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For personal use. Only reproduce with permission from The Lancet.

THE LANCET Infectious Diseases Vol 4 June 2004 http://infection.thelancet.com 337

Molecular diagnostics are revolutionising

the clinical practice of infectious disease.

Their effects will be significant in acute-

care settings where timely and accurate

diagnostic tools are critical for patient

treatment decisions and outcomes. PCR

is the most well-developed moleculartechnique up to now, and has a wide

range of already fulfilled, and potential,

clinical applications, including specific or

broad-spectrum pathogen detection,

evaluation of emerging novel infections,

surveillance, early detection of biothreat

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

automation, optimise detection

sensitivity and specificity, and expandthe capacity to detect multiple targets

simultaneously (multiplexing). This review

provides an up-to-date look at the

general principles, diagnostic value, and

limitations of the most current

PCR-based platforms as they evolve

from bench to bedside.

Lancet Infect Dis 2004; 4: 33748

Pathogen identification: scope of the problemIn the USA, hospitals report well over 5 million cases ofrecognised infectious-disease-related illnesses annually.1

Significantly greater numbers remain unrecognised, both inthe inpatient and community settings, resulting insubstantial morbidity and mortality.2 Critical and timelyintervention for infectious disease relies on rapid andaccurate detection of the pathogen in the acute-care settingand beyond. The recent anthrax-related bioterrorist eventsand the outbreak of severe acute respiratory syndrome(SARS) further underscore the importance of rapiddiagnostics for early, informed decision-making related topatient triage, infection control, treatment, and vaccinationwith life-and-death consequences for patients, healthproviders, and the public.35 Unfortunately, despite therecognition that outcomes from infectious illnesses are

directly associated with time to pathogen identification,conventional hospital laboratories remain encumbered bytraditional, slow multistep culture-based assays, which

preclude application of diagnostic test results in the acuteand critical-care settings. Other limitations of theconventional laboratory include extremely prolonged assaytimes for fastidious pathogens (up to several weeks);requirements for additional testing and wait times for

characterising detected pathogens (ie, discernment of species,strain, virulence factors, and antimicrobial resistance);diminished test sensitivity for patients who have receivedantibiotics; and inability to culture certain pathogens indisease states associated with microbial infection.2,6

The failure of either clinical judgment or diagnostictechnology to provide quick and accurate data foridentifying the pathogen infecting patients leads mostclinicians to adopt a conservative management approach.Empiric intravenous antibiotic therapy (most common in

ReviewPCR-based diagnostics

SY and RER are both at The Johns Hopkins University, School of

Medicine, Department of Emergency Medicine, Baltimore, MD, USA.

Correspondence: Dr Richard E Rothman, Department of

Emergency Medicine, Johns Hopkins School of Medicine, 1830 EMonument Street, Suite 6-100, Baltimore, MD 21205, USA.

Email [email protected]

PCR-based diagnostics for infectious diseases:

uses, limitations, and future applications inacute-care settings

Samuel Yang and Richard E Rothman

Double-stranded DNA (target)5

53

3

1 Denaturation

2 Primers annealing

5 35 3

3 Extension (polymerisation)

Each new double-stranded

DNA acts as target for new cycle

Cycles repeated exponentially:logarithmic amplification

1 Cycle

Figure 1. Schematic of PCR. The PCR reaction takes place in a thermocycler. Each PCR cycle

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

DNA polymerisation to generate new copy of the target DNA. At the end of each cycle the newly

synthesised DNA act as new targets for the next cycle. Subsequently, by repeating the cycle

multiple times, logarithmic amplification of the target DNA occurs.

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THE LANCET Infectious Diseases Vol 4 June 2004 http://infection.thelancet.com338

acute-care settings such as emergency departments andintensive care units) offers the advantages of maximum

patient safety and improved outcomes. The benefits ofconservative management may be offset, however, by addedcosts and potential iatrogenic complications associated withunnecessary treatment and hospitalisations, as well asincreased rates of antimicrobial resistance.79 A rapid reliablediagnostic assay, which allows for accurate identification ofinfected patients and informed early therapeuticintervention, would thus be invaluable for emergency andcritical care physicians.

For more than a decade, molecular testing has beenheralded as the diagnostic tool for the new millennium,whose ultimate potential could render traditional hospitallaboratories obsolete.1012 However, with the evolution ofnovel diagnostics tools, difficult questions have arisen

regarding the role of such testing in the assessment of clinicalinfectious diseases. As molecular diagnostics continue toflow from bench to bedside, clinicians must acquire aworking knowledge of the principles, diagnostic value, andlimitations of varied assays.13 Here we discuss the mostpromising molecular diagnostic techniques for infectiousdiseases in hospital-based settings: the emphasis is onPCR-based methods since they have reached greatestmaturity; existing assays, current, and future applications aredescribed. Further, a framework for describing limitationsthat have been encountered, as well as speculation regardingthe potential effect of these developments from the patient,physician, hospital, and societal perspective is provided.

Nucleic-acid-based amplification: historicalperspectiveThe first nucleic-acid-based assays used DNA probetechnology.1416 DNA probes are short, labelled, single-strandsegments of DNA that are designed and synthesised tohybridise targeted complementary sequences of microbialDNA. By contrast with traditional culture-based methodsof microbial identification, which rely on phenotypiccharacteristics, this molecular fingerprinting technique relieson sequence-based hybridisation chemistry, which confersgreater specificity to pathogen identification. Direct detectionof target microbial DNA in clinical samples also eliminatesthe need for cultivation, drastically reducing the time

required for reporting of results. In 1980, the description ofDNA hybridising probes for detecting enterotoxigenicEscherichia coli in stool samples raised hopes that nucleic-acid-based technologies would eventually replace traditionalculture techniques.17 Since that time, however, a morerestrained approach has been adopted due to recognition oftechnical limitations of the methodology; most notably, thelarge amount of starting target DNA required for analysis,which results in poor detection sensitivity.18

To attain optimum sensitivity, critical for most clinicalapplications, researchers sought to directly amplify targetmicrobial DNA. The development of the PCR technique in1985 answered this need, and provided what is now the

best-developed and most widely used method for targetDNA amplification. Other approaches, includingamplification of the hybridising probes (eg, ligase chain

reaction and Q-beta replicase amplification) and amplifi-cation of the signals generated from hybridising probes (eg,

branched DNA and hybrid capture), and transcription-basedamplification (eg, nucleic-acid-sequence-based amplificationand transcription-mediated amplification) have also beenincorporated into various detection systems.19 Detaileddescriptions of these technologies are beyond the scope of thisreview, but are well summarised elsewhere.20

PCR: basic principles and overviewPCR is an enzyme-driven process for amplifying short regionsof DNA in vitro. The method relies on knowing at least partialsequences of the target DNA a priori and using them to designoligonucleotide primers that hybridise specifically to thetarget sequences. In PCR, the target DNA is copiedby a thermostable DNA polymerase enzyme, in the presence

of nucleotides and primers. Through multiple cycles ofheating and cooling in a thermocycler to produce rounds oftarget DNA denaturation, primer hybridisation, and primerextension, the target DNA is amplified exponentially (figure1). Theoretically, this method has the potential to generatebillions of copies of target DNA from a single copy in less than1 h. For more detailed discussion of the basic principles ofPCR see references 2125.

Over the past two decades, PCR has been extensivelymodified to expand its utility and versatility. Multiplex PCRenables the simultaneous detection of several targetsequences by incorporation of multiple sets of primers.26 Toincrease sensitivity and specificity, a double amplification

step can be done with appropriately designed nestedprimers.27 Amplification may be made less specific to detectdivergent genomes by randomising portions of the primersets.28 Finally, RNA (rather than DNA) can be detected byconverting RNA into a complementary DNA copy, and thenamplifying (so-called reverse transcriptase PCR, orRT-PCR), enabling evaluation of RNA viruses or viableorganisms.27

A significant advancement in PCR technology isquantitative real-time PCR, in which amplification anddetection of amplified products are coupled in a singlereaction vessel. For purposes of clinical applicability, thisprocess represents a major breakthrough since it eliminatesthe need for laborious post-amplification processing

(ie, gel electrophoresis) conventionally needed for amplicondetection, and allows for measurement of productsimultaneous with DNA synthesis. One approach forreal-time monitoring of amplicon production is to usefluorescent DNA intercalating dyes, such as SYBR-Green I,which bind non-specifically to double-stranded DNAgenerated during amplification.29 A more popular alternativeapproach is to use a fluorescent-labelled internal DNA probewhich specifically anneals within the target amplificationregion. The choice of probe format depends onthe compatibility of its hybridisation chemistry with theexperimental design. Variations in probe format includeTaqMan (Applied Biosystems; figure 2), fluorescence

resonance energy transfer (FRET), and molecular beaconprobes.3032 Regardless of the format chosen, the internal probeemits a fluorescent signal during each amplification cycle only

Review PCR-based diagnostics

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in the presence of target sequences, withsignal intensity increasing in proportion

to the amount of amplified productsgenerated. The amount of startingtemplates in a specimen can bequantified by comparing the exact cyclenumber at which amplified productsaccumulate significantly over baselinewith a pre-derived quantitativestandard. Development of automatedinstrumentation with quantitativecapacity insures reproducibility.Practical advantages of real-time PCRover conventional PCR are thus myriadand include speed, simplicity, repro-ducibility, and quantitative capacity.

PCR-based diagnostics have beeneffectively developed for a wide range ofmicrobes. Due to its incredible sensi-tivity, specificity, and speed of ampli-fication, PCR has been championed byinfectious disease experts for identifyingorganisms that cannot be grown in vitro,or in instances where existing culturetechniques are insensitive and/or needprolonged incubation times.33,34 Appli-cation to the clinical arena, however, hasmet with variable success so far. Only alimited number of assays have been

approved by the US Food and DrugAdministration (FDA; table 1) and fewerstill have achieved universal acceptance in clinical practice.47

Furthermore, surprisingly limited effort has been focused onharnessing these time-saving diagnostics for emergencydepartment and other acute critical-care settings where time isof the essence. A discussion of the progress made and theobstacles remaining to be addressed follows.

Specific PCR diagnostics: development andclinical applicationsWith the increasing number of genomes of infectiouspathogens being sequenced, catalogues of genes can beexploited to serve as amplification targets fundamental to

the design of clinically useful diagnostic tests. As a result,over the past decade the number of PCR assays developedcommercially and in hospital-based laboratories (in-house) has continued to expand. Among the assays thathave been developed for detection of specific microbes, threeare described in more detail below, along with a discussionof their pros and cons relative to conventional diagnosticmethodologies (table 2).

One of the earliest recognised applications of PCR forclinical practice was for detection of Mycobacteriumtuberculosis.54,55 Disease characteristics favouring thedevelopment of a non-culture-based test for tuberculosisincluded week-long to month-long delays associated with

standard testing, and the public-health imperative associatedwith early recognition, isolation, and treatment of infectedpatients. Two PCR-based assays are approved by the FDA for

direct detection of M tuberculosis from clinical specimens(table 1). Although these assays result in significantimprovements in time to diagnosis, the only FDA approveduse at this time is as a diagnostic adjunct to the conventionalsmear and culture. Nonetheless, recent studies suggest thatmore widespread use of these assays may significantly affectpatient management, clinical outcomes, and cost efficacy.56,57

Potential yet unrealised applications of the assay for use inacute-care settings include earlier informed decision-makingfor appropriate use of isolation beds in high prevalence sites,regional outbreaks, or where isolation beds are scarce. The useof this and other PCR-based diagnostics in these settings,

however, will have to be balanced against costs, expertise, andtime associated with routine around-the-clock availability oftesting, as discussed in more detail below.

Another PCR-based diagnostic assay, which has gainedwidespread acceptance, is that for Chlamydia trachomatis.Conventional detection systems for this organism(ie, culture and direct antigen testing by immunoassays),have been limited by the requirement for specialised facilitiesto culture this fastidious microbe, as well as inadequatesensitivity and specificity of immunoassays relative to thegold standard culture results.58 Laboratory development andsubsequent clinical validation testing have indicatedexcellent sensitivity and specificity of the PCR assay leading

to its commercial development (table 1). Proven efficacy ofthe PCR assay for both genital and urine samples hasresulted in its application to a range of clinical settings, most


Primers and probe annealing

3 5

5 3

Polymerisation

Denatured DNA

Probe displacement by 5exonuclease of Taq polymerase

Polymerisation completed

Reporter dye

Quencher dye

Primers

Fluorescent-emittingreporter dye

Synthesised DNA

R Q

R Q

RQ

R Q

R

Q

R

Figure 2. Real-time PCR using Taqman probe. Taqman probe is a single-stranded oligonucleotide

that is labelled with two different fluorescent dyes. On the 5 terminus is a reporter dye and on the

3 terminus is a quenching dye. This oligonucleotide probe sequence is homologous to an internal

target sequence present in the PCR amplified product. When the probe is intact, the proximity of

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

polymerase thereby releasing the reporter from the quencher and producing an increase in

reporter emission intensity which can detected and quantified. As amplification continues, the

amount of reporter dye signal measured is proportional to the amount of PCR product made.

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recently routine screening of emergency department patientsconsidered at risk for sexually transmitted diseases.59,60

Although studies in the acute-care settings have not yet usedPCR assays for C trachomatison site and in real time (thusnot taking full advantage of the speed of PCR), routine useof this assay in the aforementioned studies has resulted innearly three-fold greater rates of disease detection andtreatment relative to standard care.

Distinguishing life-threatening causes of fever from morebenign causes in children is a fundamental clinical dilemmafaced by clinicians, especially when infections of the centralnervous system are being considered. Bacterial causes ofmeningitis can be highly aggressive but generally cannot bedifferentiated on a clinical basis from aseptic meningitis, abenign condition generally appropriate for outpatientmanagement.61 Culture methods often take several days to

show positive results and are confounded by poor sensitivityor false-negative findings in patients receiving empiric anti-microbials.62 One well developed assay, which has the potentialto influence the management of patients in the acute-caresetting, allows early and rapid diagnosis of diseases of viralcause. Testing and application of a PCR assay for enteroviralmeningitis has been seen to be highly sensitive. 63,64 Withreporting of results within 1 day, preliminary clinical trialshave shown significant decreases in hospital costs due todecreased duration of hospital stays and courses of antibiotictherapy.65,66 Other viral PCR assays, now routinely available,

include those for herpes simplex virus, cytomegalovirus,Epstein-Barr virus, hepatitis viruses, and HIV.67 Each has a

proven cost-saving role in clinical practice, including detectionof otherwise difficult to diagnose infections, and a newlyrealised capacity to monitor progression of disease andresponse to therapy, vital to the management of chronicinfectious diseases.68

With the increasing number of genomes of infectiouspathogens being sequenced, catalogues of genes can beexploited to serve as amplification targets. As a result, thenumber of PCR assays developed both commercially andin-house continues to expand.

Broad-ranged PCRThe notion of a universal detection system has been proposedfor the identification of classes of pathogens and speaks most

directly to the future potential effect of PCR-based assays forclinical practice in emergency and other acute critical-caresettings.69 Experimental work has focused on using sequencesof the 16S rRNA gene, an evolutionarily conserved gene seenexclusively in bacterial species.70,71 By designing primers thatare complementary to these regions, investigators can, intheory, establish the presence of any bacteria in an otherwisesterile clinical specimen (such as cerebrospinal fluid or wholeblood). Clinical applications are profound. Acute-carephysicians could rapidly identify the presence of bacteraemia.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 specificityChlamydia trachomatis Amplicor Roche PCR 93219 984119

LCX Abbott LCR >9519 >9919

AMP Gen-Probe TMA 86799219 >9919

PACE 2 Gen-Probe Hybridisation 60878135 >9935

BDProbeTec Becton Dickinson SDA 94036 >9936

Hybrid capture II CT-ID Digene Hybrid capture 95437 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 88619 97819

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 30 Bayer BDNA NA 98242

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 30 Bayer BDNA 97644

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 79491919 >9919

E-MTD Gen-Probe TMA 90995219 >9919

Neisseria gonorrhoeae Amplicor Roche PCR NA NA

LCX Abbott LCR >9519 >9919

Hybrid capture II CT/GC Digene Hybrid capture 9346 98546

BDProbeTec Becton Dickinson SDA 88936 >9936

PACE-2 Gen-Probe Hybridisation 9735 9935

Trichomonas vaginalis Affirm VIP III Becton Dickinson Hybridisation 8891 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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favour of educated practice, allowing appropriate, expeditiousdecision-making about the need for antibiotic therapy andhospitalisation. In principle, this approach could be applied toother taxonomic groups of pathogens (eg, genus of species,families of viruses, or fungi) by exploiting common features ofclasses of organisms for broad-range PCR assay design.72

Validation of this technique for eubacterial detection hasfocused on high yield clinical settings where expeditiousidentification of the presence of systemic bacterial infectionhas immediate high morbidity and mortality consequences.Notable clinical trials have included assessment of patients atrisk for infective endocarditis,7375 febrile infants at risk forsepsis,76,77 febrile neutropenic cancer patients,78 and critically illpatients in the intensive care unit.79 While several of these

studies have reported promising results (with sensitivity andspecificity for bacteraemia well above 90%), significanttechnical difficulties remain, preventing general acceptance ofthese assays in clinics and hospitals (see Limitations below).

One significant investigational role for broad-range PCRhas been its use as a molecular petri dish to identifyemerging or existing infectious causes for diseases previouslydescribed as idiopathic. The DNA amplified using thisbroad-range approach may contain intervening sequenceinformation that is phylogenetically specific to a uniquemicrobe when compared with existing microbial geneticdatabases. For example, sequencing of the 16S rRNA geneamplified via highly conserved primer sets has led to the

identification of Bartonella henselae in bacillaryangiomatosis, and Tropheryma whipplei as the unculturedbacillus associated with Whipples disease.80 Further, recent

epidemiological studies that suggest a strong associationbetween Chlamydia pneumoniaeand coronary artery diseaseserve as an example of the possible widespread, yetundiscovered, links between pathogen and host which mayultimately lead to new insights into pathogenesis anddevelopment of novel life sustaining or saving therapeutics.81

The most recent, high-profile investigational use ofbroad-range PCR was in the molecular identification of acoronavirus as the causative agent in SARS. In a variantapproach to PCR assay development, broad-based primerswith degenerate sequences designed to detect unknownviruses were used to randomly amplify the genetic contents ofinfected clinical isolates. A subset of the amplified sequencesshowed homologies to the genus of coronavirus,82,83 consistent

with other confirmatory laboratory test results. Soonafterwards, a coronavirus-specific PCR assay was developedfor rapid laboratory diagnosis of SARS.84 Notably, theseadvancements came only weeks after the first reports of thedisease surfaceda veritable tour de force bespeaking thepower of broad-range PCR.85

Antimicrobial resistance profilingWith multidrug-resistant pathogens on the rise, earlyantimicrobial resistance profiling is crucial both for timely,objective treatment of infected patients, as well as for broaderpublic-health surveillance. Conventional tests of this type arelimited by prolonged culturing time (4872 h) and poor

accuracy due to variability in inoculum size and culturingconditions. To address these shortcomings, nucleic-acid-based assays are being advanced as genetic mechanisms of


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

organismMethod Pros Cons Method Pros Cons

M tuberculosis Culture Allows susceptibility Inadequate sensitivity PCR High sensitivity (93%)/ Potential

testing Prolonged time to (TB Aamplicor) specificity (98100%)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 specifici ty Low sensit ivi ty (7080%)49 PCR Rapid detection time Limited ability for

(100%)48 Invasive specimen (Amplicor) Moderate to high multidrug-resistance

Detects only viable collection sensitivity (8090%)19 testing

organisms Prolonged time to result with high specificity Currently used as an

Allows further genotype Cold transport (95100%)19

adjunctive testor susceptibility testing High cost Probably cost effective51

Antigen detection High specificity Requires technical

method (dfa) (9899%)50 expertise

Allows assessment Moderate sensitivity

of specimen adequacy (8090%)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 (1014 days) (Amplicor EV) High specificity (94%)53

Low sensitivity (6575%)53 Rapid detection time

Multiple cell lines needed More adaptable

for isolation for serotyping

Serotyping time-consuming, Cost effective54

labour intensive, and costly

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drug resistance are elucidated. Three examples of the clinicalapplicability of resistance profiling follow.

Although the presence of a resistance gene does notnecessarily imply its expression and conferment of phenotypicresistance, its absence does establish a lack of resistancethrough that particular genetic mechanism: for example,meticillin resistance is mediated by the mecA gene.A distinctive feature of meticillin resistance is its heterogenousexpression. As such, when typical phenotypic susceptibilitytesting is used to assess resistance, meticillin-resistant strainsmay seem falsely susceptible to some -lactam antibiotics invitro.86 For this reason, direct detection of the mecAgene byPCR is more desirable. With its high detection sensitivity andspecificity, mecA PCR has gained wide acceptance and isbecoming the most reliable method of identifying meticillin-resistant Staphylococcus aureus(MRSA).87

PCR-based resistance testing in M tuberculosis has alsobeen developed for the detection of rifampicin resistance.Rifampicin resistance is well characterised and conferred bymutations within a short sequence of the rpoBgene ofM tuberculosis, which result in aminoacid substitutions in therpoB subunit of RNA polymerase.88 The Line Probe assay(LiPA; Inno-Genetics) is a commercially available PCR-basedassay that targets the mutation-prone segment of the rpoBgene.89 Correlation with standard resistance-detectionmethods has been more than 90% and is shown to provideclinicians with a drastic reduction in detection time, criticalfor treatment decisions.90 Genotypic analysis of otherM tuberculosisdrug resistance is more challenging due to the

number of mutations and genetic loci involved. Technicalinnovations (ie, multiplex PCR or DNA microarray) thatallow simultaneous amplification and analysis of multipletarget sequences will likely provide the means to surmountthis later limitation.91,92

With ever-increasing evidence supporting the prognosticvalue of identifying drug-resistant mutations, routinegenotypic resistance testing is now standard care in thetreatment of HIV-infected patients.9395 PCR followed bynucleotide sequencing is the most commonly used method.Although genotypic tests are more complex than typicalantimicrobial susceptibility tests, their ability to detectmutations at concentrations too low to affect drugsusceptibility in a phenotypic assay provides insight into the

potential for resistance to emerge. They also have theadvantage of detecting transitional mutations that do notthemselves causedrug resistance but indicate the presence ofselective drug pressure, with potential importance forindividual patient treatment decisions.

Applications in bioterrorismThe increasing threat of bioterrorism has gainedconsiderable attention in light of the anthrax outbreak thatcame after the September 11, 2001 terrorist attacks. It hasbecome increasingly apparent that responsibility for therapid recognition and accurate diagnosis of real or suspectedbioterrorism events will fall principally to front-line acute-

care physicians who will be critical in initiating appropriateresponse measures.96 Unfortunately, as was seen with the2001 anthrax episode, the clinical presentation of

bioterrorism victims may be non-specific and difficult todistinguish from commonly encountered disease processes.96

The previously described limitations of conventionalculture-based assays make such tests wholly inadequate fordetection of bioterrorism agents in suspected clinicaloutbreaks. Furthermore, traditional microbiological methods,which require prolonged incubation, increase biohazard riskat the hospital laboratory due to unnecessary propagation ofbioterrorism pathogens in culture-based systems. Widerecognition of these limitations has led to recent developmentsand refinements of PCR-based assays for a number of categoryA bioterrorism agents, including variola major, Bacillusanthracis, Yersinia pestis, and Francisella tularensis.97100 PCRdiagnostics for bioterrorism agents will likely be used both fordiagnosis of symptomatic individuals, as well as larger scalescreening of exposed victims (preclinical phase), who would

be candidates for early prophylactic therapy.Although most bioterrorism-induced illnesses resemble

natural outbreaks, there is the possibility that causativebioterrorism agents are genetically engineered to increasevirulence, acquire resistance to antibiotics or vaccines, orproduce phenotypic characteristics that resemble multiple,simultaneous infections, so-called binary agents (viainsertion of recombinant genes).101 In such cases, it is likelythat nucleic-acid-based approaches will be more invaluablethan conventional detection methods since they are themore easily adaptable and capable of uncovering detailedinformation embedded in genetic sequences.

Cost effectivenessPCR is more expensive than conventional approaches. Thedirect costs of PCR reagents, equipment, dedicated space,personnel training, and labour have been reported to be ashigh as US$125 per reaction.102 Even among PCR methods,there is variability in cost with the most expensive beingfluorogenic-based systems. Moreover, the labour intensityneeded for most assays as well as technical limitations ofmost thermocyclers to do multiple runs of PCRsimultaneously have prevented routine around-the-clocktesting in the clinical setting. On the other hand, continuedrefinement in PCR technology, as well as improvements inautomation and reproducibility via high throughputrobotics, will probably lead to increasing demand and

marked cost reductions to rates competitive with traditionalmethods. Already, this development has been reported forNeisseria gonorrhoeae and C trachomatis PCR tests whichnow cost around $9 per reaction.103

In assessing the overall benefit of PCR, however, directmonetary costs should not be the only consideration sincethe assay has several significant advantages over traditionalmethods. One study, which took a global methodologicalapproach to cost, involved assessment of perinatal screeningfor Group B streptococcus using PCR versus culturetechniques.104 Considered variables of the assays in additionto the direct monetary cost included infections averted,mortality, infant disabilities, hospital stays, and the societal

benefits of healthy infants. Overall, the authors concludedthat the benefits of PCR outweighed its cost. Notably, thisresult was reached even without inclusion of important but


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difficult-to-measure parameters, such as the societal benefitof decreased drug resistance due to targeted therapy made

possible by the PCR assay.

Limitations of PCR and emerging innovationsThe principal shortcomings in applying PCR assays to theclinical setting include false-positive results from backgroundDNA contamination; the potential for false-negative testresults; detection sensitivity exceeding clinical significance;and limited detection space of the assay or platform forsimultaneous identification of multiple species, virulencefactors, or drug resistance.

False positives

The widespread use of PCR in clinical settings has beenhampered largely by background contamination from exoge-

nous sources of DNA.105 In most pathogen-specific assays, thepredominant source of contamination is derived from carry-over products from earlier PCR reactions, which can beharboured and transmitted through PCR reagents, tubes,pipettes, and laboratory surfaces. Coupled with the robustamplification power of PCR, even very minor amounts ofcarry-over contamination may serve as substrates foramplification and lead to false-positive results. Meticulouscontrol measures such as good laboratory practices andphysical separation of preamplification and postamplificationareas can reduce contamination risks but are not foolproof.The use of enzymatic inactivation of carry-over DNA (ie,uracil N-glycosylase) can further reduce contamination risk.106

Contamination issues are most pronounced in assays thatuse universal primers, such as those targeting conservedregions of the eubacterial 16S rRNA gene. Here, theubiquitous presence of eubacterial DNA in either theenvironment or working reagents may lead to false-positivefindings. Attempts to decontaminate PCR materials haveinvolved nearly all known methods of destroying DNAincluding ultraviolet irradiation, chemical treatment, andenzymatic digestion.107,108 None of these methods has beenshown to be entirely effective without significant diminutionof assay sensitivity. We have recently reported an alternativemethod that uses a size-based ultrafiltration step for reducingcontaminating DNA from PCR reagents, primers, and DNApolymerase before amplification. Although this method of

decontamination has been shown to be effective withoutcompromising detection sensitivity in vitro,109 validation, andoptimisation of the method in clinical samples needs furtherstudy. More importantly, effective and reliable methods ofdecontamination have not yet been developed for stepsoutside the assay proper such as sample collection andpreparation. Towards this end, one promising area ofinvestigation involves development of methods to integratesample preparation, amplification and detection on a singleplatform, the so-called lab-on-a-chip. Self-containedmicrochip platforms thus hold promise for the best means ofdecontamination and overall assay efficiency.110

False negativesPCR assays for microbial detection may give false-negativeresults for two principal reasons: the relatively small sample

volume permissible for PCR reactions; and problemsassociated with PCR processing. The sample volume most

PCR assays can accommodate is quite small relative to thevolume used in conventional culture methods; as such, incases in which the concentration of infectious organisms islow, the assay may yield false-negative findings. To accountfor this, DNA extraction and purification steps are usuallyperformed before PCR amplification as a means ofconcentrating total DNA from a larger sample volume.Additional methods to optimise starting concentration oftarget DNA for the PCR reaction include: selecting specimensources (eg, cerebrospinal fluid) or specimen fractions (eg,buffy coat instead of whole blood) with the highest abundanceof microbial DNA for the DNA extraction; briefly cultivatingsamples to increase microbial load before DNA extraction;20

or introducing specific capture probes to concentrate only

microbial DNA in a given sample.111 Several sample processingobstacles may also lead to false-negative findings. Three of themost commonly encountered problems are (1) inadequateremoval of PCR inhibitors in the sample, such ashaemoglobin, blood culture media, urine, and sputum; (2)ineffective release of microbial DNA content from the cells; or(3) poor DNA recovery after extraction and purification steps.Methods to ensure best sample processing include: incorpo-rating internal amplification controls (eg, the human -globingene) to the PCR assay to monitor for presence of bothpurified sample DNA as well as potential PCR inhibitors;112

and inducing various chaotropic, enzymatic, or thermalmethods of cell lysis to effectively liberate microbial DNA

content.

20

Because of the varying effectiveness of each of thesemeasures, efforts to improve an assays detection sensitivitymay need to be individually adjusted based on the assaysclinical application and the microbial pathogen of interest.

Clinical significance of positive PCR

PCR assays may detect microbial pathogens at concentrationsbelow those of previously established gold standard referencemethods. Distinguishing whether this result represents afalse-positive finding and establishing the clinical significanceof these findings is challenging. In the past, discrepantanalysis based on the results of additional ancillary tests wasused to provide estimates of sensitivity and specificity in thepresence of an imperfect gold standard.113 One example can

be seen in assessments of novel nucleic acid-based assays indetecting C trachomatis.114,115 In these studies, false positives(DNA-amplification positive and tissue culture negative)were adjudicated by either antigen detection methods oranother well-established DNA-amplification test. Despite itspopularity, recent concerns have been raised regarding thepotential bias incurred by discrepant analysis in favour of thenew tests.116,117 Up to now, the issue has not been completelyresolved.

The complexity of the clinical interpretation of positivePCR findings is further underscored by one study thatreported that a universal PCR assay (using primers fromconserved regions of the 16S rRNA gene) amplified

eubacterial DNA in blood samples from healthy people.

118

Itis unknown whether such findings are indicative of latentdisease processes or sub-clinical colonisation. Moreover, the


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finding that microbial DNA can be detected even aftersuccessful antimicrobial treatment suggests that the assays

detect both viable and non-viable organisms.119,120

Clearly,interpretive guidelines based on the correlation of test resultswith clinical presentation and existing standards will berequired before these assays can be used for definitivediagnosis and/or treatment decisions.

One breakthrough in establishing the meaning of positivePCR results involves the development of reliable quantitativemeasures of pathogen load. While traditional PCR assays areused primarily for dichotomous outcome, innovative real-time PCR methods allow for quantitative measurement ofstarting template in the sample, which will probably be usefulin differentiating benign colonisation from either latentor active disease. Other non-PCR amplification methodswith quantitative capacities include branched DNA and

nucleic-acid-sequence-based amplification. Quantification ofpathogen load is already well established in clinical virology(eg, HIV-1, cytomegalovirus, hepatitis B virus, hepatitis Cvirus, and Epstein-Barr virus), where it has proven useful inassessing disease severity or monitoring treatmentefficacy.121126 The value and importance of PCR-based

pathogen quantifications in clinical bacteriology remainsunder investigation.127

Alternative innovations regarding PCR technologies mayhelp in differentiating viable from non-viable organisms,important for clinical practice decisions. RNA is known to berapidly degraded with a typical half-life of minutes after celldeath; thus, it has been proposed as a more accurate indicatorof viable microorganisms.128,129 In some clinical situations,detection of RNA species by RT-PCR has been shown tocorrelate well with the presence of viable organisms and hasbeen effectively used to monitor antibiotic therapy.130134

Clinical application of RNA-based approaches will needfurther improvement, however, because they have beenhampered in development by difficulties in extractingdetectable concentrations of intact RNA from small numbersof bacteria.

Limited detection space for characterising the

detected pathogen

Conventional methods for pathogen detection will not besupplanted by PCR-based assays if the latter cannotbe elaborated to further characterise detected pathogens. Asdescribed previously, genetic sequences contain rich sources ofinformation that can be analysed to ascertain pathogensspecies or strains, virulence factors, and antimicrobialsusceptibilities. However, to do so in a single reaction, simulta-neous amplification of several target genes is needed.Repeating amplifications with different primer pairs, so-calledmultiplexing, is notoriously difficult since often one or more

of the target sequences do not amplify.

91

Recent studies have shown that PCR can be used forsimultaneous reproducible amplification of multiple DNAfragments in a single reaction, provided that only a singleprimer set is used for amplification of all these fragments.Repetitive-sequence-based PCR (rep-PCR), which usesconsensus PCR primers to amplify DNA sequences locatedbetween successive repetitive elements in eubacterialgenomes, has been shown to simultaneously amplifyfragments of different sizes, allowing discrimination ofbacteria at the subspecies level.135,136 This conceptualbreakthrough has led various investigators to develop andexplore various technical approaches which harness the sameidea. By exploiting the conserved and variable sequences on

the 16S rRNA gene, we have shown through use ofquantitative PCR that a single consensus primer set canmultiplex amplify multiple species of the 16S rRNA gene withequal efficiencies.109 Similarly, ligation-dependent PCR (figure3)137 and padlock probes with rolling circle amplification(figure 4),138 which are both probe amplification methods,have also shown that multiple genetic targets can be queriedsimultaneously by using a single primer pair foramplification. Both of these assays rely on multipleoligonucleotide probes, each containing a unique targetsequence and a consensus primer sequence, that areamplifiable in the presence of their targets. Progress in theseapproaches could greatly enhance throughput in genotyping

pathogens detected, and may represent the next generation ofPCR-based assays that hold tremendous promise with regardto their clinical applications.


Hybridisationsequence

Hybridisationsequence

Stuffer sequence

Primer B

Primer Asequence

Target

Target

5

3

3 5

Primer B

Primer A

3

5

Ligated functional probe

5 hemiprobes 3 hemiprobes

sequence

A

B

C

Figure 3. Ligation-dependent PCR (LD-PCR). LD-PCR is a process in which

non-amplifiable hemiprobes for each target will be constructed as follows:

(A) For the 5hemiprobe the 5 end will be a generic primer sequence

shared by all 5hemiprobes, and the 3 end will be target-specific. The 3

hemiprobe will have a mirror symmetrical arrangement with an intervening

stuffer sequence of variable length. (B) In the presence of target sequences

the hemiprobes are juxtaposed to each other as they hybridise to their

targets. (C) A single PCR primer set based on the generic sequences on

the hemiprobes will be used for amplification of any ligated functionalprobes. The amplified product of each ligated functional probe has a

unique length that can be separated by electrophoresis.

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Even if this problem is resolved, the best way in whichto analyse the resultant PCR products remains unclear.While PCR product detection and analysis have typicallybeen achieved using gel-electrophoresis and sequencingtechniques, these approaches are laborious and time-consuming, which detracts from clinical applicability.139 Theintroduction of real-time PCR technology with the potentialuse of differentially labelled fluorescent probes forsimultaneous identification of multiple amplified productsin a single assay holds promise.140 Unfortunately, currentability to spectrally differentiate multiple fluorescent signalsis quite limited.

Another possible approach that can be used to analysemultiple amplified sequences is to incorporate microarray

technology. DNA microarrays are constructed by spatiallyisolating specific genome sequences to prearranged areason a microchip.141143 Fluorescently labelled amplificationproducts are then allowed to anneal to complementarysequences on the chip, and the resultant pattern is spectrallyanalysed. The main advantage of using microarrays forpathogen detection is the potentially large number of targetsequences the system can discriminate simultaneously. Theuse of microarray technology for pathogen detection is stillin the development phase however. Efforts to improvesensitivity, reproducibility, and to streamline approaches tocomplex data analysis are still needed before these platformscan be used clinically.

A final novel approach for analysing amplified productsis mass spectrometry. The recent advent of matrix assistedlaser description/ionisation (MALDI) technology coupled

with time of flight mass spectrometry(TOF-MS) has created a robust means

to characterise mostly proteins, butincreasingly, nucleic acid. In MALDI-TOF-MS, the organic moleculeforexample an amplified productisionised and subsequently identifiedbased on its mass-to-charge ratio(figure 5).144149 The advantagesof MALDI-TOF-MS lie in the inherentaccuracy and the high-speed (1 second)of signal acquisition, making thistechnology an attractive candidate forhigh-throughput DNA analysis.

Practical aspects of rapid and

point-of-care testingThe real-time PCR-based platformholds great promise in replacingconventional laboratory-based testingfor future point-of-care testing.With advancements in automation,integration of specimen preparationwith target identification, andminiaturisation, it will become mucheasier to bring analyses near bedsideto be done by less-trained personnel.The ability to interface with highthroughput PCR systems is already

seen in many new automated extraction instruments.Technical limitations of most PCR instruments to runoverlapping reactions in parallel have restricted analyses tobatches, thereby compromising the assays overallturnaround time. However, with the new generation ofthermocycler (eg, SmartCycler, Cepheid), separate PCRreactions, each with a unique set of cycling protocols anddata analysis, can now be done simultaneously.Furthermore, the recent introduction of hand-held battery-operated real-time PCR instruments (BioSeeq, SmithsDetection-Edgewood BioSeeq) is the latest iteration of themoving trend from laboratory to near patient testing.150

Ultimately, as lab-on-a-chip technologies mature, routinepoint-of-care testing will be realised.

The true effect of PCR development for true point-of-care testing remains to be seen. Significant practical andregulatory requirements slow and often halt the transitionof laboratory developments for bedside applications.Compliance with federal, state, and local regulations must bemet when operating point-of-care testing devices. Noveltests must go through the complex and time-consumingprocess of FDA approval. For in-house assays, strict clinicallaboratory improvement amendment requirements must bemet to define the operational characteristics of the assayrelative to current gold standards. Institutional resources,manpower issues, and cost effectiveness also have to becarefully considered when making decisions about the

practicalities of replacing traditional diagnosticmethodologies. Additional programmatic steps for truepoint-of-care testing must be developed to insure


3

5

3

5

A B

C D

P1 P1

P2

Target DNA Target DNA

Padlock probe Padlock probe

Figure 4. Padlock probe with rolling circle amplification. (A) Padlock probe hybridises to the target

sequence. (B) The padlock probe can be converted to a circle by ligation only if ends of the probe are

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

polymerase begins displacing the previously synthesised product, opening up single-stranded primer

2 (P2) binding site. (D) Each turn of displacement synthesis around the circle exposes another P2

binding site, and the resulting synthesis from the annealed P2 primers results in the displacement of

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

amplification process occurs in an isothermal reaction. Adapted from reference 138.

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effectiveness, including (1) operational turn-around time

(vs speed of the test); (2) education of practitioners ininterpretation of results; (3) development of protocols foroptimal treatment and decision-making based on resultsof novel tests; and (4) establishment of quality assuranceand quality improvement programmes. As PCR-basedtechnologies continue to mature, each of these issues willneed to be systematically addressed in order to realise theirbenefit for routine patient care.151

Continuing clinical need: how PCR diagnosticsmay revolutionise clinical carePCR technology offers a great potential in the arena ofinfectious disease. A universally reliable infectious diseasediagnostic system will certainly become a fundamental tool

in the evolving diagnostic armamentarium of the 21stcentury clinician. For front-line acute care physicians, orphysicians working in disaster settings, a quick universalPCR assay, or panels of PCR assays targeting categories ofpathogens involved in specific syndromes such asmeningitis, pneumonia, or sepsis, would allow for rapidtriage and early aggressive targeted therapy. Resources

could thus be appropriately applied, and patients with

suspected infections rapidly risk-stratified to the differenttreatment settings, depending on the pathogen andvirulence. The ability to discern species and subtype wouldallow for more precise decision-making regardingantimicrobial agents. Patients who are colonised with highlycontagious pathogens could be appropriately isolated onentry into the medical setting without delay. Targetedtherapy would diminish development of antibioticresistance, because the identification of antibiotic-resistantstrains would permit precise pharmacological intervention.Both physicians and patients would benefit from lessrepetitive testing and elimination of wait times fortraditional laboratory results. Furthermore, links with datamanagement systems, locally, regionally, and nationally,

would allow for effective epidemiological surveillance withobvious benefits for antibiotic selection and control ofdisease outbreaks.

It is certain that the individual patient will benefitdirectly from this approach. Patients with unrecognised ordifficult-to-diagnose infections could be identified andtreated promptly. Inpatient stays would be reduced with aconcomitant decrease in iatrogenic events. Societal benefitswill need to be carefully explored with attention to relativecosts of the novel diagnostics in relation to existingstandards.

Conflicts of interest

RER has served as an expert consultant to Ibis Therapeutics,

a division of Isis Pharmaceuticals (Carlsbad, CA, USA), whichdevelops diagnostic assays using both PCR and mass spectrometrytechniques.


Search strategy and selection criteria

Data for this review were identified by a search of Medline

and from the references of relevant articles. Search terms

used were: PCR, molecular diagnostics, emerging

infectious disease, bioterrorism agents, antimicrobial

resistance, DNA microarrays, and mass spectrometry.Only English language papers were considered.

Matrix

Sample molecules

Mass ion detector

Ions spread out

Lighter=faster

Heavier=slower

Pulsed laser light

Ionised sample molecules

Desorption

of matrix

Mass spectrum

Drift tube

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 intensityvsm/z value.

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