Medical Microbiology

LaboratoryDiagnosis 409

zosterblisterscanbeviewedundertheelectronmicroscope(EM).Itmustbe

remembered,however,thattheEMislesssensitivethanvirusisolationin

culturesbyafactorof 105 .Viralantigenscanbedetectedinsecretionsusing

enzymeimmunoassay(EIA),passiveagglutination,orinsmearswithimmu-

nofluorescenceperformedwithknownantibodies,forinstancemonoclonal

antibodies.Analogously,theviralgenomecanbeidentifiedbymeansoffilter

hybridization,orinsmearsortissuesectionswithin-situhybridizationusing

DNAorRNAcomplementarytotheviralgenomeasaprobe.

Samplingandtransportofdiagnosticspecimens.Transportofpatientma-

terialforthesemethodsislesscriticalthanforvirusisolation.Coldboxtrans-

portisusuallynotrequiredsincethevirusneednotremaininfectious.

— Electronmicroscopy.FornegativecontrastEM,thespecimenistrans-

portedtothelaboratorywithoutanyadditives(dilution!).

— Antigenassay.Foranimmunofluorescenceantigenassay,slideprepara-

tionsmustbemadeandfixedimmediatelyaftersampling.Specialextrac-

tionmediumsareusedinEIA.Sincecommercialkitsareusedinmost

cases,procedureandreagentsshouldbecorrelatedwiththelaboratory.

— Genomehybridization.Hereaswell,thespecimenmaterialmustmeet

specificconditionsdependingonwhetherthevirusesaretobeidentified

bythein-situmethodorafterextraction.Thismustbearrangedbefore-

handwiththelaboratory.

Significanceofresults.Apositiveresultwithadirectvirusdetectionmethod

hasthesamelevelofsignificanceasvirusisolation.Anegativetestresult

meansverylittle,particularlywithEM,duetothelowlevelofsensitivity

ofthismethod.Theantigenassayandgenomehybridizationprocedures

aremoresensitivethanEM,buttheyareselectiveanddetectonlytheviruses

againstwhichtheantibodiesorthenucleicacidprobeused,aredirected.Itis

thereforeofdecisiveimportancetoprovidethelaboratorywithdetailedin-

formation.(Seep.208f.fordefinitionsofthetermssensitivityandspecificity.)

VirusDetectionFollowingBiochemicalAmplification

Polymerasechainreaction(PCR,Fig. 7. 9 ).Thismethodprovidesahighlysen-

sitivetestforviralgenomes.First,nucleicacidisextractedfromthepatient

materialtobeanalyzed.AnyRNAvirusgenomepresentinthematerialis

transcribedintoDNAbyreversetranscriptase(seep.385f.).ThisDNA,as

wellastheDNAoftheDNAviruses,isthenreplicatedinvitrowithaDNA

polymeraseasfollows:aftertheDNAdoublestrandhasbeenseparatedby

applyingheat,twosyntheticoligonucleotidesareaddedthatarecomplemen-

tarytothetwoendsoftheviralgenomesegmentbeinglookedforandcan

hybridizetoitaccordingly.TheadjacentDNA(towardeach 50 end)isthen

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Kayser, Medical Microbiology © 2005 Thieme