PCR-based diagnostics

Review

PCR-based diagnostics for infectious diseases:

uses, limitations, and future applications in
acute-care settings
Samuel Yang and Richard E Rothman

Molecular diagnostics are revolutionising

5
Double-stranded DNA (target) 3

the clinical practice of infectious disease.
Their effects will be significant in acute- 3
5

care settings where timely and accurate 1 Denaturation
diagnostic tools are critical for patient
treatment decisions and outcomes. PCR
is the most well-developed molecular
technique up to now, and has a wide
2 Primers annealing
range of already fulfilled, and potential,
1 Cycle
clinical applications, including specific or
broad-spectrum pathogen detection, 5
3

5
3

evaluation of emerging novel infections,
surveillance, early detection of biothreat
3 Extension (polymerisation)
agents, and antimicrobial resistance
profiling. PCR-based methods may also
be cost effective relative to traditional
testing procedures. Further advancement
of technology is needed to improve Each new double-stranded
automation, optimise detection DNA acts as target for new cycle
sensitivity and specificity, and expand Cycles repeated exponentially:
the capacity to detect multiple targets logarithmic amplification
simultaneously (multiplexing). This review
provides an up-to-date look at the Figure 1. Schematic of PCR. The PCR reaction takes place in a thermocycler. Each PCR cycle
general principles, diagnostic value, and consists of three major steps: (1) denaturation of template DNA into single-stranded DNA;
(2) primers annealing to their complementary target sequences; and (3) extension of primers via
limitations of the most current
DNA polymerisation to generate new copy of the target DNA. At the end of each cycle the newly
PCR-based platforms as they evolve synthesised DNA act as new targets for the next cycle. Subsequently, by repeating the cycle
from bench to bedside. multiple times, logarithmic amplification of the target DNA occurs.

Lancet Infect Dis 2004; 4: 337–48 preclude application of diagnostic test results in the acute
and critical-care settings. Other limitations of the
Pathogen identification: scope of the problem conventional laboratory include extremely prolonged assay
In the USA, hospitals report well over 5 million cases of times for fastidious pathogens (up to several weeks);
recognised infectious-disease-related illnesses annually.1 requirements for additional testing and wait times for
Significantly greater numbers remain unrecognised, both in characterising detected pathogens (ie, discernment of species,
the inpatient and community settings, resulting in strain, virulence factors, and antimicrobial resistance);
substantial morbidity and mortality.2 Critical and timely diminished test sensitivity for patients who have received
intervention for infectious disease relies on rapid and antibiotics; and inability to culture certain pathogens in
accurate detection of the pathogen in the acute-care setting disease states associated with microbial infection.2,6
and beyond. The recent anthrax-related bioterrorist events The failure of either clinical judgment or diagnostic
and the outbreak of severe acute respiratory syndrome technology to provide quick and accurate data for
(SARS) further underscore the importance of rapid identifying the pathogen infecting patients leads most
diagnostics for early, informed decision-making related to clinicians to adopt a conservative management approach.
patient triage, infection control, treatment, and vaccination Empiric intravenous antibiotic therapy (most common in
with life-and-death consequences for patients, health
SY and RER are both at The Johns Hopkins University, School of
providers, and the public.3–5 Unfortunately, despite the
Medicine, Department of Emergency Medicine, Baltimore, MD, USA.
recognition that outcomes from infectious illnesses are
Correspondence: Dr Richard E Rothman, Department of
directly associated with time to pathogen identification, Emergency Medicine, Johns Hopkins School of Medicine, 1830 E
conventional hospital laboratories remain encumbered by Monument Street, Suite 6-100, Baltimore, MD 21205, USA.
traditional, slow multistep culture-based assays, which Email rrothman@jhmi.edu

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 Review PCR-based diagnostics

acute-care settings such as emergency departments and reaction and Q-beta replicase amplification) and amplifi-
intensive care units) offers the advantages of maximum cation of the signals generated from hybridising probes (eg,
patient safety and improved outcomes. The benefits of branched DNA and hybrid capture), and transcription-based
conservative management may be offset, however, by added amplification (eg, nucleic-acid-sequence-based amplification
costs and potential iatrogenic complications associated with and transcription-mediated amplification) have also been
unnecessary treatment and hospitalisations, as well as incorporated into various detection systems.19 Detailed
increased rates of antimicrobial resistance.7–9 A rapid reliable descriptions of these technologies are beyond the scope of this
diagnostic assay, which allows for accurate identification of review, but are well summarised elsewhere.20
infected patients and informed early therapeutic
intervention, would thus be invaluable for emergency and PCR: basic principles and overview
critical care physicians. PCR is an enzyme-driven process for amplifying short regions
For more than a decade, molecular testing has been of DNA in vitro. The method relies on knowing at least partial
heralded as the “diagnostic tool for the new millennium”, sequences of the target DNA a priori and using them to design
whose ultimate potential could render traditional hospital oligonucleotide primers that hybridise specifically to the
laboratories obsolete.10–12 However, with the evolution of target sequences. In PCR, the target DNA is copied
novel diagnostics tools, difficult questions have arisen by a thermostable DNA polymerase enzyme, in the presence
regarding the role of such testing in the assessment of clinical of nucleotides and primers. Through multiple cycles of
infectious diseases. As molecular diagnostics continue to heating and cooling in a thermocycler to produce rounds of
flow from bench to bedside, clinicians must acquire a target DNA denaturation, primer hybridisation, and primer
working knowledge of the principles, diagnostic value, and extension, the target DNA is amplified exponentially (figure
limitations of varied assays.13 Here we discuss the most 1). Theoretically, this method has the potential to generate
promising molecular diagnostic techniques for infectious billions of copies of target DNA from a single copy in less than
diseases in hospital-based settings: the emphasis is on 1 h. For more detailed discussion of the basic principles of
PCR-based methods since they have reached greatest PCR see references 21–25.
maturity; existing assays, current, and future applications are Over the past two decades, PCR has been extensively
described. Further, a framework for describing limitations modified to expand its utility and versatility. Multiplex PCR
that have been encountered, as well as speculation regarding enables the simultaneous detection of several target
the potential effect of these developments from the patient, sequences by incorporation of multiple sets of primers.26 To
physician, hospital, and societal perspective is provided. increase sensitivity and specificity, a double amplification
step can be done with appropriately designed “nested”
Nucleic-acid-based amplification: historical primers.27 Amplification may be made less specific to detect
perspective divergent genomes by randomising portions of the primer
The first nucleic-acid-based assays used DNA probe sets.28 Finally, RNA (rather than DNA) can be detected by
technology.14–16 DNA probes are short, labelled, single-strand converting RNA into a complementary DNA copy, and then
segments of DNA that are designed and synthesised to amplifying (so-called reverse transcriptase PCR, or
hybridise targeted complementary sequences of microbial RT-PCR), enabling evaluation of RNA viruses or viable
DNA. By contrast with traditional culture-based methods organisms.27
of microbial identification, which rely on phenotypic A significant advancement in PCR technology is
characteristics, this molecular fingerprinting technique relies quantitative real-time PCR, in which amplification and
on sequence-based hybridisation chemistry, which confers detection of amplified products are coupled in a single
greater specificity to pathogen identification. Direct detection reaction vessel. For purposes of clinical applicability, this
of target microbial DNA in clinical samples also eliminates process represents a major breakthrough since it eliminates
the need for cultivation, drastically reducing the time the need for laborious post-amplification processing
required for reporting of results. In 1980, the description of (ie, gel electrophoresis) conventionally needed for amplicon
DNA hybridising probes for detecting enterotoxigenic detection, and allows for measurement of product
Escherichia coli in stool samples raised hopes that nucleic- simultaneous with DNA synthesis. One approach for
acid-based technologies would eventually replace traditional real-time monitoring of amplicon production is to use
culture techniques.17 Since that time, however, a more fluorescent DNA intercalating dyes, such as SYBR-Green I,
restrained approach has been adopted due to recognition of which bind non-specifically to double-stranded DNA
technical limitations of the methodology; most notably, the generated during amplification.29 A more popular alternative
large amount of starting target DNA required for analysis, approach is to use a fluorescent-labelled internal DNA probe
which results in poor detection sensitivity.18 which specifically anneals within the target amplification
To attain optimum sensitivity, critical for most clinical region. The choice of probe format depends on
applications, researchers sought to directly amplify target the compatibility of its hybridisation chemistry with the
microbial DNA. The development of the PCR technique in experimental design. Variations in probe format include
1985 answered this need, and provided what is now the TaqMan (Applied Biosystems; figure 2), fluorescence
best-developed and most widely used method for target resonance energy transfer (FRET), and molecular beacon
DNA amplification. Other approaches, including probes.30–32 Regardless of the format chosen, the internal probe
amplification of the hybridising probes (eg, ligase chain emits a fluorescent signal during each amplification cycle only

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 PCR-based diagnostics
Review

in the presence of target sequences, with Denatured DNA

signal intensity increasing in proportion
to the amount of amplified products
generated. The amount of starting
Primers and probe annealing
templates in a specimen can be
quantified by comparing the exact cycle 3
5

number at which amplified products R 5
3
R Q
Reporter dye
accumulate significantly over baseline
Q Quencher dye
with a pre-derived quantitative Polymerisation
standard. Development of automated Primers
instrumentation with quantitative
R Fluorescent-emitting
capacity insures reproducibility. reporter dye R Q
Practical advantages of real-time PCR Synthesised DNA Probe displacement by 5

over conventional PCR are thus myriad exonuclease of Taq polymerase
and include speed, simplicity, repro-
ducibility, and quantitative capacity. R
Q
PCR-based diagnostics have been
effectively developed for a wide range of Polymerisation completed
microbes. Due to its incredible sensi-
tivity, specificity, and speed of ampli-
fication, PCR has been championed by R Q
infectious disease experts for identifying
organisms that cannot be grown in vitro,
Figure 2. Real-time PCR using Taqman probe. Taqman probe is a single-stranded oligonucleotide
or in instances where existing culture that is labelled with two different fluorescent dyes. On the 5
terminus is a reporter dye and on the
techniques are insensitive and/or need 3
terminus is a quenching dye. This oligonucleotide probe sequence is homologous to an internal
prolonged incubation times.33,34 Appli- target sequence present in the PCR amplified product. When the probe is intact, the proximity of
cation to the clinical arena, however, has the two fluorescent dyes results in quenching of the reporter dye emission by the quencher dye.
During the extension phase of PCR the probe is cleaved by 5
exonuclease activity of Taq
met with variable success so far. Only a polymerase thereby releasing the reporter from the quencher and producing an increase in
limited number of assays have been reporter emission intensity which can detected and quantified. As amplification continues, the
approved by the US Food and Drug amount of reporter dye signal measured is proportional to the amount of PCR product made.
Administration (FDA; table 1) and fewer
still have achieved universal acceptance in clinical practice.47 direct detection of M tuberculosis from clinical specimens
Furthermore, surprisingly limited effort has been focused on (table 1). Although these assays result in significant
harnessing these time-saving diagnostics for emergency improvements in time to diagnosis, the only FDA approved
department and other acute critical-care settings where time is use at this time is as a diagnostic adjunct to the conventional
of the essence. A discussion of the progress made and the smear and culture. Nonetheless, recent studies suggest that
obstacles remaining to be addressed follows. more widespread use of these assays may significantly affect
patient management, clinical outcomes, and cost efficacy.56,57
Specific PCR diagnostics: development and Potential yet unrealised applications of the assay for use in
clinical applications acute-care settings include earlier informed decision-making
With the increasing number of genomes of infectious for appropriate use of isolation beds in high prevalence sites,
pathogens being sequenced, catalogues of genes can be regional outbreaks, or where isolation beds are scarce. The use
exploited to serve as amplification targets fundamental to of this and other PCR-based diagnostics in these settings,
the design of clinically useful diagnostic tests. As a result, however, will have to be balanced against costs, expertise, and
over the past decade the number of PCR assays developed time associated with routine around-the-clock availability of
commercially and in hospital-based laboratories (“in- testing, as discussed in more detail below.
house”) has continued to expand. Among the assays that Another PCR-based diagnostic assay, which has gained
have been developed for detection of specific microbes, three widespread acceptance, is that for Chlamydia trachomatis.
are described in more detail below, along with a discussion Conventional detection systems for this organism
of their pros and cons relative to conventional diagnostic (ie, culture and direct antigen testing by immunoassays),
methodologies (table 2). have been limited by the requirement for specialised facilities
One of the earliest recognised applications of PCR for to culture this fastidious microbe, as well as inadequate
clinical practice was for detection of Mycobacterium sensitivity and specificity of immunoassays relative to the
tuberculosis.54,55 Disease characteristics favouring the gold standard culture results.58 Laboratory development and
development of a non-culture-based test for tuberculosis subsequent clinical validation testing have indicated
included week-long to month-long delays associated with excellent sensitivity and specificity of the PCR assay leading
standard testing, and the public-health imperative associated to its commercial development (table 1). Proven efficacy of
with early recognition, isolation, and treatment of infected the PCR assay for both genital and urine samples has
patients. Two PCR-based assays are approved by the FDA for resulted in its application to a range of clinical settings, most

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 Review PCR-based diagnostics

recently routine screening of emergency department patients include those for herpes simplex virus, cytomegalovirus,
considered at risk for sexually transmitted diseases.59,60 Epstein-Barr virus, hepatitis viruses, and HIV.67 Each has a
Although studies in the acute-care settings have not yet used proven cost-saving role in clinical practice, including detection
PCR assays for C trachomatis on site and in real time (thus of otherwise difficult to diagnose infections, and a newly
not taking full advantage of the speed of PCR), routine use realised capacity to monitor progression of disease and
of this assay in the aforementioned studies has resulted in response to therapy, vital to the management of chronic
nearly three-fold greater rates of disease detection and infectious diseases.68
treatment relative to standard care. With the increasing number of genomes of infectious
Distinguishing life-threatening causes of fever from more pathogens being sequenced, catalogues of genes can be
benign causes in children is a fundamental clinical dilemma exploited to serve as amplification targets. As a result, the
faced by clinicians, especially when infections of the central number of PCR assays developed both commercially and
nervous system are being considered. Bacterial causes of in-house continues to expand.
meningitis can be highly aggressive but generally cannot be
differentiated on a clinical basis from aseptic meningitis, a Broad-ranged PCR
benign condition generally appropriate for outpatient The notion of a universal detection system has been proposed
management.61 Culture methods often take several days to for the identification of classes of pathogens and speaks most
show positive results and are confounded by poor sensitivity directly to the future potential effect of PCR-based assays for
or false-negative findings in patients receiving empiric anti- clinical practice in emergency and other acute critical-care
microbials.62 One well developed assay, which has the potential settings.69 Experimental work has focused on using sequences
to influence the management of patients in the acute-care of the 16S rRNA gene, an evolutionarily conserved gene seen
setting, allows early and rapid diagnosis of diseases of viral exclusively in bacterial species.70,71 By designing primers that
cause. Testing and application of a PCR assay for enteroviral are complementary to these regions, investigators can, in
meningitis has been seen to be highly sensitive.63,64 With theory, establish the presence of any bacteria in an otherwise
reporting of results within 1 day, preliminary clinical trials sterile clinical specimen (such as cerebrospinal fluid or whole
have shown significant decreases in hospital costs due to blood). Clinical applications are profound. Acute-care
decreased duration of hospital stays and courses of antibiotic physicians could rapidly identify the presence of bacteraemia.
therapy.65,66 Other viral PCR assays, now routinely available, Previous empiric decision-making could be abandoned in

Table 1. FDA-approved nucleic-acid-based assays for detection of microbial pathogens

Organism detected Trade name Company/institution Method Clinical sensitivity Clinical specificity
Chlamydia trachomatis Amplicor Roche PCR 93·219 98·4119
LCX Abbott LCR >9519 >9919
AMP Gen-Probe TMA 86·7–99·219 >9919
PACE 2 Gen-Probe Hybridisation 60·8–78·135 >9935
BDProbeTec Becton Dickinson SDA 94·036 >9936
Hybrid capture II CT-ID Digene Hybrid capture 95·437 9937
Cytomegalovirus CMV pp67 mRNA Organon Teknika NASBA 9538 9838
Hybrid capture CMV DNA test Digene Hybrid capture 9539 9539
Gardnerella vaginalis Affirm VIP III Becton Dickinson Hybridisation 9440 8140
Group A streptococcus GP-ST test Gen-Probe Hybridisation 88·619 97·819
Group B streptococcus IDI-StrepB Infectio Diagnostics Real-time PCR 9719 10019
HCV Amplicor HCV Roche PCR 9819 NA
Versant HCV RNA qualitative assay Bayer TMA NA 9841
Versant HCV RNA 3·0 Bayer BDNA NA 98·242
HIV Amplicor HIV-1 Monitor Test Roche RT-PCR NA >9919
Trugene HIV drug resistance and Visible Genetics DNA sequencing NA NA
OpenGene DNA sequencing
NucliSens EasyQ HIV-1 bioMerieux NASBA NA >9919
Procleix HIV-1/HCV Chiron TMA >9943 >9943
Versant HIV-1 RNA 3·0 Bayer BDNA 97·644
ViroSeq Applied Biosystems DNA sequencing NA NA
HPV Hybrid capture II HPV DNA Digene Hybrid capture >9945 85-9045
M tuberculosis TB Amplicor Roche PCR 79·4–91·919 >9919
E-MTD Gen-Probe TMA 90·9–95·219 >9919
Neisseria gonorrhoeae Amplicor Roche PCR NA NA
LCX Abbott LCR >9519 >9919
Hybrid capture II CT/GC Digene Hybrid capture 9346 98·546
BDProbeTec Becton Dickinson SDA 88·936 >9936
PACE-2 Gen-Probe Hybridisation 9735 9935
Trichomonas vaginalis Affirm VIP III Becton Dickinson Hybridisation 88–91 938 10038
BDNA=branched DNA; LCR=ligase chain reaction; NASBA=nucleic-acid-sequence-based amplification; PCR=polymerase chain reaction; RT-PCR=reverse transcriptase PCR;
SDA=strand displacement amplification; TMA=transcription mediated amplification; NA=not applicable. Adapted from reference 19.

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 PCR-based diagnostics
Review

Table 2. The pros and cons of PCR-based versus conventional diagnostic methods for detection of three target organisms
Target Conventional diagnostic method PCR-based method
organism
Method Pros Cons Method Pros Cons
M tuberculosis Culture Allows susceptibility Inadequate sensitivity PCR High sensitivity (93%)/ Potential
testing Prolonged time to (TB Aamplicor) specificity (98–100%)19 contamination
High specificity with result (>2 weeks) Rapid detection time Unable to assess
nucleic-acid-based Requires further identification No transport requirement viability
identification after positive culture Allows detection from Limited ability for
High cost (direct non-invasive specimens genotype and
and indirect) associated Best for diagnosis susceptibility testing
with delayed diagnosis and screening
Acid-fast Rapid detection Inadequate sensitivity/
stain specificity
C trachomatis Culture High specificity Low sensitivity (70–80%)49 PCR Rapid detection time Limited ability for
(100%)48 Invasive specimen (Amplicor) Moderate to high multidrug-resistance
Detects only viable collection sensitivity (80–90%)19 testing
organisms Prolonged time to result with high specificity Currently used as an
Allows further genotype Cold transport (95–100%)19 adjunctive test
or susceptibility testingHigh cost Probably cost effective51
Antigen detection High specificity Requires technical
method (dfa) (98–99%)50 expertise
Allows assessment Moderate sensitivity
of specimen adequacy (80–90%)50
Rapid detection time Requires high expertise
No transport requirement
Enterovirus Viral isolation High specificity Prolonged time to result RT-PCR Higher sensitivity (98%)53
(100%)52 (10–14 days) (Amplicor EV) High specificity (94%)53
Low sensitivity (65–75%)53 Rapid detection time
Multiple cell lines needed More adaptable
for isolation for serotyping
Serotyping time-consuming, Cost effective54
labour intensive, and costly

favour of educated practice, allowing appropriate, expeditious epidemiological studies that suggest a strong association
decision-making about the need for antibiotic therapy and between Chlamydia pneumoniae and coronary artery disease
hospitalisation. In principle, this approach could be applied to serve as an example of the possible widespread, yet
other taxonomic groups of pathogens (eg, genus of species, undiscovered, links between pathogen and host which may
families of viruses, or fungi) by exploiting common features of ultimately lead to new insights into pathogenesis and
classes of organisms for broad-range PCR assay design.72 development of novel life sustaining or saving therapeutics.81
Validation of this technique for eubacterial detection has The most recent, high-profile investigational use of
focused on “high yield” clinical settings where expeditious broad-range PCR was in the molecular identification of a
identification of the presence of systemic bacterial infection coronavirus as the causative agent in SARS. In a variant
has immediate high morbidity and mortality consequences. approach to PCR assay development, broad-based primers
Notable clinical trials have included assessment of patients at with degenerate sequences designed to detect unknown
risk for infective endocarditis,73–75 febrile infants at risk for viruses were used to randomly amplify the genetic contents of
sepsis,76,77 febrile neutropenic cancer patients,78 and critically ill infected clinical isolates. A subset of the amplified sequences
patients in the intensive care unit.79 While several of these showed homologies to the genus of coronavirus,82,83 consistent
studies have reported promising results (with sensitivity and with other confirmatory laboratory test results. Soon
specificity for bacteraemia well above 90%), significant afterwards, a coronavirus-specific PCR assay was developed
technical difficulties remain, preventing general acceptance of for rapid laboratory diagnosis of SARS.84 Notably, these
these assays in clinics and hospitals (see Limitations below). advancements came only weeks after the first reports of the
One significant investigational role for broad-range PCR disease surfaced—a veritable tour de force bespeaking the
has been its use as a “molecular petri dish” to identify power of broad-range PCR.85
emerging or existing infectious causes for diseases previously
described as idiopathic. The DNA amplified using this Antimicrobial resistance profiling
broad-range approach may contain intervening sequence With multidrug-resistant pathogens on the rise, early
information that is phylogenetically specific to a unique antimicrobial resistance profiling is crucial both for timely,
microbe when compared with existing microbial genetic objective treatment of infected patients, as well as for broader
databases. For example, sequencing of the 16S rRNA gene public-health surveillance. Conventional tests of this type are
amplified via highly conserved primer sets has led to the limited by prolonged culturing time (48–72 h) and poor
identification of Bartonella henselae in bacillary accuracy due to variability in inoculum size and culturing
angiomatosis, and Tropheryma whipplei as the uncultured conditions. To address these shortcomings, nucleic-acid-
bacillus associated with Whipple’s disease.80 Further, recent based assays are being advanced as genetic mechanisms of

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 Review PCR-based diagnostics

drug resistance are elucidated. Three examples of the clinical bioterrorism victims may be non-specific and difficult to
applicability of resistance profiling follow. distinguish from commonly encountered disease processes.96
Although the presence of a resistance gene does not The previously described limitations of conventional
necessarily imply its expression and conferment of phenotypic culture-based assays make such tests wholly inadequate for
resistance, its absence does establish a lack of resistance detection of bioterrorism agents in suspected clinical
through that particular genetic mechanism: for example, outbreaks. Furthermore, traditional microbiological methods,
meticillin resistance is mediated by the mecA gene. which require prolonged incubation, increase biohazard risk
A distinctive feature of meticillin resistance is its heterogenous at the hospital laboratory due to unnecessary propagation of
expression. As such, when typical phenotypic susceptibility bioterrorism pathogens in culture-based systems. Wide
testing is used to assess resistance, meticillin-resistant strains recognition of these limitations has led to recent developments
may seem falsely susceptible to some -lactam antibiotics in and refinements of PCR-based assays for a number of category
vitro.86 For this reason, direct detection of the mecA gene by A bioterrorism agents, including variola major, Bacillus
PCR is more desirable. With its high detection sensitivity and anthracis, Yersinia pestis, and Francisella tularensis.97–100 PCR
specificity, mecA PCR has gained wide acceptance and is diagnostics for bioterrorism agents will likely be used both for
becoming the most reliable method of identifying meticillin- diagnosis of symptomatic individuals, as well as larger scale
resistant Staphylococcus aureus (MRSA).87 screening of exposed victims (preclinical phase), who would
PCR-based resistance testing in M tuberculosis has also be candidates for early prophylactic therapy.
been developed for the detection of rifampicin resistance. Although most bioterrorism-induced illnesses resemble
Rifampicin resistance is well characterised and conferred by natural outbreaks, there is the possibility that causative
mutations within a short sequence of the rpoB gene of bioterrorism agents are genetically engineered to increase
M tuberculosis, which result in aminoacid substitutions in the virulence, acquire resistance to antibiotics or vaccines, or
rpoB subunit of RNA polymerase.88 The Line Probe assay produce phenotypic characteristics that resemble multiple,
(LiPA; Inno-Genetics) is a commercially available PCR-based simultaneous infections, so-called binary agents (via
assay that targets the mutation-prone segment of the rpoB insertion of recombinant genes).101 In such cases, it is likely
gene.89 Correlation with standard resistance-detection that nucleic-acid-based approaches will be more invaluable
methods has been more than 90% and is shown to provide than conventional detection methods since they are the
clinicians with a drastic reduction in detection time, critical more easily adaptable and capable of uncovering detailed
for treatment decisions.90 Genotypic analysis of other information embedded in genetic sequences.
M tuberculosis drug resistance is more challenging due to the
number of mutations and genetic loci involved. Technical Cost effectiveness
innovations (ie, multiplex PCR or DNA microarray) that PCR is more expensive than conventional approaches. The
allow simultaneous amplification and analysis of multiple direct costs of PCR reagents, equipment, dedicated space,
target sequences will likely provide the means to surmount personnel training, and labour have been reported to be as
this later limitation.91,92 high as US$125 per reaction.102 Even among PCR methods,
With ever-increasing evidence supporting the prognostic there is variability in cost with the most expensive being
value of identifying drug-resistant mutations, routine fluorogenic-based systems. Moreover, the labour intensity
genotypic resistance testing is now standard care in the needed for most assays as well as technical limitations of
treatment of HIV-infected patients.93–95 PCR followed by most thermocyclers to do multiple runs of PCR
nucleotide sequencing is the most commonly used method. simultaneously have prevented routine around-the-clock
Although genotypic tests are more complex than typical testing in the clinical setting. On the other hand, continued
antimicrobial susceptibility tests, their ability to detect refinement in PCR technology, as well as improvements in
mutations at concentrations too low to affect drug automation and reproducibility via high throughput
susceptibility in a phenotypic assay provides insight into the robotics, will probably lead to increasing demand and
potential for resistance to emerge. They also have the marked cost reductions to rates competitive with traditional
advantage of detecting transitional mutations that do not methods. Already, this development has been reported for
themselves cause drug resistance but indicate the presence of Neisseria gonorrhoeae and C trachomatis PCR tests which
selective drug pressure, with potential importance for now cost around $9 per reaction.103
individual patient treatment decisions. In assessing the overall benefit of PCR, however, direct
monetary costs should not be the only consideration since
Applications in bioterrorism the assay has several significant advantages over traditional
The increasing threat of bioterrorism has gained methods. One study, which took a global methodological
considerable attention in light of the anthrax outbreak that approach to cost, involved assessment of perinatal screening
came after the September 11, 2001 terrorist attacks. It has for Group B streptococcus using PCR versus culture
become increasingly apparent that responsibility for the techniques.104 Considered variables of the assays in addition
rapid recognition and accurate diagnosis of real or suspected to the direct monetary cost included infections averted,
bioterrorism events will fall principally to front-line acute- mortality, infant disabilities, hospital stays, and the societal
care physicians who will be critical in initiating appropriate benefits of healthy infants. Overall, the authors concluded
response measures.96 Unfortunately, as was seen with the that the benefits of PCR outweighed its cost. Notably, this
2001 anthrax episode, the clinical presentation of result was reached even without inclusion of important but

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 PCR-based diagnostics
Review

difficult-to-measure parameters, such as the societal benefit volume permissible for PCR reactions; and problems
of decreased drug resistance due to targeted therapy made associated with PCR processing. The sample volume most
possible by the PCR assay. PCR assays can accommodate is quite small relative to the
volume used in conventional culture methods; as such, in
Limitations of PCR and emerging innovations cases in which the concentration of infectious organisms is
The principal shortcomings in applying PCR assays to the low, the assay may yield false-negative findings. To account
clinical setting include false-positive results from background for this, DNA extraction and purification steps are usually
DNA contamination; the potential for false-negative test performed before PCR amplification as a means of
results; detection sensitivity exceeding clinical significance; concentrating total DNA from a larger sample volume.
and limited detection space of the assay or platform for Additional methods to optimise starting concentration of
simultaneous identification of multiple species, virulence target DNA for the PCR reaction include: selecting specimen
factors, or drug resistance. sources (eg, cerebrospinal fluid) or specimen fractions (eg,
buffy coat instead of whole blood) with the highest abundance
False positives of microbial DNA for the DNA extraction; briefly cultivating
The widespread use of PCR in clinical settings has been samples to increase microbial load before DNA extraction;20
hampered largely by background contamination from exoge- or introducing specific capture probes to concentrate only
nous sources of DNA.105 In most pathogen-specific assays, the microbial DNA in a given sample.111 Several sample processing
predominant source of contamination is derived from “carry- obstacles may also lead to false-negative findings. Three of the
over” products from earlier PCR reactions, which can be most commonly encountered problems are (1) inadequate
harboured and transmitted through PCR reagents, tubes, removal of PCR inhibitors in the sample, such as
pipettes, and laboratory surfaces. Coupled with the robust haemoglobin, blood culture media, urine, and sputum; (2)
amplification power of PCR, even very minor amounts of ineffective release of microbial DNA content from the cells; or
carry-over contamination may serve as substrates for (3) poor DNA recovery after extraction and purification steps.
amplification and lead to false-positive results. Meticulous Methods to ensure best sample processing include: incorpo-
control measures such as good laboratory practices and rating internal amplification controls (eg, the human -globin
physical separation of preamplification and postamplification gene) to the PCR assay to monitor for presence of both
areas can reduce contamination risks but are not foolproof. purified sample DNA as well as potential PCR inhibitors;112
The use of enzymatic inactivation of carry-over DNA (ie, and inducing various chaotropic, enzymatic, or thermal
uracil N-glycosylase) can further reduce contamination risk.106 methods of cell lysis to effectively liberate microbial DNA
Contamination issues are most pronounced in assays that content.20 Because of the varying effectiveness of each of these
use universal primers, such as those targeting conserved measures, efforts to improve an assay’s detection sensitivity
regions of the eubacterial 16S rRNA gene. Here, the may need to be individually adjusted based on the assay’s
ubiquitous presence of eubacterial DNA in either the clinical application and the microbial pathogen of interest.
environment or working reagents may lead to false-positive
findings. Attempts to decontaminate PCR materials have Clinical significance of positive PCR
involved nearly all known methods of destroying DNA PCR assays may detect microbial pathogens at concentrations
including ultraviolet irradiation, chemical treatment, and below those of previously established gold standard reference
enzymatic digestion.107,108 None of these methods has been methods. Distinguishing whether this result represents a
shown to be entirely effective without significant diminution false-positive finding and establishing the clinical significance
of assay sensitivity. We have recently reported an alternative of these findings is challenging. In the past, discrepant
method that uses a size-based ultrafiltration step for reducing analysis based on the results of additional ancillary tests was
contaminating DNA from PCR reagents, primers, and DNA used to provide estimates of sensitivity and specificity in the
polymerase before amplification. Although this method of presence of an imperfect gold standard.113 One example can
decontamination has been shown to be effective without be seen in assessments of novel nucleic acid-based assays in
compromising detection sensitivity in vitro,109 validation, and detecting C trachomatis.114,115 In these studies, “false positives”
optimisation of the method in clinical samples needs further (DNA-amplification positive and tissue culture negative)
study. More importantly, effective and reliable methods of were adjudicated by either antigen detection methods or
decontamination have not yet been developed for steps another well-established DNA-amplification test. Despite its
outside the assay proper such as sample collection and popularity, recent concerns have been raised regarding the
preparation. Towards this end, one promising area of potential bias incurred by discrepant analysis in favour of the
investigation involves development of methods to integrate new tests.116,117 Up to now, the issue has not been completely
sample preparation, amplification and detection on a single resolved.
platform, the so-called “lab-on-a-chip”. Self-contained The complexity of the clinical interpretation of positive
microchip platforms thus hold promise for the best means of PCR findings is further underscored by one study that
decontamination and overall assay efficiency.110 reported that a universal PCR assay (using primers from
conserved regions of the 16S rRNA gene) amplified
False negatives eubacterial DNA in blood samples from healthy people.118 It
PCR assays for microbial detection may give false-negative is unknown whether such findings are indicative of latent
results for two principal reasons: the relatively small sample disease processes or sub-clinical colonisation. Moreover, the

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 Review PCR-based diagnostics

finding that microbial DNA can be detected even after pathogen quantifications in clinical bacteriology remains
successful antimicrobial treatment suggests that the assays under investigation.127
detect both viable and non-viable organisms.119,120 Clearly, Alternative innovations regarding PCR technologies may
interpretive guidelines based on the correlation of test results help in differentiating viable from non-viable organisms,
with clinical presentation and existing standards will be important for clinical practice decisions. RNA is known to be
required before these assays can be used for definitive rapidly degraded with a typical half-life of minutes after cell
diagnosis and/or treatment decisions. death; thus, it has been proposed as a more accurate indicator
One breakthrough in establishing the meaning of positive of viable microorganisms.128,129 In some clinical situations,
PCR results involves the development of reliable quantitative detection of RNA species by RT-PCR has been shown to
measures of pathogen load. While traditional PCR assays are correlate well with the presence of viable organisms and has
used primarily for dichotomous outcome, innovative real- been effectively used to monitor antibiotic therapy.130–134
time PCR methods allow for quantitative measurement of Clinical application of RNA-based approaches will need
starting template in the sample, which will probably be useful further improvement, however, because they have been
in differentiating benign colonisation from either latent hampered in development by difficulties in extracting
or active disease. Other non-PCR amplification methods detectable concentrations of intact RNA from small numbers
with quantitative capacities include branched DNA and of bacteria.
nucleic-acid-sequence-based amplification. Quantification of
pathogen load is already well established in clinical virology Limited detection space for characterising the
(eg, HIV-1, cytomegalovirus, hepatitis B virus, hepatitis C detected pathogen
virus, and Epstein-Barr virus), where it has proven useful in Conventional methods for pathogen detection will not be
assessing disease severity or monitoring treatment supplanted by PCR-based assays if the latter cannot
efficacy.121–126 The value and importance of PCR-based be elaborated to further characterise detected pathogens. As
described previously, genetic sequences contain rich sources of
5
hemiprobes 3
hemiprobes information that can be analysed to ascertain pathogens’
3
species or strains, virulence factors, and antimicrobial
A Primer B
susceptibilities. However, to do so in a single reaction, simulta-
sequence
5

neous amplification of several target genes is needed.
Primer A Stuffer sequence Repeating amplifications with different primer pairs, so-called
sequence multiplexing, is notoriously difficult since often one or more
Hybridisation Hybridisation
of the target sequences do not amplify.91
sequence sequence Recent studies have shown that PCR can be used for
simultaneous reproducible amplification of multiple DNA
3

fragments in a single reaction, provided that only a single
5
primer set is used for amplification of all these fragments.
Repetitive-sequence-based PCR (rep-PCR), which uses
3
5

consensus PCR primers to amplify DNA sequences located
Target
between successive repetitive elements in eubacterial
genomes, has been shown to simultaneously amplify
fragments of different sizes, allowing discrimination of
B
bacteria at the subspecies level.135,136 This conceptual
breakthrough has led various investigators to develop and
explore various technical approaches which harness the same
idea. By exploiting the conserved and variable sequences on
Target the 16S rRNA gene, we have shown through use of
quantitative PCR that a single consensus primer set can
C multiplex amplify multiple species of the 16S rRNA gene with
Primer B equal efficiencies.109 Similarly, ligation-dependent PCR (figure
Primer A 3)137 and padlock probes with rolling circle amplification
(figure 4),138 which are both probe amplification methods,
Ligated functional probe
have also shown that multiple genetic targets can be queried
Figure 3. Ligation-dependent PCR (LD-PCR). LD-PCR is a process in which simultaneously by using a single primer pair for
non-amplifiable hemiprobes for each target will be constructed as follows:
amplification. Both of these assays rely on multiple
(A) For the 5
hemiprobe the 5
end will be a generic primer sequence
shared by all 5
hemiprobes, and the 3
end will be target-specific. The 3
oligonucleotide probes, each containing a unique target
hemiprobe will have a mirror symmetrical arrangement with an intervening sequence and a consensus primer sequence, that are
stuffer sequence of variable length. (B) In the presence of target sequences amplifiable in the presence of their targets. Progress in these
the hemiprobes are juxtaposed to each other as they hybridise to their approaches could greatly enhance throughput in genotyping
targets. (C) A single PCR primer set based on the generic sequences on
the hemiprobes will be used for amplification of any ligated functional
pathogens detected, and may represent the next generation of
probes. The amplified product of each ligated functional probe has a PCR-based assays that hold tremendous promise with regard
unique length that can be separated by electrophoresis. to their clinical applications.

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 PCR-based diagnostics
Review

A B with time of flight mass spectrometry

Padlock probe Padlock probe (TOF-MS) has created a robust means
to characterise mostly proteins, but
increasingly, nucleic acid. In MALDI-
TOF-MS, the organic molecule—for
example an amplified product—is
Target DNA Target DNA ionised and subsequently identified
based on its mass-to-charge ratio
C D (figure 5).144–149 The advantages
of MALDI-TOF-MS lie in the inherent
accuracy and the high-speed (1 second)
P2 of signal acquisition, making this
technology an attractive candidate for
5
5

high-throughput DNA analysis.

Practical aspects of rapid and

3
3
point-of-care testing
P1 P1 The real-time PCR-based platform
holds great promise in replacing
Figure 4. Padlock probe with rolling circle amplification. (A) Padlock probe hybridises to the target conventional laboratory-based testing
sequence. (B) The padlock probe can be converted to a circle by ligation only if ends of the probe are
for future point-of-care testing.
matched to their target. (C) Rolling circle amplification begins when primer 1 (P1) anneals to
circularised probe and polymerase copies probe sequence, eventually copyin the entire circle. The With advancements in automation,
polymerase begins displacing the previously synthesised product, opening up single-stranded primer integration of specimen preparation
2 (P2) binding site. (D) Each turn of displacement synthesis around the circle exposes another P2 with target identification, and
binding site, and the resulting synthesis from the annealed P2 primers results in the displacement of miniaturisation, it will become much
downstream primers and products. Displacement of the downstream primers and products also
opens up additional P1 binding sites, and the process continues in an exponential cascade. The
easier to bring analyses near bedside
amplification process occurs in an isothermal reaction. Adapted from reference 138. to be done by less-trained personnel.
The ability to interface with high
throughput PCR systems is already
Even if this problem is resolved, the best way in which seen in many new automated extraction instruments.
to analyse the resultant PCR products remains unclear. Technical limitations of most PCR instruments to run
While PCR product detection and analysis have typically overlapping reactions in parallel have restricted analyses to
been achieved using gel-electrophoresis and sequencing batches, thereby compromising the assay’s overall
techniques, these approaches are laborious and time- turnaround time. However, with the new generation of
consuming, which detracts from clinical applicability.139 The thermocycler (eg, SmartCycler, Cepheid), separate PCR
introduction of real-time PCR technology with the potential reactions, each with a unique set of cycling protocols and
use of differentially labelled fluorescent probes for data analysis, can now be done simultaneously.
simultaneous identification of multiple amplified products Furthermore, the recent introduction of hand-held battery-
in a single assay holds promise.140 Unfortunately, current operated real-time PCR instruments (BioSeeq, Smiths
ability to spectrally differentiate multiple fluorescent signals Detection-Edgewood BioSeeq) is the latest iteration of the
is quite limited. moving trend from laboratory to near patient testing.150
Another possible approach that can be used to analyse Ultimately, as “lab-on-a-chip” technologies mature, routine
multiple amplified sequences is to incorporate microarray point-of-care testing will be realised.
technology. DNA microarrays are constructed by spatially The true effect of PCR development for true point-of-
isolating specific genome sequences to prearranged areas care testing remains to be seen. Significant practical and
on a microchip.141–143 Fluorescently labelled amplification regulatory requirements slow and often halt the transition
products are then allowed to anneal to complementary of laboratory developments for bedside applications.
sequences on the chip, and the resultant pattern is spectrally Compliance with federal, state, and local regulations must be
analysed. The main advantage of using microarrays for met when operating point-of-care testing devices. Novel
pathogen detection is the potentially large number of target tests must go through the complex and time-consuming
sequences the system can discriminate simultaneously. The process of FDA approval. For in-house assays, strict clinical
use of microarray technology for pathogen detection is still laboratory improvement amendment requirements must be
in the development phase however. Efforts to improve met to define the operational characteristics of the assay
sensitivity, reproducibility, and to streamline approaches to relative to current gold standards. Institutional resources,
complex data analysis are still needed before these platforms manpower issues, and cost effectiveness also have to be
can be used clinically. carefully considered when making decisions about the
A final novel approach for analysing amplified products practicalities of replacing traditional diagnostic
is mass spectrometry. The recent advent of matrix assisted methodologies. Additional programmatic steps for true
laser description/ionisation (MALDI) technology coupled point-of-care testing must be developed to insure

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 Review PCR-based diagnostics

Pulsed laser light

Drift tube Mass ion detector

Ionised sample molecules

Desorption

of matrix

Mass spectrum

Ions spread out

Sample molecules
Lighter=faster

Matrix Heavier=slower

Figure 5. MALDI-TOF mass spectrometry. Sample molecules (ie, amplified DNA products) are co-crystallised with matrix and then subjected to desorption
and ionisation by an incident laser pulse. An applied electric field accelerates the resultant ionised sample molecules across the time-of-flight (TOF) drift
tube in vacuum, and a detector at the end of the tube accurately measures the flight time from the ion source to the detector. Typically, ions with larger
mass-to-charge (M/z) ratios travel more slowly than those with smaller m/z. The data are recorded as a “spectra” that displays ion intensity vs m/z value.

effectiveness, including (1) operational turn-around time could thus be appropriately applied, and patients with
(vs speed of the test); (2) education of practitioners in suspected infections rapidly risk-stratified to the different
interpretation of results; (3) development of protocols for treatment settings, depending on the pathogen and
optimal treatment and decision-making based on results virulence. The ability to discern species and subtype would
of novel tests; and (4) establishment of quality assurance allow for more precise decision-making regarding
and quality improvement programmes. As PCR-based antimicrobial agents. Patients who are colonised with highly
technologies continue to mature, each of these issues will contagious pathogens could be appropriately isolated on
need to be systematically addressed in order to realise their entry into the medical setting without delay. Targeted
benefit for routine patient care.151 therapy would diminish development of antibiotic
resistance, because the identification of antibiotic-resistant
Continuing clinical need: how PCR diagnostics strains would permit precise pharmacological intervention.
may revolutionise clinical care Both physicians and patients would benefit from less
PCR technology offers a great potential in the arena of repetitive testing and elimination of wait times for
infectious disease. A universally reliable infectious disease traditional laboratory results. Furthermore, links with data
diagnostic system will certainly become a fundamental tool management systems, locally, regionally, and nationally,
in the evolving diagnostic armamentarium of the 21st would allow for effective epidemiological surveillance with
century clinician. For front-line acute care physicians, or obvious benefits for antibiotic selection and control of
physicians working in disaster settings, a quick universal disease outbreaks.
PCR assay, or panels of PCR assays targeting categories of It is certain that the individual patient will benefit
pathogens involved in specific syndromes such as directly from this approach. Patients with unrecognised or
meningitis, pneumonia, or sepsis, would allow for rapid difficult-to-diagnose infections could be identified and
triage and early aggressive targeted therapy. Resources treated promptly. Inpatient stays would be reduced with a
concomitant decrease in iatrogenic events. Societal benefits
will need to be carefully explored with attention to relative
Search strategy and selection criteria costs of the novel diagnostics in relation to existing
Data for this review were identified by a search of Medline standards.
and from the references of relevant articles. Search terms
used were: “PCR”, “molecular diagnostics”, “emerging Conflicts of interest
infectious disease”, “bioterrorism agents”, “antimicrobial RER has served as an expert consultant to Ibis Therapeutics,
resistance”, “DNA microarrays”, and “mass spectrometry”. a division of Isis Pharmaceuticals (Carlsbad, CA, USA), which
Only English language papers were considered. develops diagnostic assays using both PCR and mass spectrometry
techniques.

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 PCR-based diagnostics
Review

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