molecular diagnostics for the detection and characterization of...

Molecular Diagnostics • CID 2007:45 (Suppl 2) • S99

S U P P L E M E N T A R T I C L E

Molecular Diagnostics for the Detectionand Characterization of Microbial Pathogens

Gary W. ProcopDepartment 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, Universityof Miami Miller School of Medicine, Jackson Memorial Hospital (R-5), 1611 NW12th 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$15.00DOI: 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

S100 • CID 2007:45 (Suppl 2) • Procop

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

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

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

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-


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

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

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)

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


Figure 1. Examples of real-time PCR amplification reactions. The negative control specimen has produced a flat line, whereas the positive controland other template-containing samples demonstrate an exponential increase in fluorescence. The reactions that become positive earlier (i.e., that havea 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

(95�C–98�C) 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

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 categorizedhere 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

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,


Figure 3. Reverse-hybridization test demonstrating a single postam-plification assay that could be used to differentiate the majority of my-cobacteria that are clinically encountered. Here, all of the hybridizationsites are developed. The only lines that would be present if a clinicalspecimen or a positive culture sample were tested that contained a singlespecies of mycobacteria would be the lines that correspond to the species,the Mycobacterium genus, and the conjugate control position. The ab-breviations on the left side of the reverse hybridization strip correlatewith the species or groups on the right side of the strip. MAIS,Mycobacterium avium–M. intracellulare–M. scrofulaceum; MTB, M.tuberculosis.

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.

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

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 pow-

erful 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, re-

agents, 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 down-

stream 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 in-

cluding several enzymes that generate light when a nucleotide

is incorporated into the strand of DNA that is being synthe-

sized. The amount of this light is in proportion to the pyro-

phosphate released during nucleotide incorporation. For ex-

ample, if 2 of the same nucleotides (e.g., …GG) are

incorporated, then the light generated will be twice that gen-

erated 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 96-

well 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 ap-

propriately determine the sequence of homopolymers (i.e., nu-

merous nucleotides of the same type occurring one after an-

other), and the length of high-quality DNA sequence that may


Figure 4. Pyrogram (i.e., tracing derived from pyrosequencing) of a 40-bp region that has been used for the identification of the most commonlyoccurring yeast species. The sizes of the peaks are measurements of the amount of light generated, which correlates to the amount of pyrophosphategenerated during nucleotide incorporation in the process of DNA synthesis. C. parapsilosis, Candida parapsilosis.

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

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-

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


Figure 5. Limited or small-scale bioelectric microarray demonstrating the feasibility of this type of technology to differentiate most of the clinicallyimportant 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


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 andreceives royalties for licensing from Biotage and BD GeneOhm.

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