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University of Nebraska - Lincoln

DigitalCommons@University of Nebraska - Lincoln


U.S. Department of Veterans Affairs Staff
U.S. Department of Veterans Affairs
Publications

1-1-1999

Application of Polymerase Chain Reaction to the


Diagnosis of Infectious Diseases
David N. Fredricks
Stanford University School of Medicine, fredrick@cmgm.stanford.edu

David A. Relman
Stanford University School of Medicine, relman@stanford.edu

Follow this and additional works at: http://digitalcommons.unl.edu/veterans

Fredricks, David N. and Relman, David A., "Application of Polymerase Chain Reaction to the Diagnosis of Infectious Diseases"
(1999). U.S. Department of Veterans Affairs Staff Publications. Paper 4.
http://digitalcommons.unl.edu/veterans/4

This Article is brought to you for free and open access by the U.S. Department of Veterans Affairs at DigitalCommons@University of Nebraska -
Lincoln. It has been accepted for inclusion in U.S. Department of Veterans Affairs Staff Publications by an authorized administrator of
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475

STATE-OF-THE-ARTCLINICALARTICLE

Application of Polymerase Chain Reaction to the Diagnosis of Infectious Diseases


David N. Fredricks and David A. Relman From the Division of Infectious Diseases, Departmentof Medicine and
Departmentof Microbiology and Immunology,Stanford University,
Stanford,and the VeteransAffairs Palo Alto Health Care System,
Palo Alto, California

PCRandothersequence-basedmicrobialdetectionmethods, mentalstatus.On physicalexamination,she had a fever (tem-


once consideredto be only research tools, are being used peratureto 38.6?C),nuchalrigidity,andno evidenceof a rash.
increasinglyin the clinical microbiologylaboratory.As this Multiplegeneralizedseizureswere noted.Therapywith ceftri-
technologyexpandsinto the clinicalarena,clinicianswill need axoneandvancomycinwas institutedin the emergencydepart-
to learnits advantagesand limitationsso thatsoundjudgments ment for empirictreatmentof meningitis,anda CT scan of the
can be made. Astute clinicians know that results of blood brainwas obtainedpriorto a lumbarpuncture.Blood cultures
culturereports,whetherpositive or negative, must be inter- were obtainedafter antibioticshad been started,because of
preted using an understandingof the test employed and an difficultywith phlebotomy.The lumbarpuncturedemonstrated
assessmentof the clinical scenario.Similarly,infectious dis- cloudyCSF with an elevatedpressure,a WBC countof 17,500
eases practitionerswill need to expandtheirunderstandingof cells/mm3(93%neutrophils),a proteinlevel of 756 mg/dL,and
PCR-baseddiagnosticsso that these powerful tests are used a glucose level of 41 mg/dL(serumglucose level, 209 mg/dL).
appropriately. Examinationof a gram-stainedsmear of the CSF was re-
It is ourgoal to makePCR-baseddiagnosticsunderstandable portedto show many WBCs and many gram-negativediplo-
to clinicians.We will point out the limitationsof conventional cocci, suggestingthe diagnosisof meningococcalmeningitis.
diagnosticmethodsfor infectiousdiseases, discuss the advan- The patient ran a day care center, and the county health
tages and limitations of PCR-basedmethods, and mention departmentwas notifiedso thatantibioticprophylaxiscouldbe
some currentand futureapplicationsof this technology.We startedfor case contacts.The patient'svancomycinwas dis-
will not discussevery currentor pendingapplicationof PCRto continued,and penicillinwas addedto the ceftriaxonetherapy
diagnosticmicrobiology;the readeris referredto otherpubli- for optimal coverage of Neisseria meningitidis.
cations for additionaldetails [1-3]. We emphasizethe princi- On the second hospitalday, the gramstain of the CSF was
ples behindPCR-baseddiagnosis,andacknowledgea research- reviewed. The laboratoryconcluded that the gram-negative
orientedbias in our viewpoint. organismsseen on the smearhad a coccobacillarymorphology
Thelimitationsof existingdiagnosticmethodsandthepotential more consistent with Haemophilus influenzae than with
of PCR-baseddetectionand identificationmethodsare demon- N. meningitidis.The penicillinwas discontinuedand ceftriax-
stratedby a case fromStanfordUniversityMedicalCenter. one was continued.The patient'sconditionimprovedand she
was dischargedon the fifth day to completea course of out-
A Case of Meningitis patient intravenousceftriaxone.All blood and CSF cultures
were negative.Latexagglutinationtests performedon the CSF
A 53-year-oldwoman was seen in the emergencydepart- were negative for Haemophilus group b, Streptococcus pneu-
ment with a 5-hourhistoryof severe headacheand depressed moniae, group B Streptococcus, and N. meningitidis antigens.
The etiology of this patient'smeningitiswas not confirmed
using conventionalmethodsof cultivationand antigendetec-
Publicationof this State-of-the-Art
clinicalarticlehas been madepossibleby tion. To identify the bacteriumresponsiblefor this patient's
an educationalgrantfrom Roche Laboratories. illness, we used a sequence-basedapproachin our laboratory.
Received30 March1999;revised23 May 1999.
Financialsupport:D. F. is supportedby NationalInstitutesof Health(NIH)
A sampleof the patient'sCSF was centrifugedto concentrate
Physician-ScientistAward K11-AI01360.D. R. is supportedby grantsfrom bacteria,andthe pellet was digestedto liberatebacterialDNA.
(RO1-AI39587),the Departmentof Veterans Affairs (EmergingInfections This DNA was used as templatein a broad-rangePCR assay
Program),the Departmentof Defense (N65236-99-1-5428),and the Environ-
mentalProtectionAgency (InteragencyGrant). designed to copy enzymatically(amplify) a portion of the
Reprintsor correspondence: Dr. David N. Fredricks,VeteransAffairs Palo bacterial16S rRNA gene in vitro using a thermostableDNA
Alto HealthCare System 154T, 3801 MirandaAvenue, Palo Alto, California polymerase. Oligonucleotide primers complementary to
94304 (fredrick@cmgm.stanford.edu).
broadlyconservedregionsof the 16S rRNAgene were used to
ClinicalInfectiousDiseases 1999;29:475-88
? 1999by the InfectiousDiseasesSocietyof America.All rightsreserved. amplify segments of the gene that also containedvariable,
1058-4838/99/2903-0001 $03.00 phylogeneticallyinformativeDNA sequence(s)(figure1). The
This article is a U.S. government work, and is not subject to copyright in the United States.
476 Fredricksand Relman CID 1999;29 (September)

Why did the conventionalmicrobiologicaldiagnosticmeth-


s _ Complex host ods fail in this case?Althoughmicroscopy(e.g., gramstaining)
genome may suggest an etiologic agent, it rarely provides definitive
evidence of infectiondue to a particularspecies. In this case,
. Cd.
microscopyinitially misidentifiedthe organismas consistent
with N. meningitidis.Fortunately,the clinicians continued
v c
-c
----z "z- Microbial
Microbial broadspectrumantibioticswhile awaitingcultureconfirmation
.DNA of the organism,andthereforecontinuedto treatfor the H. in-
fluenzae responsiblefor this patient'smeningitis.Had ceftri-
.--. Broadrange axonebeen stoppedandpenicillinused alone,the outcomemay
have been distinctly less favorable.Clearly the microscopic
PCR microbialprimers
PCR 41.. morphologyof organismsmaybe misleading,with conclusions
influencedby the trainingand subjectiveinterpretation of the
microscopist.In othercases, the numberof organismsmay be
too low for visual detectionby microscopy.The failureof CSF
and blood culturesto providea diagnosisin this case can be
-PCR ascribedto the use of antibioticsbefore obtainingthe culture
samples.In the settingof meningitis,wherethe rapidinitiation
. ' ,~'MP,,FMF . .

of antibioticsis paramount,this scenariois not unusual,espe-


cially when a lumbarpunctureis delayedbecausea headCT is
ordered.However, there are cases of bacterialmeningitisin
regions ofmicrobial
? ......'.' ? i
DNA,:
and
:
thExpolymerase
Vf'. t .: W
generates
I
newDNA
.. which culturesfail to yield the organism,even without the
.
-?
. institutionof antibiotics,furtherdemonstratingthe limitations
of culture-basedtechnology.The CSF latex agglutinationtest
for H. influenzaegroup b antigenwas negative in this case,
?.
probablybecausethe responsibleHaemophilusspecies did not
possess groupb antigen,as has been notedfor otherbiotypeIII
? , r..
?ank : 0, . ..
isolates.

Limitations of Conventional Diagnostic Methods


Cultivation
For more than a century, the standarddiagnostic test in
infectiousdiseaseshas been in vitro cultivationusing artificial
media.Even today,clinicalmicrobiologylaboratoriesdevote a
strands,includingthe variableregionthatmay containa phylogeneti- majorityof theirefforttowardscultivationof clinical samples,
cally informative
16S rRNA sequences from bacteria present in the Gen-
gene sequence. which is a testamentto the continuedutility of cultivation
technology.Formicrobesthatareeasily tamedin the petridish,
sequence of the amplified product was then determined using the sensitivity,specificity,ability to determineantibioticsus-
an automated DNA sequencer, and aligned with other known ceptibilityand otherclinicallyrelevantbehavioralcharacteris-
tics, and intrinsicamplificationof cultivationmake this ap-
proachattractive.Certainmicrobesmay requirespecialculture
media and conditions,so failure to consider these microbes
may yield negativecultureresults.Cultivatablemicrobesmay
fingerprint, and can be used to identify an unknown agent or also fail to grow after exposure to antibioticsor after poor
infer its evolutionary relationships with other previously char- sample handling, renderinga culture-independent approach
acterized organisms. In our assay, samples of CSF and water valuablein some circumstances.Similarly,cell culturecan be
used as negative controls did not produce a PCR product, used to detectsome virusesand intracellularmicrobes,but the
indicating lack of bacterial DNA template. On the other hand, cost, labor,and time requiredfor this approachbeg for better
CSF from the patient with meningitis produced a PCR product. diagnosticmethods.
Sequencingand phylogeneticanalysisshowedthatthe organ- In contrast,other microbesare not so easily tamed in the
ism presentin the patient'sCSF matchedthatof H. influenzae laboratory.Certainpathogenssuch as Bartonellahenselae are
biotype III [4]. fastidious,andotherhumanpathogenssuch as Mycobacterium
CID 1999;29 (September) PCR and Diagnosis of Infectious Diseases 477

Proteobacteria otic selectionpressuresand the emergenceof antibioticresis-


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

largenumberof microbesmust be presentbeforethey will be then serve as anothertemplatefor furtheramplification.Mul-


visible by microscopy(e.g., 10,000 bacteriaper milliliter of tiple roundsof heatingandcooling of the reactionmixturein a
fluid). Even if the organismsare presentin sufficientlyhigh thermalcycler produceroundsof melting of double-stranded
concentrations,one mustuse the appropriatestainsand condi- DNA, annealingof primerto single-strandedtemplates,and
tions (e.g., darkfieldfor treponemes)to makethemvisible. As extensionof DNA strands,to producea logarithmicincreasein
with serology,if one fails to considera particularmicrobe,then DNA. In the ideal scenario,the primerschosenin the PCRare
one may miss that organismwhen using standardtechniques. specific for a particularmicrobial gene, and hence do not
For instance,one will have difficultyvisualizingB. henselae amplify nonspecifictargets such as human genes. Theoreti-
with a tissue gram stain, but may see the organismwith a cally, one could startwith a single copy of the targetmicrobial
Warthin-Starrysilver stain or with immunohistochemical gene presentin the reaction,and generatebillions of copies of
methods. The limited specificity of microscopy reflects our DNA from that gene.
meagerabilityto speciateorganismsbasedon morphology.To Although PCR is the best known and most widely used
identifymicrobesby directexamination,one is dependenton nucleic acid amplificationtechnology,thereare otheramplifi-
the trainingand experience of the microscopist,the correct cation technologies in use. These technologies include the
choice of stain and illumination,and the presence of large
transcription-based amplificationsystem, stranddisplacement
numbersof organisms.These multiple variablesconspire to
amplification,ligase chain reaction,and Qf3replicasesystem.
make directexaminationa poor diagnostictest in many situa- In addition,therearemethodssuch as branchedDNA technol-
tions.
ogy thatdo not amplifythe DNA, yet can detectlow levels of
In our case of meningitis,all threeconventionaldiagnostic DNA via signal amplificationfrom a probe. We will not
methodsfailedto identifythe responsibleorganism.Is thisjust discussthese othertechniquesfurther;the readeris referredto
an isolated case, or is there a problem with our diagnostic othersourcesfor more in-depthinformation[3].
armamentarium? Pneumoniais the most common infectious There are several approachesfor using PCR to detect mi-
cause of death in the United States,with 4 million cases per crobialDNA. The simplestapproachis specificPCR.Here,one
year [6, 7]. No etiologic agent can be identifiedin >35% of to a DNA targetthatis
designsprimersthatarecomplementary
cases of community-acquired pneumoniawhen using conven-
tional diagnostic methods such as cultivationand serology. specificfor the microbebeing assayed.Forinstance,by select-
Betterdiagnosticmethodsare needed. ing uniqueregions of the Whipplebacillus' 16S rRNA gene,
one can createprimersthat will amplify only the 16S rRNA
gene fromthe Whipplebacillus, Tropherymawhippelii.
In contrast,with broad-rangePCR one attemptsto detect a
PCR broadergroup of organismsby designing primers that are
PCR is an enzyme-drivenprocess for replicatingDNA in complementaryto conservedregions of a particulargene that
vitro. Using this technology,one is capableof turninga few aresharedby a given taxonomicgroup.Forinstance,one could
molecules of DNA into large quantities.Why is it useful to design primersthat are complementaryto regions of the 16S
have largeamountsof microbialDNA availablefor study?The rRNA gene that are sharedby most membersof the bacterial
levels of microbialDNA presentin clinical samples are fre- domain,with the intentionof using the more variableregions
of the amplifiedsequence for identificationor phylogenetic
quently too low for meaningfulmanipulationand measure-
ment. PCR can producesufficientamountsof DNA so that assessment[8]. In this situation,one would expect to amplify
microbescan be detectedand identified.Because each unique any bacterial16S rDNA presentin the reaction.Between the
microbehas a unique complementof DNA (or RNA), DNA extremesof specific and domain-widePCR is a large middle
can function as a molecularfingerprintto help identify mi- groundof taxon-restricted PCR.Here,one designsprimersthat
crobes.CertainDNA sequences(e.g., bacterial16S rDNA) are are complementaryto conserved regions of a gene from a
particularlyinformative,allowing one to distinguishmost mi- particulargroupof organisms;eitherthe primersare not com-
crobes from one another. plementaryto the samegene segmentin othermicrobesoutside
In the PCR, a segment of DNA is copied in vitro by a the groupor the distributionof the gene itself is limitedto that
thermostableDNA polymerase enzyme in the presence of group. For instance, primers have been designed that will
buffer, magnesium, deoxyribonucleosidetriphosphates,and amplify a segment of the DNA polymerasegene from all
primers.Oligonucleotideprimerscomplementary to regionson membersof the herpesvirusfamily,but will not amplifyDNA
the coding and the noncodingstrandof the DNA templateare polymerasegenes from otherviral families.
responsiblefor specificityin the reaction,determiningwhich Anothervariationof PCRis multiplexing,in whichmultiple
region of DNA becomes amplified.As the primersannealto specific PCR assays are run simultaneouslyin the same reac-
their complementaryregions of DNA, DNA polymerasesat- tion tube to test for multiple different DNA templates. In
tach to the primer-templatecomplexes and extend the DNA multiplexPCR,severalsets of primersareaddedto the reaction
strands,producinga copy of the DNA. Eachcopy of DNA may in orderto generateseveral differentPCR products.For in-
CID 1999;29 (September) PCR and Diagnosis of Infectious Diseases 479

stance,one couldhavea PCRassaydesignedto detectbacterial usedmethodfor detectingPCRproductsin the researchlab has


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

0.6 targetsthat are presentin large numberswithin a single mi-


crobe. One bacteriummay containthousandsof 16S rRNA
0.5
copies that are incorporatedinto ribosomesand can serve as
targetsin an RT-PCRassay.In one example,an RT-PCRassay
.0.4
was designedfor the detectionof T.pallidum 16S rRNA [12].
0.3 Using Southernhybridizationto detect the PCR product,the
investigatorswere able to detect 1/100 RNA equivalentsof an
0.2 organismin CSF. It may be possible that this RT-PCRassay
couldeven detecta singleorganismthathas lysed andliberated
0.1
0 0 0 its rRNAinto the CSF, althoughthe half-lifeof rRNAin CSF
is not known. Althoughstudies of the clinical utility of this
0 5 10 15 20 25 30 35 40 RT-PCRassay have not been published,the assay could rev-
No. of cycles olutionizethe diagnosisof neurosyphilis,since otherdiagnostic
methodsareinsensitive(CSFVDRL)or too cumbersome(rab-
Figure 3. Real time detection of the human 3-actingene using bit inoculation).
TaqManPCRreagents(PEAppliedBiosystems,FosterCity,CA) and In additionto unrivaledsensitivity,PCRoffersthe potential
a SmartCyclerhigh speedthermocycler(Cepheid,Sunnyvale,CA). Y
axis = FAM (6-carboxyfluorescein) fluorescenceintensity(volts), x for remarkablespecificity.Specificityin PCRderivesfromthe
axis = PCR cycle number.A fluorescentprobeis releasedfrom the fact that each distinct microbe has unique DNA. One can
P-actinPCRproductanddetectedduringPCR.Squares= 1,000,000; designoligonucleotideprimerscomplementary to theseunique
triangles = 100,000;open circles = 10,000;closed circles = 1,000; segments of DNA, so that only microbial DNA from the
open bar = 100;X = 10;closed bar = 0 gene copies.Higheramounts
of startingtargetDNA requirefewer PCR cycles before productis organismbeing sought is amplified.Alternatively,one can
detected(courtesyof LindaWestern,Cepheid). design oligonucleotideprimerscomplementaryto conserved
regionsof microbialDNA so as to detectmorediversegroups
of organisms.If the DNA amplifiedwith this broad-range
anchored to the surface, and subsequent fluorescence scanning approachcontains interveningsegments of DNA that are
[10, 11]. Tens or hundreds of thousands of sequences (probes) uniqueto specificmicrobes,then these microbescan be iden-
can be placed within a surface area of 1 cm2. DNA microarrays tified using techniquessuch as sequencingor restrictionen-
have been used to measure in a simultaneous, semiautomated zyme analysis.An exampleof a phylogeneticallyinformative
fashion the mRNA expressed by all Saccharomyces cerevisiae gene that can be used for both organism-specificPCR as well
genes, and -20% of all expressed human genes. In another as broad-rangePCR is the bacterial16S rRNAgene, as previ-
application not yet realized one could immobilize thousands of ously mentioned.Whenusing sequenceinformationto identify
probes (e.g., 16S rDNA) for different bacterial divisions, gen- a microbe, one avoids the need to grow the organism or
era, and species at a high density. Amplification products from maintainthe organismin a particularphysiologic state for
clinical samples subjected to broad-range 16S rDNA PCR and metabolic analysis. Although a microbe may switch certain
incorporating a fluorophore could then be analyzed by com- metabolictraitson andoff, leadingto confusionwhentryingto
parative hybridization, using this chip to determine which identify the microbe using traditionalphenotypictests, the
bacterium or bacteria are present in the sample. The speed and genetic fingerprintof the microbe remains fairly constant,
power of DNA chip technology combined with PCR should not offeringa more reliablemethodof microbialidentification.
be underestimated. Anotheradvantageto PCR-basedmicrobialdetectionand
identificationis speed. PCR can be completedin minutesto
hours. Simplemethodsto confirmthe identityof the amplifi-
Advantages of PCR Over Conventional Diagnostic cation productcan also be completedin minutes to hours.
Methods
Therefore,a turnaround time of one day is not unrealisticfor
PCR-based assays for the detection of microbial DNA can be of
many types assays. Cultivationof microbestends to take
extremely sensitive. Under the right conditions, one can am- hours to days for initial propagation,and hours to days for
plify a single copy of a microbial DNA gene or gene fragment phenotypicdiagnostictesting.Even a rapidlygrowingorgan-
from a clinical sample and detect it. If the microbe contains ism, suchas E. coli in bloodculture,requiresdaysof laboratory
multiple gene copies per organism, then one microbe may time before definitiveidentificationis made using cultivation
provide multiple targets for amplification. For instance, the methodswithphenotypictesting.We areall familiarwith cases
bacterium Escherichia coli contains seven copies of the 16S in which a patientdies soon afteran acute,nonspecificflu-like
rRNA gene per organism, so even a fraction of an E. coli (e.g., illness butbeforean organismcanbe grownin culture,suchas
a lysed organism) may be detectable by PCR. Even greater with meningococcalsepsis. These cases remind us that for
assay sensitivity can be achieved by amplifying microbial easily cultivatableorganisms,cultureis sometimestoo slow to
CID 1999;29 (September) PCR and Diagnosis of Infectious Diseases 481

be useful.Thisknowledgeinevitablyleadsto the increaseduse One approachfor monitoring amplificationproduct car-


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

tions, then we may need to definewhat constitutesa "signifi- of 1-10/IL, which are addedto a reactionvolume of 20-100
cant"level of microbialDNA for a given clinicalsituation.For jiL. If one bacteriumis presentin 1 mL of blood, then culti-
instance,what does it mean if S. pneumoniaeDNA is detected vation of 10 mL of blood has a good chance of detectingthe
in a blood sample?Althoughthe presence of pneumococcal organism(assumingthat the organismis cultivatable).PCR
DNA in blood would seem to suggest an invasive infection, thatis performedon purifiedDNA froma digest of 0.1 mL of
this may not alwaysthe case. In one study,whenblood samples the same blood concentratedto 10 juL may not detect the
from healthy subjects were examined by PCR, 17% were organism,dependingon the sensitivity of the assay and the
positive for pneumococcalDNA (samplesfrom children,not numberof gene copies of targetpresentin the bacterium.
those from adults) [16]. As with othertests, clinical correla- To increasethe sensitivityof PCR assays, microbialDNA
tions will need to be madeto see in whatsituationsPCRoffers can be concentrated,such as with sequencecapturePCR [17].
an improvementin diagnosticcapabilities,and to determine In this technique,microbialDNA is boundto complementary
what levels of microbial DNA are significant for a given captureoligonucleotides,which in turn are bound to a solid
organism,site, and situation. support.UnboundDNA and inhibitorysubstancesfrom the
samplearewashedaway. The concentrated,purifiedmicrobial
DNA is then used for PCR.
FalseNegatives

PCR assays for the detection of microbesmay be falsely


Sample Acquisition and Preparation
negative for several reasons. The sample may contain PCR
inhibitorsthat interferewith amplification.Samplesthat have One should be mindful that the sample collection process
been shown to contain PCR inhibitory substances include can have a significantimpacton the outcomeof the PCRassay.
blood (heme),blood culturemedia,urine,vitreoushumor,and Tissuesandfluidsshouldbe refrigeratedandrapidlyprocessed,
sputum. The PCR inhibitorsmust be diluted, removed, or or storedfrozenin orderto preservethe DNA for amplification.
inactivatedin order to amplify any microbialDNA present. Nucleasespresentin fluids can degradeDNA, so storagein a
DNA purificationmethods help to remove many of these magnesium-freeenvironment(e.g., with EDTA), at low tem-
inhibitors,althoughsome inhibitorspersistwhen standardpu- peratures,or in chaotropicsolutions is helpful. In the field,
rificationprotocols are used. Samples may also be falsely where freezers are not available, tissues can be stored in
negative because the digestion step has failed to release the ethanolor a chaotropicsolutionsuch as guanidineisothiocya-
microbialDNA presentor because the DNA has been lost in nate. Fixationof tissues in formaldehydeand otherpathologi-
the purificationstep. Microbeswith thick cell walls, such as cal fixativesolutionscan damageDNA, particularlywith pro-
fungi or bacterialspores, may be difficultto breakopen and longed fixationtimes. Fixationshould be avoided if samples
thereforemay requireadditionalmechanicalor enzymaticlysis are being collectedprospectivelyfor PCR analysis.
steps in orderto liberatemicrobialDNA for amplification. When using broad-rangePCR, such as with conserved16S
Amplificationof a humangene can help monitorfor PCR rDNA primers,one mustbe carefulin selectingthe tissue and
inhibitorsandcheckthe qualityof the DNA presentin a sample anatomicalsite of acquisition.Broad-rangebacterialPCRwill
of human tissue subjectedto PCR for the detection of mi- detect normalbacterialflora, and thus should be applied to
crobes. For instance,if PCR using primerscomplementaryto tissuesthatareusuallyfree of bacteriasuchas blood, CSF, and
the humanj-globin gene fails to yield a PCR productwith a brain. Broad-rangePCR using tissues that are normally in
human tissue sample, then that sample is problematic.The contact with bacteria, such as the mouth, colon, and skin,
problemsample should be checked for the presenceof PCR makes interpretation of the resultschallengingbecausemulti-
inhibitorsand DNA. If a tissue sample is 3-globinPCR neg- ple PCR productsmay be generated.When a heterogeneous
ative,thena negativeresultin a microbialDNA PCRassayhas collection of PCR productsis generated,these must be indi-
no meaning,since amplifiableDNA may not be present.If a vidually identifiedto sort out which sequencecomes from a
sample is ,3-globin PCR positive but microbial DNA PCR pathogen,andwhich sequencecomes froma normalcolonizer.
negative,then the sample is more likely a "true"negativefor There are many protocols for sample digestion and DNA
microbialDNA. Obviously,this approachwill not work with purification. Some digestion methods employ mechanical
any procedurein which human DNA is removed (e.g., see means such as freezing-thawing,sonication, agitation with
sequencecapturebelow and [17]). Some PCR kits containan glass beads or ceramicparticles,or crushingwith mortarand
internalamplificationstandardthat allows one to monitorin- pestle. Other methods use chemical or enzymaticmeans to
hibitoryactivityand test performancein each sample. break open microbes, such as using chaotropes,detergents,
PCR-basedassays may also be negativebecauseof analysis proteases,or other enzymes active on microbialcell walls.
of an inadequatesamplevolume.Largevolumesof fluidcanbe Some methods or combinationswork well for selected mi-
cultivated,such as 10-20 mL of blood. On the other hand, crobes, but there is presentlyno universalmethodoptimized
samplevolumes areusuallyvery small with PCR,in the range for digestingall microbesin all tissues. Similarly,certainDNA
CID 1999;29 (September) PCR and Diagnosis of Infectious Diseases 483

purificationprotocolswork well with certainsamples.This is isms that are prone to developingresistance,the lack of sus-
unfortunatebecauseone would like to have a universaldiges- ceptibility data is problematic.Yet PCR can play a role in
tion and DNA purificationprotocolthat one could use in the determiningantibioticsusceptibility.PCR assays have been
clinical microbiologylab for all samples destined for PCR. designed for the detection of antibioticresistancegenes in
Semiautomated DNA and RNA purificationsystemsare avail- microbes, such as the methicillinresistancegene (mecA) in
able for use in the clinical microbiologylaboratory. Staphylococcusaureusandmutationsin the rifampinresistance
gene (rpoB) in Mycobacterium tuberculosis. Although the
presence of a resistancegene in a microbe does not always
Cost imply expressionof that gene and phenotypicresistance,its
absencedoes imply a lack of resistancethroughthatparticular
PCR is expensive. For example, our clinical microbiology
geneticmechanism.In the future,multiplexPCRor microarray
laboratorycharges$125 for a herpessimplexvirus(HSV) PCR
technologymay help to identifyboth the microbeand deter-
assay using CSF. The cost of PCR reagentsand equipmentis mine antibioticsusceptibilityprofilesin one reaction.
substantial.The requirementfor separatepre-PCRand post-
PCRareasmeansthatmolecularmicrobiologylaboratoriesuse
a disproportionateshare of laboratoryspace. Furthermore,
there are trainingcosts associatedwith teachingmicrobiolo- PCR in Practice: HSV PCR for the Diagnosis of Herpes
gists to performthese moleculardiagnosticassays. Will PCR- Encephalitis
basedassays ever competewith traditionaldiagnosticmethods
An excellent example of an organism-specificPCR-based
such as cultivationand serology?
For certain diagnostic tests, such as HSV PCR for the assay is HSV PCRfor the diagnosisof herpessimplexenceph-
alitis (HSE). The previousgold standardfor the diagnosisof
diagnosisof encephalitis,PCR is currentlymore cost effective HSEwas brainbiopsywith cell culture.The cost andmorbidity
thanpreviousdiagnosticapproaches(see below and [18]). The
of this diagnostictest were high, mostlyrelatedto the need for
advantagesto PCR-basedtests, such as speed and sensitivity,
general anesthesiaand craniotomy.The significanteffort re-
may offset higherdiagnosticcosts by reducinghospitalization
and treatmentcosts. However,these indirectcost advantages quiredto make the diagnosisby brainbiopsy led some clini-
cians to treat patientsempiricallywith acyclovir ratherthan
are difficultto quantify.As the budgetsof clinical microbiol-
pursuethe diagnosis.Althoughthereis littletoxicityassociated
ogy labs continue to shrink,administratorswill look to the with acyclovir, the lack of a definitive diagnosis may have
more easily quantifiablebottomline of the laboratory,andwill
hinderedfurtherdiagnosticevaluationof patientshavingother
demandthatPCR-basedassays be cost competitivewith other
causes of encephalitis.
diagnosticmethods.The directcosts of PCR-baseddiagnostics WithHSV PCR,CSF is obtainedfromthe patientby lumbar
will likely decreaseas this technologybecomes more refined.
Some PCR assays such as the Chlamydiaand Neisseria gon- punctureand assayed,avoidingthe need for the moreinvasive
brainbiopsy. CSF is addedto a PCRreactionmixturecontain-
orrhoeaeassays have directcosts in the $8-10 rangeand are
ing primersthat are complementaryto regions in the DNA
alreadycost competitivewith culturetechnology.Miniaturiza-
tion of PCR reactionsand the use of high throughputrobotics polymerasegene or the glycoproteinB gene of HSV-1 and
HSV-2. The assay can detect about 20 gene copies of either
technologywill likely lead to substantialcost reductions.In-
creased use of PCR-basedmethods may also reduce costs herpesvirus.Thepresenceof HSV DNA in the CSF of a patient
with encephalitisis sufficientto make the diagnosisof HSE.
throughcompetitionand reducedlabor costs. For certainmi- Thistest is morerapidthancultureandis sensitiveandspecific.
crobes,PCR is the only diagnosticapproach,and thus thereis It is less costly (consideringthat the costs of surgery and
no basis for a cost comparison.For instance the Whipple
anesthesiarun into thousandsof dollars), less invasive, and
bacillus, T. whippelii,cannotbe detectedusing methodssuch
as cultureand serology, leaving PCR as the most definitive producesless morbidityand mortalitythandoes brainbiopsy.
Given the advantagesof PCR for the diagnosisof HSE, there
diagnostictest, althoughhistologyand electronmicroscopyof is now little reason to perform brain biopsies on patients
tissues may also suggest the diagnosis.
suspectedof havingthis diagnosis.
The HSV PCRassayprovidesan exampleof how difficultit
Antibiotic Susceptibility and Resistance can be to comparea new diagnostictest to a gold standard
when the gold standardis not very golden. In one study that
PCR amplification of phylogenetically informative se- comparedPCRto biopsy with culture,53 of 54 biopsy-proven
quencessuch as the 16S rRNAgene fails to providedataabout patientswith HSE were also positiveby PCR (98%) [18]. It is
the antibioticsusceptibilityof the organism.One advantageof of interestthatthreeof 47 biopsynegativepatientswere found
cultivationis that a susceptibilityprofilecan usuallybe deter- to be PCR positive (6%). How does one interpretthe results
mined in orderto help guide treatment.For organismswith when a novel test (PCR) picks up more cases than the gold
stableantibiograms,this functionis less important.For organ- standard(brainbiopsy)?Are the additionalcases false positives
484 Fredricksand Relman CID 1999;29 (September)

with the new diagnostictest, or false negativeswith the gold for monitoringresponse to antibiotictherapybecause histo-
standard?Review of the laboratoryand clinical datafromthis logic resolutionof intestinallesions may takemonthsto years,
study suggests that the positive PCR results in these biopsy whereas PCR-basedevidence of infection tends to correlate
negative patients are true positives that are due to errorsin with disease resolutionor relapse[19].
sample acquisitionthat led to negative cultureresults (e.g.,
placingthe brainin formalinbeforeculture).BecauseHSE can
be a patchyprocess,biopsy may miss areasof involvement.If Broad-Range PCR
the threePCRpositivebutbiopsynegativecases areconsidered Bacteria isolated by cultivationcan be identifiedusing a
to be truepositives,thenHSV PCRis the moresensitivetest in commerciallyavailable broad-range16S rDNA PCR assay
this study. HSV PCR for the diagnosis of HSE will likely with sequencingof the amplificationproduct.This genotypic
become the new gold standard. identificationmethodwas shown to be superiorto other(phe-
notypic)methodsof microbialidentificationwhen appliedto a
series of fastidiousaerobicgram-negativebacilli [20]. How-
Specific PCR Assays ever, broad-rangePCR for the direct detectionof microbial
DNA in clinical specimensremainsan experimentalapproach
A numberof commerciallyavailablePCR assayshave been [8]. This approachis hobbledby the presenceof contaminating
designed for the detectionof specific microbes [1, 2]. These DNA in PCRreagents,whichpreventsthe use of very sensitive
assays use primersthatare complementaryto uniquestretches PCR conditions. In addition, when multiple organismsare
of DNA present in a given microbe's genome. Assays are present in a sample, direct sequencingof the amplification
available for a variety of pathogens, including HIV, HSV, productcannotbe performedbecause there are mixed ampli-
hepatitisB virus,hepatitisC virus, cytomegalovirus,enterovi- ficationproducts.These multiplesequencetypes must be dis-
rus, Chlamydia trachomatis, M. tuberculosis, Mycobacterium tinguishedby methods such as cloning, single-strandedcon-
avium complex, T. whippelii, and Neisseria gonorrhoeae. formationalpolymorphismanalysis, or group-specificprobe
Threeof these assays are discussedbelow. hybridization.Broad-rangeconsensus PCR with direct se-
RT-PCRis used to detectviralload in an assayof HIV RNA quencingcan be successfullyappliedto clinical samplesthat
(MONITOR,Roche, Branchburg,NJ). The targetis a segment containa single organism.
of the HIV-1 gag gene. The assaynormallydetectsas few as 50 The gene targetsthathave been successfullyused in broad-
gene copies per milliliterof plasmaafterultracentrifugationof rangeconsensusPCR assays for the identificationand phylo-
the sampleto concentratevirions,and can be used to monitor genetic characterization of microbesincludethe small subunit
the effectiveness of antiretroviraltherapy.The PCR product ribosomalRNA genes (16S rDNA in prokaryotes,and 18S
binds to a probe-coatedmicrowell plate, and a colorimetric rDNA in eukaryotes),the citratesynthasegene, andheat shock
assay quantitatesthe target.A modifiedtarget,called a quan- protein genes. Phylogenetically informative gene targets
titation standard,is added to the reactionso that HIV copy shouldhave regionsof sequenceconservationfor the designof
numbercan be determined.Plasmashouldnot be collected in broad-rangeprimers,and areasof sequencediversityto distin-
heparin,as it is a PCR inhibitor. guish between organisms. The value of a gene target for
A PCR assay for C. trachomatistargets a segment of a broad-rangePCR dependsin parton the diversityand number
crypticplasmid,and is able to detect 10 plasmidcopies, or 1 of microbialsequencetypes from that targetpresentin data-
inclusion-formingunit (Amplicor, Roche). The assay is so bases, as well as the reliability of that locus in reflecting
sensitivethaturinecan be used to screenpatientsfor infection, organismalevolutionaryhistory.For the small subunitrRNA
avoidingthe need for more invasive examination,and thereby gene, there are >9,000 sequences from differentorganisms
facilitatingthe acquisitionof patients'samples.PCR is more presentin databases,makingthis a useful gene for identifying
sensitive than cell culture.A cultureshould still be obtained a microbe or determiningits close evolutionaryneighbors.
when collecting legal evidence, such as for cases of rape or Primershave been describedfor broad-rangebacterialor fun-
childabuse,as PCRresultsmay not be legally acceptableproof gal PCR assays, but there are no primersthat can detect all
of infection. Endocervicaland urethralswabs can also be groupsof viruses.Thereis too muchsequencediversityin viral
tested. The assay does not detect plasmid-freevariants of genes to design a broad-rangeviral consensus PCR assay.
C. trachomatis,andurineshouldnot be frozen,but storedin a However,one can designprimerscomplementary to conserved
refrigerator.Spermicideand surgicallubricantcan act as PCR genes segments in certain viral families, such as the DNA
inhibitors,producingfalse negativereactions. of
polymerasegene herpesviruses.
A PCR assay is available that detects a segment of the
T. whippelii 16S rRNA gene for the diagnosis of Whipple's
The Future of PCR: Technical Advances
disease (Mayo Clinic, Rochester,MN). The assay can detect
<100 copies/mLof samplefluid. PCR is more sensitivethan Advancesin nucleicacidamplificationtechnologywill make
histologyfor diagnosis.PCRis also moreusefulthanhistology futurediagnostictests fasterand less expensive[21]. PCRhas
CID 1999;29 (September) PCR and Diagnosis of Infectious Diseases 485

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

thermal cycler. PCR-based diagnostic tests offer clinicians a 15. FredricksDN, RelmanDA. Improvedamplificationof microbialDNA-
fromblood culturesby removalof the PCR inhibitorsodiumpolyanet-
powerful new weapon to add to their quivers. We are long
holesulfonate.J Clin Microbiol1998;36:2810-6.
overdue for rapid, sensitive diagnostic tests in infectious dis-
16. DaganR, ShrikerO, HazanI, et al. Prospectivestudyto determineclinical
eases that allow clinicians to make sound judgments in real relevanceof detectionof pneumococcalDNA in sera of childrenby
time. The diagnosis of Rocky Mountain spotted fever should be PCR. J Clin Microbiol1998;36:669-73.
confirmed within hours of presentation, not days later at au- 17. MangiapanG, VokurkaM, Schouls L, et al. Sequence capture-PCR
topsy, or weeks later in convalescence. PCR and other molec- improvesdetectionof mycobacterialDNA in clinicalspecimens.J Clin
Microbiol1996;34:1209-15.
ular diagnostic methods hold the hope of making rapid diag-
18. LakemanFD, Whitley RJ. Diagnosis of herpes simplex encephalitis:
nosis and directed therapy a reality.
applicationof polymerasechain reactionto cerebrospinalfluid from
brain-biopsiedpatientsand correlationwith disease. NationalInstitute
of AllergyandInfectiousDiseasesCollaborativeAntiviralStudyGroup.
Acknowledgments J Infect Dis 1995;171:857-63.
19. RamzanNN, Loftus EJ, BurgartLJ, et al. Diagnosis and monitoringof
The authorsthank Ellen Jo Baron and Richard Williams from
the Clinical Microbiology Laboratoryat StanfordUniversity Hos- Whippledisease by polymerasechain reaction.Ann InternMed 1997;
126:520-7.
pital for providing the CSF sample used in our meningitis case; 20. Tang YW, Ellis NM, HopkinsMK, SmithDH, Dodge DE, PersingDH.
and Tom White, John Sninsky, Ann Warford,and Ellen Jo Baron Comparisonof phenotypicand genotypictechniquesfor identification
for reading the manuscriptand providing input. of unusualaerobicpathogenicgram-negativebacilli. J Clin Microbiol
1998;36:3674-9.
21. WhitcombeD, Newton CR, Little S. Advances in approachesto DNA-
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