[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