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1-1-1999
David A. Relman
Stanford University School of Medicine, relman@stanford.edu
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
DigitalCommons@University of Nebraska - Lincoln.
475
STATE-OF-THE-ARTCLINICALARTICLE
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
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-
References
based diagnostics.CurrOpin Biotechnol1998;9:602-8.
1. Persing DH, Smith TF, Tenover FC, White TJ. Diagnostic molecular 22. Kopp MU, Mello AJ, Manz A. Chemicalamplification:continuous-flow
microbiology:principlesand applications.Washington,DC: American PCR on a chip. Science 1998;280:1046-8.
Society for Microbiology,1993:641. 23. MalawistaSE, BartholdSW, Persing DH. Fate of Borreliaburgdorferi
2. Even M, GoossensH. Relevanceof nucleic acid amplificationtechniques DNA in tissues of infectedmice afterantibiotictreatment.J InfectDis
for diagnosis of respiratorytract infections in the clinical laboratory. 1994;170:1312-6.
Clin MicrobiolRev 1997;10:242-56. 24. Aul JJ, AndersonKW, WadowskyRM, et al. Comparativeevaluationof
3. Tang YW, ProcopGW, PersingDH. Moleculardiagnosticsof infectious cultureand PCR for the detectionand determinationof persistenceof
diseases. Clin Chem 1997;43:2021-38. bacterialstrainsand DNAs in the Chinchillalaniger model of otitis
4. QuentinR, RuimyR, RosenauA, MusserJM, ChristenR. Geneticiden- media.Ann Otol RhinolLaryngol1998;107:508-13.
tificationof crypticgenospeciesof Haemophiluscausingurogenitaland 25. WicherK, AbbruscatoF, WicherV, Collins DN, AugerI, HorowitzHW.
neonatal infections by PCR using specific primers targeting genes Identificationof persistentinfectionin experimentalsyphilis by PCR.
coding for 16S rRNA.J Clin Microbiol1996;34:1380-5. Infect Immun1998;66:2509-13.
5. HugenholtzP, Goebel BM, PaceNR. Impactof culture-independent stud- 26. KennedyN, GillespieSH, SaruniAO, et al. Polymerasechainreactionfor
ies on the emergingphylogeneticview of bacterialdiversity.J Bacteriol assessing treatmentresponsein patientswith pulmonarytuberculosis.
1998;180:4765-74. J Infect Dis 1994;170:713-6.
6. BartlettJG, MundyLM. Community-acquired pneumonia.N Engl J Med 27. NoordhoekGT, WoltersEC, de JongeME, van EmbdenJD. Detectionby
1995;333:1618-24. polymerasechainreactionof TreponemapallidumDNA in cerebrospi-
7. PerkinsBA, RelmanD. Explainingthe unexplainedin clinical infectious nal fluid from neurosyphilispatientsbefore and after antibiotictreat-
diseases: looking forward.EmergInfect Dis 1998;4:395-7. ment. J Clin Microbiol1991;29:1976-84.
8. RelmanDA. Detectionand identificationof previouslyunrecognizedmi-
crobialpathogens.EmergInfect Dis 1998;4:382-9.
9. HollandPM,AbramsonRD, WatsonR, GelfandDH. Detectionof specific
polymerasechain reactionproductby utilizingthe 5'->3' exonuclease
activity of ThermusaquaticusDNA polymerase.Proc Natl Acad Sci The "Conflict-of-Interest
USA 1991;88:7276-80. Policy" of the Office of Con-
10. RamsayG. DNA chips: state-of-theart.Nat Biotechnol1998;16:40-4. tinuing Medical Education, UCLA School of Medicine,
11. BrownPO, BotsteinD. Exploringthe new worldof the genomewith DNA requires that faculty participatingin a CME activity
microarrays.Nat Genet 1999;21:33-7. disclose to the audienceany relationshipwith a pharma-
12. Centurion-Lara A, CastroC, ShafferJM, VanVoorhisWC, MarraCM, ceutical or equipmentcompany which might pose a
LukehartSA. Detectionof Treponemapallidumby a sensitive reverse
potential,apparent,or real conflict of interestwith re-
transcriptasePCR. J Clin Microbiol1997;35:1348-52.
13. NewcombeJ, CartwrightK, PalmerWH, McFaddenJ. PCRof peripheral gardto their contributionto the program.Dr. David A.
blood for diagnosisof meningococcaldisease. J Clin Microbiol1996; Relmanserves as a consultantfor the Applied Biosys-
34:1637-40. tems Division of the Perkin-ElmerCorporationand re-
14. MeierA, PersingDH, FinkenM, BottgerEC. Eliminationof contaminat- ceives researchreagentsfrom PE-ABD.In addition,he
ing DNA withinpolymerasechainreactionreagents:implicationsfor a serves on the ScientificAdvisoryBoardof Cepheid.
generalapproachto detectionof unculturedpathogens.J ClinMicrobiol
1993;31:646-52.