2005The future of animal models of invasive aspergillosis

MedicalMycologySupplement12005,43,S115ÁS119

ThefutureofanimalmodelsofinvasiveaspergillosisT.F.PATTERSON

DepartmentofMedicine,UniversityofTexasHealthScienceCenteratSanAntonio,SanAntonio,Texas,USAThediagnosisofinvasiveaspergillosisremainsverydifficult,coupledwithlimited

treatmentoptions.Animalmodelshavebeenutilizedtoevaluateboththediagnosis

andtreatmentofinfectionandtoassessthepathogenicityandvirulenceofthe

organism.However,animalmodelshavenotbeenstandardizedandhavebeenused

inonlyalimitedfashionforgenomicevaluationinthisdisease.Extensiveefforts

areunderway

toexpandsignificantlytheAspergillusgenomicinformation.Thus,

thestandardizationofanimalmodelsofinvasiveaspergillosisiscriticaltocreatea

unifiedplatformtoenhanceevaluationofnewergenomicinformationandallow

assessmentofpathogenicityandvirulencefactors.Proposedmodels,supportedby

arecentlyawardedNationalInstitutesofHealth/NationalInstituteofAllergyand

InfectiousDiseasescontract,willbedevelopedincloseinteractionwiththe

extendedAspergilluscommunity(includingacademiaandindustry)toanswerkey

questionsinthisdisease.Thegoalofthisworkistoprovidetheframeworkto

evaluategenomictargetsinanimalmodelsinordertoimprovethediagnosisand

treatmentofinvasiveaspergillosisthatwillultimatelyresultinimprovedoutcomes

ofpatientswiththisfrequentlyfatalinfection.

KeywordsAspergillus,invasiveaspergillosis,animalmodels

Introduction

Animalmodelshavebeenutilizedtoevaluateboththe

diagnosisandtreatmentofinvasiveaspergillosis,and

havealsobeenusedtoassesspathogenicandvirulence

featuresoftheorganism[1Á3].However,thelackof

standardizationofanimalmodelsystemsinthisdisease

haslimitedtheirvalueinassessingbothvirulenceand

pathogenicityandhamperedeffortstoidentifynew

diagnosticandtherapeutictargets.Extensiveeffortsare

underwaytosignificantlyexpandtheAspergillusgeno-

micinformation;asaresult,thereisgreatpotentialfor

identifyingnewdiagnosticandtherapeuticstrategies

criticallyneededforthisoftenlethalinfection.One

approachtoimprovingAspergillusanimalmodelshas

beentheawardofacontractbytheNationalInstitutes

ofHealth/NationalInstituteofAllergyandInfectious

Disease(NIH/NIAID)toestablishandstandardizeanimalmodelsofinvasiveaspergillosis,includingmurineandlargeranimalspecieswithbothpulmonaryanddisseminatedinfection.Thisbriefreviewwilldiscussthefutureofanimalmodelsofinvasiveaspergillosis,thelimitationsofcurrentanimalmodels,andthechallengesfacedindevelopingstandardizedmodels.Theaimsandcriticalneedsforfuturemodels,particularlyinthedevelopmentofnewdiagnostictools,willalsobeoutlined.Thegoalofthisworkistoprovidetheframeworkforevaluatinggenomictargetsinordertoimprovethediagnosisandtreatmentofinvasiveaspergillosis,whichwillultimatelyresultinimprovedoutcomesforpatients.ObjectivesoffutureanimalmodelsThefuturedevelopmentofanimalmodelsofinvasive

aspergillosishasseveralkeyobjectives,asshownin

Table1.Theseincludethedevelopmentofstandardized

modelsthatallowreproducibilitybetweenlaboratories,

andbetweenserialexperimentsconductedlongitudin-

allyinthesamelaboratorywiththesameprotocols.A

numberofvariables,suchasdifferentroutesof

challenge(rangingfromintravenoustointranasalto

DOI:10.1080/13693780400029429Correspondence:T.F.Patterson,(ProfessorofMedicine),UniversityofTexasHealthScienceCenteratSanAntonio,MailCode7881,7703FloydCurlDrive,SanAntonio,TX78229-3900,USA.Tel:'12105676680;Fax:'12105673303;E-mail:patterson@uthscsa.edu.–2005ISHAM

Table1ObjectivesofAspergillusanimalmodels

.Standardizedmodel(s)

.Recapitulateshumandisease

.Reasonablecosts

.Reproducibility

.Easeofuse

.Amenabletostudiesincluding

ÁEvaluationofnoveldiagnostics

ÁEvaluationofhostresponse

ÁEvaluationoforganismvirulencefactorsthroughmolecularmanipulations

ÁInvivoexpressionanalysis

ÁEvaluationoftherapeuticcompounds

aerosolchallenge),markedlyaffecttheoutcomeofstudies[1].Inaddition,thelackofstandardizationofvariousfactors,suchasinfectinginoculum,underlyingimmunosuppression,andvirulenceofstrainstudied,havemadecomparisonbetweenlaboratoriesdifficult

[4].Thus,amajorobjectiveforfuturemodelsistohavea‘referencestandard’modelÁor,morelikely,modelsÁthathavebeenvalidatedbetweenlaboratoriesandhavebeenshowntoproduceknowncoursesofinfectionordiseaseaswellasextentofinfectionwhichcanthenbeusedforcomparativepurposes.

Anothermajorgoalforfuturemodelsisforthecourseofdiseaseinthemodeltoaccuratelyreflectclinicalinfection.Itisparticularlyimportantfordiagnosticstudiesthatthemodelrecapitulateclinicalinfection,whichappearsmostlikelytooccurafterchallengethroughanaerosolroute.However,inmostanimalmodels,widelydisseminatedinfectionoccursmoreconsistentlywithanintravenouschallenge,andinsomesettings,suchasanimmunosuppressedrabbitmodel,pulmonaryinfectionalsoresultsfollowingintravenouschallengewhichproducesacourseofinfectionthatmimicswidelydisseminatedinfection

[5,6].Insomestudies,thesemodelsusingintravenouschallengeanddisseminatedinfectioninlargerspecies(rabbitsandguineapigs)havebeenshowntobeparticularlyusefulinevaluatingthekineticsofdiag-nosticmarkersaswellastherapeuticstudies[7Á11].

Otherissuesarepracticalandpragmatic.TheseincludecostÁsmallerspecies,suchasmice,allow

screeningstudiesbuttheserialassessmentofasmallernumberoflargerspeciesextensivelystudiedwithmultiplesurrogateendpointsmayreducetheutilizationofanimalresourcesandalsooverallcosts[12,13].Otherissuestoconsiderinfuturemodelsincludeeaseofuse,reproducibilityand,importantly,theamenabilityoftheanimalmodelstotheevaluationofnoveldiagnostics(largerspeciesofferserialsamplesbutsmalleranimalsallowmoreextensivenumberstobestudied),theevaluationofhostresponsesandassessmentoftherelationshipbetweenvirulenceoftheorganism,theeffectoftherapeuticcompoundsonresponseandcourseofinfection,andinvivogeneexpression.Becauseofthecriticalneedidentifiedforthedevelopmentofnewdiagnosticmarkersininvasiveaspergillosis,aswellastherefinementandbetterunderstandingofcurrentlyavailablemethods,theNIH/NIAIDrecentlyawardedacontract(NIH-NIAID-N01-AI-30041).Theobjectivesofthiscontractaretoestablishandstandardizeanimalmodelsofinvasiveaspergillosisthatwillallowthereliablequan-tificationofinfectiousburdenandtodefinesurrogatemarkersofinfectionanddiseaseprogression.ThesestudiesareaimedatimprovingthediagnosisandtherapyofinvasiveaspergillosisbyprovidingtheextendedAspergillusresearchcommunitywiththeresourcestoaddresssomeofthekeyquestionsinthefield,includinggeneticapproachestodiagnosis,animalmodelexperimentsandtrainingtoestablishtheseapproaches.KeyissuesforconsiderationinfuturemodelsAnumberofvariableshavebeenidentifiedascriticalforprovidingreferencestandardsforthisdisease(Table2).ThesefactorsincludestandardizationoftheinoculumÁrealizingthatanaerosolwillmostsimilarlymimicclinicalinfectionasopposedtointranasaldropletinstillation,intratrachealdelivery,orintrave-Table2Keyissuesforstandardization.Aspergillusstrainselection.InoculumstandardizationÁRouteofchallenge.ModelcharacterizationÁColonization/infectionÁLocalinfection/dissemination.HostselectionÁOutbred/inbredspeciesÁAnimalspecies(mice,guineapig,rabbit).ImmunosuppressionÁSteroidsÁNeutropeniaÁOthermethodsÁNon-immunosuppressed.Diseaseprogression.TissueburdenÁColonyformingunits(CFU)ÁSurrogatemarkerskAntigen(s):Galactomannan,otherskDNAkChitinkb-D-glucanÁOthermarkers.GeneexpressionÁEvaluationofnewgenomictargets

–2005ISHAM,MedicalMycology,43,S115ÁS119

nouschallenge.Additionally,anappropriateaerosoldeliverycanyieldamorehomogeneouspulmonaryinfectioncomparedtointranasalinstillation,asdemon-stratedthroughquantitativePCRevaluation,allowingtheentiremurinelungtobeutilizedforvariousdiagnostictestswiththeknowledgethattheinfectiousburdenisconsistentlydistributed[14].However,alter-nativeroutesmaywellhaveimportantadvantages,suchasproductionofdisseminatedinfection(forintrave-nouschallenge)orreproducibleinoculationintotheairways(forintranasalorintratrachealchallenge)

[1,15,16].Otherfeaturesincludingvirulenceofthestrainand/orspecieswillalsoneedtobeaddressed.Forexample,thecourseofinfectionchosenforstudywillvary,includingcolonizationvs.localinvasiveinfectionvs.disseminateddisease.CurrentstudiesfrequentlyutilizetheAspergillusfumigatusstrainAF293whichisthestrainusedforsequencing,butotherspeciesofferdistinctvirulencepropertiesandperhapsvaryingdiagnosticchallenges[17,18].

Anothermajorissueforconsiderationishostselec-tion.Inadditiontospeciesselection,asdiscussedabove,animalscanbeselectedasinbredstrainsvs.outbredspecies.Inbredstrainspresumablyofferlesshostgeneticvariability,buttheyareobviouslylimitedintermsofavailabilityandinanimalspeciesforwhichtheyexist.Theydoallowdistinctassessmentofsusceptibilityasdemonstratedinexperimentsdescribedbelow.However,notallinbredstrainswillbeidenticalinvariousregionsoftheworld,theyarenotalwaysreadilyavailableandaredistinctlymorecostly.Furthermore,theargumentcanalsobemadethatthediseaseunderstudyoccursinanoutbredclinicallyheterogenouspatientpopulation.Nevertheless,advan-tagesanddisadvantagesofeachcanbedescribed.Insettingswherespecifichostfactorsseemlesscriticaltooutcome,screeningstudiesmaybesufficientwithoutbredmice,sothatvalidationofthemodelinoutbredaswellasinbredstrainsisanimportantgoaloffuturemodels.

Otherhostissuesforconsiderationincludetheroleofimmunosuppression.Increasingly,infectedpatientshavelesstraditionalriskfactorsforinvasiveaspergil-losis[19Á21].Thus,futuremodelswilllikelyneedto

includeevaluationofnotonlyneutropenia(whichhasbecomelesscommonasariskfactor)butalsootherimmunosuppressiveagentsincludingcorticosteroids,asthesevariousimmunosuppressiveregimensmayimpactdiagnostictestperformanceinclinicalinfectionaswellasanimalmodels[22].

Finally,oneofthemostproblematicissuesinassessingtissueburdenofAspergillushasbeenthequantificationofthetissueburden.Semi-quantitative–2005ISHAM,MedicalMycology,43,S115ÁS119

colonycountshavebeenusedsuccessfullyinexperi-mentstoeffectivelyassesstissueburdenofAspergillus[5,23].However,theutilityoftheseculturesforhyphalorganismsislimitedastheyaredifficulttoreproducereliablybetweenlaboratories,asgrindingthehyphaemayproduceincreasedtissuecounts.Thesecountscanevenbefalselyelevatedwithantifungalagentssuchastheechinocandins,whichfragmenttheorganismtoincreasethecolonyformingunitsalthoughthetissueburdenisactuallyreduced[13,24,25].Thus,extensiveeffortsforassessingtissueburdeninfuturemodelsarefocusedontheuseofsurrogatemarkers.Theseeffortswillnotonlyprovideseveralendpointstoassesstissueburdenbutcouldalsoallowareductioninthenumbersofanimalsrequiredintheexperimentaldesignbyprovidingmultipleendpointsofassessment[12].Theseadditionalsurrogatescanincludegalactomannan,1-3,b-D-glucan,chitin,quantitativePCRtechniques,andothers[26,27].Inaddition,theincorporationofnovelmarkers,suchaslung-injuryscoresorhighresolutionchestcomputedtomography,canbeusefulintheassessmentoftissueburden[12,28].SequencingtheA.fumigatusgenomemayalsorevealnewpotentialsurrogatemarkertargets.Animportantaimforfuturemodelsisthestandardizationoftissueburdenusingthesesurrogatemarkers.FutureinhalationalmodelsTheutilityandreproducibilityofaerosolchallengewithA.fumigatusiscurrentlyundergoingevaluation[14,29].Forexample,inthemodeldevelopedbySheppardetal.[28]aneasilyreproduciblemethodofaerosolchallengeofimmunosuppressedmiceinanacrylicchamberproducedreproduciblecolonycountsandaconsistentcourseofinfection.Intra-laboratorystudiestovalidatethisapproachappearpromising,withasimilarcourseofinfectionandsimilarburdenofpulmonarydiseasebeingdemonstrated[unpublisheddata].Whilethereproducibilityofthiseasilytransportableaerosolsystemisencouraging,additionalstudiesutiliz-ingalarge-scaleinhalationalMadisonchamberarealsoinprogress.Thisinhalationalchamberprovidesaccuratedeliveryofaerosolinoculum(ofavarietyoforganisms,includingMycobacteriumtuberculosis,Ba-cillusspp.andYersenia,aswellasfungi)thatcanbedeliveredsimultaneouslytoanextensivenumberofmicebutalsomultiplelargeranimalssuchasrabbitsorguineapigs[30].Theadvantagesofsuchasystemincludeprecisedeliveryofadryaerosolinoculum,butalsosimultaneouschallengeofanimalswithanumberofvariables(suchastheroleofimmunosuppression,specificmousestrain,etc.)thatlimitsexperimental

variabilityandpotentiallyreducesthenumberofanimalsrequiredforstudy[31].

FuturegeneticstudiesusinganimalmodelsThepost-genomiceraofA.fumigatusisextremelyexcitingforthepotentialidentificationofnewdiag-nosticandtherapeutictargetsaswellasfornewinsightsintothepathogenesisandvirulenceoftheorganism.PreliminarystudiesbyZaasetal.[29,32]illustratethepotentialofthesestudies.Theseinvesti-gatorsusedanaerosolchallengeofavarietyofinbredmicetoassesstheroleofhostgeneticbackgroundonsusceptibilitytoAspergillus[32].Interestingly,ingcom-putationalgenetics,achromosomalregionwasthenidentifiedthatcorrelatedwithresistancephenotype.Candidategeneswereidentifiedfromthoseregionandstudiesarecurrentlyongoingtorevealthepotentialsignificanceofthosefindings.Theseresultsdemon-stratethepotentialofcarefullydesignedanimalmodelstoplayanimportantroleinthefutureevaluationintheexperimentalassessmentofinvasivepulmonaryasper-gillosis.

Conclusion

AnimalmodelshavebeenusefulforassessingthepathogenesisandvirulenceofAspergillusandhavesignificantlyaidedthedevelopmentofnewdiagnosticandtherapeuticmodalities.Futuremodelsofexperi-mentalaspergillosisareaimedatassistingintheidentificationanddevelopmentofnewdiagnosticandtherapeutictargetsthatmayresultfromsequencingoftheAspergillusgenome.Thesewillbeofmajorbenefittostudiesaimedultimatelyatimprovingthediagnosisandtreatmentofthisoftenlethalinfection.Acknowledgements

ThisworkhasbeensupportedwithFederalfundsfromtheNationalInstituteofAllergyandInfectiousDis-eases,NationalInstitutesofHealth,underContractNo.N01-AI-30041.

References

1AndrioleVT,MiniterP,GeorgeD,etal.Animalmodels:Usefulnessforstudiesoffungalpathogenesisanddrugef cacyinaspergillosis.ClinInfectDis1992;14(Suppl1):S134ÁS138.2PattersonTF,MiniterP,PattersonJE,etal.Aspergillusantigendetectioninthediagnosisofinvasiveaspergilosis.JInfectDis1995;171:1553Á1558.3GraybillJR.Theroleofmurinemodelsinthedevelopmentofantifungaltherapyforsystemicmycoses.DrugResistUpdate2000;3:364Á383.4NajvarLK,CacciapuotiA,HernandezS,etal.ActivityofposaconazolecombinedwithamphotericinBagainstAspergillus avusinfectioninmice:comparativestudiesintwolaboratories.AntimicrobAgentsChemother2004;48:758Á764.5PattersonT,MiniterP,RyanJ,AndrioleV.EffectofImmuno-suppressionandamphotericinBonaspergillusantigenemiainanexperimentalmodel.JInfectDis1988;158:415Á422.6SabettaJ,MiniterP,AndrioleV.Thediagnosisofinvasiveaspergillosisbyanenzyme-linkedimmunosorbentassayforcirculatingantigen.JInfecDis1985;152:946Á953.7PattersonTF,GeorgeD,IngersollR,etal.Ef cacyofSCH39304intreatmentofexperimentalinvasiveaspergillosis.AntimicrobAgentsChemother1991;35:1985Á1988.8PattersonTF,FothergillAW,RinaldiMG.Ef cacyofitracona-zolesolutioninarabbitmodelofinvasiveaspergillosis.Anti-microbAgentsChemother1993;37:2307Á2310.9PattersonTF,GeorgeD,MiniterP,AndrioleVT.Saperconazoletherapyinarabbitmodelofinvasiveaspergillosis.AntimicrobAgentsChemother1992;36:2681Á2685.10PattersonTF,MiniterP,DijkstraJ,etal.Treatmentofexperi-mentalinvasiveaspergillosiswithnovelamphotericinB/choles-trol-sulfatecomplexes.JInfectDis1989;159:717Á721.11KirkpatrickWR,McAteeRK,FothergillAW,etal.Ef cacyofvoriconazoleinaguineapigmodelofdisseminatedinvasiveaspergillosis.AntimicrobAgentsChemother2000;44:2865Á2868.12PetraitisV,PetraitieneR,SarafandiAA,binationtherapyintreatmentofexperimentalpulmonaryaspergillosis:synergisticinteractionbetweenanantifungaltriazoleandanechinocandin.JInfectDis2003;187:1834Á1843.13PetraitieneR,PetraitisV,GrollAH,etal.Antifungalef cacyofcaspofungin(MK-0991)inexperimentalpulmonaryaspergillosisinpersistentlyneutropenicrabbits:Pharmacokinetics,drugdis-position,andrelationshiptogalactomannanantigenemia.Anti-microbAgentsChemother2002;46:12Á23.14SteinbachWJ,BenjaminDK,Jr,TrasiSA,etal.Valueofaninhalationalmodelofinvasiveaspergillosis.MedMycol2004;42:417Á425.15DixonDM,PolakA,WalshTJ.Fungusdose-dependentprimarypulmonaryaspergillosisinimmunosuppressedmice.InfectImmun1989;57:1452Á1456.16AllendeMC,LeeJW,FrancisP,etal.Dose-dependentantifungalactivityandnephrotoxicityofamphotericinBcolloidaldispersioninexperimentalpulmonaryaspergillosis.AntimicrobAgentsChe-mother1994;38:518Á522.17WarnPA,MorrisseyG,MorrisseyJ,DenningDW.Activityofmicafungin(FK463)againstanitraconazole-resistantstrainofAspergillusfumigatusandastrainofAspergillusterreusdemon-stratinginvivoresistancetoamphotericinB.JAntimicrobChemother2003;51:913Á919.18SteinbachWJ,PerfectJR.Newerantifungaltherapyforemergingfungalpathogens.IntJInfectDis2003;7:5Á20.19PattersonTF,KirkpatrickWR,WhiteM,etal.Invasiveaspergillosis.Diseasespectrum,treatmentpractices,andout-comes.I3AspergillusStudyGroup.Medicine(Baltimore)2000;79:250Á260.20WaldA,LeisenringW,vanBurikJ-A,BowdenRA.EpidemiologyofAspergillusinfectionsinalargecohortofpatientsundergoingbonemarrowtransplantation.JInfectDis1997;175:1459Á1466.21MartyFM,LeeSJ,FaheyMM,etal.In iximabuseinpatientswithseveregraft-versus-hostdiseaseandotheremergingrisk

–2005ISHAM,MedicalMycology,43,S115ÁS119

factorsofnon-Candidainvasivefungalinfectionsinallogeneichematopoieticstemcelltransplantrecipients:acohortstudy.Blood2003;102:2768Á2776.

22MarrKA,BalajeeSA,McLaughlinL,etal.Detectionof

galactomannanantigenemiabyenzymeimmunoassayforthediagnosisofinvasiveaspergillosis:variablesthataffectperfor-mance.JInfectDis2004;190:641Á649.

23GraybillJR,KasterS,DrutzD.Treatmentofexperimental

murineaspergillosiswithBAYn7133.JInfecDis1983;148:898Á906.

24KirkpatrickWR,PereaS,CocoBJ,PattersonTF.Ef cacyof

caspofunginaloneandincombinationwithvoriconazoleinaGuineapigmodelofinvasiveaspergillosis.AntimicrobAgentsChemother2002;46:2564Á2568.

25BowmanJC,AbruzzoGK,AndersonJW,etal.Quantitative

OCRassaytomeasureAspergillusfumigatusburdeninamurinemodelofdisseminatedaspergillosis:Demonstrationofef cacyofcaspofunginacetate.AntimicrobAgentsChemother2001;45:3474Á3481.

26Loef erJ,KloepferK,HebartH,etal.Polymerasechainreaction

detectionofaspergillusDNAinexperimentalmodelsofinvasiveaspergillosis.JInfectDis2002;185:1203Á1206.

27OdabasiZ,MattiuzziG,EsteyE,etal.Beta-D-glucanasa

diagnosticadjunctforinvasivefungalinfections:validation,cutoff–2005ISHAM,MedicalMycology,43,S115ÁS119

development,andperformanceinpatientswithacutemyelogen-ousleukemiaandmyelodysplasticsyndrome.ClinInfectDis2004;39:199Á205.28WalshTJ,GarrettK,FeuersteinE,etal.Therapeuticmonitoringofexperimentalinvasivepulmonaryaspergillosisbyultrafastcomputerizedtomography,anovel,noninvasivemethodformeasuringresponsestoantifungaltherapy.AntimicrobAgentsChemother1995;39:1065Á1069.29SheppardDC,RiegG,ChiangLY,etal.Novelinhalationalmurinemodelofinvasivepulmonaryaspergillosis.AntimicrobAgentsChemother2004;48:1908Á1911.30McMurrayDN.Diseasemodel:pulmonarytuberculosis.TrendsMolMed2001;7:135Á137.31KirkpatrickWR,CocoBJ,PattersonTF.Anewanimalmodelofinvasivepulmonaryaspergillosis(IPA)usinglargescaleinhala-tionalchallenge.In:Abstractsofthe44thInterscienceConferenceonAntimicrobialAgentsandChemotherapy,Washington,DC,30OctoberÁ2November;2004.32ZaasA,SteinbachW,BurchL,PerfectJ,SchwartzD.Suscept-ibilitytoAspergillusfumigatus:RoleofHostGeneticBack-ground.In:Abstractsofthe7thAnnualEuropeanConferenceonFungalGenetics,CopenhagenDenmark;2004.

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