-
formation. However, it has been demonstrated that this tech-
nology can be used to detect microorganisms to which in situ
probes are hybridized [37, 38]. This combination of technol-
ogies affords the replacement of human interpretation, which
is subjective and requires advanced training and experience to
perfect, with objective, quantitative measures that are per-
formed 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 demon-
strated that the identification of the most common microor-
Molecular 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 Gen-
Probe 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 lab-
oratories 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 prod-
ucts 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 identifi-
cation of the common causes of bacteremia and fungemia [26,
42, 43]. There have, however, been some hints that this com-
pany 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 sub-
types, is also a signal amplification technology [44]. This tech-
nology has also been used for the detection of cytomegalovirus,
hepatitis B virus, Neisseria gonorrhoeae, and Chlamydia tra-
chomatis [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 spec-
ificity 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 al-
ternate technologies, such as strand-displacement amplification
and transcription-mediated amplification (i.e., nucleic acid se-
quence–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 reac-
tions that detect a single target and provide a “present or ab-
sent”–type result. For example, a Legionella pneumophila–spe-
cific PCR could be used to rapidly detect this pathogen. If used
S102 • CID 2007:45 (Suppl 2) • Procop
in conjunction with quantitative standards, these could be used
to provide quantitative information (e.g., viral loads). The com-
bination 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 limita-
tions of such assays. Several multiplex (and monoplex) assays
are commercially available as “analyte-specific reagents.” These
are commercial products that contain all the necessary com-
ponents to conduct a particular amplification reaction, which
Downloaded from https://academic.oup.com/cid/article/45/Supplement_2/S99/285931 by guest on 03 August 2021
are manufactured under Good Manufacturing Practice con-
ditions, 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 differenti-
ations the most important respiratory viral pathogens—influ-
enza A, influenza B, and respiratory syncytial virus. The real-
time 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 micro-
organisms 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 stan-
dards 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 am-
plification assays comes from the information that may be de-
rived from the amplified product (i.e., the amplicon) through
postamplification analysis. There are many methods of post-
amplification analysis, but only a few will be briefly discussed
here, given the scope of the present article. These are postam-
plification melt curve analysis, reverse hybridization, DNA se-
quencing (traditional and pyrosequencing), and solid- and liq-
uid-phase microarray analysis.
It seems that advances in laboratory diagnostics more com-
monly 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 Darwinian-
type 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 path-
ogens. This is because traditional PCR is cumbersome and slow,
especially in light of the time and process necessary to specif-
ically determine the nature of the amplified product by South-
ern blot analysis. However, all this changed with the advent of
real-time PCR, also known as “homogeneous, rapid cycle nu-
cleic acid amplification.”
The advent of real-time PCR awaited complementary ad-
vances 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 tradi-
tional 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, al-
though 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 par-
ticular 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 appli-
cations, 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 re-
actions 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. Main-
taining a closed system is important in a clinical microbiology
laboratory, because chemical contamination of the environ-
ment 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 electro-
magnetic 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 mole-
cule, while PCR is under way (thus, in real time), demonstrates
to the user that amplification is occurring. Whether this am-
plification 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 oligo-
nucleotide probes available. The 3 most commonly used types
are the hydrolysis or “TaqMan” probes, molecular beacons, and
fluorescence resonance energy transfer (FRET) probes. A pos-
itive real-time PCR result for any of these probes is demon-
strated 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)
Downloaded from https://academic.oup.com/cid/article/45/Supplement_2/S99/285931 by guest on 03 August 2021
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 de-
tection, 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 spec-
ificity 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 oligo-
nucleotide 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 broad-
range 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 epi-
demiologic purposes (e.g., the mecA cassette IV associated with
community-acquired Staphylococcus aureus infection), and the
detection of virulence factors (e.g., Panton-Valentine leukoci-
din). Although useful for the detection of certain mechanisms
of resistance, nucleic acid amplification assays for more com-
plicated and genetically diverse mechanisms of antimicrobial
resistance (e.g., the extended-spectrum b-lactamases) have re-
mained elusive and are not likely to replace traditional phe-
notypic 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
Molecular Diagnostics • CID 2007:45 (Suppl 2) • S103

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 post-
amplification 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 nu-
cleotide 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 partic-
ular melting temperature is expected for the hybridization be-
tween 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
S104 • CID 2007:45 (Suppl 2) • Procop
Downloaded from https://academic.oup.com/cid/article/45/Supplement_2/S99/285931 by guest on 03 August 2021
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 be-
tween the probe and target sequences would be detected, be-
cause the probe could not hybridize to its target sequence with
the optimal affinity and would “melt off” at a lower temper-
ature. 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 ad-
ditional data concerning the specific nature of the amplified
DNA product after real-time PCR.
This difference in melt curves created by imperfect comple-
mentarity between probes and their target hybridization sites
may be exploited to differentiate taxonomically related micro-
organisms 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 applica-
tion is quite useful, but there is some evidence that there may
be diminished sensitivity for the target that has the lower melt-
ing temperature (i.e., the imperfect complementarity) [63].
Therefore, it would be prudent to not use this type of appli-
cation 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 in-
stance, 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 hybrid-
ization 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. tu-
berculosis. 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 prod-
uct into the areas of master mix preparation or specimen prep-
aration, 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
Downloaded from https://academic.oup.com/cid/article/45/Supplement_2/S99/285931 by guest on 03 August 2021
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 assess-
ments can be performed simultaneously or in a single reaction
than if they are performed in numerous monoplex amplifi-
cation reactions. For example, it is necessary to assay for nu-
merous high-risk HPVs to appropriately diagnose or exclude
a high-risk HPV infection in a woman. It is preferable to per-
form 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 differ-
entiation 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 mul-
tiplex or a broad-range amplification reaction, is through re-
verse 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,
Molecular Diagnostics • CID 2007:45 (Suppl 2) • S105
it may be visualized through the application of a color devel-
opment 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.

Downloaded from https://academic.oup.com/cid/article/45/Supplement_2/S99/285931 by guest on 03 August 2021

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 deter-
mined. 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 com-
puter-assisted sequence analysis, afford the expanded use of
this technology to many more laboratories. Traditionally, se-
quencing 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 Figure 3. Reverse-hybridization test demonstrating a single postam-
plification assay that could be used to differentiate the majority of my-
against a genetic database that contains thousands of sequences.
cobacteria that are clinically encountered. Here, all of the hybridization
The results of a query are returned as a percentage match, which sites are developed. The only lines that would be present if a clinical
assists the molecular microbiologist with the identification of specimen or a positive culture sample were tested that contained a single
the microorganisms. There is clearly great promise for this species of mycobacteria would be the lines that correspond to the species,
approach, and commercial products (e.g., MicroSeq [Applied the Mycobacterium genus, and the conjugate control position. The ab-
breviations on the left side of the reverse hybridization strip correlate
Biosystems]) and nonpublic databases (e.g., SmartGene) have
with the species or groups on the right side of the strip. MAIS,
been established. Full-length sequencing is particularly pow- Mycobacterium avium–M. intracellulare–M. scrofulaceum; MTB, M.
erful when several nucleotide polymorphisms are needed for tuberculosis.
the characterization of a microbe and these are distant from
one another within the amplicon (e.g., some of the mutations incorporated, then the light generated will be twice that gen-
associated with HIV resistance). The drawbacks associated with erated by the incorporation of a single nucleotide (e.g., …G).
this technology would include the cost of the equipment, re- The instrument records the dispensation of the nucleotides and
agents, and access to private databases; the need to analyze long the light generated. Nucleotides that are dispensed but not
lengths of sequenced DNA, which is time and labor consuming; incorporated are hydrolyzed by apyrase, one of the enzymes in
and the inability to generate high-quality sequence just down- the mixture. Hereby, the DNA sequence is determined as it is
stream of the sequencing primer hybridization site. being synthesized. The strengths of this technology are that it
Another type of DNA sequencing that has been more recently is user friendly, the reactions occur in the commonly used 96-
described is sequencing by synthesis, or pyrosequencing [72, well plate, and the sequences immediately downstream of the
73]. This technology uses proprietary chemical reactions in- sequencing primer are readily determined, which is ideal for
cluding several enzymes that generate light when a nucleotide SNP analysis. The limitations of this approach include the cost
is incorporated into the strand of DNA that is being synthe- of the instrumentation and reagents, its limited ability to ap-
sized. The amount of this light is in proportion to the pyro- propriately determine the sequence of homopolymers (i.e., nu-
phosphate released during nucleotide incorporation. For ex- merous nucleotides of the same type occurring one after an-
ample, if 2 of the same nucleotides (e.g., …GG) are other), and the length of high-quality DNA sequence that may

S106 • CID 2007:45 (Suppl 2) • Procop

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 tra-
ditional 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
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.
examined, so that normal genetic variation may be understood
and incorporated into identification schema (i.e., some varia-
tion 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 si-
Downloaded from https://academic.oup.com/cid/article/45/Supplement_2/S99/285931 by guest on 03 August 2021
multaneously and have been a critical means of studying the
expression of multiple genes simultaneously. This technology,
regardless of its technical format, consists of numerous indi-
vidual probe-target hybridization reactions that are assayed for
simultaneously. Not surprisingly, this technology has been used
to differentiate numerous microbial pathogens, study their ex-
pression 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 resis-
tance to antimycobacterial drugs [87, 88]. Unfortunately, the
limitations of this technology, such as cost, inconsistent reac-
tions in some systems, and data management and interpreta-
tion, have surpassed its practical uses. From a practical stand-
point, 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
Molecular Diagnostics • CID 2007:45 (Suppl 2) • S107
 Downloaded from https://academic.oup.com/cid/article/45/Supplement_2/S99/285931 by guest on 03 August 2021
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 species-
specific 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 de-
scribed, including bioelectric arrays and liquid microarrays. As
with any technology, there are strengths and limitations with
each system. These systems used today have improved hybrid-
ization and reporting for the individual probe/target combi-
nations (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 im-
portant issues that require multiple results. In clinical micro-
biology, 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
identify the typical causes of invasive fungal and mycobacterial
infections (figure 5). This approach is optimized with the in-
clusion 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 ab-
sence 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

S108 • CID 2007:45 (Suppl 2) • Procop

example, one could envision a microarray that could distin-
guish between the various causes of meningoencephalitis. Such
an assay may include a complex multiplex reaction that con-
sisted 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 ex-
haustive, 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 per-
spective, 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 nu-
merous factors, such as the development of useful products,
cost, Food and Drug Administration approval, reimbursement,
and, most importantly, their performance in the clinical lab-
oratory 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 hybridi-
zation, rapid-cycle homogeneous PCR, and a variety of pos-
tamplification methods of analysis are making the genetic-based
identification and characterization of pathogenic microorgan-
isms more rapid and, in many instances, more accurate than
ever before. However, for each of these technologies, the ad-
ditions to health care costs must be weighed against the po-
tential advantages of more rapid diagnostics. The techniques
and the technologies are here. Well-controlled outcome stud-
ies are now needed to demonstrate the efficacy of these
technologies.
Acknowledgments
Supplement sponsorship. This article was published as part of a sup-
plement entitled “Annual Conference on Antimicrobial Resistance,” spon-
sored by the National Foundation for Infectious Diseases.
Potential conflicts of interest. G.W.P. is a scientific advisor for Lu-
minex, AdvanDX, Roche, Cepheid, bioMerieux, and BD GeneOhm and
receives royalties for licensing from Biotage and BD GeneOhm.
References
1. Mullis K, Faloona F, Scharf S, Saiki R, Horn G, Erlich H. Specific
enzymatic amplification of DNA in vitro: the polymerase chain reac-
tion. 1986. Biotechnology 1992; 24:17–27.
2. Shampo MA, Kyle RA. Kary B. Mullis—Nobel Laureate for procedure
to replicate DNA. Mayo Clin Proc 2002; 77:606.
3. Espy MJ, Uhl JR, Sloan LM, et al. Real-time PCR in clinical micro-
biology: applications for routine laboratory testing. Clin Microbiol Rev
2006; 19:165–256.
4. Alexander BD, Ashley ED, Reller LB, Reed SD. Cost savings with im-
plementation of PNA FISH testing for identification of Candida al-
bicans in blood cultures. Diagn Microbiol Infect Dis 2006; 54:277–82.
5. Forrest GN, Mankes K, Jabra-Rizk MA, et al. Peptide nucleic acid
fluorescence in situ hybridization-based identification of Candida al-
bicans and its impact on mortality and antifungal therapy costs. J Clin
Microbiol 2006; 44:3381–3.
6. Ramers C, Billman G, Hartin M, Ho S, Sawyer MH. Impact of a
diagnostic cerebrospinal fluid enterovirus polymerase chain reaction
test on patient management. JAMA 2000; 283:2680–5.
7. Oliveira K, Brecher SM, Durbin A, et al. Direct identification of Staph-
ylococcus aureus from positive blood culture bottles. J Clin Microbiol
2003; 41:889–91.
8. Radwanska M, Magez S, Perry-O’Keefe H, et al. Direct detection and
identification of African trypanosomes by fluorescence in situ hybrid-
ization with peptide nucleic acid probes. J Clin Microbiol 2002; 40:
4295–7.
9. Rigby S, Procop GW, Haase G, et al. Fluorescence in situ hybridization
with peptide nucleic acid probes for rapid identification of Candida
albicans directly from blood culture bottles. J Clin Microbiol 2002; 40:
2182–6.
Downloaded from https://academic.oup.com/cid/article/45/Supplement_2/S99/285931 by guest on 03 August 2021
10. Oliveira K, Procop GW, Wilson D, Coull J, Stender H. Rapid identi-
fication of Staphylococcus aureus directly from blood cultures by fluo-
rescence in situ hybridization with peptide nucleic acid probes. J Clin
Microbiol 2002; 40:247–51.
11. Oliveira K, Haase G, Kurtzman C, Hyldig-Nielsen JJ, Stender H. Dif-
ferentiation of Candida albicans and Candida dubliniensis by fluores-
cent in situ hybridization with peptide nucleic acid probes. J Clin
Microbiol 2001; 39:4138–41.
12. Hayden RT, Uhl JR, Qian X, et al. Direct detection of Legionella species
from bronchoalveolar lavage and open lung biopsy specimens: com-
parison of LightCycler PCR, in situ hybridization, direct fluorescence
antigen detection, and culture. J Clin Microbiol 2001; 39:2618–26.
13. Kempf VA, Trebesius K, Autenrieth IB. Fluorescent in situ hybridization
allows rapid identification of microorganisms in blood cultures. J Clin
Microbiol 2000; 38:830–8.
14. Hogardt M, Trebesius K, Geiger AM, Hornef M, Rosenecker J, Heese-
mann J. Specific and rapid detection by fluorescent in situ hybridization
of bacteria in clinical samples obtained from cystic fibrosis patients. J
Clin Microbiol 2000; 38:818–25.
15. Jansen GJ, Mooibroek M, Idema J, Harmsen HJ, Welling GW, Degener
JE. Rapid identification of bacteria in blood cultures by using fluo-
rescently labeled oligonucleotide probes. J Clin Microbiol 2000; 38:
814–7.
16. Drobniewski FA, More PG, Harris GS. Differentiation of Mycobacte-
rium tuberculosis complex and nontuberculous mycobacterial liquid
cultures by using peptide nucleic acid-fluorescence in situ hybridization
probes. J Clin Microbiol 2000; 38:444–7.
17. Scarparo C, Piccoli P, Rigon A, Ruggiero G, Nista D, Piersimoni C.
Direct identification of mycobacteria from MB/BacT alert 3D bottles:
comparative evaluation of two commercial probe assays. J Clin Mi-
crobiol 2001; 39:3222–7.
18. Louro AP, Waites KB, Georgescu E, Benjamin WH Jr. Direct identi-
fication of Mycobacterium avium complex and Mycobacterium gordonae
from MB/BacT bottles using AccuProbe. J Clin Microbiol 2001; 39:
570–3.
19. Alcaide F, Benitez MA, Escriba JM, Martin R. Evaluation of the BAC-
TEC MGIT 960 and the MB/BacT systems for recovery of mycobacteria
from clinical specimens and for species identification by DNA
AccuProbe. J Clin Microbiol 2000; 38:398–401.
20. Beggs ML, Stevanova R, Eisenach KD. Species identification of My-
cobacterium avium complex isolates by a variety of molecular tech-
niques. J Clin Microbiol 2000; 38:508–12.
21. Richter E, Niemann S, Rusch-Gerdes S, Hoffner S. Identification of
Mycobacterium kansasii by using a DNA probe (AccuProbe) and mo-
lecular techniques. J Clin Microbiol 1999; 37:964–70.
22. Hesselink AT, Bulkmans NW, Berkhof J, Lorincz AT, Meijer CJ, Snijders
PJ. Cross-sectional comparison of an automated hybrid capture 2 assay
and the consensus GP5+/6+ PCR method in a population-based cer-
vical screening program. J Clin Microbiol 2006; 44:3680–5.
23. Hui CK, Bowden S, Zhang HY, et al. Comparison of real-time PCR
assays for monitoring serum hepatitis B virus DNA levels during an-
tiviral therapy. J Clin Microbiol 2006; 44:2983–7.
24. Chemaly RF, Yen-Lieberman B, Castilla EA, et al. Correlation between
Molecular Diagnostics • CID 2007:45 (Suppl 2) • S109
 viral loads of cytomegalovirus in blood and bronchoalveolar lavage
specimens from lung transplant recipients determined by histology and
immunohistochemistry. J Clin Microbiol 2004; 42:2168–72.
25. Chan HL, Leung NW, Lau TC, Wong ML, Sung JJ. Comparison of
three different sensitive assays for hepatitis B virus DNA in monitoring
of responses to antiviral therapy. J Clin Microbiol 2000; 38:3205–8.
26. Chapin K, Musgnug M. Evaluation of three rapid methods for the
direct identification of Staphylococcus aureus from positive blood cul-
tures. J Clin Microbiol 2003; 41:4324–7.
27. Lefmann M, Schweickert B, Buchholz P, et al. Evaluation of peptide
nucleic acid-fluorescence in situ hybridization for identification of clin-
ically relevant mycobacteria in clinical specimens and tissue sections.
J Clin Microbiol 2006; 44:3760–7.
28. Peters RP, van Agtmael MA, Simoons-Smit AM, Danner SA, Vanden-
broucke-Grauls CM, Savelkoul PH. Rapid identification of pathogens
in blood cultures with a modified fluorescence in situ hybridization
assay. J Clin Microbiol 2006; 44:4186–8.
29. Peters RP, Savelkoul PH, Simoons-Smit AM, Danner SA, Vanden-
broucke-Grauls CM, van Agtmael MA. Faster identification of path-
ogens in positive blood cultures by fluorescence in situ hybridization
in routine practice. J Clin Microbiol 2006; 44:119–23.
30. Wilson DA, Joyce MJ, Hall LS, et al. Multicenter evaluation of a Can-
dida albicans peptide nucleic acid fluorescent in situ hybridization
probe for characterization of yeast isolates from blood cultures. J Clin
Microbiol 2005; 43:2909–12.
31. Porcheddu A, Giacomelli G. Peptide nucleic acids (PNAs), a chemical
overview. Curr Med Chem 2005; 12:2561–99.
32. Stender H. PNA FISH: an intelligent stain for rapid diagnosis of in-
fectious diseases. Expert Rev Mol Diagn 2003; 3:649–55.
33. Hayden RT, Isotalo PA, Parrett T, et al. In situ hybridization for the
differentiation of Aspergillus, Fusarium, and Pseudallescheria species in
tissue section. Diagn Mol Pathol 2003; 12:21–6.
34. Hayden RT, Qian X, Procop GW, Roberts GD, Lloyd RV. In situ hy-
bridization for the identification of filamentous fungi in tissue section.
Diagn Mol Pathol 2002; 11:119–26.
35. Hayden RT, Qian X, Roberts GD, Lloyd RV. In situ hybridization for
the identification of yeastlike organisms in tissue section. Diagn Mol
Pathol 2001; 10:15–23.
36. Kobayashi M, Urata T, Ikezoe T, et al. Simple detection of the 5S
ribosomal RNA of Pneumocystis carinii using in situ hybridisation. J
Clin Pathol 1996; 49:712–6.
37. Hartmann H, Stender H, Schafer A, Autenrieth IB, Kempf VA. Rapid
identification of Staphylococcus aureus in blood cultures by a combi-
nation of fluorescence in situ hybridization using peptide nucleic acid
probes and flow cytometry. J Clin Microbiol 2005; 43:4855–7.
38. Kempf VA, Mandle T, Schumacher U, Schafer A, Autenrieth IB. Rapid
detection and identification of pathogens in blood cultures by fluo-
rescence in situ hybridization and flow cytometry. Int J Med Microbiol
2005; 295:47–55.
39. Badak FZ, Goksel S, Sertoz R, et al. Use of nucleic acid probes for
identification of Mycobacterium tuberculosis directly from MB/BacT
bottles. J Clin Microbiol 1999; 37:1602–5.
40. Chemaly RF, Tomford JW, Hall GS, Sholtis M, Chua JD, Procop GW.
Rapid diagnosis of Histoplasma capsulatum endocarditis using the
AccuProbe on an excised valve. J Clin Microbiol 2001; 39:2640–1.
41. McNabb A, Adie K, Rodrigues M, Black WA, Isaac-Renton J. Direct
identification of mycobacteria in primary liquid detection media by
partial sequencing of the 65-kilodalton heat shock protein gene. J Clin
Microbiol 2006; 44:60–6.
42. Davis TE, Fuller DD. Direct identification of bacterial isolates in blood
cultures by using a DNA probe. J Clin Microbiol 1991; 29:2193–6.
43. Lindholm L, Sarkkinen H. Direct identification of gram-positive cocci
from routine blood cultures by using AccuProbe tests. J Clin Microbiol
2004; 42:5609–13.
44. Koliopoulos G, Arbyn M, Martin-Hirsch P, Kyrgiou M, Prendiville W,
Paraskevaidis E. Diagnostic accuracy of human papillomavirus testing
S110 • CID 2007:45 (Suppl 2) • Procop
in primary cervical screening: a systematic review and meta-analysis
of non-randomized studies. Gynecol Oncol 2007; 104:232–46.
45. Ho SK, Li FK, Lai KN, Chan TM. Comparison of the CMV Brite Turbo
assay and the Digene Hybrid Capture CMV DNA (version 2.0) assay
for quantitation of cytomegalovirus in renal transplant recipients. J
Clin Microbiol 2000; 38:3743–5.
46. Konnick EQ, Erali M, Ashwood ER, Hillyard DR. Evaluation of the
COBAS amplicor HBV monitor assay and comparison with the ultra-
sensitive HBV hybrid capture 2 assay for quantification of hepatitis B
virus DNA. J Clin Microbiol 2005; 43:596–603.
47. Van Der Pol B, Williams JA, Smith NJ, et al. Evaluation of the Digene
Hybrid Capture II Assay with the Rapid Capture System for detection
of Chlamydia trachomatis and Neisseria gonorrhoeae. J Clin Microbiol
2002; 40:3558–64.
Downloaded from https://academic.oup.com/cid/article/45/Supplement_2/S99/285931 by guest on 03 August 2021
48. Sandri MT, Lentati P, Benini E, et al. Comparison of the Digene HC2
assay and the Roche AMPLICOR human papillomavirus (HPV) test
for detection of high-risk HPV genotypes in cervical samples. J Clin
Microbiol 2006; 44:2141–6.
49. Hindiyeh M, Hillyard DR, Carroll KC. Evaluation of the Prodesse
Hexaplex multiplex PCR assay for direct detection of seven respiratory
viruses in clinical specimens. Am J Clin Pathol 2001; 116:218–24.
50. Kehl SC, Henrickson KJ, Hua W, Fan J. Evaluation of the Hexaplex
assay for detection of respiratory viruses in children. J Clin Microbiol
2001; 39:1696–701.
51. Rosey AL, Abachin E, Quesnes G, et al. Development of a broad-range
16S rDNA real-time PCR for the diagnosis of septic arthritis in children.
J Microbiol Methods 2007; 68:88–93.
52. Zucol F, Ammann RA, Berger C, et al. Real-time quantitative broad-
range PCR assay for detection of the 16S rRNA gene followed by
sequencing for species identification. J Clin Microbiol 2006; 44:2750–9.
53. Shrestha NK, Tuohy MJ, Hall GS, Reischl U, Gordon SM, Procop GW.
Detection and differentiation of Mycobacterium tuberculosis and non-
tuberculous mycobacterial isolates by real-time PCR. J Clin Microbiol
2003; 41:5121–6.
54. Sandhu GS, Kline BC, Stockman L, Roberts GD. Molecular probes for
diagnosis of fungal infections. J Clin Microbiol 1995; 33:2913–9.
55. Gould SJ. Tempo and mode in the macroevolutionary reconstruction
of Darwinism. Proc Natl Acad Sci U S A 1994; 91:6764–71.
56. Gould SJ, Eldredge N. Punctuated equilibrium comes of age. Nature
1993; 366:223–7.
57. Borst A, Box AT, Fluit AC. False-positive results and contamination
in nucleic acid amplification assays: suggestions for a prevent and
destroy strategy. Eur J Clin Microbiol Infect Dis 2004; 23:289–99.
58. Wilson DA, Yen-Lieberman B, Reischl U, Gordon SM, Procop GW.
Detection of Legionella pneumophila by real-time PCR for the mip
gene. J Clin Microbiol 2003; 41:3327–30.
59. Farrell JJ, Doyle LJ, Addison RM, Reller LB, Hall GS, Procop GW.
Broad-range (pan) Salmonella and Salmonella serotype Typhi-specific
real-time PCR assays: potential tools for the clinical microbiologist.
Am J Clin Pathol 2005; 123:339–45.
60. Odell ID, Cloud JL, Seipp M, Wittwer CT. Rapid species identification
within the Mycobacterium chelonae-abscessus group by high-resolution
melting analysis of hsp65 PCR products. Am J Clin Pathol 2005; 123:
96–101.
61. Neoh SH, Brisco MJ, Firgaira FA, Trainor KJ, Turner DR, Morley AA.
Rapid detection of the factor V Leiden (1691 G 1 A) and haemo-
chromatosis (845 G 1 A) mutation by fluorescence resonance energy
transfer (FRET) and real time PCR. J Clin Pathol 1999; 52:766–9.
62. Whiley DM, Mackay IM, Sloots TP. Detection and differentiation of
human polyomaviruses JC and BK by LightCycler PCR. J Clin Micro-
biol 2001; 39:4357–61.
63. Stevenson J, Hymas W, Hillyard D. Effect of sequence polymorphisms
on performance of two real-time PCR assays for detection of herpes
simplex virus. J Clin Microbiol 2005; 43:2391–8.
64. Levi JE, Kleter B, Quint WG, et al. High prevalence of human pap-
illomavirus (HPV) infections and high frequency of multiple HPV
 genotypes in human immunodeficiency virus-infected women in Bra-
zil. J Clin Microbiol 2002; 40:3341–5.
65. Zekri AR, El-Din HM, Bahnassy AA, et al. TRUGENE sequencing
versus INNO-LiPA for sub-genotyping of HCV genotype-4. J Med Virol
2005; 75:412–20.
66. Sturmer M, Morgenstern B, Staszewski S, Doerr HW. Evaluation of
the LiPA HIV-1 RT assay version 1: comparison of sequence and hy-
bridization based genotyping systems. J Clin Virol 2002; 25(Suppl 3):
S65–72.
67. Suffys PN, da Silva Rocha A, de Oliveira M, et al. Rapid identification
of Mycobacteria to the species level using INNO-LiPA Mycobacteria,
a reverse hybridization assay. J Clin Microbiol 2001; 39:4477–82.
68. Wilson DA, Reischl U, Hall GS, Procop GW. Use of partial 16S rRNA
gene sequencing for the identification of Legionella pneumophila and
non-pneumophila Legionella spp. J Clin Microbiol 2007; 45:257–8.
69. Simmon KE, Croft AC, Petti CA. Application of SmartGene IDNS
software to partial 16S rRNA gene sequences for a diverse group of
bacteria in a clinical laboratory. J Clin Microbiol 2006; 44:4400–6.
70. Han XY, Pham AS, Tarrand JJ, Sood PK, Luthra R. Rapid and accurate
identification of mycobacteria by sequencing hypervariable regions of
the 16S ribosomal RNA gene. Am J Clin Pathol 2002; 118:796–801.
71. Hall L, Wohlfiel S, Roberts GD. Experience with the MicroSeq D2
large-subunit ribosomal DNA sequencing kit for identification of fil-
amentous fungi encountered in the clinical laboratory. J Clin Microbiol
2004; 42:622–6.
72. Ronaghi M. Pyrosequencing sheds light on DNA sequencing. Genome
Res 2001; 11:3–11.
73. Diggle MA, Clarke SC. Pyrosequencing: sequence typing at the speed
of light. Mol Biotechnol 2004; 28:129–37.
74. Tuohy MJ, Hall GS, Sholtis M, Procop GW. Pyrosequencing as a tool
for the identification of common isolates of Mycobacterium sp. Diagn
Microbiol Infect Dis 2005; 51:245–50.
75. Kobayashi N, Bauer TW, Togawa D, et al. A molecular gram stain using
broad range PCR and pyrosequencing technology: a potentially useful
tool for diagnosing orthopaedic infections. Diagn Mol Pathol 2005;
14:83–9.
76. Kobayashi N, Bauer TW, Tuohy MJ, et al. The comparison of pyro-
sequencing molecular Gram stain, culture, and conventional Gram
stain for diagnosing orthopaedic infections. J Orthop Res 2006; 24:
1641–9.
77. Beck RC, Kohn DJ, Tuohy MJ, Prayson RA, Yen-Lieberman B, Procop
GW. Detection of polyoma virus in brain tissue of patients with pro-
gressive multifocal leukoencephalopathy by real-time PCR and pyro-
sequencing. Diagn Mol Pathol 2004; 13:15–21.
78. Gharizadeh B, Oggionni M, Zheng B, et al. Type-specific multiple
sequencing primers: a novel strategy for reliable and rapid genotyping
of human papillomaviruses by pyrosequencing technology. J Mol Diagn
2005; 7:198–205.
79. Gharizadeh B, Kalantari M, Garcia CA, Johansson B, Nyren P. Typing
of human papillomavirus by pyrosequencing. Lab Invest 2001; 81:
673–9.
Downloaded from https://academic.oup.com/cid/article/45/Supplement_2/S99/285931 by guest on 03 August 2021
80. Chittur SV. DNA microarrays: tools for the 21st Century. Comb Chem
High Throughput Screen 2004; 7:531–7.
81. Mockler TC, Chan S, Sundaresan A, Chen H, Jacobsen SE, Ecker JR.
Applications of DNA tiling arrays for whole-genome analysis. Gen-
omics 2005; 85:1–15.
82. Brentani RR, Carraro DM, Verjovski-Almeida S, et al. Gene expression
arrays in cancer research: methods and applications. Crit Rev Oncol
Hematol 2005; 54:95–105.
83. Chen T. DNA microarrays--an armory for combating infectious dis-
eases in the new century. Infect Disord Drug Targets 2006; 6:263–79.
84. Garaizar J, Rementeria A, Porwollik S. DNA microarray technology: a
new tool for the epidemiological typing of bacterial pathogens? FEMS
Immunol Med Microbiol 2006; 47:178–89.
85. Cebula TA, Jackson SA, Brown EW, Goswami B, LeClerc JE. Chips
and SNPs, bugs and thugs: a molecular sleuthing perspective. J Food
Prot 2005; 68:1271–84.
86. Butcher PD. Microarrays for Mycobacterium tuberculosis. Tuberculosis
(Edinb) 2004; 84:131–7.
87. Fukushima M, Kakinuma K, Hayashi H, Nagai H, Ito K, Kawaguchi
R. Detection and identification of Mycobacterium species isolates by
DNA microarray. J Clin Microbiol 2003; 41:2605–15.
88. Chemlal K, Portaels F. Molecular diagnosis of nontuberculous my-
cobacteria. Curr Opin Infect Dis 2003; 16:77–83.
Molecular Diagnostics • CID 2007:45 (Suppl 2) • S111