文献-Current status of fungal cell wall components in the immunodiagnostics of invasive fungal infections in humans galactomannan, m

EurJClinMicrobiolInfectDis(2007)26:755766DOI10.1007/s10096-007-0373-6REVIEWCurrentstatusoffungalcellwallcomponentsintheimmunodiagnosticsofinvasivefungalinfectionsinhumans:galactomannan,mannanand(13)-D-glucanantigensA.Kdzierska&P.Kochan&A.Pietrzyk&J.KdzierskaPublishedonline:2August2007#Springer-Verlag2007AbstractEarlydiagnosisoffungalinfectionsandtheimplementationofappropriatetreatmentrepresentmajorissuesforclinicians,nowadays.Histopathologicaldemon-strationofmicroorganismsintissuespecimensorgrowthoffungalagentsinculturemediaisstillconsideredthe“goldstandard”,butobtainingsuchspecimensmaybedifficult.Severalgroupshaveinvestigatedserologicalassaysforcellwallelementsuniquetofungalorganismsinserumorotherbodyfluidstoimprovediagnosticsinpatientswithhaematologicalmalignanciesorundergoinghaematopoieticstem-celltransplantation.Inthisreviewwehaveconcen-tratedonthecurrentlyavailableassaysallowingfordetectionofhighlyimmunogeniccomponentsoffungalcellwall:galactomannan,mannan,andalso(13)-D-glucan.Rapidserologicaltestsappeartobeusefulforscreeninghigh-riskhaematologicalpatients,sincetheyallowfortheearlydiagnosisofinvasivefungalinfections,includinginfectionswiththemostcommonpathogenssuchasAspergillusandA.KdzierskaDepartmentofClinicalMicrobiology,ChairofClinicalImmunologyandTransplantology,Polish-AmericanInstituteofPaediatrics,JagiellonianUniversityMedicalCollege,265WielickaStreet,30-663Cracow,PolandP.Kochan(*):A.PietrzykChairofMicrobiology,JagiellonianUniversityMedicalCollege,18CzystaStreet,31-121Cracow,Polande-mail:pkochancm-uj.krakow.plJ.KdzierskaDepartmentofDiagnostics,LaboratoryofMicrobiology,UniversityHospital,15bKopernikaStreet,31501Cracow,PolandCandida.Basedoncurrentliterature,factorsincreasingtheprobabilityofobtainingfalse-positiveorfalse-negativeresultsdetectedbyeachtestwerealsoanalysedandtabulated.IntroductionRecentepidemiologicstudieshaveshownincreasingnumbersofinvasivefungalinfections(IFIs)13.Mainpathogensresponsiblefortheseinfectionsareyeast-likefungifromCandidagenusandAspergillusspp.moulds.Candidaspeciesareanincreasinglyimportantcauseofinvasivecandidiasis(IC)inpatientshospitalisedininten-sivecareunits(ICUs).StudieshaveshownthattheincidenceofICinICUspeaksonthe8thto10thdayofstayintheICU.ProlongedhospitalisationintheICUisthesinglemostimportantriskfactorforICdevelopment.OtherimportantriskfactorsforICincludecentralvenouscatheterization,acuterenalfailure,diabetes,haemodialysis,andbroad-spectrumantibiotics4.RiskofICisalsohighamongpatientswhoareneutropenic.Inmostcases,episodesofICappearintheearlyphaseofneutropeniaafterintensivechemotherapyforacutemyeloidleukaemia(AML)orauto-/allogenousbone-marrowtransplant4.ICisassociatedwithhighmortalityratesreachingupto4050%2,5.AnothermajorclinicalprobleminthegroupofIFIsisinvasiveaspergillosis(IA).IApredominantlyaffectsse-verelyimmunosuppressedpatients,suchasthesewhohaveacuteleukaemiaorpatientsundergoinghaematopoieticstem-cellorsolid-organtransplantations,orpatientswithadvancedAIDSandchronicgranulomatousdisease1,3,6.AlthoughAspergillusfumigatusisthemostfrequentlyisolatedspecies,someauthorsreportanincreaseinA.756terreusandA.flavusinfections3,7,8.RiskofIAisdirectlyproportionaltothedurationofneutropeniaandreaches70%after34daysofgranulocytopenia9,10.Mostcasesofneutropenia-associatedIAoccurinpatientswhoaretreatedwithcytotoxicdrugsforhaematologicalmalignanciesandundergoinghaematopoieticstem-celltransplant6.IAmostcommonlyinvolvestherespiratorytract,reflectinginhalationastheprincipalportalofentry.Inpatientswithhaematologicalmalignancies,thissevereopportunisticfungalinfectionischaracterisedbyahighattributablemortalityraterangingfrom40to80%3,7.Earlyandprecisediagnosisofdeepmycosisisdifficultbecauseoflackofspecificsymptoms.IsolationofCandidaspp.fromasinglebloodsampleiscurrentlyenoughtostartasystemicantifungaltherapy.Innumerouscomparisonstudiesithasbeenshownthatbloodcultures,despitemonitoringonadailybasis,arepositiveinonly50%ofcases1113.Classicmycologicalexaminations,includinghistopathologicalassessment,microscopicmethodsandculturesofclinicalsamples,alsohavealimiteddiagnosticvalue1417.Serologicalmethodsusedinfungaldiag-nosticstodayaregenerallybasedondetectionofcell-wallcomponentsofselectedpathogenicfungalspecies,i.e.mannoproteins,functioningassolubleantigenicmarkers.Duringthecourseofasystemicfungalinfectiontheseantigensarepresentinthebloodstreamonlytransiently,andtheyareeliminatedbyformingimmunecomplexesaswellasviaendocytosisbyKupffercellsintheliver15.Theuseofbiologicalmarkersformonitoringofhigh-riskpatientsdependsstronglyontheprevalenceofinfection.Thereforeregularsamplingandmonitoringforthepresenceofbiologicalmarkersinthesystemicfluids(serumorplasma)isvitalforpatientswithhigh-riskfactorsfordevelopinginvasivefungaemia16,18,19.GalactomannanDetectionofcirculatingserumgalactomannan(GM)anti-genisofhighimportanceinthediagnosticsofIA.GMisahighlyimmunogenicantigen.Itspresencewasdemonstrat-edforA.fumigatusandotherAspergillusspp.(A.flavus,A.niger,A.vesicolor,A.terreus,A.nidulans,A.oryazeae).GMisacell-wallpolysaccharideofmolecularweightfrom35to100kDathatisreleasedbythefungusintoserumduringitsgrowthintissues.ItspresenceisusuallyconfirmedbyusingspecificEB-A2monoclonalratanti-body-bindinggalactofuranmoleculesofGM.Invitroantigenmaybereleasedbyfungiintheformof“pure”polysaccharideofmolecularweightof20kDa,althoughthenumberofantigenicepitopesmaychangeovertimeandaccordingtothespeciesofAspergillus16,20.EurJClinMicrobiolInfectDis(2007)26:755766PresenceofcirculatingGMintheIApatientsbloodisdeterminedbymultiplebiologicalandepidemiologicalfactors.TheirinfluenceisdifficulttoassessbecauseofthestillinsufficientknowledgeofthekineticsofGMreleaseinvivoanditspresenceinblood(penetrationandeliminationfromthebloodstream).Variablepatternsofcirculatingantigenmaybeobservedinindividualpatients20,21.Renalclearancefromthebloodalsodependsontherenalfunctionofthepatientandthesizeofthecirculatingantigen.Itwasalsoshownthattheamountofantigensreleasedinvivomaybemarkedlydecreasedbecauseoftheabsenceoforinsufficientamountofnutritiveingredientsattheinfectionsite.ThereleasedantigenmaybeusedasapotentialcarbonsourcebythedevelopingAspergillusnigerandPenicilliumfellutanummycelia.LackofglucoseandoxygenintheregionsofischaemicnecrosismayblockthereleaseofantigensbyAspergillusspp.AlowlevelofGMisalsopresentinpatientswithconfirmedpresenceofanti-Aspergillusantibodies20.GalactomannandetectionStandardtechniqueslikeimmunodiffusion,precipitationorcomplementfixationteststurnedouttobeinadequatelyspecificandnotsensitiveenoughtobeusedinthediagnosticsofIFIsinimmunodefficientpersons15,22.ImmunodiagnosticsofIAbasedonthedetectionofanti-Aspergillusantibodiesdidnotfinditsuseamongthesepatients,eventhoughitshowedhightitresofantibodiesintheearlyphaseofneutropenia4,15,17,20,23,24.Broadeningofthediagnosticnon-culturemethodsbytestingforpresenceofGMbecamepossiblethankstothetwocommerciallyavailableassaysonebeingimmunoenzy-matic(PA-EIAPlateliaAspergillus,Bio-Rad,Marnes-la-Coquette,France)andtheotherbasedonlatexagglutination(LAPastorexAspergillus,Bio-Rad).ResultsofstudiesshowedthatPA-EIAwasmoreusefulinIAdiagnostics,allowingdetectionofa10-foldlowerconcentrationofGMin1mLofbloodascomparedtothelatexagglutinationassay25.ThebenefitsanddrawbacksofthesetestsarepresentedinTable1.Single-stepELISAintroducedbyStynenetal.in1995allowedfordetectionofGMantigen26.ThistestwasfoundtobeasensitivemarkerforthepresenceofGMandwasusedforselectionofantifungaltherapyandmonitoringoftreatmentinpatientswithconfirmedandsuspectedIA27.PlateliaAspergillustesthasbeenavailableinEuropeformanyyears.IthasonlyrecentlybeenapprovedintheUnitedStatesbytheFDA19,28.ManyauthorspointtothehighsensitivityandspecificityofthistestinIAdiagnosticsinonco-haematologicalpatients16,29.Inpatientsundergoingallogeneicstem-celltransplantationEurJClinMicrobiolInfectDis(2007)26:755766Table1Diagnosticvalueofserologicaltestsindetectionofgalactomannanantigenaemia757AwidelydiscussedproblematthemomentisthechangemadebytheproduceroftheGMtestrelatingtotheopticalBenefitsImmunoenzymaticELISA(PlateliaAspergillus)GalactomannandetectioninearlyphaseofinfectionaEffectiveindetectionofIFIscausedbyAspergillusandPenicilliumGalactomannanantigendetection:0.51ng/mLofbloodHighspecificity(85%)Latexagglutinationtest(PastorexAspergillus)RapiddiagnosticsofsevereAspergillusspp.infectionsHighspecificity(90100%)DrawbacksNegativeresultdoesnotexcludeinvasiveaspergillosisNotusedindiagnosticsofzygomycosesandFusariuminfectionsNecessityofregularmonitoringofaseriesofserumsamplesbecauseofrapideliminationoftheantigenfromcirculationandforexclusionoffalse-positiveresultVariablesensitivityofgalactomannandetection(29100%)DetectionofinvasiveaspergillosisinadvancedphaseofinfectionLowdetectionthresholdofdensity(OD)indexborderline.Sofar,negativeresultshavebeenspecimenswithanindexbelow1andpositiveresultswereallsampleswithanindex1.5.Indexvaluesintherangeof11.5wereinterpretedasdubious(“greyzone”).Someauthorssuggestedloweringthecutoffvaluesbothforthenegativesamplesandforthepositiveones,explainingthattheyaretoohighandthatsuchvaluesleadtoloweringofthetestssensitivity30,33.However,ithasalsobeenreportedthatloweringofthecutoffvalueto1.0didnotimprovesensitivity29.Maertensetal.demonstratedthatatODindex0.5,theantigentestresultswerepositiveduringtheweekbeforeconventionaldiagnosticsin65%ofadulthaematologicalpatients.AdditionallyitwasshownthatloweringtheODindexfrom1.5to0.5increasedtheoverallsensitivityby21%(from76.3%to97.4%)butdecreasedtheoverallspecificityby7%(from97.5%to90.5%)28.Laietal.reportedtheirresults,indicatingthatsensitivityofthetestwas100%butspecificityonly65.7%whenODindex0.5wasused34.Atpresent,accordingtotheproducersrecommenda-tions,positivesamplesaretheoneswithODindex0.5PositiveresultforgalactomannanusingELISA(PlateliaAspergillus)galactomannanAg(15ng/mLofblood)awasobtainedatleast5daysearlierthanwithlatexagglutination(PastorexAspergillus)test25examinedforthepresenceofGMtwiceweekly,thesensitivityandspecificitywereapproximately90%.Intwo-thirdsofinfections,positiveGMresultwasobtainedbeforetheappearanceofclinicalsymptoms,radiologicalchangesorpositiveresultsobtainedwithotherdiagnosticmethods16.Ontheotherhand,therearealsoreportswhichunderlinethehighspecificityofthePlateliaAspergillustestinGMdetectionbutatthesametimeshowitslowsensitivity.OneoftheexamplesisthestudydonebyHerbrechtetal.performedonadultsandchildrenfromoncologicalandhaematologicalwards,wherethesensitiv-ityofthePlateliatestwasapproximately65,16and26%forpatientswithproven,probableandpossibleIArespectivelydefinedaccordingtocriteriaoftheEuropeanOrganizationforResearchandTreatmentofCancer/MycosisStudyGroup30.YetanotherexampleisthestudyofPinelandcolleaguesdoneonagroupof807patientsfromhaematologyandintensivecarewardswithprovenandpossibleIA.Resultsofthisstudypointtoalow,50%sensitivityofthetest31.TheusefulnessofEIAfordetectionofAspergillusGMwasalsoinitiallyconfirmedforbodyfluidsotherthanblood,includingbronchioalveo-larlavageandcerebrospinalfluid32.andthenegativeonesaresampleswithODindex0.5.ThisnewODindexiscurrentlyacceptedinEuropeandintheUnitedStates28.Theproduceralsorecommendsrepeatingthetestforallsampleswithanindex0.5usingthesamespecimenandtoalsocollectanewserumsamplefromthepatientinordertoeliminatefalse-positiveresultscomingfromcontaminationofthesamples.Twoconsecu-tivepositiveresultsforthepresenceofGMareconfirma-toryofIA20.NegativeresultsobtainedonafewretestingoccasionsarethebasisforexclusionofIAdiagnosis29.TwoconsecutivesampleswithanODindex0.5showthehighesttestaccuracy,withanimprovedpositivepredictivevalue28.Inclinicalsettings,GMdetectioninthebloodstreamisusuallyperformedinpatientswithhighriskfordevelopingIA20.Patientsbelongingtohighriskgroupsare(1)AMLpatients,(2)myelodysplasticsyndromepatients,(3)alloge-neichaematopoieticstem-cell-transplantrecipients,esp.fromnon-relateddonors,(4)patientstreatedforgraft-versus-hostdiseaseand(5)solid-organrecipients(lung,liver).Intheirstudy,Maertensetal.suggestedincludingadultpatientswithproliferativehaemocytopathieswithhighriskforIA,i.e.(1)patientsundergoingchemotherapybecauseofacutelymphocyticleukaemia,chronicmyelog-enousleukaemia,non-Hodgkinslymphoma,AMLwithneutropenia(500cells/l)expectedtolastatleast10days,advanceddysplasticsyndrome,or(2)patientsafteralloge-neicbone-marrowtransplantationorperipheralbloodstem-celltransplantation.Theysuggestnotenrollingpatients(1)758undergoingautologousbone-marrowtransplantation,(2)below16yearsofageor(3)withaplasticanaemia16.Exclusionofthe“heterogeneous”patientpopulationwithlowprobabilityfordevelopingIAeliminates,toagreatextent,thefalse-negativeresultsduringanalysisofclinicalsamplesatthesametimeraisingthesensitivityofthetestforthepresenceofGMinpatientswithconfirmedIA.FollowingtheEuropeanOrganizationforResearchandTreatmentofCancer/MycosisStudyGroupguidelines,presenceofGMinthebloodstreamisoneofthemajorcriteriaallowingconfirmationofIA,despitenegativeresultsofclassicmycologicaltests27.Itwasshownthatinasmanyas96%ofexaminedpatientsthedetectionofGMantigenaemiaprecededtheIAdiagnosis(confirmedhisto-/cytochemicallyormicrobiologically)by2110days.Furthermore,in90%ofpatients,thepresenceofGMwasdemonstratedonaverage10.5daysearlierthanpositiveresultsofcultures16.Similarly,inanotherprospectivestudy,antigenaemiawasdemonstratedbeforefirstradio-logical(8.4days)andclinical(6.9days)signsandsymptomsbegantoappearinpatients29.SensitivityofGMdetectiondependsonthesiteofinfection.Inlocalisedaspergillosis,forexampleinvasivepulmonaryaspergillosis(IPA),thesensitivityofdetectionofcirculatingantigenissignificantlylowerincomparisontothedisseminated,systemicaspergillosis10,35.DecreasinglevelsofGMmaycorrelatewithgoodprognosis;ontheotherhandabsenceofantigendoesnotexcludeinfectionwithnon-Aspergillus(Alternaria,Fusarium,Mucorales)fungi.Recently,itwasreportedthatthepresenceofGMcorrelateswithclinicallyconfirmedaspergillosisandwiththeresponsetoantifungaltherapy21.Inasmanyas76%ofconfirmedIAcases,presenceofGMcoincidedorpreceded(127days)theintroductionofantifungaltreat-ment.Intheremaining24%ofpatients,GMwasdetectedinthebloodstreamonaverageafter5.5daysofantifungaltreatment(158days).Itwasalsosuggestedthatmonitoringofserumsamplesafterinitiationoftheantifungaltherapygivesanopportunitytoestimatetheresponsetothedrugsorprovestheneedtostartsomealternativeformoftreatment16,19,23.InneutropenicpatientswithclinicalandradiologicalsuspicionofIA,someauthorsproposemonitoringtheantigeneverydayoratleasttwiceaweek,owingtoitstransientpresenceinthebloodstream16,17,24,36,37.Inpatientsundergoingtreatmentforaspergillosis,Sulahianetal.recommendmonitoringantigenaemiain2-weekintervals,butsofartheinteractionsbetweenGMkineticsinvivoandtherapyarepoorlyunderstood17.Itshouldbestressedherethattherearestillnosetdiagnosticalgorithmswhenitcomestothenumberandtothefrequencyofcollectionofserumsamples,aswellastothedurationofmonitoringoftheantigenaemiaincaseof