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CLINICAL MICROBIOLOGY REVIEWS, Jan. 2006, p. 165–256 Vol. 19, No.
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0893-8512/06/$08.00⫹0 doi:10.1128/CMR.19.1.165–256.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Real-Time PCR in Clinical Microbiology: Applications for

Routine Laboratory Testing
M. J. Espy,* J. R. Uhl, L. M. Sloan, S. P. Buckwalter, M. F. Jones, E. A. Vetter,
J. D. C. Yao, N. L. Wengenack, J. E. Rosenblatt,
F. R. Cockerill III, and T. F. Smith
Division of Clinical Microbiology, Department of Laboratory Medicine and Pathology,
Mayo Clinic, Rochester, Minnesota
INTRODUCTION .......................................................................................................................................................166
REAL-TIME PCR INSTRUMENTS ........................................................................................................................166
REAL-TIME PCR PROBE TECHNOLOGIES ......................................................................................................168
5ⴕ Nuclease (TaqMan) Probes ..............................................................................................................................168
Molecular Beacons..................................................................................................................................................169
FRET Hybridization Probes ..................................................................................................................................169
NUCLEIC ACID EXTRACTION..............................................................................................................................170
Manual Extraction..................................................................................................................................................170
Automated Extraction ............................................................................................................................................171
Auxiliary Procedures To Enhance Extraction.....................................................................................................171
REAL-TIME PCR ASSAY DEVELOPMENT .........................................................................................................172
Target Nucleic Acid Selection ...............................................................................................................................172
PCR Primer and Probe Design.............................................................................................................................173
Assay Optimization.................................................................................................................................................173
BIOSAFETY CONSIDERATIONS ...........................................................................................................................173
QUALITY CONTROL AND QUALITY ASSURANCE..........................................................................................174
Verification and Validation ...................................................................................................................................174
Positive and Negative Controls.............................................................................................................................174
Internal and Inhibition Controls..........................................................................................................................175
Reagents ...................................................................................................................................................................176
Quality Assurance...................................................................................................................................................177
Contamination.........................................................................................................................................................177
IMPLEMENTATION OF REAL-TIME PCR TESTING IN THE CLINICAL MICROBIOLOGY
LABORATORY....................................................................................................................................................177
Facilities Requirements..........................................................................................................................................177
Personnel Requirements ........................................................................................................................................177
Work Flow Design...................................................................................................................................................178
Example of work flow design: real-time PCR for detection of group A streptococci from throat
swabs.................................................................................................................................................................178
Example of work flow design: real-time PCR for detection of herpes simplex and varicella-zoster
infections ..........................................................................................................................................................179
COSTS..........................................................................................................................................................................179
Royalties ...................................................................................................................................................................179
Reagents and Instrumentation..............................................................................................................................179
Personnel..................................................................................................................................................................179
Cost Savings at the Bedside ..................................................................................................................................180
Coding and Reimbursement..................................................................................................................................180
APPLICATION OF REAL-TIME PCR FOR CLINICAL MICROBIOLOGY TESTING.................................180
BACTERIA OTHER THAN MYCOBACTERIA SPP. .............................................................................................181
General Bacteria .....................................................................................................................................................181
Slow-Growing or Poorly Culturable Bacteria .....................................................................................................181
Agents of Community-Acquired Pneumonia .......................................................................................................181
Agents of Meningitis...............................................................................................................................................189
Potential Agents of Bioterrorism ..........................................................................................................................189
Bacterial Antibiotic Resistance Genes .................................................................................................................189
MYCOBACTERIA ......................................................................................................................................................192
VIRUSES......................................................................................................................................................................192
* Corresponding author. Mailing address: Mayo Clinic, 200 First St.

SW, Hilton 470, Rochester, MN 55905. Phone: (507) 284-4682. Fax:
(507) 284-4272. E-mail: espy.mark@mayo.edu.
165
166 ESPY ET AL. CLIN. MICROBIOL. REV.
Qualitative Viral Assays.........................................................................................................................................193

Herpes simplex virus..........................................................................................................................................193
Herpes simplex virus CNS disease...................................................................................................................193
Herpes simplex virus dermal and genital disease..........................................................................................193
Varicella-zoster virus dermal disease ..............................................................................................................196
Varicella-zoster virus CNS disease...................................................................................................................196
Cytomegalovirus CNS disease...........................................................................................................................196
Epstein-Barr virus CNS lymphoproliferative disease....................................................................................198
Enterovirus CNS disease ...................................................................................................................................198
Polyomaviruses....................................................................................................................................................198
JCV CNS disease ................................................................................................................................................198
Parvovirus ............................................................................................................................................................198
West Nile virus ....................................................................................................................................................200
Respiratory Viruses ................................................................................................................................................200
Influenza viruses .................................................................................................................................................200
Rous sarcoma virus ............................................................................................................................................201
Adenovirus ...........................................................................................................................................................201
Metapneumovirus ...............................................................................................................................................201
Parainfluenza virus.............................................................................................................................................201
Severe acute respiratory syndrome coronavirus.............................................................................................201
Poxviruses ................................................................................................................................................................207
QUANTITATIVE VIRAL ASSAYS ...........................................................................................................................207
Cytomegalovirus ......................................................................................................................................................209
Epstein-Barr Virus..................................................................................................................................................209
BK Virus ..................................................................................................................................................................217
Viral Hepatitis Agents............................................................................................................................................225
Human Immunodeficiency Virus ..........................................................................................................................225
FUNGI ..........................................................................................................................................................................225
Aspergillus Species ...................................................................................................................................................225
Candida Species.......................................................................................................................................................237
Pneumocystis jiroveci................................................................................................................................................240
PARASITES .................................................................................................................................................................240
Plasmodium spp. ......................................................................................................................................................240
Babesia spp. .............................................................................................................................................................241
Trypanosoma spp. ....................................................................................................................................................241
Leishmania spp. .......................................................................................................................................................241
Toxoplasma spp. ......................................................................................................................................................241
Trichomonas spp. .....................................................................................................................................................243
Cryptosporidium, Entamoeba, and Giardia spp. ...................................................................................................243
ACKNOWLEDGMENTS ...........................................................................................................................................243
REFERENCES ............................................................................................................................................................243
INTRODUCTION is much simpler to perform than conventional PCR methods.

Additionally, accelerated PCR thermocycling and detection of
Real-time PCR has revolutionized the way clinical microbi- amplified product permits the provision of a test result much
ology laboratories diagnose human pathogens (25, 71, 73, 294, sooner for real-time PCR than for conventional PCR. The
456). This testing method combines PCR chemistry with fluo- combination of excellent sensitivity and specificity, low con-
rescent probe detection of amplified product in the same re- tamination risk, ease of performance and speed, has made
action vessel. In general, both PCR and amplified product real-time PCR technology an appealing alternative to conven-
detection are completed in an hour or less, which is consider- tional culture-based or immunoassay-based testing methods
ably faster than conventional PCR and detection methods. used in the clinical microbiology for diagnosing many infec-
Hence, for some time this technology was referred to as tious diseases. This review focuses on the application of real-
rapid-cycle real-time PCR. Other descriptions of real-time time PCR in the clinical microbiology laboratory.
PCR in the early literature included homogeneous PCR and
kinetic PCR.
Real-time PCR testing platforms provide equivalent sensi- REAL-TIME PCR INSTRUMENTS
tivity and specificity as conventional PCR combined with
Southern blot analysis. Since the nucleic acid amplification and Specifications for commercially available real-time PCR in-
detection steps are performed in the same closed vessel, the struments, including the nucleic acid probe formats supported,
risk for release of amplified nucleic acids into the environment, excitation and detection wavelengths, maximum number of
and contamination of subsequent analyses, is negligent com- samples per run, reaction volumes, and relative thermocycling
pared with conventional PCR methods. Real-time PCR instru- times are presented in Table 1. The large capacity (ⱖ96-mi-
mentation requires considerably less hands-on time and testing crowell format) instruments, which include the ABI Prism se-
167
REAL-TIME PCR
TABLE 1. Instruments available

Probe chemistries Detection No. of Reaction Rapid
Manufacturer Instrument Excitation (nm) Comments
supported (nm) samples volume (␮l) thermocycling
Roche Applied Science (http: LightCycler 1.0 Hybridization probes, LED470 530, 640, 32 10–20 Yes Special sample
//www.roche-applied-science molecular beacons, 710 containers.
.com/lightcycler-online/) TaqMan
LightCycler 2.0 Hybridization probes, LED470 530, 555, 32 10–100 Yes Special sample
molecular beacons, 610, 640, containers.
TaqMan 670, 710
Cepheid (http://www.cepheid SmartCycler II Molecular beacons, LED450–495, 510–527, 16 25–100 Yes Special sample
.com/pages/home.html) TaqMan LED500–550, 565–590, containers.
LED565–590, 606–650, Independent
LED630–650 670–750 modules.
Corbett Research Rotor-Gene Molecular beacons, LED470, LED530, 510, 550, 72 10–150 Midrange Standard
(http://www.corbettresearch 3000 TaqMan LED585, LED635 580, 610, 5–50 plastic tubes
.com/home.htm) 660 are used.
ABI (http://www.appliedbiosystems Prism 7000 TaqMan, molecular Tungsten/halogen 4-color 96 No
.com/index.cfm) beacons multiplex
Prism 7300 TaqMan, molecular Tungsten/halogen 3-color 96 No
beacons multiplex
Prism 7500 TaqMan, molecular Tungsten/halogen 4-color 96 No
beacons multiplex
Prism 7900ht TaqMan, molecular Laser 488 500–660 384 No
beacons
BioRad (http://www.bio-rad.com/) MyiQ TaqMan, molecular Tungsten/halogen Single color 96 15–100 No
beacons
iCycler iQ TaqMan, molecular Tungsten/halogen 4-color 96 50 No
beacons, multiplex
hybridization
probes
Stratagene Mx4000 TaqMan, molecular Tungsten/halogen 4-color 96 10–50 No
(http://www.stratagene.com beacons multiplex
/homepage/)
Mx3000p TaqMan, molecular Tungsten/halogen 4-color 96 25 No
beacons multiplex
MJ Research (http://www.mjr Chromo 4 TaqMan, molecular 450–490, 500–535, 515–530, 96 10–100 No
.com/) beacons 555–585, 620–730 560–580,
610–650,
675–730
Opticon TaqMan, molecular 450–495 515–545 96 10–100 No
beacons
Opticon 2 TaqMan, molecular 470–505 525–543, 96 10–100 No
beacons 540–700
Genetic Discovery Technology SynChron TaqMan, molecular 473 laser 520–720 6 10–50 Yes Electrically
(http://www.biogene.com beacons, conducting
/index.cfm) hybridization polymer
VOL. 19, 2006
probes technology.
ries (7000, 7300, and 7500), the MyiQ and iCycler, Mx4000, REAL-TIME PCR PROBE TECHNOLOGIES
MX3000p, Chromo4, Opticon and Opticon 2, and SynChron,
may be particularly useful in laboratories with large numbers One detection method for nucleic acid detection with real-
time PCR uses SYBR Green to detect the accumulation of any
of specimens. However, thermocycling on these instruments is
double-stranded DNA product. SYBR Green provides sensi-
slower than on other lower capacity instruments, including the
tive detection but is not specific. The use of SYBR Green with
LightCycler 1.0, LightCycler 2.0, and SmartCycler II. This is
instruments that can perform a melting curve analysis to de-
due to the use of a solid-phase material for heat conductance
termine the melting temperature, Tm, permits detection of
(heating block principle). The large-capacity instruments sup-
different amplification products based upon the %G⫹C con-
port high-volume testing while the rapid, lower capacity instru- tent and length of the amplification product. This is similar but
ments permit the work flow flexibility that may be especially not equivalent to agarose gel electrophoresis where the sepa-
useful for laboratories that test fewer samples. In summary, ration is based primarily on length. Because SYBR Green
work load and work flow issues may dictate which system is assays are not specific, they are often used for screening assays
best for different-sized laboratories and test volumes. where further analysis of specimens is performed to confirm
The Rotor-Gene instrument uses inexpensive standard plas- the results.
tic tubes for the PCR vessel and air for heat transfer with 72 Sensitive and specific detection is possible with real-time
reactions per run. This instrument is intermediate in speed PCR by using novel fluorescent probe technology probes.
because time is needed for heat conductance to the center of Three types of nucleic acid detection methods have been used
the tubes. The SmartCycler and LightCycler use specialized most frequently with real-time PCR testing platforms in clin-
vessels for rapid heat transfer and can complete a PCR in 30 to ical microbiology: 5⬘ nuclease (TaqMan probes), molecular
40 min. An additional few minutes are required for the melting beacons, and FRET hybridization probes (Fig. 1). These de-
curve analysis on the LightCycler. tection methods all rely on the transfer of light energy between
All the instruments support all or some of the dyes used for two adjacent dye molecules, a process referred to as fluores-
TaqMan probes and molecular beacons. Currently, only the cence resonance energy transfer (500). Collectively, these
LightCycler supports fluorescence resonance energy transfer three types of probes are frequently referred to as FRET
(FRET) hybridization probe detection with melting curve anal- probes and this general term has been used in some sections of
ysis. Quantitation of target nucleic acid is possible with any of this review. However, when specifically referring to each of
the instruments and supported detection formats. these three types of probes, only FRET appears in the name of
Recently, analyte-specific reagents (ASRs) and Food and one, i.e., FRET hybridization probes. Because FRET hybrid-
Drug Administration (FDA)-approved kits have become avail- ization probes consist of two separate probes, the term dual
able in the United States for testing on several real-time PCR FRET hybridization probes has also been used to describe this
instruments. The commercial availability of these reagents now specific type of nucleic acid detection method.
For all types of FRET probes, as the distance between ad-
make it considerably easier for many clinical microbiology lab-
jacent dye molecules increases, FRET decreases. For TaqMan
oratories to adapt real-time PCR testing platforms into their
probes or molecular beacons, the two dye molecules are at-
work flow. Because laboratory-developed (also referred to as
tached to a single probe. In contrast, for FRET hybridization
in-house developed or home brew) real-time PCR tests re-
probes, dyes are attached separately to two probes that align in
quired considerable expertise to develop and validate, they are
a head–to-tail configuration on target nucleic acid DNA. (For
generally limited to larger laboratories, especially referral lab- purposes of discussion in this review, the nucleic acid which is
oratories. The availability of ASRs and kits will also facilitate targeted for an assay is henceforth referred to as target nucleic
the development of common testing protocols and standards acid or simply the target.) The first dye is a fluorescent dye, and
so that proper comparative clinical studies can be performed the second can be either a quencher dye or another fluorescent
and ultimately reliable test results can be ensured for the dye which can absorb fluorescent light transferred from the
patient. first dye and reemit light at a different wavelength. The prox-
In addition to the usual considerations for new instrument imity of the two dyes in the probe(s) is determined by the
purchase (physical space requirement, cost of instrument, dis- nucleic acid architecture of the probe(s). However, the mech-
posables, and reagents, instrument maintenance and service, anisms to achieve a fluorescent signal with the TaqMan, mo-
reliability, upgrades, etc.), selection of a real-time PCR instru- lecular beacon, or FRET hybridization probe format are dif-
ment and real-time detection format requires consideration of ferent.
test volume, probe detection requirements, turnaround time
for results, personnel requirements, and software. Also, sample
preparation requirements must be considered as this can add 5ⴕ Nuclease (TaqMan) Probes
to the hands-on time per sample, turnaround time, and ex- The first real-time fluorescent probes developed were 5⬘
pense. Several manufacturers have developed semiautomated nuclease probes, which are commonly referred to by their
nucleic acid extraction instruments for use in tandem with proprietary name, TaqMan probes (Fig. 1A). A TaqMan probe
real-time PCR instruments. These include the MagNA Pure is a short oligonucleotide (DNA) that contains a 5⬘ fluorescent
LC and MagNA Pure Compact for use with the LightCycler dye and 3⬘ quenching dye. To generate a light signal (i.e.,
instrument, the GeneExpert for use with the SmartCycler II, remove the effects of the quenching dye on the fluorescent
and the ABI Prism 6700 for use with the ABI Prism series dye), two events must occur. First, the probe must bind to a
instruments. complementary strand of DNA at 60°C. Second, at this tem-
VOL. 19, 2006 REAL-TIME PCR 169
for detection of a specific nucleic acid sequence of a mutant

strain. Because TaqMan probes require 60°C for efficient 5⬘
nuclease activity, the PCR is usually cycled between 95 and
60°C for amplification. In addition, the cleaved (free) fluores-
cent dye accumulates after each PCR temperature cycle, and
therefore can be measured at any time during the PCR cycling,
including the hybridization step. This is in contrast to molec-
ular beacons and FRET hybridization probes, for which fluo-
rescence can only be measured during the hybridization step.
Molecular Beacons
Molecular beacons are similar to TaqMan probes but are
not designed to be cleaved by the 5⬘ nuclease activity of Taq
polymerase (Fig. 1B). These probes have a fluorescent dye on
the 5⬘ end and a quencher dye on the 3⬘ end of the oligonu-
cleotide probe. A region at each end of the molecular beacon
probe is designed to be complementary to itself, so at low
temperatures, the ends anneal, creating a hairpin structure.
This integral annealing property positions the two dyes in close
proximity, quenching the fluorescence from the reporter dye.
The central region of the probe is designed to be complemen-
tary to a region of the PCR amplification product. At high
temperatures, both the PCR amplification product and probe
are single stranded. As the temperature of the PCR is lowered,
the central region of the molecular beacon probe binds to the
PCR product and forces the separation of the fluorescent re-
porter dye from the quenching dye. The effects of the quencher
dye are obviated and a light signal from the reporter dye can be
detected. If no PCR amplification product is available for bind-
ing, the probe reanneals to itself, forcing the reporter dye and
quencher dye together, preventing fluorescent signal.
Typically, a single molecular beacon is used for detection of
a PCR amplification product and multiple beacon probes with
different reporter dyes are used for single nucleotide polymor-
phism detection. By selection of appropriate PCR tempera-
tures and/or extension of the probe length, molecular beacons
will bind to the target PCR product when an unknown nucle-
otide polymorphism is present but at a slight cost of reduced
specificity. There is not a specific temperature thermocycling
requirement for molecular beacons, so temperature optimiza-
tion of the PCR is simplified.
FIG. 1. Real-time probe technologies. (A) 5⬘ nuclease (TaqMan)
probe. (B) Molecular beacon. (C) FRET hybridization probes. (Re-
printed from reference 73 with kind permission of Springer Science FRET Hybridization Probes
and Business Media.)
FRET hybridization probes, also referred to as LightCycler
probes, represent a third type of probe detection format com-
perature, Taq polymerase, the same enzyme used for the PCR, monly used with real-time PCR testing platforms (Fig. 1C).
must cleave the 5⬘ end of the TaqMan probe (5⬘ nuclease FRET hybridization probes are two DNA probes designed to
activity), separating the fluorescent dye from the quenching dye. anneal next to each other in a head-to-tail configuration on the
A single TaqMan probe can be used for detection of ampli- PCR product. The upstream probe has a fluorescent dye on the
fied target DNA. If the intent of the assay is to differentiate a 3⬘ end and the downstream probe has an acceptor dye on the
single nucleotide polymorphism from a wild type sequence in 5⬘ end. If both probes anneal to the target PCR product,
the target DNA, then a second probe with the complementary fluorescence from the 3⬘ dye is absorbed by the adjacent ac-
nucleotide(s) to the polymorphism and a fluorescent dye with ceptor dye on the 5⬘ end of the second probe. The second dye
a different emission spectrum is utilized. Thus, TaqMan probes is excited and emits light at a third wavelength and this third
can be used to detect a specific, predefined polymorphism wavelength is detected. If the two dyes do not align together
under the probe in the PCR amplification product. For this because there is no specific DNA for them to bind, then FRET
application, two reaction vessels are required, one with a com- does not occur between the two dyes because the distances
plementary probe to detect wild-type target DNA and another between the dyes are too great. A design detail of FRET
NUCLEIC ACID EXTRACTION
A critical preanalytical step for real-time PCR assays, as well

as any assay in which nucleic acid is analyzed, is nucleic acid
extraction. Extraction methods that work for one pathogen in
a particular specimen type may not work for another pathogen
in another specimen type. For example, herpes simplex virus
DNA can be extracted relatively easily from genital swabs (115,
118), whereas extraction of DNA from vancomycin-resistant
enterococci in stool samples may be considerably more chal-
lenging (451).
A few general comments about extraction of nucleic acid
from microorganisms can be made. The thick cell wall of gram-
positive bacteria is more difficult to disrupt than the relatively
thinner cell wall of gram-negative bacteria. Substances that
may inhibit amplification such as heme in blood or bile in stool
must be removed. The released nucleic acids should be main-
FIG. 2. Melting curves obtained after PCR amplification of HSV tained in an aqueous solution to protect them from degrada-
DNA.
tion. Nucleic acids should be eluted into a small volume in
order to maximize detection.
hybridization probes is the 3⬘ end of the second (downstream) Extraction of clinical specimens can be accomplished either
probe is phosphorylated to prevent it from being used as a by manual or automated methods. A survey of the literature
primer by Taq during PCR amplification. The two probes en- demonstrates the ability of various commercially available
compass a region of 40 to 50 DNA base pairs, providing ex- methods to successfully extract a wide variety of specimens for
quisite specificity. bacterial, viral, and fungal targets (Tables 2 and 3).
FRET hybridization probe technology permits melting curve
analysis of the amplification product. If the temperature is
slowly raised, eventually the probes will no longer be able to Manual Extraction
anneal to the target PCR product and the FRET signal will be
lost. The temperature at which half the FRET signal is lost is Phenol-chloroform has been used successfully for the extrac-
referred to as the melting temperature of the probe system. tion of nucleic acids (290, 396). However, phenol is a caustic
The Tm depends on the guanine plus cytosine content and and corrosive agent, and its use should be considered a safety
oligonucleotide length. In contrast to TaqMan probes, a single hazard by clinical microbiology laboratory. A number of com-
nucleotide polymorphism in the target DNA under a hybrid- mercial manufacturers have developed manual extraction kits
ization FRET probe will still generate a signal, but the melting for use by clinical laboratories. Some of the most frequently
curve will display a lower Tm. The lowered Tm can be charac- used manual kits as reported in peer reviewed publications are
teristic for a specific polymorphism underneath the probes; presented in Table 2. These kits vary as to the method, cost,
however, a lowered Tm can also be the result of any sequence and time required for extraction (Table 2). This variability
difference under the probes. The target PCR product is de- permits the flexibility in choosing the kit that best suits the
tected and the altered Tm informs the user there is a difference needs of a specific laboratory. Manual extraction kits typically
in the sequence being detected. Generally, more than three use noncorrosive agents making them safe to use by laboratory
base pair differences under a FRET hybridization probe pre- personnel. While these kits are generally inexpensive and easy
vent hybridization at typical annealing temperatures and are to use, they have several drawbacks.
not detected. Processing of samples by manual methods requires multiple
This trait of FRET hybridization probes is advantageous manipulations. As the number of samples to be extracted in-
in cases where the genome of the organism is known to creases, so does the potential for contamination due to in-
mutate at a high frequency, such as with viruses. When a creased manipulation. In the United States, Clinical Labora-
single or limited number (⬍3) of known polymorphisms tory Improvement Amendments of 1988 (CLIA) regulations
occur between two similar targets, FRET hybridization (http://www.cms.hhs.gov/clia/) consider manual extraction
probes can also be used for discriminating strains of organ- high-complexity testing, and therefore this type of testing can
isms. An example of this application is the identification of only be performed by laboratory personnel with appropriate
herpes simplex virus type 1 (HSV-1) and HSV-2 (Fig. 2). academic credentials. In order to ensure reproducible results,
Like molecular beacons, there is not a specific thermocy- extensive training is necessary to achieve consistency among
cling temperature requirement for FRET hybridization laboratory personnel performing manual extraction. Some
probes. Molecular beacons and FRET hybridization probes, manual kits use ethanol to precipitate the nucleic acids. If not
unlike TaqMan probes, are both recycled (conserved) in properly removed, excess ethanol residues can inhibit the PCR
each round of PCR temperature cycle. Also, for Molecular (502). Finally, manual extraction is a laborious, time-consum-
beacons and FRET hybridization probes, unlike TaqMan ing process which requires the undivided attention of the tech-
probes, fluorescent signal does not accumulate as PCR nologist performing this technique in order to ensure optimal
product accumulates after each PCR cycle. results.
TABLE 2. Manual methods of nucleic acid extraction and purification for rapid real-time PCR assays
Kit (manufacturer) Technologic principlea Specimen throughput Specimen type Referencesb
High Pure (Roche Applied NA capture by glass fiber 24 samples in 1 h Serum, whole blood, plasma, 14, 18, 100, 338,
Science; www.roche fleece immobilized in a urine, stool, sterile body 401, 403
-applied-science.com) special plastic filter fluids, respiratory tract
tube and subjected to specimens, swabs (genital,
centrifugation dermal)
QIAamp (Qiagen; NA capture by silica gel 24 samples in 1 h for Respiratory tract specimens, 18, 19, 36, 120, 147,
www.qiagen.com) membrane placed in DNA; 24 samples in plasma, stool, serum, 148, 184, 193,
tube column and 1.5 h for RNA whole blood, urine, sterile 198, 210, 212,
subjected to body fluids, swabs (nasal, 214, 225, 226,
centrifugation or fecal) 234, 235, 238–242,
vacuum conditions 244, 246, 247,
250, 254, 262,
264, 266, 269,
273, 277, 279,
282, 291, 294,
304, 323, 362,
401, 402, 484,
486, 494, 515,
530, 531
IsoQuick (Orca Research; NA is partitioned into an 24 samples in 1 h for Plasma, whole blood, stool, 115–118, 120, 184,
www.bioexpress.com) aqueous phase and DNA; 24 samples in respiratory tract 213, 256, 259–261,
then precipitated with 2 h for RNA specimens, sterile body 264–266, 299, 399,
ethanol and fluids, swabs (dermal, 450, 451
resuspended in water fecal, genital)
or buffer
IsoCode Stix (Schleichar DNA bound to Matrix Processed individually Whole blood 561, A, B
& Schuell; and released by simple
www.whatman.com) water and heat elution
a
NA, nucleic acid.
b
A, A. Muyombwe, I. Lundgren, L. M. Sloan, J. E. Rosenblatt, P. G. Kremsner, S. Borrmann, and S. Issifou, Program Abstr. 52nd Am. Soc. Trop. Med. Hyg., abstr.
744, 2003; B, J. E. Rosenblatt, A. Muyombwe, L. M. Sloan, P. Petmitr, and S. Looareesuman, Program Abstr. 11th Int. Cong. Infect. Dis., abstr. 14.006, 2004.
Automated Extraction have been validated and proper maintenance procedures are in
place, quality control monitoring is less intensive than that
Automated extraction instruments are manufactured by a required for manual extraction (137).
number of different companies, and like manual methods vary While the benefits of automated extraction are considerable,
in method, cost, and time requirements for extraction. Addi- there are potential drawbacks. It is most economical when
tionally, these instruments vary as to specimen capacity per run instruments are fully loaded; therefore, a significant number of
and size (footprint) (Table 3). While these systems have not samples (50 to 100/day) should be processed in order to justify
been as widely used as manual methods, a number of studies the capital investment that is required for these instruments.
have reported their utility for extraction of a variety of speci- The footprints of automated extraction instrumentation may
men types (Table 3). Studies which compared manual and require space that is not currently available in the laboratory.
automated extraction methods have reported automated ex- In addition to the cost for equipment, costs for disposables also
traction to be equivalent and in some instances superior to need to be considered. Some vendors are now manufacturing
manual methods (116, 139, 143, 226, 446). smaller versions of earlier models of their instruments (Table
Automated extraction systems have certain inherent advan- 3). While these instruments extract significantly fewer samples
tages over manual methods. Recovery of nucleic acids from at a time, they are less expensive and have a smaller footprint
automated instruments is consistent and reproducible. Auto- than the parent instrument. These smaller versions may be
mated extraction systems keep sample manipulation to a min- viable options for smaller laboratories which process lower
imum, reducing the risk for cross contamination of samples. numbers of specimens.
Many of the instruments are closed systems, further reducing
the risk for contamination. Automated systems are typically
walk-away systems, and do not require constant attention, Auxiliary Procedures To Enhance Extraction
which permits personnel to perform other duties. The proce-
dures associated with these instruments could potentially be Recently, new products have been developed to facilitate the
classified as moderate complexity based on the the Clinical extraction of nucleic acid from clinical samples. Stool transport
Laboratory Improvement Amendments of 1988 (13). There- and recovery buffer (S.T.A.R.; Roche Diagnostics Corpora-
fore, laboratory assistants may be able to perform sample ex- tion, Indianapolis, IN) has been used successfully for the ex-
traction with these instruments. Finally once these systems traction of historically challenging specimens such as stool
114, 116, 139, 185, 208, 210,

211, 213, 215, 223, 226,
245, 253, 255, 270, 287,
Literature reference(s)
310, 506
116, 226
208, 446
143
Swabs (dermal, genital, nasal)
specimesns, swabs (dermal,
Serum, plasma, whole blood,
stool, respiratory tract
Specimen type
TABLE 3. Automated systems of nucleic acid extraction and purification for rapid real-time PCR assays
genital)
Plasma
130; 107 ⫻ 60 ⫻ 87.5
130; 107 ⫻ 65 ⫻ 77.9
26; 28.2 ⫻ 45.5 ⫻ 43
(W ⫻ D ⫻ H, cm)
140; 145 ⫻ 82 ⫻ 81
227; 142 ⫻ 84 ⫻ 99
151; 90 ⫻ 60 ⫻ 65
95; 120 ⫻ 71 ⫻ 60
90; Cylindrical (66

diam. ⫻ 79 ht.)
60; 54 ⫻ 61 ⫻ 57
20; 53 ⫻ 48 ⫻ 27
Wt (kg) and
dimensions
48 samples in 159 min

Specimen throughput
96 samples in 75 min;
FIG. 3. S.E.T.S. The inner tube will hold standard-sized swabs (ap-
4 microtiter plates
10 samples in 35–45
8 samples in 30 min
6 samples in 20 min
96 samples in 2.5 h
proximately 5 to 8 mm in diameter, 10 of 25 mm in length) during

96 samples in 2 h
spinning procedures. The collection tube is used for collection and

storage of liquid from the swab. The screw cap is for tightly closing the
in 3.5 h
collection tube.
min
(451). S.T.A.R. buffer has three important properties: infec-

tious organisms are inactivated, degradation of nucleic acids is
MagNA Pure Compact
Automated platforms
minimized, and the binding of the nucleic acids to magnetic

NucliSens Extractor
beads, as is used in the extraction process of MagNA Pure

MagNA Pure LC
ABI Prism 6100

ABI Prism 6700
MDx
9604
M48
M96
(Roche Diagnostics Corporation), is enhanced.

EZ1
The swab extraction tube system (S.E.T.S.; Roche Diagnos-

BioRobot
BioRobot
BioRobot
BioRobot
BioRobot
tics Corporation) kit, shown in Fig. 3, is a simple method for

rapidly and efficiently recovering specimen attached to and
absorbed into the fibers of a collection swab. For some organ-
isms, studies have demonstrated that specimen which is re-
NA capture by magnetic
NA capture by magnetic
bound to filter under

Technologic principlea
trieved in a microcentrifuge tube by the S.E.T.S. method, can

vacuum conditions
NA capture by silica
NA capture by silica
be directly, or after a quick lysis step (boiling), analyzed by a

fiber membrane
under vacuum
silica particles
silica particles
LightCycler real-time PCR instrument (195, 499). Alterna-

conditions
tively the centrifuged material can be extracted by the MagNA

Pure instrument to obtain a cleaner preparation of nucleic acids.
IsoCode Stix (Schleicher and Schuell, Keene, NH), a
method for stabilizing blood samples to be transported long
distances, can be used to preserve samples for later testing by
(www.Roche-Applied-Science.com;
real-time PCR. This specimen transport device has been cou-

(www.appliedbiosystems.com)
pled with real-time PCR assays for the detection of blood-

(www.biomerieux-usa.com)
borne parasites such as malaria (561). This method is not

Qiagen (www.qiagen.com)
Manufacturer
www.magnapure.com)
recommended for use with RNA assays.

Roche Applied Science
NA, nucleic acid.

Applied Biosystems
REAL-TIME PCR ASSAY DEVELOPMENT

bioMérieux
Target Nucleic Acid Selection

The target primer sequences must be unique in order to
a
identify a specific organism or an organism group, (e.g., group

A streptococcus or Mycobacterium genus screen), quantitate a BIOSAFETY CONSIDERATIONS

microbe (e.g., cytomegalovirus), or identify unique virulence
genes (e.g., verotoxin genes) or genes or mutations associated Clinical microbiology laboratories receive and process a
with antimicrobial resistance (e.g., mecA gene or mutations in wide variety of specimens, including urine, stool, whole blood,
rpoB gene associated with rifampin resistance) which can occur plasma, sputum, and swab materials. These specimens may
across strains or species. Moreover, the PCR primer must be contain a number of transmissible infectious agents including
able to identify with high efficiency and specificity the target hepatitis viruses and human immunodeficiency virus (HIV). As
primer sequences in the specimen of interest (e.g., stool or molecular testing becomes more commonplace, the question
perianal swab specimens for vancomycin-resistant entero- of at what point during the extraction process are specimens
cocci). A search for the intended primer sequence in a DNA rendered noninfectious arises. Many extraction kits contain
database such as the National Center for Biotechnology Infor- guanidinium salts as one of their compoenents. Studies have
mation (NCBI) database (http://www.ncbi.nlm.nih.gov/BLAST/) shown that guanidinium salts will disrupt cellular integrity and
may reveal cross-reactivity. However, since the databases cur- neutralize inhibitory substances (66). However, there are no
rently available represent only a small portion of the nucleic published studies that demonstrate treatment with guani-
acid sequences for microbes in complex specimen matrices dinium will ensure that specimens are not infectious.
such as stool, specimens and related organisms must also be The MagNA Pure mixes a guanidinium isothiocyanate-con-
tested to confirm the lack of cross-reactivity. The target nucleic taining lysis solution with the sample and incubates it at room
acid sequence should also be conserved in the organism to be temperature for 2 min. We have found (unpublished observa-
identified or quantitated. If sequence data of the intended tions) that this treatment renders 108 Staphylococcus aureus/ml
target area shows a significant frequency of polymorphisms a nonviable. However, further studies are required to determine
more conserved site should be chosen. if guanidinium has the same effect on other infectious agents.
Until these studies are completed individuals using real-time
PCR Primer and Probe Design PCR in clinical laboratories should practice universal precau-
tions, i.e., treating all specimens as if they were infectious (10).
PCR primers provide the first level of specificity for the PCR In the past, infectious agents, such as anthrax, have been
assay, and primers that only amplify one product will provide weaponized for use in biological warfare. The intentional re-
the best assay sensitivity. Since real-time PCR also incorpo- lease of anthrax spores in the U.S. mail system in the fall of
rates highly specific homogeneous probe detection, the anneal- 2001 emphasized the urgent need for rapid and safe laboratory
ing temperature for probes can be several degrees below the techniques for detecting Bacillus anthracis in suspicious pow-
melting temperature of the primers. PCR primers should have ders as well as human specimens (179, 289, 350). The Centers
a low potential to form secondary structures, including self and for Disease Control and Prevention (CDC) has issued guide-
crosshybridization with other oligonucleotides in the PCR.
lines for the processing and testing of specimens obtained from
This becomes increasingly more difficult as more oligonucleo-
a suspected outbreak of bioterrorism, in order to protect first-
tides are added to the reaction. Details for design of primers
line workers (direct healthcare providers and laboratory work-
and probes are beyond the scope of this review and have been
ers) (10). In the case of a smallpox outbreak, rapid and accu-
described extensively in two recent publications (191, 500).
rate laboratory detection is critical in order to quickly contain
the infection, however, this may be difficult as smallpox is a
Assay Optimization level 4 organism, and as such, must be tested at institutions
with specialized biosafety level 4 containment facilities (i.e.,
Optimization of assay conditions can be more challenging
for conventional PCR. Due to the numerous manual steps and CDC or United States Army Medical Research Institute of
time requirements for conventional PCR, the assessment of Infectious Diseases).
different testing parameters is a painstaking process. For ex- Autoclaving has been shown to be an effective way to inac-
ample, several days were frequently required to evaluate the tivate potential agents of bioterrorism, while permitting the
effects of changing a single parameter (e.g., optimal magne- nucleic acid to remain intact for analysis by PCR assays (119,
sium concentration). Because real-time PCR is more auto- 125, 179, 289, 350). The authors of these publications demon-
mated and has a shorter test turnaround time, optimization strated that autoclaving anthrax spores and vaccinia virus, a
experiments can be performed within hours instead of days. close relative of smallpox virus, destroyed their ability to be
For real-time PCR a few key components should be opti- infectious, while not affecting the integrity of their nucleic acid
mized in order to achieve maximum results (17, 35, 228, 483). so it could be detected by PCR techniques.
These factors include magnesium concentration, which allows As indicated in the preceding discussion, S.T.A.R. buffer
the polymerase enzyme to function at an optimal level; primer (Roche Diagnostics Corporation) not only stabilizes nucleic
and probe concentrations, which affect the sensitivity and spec- acid during transport at room temperatures, but can inactivate
ificity of the assay respectively; and the use of additives such as pathogens. We have observed that S.T.A.R. buffer can inacti-
dimethyl sulfoxide, which can aid in the denaturation of nucleic vate many bacteria, including such pathogens as Mycobacte-
acids with high G⫹C. The type of polymerase enzyme utilized rium tuberculosis and Escherichia coli OH157:H7 isolated from
can also play a significant role, polymerases which permit hot- culture, or present in complex matrices such as respiratory and
start PCR are preferrable. These enzymes do not function until stool specimens without damaging the integrity of the DNA
a critical maximum temperature is reached, which reduces the (unpublished data). The pathogen-inactivating properties of
generation of nonspecific sequence fragments. S.T.A.R. buffer provides laboratories an added level of safety
TABLE 4. Verification guidelines

Modified–FDA, ASR,
Verification FDA cleared Verification requirementsa
laboratory developed
Accuracy 20 positive samples, 50 negative 50 positive samples, 100 negative 90% agreement with reference
samples samples method
Precision, qualitative 1 control/day for 20 days or Same as FDA-approved assay Mean, SD, and C.V.
duplicate controls for 10 days
Precision, quantitative 20 data points at 2 to 3 Same as FDA-approved assay Mean, SD, and C.V. for each
concentrations. Within run, concentration
within day, day-to-day
Analytical sensitivity Analyze 15–20 low or no Determine mean and SD;
concentration specimens SD ⫻ 2 ⫽ detection limit
Analytical specificity Evaluate all interfering
compounds (i.e., same
chemical or genetic structure,
same source)
Reportable range, 3–5 concentrations measured in 3–5 concentrations measured in Determine upper and lower
quantitative triplicate triplicate limits of linearity
Normal values Minimum of 20 per category, Minimum of 20 per category,
100 recommended 100 recommended
a
C.V., coefficient of variation.
for processing and transporting pathogens for nucleic acid CLIA 88 document stipulates that prior to test implementa-
analysis. tion, clinical laboratories verify the manufacturer’s perfor-
mance specifications and confirm they can be replicated by
laboratory personnel when following the procedure. For labo-
QUALITY CONTROL AND QUALITY ASSURANCE
ratory developed tests or modification of test systems, labora-
Verification and Validation tories are required to establish their own performance speci-
fications prior to implementation of the new or modified test.
Clinical relevance, cost, instrumentation, and ease of per- Because nucleic acid test methods are changing and evolving
formance should be considered when evaluating a new test so rapidly, existing guidelines have been difficult to apply. The
procedure (109), but of primary importance is the verification challenges to clinical laboratories include determining the type
and validation of test performance. The ability of a laboratory of verification experiments required for a real-time PCR assay
test to consistently produce accurate and precise results is not and an acceptable number and type of specimens to evaluate.
only essential, it is the core of quality assurance programs for Providing a single set of guidelines for real-time PCR which
clinical laboratories (302). A detailed protocol for the verifi- envelops all the necessary verification and validation by all
cation of new test methods should be established by the labo- accreditation agencies would be of great benefit to laboratories
ratory prior to the verification procedure. Table 4 provides acquiring this new technology. Along with the need for a well
guidelines for verification of new test methods (333). Addition- defined quality control program for real-time PCR qualitative
ally, documentation of validation is necessary to demonstrate assays, there is need for guidelines for quantitative real-time
that a verified test continues to perform according to the lab- PCR assays. To date such guidelines only exist for a select
oratory’s requirements. These procedures help ensure the con- number of blood-borne viruses (341).
sistency of the results and that laboratory personnel remain Quality control allows the laboratory to minimize the report-
competent to perform tests and report results. ing of inaccurate results, to report results with a high degree of
Guidelines developed by regulatory agencies are not current confidence and to decrease costs by detecting errors prior to
for real-time PCR applications in clinical microbiology. The reporting results (137). One goal of the laboratory quality
Clinical Laboratory Standards Institute (formerly the National control program is to reduce the number of controls needed
Committee for Clinical Laboratory Standards) published a set for reporting acceptable results. The following information
of guidelines for molecular diagnostic methods in infectious relates to specific controls used during testing as well as the
disease testing in 1995; however, these guidelines were pro- quality control of reagents used for testing. This discussion is
vided before the introduction of real-time PCR technology not intended to be all-inclusive nor definitive, and is based to
(332). These are considered guidelines, not standards, for in- some extent on experience at Mayo Clinic with real-time PCR
fectious disease testing, and currently are undergoing revision. and our interpretation of published guidelines for generic mo-
The most recent document addressing quality control stan- lecular testing.
dards for molecular test systems is the revised CLIA 1988
document published in the Federal Register, 24 January 2003
(4). This document addresses requirements for certain quality Positive and Negative Controls
control provisions and personnel qualifications. It combines
and reorganizes requirements for test management, quality Ideally, patient specimens containing the target nucleic acid
control and quality assurance, and also changes the requisite are used as the positive control, but this is often not practical
consensus for grading proficiency testing challenges. The or feasible. An acceptable positive control is pooled negative
TABLE 5. Positive and negative control recommendations mended in both the CLIA 88 and CLSI documents. Labora-
for molecular testing tories should establish the number and frequency of controls
No. of controls based on manufacturers criteria and agency recommendations.
2003 Revision of
Assay NCCLS MM3-Aa
CLIA 88
Internal and Inhibition Controls
Positive Negative Positive Negative
An acceptable specimen should be free of inhibitory sub-
Qualitative 1 1 1 1 stances that could produce a false-negative result. Some clin-
Quantitative 2 1 2 1
Nucleic acid extraction 1 1 NR NR
ical samples may contain substances which are not always re-
moved by the extraction process and which may inhibit the
a
NR, no recommendation. PCR amplification. Inhibition of amplification can be detected
by the introduction of an internal control, also referred to as a
specimens spiked with whole organisms or if that is not avail- recovery template.
able, a representative sample of the nucleic acid to be detected. Based on the requirements of regulatory or accrediting
The positive control should be at a concentration near the agencies, individual laboratories should determine when an
lower limit of detection of the assay to challenge the detection internal control is required in an assay. For example, the 2003
system yet at a high enough level to provide consistent positive revision to CLIA 88 document does not address internal con-
results. trols nor have a requirement for assessment of inhibition of
A blank control such as water or buffer is often used as a PCR chemistry. In contrast, the College of American Pathol-
negative control. However, an optimal negative control is a ogist (CAP) molecular pathology inspection checklist indicates
sample containing nontarget nucleic acid to demonstrate that that the laboratory must determine the likelihood of the gen-
nonspecific PCR amplification and detection of amplified erated result being a false-negative result due to inhibition
product is not occurring. In addition, the negative control is when there is no amplification of product (76). If the test is
used to demonstrate that the reagents are not contaminated performed according to manufacturer instructions, published
with target nucleic acid and can be used to compensate for data containing the inhibition rate may be used for documen-
background signal generated by the reagents. The recommen- tation. Internal controls for laboratory developed assays or
dation for a negative control every fifth tube to monitor PCR modified FDA assays should be determined on a case-by-case
contamination (332) may be excessive with real-time PCR as- basis taking into account the probability that the specimen
says. The closed system for amplification and detection used source contains inhibitory substances. Specimen types such as
with real-time PCR virtually eliminates the amplicon contam- stool or sputum are generally more inhibitory to PCR chem-
ination caused by the opening and closing of reaction vessels istry than serum or plasma specimens. Also, the assay perfor-
which is problematic with conventional PCR and detection mance characteristics (sensitivity, specificity, accuracy, etc.),
methods. Even with the closed system of real-time PCR, the the implications of a false-negative result and the degree to
laboratory may still choose to add uracil-N-glycosylase to the which a clinical diagnosis depends on laboratory results, re-
PCR mix for another level of amplicon contamination control. quire consideration. Internal controls may be naturally present
The 2003 revisions to CLIA 88 rule does not specifically in the original specimen, added to the specimen prior to ex-
address real-time PCR assays but recommendations from the traction, or added to the PCR reagent mix before amplifica-
molecular testing sections can be applied to real-time PCR tion. The simplest way to establish inhibition is to add target
assays. Table 5 summarizes the 2003 revised CLIA 88 docu- nucleic acid to a portion of the sample and perform the test to
ment (4) and CLSI (332) quality control recommendations. In show that if target nucleic acid were present, the PCR would
both of these documents it is recommended that each molec- have been positive. Unfortunately, this approach increases the
ular amplification run of samples contain positive and negative cost of the assay.
controls. Additionally, in the CLIA document it is indicated Examples of the different types of internal controls that have
that a test system which includes nucleic extraction also include been used for real-time PCR assays are shown in Table 6.
a positive and negative control, with the positive control capa- Homologous and heterologous internal controls are those
ble of detecting errors in the nucleic acid extraction procedure. which do not naturally occur within the specimen source.
For a quantitative assay, two positive controls representing two These have also been referred to as exogenous controls as they
different concentrations of target nucleic acid are recom- must be added to the specimen. Homologous controls are
TABLE 6. Examples of internal controls used in real-time PCR assays

Control Gene targetsa References
Housekeeping genes Albumin, ␤-globin, 18S and 28S rRNA, ␤- and ␭-actins, cyclophilins, GAPDH, 257, 326, 489
␣- and ␤-tubulins, hypoxanthine phosphoribosyltransferase or L32
Exogenous
Heterologous control Neomycin phosphotransferase gene, phocine herpesvirus, bacteriophage ␭ DNA 432, 466, 468, 485, 516
Homologous control Control contains the same flanking nucleic acid sequence at the target nucleic 108, 257, 432, 501
acid to which the PCR primers anneal, but a different internal sequence to
which a probe anneals
a
GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
mercially available master mix components that contain stan-

dardized concentrations of reagents are available, but these do
not always include PCR primers and FRET probes.
Whether PCR primers are purchased from vendors or lab-
oratory developed, some method of chromatographic purifica-
tion should be applied. Purification recovers oligonucleotides
of the correct length. Truncated oligonucleotides can affect a
PCR by consuming reaction reagents and forming nonspecific
amplification products (159). The presence of these irregular
FIG. 4. Recovery template. The recovery template (internal con-
oligonucleotides can also falsely elevate the final concentration
trol) has the same sequence as the PCR product except the probe
region has been replaced with a sequence complementary to recovery of the working primers thus affecting the performance of the
template probes. FRET detection of the target DNA is with a probe assay. The CLSI MM3 guideline recommends that laboratories
labeled with the Red 640 dye in channel 2 of the LightCycler while the obtain a certificate of analysis from PCR primer vendors.
recovery template is detected with a probe labeled with the Red 705 These certificates may contain sequence data, base composi-
dye in channel 3. A small amount of recovery template is added to the
PCR and is amplified along with the target DNA by the same primers. tion, molecular weight of the sequence, concentration and
Thus, the two reactions compete for the primers. Normally, the recov- method of purification (332). Vendors may provide PCR
ery template is amplified in all samples, including the negative control. primer concentration, but these concentrations should be ver-
If neither the recovery template nor target DNA is amplified, then it is ified in the laboratory. Laboratories synthesizing their own
assumed that inhibition of the PCR has occurred and the test for that
oligonucleotide PCR primers should perform chromato-
sample is not valid. However, if target DNA is amplified but the
recovery DNA template is not amplified, then it is assumed that the graphic purification and determine also PCR primer concen-
target DNA is present in a proportionally greater amount. In this tration. In compliance with the CAP Molecular Pathology in-
situation, partial inhibition of the PCR may be present but the target spection checklist, each new lot of reagent should be tested in
DNA is successfully amplified or the recovery template may not be parallel with the old reagent lot using both positive and nega-
able to compete for primers and the recovery template signal may be
weak or not present. When this occurs, the positive result is valid tive patient samples ensuring the same results are obtained
because the recovery template amplification result is unnecessary. with both reagent lots (76).
Purification of FRET probes is especially important to sep-
arate both dye and oligonucleotides that have not coupled to
coamplified with target DNA using the same PCR primers.
form the FRET probes and to remove oligonucleotides with an
However, the internal sequence of the homologous control
incorrect length (554). While the quality of probe synthesis has
DNA internal to the PCR primer sites is genetically engi-
improved greatly over the past few years, quality control is
neered to be different from the target DNA such that a differ-
required to avoid probe lots with reduced performance. The
ent product signal occurs with FRET detection.
CLSI MM3 guideline for PCR primers discussed above should
An example of a homologous internal control is shown in
also apply to FRET probes. Some vendors provide a tracing
Fig. 4. Heterologous controls consist of separate amplifiable
(chromatogram, polyacrylamide gel electrophoresis analysis,
targets. Since these do not contain the target sequence, a
etc.) of the purified FRET probe as part of their quality control
separate set of PCR primers and probes are required for am-
documentation which may augment quality control. Another
plification and detection respectively. Housekeeping genes oc-
quality control service provided at a nominal charge by some
cur naturally within the specimen being tested and therefore
vendors is to determine if a particular FRET hybridization
are referred to as endogenous controls (341). The housekeep-
probe set is capable of producing FRET. The company will
ing genes occur in all human nucleated cell types and therefore
design and synthesize an oligonucleotide complementary to
these types of controls are commonly used in human genetic
both probes and a melting curve analysis is performed. The
studies. There is no single housekeeping gene that is suitable
production of a melting peak at the predicted Tm will confirm
for all experimental conditions and articles have been pub-
that the FRET hybridization probe set is capable of producing
lished on the variability of certain housekeeping genes in dif-
FRET and is acceptable to use. This probe validation process
ferent systems (326, 498).
can also be completed in the laboratory by following the
Real-time PCR assays used in microbiology require optimal
method provided on the Idaho Technology website (http:
sensitivity and the use of internal controls should not decrease
//www.idahotech.com) under probe classroom.
the sensitivity of the assay. Performing competitive assays by
Proper storage of reagents can result in an increase in shelf
amplifying serial dilutions of the target DNA with and without
life. FRET probes may arrive in a lyophilized form and the
the internal control should reveal if the sensitivity of the assay
recommendation is to store them at room temperature until
is affected (501). Generally, procedures for synthesis of homol-
resuspended. Some manufacturers state that the hydrated
gous internal controls are too complex for the clinical micro-
probes should be stored at 4°C for daily use or aliquoted into
biology laboratory (55). A number of manufacturers of real-
smaller volumes and stored at ⫺20°C. Numerous freeze-thaw
time PCR ASRs and kits are including homologous internal
cycles can be detrimental to the FRET probes. The PCR prim-
controls for their assays, which obviates this cumbersome task
ers can be stored in a similar manner as the probes. The
for the novice user of real-time PCR.
completed master mix (containing primers and probes) can be
Reagents stored at ⫺20°C for extended periods of time, without degra-
dation of the mix (452). We have also found this to be true with
The quality control of reagents is extremely important to most of our real-time PCR assay master mix reagents used at
ensure the success of real-time assays (56). Frequently, com- the Mayo Clinic. The complete mix is stored at either 4°C or
⫺20°C (assay dependent) for 1 to 6 months without loss of of facility requirements, personnel requirements, and work
activity. However, we have observed that the length and tem- flow design. These considerations are similar to those required
perature of storage are assay dependent and conditions of for implementation of any new type of testing method. A
storage require validation for each assay. The advantage of review of these requirements related to our experience at
freezing the master mix is assay reproducibility, time savings in Mayo Clinic with implementing LightCycler technology into
setting up assays, and reduced reagent contamination (452). the clinical microbiology laboratory is provided in the follow-
ing discussion. At the Mayo Clinic, some of our real-time PCR
assays have been used routinely in the clinical laboratory since
Quality Assurance
early 2000.
After implementation of the real-time PCR test it is neces-
sary to continue to monitor performance of the assay, equip-
ment, reagents, and personnel. For example, technologists Facilities Requirements
monitor patient specimen positivity rates for all real-time PCR
As previously mentioned, a physical separation of processes,
assays used in our institution on a weekly or monthly basis. If
equipment, and reagents is recommended, to minimize the risk
a sudden increase in positivity rate occurs, this could reflect
of specimen-to-specimen contamination. Four different work
seasonal variances of disease frequency (e.g., influenza virus or
areas are suggested, including a reagent preparation area to
group A streptococcus), disease outbreak, or specimen-to-
prepare PCR master mix, a sample processing area where
specimen or amplification product contamination. Daily qual-
procedures, including nucleic acid extraction, occurs, a target
ity control of reagents including positive and negative controls
loading area where the specimen is added to the PCR master
and/or extraction controls should be performed. In compliance
mix in the reaction vessel, and an amplification area where
with accrediting and regulatory agencies, comparable perfor-
thermocycling and probe detection occurs.
mance of new reagent lots compared with old reagent lots
The reagent preparation area should be kept free of all
should be verified. Instrument performance should be assessed
patient specimens and DNA extracts. Protocols for the sample
biannually when multiple instruments are used interchange-
preparation area should minimize the number of tubes that are
ably, also as required by accrediting and regulatory agencies.
simultaneously open. Each of the work areas should contain
Competency of personnel performing tests must also be eval-
dedicated working materials, reagents, and pipetting devices.
uated. Examples of competency assessment are included under
Reagents should be prepared and aliquoted into single use or
the Personnel Requirements section below.
small volumes. This ensures ease of use and less chance for
contamination.
Contamination All working surfaces should be cleaned before and after use,
preferably with a reagent that destroys nucleic acid such as a
The risk of contamination is considerably less with real-time
5% bleach solution. The manufacturer’s recommendations
PCR compared to conventional PCR, but still can occur (341).
should be followed for cleaning of instrument components
Since real-time PCR amplification is performed in a closed
(e.g., carousels with the LightCycler), processing blocks, and
system, there is no need for individual air-controlled rooms as
other instrument surfaces and parts.
is recommended for conventional PCR. In our experience with
Gloves should be changed frequently, at least before begin-
real-time PCR, specimen to specimen contamination has be-
ning each of the separate tasks required in a dedicated work
come a greater challenge than amplified product contamina-
area and should always be changed if moving from one work
tion. The most obvious situation where specimen-to-specimen
area to another work area. The use of aerosol-resistant pipette
contamination can occur is with the transfer of specimen to the
tips and pipette tips long enough to prevent specmien contact
PCR vessel or to the DNA extraction tube. Care must be taken
with the pipetter aids in the prevention of specimen contami-
to avoid contamination of the pipette device with specimen
nation (502). Enzyme contamination control systems such as
and to avoid the creation of an aerosol by blowing out the
uracil-N-glycosylase can be incorporated into the real-time
specimen from the tip.
PCR master mix as an added safeguard to sterilize amplified
Certain types of sample sources are known to contain a high
product that may be carried over to subsequent batches of
concentration of organisms that may lead to specimen-to-spec-
tests.
imen contamination, namely, viral agents. The inclusion of
negative controls and continual trend analysis of the assay are
used to recognize a contamination event. Additionally, unidi- Personnel Requirements
rectional work flow should be followed. Separation of proce-
dural steps will require separate work spaces in the laboratory, Personnel should be trained in both the preanalytical (spec-
as detailed below under the ssection on facilities requirements. imen processing and extraction) and the analytical procedures.
As with all methods performed in the laboratory, good labo- Many current laboratory professionals do not have training or
ratory practice is critical for accurate results. experience in molecular methods and also lack theoretical
knowledge of molecular microbiology. Based on our experi-
ence at the Mayo Clinic, providing a variety of methods for
IMPLEMENTATION OF REAL-TIME PCR TESTING IN
attaining this knowledge is useful. Some vendors are willing to
THE CLINICAL MICROBIOLOGY LABORATORY
provide overview presentations on molecular biology as well as
Implementation of real-time PCR testing platforms in the technical information on their specific testing platform. Ap-
clinical microbiology laboratory requires careful consideration preciation of the fundamentals will help to avoid cookbook
testing and will later allow more careful and focused trouble-
shooting.
Clinical microbiologists who have not had formal training in
molecular microbiology still possess many of the critical skills
necessary for success in performing real-time PCR testing.
Especially important is meticulous attention to detail, strict
adherence to standard operating procedures, and use of asep-
tic technique.
These skills are easily transferable from culture based con-
ventional microbiologic testing to real-time PCR testing. At
the Mayo Clinic we noted that providing training on the basics
of accurate pipetting was fundamental, especially for those
lacking experience with micropipetting.
Well-written training materials, including training checklists
and detailed standard operating procedures for each real-time
PCR test, should be available. The training checklist serves to
standardize the training of all personnel. At the Mayo Clinic, FIG. 5. Work flow algorithm for processing specimens for the lab-
we believe that identifying a technical expert to provide one- oratory diagnosis of group A streptococcal infections by real-time
on-one training for real-time PCR is critical. Technologists are PCR.
required to successfully complete a panel of unknown samples
and perform the procedure under direct observation of the
technical expert to ensure flawless manipulations throughout August 2002 we replaced a conventional testing method (rapid
the procedure. They also are required to analyze a previous antigen screen with backup culture for rapid antigen negative
run of samples with a variety of unusual results. This allows results) with the LightCycler Strep-A assay (Roche Diagnostics
them to perfect their skills manipulating the computer soft- Corporation, Indianapolis, IN) (456, 499). This real-time PCR
ware associated with the real-time PCR instrument and en- test is as sensitive as the gold standard method, culture, and
sures consistent analysis and reporting of results. Overall, our provides same-day conclusive results for all patients, whereas
technologists have been very enthusiastic about implementa- the antigen/backup culture method required up to 48 h for a
tion of real-time PCR and excited to learn the new technology. conclusive result for the majority of patients. A simple lysis and
The availability of resources for troubleshooting is a consid- extraction method of the swab sample is performed using the
eration when selecting a molecular platform for the clinical S.E.T.S. tube (Roche Diagnostics Corporation) before testing
laboratory. Laboratory-developed tests require that the tech- in the LightCycler (Fig. 5).
nical resources to resolve problems related to the assay are Prior to implementing this real-time PCR assay we worked
available within the laboratory. Use of ASRs and United States extensively with our clinical colleagues to review the perfor-
Food and Drug Administration-approved tests allow the use of mance characteristics of this novel testing method, prepare
technical support resources available from the manufacturer educational materials for patients, determine an appropriate
for troubleshooting problems related to the assay or instru- testing schedule, and clarify what health care providers and
mentation. patients should expect. One misconception of our healthcare
providers that we had to clarify was that a majority of patients
had conclusive results using the conventional antigen screen.
Work Flow Design
In fact, the sensitivity of the rapid antigen test in our hands was
After selection and successful implementation of a real-time approximately 50% compared to culture. Therefore, during
PCR testing platform into the clinical laboratory, efficiencies the height of the streptococcal pharyngitis season, when the
may be gained by the implementation of additional tests which incidence of true-positive results was ⬇30%, ⬇85% of patients
use the same methodology. Different real-time PCR tests may had to wait up to 48 h for a conclusive result by culture. This
have subtle variations (e.g., differences in nucleic acid extrac- was because with ⬇30% true-positive cases and ⬇70% true-
tion procedures), but overall the methodology is very similar. negative cases, half of the true-positive cases, or 15%, were
This attribute reduces the personnel resources required for detected by rapid antigen and the other half, or 15% of true-
training and implementation of subsequent tests. positives, as well as the 70% true-negatives required detection
Not unlike other microbiology tests, work flow and testing by culture. Cultures are held for 48 h.
schedules for real-time PCR tests are determined by the arrival In discussions with our health care providers, it became clear
times of specimens into the laboratory, clinical urgency for the that there were other considerations beyond education on test
results, and laboratory hours of operation. Many of the real- performance. These included, specimen transportation and ar-
time PCR platforms are most efficiently run in a batch mode. rival times, (especially for samples coming from more distant
Some vendors provide identical thermocycling protocols for clinics) and issues related to the expeditious provision of an-
different ASRs for the same instrument. This allows testing for tibiotics (i.e., closing times of local pharmacies). Based on
multiple analytes within the same run, which enhances the these considerations, we implemented testing five times daily
efficiency of the testing platform. (4:00 a.m., 11:30 a.m., 3:00 p.m., 5:30 p.m., and 8:30 p.m.), 7
Example of work flow design: real-time PCR for detection of days/week, with additional batches set up as needed during the
group A streptococci from throat swabs. At the Mayo Clinic in peak season.
Additionally, we have eliminated most of the follow-up pro- COSTS

cedures required by health care providers by using the follow-
Royalties
ing innovative processes with our clinical colleagues (456).
Patients identify their preferred pharmacy at the time the Developers of laboratory-developed (also called home-
throat swab is collected. A standardized prescription form (in- brewed or in-house developed) real-time PCR assays for com-
cluding the antibiotics prescribed and the patient’s preferred mercial use (i.e., the patient will be charged for the test) should
pharmacy) is completed by the healthcare provider, which then determine whether patents exist for the genetic targets they
accompanies the specimen to the laboratory. At the conclusion wish to use for their assays. If such patents exist, licenses
of the test run, results are entered into the laboratory infor- and/or royalty fees may have to be executed with the inventors
mation system and transmitted to the patient’s electronic med- or assignees of the patents. In order to avoid license and
ical record. The results from the electronic medical record are royalty fees, some developers may wish to search for alterna-
delivered to a computerized message center, allowing patients tive nucleic acid targets that are not protected by patent. In
to obtain their secure results by telephone and pick up the situations where alternative targets that are not protected by
prescription prepared for them at their selected pharmacy. patent do not exist (e.g., resistance genes associated with an-
Prescriptions are faxed to numerous local and regional phar- timicrobial resistance), licenses and/or royalties may be too
macies by the clinical microbiology laboratory staff (Fig. 5). expensive or unattainable (e.g., exclusive license agreements).
In a comparison of the personnel required for performing Laboratorians should also determine whether a separate
rapid antigen test and back up culture of antigen negative royalty is required for performing PCR. Roche Diagnostics
specimens, we realized a savings of 2.1 full time equivalents. Corporation requires that PCR royalties be paid to them if
This is based on an annual testing volume of 26,000 tests with testing is used for commercial (non-research-related) pur-
the rapid antigen test being performed at four satellite loca- poses.
tions in Rochester, Minnesota. Performance of the Roche
Strep-A ASR test allowed us to centralize testing in one loca-
Reagents and Instrumentation
tion. This results in a more efficient process that saved person-
nel effort. The cost for reagents (i.e., polymerase enzymes, PCR prim-
In summary, the introduction of this rapid real-time PCR ers, fluorescent probes, and internal, positive, and negative
assay for detection of streptococcal pharyngitis has streamlined controls) may vary according to whether they are laboratory
both the testing procedure and resulted in significant person- developed, obtained from different wholesale suppliers, or pur-
nel savings in the laboratory. Most importantly, we have also chased as ASRs or kits from a common manufacturer. Gener-
implemented new procedures in tandem with this technology ally, if reagents are purchased as ASRs or kits they are more
that facilitate the expeditious provision of appropriate antimi- expensive; however, the quality and performance characteris-
crobial therapy for our patients. tics of these reagents should be more reliable and consistent
Example of work flow design: real-time PCR for detection of because they are produced under Good Manufacturing Prac-
herpes simplex and varicella-zoster infections. In contrast to tices as mandated by the FDA. Technical support for instru-
PCR testing for group A streptococci, the support of our clin- mentation and to some extent for assay reagents should be
ical colleagues implementing this test was less of an issue. This provided by the manufacturer. In the United States, technical
was due in part to the fact that conventional PCR had been support by manufacturers may be limited for ASRs compared
used for a number of years at our institution for detection of with FDA-approved kits. FDA guuidelines prohibit manufac-
HSV in spinal fluid as well as other viruses such as hepatis C turers of ASRs to provide standard protocols to U.S. labora-
virus, and human immunodeficiency virus. tories when ASRs are used for testing. Significant capital out-
We currently use commercially available ASRs for both vari- lay for purchase of instrumentation for real-time PCR is also
cella-zoster virus (Roche Diagnostics Corporation) and HSV required, though options for creative financing (e.g., reagent
(Roche Diagnostics Corporation) testing with the LightCycler rental agreements) may be possible with some vendors.
instrument. Testing is performed three to six times daily 6 days Frequently, the cost for real-time PCR reagents is signifi-
a week. Nucleic acid is extracted from the specimen using the cantly more than the cost for culture media used for traditional
automated MagNA Pure system (Roche Diagnostics Corpora- methods. However, costs for reagents and instrumentation
tion) by laboratory assistants in our initial processing area. may be obviated by savings in labor requirements in the labo-
Real-time PCR testing is performed and results entered into ratory and cost savings at the bedside due to higher sensitivity
the laboratory information system and transmitted to the pa- and more rapid turnaround time for results for real-time PCR
tient’s electronic medical record. In comparison to viral cul- tests compared with traditional culture-based methods.
ture, which may take 14 days or longer to complete, the ma-
jority of results are available the same day the specimen is
Personnel
received (118). For these real-time PCR virology assays, effi-
ciencies were gained in faster turnaround time and improved Often the amount of labor required for performing real-time
sensitivity compared to culture. Downstream, this can lead to a PCR assays is considerably less than that required for culture-
decrease in the number of tests requested on a patient to make based assays. As previously emphasized, replacement of a
a diagnosis, and potentially shorter hospital stays. Additionally rapid antigen/culture method in our laboratory by a real-time
the personnel requirements were 2.5 times greater for the PCR assay for the detection of group A streptococcus in throat
standard viral culture method versus the real-time PCR swabs significantly reduced labor requirements. The antigen-
method. culture method required 4.0 full-time equivalents; in contrast,
TABLE 7. Reimbursement
Medicare reimbursement 2005 Mayo Clinic list price
Technique Organism CPT code
(U.S. dollars) (U.S. dollars)
Amplified probe CMV 87496 27–49 228

EBV 87798 27–49 228
HSV 87529 27–49 228
VZV 87798 27–49 228
Enterovirus 87798 27–49 228
JC and BK virus 87798 27–49 228
Group A streptococci 87651 27–49 NAa
Bordetella pertussis/Bordetella parapertussis 87801 55–98 271
Borrelia burgdorferi 87476 27–49 320
VRE 87798 27–49 271
Tropheryma whipplei 87798 27–49 321
Ehrlichia spp. 87798 27–49 222
Quantification CMV 87497 39–59 307

EBV 87799 27–59 221
a
NA, not available through the Mayo Clinic reference laboratory.
a LightCycler real-time PCR assay (LightCycler Strep-A, In a similarly designed study, Barenfanger and colleagues dem-
Roche Diagnostics Corporation) requires 1.9 full-time equiv- onstrated that provision of more rapid results for bacterial
alents. Another example relates to the detection of herpes identification and antimicrobial susceptibility decreased length
simplex virus. The personnel time requirement for shell vial of hospital stay for patients an average of 2.0 days, decreased
culture assay was 2.5 times that required for a real-time PCR the mortality rate from 9.6% to 7.9%, and resulted in an
assay (LightCycler HSV1/2, Roche Diagnostics Corporation) annual cost savings of $4,189,500 (26). Studies such as these
(456). will be important for verifying whether rapid results generated
by real-time PCR testing platforms will have similar financial
Cost Savings at the Bedside impacts.
Cost effectiveness studies are required to determine the cost

Coding and Reimbursement
savings at the bedside for real-time PCR compared with con-
ventional testing methods for diagnosis of infectious disease. Table 7 provides a list of commonly used Current Proce-
Intuitively, if a diagnosis can be provided sooner and more dural Terminology codes and respective Medicare reimburse-
reliably (higher sensitivity and specificity), patients who re- ment amounts for real-time PCR tests used for infectious dis-
quire antimicrobial therapy will receive it sooner. As well, less ease diagnosis at the Mayo Clinical Microbiology Laboratory.
auxiliary testing should be required (e.g., additional infectious Although our laboratory has not encountered problems with
diseases tests such as cultures) and patients should have less reimbursement for these tests through our regional Medicare
morbidity and therefore fewer costs related to supportive ther- carrier, laboratories are encouraged to check with their local
apy (e.g., intensive care related to a delay in diagnosis of Medicare carriers to determine whether these tests will be
sepsis). Providing a negative result sooner can have important reimbursed. In general, Medicare reimbursement is greater for
implications for the overprescription of antibiotics. For exam- real-time PCR tests than direct antigen immunoassays or cul-
ple, if a real-time PCR test result can more quickly rule out the ture-based tests.
pathogen compared with a culture-based method, then the
clinician may be less inclined to use empirical antibiotics or if
APPLICATION OF REAL-TIME PCR FOR CLINICAL
empirical antibiotics are used the duration of treatment may be
MICROBIOLOGY TESTING
shortened. In patients who are suspected of having communi-
cable diseases, such as tuberculosis, expensive infection pre- An increasing volume of published clinical studies demon-
caution (isolation) requirements may be discontinued sooner, strate the utility of real-time PCR for diagnosing microbial
if the infectious pathogen is ruled out more quickly by real- pathogens. The high sensitivity (in some instances greatly ex-
time PCR than is possible with conventional testing. ceeding sensitivity for conventional testing methods) and high
Although cost effectiveness studies have not been published specificity along with a short turnaround time for results and
for real-time PCR testing, two seminal studies indicate that ease of performance, make real-time PCR an attractive re-
more rapid provision of microbiology results can result in sub- placement method for conventional culture and antigen-based
stantial cost savings. Doern and colleagues showed that same- assays.
day versus overnight provision of results for bacterial identifi- Table 8 displays a comparison of selected real-time PCR
cation and antimicrobial susceptibility to physicians at their assays developed in our laboratory to gold standard (culture-
institution resulted in statistically significantly fewer laboratory based) assays for selected pathogens. Shown are the increases
studies ordered per patient, and a statistically significant sav- in sensitivity and time requirements for performing real-time
ings per patient hospitalization of ⬃$4,000. This represented PCR versus culture-based assays (71). It should be emphasized
an annual cost savings of $2,403,162 for their institution (97). that specimen preparation (i.e., extraction of nuclic acid) adds
TABLE 8. Comparison of LightCycler assays to culture-based Frequently, the sensitivity of these real-time PCR assays
assays for selected pathogensa equals or exceeds the standard antigen or culture method and
Sensitivity Analytical turnaround time the turnaround time for results is much shorter than the cul-
Organism (reference)
increase for ture-based method. One notable example is the time savings,
LightCycler LightCycler b Culture-based
(%) (min) assay (days) enhanced sensitivity, and reduced personnel requirements pre-
viously described in this review for detection of group A strep-
Group A streptococci (13) 7 30 1–2 tococcus from throat swabs (499). Recently, commercially pro-
Legionella spp. (5) (BALc) 0d 45 2–14
Bordetella pertussis (6) 219 50 3–7
duced analyte-specific reagents or kits have become available
Vancomycin-resistant 120 e 45 2–3 for real-time PCR for group A streptococcus detection in
enterococci (fecal throat swabs (LightCycler Strep-A assay for use with the Light-
surveillance samples) Cycler, Roche Diagnostics Corporation) and group B strepto-
(451) coccus detection in vaginal/anal swabs (IDI-StrepB assay for
Varicella-zoster virus (skin) 91 45 2–5
(117) use with the SmartCycler, Infectio Diagnostics, Inc., Sainte-
Herpes simplex virus (skin, 23 45 1–5 Foy, Quebec, Canada; LightCycler StrepB pts1, Roche Diag-
genital) (115) nostics Corporation).
Cytomegalovirus (urine) 88e 45 ⱕ1
a
Adapted and modified from reference 73 with kind permission of Springer Slow-Growing or Poorly Culturable Bacteria
Science and Business Media.
b
The time shown is for the assay only and does not include the time required
for extraction of nucleic acid, which may vary from minutes to over 1 hour Conventional PCR provided limited applications for bacte-
depending on the specimen type, organism, and whether a manual or automated rial diagnostics due to the technical difficulties required for
method is used. performing the procedure and the time delay in producing a
c
BAL, bronchoalveolar lavage.
d
Only archived culture-positive samples were available for this comparison. final result. Moreover, certain specimens, such as sputum and
e
Based on unpublished clinical studies at the Mayo Clinic, Rochester, Minn. feces, were difficult to test due to intrinsic substances that
inhibited PCR chemistry and therefore the sensitivity of the
assay was significantly compromised. As a result, conventional
to the overall time requirements for assays and the time for PCR testing methods were limited to bacteria that are difficult
specimen preparation is not accounted for in Table 8. The time to culture or grow slowly (e.g., Anaplasma phagocytophila, Bar-
requirements for extraction of nucleic acid can vary from min- tonella henselae, Bordetella pertussis, Borrelia burgdorferi sensu
utes to over an hour and depends on whether a manual or stricto, Ehrlichia spp., Legionella spp., Mycoplasma pneu-
automated method is used, the specimen type, and the organ- moniae, or Chlamydophila pneumoniae) or for which culture
ism or organisms targeted. methods did not exist (e.g., Tropheryma whipplei). A number of
The following discussion contains a comprehensive summary studies have now been conducted which demonstrate the abil-
of peer-reviewed publications and abstracts, which have eval- ity of real-time PCR for detecting these fastidious organisms as
uated real-time PCR assays in the clinical laboratory. listed in Table 9.
BACTERIA OTHER THAN MYCOBACTERIA SPP. Agents of Community-Acquired Pneumonia

Table 9 displays peer-reviewed publications, which have ap- There has been considerable interest to apply real-time PCR
plied real-time PCR testing platforms for the detection of for testing of the bacterial agents of community-acquired
bacterial pathogens or antibiotic resistance genes. For each pneumonia, especially those agents associated with atypical
publication, the specimen source(s) evaluated is listed. Addi- pneumonias. The main reason for this is that these bacterial
tionally, specific information on the real-time PCR method is pathogens, which include Chlamydophila pneumoniae, Myco-
provided including the target nucleic acid sequence, the instru- plasma pneumoniae, and Legionella spp., can be difficult to
ment and detection chemistry employed and whether the assay isolate on culture due to special growth requirements. Addi-
is available from a commercial manufacturer as an analyte- tionally, due to the slow growth of these organisms, the time
specific reagent or kit. required for a final result may be prolonged. Conventional
PCR has been demonstrated to provide excellent sensitivity for
detecting these organisms in throat swabs and respiratory se-
General Bacteria
cretions (325, 393). Real-time PCR has been shown to be as
Recent studies have demonstrated the advantages of real- effective as culture or serologic methods for detecting these
time PCR testing for bacterial agents that traditionally have pathogens, as witnessed by the relatively large number of pub-
been identified by direct immunoassay techniques (antigen lished studies presented in Table 9.
testing methods: e.g., group A streptococcus from throat Streptococcus pneumoniae, the most common agent associ-
swabs, Clostridium difficile toxin from feces, or Vero toxin or ated with typical (lobar) community-acquired pneumonia, is
Escherichia coli O157:H7 antigen from feces). Other recent easily detected by PCR in respiratory secretions. A drawback is
studies have shown the utility of real-time PCR assays for that Streptococcus pneumoniae can colonize the pharynx in the
organisms for which the routine culture method is focused on absence of disease, so qualitative detection of this organism by
identifying a single pathogen from a specimen (e.g., group A PCR may result in results that are specific for infection, but do
streptococcus from throat swabs, group B streptococcus from not necessarily connote disease (325). As a step towards solv-
vaginal/anal swabs). ing this problem, a recent study, using quantitative real-time
182
TABLE 9. Testing platforms for bacteria
Assay
Refer- Conventional Labor- Turnaround

Category Organism Specimena Sensitivity vs.
ence(s) test methodb atory time vs.
ASR Kit Target Instrument Chemistry conventional Comments
devel- conventional
method
oped method
ESPY ET AL.
General Group A 499 Throat swabs Antigen Yes pts1 gene LightCycler Dual FRET Greater Faster Real-time PCR assay
bacteria streptococcus detection/ (Roche)c hybridization required half the
(Streptococcus culture probes personnel
pyogenes)
Group B 30, 211, Anal, vaginal Culture Yes cfb gene LightCycler Dual FRET Equal Faster Susceptibility testing still
streptococcus 212 or hybridization required in patients
(Streptococcus combined probes allergic to penicillin
agalactiae) anal/vaginal
swabs
g
Abstract Anal, vaginal Culture Yes cfb gene LightCycler Dual FRET Equal Faster Susceptibility testing still
or (I.D.I.)d and hybridization required in patients
combined SmartCycler probes allergic to penicillin
anal/vaginal (LightCycler),
swabs molecular
beacons
(SmartCycler)
Shiga (Vero) 387 Feces EIA, Yes stx1, stx2, stx2e LightCycler Dual FRET Greater (EIA or Faster Differentiation of stx
toxin-producing conventional hybridization conventional (conventional genes by melting
Escherichia coli PCR assay probes PCR) PCR) curve analysis
28 Feces Culture Yes stx1, stx2 SmartCycler Molecular Greater Faster Multiple assay
beacons
Slow-growing Bartonella 559 Blood Culture, Yes ribC gene LightCycler SYBR Green I Greater Faster Serology may still be
or poorly henselae (endocar- conventional (culture or used in PCR-negative
culturable ditis) PCR assay conventional cases
bacteria PCR)
Bordetella 406 Isolates Culture Yes IS481 LightCycler Dual FRET Equal NAi Assay for IS481 also
pertussis (B. pertussis) hybridization detects but cannot
probes differentiate
Bordetella holmesii
from Bordetella
pertussis
7 Nasopha- Culture Yes IS481 (B. LightCycler Dual FRET Greater Faster Assay for IS481 also
ryngeal pertussis) hybridization detects but cannot
swabs probes differentiate
Bordetella holmesii
from Bordetella
pertussis
63 Nasopha- Culture Yes IS481 (B. ABI Prism TaqMan probes Greater Faster Assay for IS481 also
ryngeal pertussis) 7700 detects but cannot
swabs differentiate
Bordetella holmesii
from Bordetella
pertussis
Bordetella 405 Nasopha- Culture Yes IS481 (B. LightCycler Dual FRET Greater Faster Assay for IS481 also
pertussis and ryngeal, pertussis), hybridization detects but cannot
Bordetella throat or IS1001 (B. probes differentiate
parapertussis perinasal parapertussis) Bordetella holmesii
swabs, from Bordetella
BAL pertussis
CLIN. MICROBIOL. REV.
71 Nasopha- DFA, culture Yes IS481 (B. LightCycler Dual FRET Greater (DFA Faster Assay for IS481 also
ryngeal pertussis), hybridization or culture) detects but cannot
swabs or IS1001 (B. probes differentiate Bordetella
aspirates, parapertussis) holmesii from
sputa, Bordetella pertussis
VOL. 19, 2006
throat
swabs,
bronchial
washings,
pleural
fluid,
tracheal
aspirates
233 Nasopha- Culture Yes IS481 (B. LightCycler Dual FRET Greater Faster Assay for IS481 also
ryngeal pertussis), hybridization detects but cannot
swabs IS1001 (B. probes differentiate Bordetella
parapertussis) holmesii from
Bordetella pertussis
450 Nasopha- DFA, culture, Yes Yese IS481 (B. LightCycler Dual FRET Greater (DFA, Faster (DFA, Assay for IS481 also
ryngeal conventional pertussis), hybridization culture, culture, detects but cannot
swabs PCR assay IS1001 (B. probes conventional conventional differentiate Bordetella
parapertussis) PCR) PCR) holmesii from
234 Nasal, Culture Yes IS481 (B. ABI Prism TaqMan Probes Greater Faster Assay for IS481 also
pharyngeal pertussis) 7700 detects but cannot
or IS1001 (B. differentiate Bordetella
nasopha- parapertussis) holmesii from
ryngeal Bordetella pertussis
swabs,
nasopha-
ryngeal
aspirates
233 Nasopha- Culture Yes IS481 (B. ABI Prism TaqMan Probes Greater Faster Assay for IS481 also
ryngeal pertussis) 7700 detects but cannot
swabs IS1001 (B. differentiate Bordetella
parapertussis) holmesii from
486 Nasal swabs, Culture, Yes IS481 (B. iCycler Molecular Greater Faster (culture, Assay for IS481 also
sputa and conventional pertussis), beacons (culture, conventional detects but cannot
naso- PCR assay IS1001 (B. conventional PCR) differentiate Bordetella
pharyngeal parapertussis) PCR) holmesii from
aspirates Bordetella pertussis
Tropheryma 126 Lymph node, Conventional Yes 16S–23S LightCycler SYBR Green I Equal NA
whipplei duodenal test method rRNA gene
biopsy or intergenic
cardiac spacer
valve region and
rpoB gene
Agents of Chlamydophila 19 Sputa, BAL Four Yes ompA ABI Prism TaqMan probes See comments Faster Real-time PCR assay
community- pneumoniae fluid conventional 7700 produced most
aquired PCR assays consistent positive
pheumonia results when replicate
testing of samples
performed
Continued on following page

REAL-TIME PCR
183
184
TABLE 9—Continued
Assay
Refer- Conventional Labo- Turnaround

ence(s) test methodb ratory time vs.
devel- conventional
method
oped method
494 Peripheral Culture, two Yes ompA (two ABI Prism TaqMan probes See comments Faster VD4 real-time
ESPY ET AL.
blood nested domains, 7700 assay detected

mono- conventional VD2 and most positives
nuclear PCR assays VD4)
cells,
oropha-
ryngeal
swabs
536 Sputa, naso- Conventional Yes pst1 genomic ABI Prism TaqMan probes Greater Faster
pharyngeal PCR assay fragment 7700
secretions,
throat
swabs,
bronchial
aspirations,
BAL fluid,
pleural
fluid
242 Sputa, naso- Conventional Yes ompA gene ABI Prism TaqMan probes Greater Faster
pharyngeal, PCR assay 7700
throat
specimens
407 BAL fluid, Culture, Yes 16S rRNA LightCycler Dual FRET Equal Faster
bronchial serology gene hybridization
secretions, probes
tracheal
secretions,
gargle
water, and
throat
swabs
328 Nasopha- Conventional Yes pmp4 gene LightCycler Dual FRET Equal Faster
ryngeal PCR assay hybridization
aspirates probes
Legionella 536 Sputa, naso- Conventional Yes 16S rRNA ABI Prism TaqMan probes Equal Faster
pneumophila pharyngeal PCR assay gene 7700
secretions,
throat
swabs,
bronchial
aspirations,
BAL fluid,
pleural
fluid
535 BAL fluid, DFA, culture Yes 16S rRNA LightCycler Dual FRET Greater Faster Results compared
tracheal of urinary gene hybridization to combination
secretions, antigen probes of results for
bronchial DFA, culture,
secretions urinary antigen
547 Sputa, BAL DFA, culture Yes mip gene LightCycler Dual FRET Equal (culture), Faster
fluid, hybridization greater
endo- probes (DFA)
tracheal
aspirates
Legionella 175 Sputa DFA, culture Yes 23S-5S spacer ABI Prism TaqMan probes Slightly less Faster Considering only
pneumophila of urinary region 7700 sensitive positive result
and Legionella antigen for DFA, cul-
spp. ture or urinary
antigen real-
time PCR was
VOL. 19, 2006
94% sensitive,
equal to culture
396 Sputa, BAL Culture, Yes 16S rRNA LightCycler SYBR Green I Equal (culture, Faster (culture,
fluid conventional gene conventional conventional
PCR assay PCR) PCR)
391 Induced Culture Yes 16S rRNA LightCycler Dual FRET Greater Faster
sputa, BAL gene hybridization
fluid probes
408 BAL fluid Culture Yes 16S rRNA LightCycler Dual FRET Equal Faster
gene hybridization
probes
167 BAL fluid, DFA, culture Yes mip gene and LightCycler Dual FRET Equal (culture Faster (real-time
open lung for BAL, 5S gene hybridization for BAL), PCR faster
biopsy DFA, cul- probes greater (DFA than all
ture, War- for BAL), methods)
thin starry greater (DFA,
stain, in situ Warthin
hybridization starry stain
for open lung
biopsy), less
(culture and
in vitro
hybridization
for open lung
biopsy)
Mycoplasma 536 Sputa, naso- Conventional Yes 16S rRNA ABI Prism TaqMan probes Equal Faster
pneumoniae pharyngeal PCR assay gene 7700
secretions,
throat
swabs,
bronchial
aspirations,
BAL fluid,
pleural
fluid
163 Nasopha- Serology Yes 16S rRNA ABI Prism TaqMan probes Slightly less Faster (serology, 95% sensitivity
ryngeal conventional gene 7700 sensitive conventional
secretions, PCR assay (serology, PCR)
pharyngeal conventional
swabs and PCR)
blood
501 Sputa, throat Conventional Yes PI gene LightCycler Dual FRET Equal Faster
washings, PCR assay hybridization
throat probes
swabs
484 Throat swabs Serology, Yes PhHV iCycler Molecular Greater Faster
conventional sequences beacons (serology),
PCR assay equal
(conventional
PCR)
Streptococcus 148 Nasopha- Culture Yes ply gene ABI Prism TaqMan probes See comments Faster Number of
pneumoniae ryngeal 7700 organisms
swabs detected by
real-time PCR
REAL-TIME PCR
correlated with
numbers
185

TABLE 9—Continued 186
Assay

devel- conventional
method
oped method
detected by
semiqualitative
ESPY ET AL.
culture. It
remains unclear
whether these
results
correlate with
pneumococcal
lower
respiratory
tract disease
Agents of Neisseria 77 CSF, plasma, Culture Yes ctrA gene ABI Prism TaqMan probes Less (sensitivity Faster Additional cases
meningitis meningitidis serum, 7700 ⫽ 80%) detected in
whole culture-negative
blood samples
155 CSF, plasma, Culture Yes ctrA gene, ABI Prism TaqMan probes Slightly less Faster Additional cases
serum IS1106, siaD 7700 (91–93%) detected in
gene culture-negative
samples
318 CSF Culture Yes 16S rRNA LightCycler Dual FRET Equal Faster
gene, sacC, hybridization
siaD, porA probes
genes
Haemophilus 77 CSF, plasma, Culture Yes bexA gene ABI Prism TaqMan probes Equal Faster Additional cases
influenzae serum, 7700 detected in
whole culture-negative
blood cases
Streptococcus 77 CSF, plasma, Culture Yes ply gene ABI Prism TaqMan probes Slightly less Faster Additional cases
pneumoniae serum, 7700 (sensitivity ⫽ detected in
whole 92%) culture-negative
blood cases
Bacteria as- Borrelia 299 Skin biopsies Culture Yes recA gene LightCycler SYBR Green I Not provided Faster Melting curve
sociated burgdorferi (erythema analysis used to
with tick- sensu stricto migrans) identify differ-
borne dis- ent Borrelia spp.
eases
Borrelia garinii 380 Bacterial Culture Yes recA gene LightCycler SYBR Green I Equal Faster Melting curve
and Borrelia isolates analysis used to
afzelii identify differ-
ent Borrelia spp.
Ehrlichia 288 Blood, tissue N.A. Yes 16S rRNA iCycler TaqMan probes See comments Faster Previous
chaffeensis gene “positive”
samples were
all identified by
real-time PCR
Potential Bacillus anthracis 29, 119 Bacterial Culture Roche f capB, pagA LightCycler Dual FRET Equal Faster Autoclaving of
agents of isolates genes hybridization bacteria before
bioterror- probes real-time PCR
ism test did not
affect sensitivity
of assay
350 Spiked nasal Culture Yes rpoB, lef genes LightCycler Dual FRET Equal Faster
swab speci- hybridization
mens probes
98 Spiked swabs Culture Yes rpoB gene LightCycler Dual FRET Equal Faster
hybridization
VOL. 19, 2006
probes
371 Bacterial Culture Yes rpoB, pagA, LightCycler Dual FRET Equal Faster
isolates capC genes hybridization
probes
389 Bacterial Culture Yes rpoB gene LightCycler Dual FRET Equal Faster
isolates hybridization
probes
254 Bacterial capC gene LightCycler SYBR Gold Equal Faster
isolates
111 Bacterial Culture Yes rpoB, pag, ABI Prism TaqMan probes Equal Faster
isolates capC genes 7000, 7700
181 Bacterial Culture Yes Undisclosed ABI Prism TaqMan probes Equal Faster
isolates chromosomal 7700,
and pXO2, LightCycler,
pXO7 SmartCycler
targets
Yersinia pestis 493 Bacterial Culture 16S rRNA, LightCycler Dual FRET Equal Faster
isolates pla, capI, hybridization
ymt genes probes
306 Bacterial Culture Undisclosed LightCycler TaqMan probes Equal Faster
isolates target and
RAPID h
Bacterial MRSA 409 Bacterial Culture-based Yes mecA gene LightCycler Dual FRET Equal Faster
antibiotic isolates susceptibility hybridization
resistance testing probes
genes
448 Blood cul- Culture-based Yes mecA and LightCycler Dual FRET Equal Faster
tures which susceptibility sa442 genes hybridization
showed testing probes
gram-posi-
tive cocci
in clusters
472 Blood cul- Culture-based Yes mecA and LightCycler SYBR Green I Equal Faster
tures which susceptibility sa442 genes
showed testing
gram-posi-
tive cocci
in clusters
123 MRSA selec- Culture-based Yes nuc gene LightCycler Dual FRET Slight decrease Faster (selective
tive broth susceptibility hybridization (sensitivity ⫽ broth and
(oxacillin) testing probes 93.3%) real-time PCR
inoculated required ⬃24
with speci- h vs. longer
mens from periods for
wounds, conventional
abscesses, culture)
anterior
nares, peri-
neum,
urine, cath-
eter inser-
tion sites,
skin and
soft tissues,
REAL-TIME PCR
sputa, tra-
chea

187
Assay

devel- conventional
method
oped method
150 Bacterial Culture-based Yes mecA gene LightCycler Dual FRET Equal Faster
isolates susceptibility hybridization
ESPY ET AL.
testing probes
219 Bacterial Conventional Yes mecA gene ABI Prism TaqMan probes Equal Faster
isolates PCR assay 7700
130 Nasal or Culture-based Yes mecA and ABI Prism TaqMan probes Equal Faster Prior to real-time
inguinal susceptibility femA genes 7700 PCR,
swabs testing Staphylococcus
aureus cells are
selectively
removed from
swabs by a
unique
immunocapture
technique
VRE 361 Bacterial Conventional Yes vanA and LightCycler Dual FRET Equal Faster
isolates PCR assay vanB genes hybridization
probes
362 Rectal swabs Yes vanA and LightCycler Dual FRET Greater Faster
with and vanB genes hybridization
without probes
enrichment
broth
451 Culture Yes vanA and LightCycler Dual FRET Greater Faster
(Roche) vanB genes hybridization
probes
Extended 394 Bacterial Culture-based Yes blaSHV genes LightCycler Dual FRET Greater Faster
spectrum beta- isolates susceptibility hybridization
lactamases testing probes
Penicillin 214 CSF Culture-based Yes pbp2b LightCycler SYBR Green I Greater Faster Detection of
resistance in susceptibility php2b gene
Streptococcus testing associated with
pneumoniae susceptibility
Penicillin 460 Bacterial DNA Yes penA gene LightCycler Dual FRET Equal Faster penA gene codon
resistance in isolates (sequencing codon 566 hybridization 566 mutation
Neisseria of penA mutation probes associated with
meningitides gene) penicillin
resistance
detected by
melting curve
analysis
Fluoroquinolone 248 Bacterial Culture-based Yes grlA gene SmartCycler Molecular Equal Faster
resistance in isolates susceptibility mutations beacons
Staphylococcus testing
aureus
Fluoroquinolone 275 Bacterial DNA Yes gyrA gene LightCycler Dual FRET Equal Faster Mutations
resistance in isolates sequencing mutations hybridization detected by
Yersinia pestis of gyrA gene probes melting curve
analysis
Obligately Clostridium 27 Feces Cytotoxicity Yes tcdA and tcdB SmartCycler Molecular Slightly less Faster
anaerobic difficile assay genes beacons (sensitivity ⫽
bacteria 97%)
PCR, demonstrated that the numbers of Streptococcus pneu-

moniae organisms detected by real-time PCR in nasopharyn-
geal secretions correlated with the numbers detected by semi-
quantitative cultures (150). Other prospective clinical studies
are required to support these findings. It could be argued that
real-time PCR could likewise detect patients colonized but not
infected with group A streptococcus. However, a study previ-
ously described in this review showed that all patients with
group A streptococcus detected by real-time PCR had clinical
criteria for streptococcal pharyngitis (499).
RAPID refers to “ruggedized” advanced pathogen identification device, which is a field-deployable real-time PCR assay platform (Idaho Technology, Salt Lake City, Utah).
Agents of Meningitis
C. Ménard, F. J. Picard, J. Frenett, M. Gagnon, D. B. Ke, M. Ouellette, P. H. Roy, and M. G. Bergeron, Am. Soc. Microbiol. 100th Annu. Meet., abstr. C-193, 2000.
The significant mortality and morbidity associated with bac-

terial meningitis requires rapid diagnosis. Real-time PCR pro-
vides a much more rapid result than culture, which is the gold
standard. Additionally, the sensitivities for detecting the major
bacterial pathogens associated with meningitis (Neisseria men-
ingitidis, Streptococcus pneumoniae, and Haemophilus influen-
zae) for most studies listed in Table 9 equal culture. Impor-
tantly, in cases of meningitis where antibiotics are provided
Prototype assay evaluated for this study now available as ASR: LightCycler Bordetella IS481/1001 assay (Roche Diagnostics Corporation).
before cultures are obtained, PCR may be particularly advan-

tageous as it can be positive, whereas culture is negative.
IDI-Strep B Assay (Infectio Diagnostics, Inc., Sainte-Foy, Quebec, Canada) for use with SmartCycler (Cepheid, Sunnyvale, CA).
Potential Agents of Bioterrorism

The intentional release of anthrax spores in the U.S. mail
system in the fall of 2001 catapulted the development of real-
time PCR assays for the rapid detection of potential agents of
bioterrorism both in human specimens and environmental
samples. Scientists at the U.S. Centers for Disease Control as
well as scientists in the private sector have developed a number
of highly sensitive and specific real-time PCR assays for detec-
tion of these agents, including Bacillus anthracis and Yersinia
pestis, as shown in Table 9. Importantly, these assays provide a
LightCycler Bacillus anthracis detection kit (Roche Applied Science, Indianapolis, IN).
result much sooner than standard culture methods. Addition-

EIA, enzyme immunoassay; DFA, direct fluorescent antibody; N.A., not applicable.
ally, at least two papers have described the biosafety advan-

LightCycler Strep-A assay (Roche Diagnostics Corporation, Indianapolis, IN).
tages of using a real-time PCR testing platform versus culture

for detection of B. anthracis. In these studies the sensitivity of
a real-time PCR assay (119) or conventional PCR assay (125)
was not affected if samples were autoclaved before testing; the
biohazard concern for exposure to Bacillus anthracis was obvi-
ated because cultures of autoclaved samples were negative. A
limitation of real-time PCR studies for agents of bioterrorism
BAI, bronchoalveolar lavage; CSF, cerebrospinal fluid.
has been the lack of testing human specimens. All of the

studies listed in Table 9 for Bacillus anthracis and Yersinia
pestis evaluated isolates or spiked human specimens.
Bacterial Antibiotic Resistance Genes

Infections caused by methicillin (oxacillin)-resistant Staphy-
lococcus aureus (MRSA) and vancomycin-resistant Enterococ-
cus spp. (VRE) have worse outcomes and higher associated
costs than infections caused by methicillin (oxacillin)-suscepti-
NA, not applicable.
ble Staphylococcus aureus or vancomycin-susceptible Entero-

coccus spp. (74). Unfortunately, the rates of MRSA and VRE
continue at an accelerating pace in U.S. hospitals (96). Guide-
lines published in May 2003 by the Society of Healthcare Ep-
idemiologists of America advise active surveillance programs
h
d
a
b
c
g
f
in health care institutions for detection of MRSA and VRE

TABLE 10. Real-time PCR methods for mycobacteriologya 190
Clinical Turnaround
Method or sensitivity vs. time vs.
Specimen Technology Target Status Conventional method Reference(s) Comments
species conventional conventional
method method
Genus screen Culture LightCycler FRET 16S rRNA HB Culture NA Faster 243 3 primer/probe sets to detect
HP Mycobacterium spp.,
Mycobacterium avium,
ESPY ET AL.
Mycobacterium tuberculosis
Genus screen Culture ABI 7700 molecular 16S rRNA HB Culture NA Faster 449 Also differentiation of Mycobacterium
beacons tuberculosis and comparison w/
Amplicor PCR
Genus screen Respiratory and ABI 7700 molecular IS6110, senX3, HB Culture Greater Faster 50 Genus screen and Mycobacterium
nonrespiratory beacons regX3 tuberculosis detection and
quantitation
Genus screen Respiratory ABI 7700 molecular 16S rRNA HB Culture Lower Faster 136 Genus screen and Mycobacterium
beacons tuberculosis detection
Genus screen Fine-needle aspirates, iCycler TaqMan ITS HB Culture Greater Faster 51 1 primer set/3 probes to detect
tissue Mycobacterium spp.,
Mycobacterium Culture LightCycler FRET hsp65, 16S HB Culture, biochemicals NA Faster 443 Multiplex assay to differentiate
abscessus HP rRNA or hsp65 RFLP Mycobacterium abscessus and
Mycobacterium chelonae
Mycobacterium Culture LightCycler FRET 16S rRNA HB Culture NA Faster 243 3 primer/probe sets to detect
avium complex HP Mycobacterium spp.,
Fine-needle aspirates, iCycler molecular ITS HB Culture, nucleic acid Greater Faster 51 1 primer set/3 probes to detect
tissue beacons probe Mycobacterium spp.,
Tissue ABI 7700 TaqMan hsp65 Expmtl Culture, nucleic acid Lower Faster 481 Research in birds
probe
Mycobacterium Culture LightCycler SYBR IS900 HB Culture NA Faster 353
avium subsp.
paratuberculosis
Fixed tissue GeneAmp 5700 IS900 HB Culture NA Faster 107 Investigation of sarcoid etiology
TaqMan
Mycobacterium Culture LightCycler FRET oxyR, narG, HB Culture, biochemicals NA Faster 462 Differentiation of Mycobacterium
bovis HP RD1 tuberculosis,
Mycobacterium bovis,
Mycobacterium bovis BCG
Mycobacterium Tissue LightCycler FRET IS6110 Expmtl Culture, histopathology Lower Faster 478 Research in cattle
bovis HP
bovis BCG HP RD1 tuberculosis,
Mycobacterium Culture LightCycler FRET hsp65, 16S HB Culture, biochemicals, NA Faster 443 Multiplex assay to differentiate
chelonae HP rRNA or hsp65 RFLP Mycobacterium abscessus and
Mycobacterium chelonae
Mycobacterium Tissue ABI 7700 TaqMan hsp65 Expmtl Culture/nucleic acid Lower Faster 481 Research in birds
genavense probe
Mycobacterium Tissue LightCycler FRET pra HB Microscopy Lower Equal 235
leprae HP
tuberculosis HP RD1 tuberculosis,
Mycobacterium Culture LightCycler FRET 16S rRNA HB Culture NA Faster 243 3 primer/probe sets to detect
tuberculosis HP Mycobacterium spp.,
complex Mycobacterium avium,
Culture LightCycler FRET ITS HB Culture NA Faster 236
VOL. 19, 2006
HP
Culture, sputum LightCycler FRET IS6110 HB Culture Lower Faster 169 Idaho Technology LightCycler
HP
Culture, respiratory LightCycler FRET ITS HB Culture Lower Faster 315 Also compared with Amplicor
HP PCR test
Culture, respiratory ABI 7700 molecular 16S rRNA HB Culture Lower Faster 449 Differentiation of Mycobacterium
and nonrespiratory beacons tuberculosis from NTMs;
comparison w/Amplicor PCR
Respiratory and ABI 7700 molecular IS6110, HB Culture Greater Faster 50 Genus screen and Mycobacterium
nonrespiratory beacons senX3, tuberculosis detection and
regX3 quantitation
Respiratory SmartCycler IS6110 HB Culture Equal Faster 70 Also compared with Amplicor PCR
TaqMan test
Respiratory and ABI 7700 molecular 16S rRNA HB Culture Lower Faster 136 Genus screen and Mycobacterium
nonrespiratory beacons tuberculosis detection
Respiratory and ABI 7000 TaqMan IS6110 HB Culture NR Faster 260 Comparison with AMTD PCR test
nonrespiratory
Sputum ABI 7700 molecular IS6110 HB Microscopy and NA Faster 94 Mycobacterium tuberculosis
beacons culture quantitation
Fine-needle aspirates, iCycler TaqMan ITS HB Culture Greater Faster 51 1 primer set/3 probes to detect
tissue Mycobacterium spp.,
Fixed tissue ABI 7700 molecular GM-CSF, Expmtl Conventional PCR NA Faster 562 Gene expression in mouse
beacons IFN-␥, etc. granulomas
Mycobacterium Culture ABI 7700 molecular gyrA, katG LD IS6110 RFLP NA Faster 412 Genotyping of Mycobacterium
tuberculosis beacons tuberculosis complex
complex
genotyping
Mycobacterium Culture LightCycler FRET katG, rpoB LD Broth dilution NA Faster 135, 300, INH and RIF resistance
tuberculosis HP 495, 496
complex
susceptibility
Culture LightCycler FRET rpoB LD Broth dilution NA Faster 106 RIF resistance
HP
Culture LightCycler SYBR 16S rRNA LD Broth dilution NA Faster 416 RIF resistance; 2 clinical specimens
Culture ABI 7700 molecular ahpC, oxyR, LD Broth dilution NA Faster 378 INH and RIF resistance
beacons inhA, kasA
Culture, sputum ABI 7700 molecular rpoB LD Broth dilution Equal Faster 110, 379 RIF resistance
beacons
Culture, sputum iCycler TaqMan katG LD Broth dilution Lower Faster 505 INH resistance
Culture, smear-positive iCycler molecular katG, inhA, LD Broth dilution Equal Faster 274 INH and RIF resistance
clinical specimens beacons rpoB
Respiratory ABI 7700 TaqMan rpoB, katG, LD Broth dilution Equal Faster 523 8 probes to detect INH, RIF, EMB
embB resistance
Mycobacterium Tissue ABI 7700 TaqMan IS2404 LD Culture, conventional Greater Faster 417
ulcerans PCR
a
Abbreviations: EMB, ethambutol; Expmtl, experimental; HP, hybridization probe; INH, isoniazid; LD, laboratory developed; NA, not applicable; NR, not reported; RIF, rifampin; RFLP, restriction fragment length
polymorphism; NTM, nontypeable mycobacteria; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN, interferon; HB, home brew; AMTD, amplified Mycobacterium tuberculosis direct test.
REAL-TIME PCR
191
carriers (327). Numerous studies have shown that surveillance comprehensive studies, Lachnik et al. designed genus-specific
for and isolation of carriers of MRSA and VRE can signifi- primers to target the 16S rRNA gene and were able to detect
cantly reduce the incidence of nosocomial infections by these 33 species of mycobacteria from culture (243). Two sets of
organisms and be cost-saving (327). sequence-specific FRET hybridization probes enabled further
Broad-based surveillance for MRSA or VRE, using culture- differentiation of the Mycobacterium tuberculosis complex and
based methods, may be especially demanding if not impossible two members of the Mycobacterium avium complex (Mycobac-
for most clinical microbiology laboratories. Moreover, the time terium avium and Mycobacterium avium subsp. paratuberculo-
required for a final result may take several days. Real-time sis) from the other 30 species analyzed. Mycobacterium intra-
PCR testing methods for both MRSA and VRE show great cellulare, another member of the Mycobacterium avium
promise for simplifying this process and providing same day complex, was not reliably identified using the assay because it
results. Notably with VRE, nearly all studies, which use either exhibited a melting temperature that was indistinguishable
conventional PCR or real-time PCR, show improved sensitiv- from that of several other species examined.
ities for detecting this pathogen from fecal specimens com- Detection of antitubercular drug resistance is vital to effec-
pared with culture. We recently demonstrated a 120% increase tive patient management. Real-time PCR offers the potential
in sensitivity using a commercially available ASR for detection to detect gene mutations responsible for drug resistance within
of VRE in perianal swabs versus culture (451). Two manufac- hours from patient specimens compared with the average of 2
turers have ASRs or kits available for use for VRE or MRSA weeks required for traditional susceptibility test methods. The
testing with real-time PCR testing platforms. Infectio Diagnos- rpoB and katG genes are the most common Mycobacterium
tic, Inc., has recently received FDA approval for a kit that can tuberculosis targets utilized in real-time PCR methods and
directly screen nasal swabs for MRSA using the SmartCycler well-known mutations in these genes correlate with resistance
instrument (IDI-MRSA). Roche Diagnostics Corporation pro- to rifampin and isoniazid, respectively (106, 110, 135, 353, 379,
vides separate ASRs for VRE detection (LightCycler vanA/ 495, 496, 505). The significance of other gene targets such as
vanB detection assay) and MRSA (LightCycler mecA detection kasA, ahpC-oxyR, and inhA for the prediction of isoniazid
assay), using the LightCycler instrument. resistance is still somewhat controversial (378). Torres et al.
used two sets of FRET hybridization probes to detect rpoB
mutations in 24 rifampin-resistant strains of Mycobacterium
MYCOBACTERIA
tuberculosis and another set of FRET hybridization probes to
The traditional approach for diagnosing mycobacterial in- detect katG mutations in 15 isoniazid-resistant Mycobacterium
fection relies upon the use of stains for detection of acid-fast tuberculosis strains (496). Additionally, Garcia de Viedma et al.
bacilli and growth in culture on solid and/or liquid media. used two sets of rpoB probes and one set of katG probes to
Mycobacteria isolated from cultures are identified using bio- detect rpoB and katG mutations, but in a single tube, for 29
chemical analysis, nucleic acid probes, or 16S rRNA gene se- resistant Mycobacterium tuberculosis isolates (135). Since not
quencing. This culture and identification process is time-con- all gene mutations conferring drug resistance are well charac-
suming, labor intensive, and in some cases lacks sensitivity or terized and are thus not amenable to PCR assay development,
specificity (236). Real-time PCR has the potential to signifi- traditional culture-based susceptibility testing methods are still
cantly change the current paradigm for mycobacteria identifi- required. However, the ability to predict rifampin and isonia-
cation by decreasing turnaround time for identification from zid resistance up to 2 weeks sooner than current methods for
weeks to hours while maintaining or improving upon diagnos- some isolates should have significant benefit for patient care.
tic sensitivity and specificity. A number of Mycobacterium tuberculosis real-time PCR as-
The majority of real-time PCR methods reported to date for says have been performed directly from patient specimens
mycobacteria focus on detection of the Mycobacterium tuber- rather than from culture (Table 10). Extraction and amplifica-
culosis complex and do not differentiate between the species tion of nucleic acids directly from patient specimens can de-
within the complex. Miller et al. developed a real-time PCR crease identification turnaround time from weeks to hours.
assay that rapidly and specifically detected the Mycobacterium Additional studies focused on extraction optimization from
tuberculosis complex directly from acid-fast smear-positive re- difficult specimen matrices (i.e., sputum, stool) will be required
spiratory specimens and from BacT/ALERT MP culture bot- to insure sufficient assay sensitivity when compared with cul-
tles (315). The same group then demonstrated that a similar ture. Presently there are approximately 129 currently recog-
Mycobacterium tuberculosis complex real-time assay tested on nized species and subspecies of Mycobacteria (http://www.dsmz
366 acid-fast smear-positive respiratory specimens had sensi- .de/species/gn250376.htm), and most have been implicated as
tivity and specificity equal to the AMPLICOR PCR assay human pathogens in the literature. Importantly, in many of the
(Roche Diagnostics Corporation) and required one-half the real-time PCR methods published to date, only a fraction of
time (3 h versus 6 h) to complete (69). Stermann et al. suc- clinically significant mycobacteria species have been tested to
cessfully designed sets of sequence specific primers and FRET determine whether they might be detected or might cross-react
hybridization probes to target polymorphisms within the narG, when looking for a specific target organism such as Mycobac-
oxyR, and RD1 loci of the Mycobacterium tuberculosis complex terium tuberculosis.
that allowed differentiation of Mycobacterium tuberculosis, My-
cobacterium bovis, and Mycobacterium bovis BCG, respectively VIRUSES
(462).
Several publications address the detection of mycobacteria The earliest applications of real-time PCR for testing in the
at the genus level (50, 136, 243, 449). In one of the most clnical microbiology laboratory were reported for the detection
of viruses. This was not unexpected as conventional PCR as- virus 6 (n ⫽ 2), and Epstein-Barr virus (EBV) (n ⫽ 1) DNA.
says were already recognized as the method of choice for Interestingly, of 22 of these patients with clinically diagnosed
detecting or quantifying some viruses, (e.g., detection of herpes encephalitis, two of three patients coinfected with HSV and
simplex virus in cerebrospinal fluid (CSF) or quantification of human herpesvirus 6 died, compared to 1 of 19 (5%) patients
cytomegalovirus in blood or plasma). As a result, extensive infected with only HSV (476).
literature exists describing the application of real-time PCR for A recent report indicated the value of using a comprehen-
detection and quantification of viral pathogens in human spec- sive menu of real-time PCR assays (cytomegalovirus [CMV],
imens. Therefore, this section represents the largest section in EBV, HSV-1, HSV-2, and varicella-zoster virus [VZV]) for
this review for any group of pathogens for which real-time testing CSF specimens by using a single LightCycler program
PCR has been applied. (466). Compared to conventional PCR, these real-time (Light-
Cycler) assays were rapid, simple, and convenient for testing
for herpesvirus DNA in the routine laboratory (467). Because
Qualitative Viral Assays
of overlapping clinical symptomatology produced by many of
Herpes simplex virus. Herpes simplex virus (HSV) produces the herpesviruses, in addition to other microbial targets, future
a wide spectrum of clinical manifestations; including genital, testing of CSF samples may incorporate assays for several
dermal, and central nervous system disease. It is the most targets, rather than for a single unique sequence of one organ-
common etiologic agent of sporadic focal central nervous sys- ism (3, 224). This would seem both economically and techni-
tem (CNS) disease; the mortality rate in untreated patients is cally feasible since the most labor and time-consuming event is
almost 70% but can be reduced to 20% with prompt antiviral generally the nucleic acid extraction step. Several target assays
therapy with acyclovir (447). Several gene targets have been can be performed after the extraction of a single specimen.
selected for the detection of HSV DNA by real-time PCR, We have used the Roche HSV LightCycler assay (LightCy-
including genes coding for glycoproteins B, C, D, and G, thy- cler herpes simplex virus 1/2 primer/hybridization probes;
midine kinase, DNA polymerase, and DNA binding protein LightCycler HSV 1/2 Template DNA) since its introduction to
(317). (Tables 11 and 12). the market in early 2003. All CSF specimens are processed and
Herpes simplex virus CNS disease. Several studies pub- assayed separately from genital and dermal sources to decrease
lished in the early to mid-1990s established PCR as the pre- the possibility of specimen to specimen contamination. In ad-
ferred method for diagnosing CNS disease (244, 384, 420). dition to primers and probes for PCR detection of HSV target
Collectively, these reports provided the credibility for the rou- nucleic acid (template DNA), probes specific for an internal
tine molecular diagnosis of CNS disease caused by an infec- control (also called the recovery template) target are also in-
tious agent and were the break-through evidence that PCR cluded in the reaction master mix (LightCycler HSV 1/2 re-
amplification technology could be applied for the detection of covery template, Roche Diagnostic Corporation). The internal
target sequences of other viruses and microbial agents which control is amplified by the same PCR primers which are used
could not be optimally detected by culture-based methods. for HSV target DNA. However, the internal control consists of
Other laboratories, including ours, confirmed these findings in target nucleic acid which is detected by a second pair of FRET
clinical evaluations and recognized that molecular amplifica- hybridization probes. These probes do not anneal with HSV
tion of HSV DNA (replacing brain biopsy inoculation in cell target DNA.
culture) was the new gold standard for the laboratory diagnosis Theoretically, the internal control added to a sample may be
of these infections (23, 90, 386, 431). Indeed, PCR technology, preferentially amplified especially in CSF specimens with low
including real-time PCR methods, facilitated our understand- copy levels of HSV DNA. To evaluate this possibility we com-
ing of the clinical spectrum of HSV CNS disease, which can pared the detection of HSV DNA in CSF in the presence and
vary from mild meningitis (Mollaret’s) to severe necrotizing absence of the internal control reagents. In the presence of an
encephalitis (224, 294, 317, 436, 475, 550). internal control, we found no difference in the detection of
General experience with these PCR assays indicate that CSF dilutions of HSV DNA from clinical specimens. Importantly,
specimens positive for HSV DNA were obtained from neo- both nucleic acid targets were detected with a range of 1 and
nates to elderly adults, although individuals 30 to 69 years of 2,000 copies of HSV DNA per reaction. At the two higher copy
age infected with this virus predominated (3, 317). The prev- levels excess HSV DNA was preferentially amplified, but not
alence of HSV-1 versus HSV-2 is likely dependent on the internal control target, by PCR (Table 13).
laboratory practice i.e., whether specimens are submitted to a HSV is detectable in CSF as early as 1 day after onset of
commercial laboratory (likely more severe CNS disease asso- clinical signs and symptoms. In most cases, DNA is present for
ciated with HSV-1) or a local community-based population an average of 4 days in CSF specimens of patients with CNS
from which specimens from a wider spectrum of HSV CNS disease; however, HSV DNA may persist for up to 30 days
infections are submitted for diagnostic evaluation (3, 317, 374, after the onset of CNS disease in some patients who have
477). received antiviral therapy. Persistence of HSV DNA may ac-
The molecular detection by PCR of coinfection due to other tually be fortuitous especially in cases that receive empirical
herpesviruses and microbial agents in CSF specimens of pa- treatment before PCR testing is performed (317, 477).
tients with CNS disease may have important medical implica- Herpes simplex virus dermal and genital disease. Recogni-
tions. Coinfections in the CNS may be associated with more tion of conventional PCR as the gold standard for detection of
severe disease in patients compared with infection with a single HSV DNA in CSF specimens was readily accepted by micro-
agent. For example, of 30 CSF specimens containing HSV biologists since the diagnosis of this virus infection was rarely
DNA, three samples also had coinfection with human herpes- obtained by cell culture techniques with these samples. Con-
TABLE 11. Detection of herpes simplex virus DNA by real-time PCR in cerebrospinal fluid specimens
No. of
Reference No. positive (%) Test platform/gene target Comparison studies Comments
specimens
Ryncarz et al. 380 42 (11.1) ABI Prism/glycoprotein B Of 58 total positive Probes specific for HSV-1
1999 (422) specimens, 13 were or HSV-2 were used to
positive only by differentiate genotypes
conventional PCR; 3 in separate reactions.
were positive only by
real-time PCR.
Kessler et al. 59 20 (33.9) LightCycler/DNA 20 positive samples The real-time PCR assay
2000 (218) polymerase were detected by both on the LightCycler
conventional and instrument proved to be
real-time PCR very quick and
methods. Four laborsaving (⬍1 h)
samples yielded compared with
discrepant results: conventional PCR (4 h).
two positive by
conventional PCR
only; two positive by
real-time PCR only.
Peter et al. 3,200 62 (1.6)a ABI Prism/DNA A preponderance of No inhibitors of PCR

2001 (374) polymerase HSV-2 over HSV-1 detected.
infections was
obtained in specimens
submitted for routine
diagnostic testing.
Aberle et al. 576 153 (26.6) ABI Prism/HSV-1 DNA Amount of virus varied Overall broad testing for
2002 (3) binding protein; HSV-2 among the individual different viruses in CSF
glycoprotein G diseases, associated clearly leads to a
with HSV-1, HSV-2, significant increase in
VZV, and CMV. Low the detection rate of
levels of EBV and viral CNS infections.
human herpesvirus 6
DNA were detected
in CSF specimens.
Kimura et al. 28 (from 20 (71.4)b ABI Prism/DNA A real-time PCR Patients with CNS
2002 (220) neonates) polymerase assay was applied to infection had the
quantitate the highest viral loads in
viral load in the CSF. Patients with
conventional HSV-2 infection had
PCR-positive more CNS involvement
specimens. and neurological
impairment and
higher viral load in CSF
than did patients with
HSV-1.
Stöcher et al. 30 8 (26.6) LightCycler/DNA Detection limits with Four LightCycler PCR
2003 (467) polymerase herpesvirus type- tests were developed
specific DNA spiked (CMV, EBV, HSV-1
into CSF, serum, or and HSV-2, VZV) with
plasma was 250 or standard test
500 DNA copies/ml. parameters.
a
Totals, 26 HSV-1 and 36 HSV-2.
b
Results obtained by conventional PCR: 6 of 7 CSF samples from patients with disseminated HIV disease; 11 of 11 patients with CNS infection; 3 of 10 CSF samples
with skin, eye, or mouth infection.
ventional PCR was not adapted for the detection of HSV in dermal and genital specimens for HSV with considerable sen-
dermal or genital sources, because cell culture or direct stain- sitivity and specificity and results can be available in less than
ing techniques (e.g., fluorescent antibody staining) were rela- one hour subsequent to nucleic acid extraction.
tively more sensitive for detecting HSV in these specimens and HSV is likely the most common virus recovered in cell cul-
conventional PCR would have been too work intense and ex- tures in the diagnostic virology laboratory; this virus accounts
pensive and require considerable time for a result. In contrast, for over 70% of the total virus isolates at the Mayo Clinic (454,
real-time PCR platforms now make it relatively easy to test 456). Combined data obtained for the detection of HSV using
195
TABLE 12. Comparison of cell culture and real-time PCR for the laboratory detection of herpes simplex virus infections from dermal and genital specimens
REAL-TIME PCR
Culture Real-time PCR

Reference No. positive No. of No. positive Comments
No. of specimens Cells Test platform/gene target
(%) specimens (%)
Ryncarz et al. 335 (genital tract) 162 (48.4) Human diploid fibroblast 335 248 (74.0) ABI Prism/glycoprotein B First study of laboratory diagnosis of genital
1999 (422) (24-well microtiter tract specimens by real-time PCR
plates) rapid culture
technique
Espy et al. 200 (genital, 160; 69 (34.5) MRC-5 (shell vials) 200 88 (44) LightCycler/DNA polymerase, Feasibility study performed by
2000 (118) dermal, 38; thymidine kinase developmental technologists
ocular, 2)
Espy et al. 500 (genital, 288; 158 (31.6) MRC-5 (shell vials) 500 225 (45) LightCycler/DNA polymerase, Implementation study performed by
2000 (115) dermal, 192; thymidine kinase technologists performing laboratory
ocular, 20) testing for diagnostic purposes
Koenig et al. 104 (genital and 43 (41.3) Rabbit kidney (tubes) 104 55 (52.9) LightCycler/glycoprotein D SYBR Green I dye detection system
2001 (229) dermal)
Aldea et al. 118 (genital tract) 28 (23.7) A-549; MRC-5 (tubes) 118 34 (28.8) LightCycler/DNA polymerase, SYBR Green I dye detection system
2002 (4) thymidine kinase
Burrows et al. 262 36 (13.7) 262 75 (28.6%) LightCycler/DNA polymerase LightCycler PCR detected and subtyped
2002 (57) HSV in 99% (66/67) of HSV-positive
specimens, compared to 81% (54/67) by
rapid antigen or 57% (36/63) by culture
van Doornum 668 anogenital 199 (29.8) Human embryonic lung 688 240 (34.9) ABI Prism/glycoprotein D, 17 (culture) and 27 (PCR) specimens were
et al. 2003 (dermal, ocular, fibroblasts (24-well glycoprotein G positive for VZV
(506) oral, microtiter plates),
bronchoalveolar rapid culture
lavage) technique
Wald et al. 36,471 specimens 1,087 (2.9) Human diploid 36,461 4,415 (12.1) ABI Prism/glycoprotein B Of 4,464 samples positive by more than one
2003 (526) (mucosal swabs fibroblasts (microtiter test (cell culture and PCR) 3,377 (75.7%)
obtained from plates), rapid culture were positive by PCR only; 49 (1.1%)
a variety of technique were positive by culture only. This study
anatomic sites) is the most extensive comparison between
from 296 virus isolation and detection of a
subjects pathogen by PCR. Transport of
specimens during summertime likely
reduced the detection rate of HSV in cell
cultures compared to molecular
amplification. This conclusion is
consistent with the results of a
VOL. 19, 2006
publication demonstrating the stability of

nucleic acids after storage (201)
TABLE 13. Detection of herpes simplex virus DNA by real-time senting a 17.2% increase compared with shell vial cell cultures
PCR in CSF with and without an internal control (455).
No. of DNA copies Varicella-zoster virus dermal disease. Varicella-zoster virus
Specimen Internal
no. control result causes both varicella (primary infection, chickenpox) and zos-
With Without
ter (reactivated infection, shingles). VZV produces a general-
1 117,300 108,500 Negative ized vesicular rash on the dermis (chickenpox) in unimmunized
15,820 16,500 Negative normal children, usually before 10 years of age. After primary
1,959 2,220 Positive
239 206 Positive infection with VZV, the virus persists in latent form and may
32 24 Positive emerge (usually in adults aged 50 years and older) clinically to
1 Negative Positive cause a unilateral vesicular eruption, generally in a dermato-
mal distribution (shingles). Traditionally, VZV has been de-
2 48 48 Positive
tected in the laboratory by the rather slow (2 to 5 days in shell
4 10 Positive
vial cell culture) replication of the virus in cell culture; how-
3 1 6 Positive ever, these infections have been more rapidly diagnosed by
immunofluorescence and conventional PCR methods (75,
430).
Real-time PCR techniques permit highly sensitive same-day
shell vial cell culture and real-time PCR was highly significant detection of VZV in clinical specimens. At the Mayo Clinic, we
in demonstrating the increased sensitivity and specificity of compared a LightCycler PCR assay with shell vial cell culture
real-time PCR compared to the cell culture assay (P ⱕ 0.0001) methods for the detection of VZV from dermal specimens in
(Table 12). On the basis of these developmental results, the the routine clinical laboratory. This assay served as the proto-
real-time assay replaced the shell vial cell culture assay in May type for the ASR developed by Roche Diagnostics Corpora-
2000 in our laboratory for the routine detection of HSV infection (LightCycler VZV ORF29 primer/hybridization probes).
tions from these specimens (456). This assay served as a pro- VZV DNA was detected in 44 of 253 (17.4%) by real-time
totype for the ASR assay later commercially developed by PCR, but only 23 isolates of VZV were cultured from these
Roche Diagnostics Corporation and introduced into our lab- specimens (117) (Table 14). This initial comparison demon-
oratory. strated a 91% increase in the laboratory diagnosis of VZV
Subsequent trend analysis of the Roche ASR and the shell infections by real-time PCR compared with cell culture tech-
vial cell culture has demonstrated increased sensitivity (genital niques. Subsequent trend analysis of two studies (each span-
specimens, 12%; dermal specimens, 17%) of the Roche Light- ning a year’s period of time), which compared PCR with cell
Cycler ASR compared to conventional culture (455, 456). Us- culture confirmed these initial results: 71% increase (455) and
ing the Roche ASR and the melting curve feature of the 161% increase (456). Two additional studies performed by the
LightCycler PCR instrument allows differentiation of the two ABI TaqMan technology (58.8%) and by LightCycler (240%)
genotypes of HSV. With this HSV assay, about 5% of positive methods reported increased detection of VZV DNA com-
specimens from dermal and genital sources have polymor- pared with cell culture recovery of the virus (437, 506) (Table
phisms present so that an intermediate melting curve peak 14). A real-time PCR assay was developed to differentiate
occurs approximately in the middle of the two peaks produced VZV infection due to wild-type virus or vaccine strains of the
by typical HSV-1 and HSV-2 viruses. We have designated virus using melting curve analysis (491) (Table 14).
these intermediate strains type A (one polymorphism is Varicella-zoster virus CNS disease. Neurologic complica-
present compared with the prototype HSV-1 DNA) and type B tions after VZV infection, occur most commonly, although not
(three polymorphisms are present in the probe region com- exclusively, in immunocompromised patients, especially those
pared with HSV-2 DNA) (196). These polymorphisms resulted with AIDS and particularly in individuals with a history of or
in an altered FRET probe melting curve, with a peak Tm of concomitant herpes zoster (92, 224). VZV is a recognized
61.8°C for type A and 62.7°C for type B. These fall between the cause of encephalitis, myelitis, radiculitis, and acute meningitis
Tm of HSV genotype 1 (55.3°C) and the Tm of genotype 2 in immunocompromised patients. In a retrospective study,
(69.7°C) (Fig. 2). These results are consistent with those re- VZV DNA was detected from 5% of CSF specimens (3).
ported by Anderson and colleagues (8). These intermediate Interestingly, real-time LightCycler PCR was positive for VZV
strains obviously represent a unique population of HSV and DNA (most prevalent herpesvirus detected) in 128 of 1,079
may have epidemiologic and pathogenic significance compared (11.9%) CSF specimens at the Mayo Clinic during a 2-year
with wild-type strains of this virus. From a diagnostic stand- period (454).
point, it is important to recognize that these intermediate Cytomegalovirus CNS disease. CMV infection can occur in
strains are identified as HSV but cannot be denoted as either the CNS and clinical presentations are generally in the form of
HSV-1 or HSV-2 by this assay without additional testing with encephalitis, although myelitis has also been described (224).
intermediate HSV control strains. In one study, CMV DNA was detected by conventional PCR in
Data from at least eight publications, five LightCycler and the CSF of HIV patients more frequently than any other her-
three ABI and TaqMan, have shown increased detection rates pesvirus (316). CMV DNA is rarely detected in HIV-infected
by real-time PCR (range, 20% to 300%) over cell culture patients without clinical neurological disease. Recently, using a
methods for diagnosis of HSV infections (Table 12). In our real-time PCR assay, the viral load of herpesviruses, including
experience, PCR produced a 4.1% increase in the rate of CMV, was assessed in the CSF (3). Although unclear at this
detection of HSV from over 2,500 dermal specimens, repre- time, assessment of viral load in the CNS may have prognostic
TABLE 14. Detection of varicella-zoster virus DNA in clinical specimens by real-time PCR
197
Cell culture Real-time PCR

REAL-TIME PCR
Reference Specimen(s) No. of No. positive No. of No. positive Test platform/ Comments
specimens (%) specimens (%) gene target
Espy et al. Dermal 253 23 (9.1) 253 44 (17.4) LightCycler/gene 28, Real-time PCR was 91% more sensitive
2000 (117) DNA polymerase; than shell vial cell culture assay for
gene 29, DNA detection of VZV DNA.
binding protein
Furuta et al. Saliva (Ramsay 25 13 (52) ABI Prism/gene 29, Analyzed VZV DNA copy number in saliva
2001 (134) Hunt DNA binding samples. VZV load in saliva from patients with
syndrome) protein Ramsay Hunt syndrome peaked near the day of
appearance of zoster.
Saliva (zoster 31 17 (55)
sine herpete)
Aberle et al. CSF 576 29 (50) ABI Prism/gene 31, Overall broad testing for different herpesvirus
2002 (3) glycoprotein B from CSF has led to an increase in the
detection rate of those viruses, especially in
relation to VZV-associated CNS disease.
Dworkin et al. Ocular 10 9 (90) ABI Prism Used for detection of VZV DNA from infectious
2002 (103) posterior uveitis. PCR target not given.
van Doornum Dermal 366 17 (4.6) 366 27 (7.4) ABI Prism/gene 38 Real-time PCR was 53.8% more sensitive than
et al. 2003 cell culture (microtiter plates) for detection of
(506) VZV infections.
Wiedmann et CSF, vitreous 56 (19 CSF, 6 vitreous, 54 (96%) LightCycler/gene 28, Real-time and laboratory-developed nested
al. 2003 fluid, dermal, 22 dermal swabs, 9 DNA polymerase methods had equal sensitivity for detecting
(530) tissue tissue) VZV DNA.
Stöcher et al. CSF 30 7 (23.3) LightCycler/gene 28, PCR tests were also performed for detection of
2003 (467) DNA polymerase DNA of CMV, EBV, and HSV-1/2.
O’Neill et al. Dermal 68 (some archived 29 (42.6) LightCycler/gene 38 Real-time nested multiplex assay was equal in
2003 (354) specimens used) sensitivity to nested laboratory-developed
conventional PCR.
Tipples et al. Clinical isolates, 18 (14 wild type; 4 LightCycler/gene 38 Differentiation of VZV wild-type from vaccine
2003 (491) Eileen strain, Oka vaccine strain) strains was obtained by melting curve analysis
and vaccine of amplified products.
strain
Schmutzhard Dermal 110 15 (14) 110 51 (46) LightCycler/gene 4, Real-time PCR provided 240% increase in
et al. 2004 transactivator, detection of VZV infections compared
(437) tegument protein with cell culture.
system
VOL. 19, 2006
Campsall et ABI Prism/open Assay distinguishes the vaccine strain of VZV

al. 2004 reading frame 62 (Oka) from wild-type VZV. The assay was
(58) 100% concordant with two standard PCR-
restriction fragment length polymorphism
methods for 136 VZV strains.
implications, may predict distinct CNS manifestations, and urine of a renal transplant patient and JCV was from the brain
may be useful for differentiating between real infection and tissue of a patient with Hodgkin’s lymphoma complicated by a
nonspecific presence of virus in the CSF, especially in severly demyelinating disease, progressive multifocal leukoencepha-
immunocompromised individuals. lopathy (298). Present evidence indicates that BKV is strongly
Epstein-Barr virus CNS lymphoproliferative disease. Ep- associated with nephropathy, especially in kidney transplant
stein-Barr virus has been implicated in the development of patients, as well as patients with hematuria and ureteral ste-
lymphomas particularly in immunocompromised patients. A nosis (238, 270). The association of JCV with progressive mul-
review of 26 lymphomas involving the CNS revealed that 9 of tifocal leukoencephalopathy in immunocompromised patients,
26 (34.6%) occurred in immunocompromised patients after especially those with AIDS, is well documented (102, 272, 278,
renal transplantation, HIV infection, leukemia, and Wiskott- 521). The clinical significance of these viruses in other diseases
Aldrich syndrome. EBV sequences were detected in all nine such as colorectal cancers and kidney tissues from healthy
lymphomas, but only 2 of 17 lymphomas occurred in immuno- individuals is controversial (336, 349).
compromised patients (330). In another study, seven of eight JCV CNS disease. In the early 1990s, conventional PCR of
patients with posttransplant primary CNS lymphoma had EBV JCV target DNA sequences in CSF specimens replaced histo-
sequences detected by in situ hybridization (377). EBV DNA logic examination of brain biopsy tissue for rapid, noninvasive
was detected by conventional PCR in CSF samples from 14 of laboratory diagnosis of these infections (479, 480). The simul-
49 (27%) of AIDS patients. Eight of the 13 cases had primary taneous qualitative, differential detection of JCV or BKV by
CNS lymphoma (49). More recently, real-time PCR assays melting curve analysis of a common target sequence in the VP2
have been formatted to detect EBV target DNA for the de- gene was developed with the LightCycler instrument and dem-
tection of AIDS-related brain lymphoma (3, 39, 488). In one onstrated to have performance characteristics comparable to
study, of 42 patients, 20 had primary CNS lymphoma and 22 conventional PCR (540). Present evidence indicates that im-
had non-Hodgkin’s lymphoma. EBV DNA was detected in the plementation and reporting of qualitative real-time PCR re-
CSF from 16 of 20 (80%) patients with primary CNS lym- sults for JCV in CSF is appropriate even though BKV was
phoma, 7 of 22 (32%) with systemic non-Hodgkin’s lymphoma, found in the CNS of an AIDS patient (43). Nevertheless,
and 8 of 12 (67%) with CNS non-Hodgkin’s lymphoma (39). additional experience with 400 CSF specimens from immuno-
Enterovirus CNS disease. Enteroviruses such as coxsackievi- suppressed individuals with neurological symptoms has not
ruses A and B, echoviruses, and parechoviruses (previously revealed the presence of BKV DNA by PCR (43).
echoviruses 22 and 23), and poliovirus, are estimated to cause Simian virus 40 virus, another polyomavirus related to JCV
14% to 21% of all respiratory tract infectious, especially in the and BKV, has been detected in CNS tissue specimens and may
summer and autumn months (67). Collectively, these viruses be of more diagnostic significance (perhaps as a coinfection)
are associated with diverse clinical manifestations ranging from than BKV in these infections (270, 492).
mild febrile illness to CNS (aseptic meningitis, encephalitis), Parvovirus. B19, previously classified as a parvovirus, is now
myocarditis, neonatal systemic enteroviral disease, and para- included in the genus Erythrovirus based on preferential repli-
lytic poliomyelitis (12, 320). Recovery of enteroviruses in cell cation of this virus in erythroid progenitor cells, as extensively
cultures is limited by low sensitivity as well as the poor growth reviewed by Heegaard and Brown (168). Infection with B19
characteristics of many serotypes (279). Rotbart described the occurs early in life, and the virus is transmitted by respiratory
utility of PCR methods over cell culture methods for rapidly secretions and occasionally by blood products; antibody prev-
detecting CNS enterovirus infection (419). Several publica- alence ranges from 2% to 15% in early childhood to 85% in
tions have confirmed these results; implementation of this elderly adults (87, 168). B19 may result in an asymptomatic
technology in diagnostic virology laboratories has been shown infection or produce a wide spectrum of disease ranging from
to reduce medical costs incurred by patients by reducing hos- erythema infectiosum (synonyms include slapped cheek syn-
pitalization and hospital stays and the use of unnecessary an- drome and 5th disease) in children to arthropathy, severe ane-
tibiotics and antiviral drugs (127, 343, 392, 415, 418). mia, and systemic manifestations involving the CNS, heart, and
The recent availability of real-time PCR methods has facil- liver dependent on the immune competence of the host (54,
itated the rapid and sensitive detection of enterovirus in the 410). Infection with B19 in pregnant women may cause hy-
CSF, which is critical for patient care. For example, of 104 CSF drops fetalis, congenital anemia, abortion, or stillbirth of the
specimens, 22 enteroviruses (21.2%) were recovered by cell fetus (522).
culture methods, whereas 61 (58.7%) (177% increase) were Most acute infections with B19 are diagnosed in the labora-
detected by real-time PCR (319) (Table 15). Real-time PCR tory by serologically detecting immunoglobulin M (IgM) and
assays are directed to amplify conserved target nucleic acid IgG class antibodies with enzyme-linked immunosorbent assay
sequences in the 5⬘-nontranslated region of the virus. How- (ELISA) testing. PCR detection of target DNA of B19 has had
ever, human parechovirus type 1 (formerly echovirus 22) may application in the control of transmission of the virus present
not be detected by all PCR assays that use this target (258). in blood or blood products such as plasma pools (532). A few
Sensitivity for detecting enterovirus cDNA has been shown to real-time PCR assays (ABI TaqMan and LightCycler) have
be comparable in sensitivity to conventional PCR assays but been developed with diagnostic application for detecting B19
real-time instruments for PCR were less labor intensive and DNA in association with infection during pregnancy or assess-
easier to implement in the clinical laboratory (207, 390) (Table ing the prevalence of the virus nucleic acid in blood products
15). (2, 164, 227, 232, 435, 438).
Polyomaviruses. JC virus (JCV) and BK virus (BKV) were In one study of 164 nonscreened pools of plasma, 92 (56%)
recovered in cell cultures in 1971; BKV was derived from the contained B19 DNA as detected by LightCycler DNA; 13 of
TABLE 15. Detection of enterovirus cDNA by real-time PCR
199

REAL-TIME PCR
Reference No. of No. positive No. of No. positive Test platform/ Comments
Verstrepen et al. 70 17 (24.3) 70 19 (27.1) ABI Prism/5⬘ nontranslated Sensitivity of real-time PCR was 100%
2001 (515) region compared with cell culture.
Read et al. 2001 50 (originally found 49 (98) LightCycler/5⬘ nontranslated Publication described conversion of a
(403) positive by region conventional multiplex PCR assay that
conventional PCR) detects HSV-1, HSV-2, VZV, and
enterovirus with the LightCycler
system.
Corless et al. 2002 200 (97, CSF; 103, 97 (CSF), 103 (throat An additional 33 (15.9) ABI Prism/5⬘ nontranslated Real-time PCR was 11.5% more sensitive
(78) throat swabs) swabs) enterovirus and 2 region than cell cultures for the diagnosis of
giving previously (1) parechoviruses enterovirus infections using CSF
negative results (formerly specimens. Based on limiting dilutions,
in cell culture echoviruses 22 and the TaqMan enterovirus and
23) identified by parechovirus PCR showed an increase
PCR of two orders of magnitude compared
to cell culture with a sensitivity of
100% when assessed using enterovirus
cell culture-positive samples.
Watkins-Riedel et 60 (stool, 12; CSF, 6/12 feces (50), 60 6/12 feces (50), 2/38 LightCycler 5⬘ nontranslated Sensitivity of the real-time PCR was 10–
al. 2002 (528) 38; serum, 8; 2/38 CSF CSF (5.3) region 100-fold higher compared to
throat swabs, 2) (5.3) AMPLICOR EV test.
Monpoeho et al. 104 (CSF) 22 (21.2) 104 (CSF) 61 (58.7) ABI Prism/5⬘ nontranslated Real-time PCR allows a large number of
2002 (319) region samples to be screened rapidly during
an epidemic and its sensitivity,
simplicity, and reproducibility make it
a highly reliable and suitable tool in
the clinical laboratory.
Nijhuis et al. 2002 41 (feces), 8 9 (22.0), 41 (feces), 8 (CSF), 43 10 (24.4), 4 (50.0), ABI Prism/5⬘ nontranslated Real-time assay was robust and easily
(344) (CSF), 43 3 (37.5), 0 (bronchoalveolar 2 (4.7) region standardized, which make it an
(bronchoalveolar lavage) excellent alternative for conventional
lavage) time-consuming viral culture.
Rabenau et al. 109 (CSF) 23 (21) LightCycler/5⬘ nontranslated Performance characteristics of real-time
2002 (390) region PCR were comparable to those using a
conventional PCR assay. Compared
with the conventional laboratory-
developed assay real-time PCR was
less labor intensive and easy to use.
Kares et al. 2003 32 12 (37.5) 55 (CSF, 21; stool, 32; 32 (58.1) LightCycler/5⬘ nontranslated Real-time assays were of equal sensitivity
(207) nasopharyngeal region, SYBR Green I to laboratory developed developed
aspirate, 3) dye detection real-time PCR test. Fifteen of 20
samples which were negative in cell
culture were positive by real-time
PCR.
VOL. 19, 2006
Verboon-Maciolek 19 (CSF) 4 (21.1) 19 (CSF and serum) 5 CSF (26.3), 9 serum ABI Prism 5⬘ nontranslated Study population was infants ⱕ60 days
et al. 2003 (514) (47.4) region old who received a clinical diagnosis of
sepsis. Enterovirus infections are an
important cause of sepsis in infants
admitted to the hospital.
these pools contained more than 104 international units WNV reverse transcription-PCR kit, Artus; LightCycler WNV
(IU)/ml of the Erythrovirus genome. Further, of more than Detection kit, Roche Applied Science) (72).
503,000 blood donations, 29 contained more than 5 ⫻ 106
IU/ml of B19 DNA (232). Of two real-time PCR kits available Respiratory Viruses
commercially (Real Art Parvo B19 LC; Roche Diagnostics)
only the Real Art test detected all three genotypes of parvo- Acute respiratory tract infections are a significant cause of
virus. However, of 140,160 blood units, genotype 1 (detected morbidity and mortality particularly in the very young and
by both assays), but not genotype 2 or 3, was detected in these elderly and in immunocompromised patients (33). Predictably,
plasma specimens (182). Certainly, real-time PCR tests capa- these viruses occur predominantly in the winter and spring
ble of detecting and distinguishing the genotypes of parvovirus seasons of the year (104). In addition to their role in causing
B19 will be necessary to determine their clinical imporatance. common infection of pharynx, eye, and middle ear, these vi-
A recent example is the V9 variant of the virus recovered from ruses can cause severe systemic complications associated with
skin biopsies from patients with B19-unrelated skin disease lower respiratory tract disease, especially in individuals with
(183). risk factors such as heart and lung disease and other chronic
West Nile virus. West Nile virus (WNV), a flavivirus, is conditions such as diabetes, kidney disease, asthma, anemia,
transmitted from birds to humans primarily by the Culex spe- and other blood disorders.
cies of mosquitoes and is responsible for CNS disease, partic- The classic respiratory viruses have been traditionally iden-
ularly in immunocompromised and elderly patients, with a tified by inoculation of specimens into a variety of cell cultures.
fatality rate of 7% to 10% (375, 424, 490). Several modes of Although early antigenic components of these viruses can be
transmission of WNV have been recognized: blood product detected as early as 24 h after inoculation of shell vial cultures
transfusion, organ transplantation, and occupational exposure using monoclonal antibodies, the performance of these tests is
in laboratory workers (4, 5, 424). Serologic detection of IgM dependent on many variables, the most important of which is
(CSF) and IgG (serum) class antibodies to WNV is the stan- the lability of these viruses in transit to the laboratory (259,
dard laboratory procedure for diagnosis infection with this 464). Over a 5-year period at the Mayo Clinic, adenovirus,
influenza virus types A and B, and parainfluenza virus repre-
virus, especially in immunologically competent hosts (207,
sented only 4.2% of the total viruses recovered in this predom-
216). Traditional recovery of the virus in cell cultures for rou-
inantly tertiary-care medical practice. Nevertheless, these vi-
tine laboratory diagnosis is not recommended because of poor
ruses required 35% of our total cell culture requirements in the
sensitivity and safety concerns with the procedures (245, 375).
diagnostic virology laboratory (454). Even with the most sen-
Detection of target RNA (cDNA) of WNV in CSF or serum
sitive and rapid cell culture system, an average of 2 to 3 days
specimens can be a valuable adjunctive assay to a serologic
were required to detect common viral respiratory infections
diagnosis of infection, especially in patients who do not de-
(101). Because several published comparisons have shown sub-
velop detectable antibodies to the virus (178). In a study of 28
stantial increases in sensitivity of PCR compared with cell
CSF specimens collected during the first 2 weeks of illness
culture technology, based on economic factors (expense and
from patients with serologically confirmed WNV infections, 16
labor intensive technology associated with cell culture), and
(57%) were positive by real-time PCR; only 4 of 28 (14%) certainly on the performance characteristics of the tests (PCR
serum samples from the same patients had detectable WNV and culture), laboratories should strongly consider implemen-
RNA (cDNA) (245). In a related study of 10 CSF specimens tation of molecular tests for these respiratory viruses (454,
from confirmed cases of WNV infection, seven were positive 533).
by real-time PCR; four of five of these patients died. In this Influenza viruses. Rapid laboratory diagnosis of influenza is
report, no correlation was found between PCR results and critical for infection control, especially in hospital and nursing
either the duration of illness at the time of CSF collection or home settings. Because of the life-threatening implications of
the presence of IgM class antibody to WNV in that specimen the predicted seasonal occurrence of influenza virus infections,
(47). a rapid and accurate identification of both influenza A and B
Even though peak titers of virus in both CSF and serum may virus genotypes provides the opportunity for intervention with
be present in the early acute stages of infection and disease, effective antiviral treatment if provided to the patient in the
detection of WNV RNA (cDNA) may provide a rapid and early stages of this viral disease (339). Real-time PCR is con-
early laboratory diagnosis of infection compared with serologic siderably more sensitive than cell culture for the detection of
testing (190, 424). Nevertheless, occasionally target nucleic influenza virus type A (range, 45.7% to 121% increase) (36,
acid of WNV can be detected in blood and CSF specimens 434).
several days after the onset of disease symptomatology (245). A recent report from our laboratory indicated that real-time
Further, of 15 blood units tested in a look-back evaluation of PCR detected 92 of 557 (16.5%) compared to 51 of 557 (9.2%)
blood donors, three were PCR-positive, but all samples were respiratory specimens inoculated into cell culture. R-mix cell
IgM and culture negative for WNV (166). cultures (combined monolayers of human lung carcinoma
Detection of WNV target nucleic acid in high volumes of [A549] and mink lung [Mv1Lu] cells) were stained with mono-
specimens such as insect pools, avian tissues, serum from blood clonal antibodies between 24 and 48 h postinfection rather
donors, and CSF from patients has been formatted using Taq- than the recommended 24 h. Specimens are batched for sev-
Man real-time PCR technology (47, 166, 245, 446). At least eral test runs by real-time PCR during the day. The report
two commercial sources offer analytic specific reagents or kits turnaround time for the real-time PCR method is just a few
for real-time PCR using the LightCycler platform (RealArt hours, compared with 24 to 48 h with cell culture technology
(M. J. Espy, S. K. Schneider, P. A. Wright, S. Kidiyala, M. F. number of positive specimens containing metapneumovirus. In
Jones, and T. F. Smith. Program Abstr. 20th Annual Clinical addition, cytopathic effects are not detected in any of the cells
Virology Symposium and Annual Meeting of the Pan Ameri- until 10 to 12 days after inoculation. Sometimes subculture
can Society for Clinical Virology, abstr. M51. 2004). (subpassage) is required to confirm cytopathic effects in cell
Rous sarcoma virus. Rous sarcoma virus (RSV) is a major cultures (63).
cause of serious lower respiratory tract disease in infants and in Molecular detection has indicated that this virus can infect
adults with underlying cardiopulmonary disease and severely all age groups, producing substantial clinical and economic
immunocompromised patients especially those individuals impact (307). Studies in the Netherlands indicated that by 5
with bone marrow transplants and leukemia patients (42, 541). years of age, nearly all individuals have been exposed to meta-
In one study, RSV was the most common virus detected by pneumovirus; worldwide, this virus may account for at least 5%
PCR among children hospitalized for bronchiolitis, pneumo- to 75% of respiratory tract infections in hospitalized children
nia, or croup (174). (203, 504). Real-time PCR assays were found to be as sensitive
Two major subgroups of RSV are recognized, A and B as conventional PCR for detection of metapneumovirus cDNA
(538). Similar to PCR for influenza virus, real-time PCR assays (84, 295, 296) (Table 16). Because of the technical advantages
for RSV have been shown to be more sensitive compared with of using real-time PCR for the rapid and sensitive detection of
direct antigen detection (TestPack) (Table 16). In addition, for metapneumovirus, this method would be preferable to the
three studies, the sensitivity of real-time PCR was 23.6% to variable diagnostic results produced by cell culture isolation
225% greater than that of cell culture systems (122, 154, 508) and identification of this virus.
(Table 16). Immunofluorescence detection of RSV antigen in Parainfluenza virus. Parainfluenza viruses (types 1 to 4)
epithelial cells from the respiratory tract has been an impor- have traditionally been associated with croup, bronchiolitis,
tant rapid diagnostic test procedure in the clinical laboratory. and pneumonia in infants and children; however, they also
For one study evaluating 175 nasopharyngeal specimens, real- produce significant disease in elderly and immunocompro-
time PCR detected 36 (20.6%) RSV-positive samples com- mised patients (20, 33, 160, 483, 537). Ideally, molecular am-
pared with 32 (18.3%) diagnosed using immunofluorescence plification tests for community acquired pneumonia due to the
(189). In another study, of 75 nasal aspirates from children usual viruses (and bacteria) which cause lower respiratory tract
hospitalized for acute respiratory tract disease, 31 (41.3%) involvement with overlapping clinical features, need to be bun-
were positive by immunofluorescence and 42 (56%) were pos- dled according to clinical practice guidelines using test algo-
itive by real-time PCR (154). These data suggest that PCR rithms developed by clinical and laboratory practice personnel.
could replace cell culture methods and even direct detection of Recent publication of a rapid and sensitive multiplex real-time
RSV by immunofluorescence for the routine detection of re- PCR assay for the laboratory diagnosis of influenza viruses A
spiratory tract infection caused by RSV (173). and B, RSV, and four serotypes of parainfluenza viruses dem-
Adenovirus. Limited publications exist which have compared onstrated a 30% increase of these respiratory tract viruses
real-time PCR to cell culture for the detection of adenovirus in compared with cell culture recoveray of those agents (483).
human specimens (121, 187). Another report indicated the Severe acute respiratory syndrome coronavirus. Severe
utility for quantifying adenovirus DNA for guiding clinical acute respiratory syndrome coronavirus (SARS-CoV) causes a
intervention and assessing response of patients to antiviral highly contagious atypical pneumonia which is spread by re-
therapy (246, 247). Specifically, the real-time PCR assays were spiratory secretions and airborne transmission. From Novem-
designed to detect adenovirus type 4 (subgroup E) in military ber 2002 to July 2003, a total of 8,464 cases were reported,
personnel. The clinical applicability of a real-time PCR assay resulting in 799 deaths and a fatality rate of 9.4% (382, 425).
for adenovirus will require target DNA homologous to the Recovery and identification of SARS-CoV in cell cultures is
other subgroups of the virus (51 serotypes classified into six hazardous for routine clinical laboratories because of the risk
groups, A to F) to detect strains associated with respiratory, of laboratory-acquired infections with this virus and biosafety
ocular, and the several other anatomical areas of infection with level 3 laboratory facilities are required for cell culture recov-
adenoviruses in immunocompromised patients (230, 414, 513). ery and identification of this virus (548).
These retrospective studies demonstrated almost 100% corre- Experience from the Chinese University of Hong Kong in-
lation of real-time PCR (ABI and SmartCycler platforms) and dicated that the yield of diagnostic virus isolation was much
cell culture methods with known adenovirus containing speci- lower than by PCR testing. No specimen was positive by cul-
mens (Table 16). Two studies reported equal or greater ture but negative by PCR (64). Alternatively, inactivation of
(35.9%) sensitivity of real-time PCR versus conventional PCR the SARS-CoV by autoclaving prior to testing by real-time
for the detection of adenovirus DNA (151, 170) (Table 16). PCR may provide the potential for the safe processing of the
Metapneumovirus. In 2001, a new virus from children and specimen by laboratory personnel (119). Early recognition and
adults with acute respiratory tract infections was identified containment of a reemergent outbreak of SARS-CoV depends
(503, 545). Metapneumovirus is classified among the on the vigilance and awareness of physicians and allied health
Paramyxoviridae, subfamily Pneumovirus, and is closely related personnel to recognize the clinical, epidemiologic, and labora-
to RSV phylogenetically and may have overlapping symptom- tory criteria compatible with the published criteria of a case
atology with this virus (63). Reliable detection of this virus may definition of possible infection with this infection (16).
require lengthy incubations times (up to 17 days) after inocu- The laboratory can play a critical role to document the
lation of cell cultures such as tertiary monkey kidney or LLC- etiology of the respiratory tract infection recognizing the over-
MK2 cells (161). Generally, several types of cells (HEp-2, lapping clinical features of SARS-CoV with other viruses such
LLC-MK-2, and MDCK) are required to detect the maximum as influenza virus A and B which may be circulating in popu-
TABLE 16. PCR for viruses 202
Virus Reference No. of No. positive No. of No. positive Test platform/ Comments
Cells or antigen
Influenza Ward et al. 233 throat swabs 50 (21.5) MDCK, tertiary 233 140 (60.0) ABI Prism/M1 matrix Real-time assays were designed for
virusa 2004 cynomolgus gene both A and B viruses. Large-
(527) scale screening and
ESPY ET AL.
identification of influenza virus

using real-time PCR was carried
out as part of the development
of zanamivir.
van Elden et Tertiary rhesus 27 reference strains A, 36 (37), ABI Prism/matrix protein Influenza virus could be detected
al. 2001 monkey (A), 9 reference B, 4 (4) (A), hemagglutinin in nasal wash specimens up to 7
(507) kidney cells strains (B), and gene (B) days after initial presentation of
other isolates influenza-like symptoms.
98 clinical 22 (22.4) Shell vial 98
specimens
Spackman 1,550 cloacal, 266 (17.2) Embryonated 1,550 303 (19.6) ABI Prism/matrix gene Recovery of influenza virus
et al. tracheal, or eggs (conserved for all A requires 1–2 weeks in
2002 environmental matrix genes; in embryonated eggs.
(458) swabs from addition, primer sets
various avian were developed to H5
species and H7 strains of avian
influenza virus; broad-
range detection
including both North
American and Eurasian
lineage avian viruses
and isolates of human,
equine, and swine
origin influenza A virus
Smith et al. 58 throat and nasal 35 (60.3) (25 A; Rhesus monkey 58 51 (88.0) (41 A; LightCycler/A matrix (300 4 LightCycler (positive only)
2003 swabs 10 B) kidney cells/ 10 B) bp) protein (5⬘ end of results sequenced. Analysis of
(453) tube culture the matrix gene M1), B fragment matched the M1 gene
nucleoprotein (184 bp); of influenza virus A. B strains,
LC run parameters frozen supernatants previously
were identical for A known to contain influenza virus
and B B assay tested with H3N2 and
H1N1 strains and two influenza
B viruses (Beijing-like).
Boivin et al. Nasal and LightCycler/hemagglutinin Study assessed kinetics of the

2003 (37) pharyngeal swabs gene influenza virus load in
respiratory tract samples of
infected individuals receiving
early treatment with
neuraminadase inhibitors
compared to those receiving
deferred treatment. The mean
pretreatment virus load was
significantly lower in 24 patients
who initiated treatment within
24 h of the onset of symptoms
than it was in 26 patients who
initiated treatment between 24
and 48.
Boivin et al. 172 (subset of A: 19 (11.0), B: 0 Compared to 172 42 (24.4) LightCycler/matrix genes Melting curve feature of
2004 (36) nasopharyngeal rapid antigen of both A and B LightCycler instrument was used
aspirates from assays for A to differentiate influenza strains
hospitalized and B from RSV. Pediatric study ⱕ3
children) (Becton yr of age.
VOL. 19, 2006
Dickinson)
Frisbie et al. 75 children ⱕ4 yr; 22 (29.3) archived Rhesus monkey 75 18 (24) ABI Prism/A matrix gene; Repeat testing by culture of 21 of
2004 archived nasal nasal aspirates kidney cells B hemagglutinin gene 22 positive archival specimens
(132) aspirates revealed only 11 positive results;
all 21 were PCR positive by
repeat testing.
Espy et al.b 557 respiratory 51 (9.2%) R-mix R-mix 557 92 (16.5%) LightCycler/matrix gene LightCycler PCR was rapid (3 h
2004 tract specimens 24 (4.3%) extraction and analytic time)
(throat swabs, BINAX and more sensitive than R-mix
nasal washes, antigen test cell cultures and BINAX for the
bronchoalveolar detection of influenza virus type
lavage, sputum, A (H3N2) strains from clinical
nasal swabs) specimens.
Respiratory Boivin et al. 204 nasopharyngeal 94 (46) RSV TestPack 204 104 (51) ABI Prism/F gene Authors developed a multiplex test
syncytial 2003 (36) aspirates from antigen test to differentiate RSV and
virus children ⬍3 yr influenza A and B virus
of age amplicons by melting curve
analysis.
Borg et al. 62 acute respiratory 29 (46.8) ABI Prism/F1 gene Real-time (quantitative) assay
2003 (38) tract infection (subunit of fusion specific for subgroup A was
(children); protein) developed. The median viral
nasopharyngea1 load of the specimens from
secretions patients with chronic obstructive
125 adults with 35 (28) pulmonary disease was 6.1 ⫻ 10
chronic copies/ml compared with
obstructive median of 1.2 ⫻ 107 copies/ml
pulmonary in children with respiratory tract
disease, nasal infection.
lavage fluid and
induced sputum
Falsey et al. 169, 13 adult 58 (34) HEp-2 169 73 (43) ABI Prism/F gene Real-time assay detects both RSV
(122) volunteers, nasal A and B subgroups.
washes
Gueudin et 75 nasal aspirates 34 (45.3) MRC-5 and 75 42 (56%) LightCycler/N gene Direct immunofluorescence was
al. 2003 A-549 positive in 31/75 (41.3%) of
(154) samples.
Hu et al. 175 nasopharyngeal 21 (12), immuno- 175 36 (20.3) (10 RSV ABI Prism/N gene Assay detects RSV A and B
2003 aspirates from fluorescence A, 26 RSV B) subgroups. Immunofluorescence
(189) children technique identified 32/75
(42.7%) RSV-positive samples.
Mentel et 71 25 (35.2%) iCycler/F gene 71 consecutive specimens were

al. 2003 71 (Real-Time) processed and tested by PCR
(313) 71 19 (26.7%) Nested, from hospitalized children with
conventional clinical symptoms of acute
PCR respiratory distress to obtain a
10 (14.1) Antigen rapid laboratory diagnosis of
REAL-TIME PCR
ELISA RSV infection.

(Virion\Serion)

203
204
TABLE 16—Continued
Virus Reference No. of No. positive No. of No. positive Comments
Cells or antigen Test platform/gene target
specimens (%) specimens (%)
van Elden et 168 (during period 4 (2.4%) Shell vial 168 13 (4.9%) ABI Prism/N gene Real-time assay detects both RSV
ESPY ET AL.
al. 2003 of symptoms), rhesus (Real-time A and B subgroups. Detection

(508) combined nose monkey PCR) of RSV cDNA by nested PCR
and throat swabs kidney cells 13 (4.9%) and real-time PCR is equivalent.
from immuno- Laboratory
compromised developed
adults nested PCR
(autologous or
allogenic stem
cell transplant
patients (n ⫽
73), hematologic
malignancies
(n ⫽ 17)
Adenovirus Houng et al. 96 throat swabs 72 (previously A-549 cell 96 specimens 71 ABI Prism/Ad4 hexon Quantitative real-time PCR assay
2002 from military positive Ad4 cultures selected for gene used to detect Ad4 (subgroup
(187) personnel with strains isolated (human lung positive results E) DNA in specimens from
acute respiratory in cell cultures) carcinoma) by previous military recruits during an acute
tract disease inoculation in respiratory disease outbreak.
cell cultures The assay did not crossreact
from 1953 with representative members of
through 1998 adenovirus subgroups A, B, C,
and then tested D, and F.
by PCR in 2001
Gu et al. 45 20 (44.4) ABI Prism/hexon gene Real-time quantitative PCR was
2003 designed to detect adenovirus
(151) DNA from all major subgroups
of the virus.
45 (conventional 20 (44.4)
PCR)
Heim et al. 234 (real-time 53 (22.6) LightCycler/hexon gene Adenovirus DNA was detected in
2003 PCR) blood by real-time (quantitative)
(170) PCR in 4/27 (14.8%) pediatric
and 8/93 (8.6%) of adult stem
cell transplant patients but only
in 5/306 healthy blood donor
controls (1.6%). Detection of
234 (conventional 39 (16.6)
high virus loads in blood holds
PCR)
promise for simplified and
earlier diagnosis of disseminated
adenovirus disease in
immunosuppressed patients.
Lion et al. 132 (patients) 36 (27.2) ABI Prism/hexon gene In this series, 8/11 (73%) children
2003 (patients); with adenovirus DNA in blood
(277) positive results specimens, but none of the
from at least patients with adenovirus DNA
one specimen detectable at sites other than
blood, developed fatal
disseminated adenoviral disease.
Faix et al. 140 throat swabs 99 (70.7) A-549 cell 140 98 (70%) SmartCycler/Ad4 hexon Assay developed by Houng et al.
2004 from 86 subjects cultures gene (187) was adapted to the
(121) SmartCycler instrument for the
detection of adenovirus DNA in
specimens from military recruits
VOL. 19, 2006
during an acute respiratory

disease outbreak. Overall, rapid
PCR results had a sensitivity of
100% and a specificity of 100%
compared with viral culture.
Leruez-Ville 44 plasma 8 (18.1) ABI Prism/hexon gene All 8 patients for whom PCR
et al. detected adenovirus DNA in
2004 blood samples had disseminated
(261) adenovirus infection.
Lankester et iCycler IQ/hexon gene Four allogeneic stem cell

al. 2004 transplant patients were treated
(246) with ribavirin and subsequently
with cidofovir. Quantitative real-
time PCR determinations
indicated that these antiviral
drugs did not reduce levels of
adenovirus DNA.
Meta- Côté et al. LLC-MK2 20 specimens 20 (100%) with LightCycler/nucleoprotein, PCR is method of choice for
pneumo- 2003 (84) (continuous selected as prob- nucleoprotein, matrix, fusion, metapneumovirus laboratory
virus monkey able positive cul- N gene target phosphoprotein, diagnosis because of poor
kidney cell tures for meta- polymerase gene replication of the virus in cell
line) pneumovirus targets culture.
10 nasopharyngeal 10 (100%) with
aspirates nucleoprotein, N
gene target
Mackay et 62 nasopharyngeal 12 (19.4) real-time LightCycler/nucleoprotein Real-time PCR was the preferred
al., 2003 aspirates PCR gene target choice for detecting
(295) 6 known PCR- 6 (9.7) metapneumovirus and was
positive and 56 Conventional considerably more reliable than
known PCR- PCR cell culture in the routine
negative clinical laboratory.
specimens
(conventional
methods)
Maertzdorf 38 clinical samples 38 (100) ABI Prism/nucleoprotein Human metapneumoviruses can be

et al., found positive by gene target divided into two main genetic
2004 conventional lineages (A and B) representing
(296) PCR two serotypes and each
54 clinical samples 0 comprising two sublineages (A1,
known as A2, B1, B2). This assay detects
negative by all strains of the virus.
conventional
PCR
Para- Templeton 358 11 (3.1%) HEp-2, HEL, 358 4 (1.1) iCycler IQ/accession nos. Rapid real-time multiplex PCR
influenza et al. and PI-1-70948, PI-2- assay was developed for the
virus 2004 LLCMK2 AF213352, PI-3- detection of influenza A and
(483) M18760, PI-4-M55976 influenza B viruses, RSV, and
REAL-TIME PCR
parainfluenza viruses 1, 2, 3,
and 4.

205
Virus Reference No. of No. positive No. of No. positive Test platform/ Comments
Cells or antigen
Templeton Case report: para- Parainfluenza virus 1 PCR was found to be far more
et. al. influenza virus (accession no. 70948); sensitive than culture or
2004 type 3 detected parainfluenza virus 2 immunofluorescence in an
(482) by direct immu- (AF213352); immunocompromised host
ESPY ET AL.
nofluorescence parainfluenza virus 3 (post-stem cell transplantation)

from nasal wash (M18760); and results of this assay (real-
specimens. parainfluenza virus 4 time PCR) could improve
(M55976); iCycler IQ management of patients.
(molecular beacon
technology)
Parainfluenza
virus type 3
detected by
culture (child
with acute
lymphoblastic
leukemia who
received a
hematopoietic
stem cell
transplant)
SARS-CoV Poon et al. 98 nasopharyngeal 43 (44) ABI/TaqMan/ORF16

2004 aspirates
(382)
36 stool samples 21 (57)
Drosten et 66 samples from 29 47 (70.8) LightCycler/replicase gene

al. 2004 confirmed SARS (Real/Art LC kit)
(99) patients; 31
respiratory
specimens
35 stool and other 44 (67.1) LightCycler/nucleocapsid
specimens gene
Chan et al. 531 respiratory 45 (8.4) Vero E6 471 respiratory 122 (25.9) No specimen was positive by
2004 (64) tract tract culture and negative by PCR.
526 nonrespiratory 4 (0.76) 365 nonrespiratory 83 (22.7)
Ng et al. 36 plasma 15 (41.6) ABI/TaqMan/ Samples collected over 2 weeks.

2003 polymerase gene
(337) 23 serum 18 (78.2) ABI/TaqMan/ Samples collected on day of
nucleocapsid gene hospital admission.
a
In this section, A and B refer to influenza virus types A and B, respectively.
b
M. J. Espy, S. K. Schneider, P. A. Wright, S. Kidiyala, M. F. Jones, and T. F. Smith, Program Abstr. 20th Clin. Virol. Symp., abstr. M51, 2004.
lations at the same time (16, 68, 72). Currently, real-time PCR ment was particularly adaptable for the differentiation of sev-
reagents are available from at least two commercial manufac- eral members of the orthopoxvirus genes but particularly for
turers (Artus: RealArt HPA-Coronavirus RT PCR Kits for the specific identification of variola virus from cowpox and
LightCycler; ABI Prism 7000, 7700, 7900H; and RotorGene; vaccinia virus; this test served as the first real-time assay for the
and LightCycler SARS-CoV, Roche Diagnostics) (72). Early detection of those viruses (114) (Table 17). For this particular
identification and documentation of SARS CoV infection assay it is still important to consider clinical and epidemiologic
(based on a firm laboratory diagnosis) on a global basis may characteristics of the patient to associate vaccinia (previous
control the transmission of this highly contagious infection by immunization) or cowpox (animal contact) to the infection
effective use of isolation and quarantine measures for patients since the melting temperature differed for those two viruses by
and area contacts (11, 252). less than 1°C. Subsequently, in a recent study, one LightCycler
Compared to serology, the use of real-time PCR technology assay was able to resolve all nonvariola orthopoxviruses by the
is critical since target nucleic acid of the virus can be detected simultaneous use of four hybridization probe-based real-time
in specimens from patients in the early stages of infection PCR assays (345). Separate reaction vessels with specific prim-
(Table 16). Poon et al. found that of 50 nasopharyngeal aspi- ers and probes would be required to achieve this level of
rates collected 1 to 3 days after onset of disease, 40 (80%) were identification of orthopoxviruses with real-time platforms
positive for SARS-CoV target nucleic acid (383). However, which do not have melting curve features.
SARS-CoV has been found in sputum, throat swabs, serum, Because of the laboratory safety concerns of infection to
lung, kidney, bone marrow, and feces by real-time PCR tar- individuals processing specimens for the diagnosis of possible
geting sequences in the nucleocapsid and RNA polymerase variola virus infection from high-risk patients, specific tests to
(ORF1b) genes of the virus (46, 240, 252, 297, 337). Specimens identify this virus should be carried out by trained personnel in
(especially feces) obtained about 10 days from symptom onset a biosafety level 4 facility such as exists at the Centers for
are associated with the highest yield for all specimen types, Disease Control. However, for clinically evaluated low-risk
which correlates with the timing of peak virus loads (68). Nev- patients for variola virus infection, specimens could be pro-
ertheless, the relative productivity of each specimen type for cessed in appropriate facilities (Laboratory Response Network
detection of SARS-CoV needs to be assessed before negative Laboratories) for viruses such as HSV, VZV, enteroviruses,
results by real-time PCR assays can be used to rule out the and vaccinia virus. As an additional safeguard for the labora-
presence of this viral infection (474). tory, the specimens can be autoclaved, under controlled con-
Importantly, the performance of one commercial (RealArt ditions, before testing the sample for the presence of viral
HPA) and six laboratory-developed conventional and real- target nucleic acids by real-time PCR (119). Autoclaving had
time (LightCycler) PCR assays were compared for the detec- no detrimental affect on the amplification of target DNA from
tion of SARS-CoV in clinical specimens (297). Of 68 clinical HSV, VZV, and vaccinia virus (119).
specimens (17 respiratory tract specimens, 29 urine samples,
and 22 stool or rectal swabs specimen), six of seven assays QUANTITATIVE VIRAL ASSAYS
detected at least 17 of 18 positive results (defined as positive in
at least two assays), and two of the assays had a sensitivity of Real-time PCR provides a tool in the clinical laboratory for
100%. There was no significant difference in the sensitivity providing quantitative results of viral target nucleic acid
between the assays (P ⫽ 0.5). In another study, sensitivities of present in a clinical sample. The results of quantitation of a
70.8% (Artus) and 67.1% (Roche) were obtained with 66 spec- viral nucleic acid target determination in a blood specimen, for
imens from patients with confirmed SARS. The authors em- example, may be applicable for assessing the relationship be-
phasized that PCR should not be used to comprehensively rule tween the viral load (i.e., copy level) of a viral target and the
out SARS (99). prediction of the progression of infection to clinical disease.
Quantitative test results for nucleic acid targets have become
especially relevant with serial specimens from transplant pa-
Poxviruses
tients to monitor for evolving sypmptomatic infection or for
Variola virus is a large, brick-shaped particle containing assessing the effectiveness of antiviral therapy.
DNA and belongs to the Orthopoxvirus genus of the family Technically, quantitative real-time PCR is performed by the
Poxviridae (114). Other members of this genus include mon- addition of standards which have known specified or calibrated
keypox, cowpox, racoonpox, skunkpox, and ectromelia viruses; levels of target nucleic acid. Three to five dilutions of a stan-
although very uncommon, recent reports indicate that these dard are included in each test run of each quantitative real-
infection can occur, especially after human contact with in- time PCR determination. Using the known copy level of the
fected animals (95, 131, 267, 322, 404). Almost all dermal standard reagent, the software of the instrument generates a
lesions due to viruses in routine laboratory practice are caused standard curve in a plot that relates fluorescence (measure of
by HSV and VZV; however, immediate recognition of the amplified product) and the cycle number in which the nucleic
clinical features of smallpox and differentiation of variola virus acid target is detected. Quantitative detection of viral nucleic
from other virus infections involving the skin is of paramount acid is determined by comparing the cycle number (crossover
importance. Most importantly, the finding of a suspected case point or Cp) of the specimen with the standard curve generated
of smallpox must be considered as an international health with known levels of the target nucleic acid. Quantitative stan-
emergency and be brought to the attention of national official dards (e.g., EBV DNA) from commercial sources are helpful
through local and state health laboratories (171). for developing quantitative tests for viral load levels. Alterna-
The melting curve feature of the LightCycler PCR instru- tively, nucleic acid from viruses cultivated in cell cultures (or
TABLE 17. Detection of othopoxvirus DNA by real-time PCR 208
Reference No. of No. positive Comments
Cells No. of specimens No. positive (%) Test platform/gene target
specimens (%)
Espy et al. Reference strains. American Type Culture LightCycler Variola virus (Tm, 62.45°C) could
2002 (114) Collection: vaccinia virus (vr-117); PCR/hemagglutinin gene be differentiated from vaccinia
cowpox virus (vr-302); monkeypox virus (Tm, 56.72°C) and monkeypox
ESPY ET AL.
(vr 267). Centers for Disease Control: (Tm, 56.24°C) viruses.

vaccinia virus; variola virus (smallpox);
plasmid insert of a 300-bp segment of
the hemagglutinin gene
Sofi Ibrahim Centers for Disease Control: variola virus, LightCycler, SmartCycler/ Both PCR platforms were
et al. 2003 (smallpox): 48 strains of camelpox, hemagglutinin gene considered acceptable for the
(457) cowpox, ectromelia, gerbilpox, detection of variola virus
monkeypox, rabbitpox, raccoonpox, DNA.
skunkpox, vaccinia, herpes simplex
virus, and varicella-zoster virus
322 coded samples; 48 different stains SmartCycler: 2 samples with false-positive
(above) results among 116 samples not
containing variola virus (specificity,
98.3%): LightCycler: 5 samples with
false-positive results among 116
samples not containing variola virus
(specificity 95.7%)
Kelly et al. ABI Prism, Laboratory Publication reports the detection

2004 (217) Response Network assays of vaccinia virus-specific DNA
for vaccinia virus and for in lesions of patients
varicella-zoster virus. previously immunized against
smallpox.
Kulesh et al. 16 coded samples: 8 variola virus LightCycler, SmartCycler/ Both LightCycler and
2004 (241) (smallpox); 5 non-variola virus, hemagglutinin gene. SmartCycler had 100%
orthopox virus isolates; 2 varicella- SmartCycler detected sensitivity with both platforms
zoster virus; 1 herpes simplex virus 92.3% of the with samples above the limit
orthopoxvirus DNAs and of detection (ⱖ12 gene
between 95% and 93.8% copies).
of the variola virus DNAs.
LightCycler detected
96.2% of the orthopox
DNAs and 93.8% of the
variola virus DNAs.
Nitsche et al. LightCycler/genes: A13L, A complete set of four

2004 (345) rpo18, VETF hybridization probe-based
real-time PCR assays provide
for the specific detection of
orthopoxvirus DNA by melting
point analysis.
Olson et al. LightCycler/fusion protein PCR primers were designed to

2004 (352) gene detected all Eurasian-African
species of orthopoxvirus. A
single nucleotide mismatch in
the target sequence allowed
differentiation of variola virus
from other orthopoxviruses by
melting-curve analysis.
target nucleic inserted into a plasmid) may also be used to rum, plasma, leukocytic) which may yield maximal copy num-
generate standard curves for quantitative assays; however, bers of CMV DNA at different stages of this viral infection.
these reagents should ideally be obtained from commercial Finally, probit regression analysis, (probability of achieving
sources to ensure uniformity of results. e.g., 95% positive results at a low copy level of target DNA),
and an electronic display of trend analysis results for ease of
interpretation by attending physicians should be integrated
Cytomegalovirus
into this laboratory practice (149). It is recognized, however,
At the present time, many real-time quantitative tests for that graphic trend analysis of sequential quantitative results is
CMV DNA have been formatted on either the ABI or the an unmet challenge for most laboratory information systems.
LightCycler instruments. Choice of either instrument depends
on work flow issues in the laboratory rather than specific per- Epstein-Barr Virus
formance characteristics of the platforms. These platforms of-
fer unique performance features of precision and reproducibil- Several malignancies have been associated with EBV infec-
ity of test results; nevertheless, the customized formatting of tions, especially in immunosuppressed patients who lack anti-
test procedures using these instruments is highly variable body to the virus. These include posttransplant lymphoprolif-
among laboratories (Table 18). For example, of 26 articles in erative disorders, Burkitt’s lymphoma, Hodgkin’s disease,
which TaqMan probes were used, there were 10 different gene nasopharyngeal carcinoma, gastric carcinoma, breast cancer,
targets, at least three different units of result reports (CMV and hepatocellular carcinoma (158, 239). As previously dis-
DNA/␮l, CMV DNA/105 peripheral blood leukocytes, CMV cussed, some EBV-associated malignancies can occur in the
DNA/␮g of human DNA), and four different specimen com- central nervous system especially in HIV patients. A recent
partments of blood (whole blood, plasma, peripheral blood report indicates that EBV may contribute to the pathobiology
leukocytes, and white blood cell-reduced blood). Nevertheless, of multiple sclerosis in children (6).
compared to conventional PCR, optimization of these impor- Quantitation of EBV DNA in these patients provides the
tant variables can be achieved by real-time instrumentation potential for the designation of viral load (threshold) levels
with common operational profiles, reagents, and standards. generally associated with healthy or subclinical carriers of EBV
The compartment of blood used as the optimal specimen may (reactivated infection) compared with those levels of virus that
vary according to the stage of viral replication in an individual produce disease states such as posttransplant lymphprolifera-
patient (400). For example, the presence of CMV DNA in tive disorder in transplant patients (158). Viral load levels
plasma may be associated with active viral replication and obtained during the posttransplantation course may also pro-
disease development relative to other specimens (149). vide the clinician with information for initiating and monitor-
Several publications using either LightCycler or ABI Prism ing response to therapy. From a clinical perspective, quantita-
7700 (TaqMan) have reported comparisons for the detection tive viral load information may guide a preemptive strategy to
of pp65 matrix protein of CMV with real-time PCR (Table 18). reduce the incidence and level of EBV reactivation in trans-
Molecular amplification has several important advantages to plant patients by administration of antiviral agents when target
detection of CMV antigen (the antigenemia test) even though EBV DNA or significant levels of EBV DNA are detected.
some studies indicated general agreement between the two Similar to quantitative CMV assays, the majority of real-time
methods. In general, quantitative real-time PCR has several PCR quantitative assays for detection of EBV DNA, have been
advantages to the antigenemia test including increased sensi- developed and formatted using the ABI 7700 Prism instrument
tivity for early detection of CMV infection or reactivation, and TaqMan probes. Dilution of target EBV DNA (mostly
utility for patients with neutropenia, stability of target DNA in DNA polymerase, BALF-5) inserted into plasmids have been
blood specimens, wide detection range (7 to 8 log10) of CMV prepared and amplified by real-time PCR to produce the stan-
DNA, ability to process large number of specimens, flexibility dard curves for quantitative assays. The expected linear range
of time of transport and processing of specimens, and the of detection by either the ABI or LightCycler real-time PCR
potential for increased accuracy of results through precision platform spans 107 to 108 log10 copies/ml.
instrumentation (133, 309, 346, 549). Practical clinical applications of EBV viral load determina-
Standardization, implementation, and result interpretation tions by real-time PCR to reduce the incidence of EBV reac-
and reporting will ultimately depend on uniform guidelines tivation and replication and the subsequent development of
and availability of commercial products to obtain uniformity EBV related lymphomas (posttransplant lymphproliferative
among laboratories. Among the important variables are disorder) have been demonstrated. Generally, quantitative as-
threshold copy levels of CMV DNA significant for low- and says are oftentimes performed three to five times each week
high-risk patients to guide antiviral treatment regimens and for enabling the investigators to determine the patterns and trends
different patient populations of organ transplantation patients of EBV replication in solid organ transplant patients (342,
who have unique demographic and medical management char- 509). For example, several publications from the University
acteristics. Technically, the calculation of the concentration Medical Center, Rotterdam, The Netherlands, have shown a
(viral load) is critically dependent on the accuracy of both the correlation between EBV viral DNA loads and the likelihood
copy number of the CMV target in a plasmid standard used to of development of posttransplant lymphproliferative disorder
establish a standard curve for quantitation, but also on the (341, 342, 509–511). A threshold of 1,000 copies of EBV
calculations of CMV DNA/ml of specimen introduced into the DNA/ml plasma was chosen to begin each treatment with
PCR mixture prior to amplification. Interpretation of results rituximab, a monoclonal antibody directed against the CD20
need to be guided by the compartment of blood sampled (se- receptor binding site for EBV (510). This resulted in a com-
TABLE 18. Quantitative detection of cytomegalovirus DNA by real-time PCRa 210
Comparison to other
Platform Reference Specimen Target Quantitative standard Reporting units Comments
systems
ABI Prism Machida et al. 16 patients (bone US17 gene Range: 10 to 107 CMV DNA Copies of CMV DNA/ Real-time PCR compared CMV DNA was not detected in
2000 (293) marrow transplant); copies/well 500 mg of DNA with antigenemia assay blood specimens of the 55
136 blood samples (blood), copies CMV and by shell vial cell healthy patients seropositive
from patients: 70 DNA/100 ␮l plasma culture. for CMV. The results of PCR
blood specimens (plasma) did not correlate
ESPY ET AL.
from healthy well with those obtained by

volunteers the antigenemia test; however,
PCR (nucleated cells from
blood) had high correlation
with the antigenemia test
results.
Nitsche et al. Plasma; bone marrow Major immediate- As a positive control, a Copies CMV genome/ CMV load was higher in CMV DNA was detected from
2000 (347) transplant patients early gene plasmid containing the ml plasma CMV antigen-positive 5/27 healthy blood donors
target sequence from the (antigenemia) patients seronegative for CMV.
target gene was used with than in antigen-
101 to 107 plasmids/assay negative patients.
Tanaka et al. Peripheral blood Immediate-early Range from 6 to over 106 Copies CMV DNA/106 Real-time PCR compared The correlation between the
2000 (473) leukocytes, plasma gene copies of CMV DNA, cells with antigemia assay. CMV DNA copy number and
Plasmid containing the IE the pp65-positive cell count
gene used to develop a (antigenemia test) was
standard curve for statistically significant (P ⫽
quantitative results. 0.01). CMV DNA copy level/
106 cells was higher in
symptomatic patients (blood,
plasma specimens) than in
asymptomatic patients.
Funato et al. Whole-blood samples Major immediate- A plasmid containing the Copies CMV DNA/␮g Conventional PCR and No CMV DNA was detected in
2001 (133) from infants with early gene target gene of CMV was DNA real-time PCR were 97 healthy patients by
hepatitis constructed and used as a compared for the conventional or by real-time
quantitative standard. detection of CMV PCR methods. Eighteen
Reference value for the DNA in this patient samples tested negative by
CMV DNA copy numbers population. conventional PCR: 14 samples
was determined as 109 were negative; four samples
molecules/3.7 ng/␮l based contained CMV DNA with
on the molecular weight of low copy levels of CMV DNA
the DNA inserted into the (10–80).
plasmid.
Gault et al. Blood (peripheral UL83 (pp65 gene) Plasmid containing one copy of Copies CMV DNA/2 ⫻ Known pp65 antigenemia- The results of the real-time PCR
2001 (138) blood leukocytes) the UL83 target sequence 105 leukocytes positive (PBL samples) assay correlated with those of
(46 samples) used as a quantitative collected from solid the antigenemia assay (P ⬍
standard plasmid containing organ transplant 0.0001).
human genomic DNA patients were amplified
(albumin gene) coamplified by real-time PCR.
with specimen DNA.
Griscelli et al. Peripheral blood UL83, pp65 gene A plasmid containing both the Copies CMV DNA/ Real-time PCR was Study quantified CMV DNA in
2001 (149) leukocytes of bone (phosphorylated CMV and the 200,000 leutkocytes compared to the the glyceraldehyde-3-phos-
marrow transplant matrix protein) glyceraldehyde-3-phosphate antigenemia test. phate dehydrogenase gene
patients dehydrogenase genes was using a plasmid containing
used as a standard. both sequences of 16 patients,
CMV DNA was detected by
PCR in 13/16 (91.3%), a mean
of 15 days prior to the appear-
ance of antigenemia.
Guiver et al. Blood, 362 samples Glycoprotein B Plasmid standard of CMV Copies CMV DNA/ml Real-time PCR was Viral loads of real-time PCR
2001 (156) from 25 patients; gene DNA diluted to contain 10 (limit of sensitivity compared to CMV showed a highly significant
12 single lung, 13 to 106 copies/PCR was 500 copies/ml antigenemia test. linear correlation with
heart transplant blood based on antigemia levels.
VOL. 19, 2006
sampling volume of 2
␮l of EDTA treated
blood following
extraction.
Limaye et al. Plasma samples from Immediate-early Copies CMV DNA/ml CMV DNA was detected a
2001 (271) stem cell transplant gene median of 13 days before
patients onset of CMV disease (range,
0–35 days). Monitoring plasma
CMV DNA loads may be
useful in the prevention of
pre-engraftment CMV disease.
Najioullah et Blood (serum) HXFL4 gene Plasmid dilutions from 5 ⫻ 102 Copies CMV DNA/␮g Real-time PCR and The qualitative PCR was positive
al. 2001 (positive for CMV to 2 ⫻ 108 copies of CMV DNA conventional PCR in 48 samples and the
(329) in cell cultures) DNA/ml were tested. (four compared with positive quantitative real-time assay
from transplant dilutions were used to cell culture results was positive in 46 samples
patients develop a standard curve in obtained with blood from a total of 50 cell culture-
the quantitative test). CMV specimens. positive specimens.
DNA obtained from
Advanced Biotechnologies
used as a positive control
for real-time PCR.
Sanchez et al. Blood specimens from DNA polymerase Copies CMV DNA/␮g Risk of CMV pneumonitis after
2001 (426) lung transplant gene, human human DNA lung transplantation is related
recipients apoprotein B to the level of CMV DNA in
gene blood.
Satou et al. Whole blood Major immediate- Target gene inserted into a Copies CMV DNA/␮g Results of real-time PCR Forty specimens from normal
2001 (429) early gene plasmid. DNA correlated with subjects were negative for
conventional PCR and CMV DNA. Real-time PCR
with serology in several was useful for the quantitative
specimens from one detection of CMV DNA from
liver transplant patient. serial samples of blood from a
patient post-liver
transplantation.
Dworkin et al. Aqueous vitreous Not provided CMV DNA obtained from CMV genome/␮l Real-time PCR may be applied
2002 (103) specimens from Advanced Biotechnologies, vitreous to infectious agents
patients with Inc., Columbia, MO; SYBR responsible for posterior
posterior uveitis Green I dye fluorescein uveitis; the technique will
detection system likely be useful for the
diagnosis of this entity and the
linkage of viral pathogens to
the disease.
Greenlee et Blood (RBC, WBC- Immediate-early ABI standard at stock Copies CMV DNA/ml CMV DNA was not detected in
al. 2002 reduced), 100 gene of CMV concentration of 104 copies/ any of the RBC samples.
(146) blood donors ml. The standard was used
seroprositive for as a template for the
CMV; 93 blood TaqMan assay at 5, 50, 500,
donors and 50,000 copies/50 ␮l
seronegative for PCR.
REAL-TIME PCR
CMV

211
Comparison to other
systems
Mori et al. Plasma allogeneic US17 gene A standard ranging 101 to 107 Copies CMV DNA/100. Qualitative results of the Authors used real-time PCR
2002 (321) hematopoietic stem copies/well was used for a ␮l real-time PCR were results to guide preemptive
cell transplant standard curve for compared with the therapy (ganciclovir) for CMV
patients quantitation of CMV DNA determinations disease. Qualitative results of
obtained with the real-time PCR were detected
ESPY ET AL.
antigenemia test. earlier than antigenemia in

the posttransplantation period
in 18/30 (60%) patients.
Antigenemia was detected
before PCR results in only
3/30 (10%) patients.
Sanchez et al. Plasma and leukocyte DNA polymerase CMV quantitation was linear Copies CMV DNA/ml Authors created a multi- Applied to whole blood, the
2002 (427) lysate preparations gene. Primers over a range of 101 to 106 plex, quantitative real- assay provides a measurement
and probes for copies. time PCR assay that of CMV DNA in reaction to
amplification of amplifies CMV DNA cellular content without a
the human apo- and human DNA in need for cell-counting
protein gene the same reaction tube. procedures.
were included in
the assay.
Yakushiji et Plasma from stem cell US17 CMV DNA copies/ml CMV antigenemia and The number of CMV antigen-
al. 2002 transplant patients quantitative real-time positive cells by the
(549) PCR were compared antigenemia assay and the
for monitoring CMV level of CMV DNA by real-
reactivation after time PCR correlated well.
allogenic stem cell
transplantation.
Hänfler et al. Buffy coat leukocytes pp150 IL-32 gene Linear range 101 to 107 copies CMV DNA copies/2-␮l Authors developed a A high degree of conformity was
2003 (162) standard curve data sample duplex real-time PCR attained between PCR viral
prepared from a plasmid that was capable of load and antigenemia tests
with target insert diluted quantifying CMV DNA using leukocyte specimens.
from 0.5 ⫻ 101 to 0.5 ⫻ 107 and ␤-actin DNA as
copies/␮l internal control
simultaneously within
one reaction.
Ikewaki et al. Plasma US17 gene Plasmid containing the target CMV DNA/ml Study compared real-time Real-time PCR was more sensi-
2003 (193) sequence was diluted (seven PCR assay for tive than the antigenemia and
10-fold dilutions) detection of CMV nested PCR assays. In addi-
corresponding to 101 to 107 DNA with nested tion, real-time PCR was able
plasmid copies/reaction. conventional PCR and to detect CMV reactivation
Dynamic range: 5 ⫻ 102 to antigenemia tests. earlier in the clinical course
5 ⫻ 108 copies/ml than the antigenemia and
nested PCR. CMV viral loads
of 5,000 copies/ml were pro-
posed as the cutoff for initiat-
ing therapy in adult T-cell leu-
kemia-lymphoma patients.
Li et al. 2003 Whole blood Glycoprotein B Plasmid standard concentra- Copies CMV DNA/ml A laboratory-developed High correlation between
(269) tion calibrated by spectro- PCR assay and a semi- antigenemia value and CMV
photometry at 260 nm. quantitative pp65 anti- DNA loads. Antigenemia
genemia assay were values of 11 to 100, 101 to
compared to a quanti- 1,000, and over 1,000 positive
tative real-time assay cells/2 ⫻ 105 leukocytes
for the detection of corresponded to median CMV
CMV. DNA levels of 1,000, 4,000,

and 10,000 copies/ml and are
proposed as cutoff points for
initiating antiviral therapy in
patient groups with high,
intermediate, and low risk of
VOL. 19, 2006
CMV disease, respectively.
Nitsche et al. Blood leukocytes Major immediate- Authors compared real- The PCR and antigenemia tests
2003 (346) early gene time PCR with were positive in 19 of 77
antigenemia test using patients. An additional 26%
blood specimens from (22 patients) were positive
patients who exclusively by PCR. in 5/20
underwent stem cell patients. Febrile episodes in
transplantation. two patients with fever of
unknown origin may have
been caused by a reactivation
of CMV. These results imply
that CMV infection can be
expected not only in
transplant patients but also in
chemotherapy treated
neutropenic patients.
Persson et al. Neutropenic patients UL65 (pp67 CMV DNA genomes/ml CMV DNA but not HHV-6 (or
2003 (373) following protein) HHV-7) was detected
chemotherapy
Boeckh et al. A double primer Copies CMV DNA/ml The double-primer

2004 (35) assay was de- (UL55/UL123-exon 4)
signed consisting was superior to pp65
of two sets of antigenemia and
primers and viremia by culture with
probes to amplify regard to sensitivity,
a portion of the specificity, and
gB region predictive values.
(UL55) and
UL123-exon 4
region
Nesbitt et al. Plasma, 78 EDTA- Junction between Copies CMV DNA/ml Tested the effects of Study indicated that valid results
2004 (335) anticoagulated glycoprotein B storing whole blood for can be obtained from EDTA-
whole blood and UL123 24 h before separating anticoagulated blood stored at
samples plasma room temperature for at least
24 h before separation of
plasma.
Visconti et al. Peripheral blood Immediate early Linear range over 4 log10 CMV DNA genome Authors experimentally CMV DNA load reduced but
2004 (520) mononuclear cells copies/ml determined the effi- was not eliminated in whole
experimentally ciency of removal of blood and platelets after
infected with CMV CMV using leukocyte treatment with leukocyte
depletion filters in a depletion filters.
model system of PB-
MC’s infected with the
virus.
LightCycler Nitsche et al. Major immediate- Linear range: 101 to 107 CMV CMV DNA genome LightCycler and ABI Prism 7700
1999 (348) early gene DNA genome equivalent/ equivalent/assay were compared for the
assay. MIE gene inserted detection of CMV DNA. The
REAL-TIME PCR
into plasmid vector; DNA ABI Prism 7700 appears to be

concentration determined by useful for the processing of

213
Comparison to other
systems
a spectrophotometer at 260 large numbers of samples
nm and the corresponding under standard conditions,
copy number calculated. whereas the LightCycler has
its strength in smaller sample
numbers and the use of
ESPY ET AL.
various reaction conditions.

Schaade et al. Plasma, urine Glycoprotein B Amplicon cloned in plasmid. DNA copies/ml LightCycler detected higher
2000 (433) gene Concentration of DNA levels of CMV DNA than the
calibrated by spectrometry COBAS instrument; however,
at 260 nm. Dynamic range, both tests had comparable
102 to 108 plasmid copies of sensitivities for detecting
CMV DNA/ml CMV DNA in clinical
specimens.
Kearns et al. Glycoprotein B Range: 2 ⫻ 103 to 5 ⫻ 108 LightCycler assay Data confirm and extend those
2001 (213) gene CMV DNA copies/ml compared to TaqMan of Schaade (433). The
(copies/reaction converted real-time PCR. Of 50 LightCycler assay was shown
to copies/ml) samples, the majority to be more sensitive than the
of results (36/50, 72%) detection of early antigent
were within 0.5–1 log10 fluorescent foci testing for
and 3 (14%) by log10. urine and respiratory
specimens.
Kearns et al. Blood Glycoprotein B Range: 10 to ⬎2 ⫻ 105 CMV DNA copies/␮l LightCycler results com- PCR product identity confirmed
2001 (215) gene DNA copies EcoRI plasmid parable to TaqMan by melting curve analysis.
quantified and linearized (blood). TaqMan could Runs acceptable if the
and used as quantitative reproducibly detect external quantitative standard
standard. A series of five down to 20 plasmid within 0.5 log10 of the target
log10 dilutions correspond- copies. Detection level value and the standard curve
ing to 2 ⫻ 101 to 2 ⫻ 105 of LC, ⱕ10 copies gave a mean squared value of
copies/2 ␮l were prepared CMV DNA. Of 51 ⬎10⫺2.
and run as external stan- positive samples
dards. (blood) the quantita-
tive results using Taq-
Man ranged from 102
to 3 ⫻ 106 copies/ml.
LightCycler results
ranged from 1.1 ⫻ 102
to 1.7 ⫻ 106 copies/ml.
Ando et al. Aqueous humor; 6 Glycoprotein B Range: 101 to 104 copies/␮l. SYBR Green I dye CMV DNA
2002 (9) patients with gene Plasmid with target insert was detected at levels up to
clinically diagnosed used to develop standard for 1.6 ⫻ 104 copies/␮l of
CMV retinitis quantitation aqueous humor obtained from
patients with retinitis. Study
revealed correlation between
levels of CMV DNA and the
extent of the area affected by
CMV retinitis before antiviral
treatment and the prolonged
retention of CMV genome
after antiviral treatment.
Kearns et al. Urine and respiratory Glycoprotein B Range: 2 ⫻ 103 to 5 ⫻ 108 DNA copies/ml LightCycler PCR provided a 3-
2002 (216) samples gene CMV DNA copies/ml. Note: fold increase in sensitivity
copies/reaction (215) compared to detection of
converted to copies/ml in CMV early antigen in cell
the publication culture (urine samples). Urine
viral loads were higher in con-
genitally CMV-infected infants
(median, 1.6 ⫻ 105 copies/ml)
compared with 15 transplant
recipients (median, 9 ⫻ 103
VOL. 19, 2006
copies/ml). Urine samples did

not require extraction prior to
PCR testing.
Stöcher et al. US17 gene of CMV Linear range: 500 to 50,000 CMV DNA Authors developed a normalized
2002 (465) CMV DNA copies/ml copies/capillary quantitative competitive real-
time PCR assay using a PCR
competitor as heterologous
DNA. The results obtained
with conventional real-time
quantification on the
LightCycler instrument were
almost identical to those
obtained with the normalized
based quantification assay.
Cortez et al. Whole blood from 51 Plasmid with target CMV DNA copies/ml Real-time PCR results were
2003 (79) stem cell transplant insert used to positive earlier than
recipients develop standard antigenemia results in 30/39
for quantitation (77%) episodes of CMV
(5000, 500, 50, 5 infection detected by
copies/reaction). antigenemia. Real-time PCR
Conversion to remained positive after
copies/ml: DNA treatment was discontinued in
extracted from 14/39 (36%) episodes and
200 ␮l whole predicted the return of CMV
blood and then reactivation in 4/13 (31%)
suspended in 50 episodes.
␮l water; 5 ␮l
was used in each
of two reactions.
The sum of these
two reactions
represented 50
␮l of whole
blood for which
the sum was mul-
tiplied by 25 to
obtain copies/ml.
Mengelle et 14 patients (solid UL83 gene (codes Plasmid with CMV target CMV DNA copies/ml CMV DNA was detected before
al. 2003 organ transplant), for lower matrix sequence used to develop pp65 antigen in three patients,
(309) 198 blood protein pp65) standard for quantification. whereas the two tests were
specimens Detection limit was 1 log10 positive simultaneously for
(leukocytes) genome copy/capillary eight patients. Molecular
Range: 1 log10–5 log10 method of real-time PCR
copies/reaction. could be useful for monitoring
infections and antiviral
treatment in recipients of
solid organ transplants.
Pang et al. 404 plasma specimens Glycoprotein B Range: 2.5 ⫻ 102 to 107 DNA CMV DNA copies/ml LightCycler PCR was The costs per sample were
2003 (365) from 66 solid organ gene copies/ml plasma. Positive plasma most sensitive (54% of highest with COBAS ($104.7)
transplant patients control was prepared (106 specimens positive) compared with LightCycler
REAL-TIME PCR
genome copies/tube) from compared to the ($39.8) and antigenemia

CMV-infected cell cultures. COBAS Amplicor ($35.8) (Canadian dollars).
215

Comparison to other
systems
CMV monitor (48.6%) Because of its sensitivity,
and the pp65 specificity, cost effectiveness,
antigenemia (26%) and simplicity, the LC-PCR
assay. assay would replace the
antigenemia and COBAS
ESPY ET AL.
assay as the preferred

technique for the surveillance,
diagnosis, and monitoring of
response of CMV diseases in
high-risk populations.
Gouarin et al. Whole blood from 21 UL83 gene (codes Plasmid with target insert used CMV DNA copies/ml CMV DNA was detected earlier
2004 (145) renal transplant for lower matrix to develop standard for than antigenemia in the
patients; 248 protein pp65) quantitation. Range: 5 to 5 posttransplantation period.
specimens ⫻ 106 copies/reaction Real-time PCR with whole
blood can be used to monitor
renal transplant patients at
risk of developing CMV
disease and to assess response
to antiviral therapy. Study
found that ⬎4 log10 copies/mL
of whole blood indicates
extensive CMV infection and
leads to initiation of antiviral
treatment.
Hong et al. 147 plasma specimens Glycoprotein B or ␤-Globulin gene was amplified CMV DNA copies/ml gB target provided a The combination of automated
2004 (186) from bone marrow EcoRID region in parallel to control for the plasma sensitivity of 96% and DNA preparation and real-
and stem cell efficiency of nucleic acid specificity of 100% time PCR detection allows a
transplant patients extraction and amplification compared to the sensitive, precise, accurate
steps. Range: 125 copies/ml EcoRID target (real- high-throughput assay of
to 5 ⫻ 109 copies/ml. A time) and conventional CMV viral load that can be
CMV gB plasmid containing (gel and Southern blot) used as a laboratory trigger
the whole 2.7-kb gB gene for preemptive antiviral
subcloned into a pCR2.1 therapy.
vector was used as a
quantification standard for
the assay.
iCycler iQ Aberle et al. CSF us 17 gene Copies CMV DNA/ml Virus load was 2.0 ⫻ 102 to 1.9
2002 (3) (corresponding to 2 ⫻ 106 CMV DNA/ml in
copies of DNA per patients with encephalopathy
TaqMan PCR in immunosuppressed patients.
mixture). Quantitation of viral loads in
CSF may be important
regarding prognosis of disease
and prediction of distinct CNS
manifestation.
RotorGene Herrmann et 138 plasma specimens DNA polymerase Linear range of 103 to 108 CMV DNA copies/ml Of 138 samples, 105 were Two quantitative CMV assays
al. 2004 from 44 patients gene; copies/ml. Target DNA used positive by real-time with different gene targets
(176) with suspected glycoprotein B for standard curves was PCR and 71 were posi- were developed. The duplex
CMV infection gene purified genomic DNA from tive by COBAS assay real-time PCR system had a
CMV strain AD169. (34 samples exclusively higher sensitivity than the
positive by real-time COBAS Amplicor Monitor
PCR) (PCR was 48% test system and the linear
more sensitive than measure level was at least 3
COBAS). orders of magnitude higher.
plete abrogation of posttransplant lymphproliferative disorder
SCT) at risk for CMV disease.

in plasma. A CMV DNA level
CMV DNA load in whole blood
sensitivity and 90% specificity

in plasma of 10,000 copies/ml
transplant patients (SOT and

provided a threshold of 81%
significantly higher than that
mortality after 6 months of therapy (341, 342, 510). Further,

tends to be slightly but not
EBV DNA was not detected in 14 of 17 (82.4%) of these

for initiating treating in patients posttreatment. In another report, nine transplant pa-
tients (eight bone marrow and one kidney) developed post-
transplant lymphproliferative disorder associated with a rapid
rise in EBV viral load exceeding 105 EBV genomes/␮g of
peripheral blood mononuclear cell-derived DNA compared
Abbreviations: IE, immediate-early; RBC, red blood cells; WBC, white blood cells; IL, interleukin; HHV, human herpevirus; MIE, major immediate-early; LC-PCR, Light Cycler PCR. with ⱕ104 EBV genomes/peripheral blood mononuclear cell in
patients who did not have posttransplant lymphproliferative
disorder (355).
appeared to be capable
assay, quantification of
CMV DNA in plasma
Reports of real-time PCR assay for detection of EBV DNA

with the pp65 antigen
of guiding the clinical

Based on a comparison
transplant patients.
have appeared mainly in the last 6 years; over 80% were pub-
management of
lished from 2001 to 2004 (Table 19). The focus of these reports
has been the development of individual assays to provide quan-
titative EBV DNA results to support specific medical practices.
Consequently, these laboratory developed assays in each insti-
tution have been customized and the results evaluated in pa-
tient populations (e.g., solid-organ transplant patients) which
may be unique regarding demographic characteristics (age and
CMV DNA copies/ml
gender), pretransplant diseases, type of transplant (lung, kid-

ney, heart, pancreas), and immunosuppression regimen and
other medications. In contrast to assays based totally on bio-
logical variables, real-time PCR instrumentations provide the
plasma
basis to develop and standardize the many technical compo-

nents of these platforms. For example, sample extraction could
be monitored to achieve maximum yields of nucleic acids and
phocine herpesvirus DNA as
provide for effective removal of PCR inhibitions.

Clinical samples were spiked
For the assay, the idealized PCR target gene could be se-
with a fixed amount of
lected that would allow maximum efficiency of the amplifica-

an internal control.
tion process. Further, a plasmid insert of this gene with appro-

priate calculations to determine nucleic seed and target
concentration could be used as a quantitative standard and the
units of reporting would be the same in all laboratories and
obviously dependent on the analysis of a common sample com-
partment of blood (whole blood, peripheral block, mononu-
clear cells, plasma, or serum). Real-time PCR assays have the
potential for controlling these technical variables in the labo-
ratory. Ultimate utility of these assays for EBV quantitation as
Immediate-early
antigen gene
well as quantitation for other viruses such as CMV will be the

application of accurate, reproducible results in each patient
population. While empirically establishing local practice guide-
lines such as beginning antiviral treatment according to thresh-
old levels of DNAemia are practical and necessary for appro-
(SCT) patients; 295
stem cell transplant
priate medical management of patients, it is also important to

to address the cor-
ples were selected
CMV DNA loads

whole-blood sam-
solid organ trans-
409 specimens from
relation between
plant (SOT) and
acknowledge that each patient may have their own individual

corresponding
in plasma and
whole blood
set point, that is, the viral load level which leads to symptom-
atic infection.
BK Virus
As previously mentioned under Qualitative Viral Assays, BK
2004 (205)
Kalpoe et al.
virus can cause tubulointerstitial nephritis and ureteric stenosis

in renal transplant recipients and hemorrhagic cystitis in pa-
tients who have undergone bone marrow transplantation
(359). Renal biopsy specimens may be examined histologically
for the presence of BKV inclusion bodies which is a more
specific diagnostic criterion compared with detection of the
virus in urine specimens. In addition, the presence of charac-
a
teristic decoy cells in the urine is a morphological marker for

TABLE 19. Quantitative detection of Epstein-Barr virus DNA by real-time PCRa 218
Author Comparison to other
Platform Specimen Target Quantitative standard Reporting units Comments
(reference) systems
ABI Prism Kimura et al. Peripheral blood BALF5 gene Linear range, 2 to 107 copies Copies EBV DNA/␮g Real-time PCR detection The virus load in peripheral
1999 (221) mononuclear (DNA poly- EBV DNA/␮g DNA of PBMC DNA of EBV DNA was mononuclear cells was
cells (PBMC) merase) compared with in situ 103.7 copies/␮g of DNA
hybridization tech- in patients with EBV-
niques. related lymphoprolifera-
ESPY ET AL.
tive disorders, 104.1 cop-

ies/␮g DNA in patients
with chronic active EBV
infections, and 102.3 cop-
ies/␮g DNA in patients
with infectious mononu-
cleosis. The copy num-
bers of EBV DNA in
PBMC from symptomatic
EBV infections was cor-
related with the EBV-
positive cell number de-
termined by the in situ
hybridization assay (P ⬍
0.0001).
Lo et al. Plasma BamHI-W; A calibration curve was run in Copies EBV DNA/ml Using real-time PCR cell-
1999 (281; EBNA-1 parallel and in duplicate free EBV DNA was de-
also 265, with each analysis, using tectable in 55/57 (96%)
280) DNA extracted from the median concentration,
EBV-positive cell line Na- 21,058 copies of naso-
malwa (ATCC) as a stan- pharyngeal carcinoma
dard. A conversion factor (NPC) patients and 3/43
of 6.6 pg of DNA/diploid (7%) controls (median
cell was used for copy concentration, 0 copies/
number calculation. ml). Results suggest
quantitative analysis of
plasma EBV DNA may
be a useful clinical and
research tool in the
screening and monitoring
of patients with nasopha-
ryngeal carcinoma.
Niesters et Plasma BNRF1 p143 A standard containing 6.68 ⫻ Copies EBV DNA EBV DNA could be de-
al. 2000 gene (nongly- 109 EBV particles/ml (Ad- (genome equiva- tected in all transplant
(342) cosylated vanced Biotechnologies) lents)/ml patients diagnosed with
membrane was used as a standard. PTLD, with a mean load
protein) Serial half-log dilutions of of 544,570 copies/ml. No
this standard ranging from EBV DNA could be de-
107 to 10 copies/ml were tected in healthy individ-
made to characterize lin- uals and in immunosup-
earity, precision, specificity, pressed control groups.
and sensitivity of the assay. A mean of 6,400 cop-
ies/ml could be detected
in patients with infec-
tious mononucleosis.
Orii et al. 22 transplant BALF5 A plasmid that contained Copies EBV/␮g DNA Quantitative real-time Real-Time PCR and
2000 (356) patients BALF5 was constructed PCR was compared to EBER-1 (in situ hybrid-
and diluted to prepare a qualitative DNA in ization) results exceeded
standard source curve for plasma by PCR and the cut-off level of 102.5
quantitation. EBV-encoded mRNA copies/␮g DNA

(EBER1) by in situ
hybridization for the
detection of
posttransplant
lymphoproliferative
VOL. 19, 2006
disease
Dehee et al. Blood, periph- BNRF1 gene A plasmid with the target Copies EBV DNA/ Significantly higher EBV
2001 (91) eral blood gene was developed to pro- 106 PBMC loads were found in
mononuclear duce a standard curve for HIV-infected patients
cells quantitative determina- compared with EBV-
tions. All virus DNA quan- seropositive healthy
tification was carried out group (P ⬍ 0.00001).
simultaneously in order to EBV loads were not
determine the input cellu- correlated with the
lar DNA for each sample clinical stages of HIV
and was used as an endog- infection or HIV
enous reference to normal- replication
ize the variations due to
differences in the PBMC
count or DNA extraction.
The Namalwa cell line
(ATCC CRL1432) contain-
ing two integrated copies
of the EBV genome/cell
was used as a positive con-
trol.
Jabs et al. Peripheral blood BamHI-K A pCMV EBNA plasmid Copies of EBV DNA/ Aim of study was to Single-tube coamplification
2001 (198) mononuclear (BKRF1) which carries the complete ␮g of PBMC DNA develop a rapid and of EBV and genomic
cells encoding EBNA nuclear gene reliable PCR protocol coreactive proteins
EBNA-1 (EBNA-1) was used for for the quantification allowed normalization of
calibration of the Namalwa of cell-associated EBV EBV DNA copy levels
DNA standard (CRL-1432, DNA associated with that provided a more
ATCC) PTLD in transplant accurate quantification
recipients. (than amplification of
EBV DNA alone) of cell
bound target DNA.
Lo et al. Serum BamHI-W A calibration curve was run in Copies EBV DNA/ml Patients with gastric Serum EBV DNA reflects
2001 (282) parallel and in duplicate carcinoma, gastritis, tumoral EBER status
with each analysis, using and healthy controls and presents the
DNA extracted from the were evaluated for the possibility that circulating
EBV-positive cell line presence of EBV EBV DNA may be used
Namalwa as a standard. A DNA in serum by as a tumor marker for
conversion factor of 6.6 pg real-time PCR. the EBER-positive
of DNA/diploid cell was Resected tumor gastric carcinomas.
used for copy number specimens from
calculation (281). patients with gastric
carcinoma were tested
for EBER (in situ)
and by real-time PCR
(serum)
van Esser et Plasma (stem BNRF1 p143 A standard containing 6.68 ⫻ Copies EBV DNA Qualitative monitoring of
al. 2001 cell transplant nonglycosylated 109 EBV particles/ml (Ad- (genome EBV DNA levels from
(509) patients) membrane vanced Biotechnologies) equivalents)/ml the start of and during
protein gene was used as a standard. therapy for EBV-
REAL-TIME PCR
Serial half-log dilutions of lymphoproliferative

this standard, ranging from disease rapidly and

219
(reference) systems
107 to 10 copies/ml were accurately predicts for

made to characterize lin- response to therapy as
earity, precision, specificity, early as within 72 h.
and sensitivity of the assay.
ESPY ET AL.
Wagner et al. Blood, periph- BamHI-K region The EBV-positive Burkett’s EBV genomes/␮g Patients with PTLD had a
2001 (525) eral blood coding for lymphoma cell line Nama- PBMC DNA median viral load of
mononuclear EBNA1 lwa was used as a standard 19,200 EBV genomes/␮g
cells plasma BamHI-W for quantification of EBV PBMC DNA or 3,225
DNA. Namalwa cells con- EBV genomes/100 ␮l
tain two integrated EBV plasma. Although both
copies/cellular genome. PBMC and plasma were
Other cell lines (Daudi and useful specimens for
Raji) were also used as laboratory diagnosis of
positive controls. PTLD, the specificity was
higher if the EBV viral
load was determined in
plasma.
Leung et al. Whole blood BALF5 gene Raji cells used as a positive Copies EBV DNA/␮g Aim was to develop a The mean copy levels of
2002 (264) (DNA control for detection and amplified DNA rapid and reliable EBV DNA/␮g amplified
polymerase) preparation of a standard (EBV DNA copy PCR protocol for DNA in patient popula-
curve for EBV DNA number was quantitation of the tions were: healthy con-
(dilutions to 7.5 105 normalized against cell-associated EBV trols, 0.5; hemodialysis,
genome/reaction; ␤- ␤-globulin) genome. 27; renal transplant, 40;
globulin used as a and infectious mononu-
housekeeping gene. cleosis, 782. The percent-
age of individuals whose
EBV levels were ⬎35
copies/␮g amplified
DNA: healthy, 0; hemo-
dialysis, 38; renal trans-
plant, 40%; infectious
mononucleosis, 87%.
Matsukura et Blood (PBMC) BALF5 gene A standard curve for quanti- Copies EBV DNA/␮g Aim of study was to In 15 patients, the mean
al. 2002 (DNA tative detection of EBV DNA demonstrate the values of the highest
(305) polymerase) DNA was obtained by significance of serial EBV DNA levels from
measuring dilutions of a monitoring of EBV patients who had the
plasmid containing the tar- DNA by real-time following clinical
get gene. PCR after liver features: fever (36,232);
transplantation. upper respiratory
syndrome (16,040);
diarrhea (15,968); ascites
(2,485);
lymphadenopathy
(336,858); and PTLD
(60,486).
Teramura et Plasma; serum BALF5 gene A standard curve for quanti- Copies EBV DNA/ml Study compared the de- EBV DNA copy levels from
al. 2002 (DNA tative detection of EBV tection of EBV DNA serial specimens from 10
(487) polymerase) DNA was obtained by in specimens from pa- patients demonstrated
measuring dilutions of a tients with hemoph- decreasing or undetect-
plasmid containing the tar- agolytic lymphohistio- able levels of target
get gene. cytosis with patients DNA following appropri-
with infectious mono- ate therapy.
nucleosis.
van Esser et Plasma (stem BNRF1 p143 A standard containing 6.68 ⫻ Copies EBV DNA Authors studied whether Preemptive therapy was
al. 2002 cell transplant gene 109 EBV particles/ml (genome equiva- preemptive therapy given to patients with
(510) patients) (nonglycosylated (Advanced lents)/ml with rituximab viral reactivation more
membrane Biotechnologies) was used prevents EBV than or equal to 1,000
protein) as a standard. Serial half- lymphoproliferatifive copies of EBV DNA
VOL. 19, 2006
log dilutions of this disease in patients (genome equivalents)/ml;

standard, ranging from 107 undergoing stem cell 14/17 (82.4%) patients
to 10 copies/ml were made transplantation. treated with rituximab
to characterize linearity, had complete clearance
precision, specificity, and of EBV DNA from
sensitivity of the assay. plasma.accurate
quantification (than
amplification of EBV
DNA alone) of cell
bound target DNA.
Orentas et Blood, EBER1 gene A plasmid containing the EBV genomes/␮g In 9 patients (8 bone
al. 2003 peripheral EBER1 gene was used to PBMC marrow, 1 kidney
(355) blood prepare a standard curve transplant), PTLD was
mononuclear for the quantitative associated with a rapid
cells detection of EBV DNA. rise in viral load
exceeding 105 EBV
genomes/␮g of PBMC-
derived DNA. The
threshold for normal
EBV viral load
(compared to the levels
in patients with PTLD)
based on a combined
experience with viral load
analysis is defined as 104
EBV genomes/␮g
PMBC.
Pitetti et al. Serum, Patient BALF5 gene Plasmid with target insert was Copies EBV DNA/ml None of the purified DNA
2003 (381) population (DNA used to provide sample inhibited the
was children polymerase) quantitation standards. amplification of the
(mean age, 9 internal positive control.
yr) with pri- Twenty-one (75%) of the
mary EBV patients with primary
infection (n ⫽ EBV, one (4%) of the
28); EBV se- seronegatives, and none
ronegative of the seropositives had
patients (n ⫽ detectable EBV DNA
25); EBV- loads. Viral loads varied
seropositive widely in patients with
patients (n ⫽ primary EBV infection
26) (101 to 103). EBV
posttransplant
lymphoproliferative
disease was diagnosed in
a seronegative patient
with an EBV DNA load
of 1,450 copies/ml.
Wadowsky et Whole blood; BALF5 gene The gene target was inserted Plasma: copies EBV Whole blood: EBV DNA Comparing real-time PCR
al. 2003 plasma from (DNA into a plasmid and DNA/ml copies/ml, EBV and conventional PCR,
(524) 44 transplant polymerase) determinations made in copies/␮g DNA based whole-blood PBL loads
REAL-TIME PCR
patients 10-fold increments ranging on spectrophotometric correlated highly (r2 ⫽

221
(reference) systems
from 20 to 2,000,000 measurement, and 0.900), whereas plasma

copies/reaction EBV copies DNA/105 and PBL loads correlated
PBLs based on poorly (r2 ⫽ 0.512). Data
absolute count. EBV provide outoff levels of
DNA load levels were EBV DNA in blood
ESPY ET AL.
measured in whole compartments (whole

blood (n ⫽ 60), blood, plasma, PBLs) for
plasma (n ⫽ 59), and various risk groups of
samples of peripheral patients.
blood lymphocytes
(n ⫽ 6) by competitive
PCR.
Leung et al. Plasma BamHI-W A calibration curve was run in Copies EBV DNA/ml The sensitivities and The sensitivities of EBV
2004 (266) EBNA-1 parallel and in duplicate specificities of IgA- DNA and IgA-VCA for
with each analysis, using VCA and EBV DNA diagnosis of NPC were
DNA extracted from the for diagnosis of NPC 95% and 81%,
EBV-positive cell line were determined in respectively. The
Namalwa as a standard. A 139 new cases of NPC specificities of EBV
conversion factor of 6.6 pg and 178 healthy DNA and IgA-VCA
of DNA/diploid cell was individuals. were 98% and 96%,
used for copy number respectively.
calculation (281).
Lin et al. Plasma speci- ABI Prism/ ␤-Globulin gene served as a EBV DNA/ml plasma The median concentrations
2004 (273) mens from 99 BamHI-W control for activity of Taq of plasma EBV DNA
patients with polymerase. A calibration were 681 copies/ml
biopsy-proven curve was obtained using among 25 patients with
nasopharyn- DNA extracted from the stage III disease, 1,703
geal carci- EBV-positive cell line copies/ml among 74 pa-
noma Namalwa as the standard. tients with stage IV dis-
ease, and 291,940 cop-
ies/ml among 19 control
patients with distant me-
tastasis. Patients with
relapse had a signifi-
cantly higher plasma
EBV DNA concentration
before treatment than
those who did not have a
relapse. Quantification of
plasma EBV DNA was
useful for monitoring
patients with nasopharyn-
geal carcinoma and pre-
dicting the outcome of
treatment.
Yu et al. Plasma EBER All plasma DNA samples Copies EBV DNA/ml Tumor tissue samples Plasma EBV DNA concen-
2004 (555) were also subjected to real- were tested for the trations in patients with
time PCR analysis for the presence of EBV by in EBV-encoded RNA
␤-globin gene. situ hybridization and (EBER)-positive tumors
compared to the nonnasopharyngeal and
detection of EBV neck carcinomas (squa-
DNA in plasma by mous cell carcinoma;
real-time PCR. lymphoepithelial carcino-
mas) (median, 3,827 cop-
ies) were significantly
higher than those in the
controls (median, 0 copy/
ml, P ⫽ 0.0001). Plasma
EBV DNA was detected
in all of the patients with
VOL. 19, 2006
EBER-positive tumors.
EBV LightCyder Aritaki et al. Peripheral blood GenBank Raji cells that contained 50 Copies/␮g of EBV Other herpesvirus targets No EBV-related disease
(21) cells; 16 accession no. copies EBV genome/cell DNA (HHV-6 and CMV) was found in the study
patients V01555 used as standard were measured in this patients. Viral loads were
undergoing study. low (⬍103 copies/␮g of
stem cell EBV DNA). High num-
transplantation. bers of CMV genome
were detected in 3/13
patients after transplant
and reactivation of
HHV-6 was frequently
seen.
Brengel- Peripheral blood BXLF-1 gene BXLF-1 cloned into plasmid PBMC: copies EBV LightCycler results were 12/32 (37.5%) PBMC from
Pesce et mononuclear (thymidine and quantitative standard DNA genome/␮g compared with a healthy EBV-seropositive
al. (45) cells from 88 kinase) run from 2 to 2 ⫻ 107 DNA. Serum: routinely used ELISA- individuals were positive
patients: 32 copies. copies EBV DNA/ PCR of 150 samples at very low copy levels.
healthy EBV- ml. and a good correlation EBV DNA was detected
seropositive; was found (R ⫽ in 80% of classical infec-
34 EBV- 0.956) tion mononucleosis pa-
associated tients (mean, 288 cop-
disease; 22 ies/ml of serum). In 5
HIV infected. cases of EBV-related
posttrans plantation lym-
phoproliferative disease,
the viral load was
⬎10,000 copies EBV
DNA/␮g DNA at the
diagnosis of lymphoma.
Stevens et al. Whole blood EBNA-1 gene EBNA was inserted into Copies EBV DNA/ml Real-time PCR was com- Aim of study was to de-
(463) plasmid DNA and pared with quantita- velop a LightCycler-
quantified in duplicate in tive competitive PCR based real-time PCR as-
each PCR run. For a for EBV DNA. say for monitoring EBV
quantitative standard the load in whole blood. In
plasmid was used in 253 blood samples from
concentrations ranging patients with Burkitt’s
from 10 to 104 lymphoma, infectious
copies/reaction. mononucleosis, or human
immunodeficiency virus
infection, a weak but sig-
nificant correlation be-
tween the two methods
was found (P ⬍ 0.001).
Patel et al. Whole blood; BZLF-1 gene, Namalwa EBV cell line Copies CMV DNA/ Intra- and interassay Quantitative EBV DNA
(370) plasma which codes (ATCC 1432) used as a ml variability studies assay was developed to
for the standard, which contains using external investigate the natural
ZEBRA two copies EBV genome/ quantitative standards history of EBV infection
protein cell. BZLF-1 gene was and DNA extracted in immunosuppressed
incorporated into a plasmid from whole blood and patients to identify those
for use as a quantitative plasma samples were at greatest risk of devel-
REAL-TIME PCR
control (100 to 109 copies/ shown to be within 0.5 oping disorders and mon-
reaction). log10. itor response to therapy.

223
(reference) systems
Stöcher et al. Plasma GenBank Copies EBV DNA/ml Internal control DNA Authors developed a set of
(466, 467) accession contained a stretch of automated LightCycler
numbers the neomycin PCR assays for the
109336–109351 phosphotransferase detection of CMV, EBV,
(EBV). Note: gene which was HSV-1/2, and VZV DNA
ESPY ET AL.
numbers for flanked by the four in plasma samples and

CMV, HSV-1/ forward and reverse complementation of the
2 and VZV primer binding sites assays with internal
also included. that were specific for amplification controls.
each herpesvirus type-
specific PCR of the
set.
LightCycler Balandraud Peripheral blood IR-1 Raji cell line, which harbors Copies EBV/500 ng In patients with rheumatoid
et al. (24) mononuclear 50 copies of EBV genome DNA (1.5 ⫻ 105 arthritis, the EBV DNA
cells per cell, was used as an cells) load in PBMCs is
external EBV standard. increased almost 10-fold
EBV copy number was compared with that in
calibrated by serial normal controls.
dilutions of Raji cell DNA
and ranged from 105 to
0.064 copies/ml of standard
EBV DNA dilution.
iCycler iQ Yuge et al. Blood, PBMC, BALF5 gene Linear range, 2 to 107 copies Copies EBV DNA/␮g Case report of a 3-year- Viral loads of EBV DNA in
(556) and plasma (DNA EBV DNA/␮g DNA. DNA old, previously healthy PBMC and plasma
polymerase) boy who developed a remained high with
chronic active EBV therapeutic interventions.
infection. PCR of a liver tissue
sample was positive for
EBV DNA CD56⫹ cells
infected with EBV.
Jeblink et al. Whole blood EBER gene Both EBV and CMV assays EBV DNA copies/ml EBV and CMV viral The real-time PCR assays
(200) (glycoprotein were able to detect viral loads in patient showed increases in viral
B gene for DNA over a linear range samples obtained by load before clinical
CMV) of 101 to 107 copies/well, gel-based and by real- measures of viral disease
which was equivalent to time PCR were very and decreases in viral
103 to 109 copies/ml similar. load during antiviral
therapy in two of six
pediatric patients.
a
Abbreviations: ATCC, American Type Culture Collection; PTLD, posttransplant lymphoproliferative disease; HIV, human immunodeficiency virus; PBL, peripheral blood leukocytes; IgA, immunoglobulin A; VCA,
viral capsid antigen.
viral replication (359). Although PCR detection of BKV DNA Human Immunodeficiency Virus
in urine specimens of patients with clinically suspect nephritis
is a sensitive test, a positive test does not necessarily reflect the HIV-1 and HIV-2 RNA levels in the plasma of infected
etiology of this condition since asymptomatic reactivated in- individuals can be determined reliably by laboratory-developed
fection may occur in 10% to 45% of kidney transplant patients. quantitative rapid real-time PCR assays (Table 21). These as-
Conversely, a negative PCR result can be informative to re- says differ in the probe chemistry and amplification/detection
duce or eliminate the likelihood of BKV-associated nephritis. systems used, and dynamic ranges of results and assay preci-
Randhawa et al. developed a real-time PCR LightCycler sion are comparable to those of commercially available assays.
assay to quantitate BKV DNA in renal transplant patients Quantitative assays for measurement of HIV-1 and HIV-2
(395). Viral loads were measured in urine, plasma, and kidney proviral DNA have also been developed. Qualitative rapid
biopsy specimens in three clinical conditions: (i) patients with real-time PCR assays developed for HIV-1 and HIV-2 have
asymptomatic BKV viruria, (ii) patients with active BKV allo- been reported for the detection of proviral DNA. They are
graft nephropathy, and (iii) patients with resolved BKV ne- highly sensitive, and the analytical sensitivities of the HIV-1
phropathy. Active BKV nephropathy was associated with proviral DNA assays were comparable to that of the commer-
quantitative levels of 5 ⫻ 103 copies/ml plasma of BKV DNA. cially available Amplicor HIV-1 DNA test, v1.0 (Roche Mo-
All of these active cases had BKV target DNA at levels greater lecular Systems, Inc., Branchburg, NJ).
than 107 copies/ml of urine. Resolution of nephropathy was
correlated with decreased viruria levels, disappearance of viral
inclusions, and persistent but low-level target DNA in biopsy FUNGI
specimens. Viral loads in patients with asymptomatic viruria Aspergillus Species
were generally lower but sometimes overlapped with levels
typical for patients with BKV nephritis (395). Of all the fungal genera, Aspergillus has been the one most
Establishment of general threshold levels of BKV DNA in extensively targeted for the development of real-time PCR
urine may be useful. For example, the occurrence of hemor- assays. The rationale behind this effort is that timely detection
rhagic cystitis in allogeneic bone marrow transplant patients of Aspergillus spp. may decrease the extreme morbidity and
was associated with BKV DNA levels in urine above 104 cop- mortality associated with invasive aspergillosis. Currently,
ies/␮l; similarly, all four patients with acute BKV-related ne- there are at least 167 recognized species and species variants of
phropathy had copy levels of ⬎ 105 copies/␮l (263, 512). BKV Aspergillus (http://www.ncbi.nlm.nih.gov/Taxonomy) but most
was also detected in the plasma of three of four (75%) patients cases of aspergillosis are attributed to Aspergillus fumigatus,
(512). The quantitative levels of BKV DNA in urine and blood Aspergillus flavus, and Aspergillus niger. A few other species,
may not always be directly correlated; this difference may re- including Aspergillus nidulans, Aspergillus terreus, and Aspergil-
flect independent reactivation of the virus in different tissues lus versicolor, have been reported to cause clinically significant
during immunosuppression (263). disease (413).
The vast majority of real-time PCR methods reported to
date for Aspergillus target Aspergillus fumigatus. Second in fre-
Viral Hepatitis Agents
quency are reports describing real-time PCR methods for As-
Almost all of the reported rapid real-time PCR assays for pergillus flavus. The variety of extraction methods, targets,
hepatitis viruses are quantitative tests that measure viral load primers and probes, source material, and amplification proto-
in serum or plasma for monitoring therapeutic responses of cols makes direct comparison of the methods difficult. In ad-
patients with hepatitis A, B, C, D, or E infection. These assays dition, the number of specimens examined from patients with
were laboratory developed and showed various dynamic ranges proven or probable invasive aspergillosis is low, making the
of results and reproducibility (Table 20). The chemistries used assessment of method sensitivity and specificity difficult. Nota-
include SYBR Green I, FRET hybridization, TaqMan, and bly, Rantakokko-Jalava et al. (397) and Pryce et al. (385)
molecular beacon probes used with the LightCycler, ABI compared the results of their real-time PCR assays for As-
PRISM sequence detection system, and Stratagene Mx4000. pergillus fumigatus in bronchoalveolar lavage fluid, tissue bi-
Assays to quantify hepatitis B and C virus load in liver tissue opsy specimens, or blood to a clinical diagnosis for invasive
have also been described (542, 558). Presently, there is only aspergillosis that was based on recently published consensus
one commercially available quantitative assay, which has a criteria (22).
research use-only label for the measurement of hepatitis A Rantakokko-Jalava et al. used a LightCycler assay targeting
viral DNA in serum or plasma. Assays for qualitative detection a mitochrondrial gene for Aspergillus fumigatus and observed
of hepatitis B and C viruses in serum and plasma have been positive PCR results from bronchoalveolar lavage fluid in six of
reported with high analytical sensitivity necessary for screening seven, two of four, and four of five patients with proven, prob-
of blood donors (314) and diagnosis of infection prior to the able, and possible invasive pulmonary aspergillosis, respec-
appearance of serologic markers. However, many of the quan- tively. The diagnostic sensitivity of the assay was reported to be
titative assays for hepatitis B and C viruses are as sensitive as 73% with a specificity of 93% and positive and negative pre-
these qualitative assays and may be applicable for diagnostic dictive values of 73% and 95%, respectively. Use of a crossing
purposes. Some qualitative assays were developed to deter- point ⬎ 35 cycles as a cutofffor a positive result improved the
mine hepatitis B virus polymerase gene mutants at various ability to discriminate between colonization and invasion but
levels of subpopulation and analytical sensitivity (60, 388, 539, decreased the sensitivity of the assay to 45%. Importantly, this
544, 553, 560). report established analytical specificity by testing against a
TABLE 20. Comparison of rapid-cycle real-time PCR assays for the detection of hepatitis virus types A, B, C, D, and E in clinical specimensa 226
Hepatitis Test platform/probe Specimen Range of
Reference Specimen Target Comparison to other assays Comments
virus type chemistry volume input quantitative results
HAV Costa-Mattioli et al. Serum ABI Prism 7700/TaqMan 87-bp fragment 140 ␮l 360 to 3.6 ⫻ 108 100% sensitivity and specificity CV varied from
2002 (83) probe in 5⬘-non- copies/ml in 41 patient sera and 200 0.98% to 6.46%;
coding re- healthy blood donor sera HAV RNA re-
gion tested by anti-HAV IgM. mained detect-
ESPY ET AL.
able for ⬎60

days after clinical
diagnosis.
Rezende et al. 2003 Serum LightCycler/TaqMan 77-bp fragment 140 ␮l 100 to 108 cop- 72% sensitivity in 50 patient
(411) probe in 5⬘ non- ies/ml sera tested by anti-HAV
coding re- IgM.
gion
Roche Applied Plasma, serum LightCycler/FRET 5⬘ noncoding 300 ␮l serum 5,000 to 108 Commercially avail-
Science (http: hybridization probes region geq/ml able for research
//www.roche use only.
-applied-science
.com/lightcycler
-online/)
HBV Abe et al. 1999 (1) Serum ABI Prism 7700/TaqMan 174- or 241-bp 100 ␮l 200 to 2 ⫻ 109 Correlated with Quantiplex Interassay CV var-
probe fragment of copies/ml HBV DNA 1.0 Assay (R2 ied from 0.7% to
S gene or ranged from 0.93 to 0.97 for 9.1%.
331-bp frag- the three targets) and quan-
ment of X tified eight clinical sera
gene more than Quantiplex
among 46 patient sera;
100% specificity in 23
healthy donor sera.
Aliyu et al. 2004 (5) Serum LightCycler/FRET 259-bp frag- 200 ␮l 250 to 5 ⫻ 108 Detected 26 positive sera Intra- and interas-
hybridization probes ment of S copies/ml more than conventional lab- say CV varied
gene oratory-developed PCR as- from 2.6% to
say among 89 patient sera 75.3% and from
9.2% to 70.2%,
respectively.
Brechtbuehl et al. Plasma, serum LightCycler/SYBR 100-bp frag- 150 ␮l 1.3 ⫻ 104 to 1.3 Quantification of HBV DNA Interassay CV
2001 (44) Green I ment of S ⫻ 1010 cop- levels in 21 (58%) of 36 ranged from
gene ies/ml for sin- HBsAg-positive blood donor 1.2% to 2.9%.
gle-round sera were higher than those
PCR; 400 to obtained by Amplicor HBV
1.3 ⫻ 108 Monitor test, with mean
copies/ml for titer difference of 0.64 log
nested PCR (range, 0.14 to 0.96 log)
among samples of ⬎106
copies/ml.
Candotti et al. 2004 Plasma Stratagene 81-bp fragment 200 ␮l 30 (95% detec- Correlated with single-virus Intra- and interas-
(59) Mx4000/TaqMan of S gene tion rate) to real-time PCR assay (R2 ⫽ say CV ranged
probe 105 IU/ml 0.94) among 24 samples from 0.8% to
with HBV DNA titers 1% and from
ranging from 15 to 105 IU/ 3.4 to 3.6%,
ml; 100% specificity among respectively;
266 healthy blood donor equivalent
VOL. 19, 2006
sera tested by HBsAg EIA. detection of

HBV genotypes
A to F; a
multiplex assay
which also
detects HCV
RNA and HIV-
1 RNA.
Cane et al. 1999 Serum LightCycler/FRET 104-bp frag- 10 ␮l 105 copies/ml 100% agreement with direct Detect polymerase
(60) hybridization probes ment of minimum DNA sequence analysis gene mutations
polymerase and/or cloning assay in 20 encoding resis-
gene sera from 8 patients (HBV tance to lamivu-
genotypes A to H). dine in HBV
minor species
comprising 20%
of total popula-
tion.
Hennig et al. 2002 Plasma ABI Prism 7700/TaqMan 132-bp frag- 1 or 2 ml 28 IU/ml for Detected 14 of 16 (88% sensi- Developed for
(172) probe ment of C 1-ml sample; tivity) HBsAg-positive sera screening of
gene 14 IU/ml for and 3 of 189 (98.4% speci- blood donors.
2-ml sample ficity) HBsAg-negative sera
among 205 anti-HBc-posi-
tive patients
Ho et al. 2003 Serum LightCycler/FRET 130-bp frag- 200 ␮l 250 to 2.5 ⫻ Detected 114 of 120 (95% Intra- and interas-
(180) hybridization probes ment of C 1010 copies/ml sensitivity) HBsAg-positive say CV, were
gene sera and none of 45 (100% 6% and 16%,
specificity) HBsAg-negative respectively.
sera; correlated with Digene
Hybrid Capture II HBV
DNA test (R2 ⫽ 0.90) on 67
patient sera.
Ide et al. 2003 Serum ABI Prism 7700/SYBR 52-bp fragment 150 ␮l 50 to 316 cop- Utilizes amplified
(192) Green I of C gene ies/ml products from
the Amplicor
HBV Monitor
test as templates;
intra-assay CV
was 1.4% at 316
copies/ml.
Jardi et al. 2001 Serum LightCycler/FRET 144-bp frag- 200 ␮l 103 to 108 cop- Detected 128 of 193 (66%) Intra- and interas-
(199) hybridization probes ment of C ies/ml HBsAg-positive sera vs. 84 say CV, varied
gene detected by Quantiplex HBV from 2% to 4%
DNA 1.0 assay; correlated and from 4% to
6%, respec-
REAL-TIME PCR
with Quantiplex assay (R2 ⫽

0.94) in 30 patient sera. tively.
227

Leb et al. 2004 Plasma LightCycler/FRET 139-bp 200 ␮l 200 to 8 ⫻ 108 Quantified 107 vs. 100 samples Intra- and interas-
(253) hybridization probes fragment of copies/ml by COBAS Amplicor HBV say CV, were
C gene Monitor test among 123 0.7% and 1.5%,
clinical plasma simples, with respectively.
ESPY ET AL.
good correlation (R2 ⫽

0.95) between the two
assays in 97 samples.
Loeb et al. 2000 Serum ABI Prism 7700/TaqMan 105-bp frag- 200 ␮l 10 to 109 cop- Quantified 119 vs. 55 sera by Detected HBV
(283) probe ment of ies/ml Quantiplex HBV 1.0 assay DNA in 10
overlapping among 157 patient sera; (9%) of 109
region be- 100% specificity in 119 sera patients who
tween X and negative for all HBV sero- were positive
polymerase logic markers. only for anti-
genes HBc total anti-
bodies in 84
(78%) of 108
HBsAg-positive/
HBeAg-negative
patients.
Mercier et al. 1999 Plasma, serum ABI Prism 7700/TaqMan 113-bp frag- 200 ul 50 copies/ml 100% sensitivity and specificity A multiplex assay
(314) probe ment of C (analytical in 50 patients and 50 blood which also de-
gene sensitivity) donors tested by Quantiplex tects HCV
HBV 1.0 (bDNA) assay; RNA
1.5% false-positive rate in
screening 274 healthy blood
donors.
Mukaide et al. 2003 Serum ABI Prism 7700/TaqMan 120-bp frag- 300 ul 3 to 108 IU/ml Correlated with Amplicor Intra- and interas-
(324) probe ment of HBV Monitor test at R2 ⫽ say CVs varied
overlapping 0.94 with a commercially from 1.1% to
region be- available transcription-medi- 7.8% and from
tween core ated amplification-hybridiza- 1.6% to 4.8%,
and poly- tion protection assay (Chu- respectively.
merase gai Diagnostics Science,
genes Tokyo, Japan) at R2 ⫽ 0.94
in 156 and 100 patient sera,
respectively.
Pang et al. 2004 Serum ABI Prism 7700/3⬘ minor 108-bp frag- ? 200 to 109 cop- Correlated with COBAS Am- Capable of quanti-
(364) groove binder- ment of core ies/ml plicor HBV Monitor Test in fying HBV
conjugated TaqMan promoter 110 sera containing wild- DNA in sam-
probe gene, or type/core promotor mutant ples containing
107-bp frag- HBV (R2 ⫽ 0.86) and in 71 from 10% to
ment of pre- sera containing wild-type/ 90% core pro-
core gene precore mutant HBV (R2 ⫽ motor mutants
0.85). or samples with
20% to 90%
precore mutants
Paraskevis et al. Serum LightCycler/FRET 156-bp seq- 200 ␮l 250 to 1011 cop- Quantified 292 vs. 282 sera by Intra- and interas-
2002 (367) hybridization probes ment of ies/ml Amplicor HBV Monitor test say CVs varied
overlapping among 302 HbsAg-positive from 4.6% to
region of patients (R2 ⫽ 0.86); quanti- 30.1% and from
polymerase fied 63 vs. 36 sera by Quan- 5.1% to 53%,
VOL. 19, 2006
and S genes tiplex HBV DNA 1.0 assay respectively.

among 66 HbsAg-positive
patients (R2 ⫽ 0.88).
Pas et al. 2002 Serum ABI Prism 7700/TaqMan 90-bp fragment 200 ␮l 46 to 1.25 ⫻ 109
(369) probe of pre-S gene IU/ml
Punia et al. 2004 Serum LightCycler/SYBR- rt204- and 100 ␮l - Detected HBV rt204 and Detect and quan-
(388) Green I rt180-con- rt180 mutants in two more tify HBV rt204
taining frag- patients than conventional and rt180 mu-
ments of DNA sequencing assay tant populations
polymerase among 10 patients treated at 0.01% of 105
gene with lamivudine. to 109 copies of
wild-type HBV
DNA using am-
plification-refrac-
tory mutation
system PCR.
Schaefer et al. 2003 Serum LightCycler/SYBR- 336-bp frag- 200 ␮l 20 to 2 ⫻ 109 Yielded quantification differ- Primers amplified
(434) Green I ment of IU/ml ences of ⬍0.5 log IU/ml in S gene frag-
S gene 25 of 30 (83.3%) sera tested ments of all
by a similar LightCycler as- well-character-
say amplifying a X gene tar- ized HBVs in
get. humans, pri-
mates, squirrels,
and woodchuck.
Stelzl et al. 2004 Serum LightCycler/FRET 120-bp frag- 200 ␮l 2.5 ⫻ 102 to 109 100% sensitivity and specificity Commercially as
(461) hybridization probes ment of IU/ml compared to COBAS Am- RealArt HBV
HBV plicor HBV Monitor test LC PCR. Re-
genome among 117 clinical sera; reagents available
(? gene) sults of 76% (37 of 49) of from Artus
positive sera were within GmbH, Ham-
⫾0.5 log IU/ml by both tests burg, Germany;
and 18%, respectively. intra- and inter-
assay variation
ranged from 9%
to 40% and from
16% to 73%,
respectively.
Sum et al. 2004 Serum ABI Prism 112-bp frag- 200 ␮l 102 to 109 cop- Detected 128 (73%) vs. 119 ⬍7% intra-assay
(469) 7000/molecular beacon ment of ies/ml (68%) positive sera by CO- and ⬍5% inter-
probe S gene BAS Amplicor HBV Moni- assay variation
tor test among 175 HBsAg- in testing HBV
positive patients (R2 ⫽ ad and ay sub-
REAL-TIME PCR
0.90). type DNA stan-

dards.
229


Takaguchi et al. Serum, liver ABI Prism 7700/TaqMan 272-bp frag- 100 ␮l se- 200 copies/ml Detected HBV DNA in 12 Unknown analyti-
2002 (470) tissue probe ment of rum, 10 (analytical (33%) of 36 patients with cal sensitivity of
X–pre-C mg tissue sensitivity for HBsAg- and anti-HCV Ab- assay for liver
gene region serum) negative chronic liver dis- tissue.
ESPY ET AL.
ease; 5 of 6 serum HBV

DNA-positive patients also
had detectable HBV DNA
in liver tissue.
Weinberger et al. Serum ABI Prism 7700/TaqMan 81-bp fragment 200 ␮l 102 to 109 Good correlation of results
2000 (534) probe of S gene geq/ml with laboratory-developed
semiquantitative conven-
tional PCR assay (R2 ⫽
0.83).
Whalley et al. 2001 Serum LightCycler/FRET 130-bp frag- 200 ␮l 400 copies/ml Good agreement with results Utilize melting
(539) hybridization probes ment of (analytical of direct DNA sequence curve analysis to
polymerase sensitivity) analysis. detect and dif-
gene ferentiate
rtM204 mutants
at ⱖ25% level
of minor popu-
lation.
Wightman et al. Serum ABI Prism rtM204-con- 200 ␮l 102 copies/ml Good agreement with results Able to detect 1%
2004 (544) 7700/molecular beacon taining frag- (analytical of direct DNA sequence minor popula-
probe ment in C sensitivity) analysis. tions of
domain of rtM204V and
polymerase rtM204I mu-
gene tants.
Yeh et al. 2004 Serum LightCycler/FRET 340-, 368-, and 200 ␮l 102 to 1011 Good correlation with quanti- Intra- and interas-
(553) hybridization probes 416-bp frag- copies/ml tative results obtained by say CVs were
ments of Amplicor HBV Monitor test 8.9% and 14.3%,
S gene (R2 ⫽ 0.99), NGI Super- respectively, for
Quant assay (R2 ⫽ 0.98), quantification of
and Quantiplex HBV bDNA HBV DNA; able
assay (R2 ⫽ 0.99); good to differentiate
agreement of genotype HBV genotypes
results with direct DNA A to G and de-
sequence analysis. tect mixed geno-
type infections at
10% level of
minor popula-
tion.
Zanella et al. 2002 Serum GeneAmp 5700/TaqMan 123-bp frag- 200 ␮l 102 to 1010 cop- Detected HBV DNA in 38 Intra- and interas-
(557) probe ment of ies/ml (35%) more samples than say CVs ranged
S gene Digene Hybrid Capture from 8.4% to
HBV DNA test (R2 ⫽ 0.76) 35.8% and from
among 108 HBsAg-positive 6.2% to 53.1%,
sera; 100% specificity on respectively.
sera from 20 healthy sero-
negative blood donors.
VOL. 19, 2006
Zanella et al. 2002 Liver tissue GeneAmp 5700/TaqMan 123-bp frag- 200 mg 50 to 107 No comparison with other Higher HBV
(558) probe ment of tissue copies/ml assays. DNA titers in
S gene liver tissues
from HBsAg-
positive than
HBsAg-ngative
patients (P ⬍
0.01).
Zhang et al. 2002 Serum LightCycler/FRET 217-, 586-, and 200 ␮l 100 copies/ml Good agreement with results Utilize melting
(560) hybridization probes 650-bp frag- (analytical of direct DNA sequence curve analysis to
ments of sensitivity) analysis. detect amd dif-
basal core ferentiate core/
promoter, precore and
pre-C, and HBsAg mutants
S genes, re- at 5% level of
spectively minor popula-
tion
HCV Bullock et al. 2002 Serum LightCycler/FRET 324-bp frag- 200 ␮l 2 ⫻ 104 cop- Concordance with line probe HCV genotyping
(53) hybridization probes ment in ies/ml mini- assay (INNO-LiPA) in 110 assay differenti-
5⬘ noncoding mum (99%) of 111 patient sera. ating among
region for genotypes 1, 1a,
initial PCR; 1b, 2, 2a/c, 2b,
284-bp frag- 3a, and 4.
ment for
second-
round PCR
Candotti et al. 2004 Plasma Stratagene 68-bp fragment 200 ␮l 167 (95% detec- Correlated with single virus Intra- and interas-
(59) Mx4000/TaqMan in 5⬘ non- tion rate) to real-time PCR assay (R2 ⫽ say CVs ranged
probe coding 105 IU/ml 0.95) among 14 samples from 0.8% to
region with HIV-1 RNA titers 2.4% and from
ranging from 300 to 105 IU/ 2.6 to 2.9%,
ml; 99% specificity among respectively;
266 healthy blood donor equivalent de-
sera tested by anti-HCV tection of HCV
EIA. genotypes 1 to
6; a multiplex
assay which also
detects HBV
DNA and
HIV-1 RNA.
Enomoto et al. 2001 Serum ABI Prism 7700/TaqMan 161-bp frag- 250 ␮l 10 to 108 Correlated with Quantiplex
(112) probe ment in copies/ml HCV 2.0 (R2 ⫽ 0.84), Am-
REAL-TIME PCR
5⬘ noncoding plicor HCV Monitor v2.0

region (R2 ⫽ 0.85), and HCV core
231

Ag EIA (R2 ⫽ 0.55) in 50

patient sera.
Kawai et al. 1999 Serum ABI Prism 7700/TaqMan 199-bp 50 ␮l 2 ⫻ 103 to 2 ⫻ Correlated with Amplicor
ESPY ET AL.
(209) probe fragment in 108 IU/ml HCV Monitor v1.0 (R2 ⫽

5⬘ noncoding 0.81) in 138 patient sera;
region 10- to 100-fold higher titers
than those of Amplicor
HCV Monitor.
Kleiber et al. 2000 Plasma, serum ABI Prism 7700/TaqMan 250-bp 200 ␮l 64 to 107 IU/ml 100% specificity in 100 HCV- CVs varied from
(222) probe fragment in seronegative blood donor 21.6% to 30.4%;
5⬘ noncoding sera tested by Cobas equivalent
region Amplicor HCV test, v2.0. detection of
HCV genotypes
1 to 5.
Komurian-Pradel et Serum LightCycler/SYBR 220-bp frag- 200 ␮l 4 to 4.05 ⫻ 106 Correlated with Quantiplex CVs varied from
al. 2001 (231) Green I ment in IU/ml HCV RNA 2.0 assay (R2 ⫽ 0.7% to 3.7%.
5⬘ noncoding 0.79) and quantified 2 more
region clinical serum specimens
than Quantiplex among 33
patient sera.
Martell et al. 1999 Serum ABI Prism 7700/TaqMan 194-bp frag- 140 ␮l 330 to 107 Correlated with Quantiplex CVs varied from
(304) probe ment in copies/ml HCV RNA 2.0 assay (R2 of 1% to 6.2%.
5⬘ noncoding 0.71 to 0.88) and National
region Genetics Institute Super-
quant assay (R2 of 0.74 to
0.99) in 79 patient sera.
Mercier et al. 1999 Plasma, serum ABI Prism 7700/TaqMan 168-bp frag- 200 ␮l 50 copies/ml 100% sensitivity and specificity CVs varied from
(314) probe ment in (analytical in 50 patients and 50 blood 8.7% to 74.7%;
5⬘ noncoding sensivity) donors tested by Quantiplex a multiplex as-
region HBV 1.0 assay; 0.7% false- say which also
positive rate in screening detects HBV
274 healthy blood donors. DNA.
Ratge et al. 2002 Serum LightCycler/FRET 235-bp frag- 200 ␮l 109 to 2,500 100% agreement in results CVs varied from
(398) hybridization probes ment in IU/ml with a qualitative conven- 11.5% to 12.9%;
5⬘ noncoding tional assay for 156 patient two rounds of
region sera. rapid-cycle real-
time PCR in
this assay.
Schroter et al. 2001 Serum LightCycler/SYBR 124-bp frag- 200 ␮l 900 to 7 ⫻ 106 Correlated with Amplicor
(440) Green I ment in copies/ml HCV Monitor v1.0 and in-
5⬘ noncoding house conventional PCR
region assays in 81 patient sera and
30 healthy blood donor sera.
Schroter et al. 2002 Serum LightCycler/FRET 143-bp frag- 200 ␮l 103 IU/ml (ana- 100% concordance with con- HCV genotyping
(439) hybridization probes ment in lytical sensi- ventional HCV NS5B gene assay differenti-
5⬘ noncoding tivity) sequencing assay in 190 pa- ating among
region tient sera. genotypes 1 to 4
(not subtypes).
VOL. 19, 2006
Takeuchi et al. 1999 Serum ABI Prism 7700/TaqMan 161-bp vs. 250 ␮l 10 to 108 256-bp target amplification CVs varied from
(471) probe 256-bp frag- copies/ml correlated with Quantiplex 0.4% to 4.7%;
ment in HCV 1.0 assay (R2 ⫽ 0.84) 161-bp target
5⬘ noncoding and Amplicor HCV Monitor amplification
region v1.0 (R2 ⫽ 0.61) in 15 pa- showed higher
tient sera; 10- to 100-fold sensitivity (e.g.,
more sensitive than and higher RNA
Amplicor HCV Monitor titers) than
v1.0, and 100% specificity in 256-bp target
50 patient sera. amplification.
White et al. 2002 Serum, liver LightCycler/SYBR 244-bp frag- 200 ␮l se- 102 to 106 Correlated with Amplicor Assay conisted of
(542) biopsy Green I ment in rum; 0.8 to copies/ml HCV Monitor v2.0 (R2 ⫽ an initial 15-
tissue 5⬘ noncoding 8.1 mg serum or 0.86) in 16 patient sera. cycle real-time
region liver tissue copies/ug of PCR followed
total RNA in by 40-cycle real-
tissue time PCR.
Yang et al. 2002 Plasma, serum ABI Prism 182-bp frag- 100 ␮l 104 (60% detec- 100% sensitivity and specificity Equivalent detec-
(552) 7700/molecular beacon ment in tion rate) to in 17 patients and 14 tion of HCV
5⬘ noncoding 109 copies/ml healthy blood donors tested genotypes 1 to 6.
region by anti-HCV EIA.
HDV Yamashiro et al. Serum LightCycler/SBYR 134-bp frag- 150 ␮l 100 to 106 Good correlation with HBV
2004 (551) Green I ment; 98% copies/ml DNA levels in 48 patients.
homologous
for geno-
types I and
IIa, 100%
homologous
for genotype
Iib
HEV Orru et al. 2004 Feces LightCycler/SYBR 77-bp fragment 300 ␮l of 10 to 106 copies/ Correlated with anti-HEV an-
(357) Green I in ORF2 10% stool PCR tibody by EIA and conven-
region suspension tional real-time PCR in one
in PBS clinical sample.
a
HAV, HBV, HCV, HDV, and HEV, hepatis virus type A, B, C, D, and E, respectively; CV, coefficient of variation; geq, genome equivalent; EIA, enzyme immunoassay; Ab, antibody; PBS, phosphate-buffered saline.
REAL-TIME PCR
233
TABLE 21. Comparison of rapid-cycle real-time PCR assays for the detection of HIV-1 and HIV-2 in clinical specimensa 234
Range of
Test platform/probe Specimen volume Comparison to other
HIV type Reference Specimen type(s) Target quantitative Comments
chemistry input assays
results
HIV-1 Brussel et al. 2003 PBMC in whole blood LightCycler/FRET Overlapping region 106 PBMC 6 to 105 copies No comparison with A nested PCR assay
(52) hybridization between LTR per 5 ⫻ 104 other assays. designed to quan-
probes and chromo- PBMC tify HIV-1 provi-
somal Alu ele- ral DNA for in
ESPY ET AL.
ment in initial vitro infectivity

PCR; LTR gene experiments.
in second round
of PCR
Candotti et al. Plasma Stratagene 79-bp fragment in 200 ␮l 680 (95% de- Correlated with sin- Intra- and interassay
2004 (59) Mx4000/Taq- LTR region tection rate) gle virus real-time CV ranged from
Man probe to 2 ⫻ 105 PCR assay (R2 ⫽ 1.6% to 3.4% and
IU/ml 0.87) among 39 from 3.4 to 7%,
samples with respectively;
HIV-1 RNA ti- equivalent detec-
ters ranging from tion of HIV-1
200 to 2 ⫻ 105 group M subtypes
IU/ml; 100% A to G; a multi-
specificity among plex assay which
266 healthy blood also detect HBV
donor sera tested DNA and HCV
by anti-HIV-1/2 RNA.
EIA.
Desire et al. 2001 PBMC in whole blood ABI Prism 7700/ 199-bp fragment of 5 ⫻ 106 PBMC 5 to 105 copies No correlation with Quantify HIV-1
(93) TaqMan probe pol gene per 106 HIV-1 RNA lev- proviral DNA;
PBMC els in 21 clinical intra- and interas-
plasma samples say CVs were
tested by Ampli- 13% and 27%,
cor HIV-1 Moni- respectively.
tor v1.5 or Quan-
tiplex HIV RNA
2.0 assay.
Eriksson et al. PBMC in whole blood ABI Prism 7700/ 89-bp fragment of 5 ⫻ 106 CD4⫹ 2 to 105 copies No comparison with Quantify HIV-1
2003 (113) TaqMan probe pol gene cells per 104 other assays. proviral DNA;
CD4⫹ cells intra-assay %CV
ranged from 30%
to 34%
Ghosh et al. 2003 Breast milk ABI Prism 7700/ Fragment of LTR 1 ml 102 to 107 cop- No comparison with Quantify HIV-1
(140) TaqMan probe gene ies/ml other assays. proviral DNA in
breast milk.
Gibellini et al. Plasma LightCycler/SYBR 142-bp fragment of 1 ml 50 to 5 ⫻ 105 Detected 4 (7%) Both intra- and
2004 (141) Green gag gene copies/ml more positive interassay CVs
samples than were ⬍4%.
Quantiplex HIV-1
RNA 3.0 bDNA
assay (R2 ⫽ 0.91)
among 56 plasma
specimens from
HIV-infected
patients; 100%
VOL. 19, 2006
specificity on 25
samples from
seronegative
blood donors.
Gibellini et al. PBMC in whole blood LightCycler/SYBR 266-bp fragment of 5 ⫻ 106 PBMC 5 to 104 copies No correlation with Quantify HIV-1
2004 (142) Green pol gene per 106 HIV-1 RNA lev- subtype B provi-
PBMC els in 50 clinical ral DNA; intra-
plasma samples and interassay
tested by Quanti- CVs were ⬍5%.
plex HIV RNA
3.0 assay; 100%
sensitivity and
specificity on 50
HIV-1-seroposi-
tive and 20 sero-
negative clinical
sera tested by
conventional PCR
assay.
Gueudin et al. Plasma LightCycler/Taq- 111-bp fragment of 200 ␮l 1.5 ⫻ 103 to 5 No comparison with Specifically quantify
2003 (153) Man probe LTR gene ⫻ 105 cop- other assays. HIV-1 group O
ies/ml RNA; both intra-
and interassay
CVs were ⬍20%.
Ito et al. 2003 Lymphoid tissue (ton- ABI Prism 7700/ Fragments of vari- 5 mg 10 to 105 cop- Good correlation Quantify HIV-1
(197) sillar tissue) SYBR Green ous bp sizes in ies/reaction with levels of RNA in lymphoid
V3–V5 region of HIV-1 p24 Ag in tissue; both intra-
gp120 gene lymphoid tissue and interassay
(R2 ⫽ 0.99). CVs were ⬍10%.
Palmer et al. 2003 Plasma ABI Prism 7700/ Fragment of gag 1 to 7 ml 102 to 108 cop- Correlated with re- Intra-assay variabil-
(363) TaqMan probe gene ies/ml sults of VER- ity ranged from
SANT HIV RNA 13% to 37%.
3.0 bDNA assay
(R2 ⫽ 0.89) on
22 clinical speci-
mens, but de-
tected titers in
15 additional
samples that were
negative by
bDNA test; 100%
specificity on
samples from se-
ronegative blood
REAL-TIME PCR
donors.
235
Range of
Test platform/probe Specimen volume Comparison to other
HIV type Reference Specimen type(s) Target quantitative Comments
chemistry input assays
results
Saha et al. 2001 Plasma LightCycler/scor- 104-bp fragment of ? 1 to 108 cop- Good correlation Intra- and interassay
(423) pion probe pol gene ies/reaction with results of variability ranged
rapid PCR assay from 1% to 46%
using molecular and from 5% to
ESPY ET AL.
beacon probe 20%, respectively.

(R2 ⫽ 0.99).
Vet et al. 1999 Plasma ABI Prism 7700/ Fragment of gag ? 10 to 106 cop- Correlated with re- A multiplex assay
(518) molecular bea- gene ies/reaction sults of Amplicor which also quanti-
con probe HIV-1 Monitor fies HIV-1,
test v1.0. HTLV-I, and
HTLV-II RNA
levels.
Victoria et al. PBMC and lymphoid Smart Cycler/ Fragment of LTR 5 ⫻ 106 cells 10 to 5 ⫻ 104 No comparison with Quanitfy HIV-1
2003 (519) cell lines SYBR Green gene copies per other assays. RNA for in vitro
106 cells infectivity experi-
ments.
Weber et al. 2003 Cell culture superna- Smart Cycler/ 199-bp fragment of 200 ␮l 10 to 107 No comparison with Quantify HIV-1
(529) tant SYBR Green pol gene copies/reac- other assays. proviral DNA for
tion in vitro infectivity
experiments.
HIV-2 Damond et al. Whole blood LightCycler/ 160-bp fragment of 1 ml 5 (100% de- HIV-2 proviral DNA Requires nested
2001 (88) TaqMan probe gag gene tection rate) levels correlated RT-PCR and
to 500,000 with positive cell quantifies HIV-2
copies/105 cultures for HIV-2 proviral DNA,
PBMC virus (U ⫽ 26; P with intra- and
⬍ 0.02) but not inter assay CVs
with rate of ranging from
plasma HIV-2 11.8% to 41%
RNA detection (U and 30% to 40%,
⫽ 61; P ⬍ 0.07) respectively;
among 29 HIV-2 equivalent detec-
antibody-positive tion of HIV-2
patients. subtypes A and B.
Damond et al. Plasma LightCycler/ 89-bp fragment of 1 ml 250 to 5 ⫻ 105 HIV-2 RNA de- Intra- and interassay
2002 (89) TaqMan probe gag gene copies/ml tected at ⬎250 CVs varied from
copies/ml in 21 1% to 4.4%;
(54%) of 39 pa- equivalent detec-
tients with detect- tion of HIV-2
able HIV-2 provi- subtypes A and B.
ral DNA, with
100% specificity
among 25 HIV-
negative blood
donors and 25
HIV-1 antibody-
positive patients.
broad-range panel of potentially cross-reacting organisms, all
subtypes A and B.
Intra- and interassay
which also quanti-

0.2% to 5.6% and
Interassay CVs var-
HIV-2 subtype A
ly; equivalent de-
7.5%; differential
PBMC, peripheral blood mononuclear cells; LTR, long terminal repeat; EIA, enzyme immunoassay; CV, coefficient of variation; Ag, antigen; HTLV, human T-cell lymphotropic virus; RT, reverse transcription.
3.1%, respective-
tection of HIV-2
CVs varied from
quantification of
ied from 2% to
of which showed negative results.
A multiplex assay
HTLV-II RNA
and B RNAs.
from 0.7% to
HTLV-I, and
The assay developed by Pryce et al. (385), targeted the 18S
fies HIV-1,
rRNA gene of Aspergillus fumigatus isolated from whole blood
levels.
samples and compared the results to the clinical features of
eight patients at high-risk of invasive aspergillosis. Their assay
was positive for 1 patient with clinically proven invasive pul-
monary aspergillosis and 1 patient with probable invasive pul-
positive (by West-

ern blot) patients.
copies/ml in 12 of
qualitative results
monary aspergillosis. The PCR assay was also positive in 2
HIV-2 antibody-
HIV-1 antibody-
HIV-2 antibody-
positive patients
but none in two
antibody assay.
tected at ⬎250
HIV-2 RNA de-
HIV-2 RNA de-

patients with no clinical evidence of fungal infection and there-
positive and 1
of anti-HIV-2
HIV-negative
Correlated with
13 untreated
(81%) of 16
tected in 13
fore it was not possible to distinguish between a false-positive

PCR result and subclinical fungemia in these patients. The
sera.
PCR results were negative for 1 patient with proven dissemi-

nated invasive Aspergillus terreus infection, highlighting the lim-
itations of assays targeting only Aspergillus fumigatus.
A number of studies have compared the sensitivity of real-
107 copies/ml
rate) to 5 ⫻
10 to 106 cop-
ies/reaction
time PCR methods to the galactomannan ELISA test for As-

250 to 2.5 ⫻
detection
500 (100%
106 cop-
pergillus antigen recently approved by the FDA. Kami et al.

ies/ml
(206) compared a real-time PCR assay targeting the 18S rRNA

gene to the galactomannan test (Platelia Aspergillus, Pasteur
Diagnostic) and a test for (133)-␤-D-glucan (Fungi-Tec,
Seikagaku Corporation), which serves as a marker of fungal
infection. They examined 323 blood samples from 122 patients
with hematologic malignancies, including 33 patients with in-
vasive pulmonary aspergillosis and 89 control patients. The
400 ␮l
1 ml
reported sensitivity for the PCR, galactomannan, and (133)-

?
␤-D-glucan assays for the diagnosis of invasive pulmonary as-

pergillosis were 79%, 58%, and 67% respectively; specificities
173-bp fragment in
LTR and gag/pol

61-bp fragment of
fragment of gag
region between
ORF, or 67-bp
were 92%, 97%, and 84%. The positive PCR findings preceded
Fragment of env
LTR region
those of galactomannan and (133)-␤-D-glucan measurements

by 2.8 ⫾ 4.1 and 6.5 ⫾ 4.9 days, respectively. Other studies
comparing the galactomannan ELISA to laboratory developed
gene
gene
real-time PCR assays suggest that a combination of the two

methods may provide improved diagnosis of invasive aspergil-
losis (61, 80, 428)
LightCycler/SYBR
TaqMan probe
The molecular mechanisms of drug resistance in fungi have

molecular bea-
ABI Prism 7700/
ABI Prism 7700/
traditionally been difficult to elucidate due to the cumbersome

con probe
nature of susceptibility testing for these organisms. Nasci-

Green I
mento et al. (331) developed a real-time PCR method to detect

Aspergillus fumigatus mutations that confer high-level resis-
tance to itraconazole. Their results demonstrated that overex-
pression of two genes, AfuMDR3 and AfuMDR4, which encode
drug efflux pumps, and the selection of drug target site muta-
tions can be linked to high-level itraconazole resistance.
Candida Species
Plasma
Plasma
Plasma
The majority of real-time PCR assays developed to date for

Candida species have focused on the identification of the six or
seven most common species isolated from clinical specimens
and most assays analyzed isolates growing in pure culture (157,
Ruelle et al. 2004
188, 268). If separation of the various species was attempted, it

Vet et al. 1999
Schutten et al.
2000 (442)
required multiplexed sets of primers and probes or multiple

sets of species-specific probes.
(421)
(518)
Candida species are the fourth leading cause of nosocomial

bloodstream infections and are associated with a mortality rate
of 40 to 50% so rapid and reliable detection of candidemia has
attracted significant interest (105, 152, 366). Selvarangan et al.
(444) reported the identification of six Candida spp. directly
a
from growing blood cultures using the internal transcribed

TABLE 22. Real-time PCR methods for fungia 238
Clinical Turnaround
sensitivity vs. time vs. Refer-
Genus Specimen Technology Target Status Conventional method Comments
conventional conventional ence(s)
method method
Aspergillus BAL fluid iCycler TaqMan 18S rRNA HB Culture, histopathology Greater Faster 428 Compared with antigen detection
BAL fluid, LightCycler FRET HP 18S rRNA HB NA NA Faster 287 MagNA Pure vs. manual
blood extraction
ESPY ET AL.
BAL fluid, LightCycler FRET HP ITS Expmt1 Culture, histopathology Greater Faster 358 Research in rabbits
lung tissue
BAL fluid, LightCycler FRET HP mito. tRNA HB Culture, histopathology Greater Faster 397
tissue
BAL fluid, LightCycler FRET HP cyto. b HB Culture, histopathology NR Faster 459 Quantitative; nested PCR was
blood more sensitive
Blood, ABI 7700 TaqMan 5.8S rRNA HB Culture, histopathology Lower Faster 376 Quantitative
serum
Blood, ABI 7700 TaqMan 18S rRNA HB Culture, histopathology Greater Faster 206, 210 Compared with antigen detection
plasma
Blood, ABI 7700 TaqMan 28S rRNA HB Culture, histopathology Greater Faster 61 Compared with antigen detection
serum
Serum, ABI 7700 TaqMan fks HB Culture, histopathology NR Faster 81 Evaluation of serum, white cell
plasma, pellet, plasma
cell pellet
Blood LightCycler FRET HP 18S rRNA HB Culture, histopathology NR Faster 285, 385 Quantitative
Blood, tissue LightCycler FRET HP 18S rRNA Expmt1 Culture, histopathology Greater Faster 286 Research in mice and rabbits
Serum LightCycler FRET HP mito. rRNA HB Culture Greater Faster 80 Comparison with antigen
detection
Culture ABI 7700 TaqMan atr HB Conventional PCR NA Faster 445 Monitoring of efflux pump
transcript levels
Culture ABI 7700 Molecular mdr HB Conventional PCR NA Faster 331 Monitoring of expression levels
Beacons
Tissue ABI 7700 TaqMan 18S rRNA Expmt1 CFU quantitation NA Faster 41 Caspofungin efficacy monitoring
in mice
Tissue LightCycler FRET HP 18S rRNA HB Culture, histopathology Equal Faster 194 3 clinical specimens
Candida Blood ABI 5700 TaqMan ITS2 HB Culture Greater Faster 290–292 Quantitative
Blood LightCycler FRET HP 18S rRNA HB Culture NR Faster 285, 385 Quantitative
Blood LightCycler FRET HP 18S rRNA HB Culture Greater Faster 543 Detects seven species with one
probe set
Blood LightCycler FRET HP ITS HB Culture Equal Faster 444 Detects/differentiates six species
with four probe sets
Culture ABI 7700 TaqMan ITS2 HB Culture NA Faster 157 Detects/differentiates six species
with three probe sets
Culture ABI 7700 TaqMan erg11 HB Broth dilution NA Faster 65, 144 Fluconazole resistance
genotyping
Culture ABI 7700 molecular ITS2 HB Culture, phenotypic NA Faster 368 C. dubliniensis identification
beacons assays
Culture LightCycler FRET HP erg11 HB Broth dilution NA Faster 284 Fluconazole resistance
genotyping
Culture LightCycler FRET HP erg11, cdr, HB Broth dilution NA Faster 129, 256 Fluconazole resistance
mdr genotyping
Culture LightCycler FRET HP act Expmt1 Culture NA Faster 351 Cutaneous C. albicans viability
model
Culture LightCycler SYBR 5.8S rRNA HB Culture, biochemicals NA Faster 188 Detects/differentiates six species
with six primer sets
Oral rinse LightCycler SYBR 18S rRNA HB Culture Lower Faster 543
Tissue LightCycler FRET HP 18S rRNA HB Culture Greater Faster 194 1 clinical specimen
Coccidioides Culture LightCycler FRET HP Ag2/PRA HB Culture, nucleic acid NA Faster 31 Extraction procedure safety
probe evaluated
Conidiobolus Tissue LightCycler FRET HP 18S rRNA HB Culture, microscopy Equal Faster 194 1 clinical specimen
VOL. 19, 2006
Cryptococcus Culture LightCycler SYBR 5.8S rRNA HB Culture, biochemicals NA Faster 188
Tissue LightCycler FRET HP 18S rRNA Expmt1 Culture Lower Faster 32 Research in mice; quantitative
Histoplasma Culture, LightCycler FRET HP ITS LD Culture, nucleic acid Equal Faster 303 3 clinical specimens
BAL fluid, probe except
tissue, for bone
bone marrow
marrow
Tissue LightCycler FRET HP 18S rRNA LD Culture, nucleic acid Equal Faster 194 1 clinical specimen
probe
Paracoccid- Culture ABI 7700 TaqMan 20 genes LD Conventional PCR NR Faster 301 Yeast and hyphal phases
ioides differential gene expression
Pneumocystis Oral swabs, iCycler SYBR mt LSU Expmt1 Stains, microscopy NA Equal 276 Quantitative monitoring of levels
tissue in rats
Tissue LightCycler FRET HP dhfr Expmt1 Stains, microscopy NR Equal 250 Quantitative; research in rats
BAL fluid ABI 7700 TaqMan mt LSU LD Stains, microscopy Greater Equal 308 Quantitative
BAL fluid, LightCycler FRET HP msg LD Stains, microscopy Equal Equal 128, 251 Quantitative
oral
washes,
sputum
Pneumocystis BAL fluid LightCycler SYBR mt SSU LD Stains, microscopy NR Equal 204 Quantitative
Nasopharyn- ABI 7700 TaqMan mt LSU LD Stains, microscopy NR Equal 497 Comparison with nested PCR,
geal aspi- noninvasive
rate
Plasmid ABI 7700 SYBR dhps LD Conventional PCR, NA Faster 334 Genotyping of dhps gene
sequencing
Oral wash LightCycler FRET HP msg HB Stains, microscopy Lower Equal 249 Quantitative, noninvasive
Sputum, LightCycler SYBR 5S rRNA LD Stains, microscopy NR Equal 360 Comparison with conventional
bronchial PCR
wash
Stachybotrys Culture ABI 7700 TaqMan 18S rRNA LD Culture, microscopy NA Faster 85 Quantitative
a
Abbreviations: ASR, analyte-specific reagent; LD, laboratory developed; NA, not applicable; NR, not reported; cyto., cytochrome; Expmtl, experimental; HP, hybridization probe; Mito. or mt, mitochondrial; BAL,
bronchoalveolar lavage; HB, home brew; ITS, internal transcribed spacer; LSU, large subunit; SSU, small subunit.
REAL-TIME PCR
239
spacer 1 (ITS1) and ITS2 regions flanking the 18S, 5.8S, and this cutoff value also raised the number of false negative re-
28S rRNA genes and four sets of sequence-specific FRET sults. See Table 22 for a literature review.
hybridization probes. The assay was 100% sensitive and spe-
cific for 62 blood culture isolates containing yeasts compared
with culture and identification using phenotypic and biochem- PARASITES
ical methods. Plasmodium spp.
Maaroufi et al. (292) developed a TaqMan-based real-time
PCR assay for detection of Candida spp. from blood samples Among parasitic infections, real-time PCR has been applied
that featured a Candida genus-specific probe and a Candida most vigorously in the diagnosis of malaria. Conventional di-
albicans species-specific probe. One-hundred twenty-two blood agnosis is based on microscopic examination of peripheral
samples from 61 patients with clinically proven or suspected blood smears, and accuracy is dependent upon the training and
systemic Candida infections were evaluated using the assay and experience of the preparers and readers of the slides. Expertise
the sensitivity and specificity for Candida albicans detection is often lacking. On the other hand, microscopy is inexpensive,
was reported as 100 and 97%, respectively. The Candida ge- does not require complex equipment, and is relatively rapid.
nus-specific probe cross-reacted with a number of other fungal Although the startup costs for real-time PCR are high, the test
organisms and the sensitivity and specificity of the genus-spe- reagents are inexpensive, interpretative subjectivity is elimi-
cific probe was reported to be 100 and 72%, respectively. nated, and no special expertise is required by technologists.
Two real-time PCR methods have been described to detect This methodology may not be usable in remote field areas of
point mutations in the erg11 gene that are associated with countries where malaria is endemic, where electricity would
fluconazole resistance in Candida spp. (256, 284). not be available, but could be valuable in regional clinics for
rapid detection of Plasmodium, and importantly for proper
treatment, accurate determination of the infecting species. The
Pneumocystis jiroveci latter is especially critical since Plasmodium falciparum infec-
tion has a significant mortality rate and treatment is different
Laboratory detection of Pneumocystis jiroveci (formerly than for other species. Resistance to antimalarials is also a
Pneumocystis carinii f. sp. hominis) has traditionally been problem and real-time PCR has the potential to rapidly detect
achieved by examination of a fluorescent smear or through the the resistance genes, although this application has not yet been
use of a direct fluorescent antibody. The smear is relatively developed.
quick and inexpensive but has the disadvantages of being in- The RealArt Malaria LC PCR assay (Artus GmbH, Ham-
sensitive and highly dependent upon reader expertise. The burg, Germany) is a commercially available kit which was de-
direct fluorescent antibody test has been problematic of late veloped for use on the LightCycler (Roche Diagnostics). It
due to the discontinuation of control materials by kit manu- targets the 140-bp region of the 18S rRNA genes of the four
facturers and the lack of readily available laboratory developed species of Plasmodium which infect humans. This assay detects
controls at many institutions due to a decline in the number of the presence of Plasmodium in blood but does not determine
Pneumocystis jiroveci-infected patients in this era of highly ac- which species is present, which is a significant limitation. In a
tive antiretroviral therapy. study of 259 travelers to areas where malaria is endemic, the
Pneumocystis jiroveci detection is likely to be one of the few Artus kit was 99.5% sensitive and 100% specific in the detec-
instances in which real-time PCR is slower than the conven- tion of Plasmodium compared to a nested PCR method (124).
tional method. Performing both a fluorescent stain and reading A limited evaluation of the quantitation of parasitemia was
the slide takes approximately 30 min while the extraction and performed but there was only a low to moderate correlation
real-time PCR assay has an analytical turnaround time of ap- with gene copy number and microscopic determinations.
proximately 3 h. However, the enhanced sensitivity and objec- Laboratory-developed assays have also been developed for
tive nature of the real-time PCR assay make this method more the LightCycler and FRET technology using either SYBR
appealing than direct staining. green (R. U. Manson, K. A. Mangold, R. B. Thomason, Jr., E.
The role of quantitative PCR for Pneumocystis jiroveci has Koay, L. R. Peterson, and K. Kaul, Program Abstr. 43rd In-
received attention since Pneumocystis jiroveci can colonize tersci. Conf. Antimicrob. Agents Chemother., abstr. P-414,
healthy individuals (neonates, pregnant woman, etc.). Larsen 2003) or LC Red 640 as the acceptor dye. The latter method,
et al. (251) describe a quantitative, touch-down, real-time PCR in conjunction with melting curve analysis, was used to evalu-
assay for the diagnosis of Pneumocystis carinii pneumonia ate blood transported on IsoCode Stix from patients from
(PCP). The authors examined lower respiratory tract and oral Gabon and Thailand suspected of having malaria (A.
washes from PCP and non-PCP patients, targeting the major Muyombwe, I. Lundgren, L. M. Sloan, J. E. Rosenblatt, P.G.
surface glycoprotein (msg) gene of P. jiroveci. They found that Kremsner, S. Borrmann, and S. Issifou, Program Abstr. 52nd
lower respiratory tract samples from the PCP and non-PCP Am. Soc. Trop. Med. Hygiene, abstr. 744, 2003; J. E. Rosen-
patients contained a median of 938 (range, 2.4 to 1,040,000) blatt, A. Muyombwe, L. M. Sloan, P. Petmitr, and S. Looaree-
and 2.6 (range, 0.3 to 248) copies of msg per tube, respectively. suman, Program Abstr. 11th Int. Cong. Infect. Dis., abstr
Similarly, the oral washes from PCP and non-PCP patients 14.006, 2004). In general, this method was equivalent to con-
contained a median of 49 (range, 2.1 to 2,595) and 6.5 (range, ventional microscopy in the detection and identification of
2.2 to 10) copies per tube, respectively. The authors suggest species of Plasmodium present. Other real-time PCR labora-
that applying a cutoff value of 10 target copies per reaction tory developed assays also targeting the 18S rRNA gene have
reduces the number of false-positive results. However, use of been developed using fluorescence-based 5⬘ nuclease TaqMan
technology (Roche Molecular Diagnostics) and either the iCy- however, cautioned that because kinetoplast DNA has a high
cler (Bio-Rad Labs) (255) or the ABI 7700 (Applied Biosys- degree of polymorphism, appropriate internal biprobes or al-
tems) (372). Again, detection of Plasmodium by these methods ternative gene targets will have to be identified for this method
compared well with microscopy, but they do not allow identi- to find practical diagnostic use.
fication of the particular species present in a single test format In fact, Schulz and colleagues (441) designed primers for
as can be accomplished by melting curve analysis using the amplification of an 18S rRNA leishmanial segment for detec-
LightCycler. tion and differentiation of species of Leishmania using cultured
parasites and blood, bone marrow, and tissues from infected
Babesia spp. patients. Their laboratory-developed assay used FRET and LC
Red 640 dye and the LightCycler with melting curve analysis of
A laboratory-developed real-time PCR assay using FRET
amplicons. Parasites were detected in 12 clinical samples and
technology and LC-Red 640 dye has been developed for use
the analytical sensitivity (94 parasites per ml of blood) was
with the LightCycler in the detection of Babesia microti in
within a range which would facilitate the diagnosis of visceral
blood. This assay is a modification of a conventional PCR assay
leishmaniasis from peripheral blood. This assay allows discrim-
for Babesia microti developed in our laboratories at the Mayo
Clinic (237). In studies of patients on Block Island, R.I., PCR ination of three clinically relevant Leishmania groups (Leish-
was more sensitive and at least as specific as blood smear and mania donovani complex, Leishmania braziliensis complex, and
hamster inoculation for the diagnosis of acute babesiosis. PCR others).
may be particularly useful during acute infection before serol- Bossolasco et al. also used an 18S rRNA target with ABI
ogy becomes positive or when blood smears are negative or Prism technology to develop a real-time PCR assay for moni-
intraerythrocytic forms are difficult to differentiate from Plas- toring HIV-infected patients with visceral leishmaniasis (40).
modium. Epidemiology may help in this determination, but They detected decreasing levels of Leishmania DNA in the
occasionally confounding circumstances (such as prior travel to peripheral blood of patients after treatment with liposomal
areas where it is endemic or blood transfusion) may be present. amphotericin B. Moreover, elevated parasite levels were de-
PCR may also be helpful in the recognition of coinfection with tected in patients who relapsed following discontinuation of
other tick-transmitted organisms, such as Ehrlichia and Borre- therapy.
lia spp.
Trypanosoma spp. Toxoplasma spp.
Although no real-time PCR assay has yet been developed Several laboratories have developed real-time PCR assays
for the detection of Trypanosoma spp. in human blood, an for the detection of Toxoplasma gondii in blood, serum, CSF,
investigational FRET/LightCycler method has been used to and amniotic fluid (82, 312; L. M. Sloan, P. S. Mitchell, R.
detect Trypanosoma cruzi in experimentally infected mouse Patel, and J. E. Rosenblatt, Program Abstr. 101st Annu. Meet.
tissues (86). SYBR green and primers targeting a kintoplast Am. Soc. Microbiol., abstr. C-312, 2001). Each of these used
minicircle sequence or a 195-bp satellite DNA sequence were the B1 gene of Toxoplasma gondii as a target and the Light-
used. The assay was used to quantitate the parasite burden Cycler with FRET technology. Two studies found real-time
during acute and chronic phases of infection and the analytical PCR to be equivalent to PCR-ELISA with CSF and amniotic
sensitivity was determined to be 0.1 to 0.01 parasite equiva- fluid (L. M. Sloan, P. S. Mitchell, R. Patel, and J. E. Rosenb-
lents. These workers hope to apply this technology to diagnosis latt, abstr. C-312) or serum, buffy coat, and CSF in a stem cell
of Trypanosoma cruzi in tissues of infected humans, which transplant patient with CNS toxoplasmosis (312). In the latter
would be very useful since there is no specific method for case, detection was earlier and more persistent in buffy coats,
identifying these organisms by tissue microscopy. Conventional suggesting that this is the optimum type of blood specimen for
PCR has been used to identify Trypanosoma cruzi in the blood real-time PCR.
of patients with Chagas’ disease (48, 177). Future development
Another study used the assay in serum for diagnosis and
of real-time PCR methods will be a welcome advance since
follow-up of four stem cell transplant patients (82). They com-
Trypanosoma cruzi are difficult to detect in blood smears and
pared their results with a conventional PCR but also were able
serology may not be readily available or be positive in acute
to quantitate parasitemia using real-time PCR by correlating
infections. Quantitation of parasitemia will also be useful in
extracted DNA assay crossing points with corresponding
following response to antitrypanosomal therapy.
tachyzoite counts of cultured Toxoplasma gondii. They were
able to correlate low parasitemias with clinical improvement
Leishmania spp. following treatment in three patients and increasing parasite
An investigational assay using FRET, SYBR green, and the counts in one patient who developed CNS toxoplasmosis.
LightCycler has been used to detect and differentiate cultured These studies illustrate the special utility of real-time PCR in
strains of Old World Leishmania spp. (Leishmania major, the diagnosis of toxoplasmosis in immunosuppressed patients
Leishmania donovani, Leishmania tropica, and Leishmania in- and pregnant women or neonates. This is an important ad-
fantum) (340). Primers were chosen to amplify a 120-bp con- vance since alternative diagnostic methods such as culture or
served region of kinetoplast DNA minicircles and the detec- serology are not routinely available or difficult to interpret in
tion limit was 0.1 to 1.0 parasite per reaction. The authors, these types of patients.
242
ESPY ET AL.
TABLE 23. Real-time PCR assays for parasites

Turnaround
Sensitivity vs.
Conventional time vs.
Species Specimen Technology Targeta Statusb conventional Reference(s)e Commentsd
method conventional
method
method
Plasmodium Bloode LightCycler FRET 18s rRNA ASR, HB Microscopy Equal Equal 124, A, B, C ASR does not identify
species
Blood iCycler TaqMan 18s rRNA HB Microscopy Equal Equal 255 Does not identify spe-
cies in one test
Blood ABI 7700 TaqMan 18s rRNA HB Microscopy Equal Equal 372
Babesia Blood LightCycler FRET HB Microscopy, serology Greater, greater Equal, faster 237 (modified) Serology total AB
Trypanosoma Tissue LightCycler FRET kDNA Expmtl Microscopy Greater Faster 86 Research in mice
Leishmania Cultures LightCycler FRET kDNA Expmtl Microscopy Greater Faster 340 Identify cultured para-
sites
Blood, bone marrow, LightCycler FRET 18S rRNA HB Microscopy culture Greater Faster 441 Identifies by MCA
tissue
Blood ABI Prism TaqMan 18S rRNA HB Culture Greater Faster 40 Quantitation of para-
sitemia
Toxoplasma Blood, serum, CSF, LightCycler FRET B1 gene HB Culture, serology Equal, greater Faster, faster 82, 312, D Good for acute toxo-
amniotic fluid plasmosis
Trichomonas Urine Light-Cycler FRET ␤-Tubulin HB Microscopy, culture Greater, equal Equal, faster 165 Urine vs. vaginal swabs
gene a question
Giardia, Cultures, cysts, stool iCycler TaqMan SSU HB Microscopy, antigen Greater, equal Equal, equal 34, 516, 517 Multiplex assay, lim-
Entamoeba, rRNA detection ited data
Cryptosporidium
Entamoeba Stool LightCycler FRET rRNA HB Microscopy, antigen Greater, equal Equal, equal 34 Separates E. dispar and
detection E. histolytica
Encephalitozoon Stool LightCycler FRET 16S rRNA HB Microscopy (cannot Greater Equal 548 Identifies by MCA
identify species)
Enterocytozoon Stool LightCycler FRET 16S rRNA HB Microscopy (cannot Greater Equal E Identifies E. bieneusi
identify species) by MCA
Stool ABI Prism 7700 SSU HB Microscopy (cannot Greater Slowe 311 Quantitates E. bieneusi
TaqMan rRNA identify species)
a
kDNA, kinetoplast DNA; SSU, small subunit.
b
HB, home brew; Expmtl, experimental.
c
A, R. U. Manson, K. A. Mangold, R. B. Thomason, Jr., E. Koay, L. R. Peterson, and K. Kaul, Program Abstr. 43rd Intersci. Conf. Antimicrob. Agents Chemother, abstr. P-414, 2003; B, A. Muyombwe, I. Lundgren,
L. M. Sloan, J. E. Rosenblatt, P. G. Kremsner, S. Borrmann, and S. Issifou, Program Abstr. 52nd Meet. Am. Soc. Trop. Med. Hyg., abstr. 744, 2003; C, J. E. Rosenblatt, A. Muyombwe, L. M. Sloan, P. Petmitr, and S.
Looareesuman, Program Abstr. 11th Int. Cong. Infect. Dis., abstr. 14.006, 2004; D, L. M. Sloan, P. S. Mitchell, R. Patel, and J. E. Rosenblatt, Program Abstr. 101st Annu. Meet. Am. Soc. Microbiol., abstr. C-312, 2001;
E, N. L. Wengenack, D. M. Wolk, S. K. Schneider, L. M. Sloan, S. P. Buckwalter, and J. E. Rosenblatt, Program Abstr. 103rd Annu. Meet. Am. Soc. Microbiol., abstr. C-283, 2003.
d
MCA, melting curve analysis.
e
Whole blood or spotted on filter paper or IsoCode Stix.
Trichomonas spp. differentiation of Entamoeba histolytica and Entamoeba dispar.

The assay was not compared to antigen detection assays which
Real-time PCR was used to detect Trichomonas vaginalis in also differentiate these amoeba and therefore their relative
the urine of sexually active high school students (165). The efficiencies in diagnosing amoebiasis could not be determined.
nucleic acid target was a 112 bp segment of the ␤-tubulin gene Microsporidia are difficult to detect in stools because of their
and FRET/LightCycler technology was employed. The assay small size, somewhat nonspecific staining characteristics, and
consistently detected one to four Trichomonas vaginalis per lack of experience of most diagnostic laboratories in identify-
PCR run and approached the sensitivity (97.8%) and specific- ing this infrequently recognized protozoan. A real-time PCR
ity (97.4%) of a TaqMan-based PCR using vaginal swabs (202). method has been described for the detection of Encephalito-
It was not directly compared to culture either of urine or zoon intestinalis in stools (547). Primers were designed to am-
vaginal swabs which would have added important information plify a 268-bp region of the 16S rRNA gene of Encephalitozoon
since culture is generally considered the gold standard. The spp. (Encephalitozoon intestinalis, Encephalitozoon hellem and
commercial availability of this laboratory developed assay Encephalitozoon cuniculi). FRET/LT Red 640 dye technology
would be significant since culture of vaginal samples is consid- was used with the LightCycler and melting curve analysis was
ered to be too complex and time consuming for routine use performed to determine species identification. The assay was
and microscopy is insensitive. evaluated by spiking normal stools with various dilutions of
Encephalitozoon spores and comparing PCR results with mi-
Cryptosporidium, Entamoeba, and Giardia spp. croscopy using trichrome blue stain. Real-time PCR was sig-
nificantly more sensitive than microscopy and the three En-
A number of real-time PCR assays have been developed for cephalitozoon species were accurately differentiated, which
the detection of protozoan pathogens in stools (34, 311, 516, cannot be accomplished by microscopy.
517, 547; N. L. Wengenack, D. M. Wolk, S. K. Schneider, L. M. Subsequently, the same laboratory has described a similar
Sloan, S. P. Buckwalter, and J. E. Rosenblatt, Program Abstr. LightCycler assay for the detection of the microsporidium
103rd Annu. Meet. Am. Soc. Microbiol., abstr. C-283, 2003). which is most frequently associated with intestinal infection,
Verweij et al. initially described a specific assay for a 62-bp Encephalitozoon bieneusi (N. L. Wengenack, D. M. Wolk, S. K.
fragment of the small-subunit rRNA of Giardia lamblia using Schneider, L. M. Sloan, S. P. Buckwalter and J. E. Rosenblatt,
TaqMan probes and the iCycler real-time detection system abstr. C-283). Using spiked stools and five Encephalitozoon
(Bio-Rad). This assay was as sensitive as an antigen detection bieneusi clinical specimens, the method was shown to detect as
method (ELISA, Alexon-Trend) and more sensitive than mi- few as 1 to 10 targets per PCR run and Encephalitozoon bie-
croscopy of stool concentrates (517). Subsequently, the same neusi could be accurately identified by melting curve analysis.
laboratory described a multiplex real-time PCR assay for the Menotti et al. used a real-time PCR assay to quantitatively
simultaneous detection of Giardia lamblia, Entamoeba histo- follow Encephalitozoon bieneusi infection in immunosup-
lytica, and Cryptosporidium parvum (516). The target for Enta- pressed patients who were being treated with fumagillin (311).
moeba histolytica was a 172 bp fragment of small-subunit They amplified a 102-bp fragment of the small-subunit rRNA
rRNA (differentiates from Entamoeba dispar) and that for gene using a TaqMan probe with the ABI Prism 7700 sequence
Cryptosporidium parvum was a 138-bp fragment inside the detection system and compared PCR results with microscopy
Cryptosporidium parvum-specific 452-bp fragment. TaqMan using Uvitex 2B and trichrome blue stains. They correlated
probes were used with the iCycler technology. The assay was microscopic counts with PCR copy numbers derived from di-
performed on species-specific DNA controls of cultures of lutions of plasmid controls and determined that real-time PCR
Entamoeba histolytica and isolated cysts of Giardia lamblia and performed better than did semiquantitative counts by micros-
Cryptosporidium parvum and patient specimens were analyzed copy of parasite burden in response to therapy.
by microscopy and/or antigen detection tests. The multiplex See Table 23 for a literature review.
assay was described as being 100% sensitive and specific, but
only 20 positives for each organism were examined and antigen ACKNOWLEDGMENT
tests were not performed for Cryptosporidium parvum and En- JoAnn Brunette is thanked for her assistance in preparation of the
tamoeba histolytica and Entamoeba dispar (these two Entam- manuscript.
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CLINICAL MICROBIOLOGY REVIEWS, Jan. 2006, p. 165–256 Vol. 19, No.

Real-Time PCR in Clinical Microbiology: Applications for

* Corresponding author. Mailing address: Mayo Clinic, 200 First St.

Qualitative Viral Assays.........................................................................................................................................193

INTRODUCTION is much simpler to perform than conventional PCR methods.

TABLE 1. Instruments available

for detection of a specific nucleic acid sequence of a mutant

NUCLEIC ACID EXTRACTION

A critical preanalytical step for real-time PCR assays, as well

114, 116, 139, 185, 208, 210,

90; Cylindrical (66

48 samples in 159 min

proximately 5 to 8 mm in diameter, 10 of 25 mm in length) during

spinning procedures. The collection tube is used for collection and

(451). S.T.A.R. buffer has three important properties: infec-

minimized, and the binding of the nucleic acids to magnetic

beads, as is used in the extraction process of MagNA Pure

ABI Prism 6100

(Roche Diagnostics Corporation), is enhanced.

The swab extraction tube system (S.E.T.S.; Roche Diagnos-

tics Corporation) kit, shown in Fig. 3, is a simple method for

bound to filter under

trieved in a microcentrifuge tube by the S.E.T.S. method, can

be directly, or after a quick lysis step (boiling), analyzed by a

LightCycler real-time PCR instrument (195, 499). Alterna-

tively the centrifuged material can be extracted by the MagNA

real-time PCR. This specimen transport device has been cou-

pled with real-time PCR assays for the detection of blood-

borne parasites such as malaria (561). This method is not

recommended for use with RNA assays.

NA, nucleic acid.

REAL-TIME PCR ASSAY DEVELOPMENT

Target Nucleic Acid Selection

identify a specific organism or an organism group, (e.g., group

A streptococcus or Mycobacterium genus screen), quantitate a BIOSAFETY CONSIDERATIONS

TABLE 4. Verification guidelines

TABLE 6. Examples of internal controls used in real-time PCR assays

mercially available master mix components that contain stan-

Additionally, we have eliminated most of the follow-up pro- COSTS

Amplified probe CMV 87496 27–49 228

Quantification CMV 87497 39–59 307

Cost effectiveness studies are required to determine the cost

BACTERIA OTHER THAN MYCOBACTERIA SPP. Agents of Community-Acquired Pneumonia

Refer- Conventional Labor- Turnaround

Continued on following page

Refer- Conventional Labo- Turnaround

blood nested domains, 7700 assay detected

Continued on following page

Refer- Conventional Labo- Turnaround

Continued on following page

Refer- Conventional Labo- Turnaround

PCR, demonstrated that the numbers of Streptococcus pneu-

The significant mortality and morbidity associated with bac-

before cultures are obtained, PCR may be particularly advan-

Potential Agents of Bioterrorism

result much sooner than standard culture methods. Addition-

ally, at least two papers have described the biosafety advan-

tages of using a real-time PCR testing platform versus culture

has been the lack of testing human specimens. All of the