Application of Polymerase Chain Reaction to the Diagnosis of Infectious Diseases

Author(s): David N. Fredricks and David A. Relman
Source: Clinical Infectious Diseases, Vol. 29, No. 3 (Sep., 1999), pp. 475-486
Published by: Oxford University Press
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Stanford University. phylogeneticallyinformativeDNA sequence(s)(figure1).111.Latexagglutinationtests performedon the CSF were negative for Haemophilus group b.500 cells/mm3(93%neutrophils). David N. discuss the advantages and limitations of PCR-basedmethods.nuchalrigidity. group B Streptococcus. Palo Alto. and mention some currentand futureapplicationsof this technology. This DNA was used as templatein a broad-rangePCR assay designed to copy enzymatically(amplify) a portion of the bacterial16S rRNA gene in vitro using a thermostableDNA polymerase.Blood cultures were obtainedafter antibioticshad been started.a WBC countof 17.whetherpositive or negative.and penicillinwas addedto the ceftriaxonetherapy for optimal coverage of Neisseria meningitidis.6?C). Departmentof Medicine and Departmentof Microbiology and Immunology.The penicillinwas discontinuedand ceftriaxone was continued. F. It is ourgoal to makePCR-baseddiagnosticsunderstandable to clinicians. The patient ran a day care center. 1058-4838/99/2903-0001 $03. Oligonucleotide primers complementary to broadlyconservedregionsof the 16S rRNAgene were used to amplify segments of the gene that also containedvariable. A sampleof the patient'sCSF was centrifugedto concentrate bacteria. meningitidis. must be interpreted using an understandingof the test employed and an assessmentof the clinical scenario. We emphasizethe principles behindPCR-baseddiagnosis.she had a fever (temperatureto 38.and a glucose level of 41 mg/dL(serumglucose level. California 94304 (fredrick@cmgm.clinicianswill need to learnits advantagesand limitationsso thatsoundjudgments can be made.The patient'svancomycinwas discontinued.All blood and CSF cultures were negative. The laboratoryconcluded that the gram-negative organismsseen on the smearhad a coccobacillarymorphology more consistent with Haemophilus influenzae than with N.the readeris referredto otherpublications for additionaldetails [1-3]. 02 Nov 2015 09:50:11 UTC All use subject to JSTOR Terms and Conditions . To identify the bacteriumresponsiblefor this patient's illness.475 STATE-OF-THE-ARTCLINICALARTICLE Application of Polymerase Chain Reaction to the Diagnosis of Infectious Diseases David N. Financialsupport:D. Examinationof a gram-stainedsmear of the CSF was reportedto show many WBCs and many gram-negativediplococci.All rightsreserved.D.Similarly. On the second hospitalday. Astute clinicians know that results of blood culturereports.29:475-88 ? 1999by the InfectiousDiseasesSocietyof America.42 on Mon.We will point out the limitationsof conventional diagnosticmethodsfor infectiousdiseases.We will not discussevery currentor pendingapplicationof PCRto diagnosticmicrobiology. The This content downloaded from 128.On physicalexamination.As this technologyexpandsinto the clinicalarena.because of difficultywith phlebotomy.The patient'sconditionimprovedand she was dischargedon the fifth day to completea course of outpatient intravenousceftriaxone. ClinicalInfectiousDiseases 1999. meningitidis antigens. Received30 March1999. Multiplegeneralizedseizureswere noted.VeteransAffairs Palo Reprintsor correspondence: Alto HealthCare System 154T.and the EnvironmentalProtectionAgency (InteragencyGrant). Stanford.Therapywith ceftriaxoneandvancomycinwas institutedin the emergencydepartment for empirictreatmentof meningitis.andacknowledgea researchorientedbias in our viewpoint. is supportedby NationalInstitutesof Health(NIH) Physician-ScientistAward once consideredto be only research tools. the gramstain of the CSF was reviewed.andno evidenceof a rash. California mentalstatus. Dr.revised23 May 1999.stanford. 3801 MirandaAvenue. 209 mg/dL).and the VeteransAffairs Palo Alto Health Care System. are being used increasinglyin the clinical microbiologylaboratory. and the county health departmentwas notifiedso thatantibioticprophylaxiscouldbe startedfor case contacts.infectious diseases practitionerswill need to expandtheirunderstandingof PCR-baseddiagnosticsso that these powerful tests are used appropriately. The etiology of this patient'smeningitiswas not confirmed using conventionalmethodsof cultivationand antigendetection.The lumbarpuncturedemonstrated cloudyCSF with an elevatedpressure. we used a sequence-basedapproachin our laboratory. Relman PCRandothersequence-basedmicrobialdetectionmethods. Thelimitationsof existingdiagnosticmethodsandthepotential of PCR-baseddetectionand identificationmethodsare demonstratedby a case fromStanfordUniversityMedicalCenter.a proteinlevel of 756 mg/dL. Fredricks. is supportedby grantsfrom (RO1-AI39587). Palo Alto.anda CT scan of the brainwas obtainedpriorto a lumbarpuncture. suggestingthe diagnosisof meningococcalmeningitis.the Departmentof Defense (N65236-99-1-5428).andthe pellet was digestedto liberatebacterialDNA.the Departmentof Veterans Affairs (EmergingInfections Program). Fredricks and David A. and N. A Case of Meningitis A 53-year-oldwoman was seen in the emergencydepartment with a 5-hourhistoryof severe headacheand depressed clinicalarticlehas been madepossibleby Publicationof this State-of-the-Art an educationalgrantfrom Roche Laboratories.121.00 From the Division of Infectious Diseases. R. Streptococcus pneumoniae.

and intrinsicamplificationof cultivationmake this approachattractive.121. probablybecausethe responsibleHaemophilusspecies did not possess groupb antigen. .~'MP.and time requiredfor this approachbeg for better diagnosticmethods. -? . r. sequences from bacteria present in the Gencally sequence of the amplified product was then determined using an automated DNA sequencer. gramstaining) may suggest an etiologic agent.. and aligned with other known fingerprint.. which is a testamentto the continuedutility of cultivation technology. .includingthe variableregionthatmay containa phylogeneti16S rRNA informative gene sequence.The CSF latex agglutinationtest for H.DNA . and can be used to identify an unknown agent or infer its evolutionary relationships with other previously characterized organisms.Cultivatablemicrobesmay also fail to grow after exposure to antibioticsor after poor sample handling.In othercases.. microscopyinitially misidentifiedthe organismas consistent with N. Limitations of Conventional Diagnostic Methods Cultivation strands.Had ceftriaxonebeen stoppedandpenicillinused alone. ' .42 on Mon.. ? .andthereforecontinuedto treatfor the H.111.wherethe rapidinitiation of antibioticsis paramount..: W I .this scenariois not unusual. it rarely provides definitive evidence of infectiondue to a particularspecies. Cd.the outcomemay have been distinctly less favorable.Certainpathogenssuch as Bartonellahenselae are fastidious.Clearly the microscopic morphologyof organismsmaybe misleading. .. PCR PCR 41.However. samples of CSF and water used as negative controls did not produce a PCR product.Similarly. On the other hand.FMF .Formicrobesthatareeasily tamedin the petridish. the standarddiagnostic test in infectiousdiseaseshas been in vitro cultivationusing artificial failure to consider these microbes may yield negativecultureresults.. there are cases of bacterialmeningitisin which culturesfail to yield the organism.cell culturecan be used to detectsome virusesand intracellularmicrobes.specificity. ?ank : 0.ability to determineantibioticsusceptibilityand otherclinicallyrelevantbehavioralcharacteristics.29 (September) Why did the conventionalmicrobiologicaldiagnosticmethods fail in this case?Althoughmicroscopy(e.' ? i Vf'.In the settingof meningitis.Even today. In contrast. ?.'. labor. For more than a century. the numberof organismsmay be too low for visual detectionby microscopy.but the cost. the sensitivity. influenzae responsiblefor this patient'smeningitis.especially when a lumbarpunctureis delayedbecausea headCT is ordered. ofmicrobial and newDNA DNA... has been notedfor otherbiotypeIII isolates.furtherdemonstratingthe limitations of culture-basedtechnology.other microbesare not so easily tamed in the laboratory.Fortunately. regions thExpolymerase generates : : ? .with conclusions influencedby the trainingand subjectiveinterpretation of the microscopist. t . v "z- c Microbial Microbial . . renderinga culture-independent approach valuablein some circumstances.476 Fredricksand Relman s _ Complex host genome . influenzae biotype III [4]. 02 Nov 2015 09:50:11 UTC All use subject to JSTOR Terms and Conditions . CID 1999.the clinicians continued broadspectrumantibioticswhile awaitingcultureconfirmation of the organism. Broadrange -c ----z microbialprimers -PCR .--.Certainmicrobesmay requirespecialculture media and conditions.The failureof CSF and blood culturesto providea diagnosisin this case can be ascribedto the use of antibioticsbefore obtainingthe culture samples. indicating lack of bacterial DNA template. In our assay.. CSF from the patient with meningitis produced a PCR product..andotherhumanpathogenssuch as Mycobacterium This content downloaded from 128. In this case.even without the institutionof antibiotics.g. influenzaegroup b antigenwas negative in this case.clinicalmicrobiologylaboratoriesdevote a majorityof theirefforttowardscultivationof clinical samples. Sequencingand phylogeneticanalysisshowedthatthe organism presentin the patient'sCSF matchedthatof H.

Percentageof cultivatedand uncultivatedbacteriafrom several selected bacterial divisions present in the ARB sequence database. the delay between obtaining a culture and the generation of a result necessitates empiric antibiotic therapy. Unfortunately. Serologicdetectionof antibodiesmay not be helpfulin the very acutestage of illness.and they are not always reproFor instance. sometimes lasting for months. Serology Serologic assays based on the detection of host-derived antibodiesor microbe-derivedantigens have several limitations. and low G+C gram positives that contain known human pathogens. For rapidly evolving diseases. a cultivation-independent method would offer the potential for rapid diagnosis.PCR and Diagnosis of Infectious Diseases CID 1999. Detectionof microbial antigensrequiresa relativelylarge microbialburden. and should not be surprised when sequencebased methods reveal novel microbes associated with human disease. which in turn could help reduce antibi- causes of outbreaksamongworkersin the clinical microbiology laboratory. Other pathogens. such as mycobacteria and fungi. even in those divisions such as the proteobacteria.From [5] (used with permission).these phenotypic tests have limited discriminatorypower in identifying microbes. There are several potential advantages to a speedy diagnosis.which limitsassaysensitivity.The immunocompromised host may never mount an appropriateantibodyresponse to infection. 13 divisions are composed entirely of uncultivated organisms [5].Unlikecultivation. It is estimated that <1% of the bacteria present on earth have been described to date using cultivation technology. will grow in the laboratory but may require prolonged periods of cultivation. In many cases.29 (September) otic selectionpressuresand the emergenceof antibioticresistance. we should be mindful of the limitations of cultivation technology.the ducibleandthey areusuallynonquantitative. these organisms tend to be previously uncharacterized. Second.whichdetectsbroad groupsof microbes.serologicassaysmustbe orderedindividually and target narrow groups of organisms. First. However.traditionally by using phenotypic tests such as the ability to metabolizesugarsor growthin the presenceof certainchemicals or antibiotics. becausethe host may not have time to mount an antibodyresponse.and.In is likely thatantibioticcosts woulddropandclinical outcomeswould improve. Efforts to grow viruses such as human papillomavirus and hepatitis C virus in cell culture have been equally frustrating. again limitingthe utility of serologicassays.the diagnosisis not made.Organisms isolatedon artificialmediamust still be identified. therefore.Thesehighly infectiousmicrobesmustbe handled in biological safety hoods or sent out to referencelaboratories where such facilities exist. Goebel. since samples can be treatedto kill microbeswhile preservingnucleic acid for analysis. Anotherproblemwith cultivationin the laboratoryis that certainorganismsconstitutea healththreatto laboratoryworkers who attemptto propagatethem.the resultsfor a given microbemay vary depending on the state of the organism. Therefore.111.If the cliniciandoes notthink of the correctserologictest to order. Organismssuch as Fran- Proteobacteria Actinobacteria Low G+C gram _ positives Cytophagales Acidobacterium Verrucomicrobia 477 E cisella tularensis and Coccidioides immitis are notorious Green non- sulfur bacteria OP 11 100 50 0 50 100 Percentage representationin ARBdatabase Figure 2.whichmaynotbe available.A relatively This content downloaded from 128. Microscopy/Histology The direct detection of microbes in tissues or fluids by microscopyhas limitedsensitivityand specificity.these microbes are sometimes isolated from patientswho are not suspectedof harboringsuch highly infectiouspathogens. For these slow-growing microbes. Finally. and Pace in their review. one might reduce the use of empiric antibiotics. 02 Nov 2015 09:50:11 UTC All use subject to JSTOR Terms and Conditions . A sequencebased diagnosticmethod could identify these hazardousmicrobes withoutrisk.These issues need to be addressed with carefulstudies. serologic assays requirespecific and reliableantiseraor antigens. cell wall compositionof an organismmay vary dependingon the selectionof growthmedia.Why do these microbes resist our cultivation attempts? Answers to this question remain obscure.the host may succumbto infectionbeforeantibodies can be produced. When environmental niches are sampled to determine the bacterial census.121.successfulcultivationof a microbedoes not necessarily imply successful identificationof the microbe. leprae and Treponemapallidum continue to defy our attempts at cultivation on artificial media. sequence-based techniques usually reveal large numbers of microbes that fail to grow using standard cultivation techniques. actinobacteria. a more appropriate question may be "why are we successful in getting so many human pathogens to grow in the laboratory?" It seems less surprising that there are culture-resistanthuman pathogens when one considers the situation in environmental microbiology. Figure 2 illustrates the relatively high percentage of uncultivated bacteria present in several selected cosmopolitan bacterial divisions.appropriateprecautionsare not used.Althoughusually successful. Of 36 bacterial divisions noted by Hugenholtz. with more specifically directedantimicrobial therapy.42 on Mon.

the readeris referredto othersourcesfor more in-depthinformation[3]. Theoretically.producinga copy of the DNA. These technologies include the transcription-based is capableof turninga few molecules of DNA into large quantities. No etiologic agent can be identifiedin >35% of cases of community-acquired pneumoniawhen using conventional diagnostic methods such as cultivationand serology.Oligonucleotideprimerscomplementary the coding and the noncodingstrandof the DNA templateare responsiblefor specificityin the reaction. Even if the organismsare presentin sufficientlyhigh concentrations.g.42 on Mon.478 Fredricksand Relman largenumberof microbesmust be presentbeforethey will be visible by microscopy(e. Eachcopy of DNA may CID 1999. In multiplexPCR. yet can detectlow levels of DNA via signal amplificationfrom a probe.but will not amplifyDNA polymerasegenes from otherviral families. Although PCR is the best known and most widely used nucleic acid will have difficultyvisualizingB.and to regionson primers. Using this technology.and the presence of large numbersof organisms.Multiple roundsof heatingandcooling of the reactionmixturein a thermalcycler produceroundsof melting of double-stranded DNA. There are several approachesfor using PCR to detect microbialDNA. 7].determiningwhich region of DNA becomes amplified.if one fails to considera particularmicrobe.Forinstance. a segment of DNA is copied in vitro by a thermostableDNA polymerase enzyme in the presence of buffer.eitherthe primersare not complementaryto the samegene segmentin othermicrobesoutside the groupor the distributionof the gene itself is limitedto that to a DNA targetthatis designsprimersthatarecomplementary specificfor the microbebeing assayed.Here.and Qf3replicasesystem.121. darkfieldfor treponemes)to whichmultiple specific PCR assays are run simultaneouslyin the same reaction tube to test for multiple different DNA templates.29 (September) then serve as anothertemplatefor furtheramplification. deoxyribonucleosidetriphosphates.. one could startwith a single copy of the targetmicrobial gene presentin the reaction..g. For instance.CertainDNA sequences(e. In this situation. annealingof primerto single-strandedtemplates. We will not discussthese othertechniquesfurther. In our case of meningitis.111. or is there a problem with our diagnostic Pneumoniais the most common infectious armamentarium? cause of death in the United States.with 4 million cases per year [6.Is thisjust an isolated case. 02 Nov 2015 09:50:11 UTC All use subject to JSTOR Terms and Conditions .Why is it useful to have largeamountsof microbialDNA availablefor study?The levels of microbialDNA presentin clinical samples are frequently too low for meaningfulmanipulationand measurement. Tropherymawhippelii. Betterdiagnosticmethodsare needed. DNA can function as a molecularfingerprintto help identify microbes.therearemethodssuch as branchedDNA technology thatdo not amplifythe DNA. henselae with a tissue gram stain.These multiple variablesconspire to make directexaminationa poor diagnostictest in many producea logarithmicincreasein DNA. PCR can producesufficientamountsof DNA so that microbescan be detectedand identified. magnesium.with the intentionof using the more variableregions of the amplifiedsequence for identificationor phylogenetic assessment[8]. stranddisplacement amplification.As the primersannealto their complementaryregions of DNA. In contrast. The limited specificity of microscopy reflects our meagerabilityto speciateorganismsbasedon morphology.Because each unique microbehas a unique complementof DNA (or RNA).To identifymicrobesby directexamination. DNA polymerasesattach to the primer-templatecomplexes and extend the DNA strands.and generatebillions of copies of DNA from that gene.For in- This content downloaded from could design primersthat are complementaryto regions of the 16S rRNA gene that are sharedby most membersof the bacterial domain.the correct choice of stain and illumination. but may see the organismwith a Warthin-Starrysilver stain or with immunohistochemical methods.with broad-rangePCR one attemptsto detect a broadergroup of organismsby designing primers that are complementaryto conservedregions of a particulargene that aresharedby a given taxonomicgroup. For instance.severalsets of primersareaddedto the reaction in orderto generateseveral differentPCR products. primers have been designed that will amplify a segment of the DNA polymerasegene from all membersof the designsprimersthat groundof taxon-restricted are complementaryto conserved regions of a gene from a particulargroupof organisms. In the ideal scenario.Between the extremesof specific and domain-widePCR is a large middle PCR.allowing one to distinguishmost microbes from one another.then one may miss that organismwhen using would expect to amplify any bacterial16S rDNA presentin the selecting uniqueregions of the Whipplebacillus' 16S rRNA gene. Anothervariationof PCRis multiplexing. As with serology. PCR PCR is an enzyme-drivenprocess for replicatingDNA in vitro.all threeconventionaldiagnostic methodsfailedto identifythe is dependenton the trainingand experience of the microscopist. In mustuse the appropriatestainsand conditions (e.ligase chain reaction. In the PCR.and extensionof DNA strands. and hence do not amplify nonspecifictargets such as human genes.the primerschosenin the PCRare specific for a particularmicrobial gene. The simplestapproachis specificPCR.thereare otheramplification technologies in use. 10. bacterial16S rDNA) are particularlyinformative. one can createprimersthat will amplify only the 16S rRNA gene fromthe Whipplebacillus.000 bacteriaper milliliter of fluid).

and thus must be performed with extreme care and interpretedwith even greater caution. with primerpairs directedtowardS.Themost commonly 479 usedmethodfor detectingPCRproductsin the researchlab has been gel whichthe primersannealto sites in the genome (humanor microbial)otherthanthe intendedtargetsequences.g. or high densityDNA microarrays. and the group B Streptococcus. some method of postamplification analysisis neededto determinewhich organism is representedin a positive reaction.annealingof a specific oligonucleotideprobe to a regionof the amplificationproductthatspansthe primingsites (e.the size of the PCR amplificationproducts can be estimated after the DNA is visualized ( can be amplifiedas in standardPCR. How does one detect an RNA target. such as filtersor glass slides. Southernhybridization. Probebasedconfirmationmethodsalso have the advantageof detecting small quantitiesof amplificationproducts. When DNA fragmentsof known size are run in the same gel (as size standards).one can quantitate and characterizefluorescentlylabeled mRNA or DNA by allowing it to hybridizeto a complementaryDNA sequence(s) This content downloaded from 128.Unfortunately.111. pneumoniae.probeELISA.Most commercialmethodsare likely to use an oligonucleotideprobein an ELISAformat. Nesting of PCR increasesassay sensitivityand can increase specificityas well.and thereforereduces assay time..thereare examplesin which a PCRproduct of the appropriatesize is generated.The TaqMansystem(Perkin-Elmer Applied Biosystems. such as rRNA or a segmentfromthe genomeof an RNA virus?A modificationof PCR called reversetranscriptasePCR (RT-PCR)can be used. The firstPCRreactionamplifiesa microbialDNA targetusing primerscomplementaryto the organismor groupof organisms being assayed.CID 1999. but at sites internalto the previouspriming sites. H.Additionalspecificityresultsfromthe set of specific primers employed in the second round. For instance. and generatea PCRproductthathappensto be similarin size to the intendedproduct.A given set of primersshouldgeneratea PCRproductof a particularsize.42 on primerfromthe first roundis used in the second roundreactionas well.In the second round. Oncethe DNA templateforms. In nested PCR. Because nonspecificamplificationproductsmay be generatedin a PCR. and hybridizationprotection assays).(Inhemi-nestedPCR. the nonspecificamplification productwill probablynot participateas templatein the second roundbecause it is unlikely to have regions of DNA complementaryto the second set of specific primers.121. with subsequentdilution of other DNA and inhibitors.In such an assay. an electrical gradientis applied througha buffer solution.slot blotting. influenzae..a sampleof the firstPCR is addedto freshreactionmixturefor a secondPCRusing a set of primersthatannealto regionsof the same is also the most time consumingand laborious.The contentsof the PCR are loaded into an agarose or acrylamidegel. N.and the second roundmay producea 200-bp productthat is a subset of the 400-bpproduct.the first roundreactionmay producea 400-bp product. The problemwith nested PCR is thatit is highly proneto contamination with amplificationproducts.and thus offer superiorsensitivity. one uses the productof a primaryPCR reactionas templatein a second PCR reaction. and the productsmigrate throughthe gel matrix.confirm. With so-calledDNA chip technology.but the productis not the intended amplificationproduct.This occurs because of mispriming. 02 Nov 2015 09:50:11 UTC All use subject to JSTOR Terms and Conditions .one couldhavea PCRassaydesignedto detectbacterial DNA in CSF thatuses five differentspecificPCR reactionsin one tube. then gel electrophoresiswill providean initialidea of which PCRreaction(s)took place.andquantifythe PCRproduct as it is being generatedin real time (figure3) [9].or restrictionenzyme cleavage of the amplificationproduct(using an enzymeknownto cut a specific sequencewithin the intendedproduct)with gel electrophoresisof the digest (restrictionfragmentlengthpolymorphism [RFLP] analysis). with smallerproductstraveling fartherin the gel becausethey experienceless resistance. Anothermethodused to characterizeandidentifyPCRproducts employsnucleotidesequencesattachedto solid supports.) Increased sensitivityis obtainedbecausethe targetis enrichedin the first round of this will providea rapidyet highly specific method of detectinga PCR product andconfirmingits identity. Listeria monocytogenes. meningitidis. CA) uses a fluorescently labeledprobeto mustdetermineif the intended PCRamplificationproductwas generated. Even if nonspecific amplificationoccurs in roundone.the identityof the productsshouldbe confirmed. OpeningPCR reactiontubes afterround one and transferringamplificationproductsto new tubes are conduciveto contamination. Oligonucleotideprimerscatalyzeconversionof a specific segment of RNA into DNA. Although sequencing of the PCR productis the most rigorousmethodof confirmingamplification productidentity.The usual efforts to inactivateamplificationproducts in orderto preventcontaminationdo not workwithnestedPCR because one needs to use amplifiabletemplatefrom the first roundin roundtwo. Foster RNA templateis the initialtarget. The amplificationproductsmigrate thoughthe gel accordingto size.g.and reverse transcriptasecreatesa complementaryDNA copy of the RNA. methodsincludesequencingof the amplification Confirmatory product.If the amplification productsdifferin size. Confirmationand Identificationof PCR Products Aftercompletinga PCR. single-strandedconformational polymorphismanalysis.29 (September) PCR and Diagnosis of Infectious Diseases stance. In RT-PCR. using ethidiumbromide staining and illuminationwith ultraviolet light). This system obviates the need for postamplificationdetectionand confirmation of product. and the appearanceof an amplificationproduct of the appropriatesize in a gel is consistentwith a positive PCR. This approach is sometimeshamperedby interferencebetweenprimerswithin the same reaction.

PCRoffersthe potential for remarkablespecificity.000.the assay could revolutionizethe diagnosisof neurosyphilis.Alternatively. leadingto confusionwhentryingto identify the microbe using traditionalphenotypictests. anchored to the surface. 02 Nov 2015 09:50:11 UTC All use subject to JSTOR Terms and Conditions .Squares= 1. a lysed organism) may be detectable by PCR.requiresdaysof laboratory time before definitiveidentificationis made using cultivation methodswithphenotypictesting. offeringa more reliablemethodof microbialidentification. If the microbe contains multiple gene copies per organism. althoughthe half-lifeof rRNAin CSF is not known.0. hours to days for initial propagation. as previously can design oligonucleotideprimerscomplementaryto conserved regionsof microbialDNA so as to detectmorediversegroups of organisms.If the DNA amplifiedwith this broad-range approachcontains interveningsegments of DNA that are uniqueto specificmicrobes. and subsequent fluorescence scanning [10.000. PCR can be completedin minutesto hours. Although a microbe may switch certain metabolictraitson andoff.CA). Anotheradvantageto PCR-basedmicrobialdetectionand identificationis speed.cultureis sometimestoo slow to This content downloaded from 128. Y fluorescenceintensity(volts).An exampleof a phylogeneticallyinformative gene that can be used for both organism-specificPCR as well as broad-rangePCR is the bacterial16S rRNAgene. DNA microarrays have been used to measure in a simultaneous. Using Southernhybridizationto detect the PCR product. genera. open bar = 100.1 0 0 0 5 10 0 15 20 25 30 35 40 No. For instance..g. These cases remind us that for easily cultivatableorganisms.X = 10. In additionto unrivaledsensitivity.Cepheid). coli (e. x axis = FAM (6-carboxyfluorescein) axis = PCR cycle number. triangles = 100. one avoids the need to grow the organism or maintainthe organismin a particularphysiologic state for metabolic analysis. Even greater assay sensitivity can be achieved by amplifying microbial CID 1999.In one example. one can amplify a single copy of a microbial DNA gene or gene fragment from a clinical sample and detect it.000. Tens or hundreds of thousands of sequences (probes) can be placed within a surface area of 1 cm2.2 0.nonspecificflu-like illness butbeforean organismcanbe grownin culture. One can to theseunique designoligonucleotideprimerscomplementary segments of DNA. Simplemethodsto confirmthe identityof the amplification productcan also be completedin minutes to hours.Higheramounts of startingtargetDNA requirefewer PCR cycles before productis detected(courtesyof LindaWestern.the investigatorswere able to detect 1/100 RNA equivalentsof an organismin RT-PCRassay was designedfor the detectionof T. and -20% of all expressed human genes. The speed and power of DNA chip technology combined with PCR should not be underestimated.3 circles = 10.121. the genetic fingerprintof the microbe remains fairly constant.6 0. In another application not yet realized one could immobilize thousands of probes (e.and hours to days for phenotypicdiagnostictesting.000.since otherdiagnostic methodsareinsensitive(CSFVDRL)or too cumbersome(rabbit inoculation).Specificityin PCRderivesfromthe fact that each distinct microbe has unique DNA.a turnaround of of microbestends to take Cultivation many types assays. Advantages of PCR Over Conventional Diagnostic Methods PCR-based assays for the detection of microbial DNA can be extremely sensitive. time of one day is not unrealisticfor Therefore. One bacteriummay containthousandsof 16S rRNA copies that are incorporatedinto ribosomesand can serve as targetsin an RT-PCRassay. Under the right conditions. so even a fraction of an E.Fredricksand Relman 480 0. Althoughstudies of the clinical utility of this RT-PCRassay have not been published. the bacterium Escherichia coli contains seven copies of the 16S rRNA gene per organism.We areall familiarwith cases in which a patientdies soon afteran acute. of cycles Figure 3.A fluorescentprobeis releasedfrom the P-actinPCRproductanddetectedduringPCR.4 0.Sunnyvale.then these microbescan be identified using techniquessuch as sequencingor restrictionenzyme analysis. It may be possible that this RT-PCRassay couldeven detecta singleorganismthathas lysed andliberated its rRNAinto the CSF.closed circles = 1.Even a rapidlygrowingorganism.42 on Mon. then one microbe may provide multiple targets for amplification. 16S rDNA) for different bacterial divisions.000. and species at a high density. so that only microbial DNA from the organismbeing sought is amplified.pallidum 16S rRNA [12]..111. coli in bloodculture.Whenusing sequenceinformationto identify a microbe.29 (September) targetsthat are presentin large numberswithin a single microbe. suchas E. 11].CA) and a SmartCyclerhigh speedthermocycler(Cepheid. using this chip to determine which bacterium or bacteria are present in the sample.FosterCity.suchas with meningococcalsepsis.closed bar = 0 gene copies. Real time detection of the human 3-actingene using TaqManPCRreagents(PEAppliedBiosystems. Amplification products from clinical samples subjected to broad-range 16S rDNA PCR and incorporating a fluorophore could then be analyzed by comparative hybridization. semiautomated fashion the mRNA expressed by all Saccharomyces cerevisiae genes.5 .

this method cannot monitorfor episodic contaminationfrom items such as gloves or pipettes.thenfalse positive reactions may ensue. running samples replicate and repeatingPCR assays shouldreveal problemswith episodic contamination.and if this test had a high negativepredictivevalue [13].once a PCR reaction is set up in the pre-PCRarea. and is used as template for new uracil-containingamplification products.29 (September) PCR and Diagnosis of Infectious Diseases be useful. Amplification productinactivationwill not controlthis problembecause the contaminatingDNA has not been previouslyamplified.All materialsand personnelare supposedto flow one way. If even a submicroscopic portionof a positive amplificationreactiongets into the environmentwheresubsequentPCRreactionsaremixed.42 on Mon.A single PCR can generatebillions of DNA copies. each of which is capable of acting as target for a future PCR reaction. pens.When a sample is positive. especially from the polymerase enzyme.. Unfortunately. when amplifyingthe bacterial16S rRNA gene with consensus primers. DNA contaminatessamples throughseveralmeans.Amplification with the signaturesequence primersproves that the sample containspreviouslyamplifiedtarget. If one looks carefully. Bases at the 3' endsof the primersbindto theircomplementary bases in the targetDNA. Thymidineremainsintactin the sampleDNA. The most importantmeansof contaminationis throughamplificationproductcarryover. it is possible to detect small fragmentsof bacterial16S rDNA in many "sterile"solutions.even for illnesses thatultimatelyprove to be viral in origin.111. or fungal 18S rDNA sequencesandhighly sensitivereactionconditions. which may introduceamplificationproductsinto a reaction withoutdirectlycontaminating the originalsampleor solutions.CID 1999.. tube racks. the reactionmixturesare treated with the enzyme uracilN-glycosylaseto renderany contaminating(i.A morerapidand sensitivediagnostictest for infection. suchas PCR. Amplificationproductcarryovercontaminationcan also be eliminatedor reducedby using some inactivationtechniques.A clinical samplemay have largequantitiesof target DNA present. In one method.the PCR reactionscontaindTTP and isopsoralen. It is very difficultto eliminateall contaminatingDNA.Thus.This problemcan be minimizedby changing gloves between handling of samples. providingspecificityin the PCR. 481 One approachfor monitoring amplificationproduct carryovercontaminationin samplesor solutionsemploysprimers thatcontainadditionalsignatureoligonucleotidesat the 5' end of each primerin orderto maketaggedamplificationproducts.deoxyuridinetriphosphate(dUTP)is used as a substrate in PCR instead of deoxythymidinetriphosphate (dTTP).such as waterand Taqpolymerase.namelythe incredible sensitivity of enzymatic amplification. PCR reagents.since native targetdoes not contain this signaturesequence and thereforewill not amplify.g.e.such as water for injection. Some laboratoriesalso have a separateroom for specimendigestionand processing.121. duplicating sampleanalysis. False positive reactionsmay also be causedby intersample contamination. in However.Some labs have separategowns or disposablegowns for each area. These methods do not work well for PCR productsof -100 bp or less in size.The negativecontrols arepositivebecausethe PCRreagents. The uracilN-glycosylaseis then inactivatedbeforeproceeding with PCR. DNA may contaminategloves or otheritems in the environment. one may detectmicrobialDNA uniformly.false positive reactionsare the Achilles' heel of PCRand stem fromits greateststrength. tubes.and doorhandles(almostany object) are capable of harboringor transmittingPCR amplification products. A more subtleproblemwith false positives may arise from the detectionof small quantitiesof microbialDNA in clinical samples. e.If PCR is more sensitivethan culturein some situa- This content downloaded from 128.leading to the inadvertentintroductionof DNA into otherPCR reactions whereit is amplified.pipettes. uracilcontaining)DNA incapableof amplification.even when thatDNA is presentin infinitesimallysmall amounts. it can be re-testedin a PCR assay using primersthat are complementaryto the signaturesequences.With primersthat are complementaryto highly conservedbacterial16S.and uninoculatedblood culturemedia [15].andavoidingaerosolgeneration. it is moved to the post-PCRarea where amplificationand productanalysis are performed.Thisknowledgeinevitablyleadsto the increaseduse of empiricantibiotics.Thisproblem canbe detectedby interspersingnegativecontrolreactionswith the test samplesto see if these controlsare positive.False positive results occur because PCR may amplify "contaminating" DNA that finds its way into a sample. Thyminedimersformbetweenthymidine bases.For highly sensitive. Justbecausea solutionis steriledoes not meanthatit is free of microbialDNA.The signaturesequences at the 5' end become incorporatedinto PCR productsbut do not annealto target. Before each PCR.containsmallamountsof bacterialDNA. broadrangePCRapplications.To reducethe risks of false positive reactionsfrom amplificationproductcarryover.hands. Anothercauseof falsepositivereactionsoccursin the setting of broad-rangePCR. Problems and Limitations of PCR False Positives Ironically.we may need to createa new standard for cleanlinessin our reagentsthat measuresmicrobialDNA insteadof colony-formingunits.When opening this sample.laboratoriesare usuallyphysically dividedinto pre-PCRand post-PCRrooms.and are treatedwith ultravioletlight afterthe amplificationstep [14]. 02 Nov 2015 09:50:11 UTC All use subject to JSTOR Terms and Conditions .In anothermethod. only that no microbes can be cultivated.mightreversethis trendtowardempiricismif the correctmicrobescould be identifiedearly in the infection. frompre-PCRto post-PCRrooms.and to the emergenceof antibioticresistance.Materialsare not allowed into the pre-PCRroom unless they are new or have been decontaminated. renderingthe DNA incapableof furtheramplification.

482 Fredricksand Relman tions.29 (September) of 1-10/IL.1 mL of the same blood concentratedto 10 juL may not detect the organism. where freezers are not available.3-globin PCR positive but microbial DNA PCR negative.thena negativeresultin a microbialDNA PCRassayhas no meaning. colon. 02 Nov 2015 09:50:11 UTC All use subject to JSTOR Terms and Conditions .111. DNA purificationmethods help to remove many of these inhibitors.. When using broad-rangePCR.blood culturemedia.whenblood samples from healthy subjects were examined by PCR. FalseNegatives PCR assays for the detection of microbesmay be falsely negative for several reasons. Amplificationof a humangene can help monitorfor PCR inhibitorsandcheckthe qualityof the DNA presentin a sample of human tissue subjectedto PCR for the detection of microbes. Broad-rangePCR using tissues that are normally in contact with bacteria.then the sample is more likely a "true"negativefor microbialDNA. such as fungi or bacterialspores. see sequencecapturebelow and [17]). Samples may also be falsely negative because the digestion step has failed to release the microbialDNA presentor because the DNA has been lost in the purificationstep.or in chaotropicsolutions is helpful. which are addedto a reactionvolume of 20-100 jiL.microbialDNA is boundto complementary captureoligonucleotides. Microbeswith thick cell walls. The PCR inhibitorsmust be diluted. PCR-basedassays may also be negativebecauseof analysis of an inadequatesamplevolume. agitation with glass beads or ceramicparticles. or inactivatedin order to amplify any microbialDNA present. then cultivation of 10 mL of blood has a good chance of detectingthe organism(assumingthat the organismis cultivatable).if PCR using primerscomplementaryto the humanj-globin gene fails to yield a PCR productwith a human tissue sample. Other methods use chemical or enzymaticmeans to break open microbes. CID 1999.this approachwill not work with any procedurein which human DNA is removed (e. In this technique. then we may need to definewhat constitutesa "significant"level of microbialDNA for a given clinicalsituation.If a sample is . tissues can be stored in ethanolor a chaotropicsolutionsuch as guanidineisothiocyanate. microbialDNA can be concentrated.The problemsample should be checked for the presenceof PCR inhibitorsand DNA. but there is presentlyno universalmethodoptimized for digestingall microbesin all tissues. In one study.For instance. On the other hand. at low temperatures. Similarly. or storedfrozenin orderto preservethe DNA for amplification. The concentrated.since amplifiableDNA may not be present. The sample may contain PCR inhibitorsthat interferewith amplification.or other enzymes active on microbialcell the range Sample Acquisition and Preparation One should be mindful that the sample collection process can have a significantimpacton the outcomeof the PCRassay. 17% were positive for pneumococcalDNA (samplesfrom children. CSF. such as using chaotropes.certainDNA This content downloaded from mustbe carefulin selectingthe tissue and anatomicalsite of acquisition.g.such as 10-20 mL of blood.not those from adults) [16]. Some digestion methods employ mechanical means such as freezing-thawing.PCR thatis performedon purifiedDNA froma digest of 0.such as with sequencecapturePCR [17]. samplevolumes areusuallyvery small with PCR.these must be individually identifiedto sort out which sequencecomes from a pathogen.which in turn are bound to a solid support. and Some PCR kits containan internalamplificationstandardthat allows one to monitorinhibitoryactivityand test performancein each sample. such as the mouth.UnboundDNA and inhibitorysubstancesfrom the samplearewashedaway.. then that sample is problematic.Largevolumesof fluidcanbe cultivated.what does it mean if S. As with othertests. Some methods or combinationswork well for selected microbes.Broad-rangebacterialPCRwill detect normalbacterialflora. Tissuesandfluidsshouldbe refrigeratedandrapidlyprocessed.and to determine what levels of microbial DNA are significant for a given organism. To increasethe sensitivityof PCR assays.althoughsome inhibitorspersistwhen standardpurificationprotocols are used.urine.121. makes interpretation of the resultschallengingbecausemultiple PCR productsmay be generated. such as with conserved16S rDNA primers. clinical correlations will need to be madeto see in whatsituationsPCRoffers an improvementin diagnosticcapabilities. this may not alwaysthe case. Fixationshould be avoided if samples are being collectedprospectivelyfor PCR analysis. and brain. If one bacteriumis presentin 1 mL of blood.vitreoushumor. with EDTA).dependingon the sensitivity of the assay and the numberof gene copies of targetpresentin the bacterium.and sputum. Obviously. If a tissue sample is 3-globinPCR negative. so storagein a magnesium-freeenvironment(e.42 on Mon. Fixationof tissues in formaldehydeand otherpathological fixativesolutionscan damageDNA. pneumoniaeDNA is detected in a blood sample?Althoughthe presence of pneumococcal DNA in blood would seem to suggest an invasive infection. Nucleasespresentin fluids can degradeDNA.sonication. proteases. removed.Samplesthat have been shown to contain PCR inhibitory substances include blood (heme).detergents.When a heterogeneous collection of PCR productsis generated. and skin. and thus should be applied to tissuesthatareusuallyfree of bacteriasuchas blood.or crushingwith mortarand pestle. For instance. may be difficultto breakopen and thereforemay requireadditionalmechanicalor enzymaticlysis steps in orderto liberatemicrobialDNA for amplification. In the field. There are many protocols for sample digestion and DNA purification.purifiedmicrobial DNA is then used for PCR.andwhich sequencecomes froma normalcolonizer. particularlywith prolonged fixationtimes.

DNA and RNA purificationsystemsare availSemiautomated able for use in the clinical microbiologylaboratory. such as HSV PCR for the diagnosisof encephalitis. T.The directcosts of PCR-baseddiagnostics will likely decreaseas this technologybecomes more refined.and thus thereis no basis for a cost comparison.avoidingthe need for the moreinvasive brainbiopsy. It is less costly (consideringthat the costs of surgery and anesthesiarun into thousandsof dollars).As the budgetsof clinical microbiology labs continue to shrink. leaving PCR as the most definitive diagnostictest. Antibiotic Susceptibility and Resistance PCR amplification of phylogenetically informative sequencessuch as the 16S rRNAgene fails to providedataabout the antibioticsusceptibilityof the organism. the lack of a definitive diagnosis may have hinderedfurtherdiagnosticevaluationof patientshavingother causes of encephalitis.Thepresenceof HSV DNA in the CSF of a patient with encephalitisis sufficientto make the diagnosisof HSE. Furthermore.For organismswith stableantibiograms.PCR is currentlymore cost effective thanpreviousdiagnosticapproaches(see below and [18]). mostlyrelatedto the need for general anesthesiaand craniotomy. The cost of PCR reagentsand equipmentis substantial.In the future. althoughhistology and electronmicroscopyof tissues may also suggest the diagnosis. Thistest is morerapidthancultureandis sensitiveandspecific.For organ- presence of a resistancegene in a microbe does not always imply expressionof that gene and phenotypicresistance. For certainmicrobes.111.administratorswill look to the more easily quantifiablebottomline of the laboratory.PCR assays have been designed for the detection of antibioticresistancegenes in microbes.42 on Mon.The requirementfor separatepre-PCRand postPCRareasmeansthatmolecularmicrobiologylaboratoriesuse a disproportionateshare of laboratoryspace.One advantageof cultivationis that a susceptibilityprofilecan usuallybe determined in orderto help guide treatment. However. The advantagesto PCR-basedtests. there is now little reason to perform brain biopsies on patients suspectedof havingthis diagnosis. whippelii.Miniaturization of PCR reactionsand the use of high throughputrobotics technologywill likely lead to substantialcost reductions. Given the advantagesof PCR for the diagnosisof HSE. less invasive. may offset higherdiagnosticcosts by reducinghospitalization and treatmentcosts.PCR is the only diagnosticapproach. WithHSV PCR.Althoughthereis littletoxicityassociated with acyclovir.this functionis less important. CSF is addedto a PCRreactionmixturecontaining primersthat are complementaryto regions in the DNA polymerasegene or the glycoproteinB gene of HSV-1 and HSV-2.cannotbe detectedusing methodssuch as cultureand serology.This is unfortunatebecauseone would like to have a universaldigestion and DNA purificationprotocolthat one could use in the clinical microbiologylab for all samples destined for PCR. such as speed and sensitivity.Increased use of PCR-basedmethods may also reduce costs throughcompetitionand reducedlabor costs. 02 Nov 2015 09:50:11 UTC All use subject to JSTOR Terms and Conditions .multiplexPCRor microarray technologymay help to identifyboth the microbeand determine antibioticsusceptibilityprofilesin one reaction.121. In one study that comparedPCRto biopsy with culture. our clinical microbiology laboratorycharges$125 for a herpessimplexvirus(HSV) PCR assay using CSF.Yet PCR can play a role in determiningantibioticsusceptibility. The HSV PCRassayprovidesan exampleof how difficultit can be to comparea new diagnostictest to a gold standard when the gold standardis not very golden. such as the methicillinresistancegene (mecA) in Staphylococcusaureusandmutationsin the rifampinresistance gene (rpoB) in Mycobacterium tuberculosis.PCR and Diagnosis of Infectious Diseases CID 1999.andwill demandthatPCR-basedassays be cost competitivewith other diagnosticmethods. The assay can detect about 20 gene copies of either herpesvirus. It is of interestthatthreeof 47 biopsynegativepatientswere found to be PCR positive (6%).The cost andmorbidity of this diagnostictest were high.For instance the Whipple bacillus.the lack of susceptibility data is problematic. 483 isms that are prone to developingresistance. Some PCR assays such as the Chlamydiaand Neisseria gonorrhoeaeassays have directcosts in the $8-10 rangeand are alreadycost competitivewith culturetechnology. and producesless morbidityand mortalitythandoes brainbiopsy. Although the Cost PCR is expensive. For example.its absencedoes imply a lack of resistancethroughthatparticular geneticmechanism.The significanteffort requiredto make the diagnosisby brainbiopsy led some clinicians to treat patientsempiricallywith acyclovir ratherthan pursuethe diagnosis.these indirectcost advantages are difficultto quantify. PCR in Practice: HSV PCR for the Diagnosis of Herpes Encephalitis An excellent example of an organism-specificPCR-based assay is HSV PCRfor the diagnosisof herpessimplexencephalitis (HSE). there are trainingcosts associatedwith teachingmicrobiologists to performthese moleculardiagnosticassays. Will PCRbasedassays ever competewith traditionaldiagnosticmethods such as cultivationand serology? For certain diagnostic tests.CSF is obtainedfromthe patientby lumbar punctureand assayed.53 of 54 biopsy-proven patientswith HSE were also positiveby PCR (98%) [18]. How does one interpretthe results when a novel test (PCR) picks up more cases than the gold standard(brainbiopsy)?Are the additionalcases false positives This content downloaded from 128.29 (September) purificationprotocolswork well with certainsamples. The previousgold standardfor the diagnosisof HSEwas brainbiopsywith cell culture.

as PCRresultsmay not be legally acceptableproof of infection.121. andheat shock protein genes. but there are no primersthat can detect all groupsof viruses. PCR is more sensitivethan histologyfor diagnosis. In addition. Roche).Spermicideand surgicallubricantcan act as PCR it is a PCR inhibitor. PCRhas This content downloaded from 128. Mycobacterium avium complex. Chlamydia can designprimerscomplementary in viral such as the DNA certain families. Branchburg.and areasof sequencediversityto distinguish between organisms. These assays use primersthatare complementaryto uniquestretches of DNA present in a given microbe's genome. to conserved However. However.hepatitisC virus. The assay does not detect plasmid-freevariants of C. whippelii 16S rRNA gene for the diagnosis of Whipple's disease (Mayo Clinic. direct sequencingof the amplification productcannotbe performedbecause there are mixed amplificationproducts.whichpreventsthe use of very sensitive PCR conditions. there are >9.For the small subunitrRNA gene. or 1 inclusion-formingunit (Amplicor.called a quantitation standard.the citratesynthasegene. tuberculosis. trachomatistargets a segment of a crypticplasmid. or false negativeswith the gold standard?Review of the laboratoryand clinical datafromthis study suggests that the positive PCR results in these biopsy negative patients are true positives that are due to errorsin sample acquisitionthat led to negative cultureresults (e.and thereby facilitatingthe acquisitionof patients'samples.Plasmashouldnot be collected in heparin.000 sequences from differentorganisms presentin databases. The assaynormallydetectsas few as 50 of gene copies per milliliterof plasmaafterultracentrifugation the sampleto concentratevirions. Primershave been describedfor broad-rangebacterialor fungal PCR assays. 02 Nov 2015 09:50:11 UTC All use subject to JSTOR Terms and Conditions . genes segments of polymerasegene herpesviruses. including HIV. HSV. when multiple organismsare present in a sample. broad-rangePCR for the direct detectionof microbial DNA in clinical specimensremainsan experimentalapproach [8].A modifiedtarget.biopsy may miss areasof involvement. cytomegalovirus..The PCR product binds to a probe-coatedmicrowell plate.If the threePCRpositivebutbiopsynegativecases areconsidered to be truepositives.42 on Mon. Phylogenetically informative gene targets shouldhave regionsof sequenceconservationfor the designof broad-rangeprimers. A PCR assay is available that detects a segment of the T.andurineshouldnot be frozen.29 (September) for monitoringresponse to antibiotictherapybecause histologic resolutionof intestinallesions may takemonthsto years.PCRis also moreusefulthanhistology CID 1999.but storedin a refrigerator. Broad-Range PCR Bacteria isolated by cultivationcan be identifiedusing a commerciallyavailable broad-range16S rDNA PCR assay with sequencingof the amplificationproduct. Assays are available for a variety of pathogens. The assay is so sensitivethaturinecan be used to screenpatientsfor infection. T. as well as the reliability of that locus in reflecting organismalevolutionaryhistory. placingthe brainin formalinbeforeculture).Roche. and Neisseria gonorrhoeae. avoidingthe need for more invasive examination.BecauseHSE can be a patchyprocess. Endocervicaland urethralswabs can also be tested. whippelii. The gene targetsthathave been successfullyused in broadrangeconsensusPCR assays for the identificationand phyloof microbesincludethe small subunit genetic characterization ribosomalRNA genes (16S rDNA in prokaryotes. single-strandedconformationalpolymorphismanalysis. such as for cases of rape or added to the reactionso that HIV copy numbercan be determined. whereas PCR-basedevidence of infection tends to correlate with disease resolutionor relapse[19].PCR is more sensitive than cell culture. The value of a gene target for broad-rangePCR dependsin parton the diversityand number of microbialsequencetypes from that targetpresentin databases. This approachis hobbledby the presenceof contaminating DNA in PCRreagents. RT-PCRis used to detectviralload in an assayof HIV RNA (MONITOR. HSV PCR for the diagnosis of HSE will likely become the new gold standard. M.Broad-rangeconsensus PCR with direct sequencingcan be successfullyappliedto clinical samplesthat containa single organism.producingfalse negativereactions.makingthis a useful gene for identifying a microbe or determiningits close evolutionaryneighbors. hepatitisB virus. or group-specificprobe hybridization.MN).111. and a colorimetric assay quantitatesthe target.enterovirus. Rochester.This genotypic identificationmethodwas shown to be superiorto other(phenotypic)methodsof microbialidentificationwhen appliedto a series of fastidiousaerobicgram-negativebacilli [20]. The assay can detect <100 copies/mLof samplefluid. Threeof these assays are discussedbelow.These multiplesequencetypes must be distinguishedby methods such as cloning. A PCR assay for C. 2].thenHSV PCRis the moresensitivetest in this study. The targetis a segment of the HIV-1 gag gene. The Future of PCR: Technical Advances Advancesin nucleicacidamplificationtechnologywill make futurediagnostictests fasterand less expensive[21].484 Fredricksand Relman with the new diagnostictest.g. Specific PCR Assays A numberof commerciallyavailablePCR assayshave been designed for the detectionof specific microbes [1.A cultureshould still be obtained when collecting legal evidence.and is able to detect 10 plasmidcopies.NJ).and 18S rDNA in eukaryotes).Thereis too muchsequencediversityin viral genes to design a broad-rangeviral consensus PCR assay. trachomatis.and can be used to monitor the effectiveness of antiretroviraltherapy.

but that PCR results do correlatewith clinical responseto antibiotics andalso canpredictrelapse. sensitive.the TaqMansystem [9] uses a fluorescentlylabeledprobeandthe exonucleaseactivity of Taqpolymeraseto monitorthe formationof productas it is being generated(figure3). and that the PCR resultscorrelatedwith culture results[23]. Real time detectionmethodscan be combined with miniaturized. This limitationcan be overcomeby samplepreparationmethodsthatconcentratemicrobialnucleic acids.PCR and Diagnosis of Infectious Diseases CID 1999. small-volume. As the cost of PCR reagents decline. 02 Nov 2015 09:50:11 UTC All use subject to JSTOR Terms and Conditions . Using this microdevice.the episodicallypositive PCRresultsfromthis studymay insteadbe due to false positive reactions.42 on Mon. replacingculture media with PCR reaction mix. or by continuousflow/multiplesamplingmethodsthat increase the volume of sample analyzed.20 cycles of PCR could be performedin as little as 90 seconds. such as with use of cell wall active antibiotics?Is the presence of bacterialRNA a betterindicatorof currentinfectionwith viable organismsthan bacterialDNA? Whatis a clinicallysignificantlevel of microbial DNA at a particularsite? How can PCR distinguishbetween colonization. and specific.Panels of PCR assays are likely to be developed. In a rabbit model of syphilis in which live or heat-killed T. and the incubatorwith the This content downloaded from 128.Ratherthanhavingto growan intactmicrobe. For instance.rapidPCRhas beenperformedin microcapillary tubes and micromachinedsilicon chip-based reactionchambers. and characterizea microbialDNA targetwithin minutes. Humanstudieslookingat the persistenceof microbialDNA by PCRare few. influenzae. Answers to these questionswill requireclinicians and researchersto comparecarefullyPCR-basedmicrobialdetection methodswith existing diagnosticmethods.121.and false negativereactionsdue to the presenceof PCR inhibitorsin the sample.Only with further experiencewill the benefits and limitationsof PCR become fully apparent. investigatorsusing a chinchillamodel of otitis media found a 485 strong correlationbetween PCR results and culture results when animalswere injectedwith viable H. but includea studyof pulmonarytuberculosis treatmentthat showedthat sputumsmearsand culturesfor M. targetingmicrobes involvedin specificsyndromessuchas pneumonia. and relapsing infection? Is microbial DNA routinely found in "sterile"sites sampledfrom normalindividuals? A few animalstudieshave attemptedto addressthese questions. Questions without Answers The applicationof PCR to the detection of microbes in clinical samples raises several is possible to design a microchip or microcapillaryPCR apparatusthat can amplify.since the investigatorsused a nested PCR assay with its high potential for contamination.or due to amplifiableDNA from nonviable organisms[26].but that the DNA from different microbes may have different eliminationkineticsat differentsites.It is not clearif the persistentPCR signal seen in some of these patientsis due to low levels of viable organisms.For instance.and the ability to analyzenumerousaliquotsfrom a clinical sample so that multipletests can be performedon a limitedvolume of tissue or fluid. high speed.111.latent infection. tuberculosisconvertto negative before PCR results. Conclusions PCR is a powerfultechniquethat is increasinglyappliedto the diagnosisof will be able to "grow"a segmentof its DNA. the clinical microbiologylaboratoryof the future will look increasinglylike a molecularbiology laboratory. whereas viable organismswere detected by PCR for months [25]. active infection.A study of HSV PCR for herpesencephalitis suggested that HSV DNA may persist in CSF for 2-3 weeks afterinitiationof effective treatment[18].rapid PCR technologies.The advantagesof small-volumePCRincludereducedcost fromthe use of fewer reagents.PCR-basedassays can be fast.meningitis. pallidum was injectedinto the skin and testes.When animals were injected with purifiedbacterialDNA or with killed bacteria.investigatorswere able to show by PCR that Borrelia burgdorferiDNA disappearedfromtissues immediatelyaftera 5-day antibiotictreatment course. This studysuggeststhatthe responseto antibiotics for Lyme disease might be monitoredby PCR. Similarly. Anotheradvancein PCR-baseddiagnosticsis real time detectionof PCRproducts. Witha mousemodelof Lymedisease. The disadvantageof smallvolume PCR for the detection of microbesis thatorganismspresentin low concentrationsin a samplemay be missed.pallidum in CSF from patients with neurosyphilissuggests that bacterialDNA may persistfor years afterantibiotictreatment [27]. detect. These studies suggest that bacterial DNA is clearedfromthe tissuesites of animalsafterbacterialdeath.29 (September) been miniaturizedso that nanoliterquantitiesof sample are processed within a few minutes. PCR-basedassays detect microbialnucleic acid in clinical samplesand do not require growthof the organism. Similarly. anddiarrhea.How long does microbialDNA persistin tissues afterdisease resolutionor antibiotic treatment?Does microbialDNA from some microbes persistin certaintissues or body fluids afterviable organisms aregone?Does microbialDNA increasein the blood afterlysis of organismsat a distanttissue site. However. continuousflow PCRhas been performedon a glass microchip in which the sample is moved rapidlybetween thermostated temperaturezones [22]. With currentlyavailabletechnology. A study of PCR for the detectionof T.andas the numberof PCR-basedapplicationsincrease. heat-killed treponemeswere no longer detectableby PCR after 15-30 days.the amplifiableDNA rapidlydisappeared[24]. but may also be associatedwith technical problemssuch as false positive reactionsdue to sample contamination.

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J Clin Microbiol1991. et al. sensitive diagnostic tests in infectious diseases that allow clinicians to make sound judgments in real time.J Bacteriol 1998. J Clin Microbiol 1998. J Infect Dis 1995. Eliminationof contaminating DNA withinpolymerasechainreactionreagents:implicationsfor a generalapproachto detectionof unculturedpathogens.280:1046-8. CastroC.42 on Mon. HazanI. or weeks later in convalescence. PCR and other molecular diagnostic methods hold the hope of making rapid diagnosis and directed therapy a reality. Detectionof Treponemapallidumby a sensitive reverse transcriptasePCR. 11.10:242-56.88:7276-80. 17. CID 1999.Washington. Loftus EJ. LukehartSA. Exploringthe new worldof the genomewith DNA microarrays. and Tom White. Chemicalamplification:continuous-flow PCR on a chip. HollandPM.9:602-8. McFaddenJ.or real conflict of interestwith regardto their contributionto the program. Education. MundyLM. 23. 10. Centurion-Lara A. J Clin Microbiol1997.171:857-63. 7. BartlettJG. 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