Susceptibility Testing of Fungi to Antifungal Drugs

16 Pages·2017·1.21 MB·English

Journal of
Fungi
Review
Susceptibility Testing of Fungi to Antifungal Drugs
MaurizioSanguinetti1,*andBrunellaPosteraro2
1 InstituteofMicrobiology,FondazionePoliclinicoUniversitarioA.Gemelli,IstitutodiRicoveroeCuraa
CarattereScientifico(IRCCS),UniversitàCattolicadelSacroCuore,00168Rome,Italy
2 InstituteofMedicalPathologyandSemeiotics,FondazionePoliclinicoUniversitarioA.Gemelli,IRCCS,
UniversitàCattolicadelSacroCuore,00168Rome,Italy;[email protected]
* Correspondence:[email protected];Tel.:[phone]
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:15July2018;Accepted:13August2018;Published:15September2018
Abstract: Susceptibilitytestingoffungiagainstantifungaldrugscommonlyusedfortherapyisakey
componentofthecareofpatientswithinvasivefungalinfections. Antifungalsusceptibilitytesting
(AFST)hasprogressedinrecentdecadestofinallybecomestandardizedandavailableasbothClinical
andLaboratoryStandardsInstitute(CLSI)andEuropeanCommitteeonAntimicrobialSusceptibility
Testing(EUCAST)referencemethodsandincommercialmanual/automatedphenotypicmethods.
Inclinicalpractice,theSensititreYeastOneandEtestmethodsarewidelyusedforAFST,particularly
forsterilesiteisolatesofCandida. Nevertheless,AFSTismovingtowardnewphenotypicmethods,
suchasmatrix-assistedlaserdesorptionionizationtime-of-flightmassspectrometry(MALDI-TOF
MS),thatarecapableofprovidingrapid,andpotentiallymoreactionable,resultsforthetreating
clinician. OurobjectiveistosummarizeupdateddataonphenotypicmethodsforAFSTofCandida
andAspergillusspeciesandtoassesstheirsignificanceinviewofopposing,butemerging,molecular
genotypicmethods.
Keywords: antifungalsusceptibility;Candida;Aspergillus;CLSI;EUCAST;MALDI-TOFMS
1. Introduction
Invasivefungalinfections,especiallythosecausedbythespeciesCandidaandAspergillus,continue
toriseinfrequency[1]and,alarmingly,areassociatedwithantifungalresistance[2],whichmakesthe
managementofpatientswithsuchinfectionsparticularlychallenging[3,4]. Exceptforinfectionsdue
toinherentlyantifungal-resistantspecies,themajorityoftheseinfectionsareclinicallytreatableby
threecurrentlyavailableantifungaldrugclasses: triazoles(fluconazole,itraconazole,voriconazole,
posaconazole,isavuconazole),echinocandins(anidulafungin,caspofungin,micafungin),andpolyenes
(amphotericinB-deoxycholatewithitslipidandliposomalformulations)[5,6].Parallelingtherelatively
recent introduction of new antifungal drug classes [7] and the discovery of novel agents [8] for
thetreatmentofinvasivefungalinfections,thefieldofantifungalsusceptibilitytesting(AFST)has
progressedtremendouslyinthepastseveralyears[9]. Apartfrompractical,reliable,andreproducible
laboratorymethodsforAFST,therehasbeenaconsiderablepushtowardmethodsabletocorrelate
invitro laboratory tests with clinical outcome and identify new, clinically important resistance
mechanisms,ashasbeendoneforsusceptibilitytestingofbacteria[10].
The goal of performing AFST is to produce actionable data for the treating clinician on
the susceptibility, intermediate (or dose-dependent) susceptibility, or resistance phenotype for an
organism–antifungal agent combination. In a viewpoint article published ~15 years ago, Rex and
Pfaller[11],whilediscussingtheaccuracyofthe“90–60rule”bywhichAFSTcanpredicttheoutcome
oftreatment,saidthatAFST“hasindeedcomeofageasausefulclinicaltool.”Thisultimatelycame
truelaterthroughnotonlyrefinementoftheClinicalandLaboratoryStandardsInstitute(CLSI)and
J.Fungi2018,4,110;doi:10.3390/jof4030110 www.mdpi.com/journal/jof
J.Fungi2018,4,110 2of16
EuropeanCommitteeonAntimicrobialSusceptibilityTesting(EUCAST)referencemethodscurrently
inplace[12–15],butalsoexpansionofcommercialandautomatedmethodsforAFST[16].
All of these achievements increased the likelihood that testing several organism–drug
combinations(mostnotablyCandidaspeciesandtheazoleantifungalagents)couldusefullyinfluence
the selection of therapy, thus aiding clinicians in the management of difficult-to-treat fungal
infections[17]. Meanwhile,betterconsistencyandaccuracyoftesting,alongwithclinicaloutcomes
andpharmacokinetic/pharmacodynamicdata,ledtothecreationofwell-validatedclinicalbreakpoints
(CBPs),atleastforazolesandcommonCandidaspecies[9]. However,inlieuofCBPs,establishing
epidemiologicalcutoffvalues(ECVs)helpedtodistinguishwild-type(WT)isolatesfromthosethat
mayharboranacquiredresistancemechanismandarelesslikelytorespondtoagivenantifungal
agent(non-WT)[18].
TheobjectivesofthispaperaretoprovideupdatesonnewdatafromAFSTstudiesandtodiscuss
howAFSTmightimproveoutcomesofinvasivefungalinfections. Inviewoftheirclinicalimportance,
wewillfocusonAFSTofCandidaandAspergillusspecies.
2. ConventionalPhenotypicAssaysforTestingFungalSusceptibility
2.1. ReferenceAFSTMethods
Currently, phenotypic assays to perform invitro AFST for either yeasts or filamentous fungi
(also termed molds) include two universally recognized standard methods, CLSI [12,13] and
EUCAST [14,15], which apply the broth microdilution method (BMD). Both measure antifungal
activity, expressed as the minimum inhibitory concentration (MIC) of an antifungal drug, which
indicatestheminimaldrugconcentrationthatinhibitsfungalgrowth. Despitesomemethodological
differences(e.g.,glucoseconcentration,inoculumsize,readingendpoint,etc.) betweenthetwo[16],
CLSIandEUCASThavebeenproventoyield,uponcompletionoftesting,comparableMICdatafor
allclassesofantifungalagents[19,20]. Inparticular,theEUCASTmethodusesahigherpercentageof
glucose(2%)inthetestmediumtofacilitateincreasedfungalgrowth[14,15],whichmaybeparticularly
advisedwhentestingmolds. ToacceleratethetimetoAFSTresults,bothmethodsinclude(atleast
forCandidaspecies)arecommendedincubationtimeof24h[20]. BasedondifferentabsoluteMIC
values generated, EUCAST and CLSI have established divergent CBPs (i.e., MIC thresholds used
toclassifyisolatesassusceptibleorresistant)forCandidaspecies[21,22]. ContrarytoCLSI,which
has not set CBPs against any molds (including Aspergillus species) [23], EUCAST provides mold
CBPs, with species-related CBPs determined for A. fumigatus, A. flavus, A. nidulans, A. niger, and
A.terreus[21]. Thetwostandardsproposetheconceptofminimumeffectiveconcentration(MEC)
forreadingechinocandinAFSTresultsofmolds. However,MECdeterminationisnotalwayseasy
becauseofitsrelianceonassessingthetransitionpointofhyphaefromnormaltoaberrantforms,which
oftenrequiresmicroscopicobservation. Ingeneral,referenceBMDassaysaretechnicallydemanding
and not intended for routine laboratory practice. Additionally, the interlaboratory variability in
caspofungin MICs noted with Candida species may significantly hinder the use of both CLSI and
EUCASTmethods[24]. Nonetheless,theyholdgreatvalueasindispensablecomparatorsinevaluating
performancestudiesofcommercialmethods[25,26],suchasthosediscussedbelow.
Inonelargestudypublishedrecently[27],goodcorrelationwasobtainedbetweenEUCAST(EDef
7.2) and CLSI (M27-A3) for amphotericin B, flucytosine, anidulafungin, caspofungin, micafungin,
fluconazole, isavuconazole, itraconazole, posaconazole, and voriconazole among 357 isolates of
Candidaspecies,showing>93%categoricalagreementforallantifungalagentstested. Lowagreement
mainly regarded testing of amphotericin B, anidulafungin, and isavuconazole against C. glabrata,
and caspofungin against C. parapsilosis, C. tropicalis, and C. krusei, leading to further calls for
moreharmonization.
J.Fungi2018,4,110 3of16
2.2. CommercialAFSTMethods
Aspreviouslyreviewed[16],eitherBMDmethods,whichusecolorendpointsduetometabolic
dye(e.g.,AlamarBlue)incorporatedintogrowthmedia(e.g.,SensititreTMYeastOneTM(SYO;Thermo
FisherScientific,Waltham,MAUSA)),oragar-basedmethods,whichuseconcentrationgradientsof
antifungalsthatdiffuseintogrowthmedia(e.g.,Etest®;ABBiodisk,Solna,Sweden),aremodifications
oftheCLSI/EUCASTreferencemethods. Avalidalternativetothese(manual)assaysistoperform
AFSTthroughautomatedmeans(e.g.,VITEK®2system;bioMérieux,Marcy-l’Étoile,France). These
phenotypicmethodsareperselimitedbyrequiringapurecultureoftheinfectingorganismbefore
testing. Nevertheless,SYO,Etest,andVITEK2arecommercialmethodswidelyusedforinvitroAFST
of Candida and/or Aspergillus species, and are thought to be superior to reference methods in use,
convenience,andflexibility[16].
Since its introduction in routine microbiology laboratories, the SYO microdilution antifungal
panel—allowing simultaneous testing of amphotericin B, echinocandins, and triazoles—has been
extensively evaluated for yeasts, becoming the focus of large hospital studies [28–30]. For CLSI
CBPs/ECVstoassignsusceptibility(ortheWTphenotype)tosystemicallyactiveantifungalagents
(theSYO’smanufacturerrecommendsusingCLSICBPs),tworecentstudiesreportedonSYOMIC
resultsforCandidaspecies.InthestudybyPosteraroetal.[29],susceptibility/WTratestoamphotericin
Bandflucytosinewereover97%inallyeastisolates(n=1250,includingCandidaandnon-Candida
species). Ratesforfluconazole(excludingC.krusei), itraconazole, andvoriconazolewere98.7%in
C. albicans, 92.3% in the C. parapsilosis species complex, 96.1% in C. tropicalis, 92.5% in C. glabrata,
and 100% in both C. guilliermondii and C. krusei. Rates for echinocandins were 99.7% to 99.8%
in all Candida species. Similarly, Xiao et al. [30] found that over 99.3% of the isolates (n = 1072,
including all common non-albicans Candida species) had a WT phenotype to amphotericin B and
flucytosine. Susceptibility/WTratesforazolesamongC.parapsilosisspeciescomplexisolateswere
≥97.5%. Among ~14.3% of fluconazole-resistant C. glabrata isolates, 11.6% were cross-resistant to
fluconazoleandvoriconazole.AllC.kruseiisolatesweresusceptible/WTtovoriconazole,posaconazole,
and itraconazole, whereas 97.7% to 100% of isolates were susceptible to caspofungin, micafungin,
andanidulafungin.
Echinocandinsrepresentfirst-linetreatmentofinvasiveCandidainfections[5,31,32]. Acquired
echinocandinresistanceismainlyobservedamongC.albicansandC.glabrataandisassociatedwith
mutationsintwonarrowhotspot(HS)regions(HS1andHS2)ofFKS1(C.albicansandC.glabrata)and
FKS2(C.glabrataonly)genes[33]. Eschenaueretal.[28]underscoredthatadoptingCLSICBPsfor
caspofunginmayoverstatetheratesatwhichisolatesofC.glabrataandC.kruseiarenonsusceptibleto
caspofungin. Whileclinicalmicrobiologylaboratoriesshouldusemicafunginandanidulafunginas
surrogatemarkerstopredictsusceptibilityorresistancetocaspofungin[34,35],someauthorshave
triedtoestablishECVsusingtheSYOmethod[36,37]. Espinell-Ingroffetal.[37]calculatedSYOECVs
for echinocandins and eight Candida species. Notably, SYO ECVs for anidulafungin, caspofungin,
and micafungin correctly classified as non-WT 88.9% (72/81), 91.4% (74/81), and 93.8% (76/81),
respectively,ofCandidaisolateswithknownFKSmutations.Despitetheirsensitivityforidentifyinghot
spotmutations,thepositiveandnegativepredictivevaluesoftheseECVsinroutineclinicalapplication
werenotdetermined. Veryrecently,Kritikosetal.[38]calculatedechinocandinECVsforC.albicans
(n=1277)andC.glabrata(n=347)testedbySYOandassessedtheirabilitytoidentifyFKSmutants
ina10-yearcandidemiasurveyfromtheFUNGINOSnetwork. Among70isolateswithMIC≥ECV
foranyechinocandinandthensequenced,noFKSmutationwasfoundinthe52“limitWT”isolates
(MIC=ECVforatleastoneechinocandin),suggestinganexcellentnegativepredictivevalueofthese
ECVs. Amongthe18“non-WT”isolates(MIC>ECVforatleastoneechinocandin),FKSmutations
werefoundintheonlytwoisolateswithMIC>ECVforallthreeechinocandins,butnotintheisolates
havinga“non-WT”phenotypeforonlyoneortwoechinocandins. However,validatingtheseSYO
EVCsinsettingswithhigherratesofechinocandinresistanceremainstobedone.
J.Fungi2018,4,110 4of16
Despitebeingararity,echinocandinnon-WTclinicalisolatesofAspergillusspecieswithMECs
above the ECV were identified [39]. Reduced susceptibility of Aspergillus fumigatus isolates to
echinocandins invitro can develop via FKS-dependent and FKS-independent mechanisms [40,41].
Aspreviouslymentioned,SYOisacolorimetricadaptionoftheCLSIBMDmethod,whichisbased
on either the M27-A3 standard for yeasts or the M38-A2 standard for molds [12,13]. SYO color
endpointsaredeterminedvisuallyastheAlamarBluecolorchangesfromblue(0%growth)tored
(100%growth),andonlyanintermediatepurplecolorcanbeindicativeof50%growth. Untilnow,only
onepublishedstudyhasevaluatedtheperformanceofSYOversusthereferenceCLSIBMDmethodfor
invitrosusceptibilitytestingofAspergillusspeciestoechinocandins. Siopietal.[42]tested39isolates
ofA.fumigatus, A.flavus, andA.terreus, includingtwoechinocandin-resistantA.fumigatusstrains.
Overall,thebestagreementwiththeCLSIM38-A2methodwaswithaninoculumof104 CFU/mL,
incubationtimesof20hforA.flavusand30hforA.fumigatusandA.terreus,andreadingthefirst
purplewell. However,theagreementwaspoorforcaspofungin(0to54%)butgoodformicafungin(77
to100%),whereasitwasinconclusiveforanidulafunginbecauseallAspergillusisolatesyieldedoff-scale
SYOcolorendpoints. Interestingly,bothresistantisolatesconvertedAlamarBluewithin24hathigh
concentrations,indicatingthatdetectionofechinocandin-resistantAspergillusisolatesmaybefeasible
withtheSYOmethod. Hence,theauthorsnoticedthatthecurrentAlamarBlueconcentrationmay
besufficientfortheslower-growingA.fumigatusandA.terreusbutnotforthefast-growingA.flavus,
highlightingtheneedforfurtherexplorationandvalidationoftheoptimalAlamarBlueconcentration.
Bytheauthors’opinion[42],inanaverageclinicallaboratory,agoodcompromisecouldbeinoculating
A.flavusisolatesintheafternoonandreadingthemthenextmorning,andinoculatingA.fumigatusand
A.terreusisolatesinthemorningandreadingthemthenextafternoon. Therefore,theauthorsadvised
notconsidering48hofincubationforSYOandechinocandins,insteadproposingsusceptibilitytesting
ofAspergillusspeciesagainstamphotericinB,itraconazole,posaconazole,andvoriconazole.
Consistent with the role of triazoles in the treatment and/or prophylaxis of invasive
aspergillosis [43], CLSI-based ECVs for itraconazole, posaconazole, voriconazole, and, lately,
isavuconazole were established for Aspergillus species (A. fumigatus, A. flavus, A. terreus, A. niger,
A. nidulans, and A. versicolor) to aid in the early identification of clinical isolates with acquired
resistance mechanisms [44,45]. Different patterns of azole resistance (i.e., multi-azole, pan-azole,
or single-azole resistance) in A. fumigatus are, to various extents, associated with mutations in the
codingregionofthesterol14α-demethylase-encodinggeneCYP51A,aswellastandemrepeatsof34,
46,and53bpupstreaminthepromoterregionofCYP51A[46].Themostfrequentlyobservedmutation
isTR /L98H,whichconsistsofa34bptandemrepeat(TR )inthegenepromoterregioncombined
34 34
withthesubstitutionofleucine98forhistidine(L98H)[47]. Melloetal.[48]comparativelyevaluated
the invitro activity of posaconazole, voriconazole, and itraconazole against clinical isolates from
common(n=59)anduncommon(n=27)AspergillusspeciesusingtheSYOandCLSIM38-A2methods.
TheoverallessentialagreementbetweenSYOMICsandCLSIMICswas100%forvoriconazoleand
96.5%forbothitraconazoleandposaconazole. CalculatingonlyforisolatesofA.fumigatus,A.flavus,
A.terreus,andA.niger,theessentialagreementvaluewasunchangedforvoriconazole(100%),but
increasedforposaconazole(98.3%)anddecreasedforitraconazole(94.9%). WheninterpretingMICs
accordingtoCLSIECVsforthe21A.fumigatus,19A.flavus,12A.terreus,7A.niger,and5A.nidulans,
thecategoricalagreementbetweenthemethodswas96.9%(62/64isolates)forposaconazole,98.4%
(63/64 isolates) for voriconazole, and 93.7% (60/64 isolates) for itraconazole. Of 10 A. fumigatus
isolates with CYP51A alterations, all but two isolates exhibited non-WT phenotypes (MIC > ECV)
forposaconazoleandvoriconazole(oritraconazole)obtainedwithboththeSYOandCLSImethods.
Interestingly,oneofthetwoisolates(bothharboringtheTR /L98Hmutation)hadaposaconazole
34
non-WTphenotypethatwasdeterminedbytheCLSImethodbutnotbytheSYOmethod. Theother
isolatehadavoriconazolenon-WTphenotypethatwasdeterminedbytheSYOmethodbutnotby
the CLSI method. Posaconazole MICs ≤0.5 µg/mL provided separation between WT isolates of
A.fumigatusandthoseharboringmutationsintheCYP51Agene. Basedonthesefindings,SYOand
J.Fungi2018,4,110 5of16
CLSIareequivalentmethodsfortestingtriazolesusceptibilityinAspergillusspecies. Itisworthnoting
that, inlinewithSiopietal.’sstudy[42], Melloetal.[48]performedvisualreadingsofSYOMICs
regardlessofcolorchanges,whichwerenecessarybecauseofprolongedincubationtimes(>24h)of
theSYOpanels.
ComparedtoSYO,gradientdiffusionassayssuchasEtest/MICTestStripsareeasytoperformbut
expensivewhenusedonalargerscale[46]. However,inthecontextofazoleresistance,recentreports
showthereliabilityofgradientMICapproachesthatrepresentalternativemeansofquantitatively
determininginvitrosusceptibilitytoantifungalagents[49,50]. Arendrupetal.[49]evaluatedtheMIC
StripIsavuconazoletest(Liofilchem, RosetodegliAbruzzi, Italy), theonlycommerciallyavailable
isavuconazolesusceptibilitytest,againstEUCASTEDef9.3byusing40WTand39CYP51Amutant
isolatesofA.fumigatus. MICsweredeterminedbytwoindependentreadersandwereinterpreted
accordingtotheEUCASTisavuconazoleepidemiologicalcutoffvalue(ECOFF;2µg/mL)andclinical
breakpoint(1µg/mL).Usingthestrip’sfullinhibitionendpoint,theessentialagreementswiththe
EUCASTreferencemethod(at±1and±2twofolddilutions)were73–75%and89–90%[49],which
werebetterthanpreviouslyfoundfortheisavuconazoleEtest(nolongeravailable)whencompared
withtheCLSIreferencemethod[51]. Thecategoricalagreementwas>91%,with6.3–8.9%verymajor
errors(definedasisolatecategorizedasresistantbytheEUCASTreferencemethodbutsusceptibleby
thestriptest)and0–1.3%majorerrors(definedasisolatecategorizedassusceptiblebytheEUCAST
referencemethodbutresistantbythestriptest). Interestingly,verymajorerrorsincludedfourisolates
withaWTCYP51Agenotypethateithermayhavebeenharboringotherresistancemechanismsormay
havebeenisolatesmisclassifiedasresistantbytheEUCASTreferencemethodbecauseoftherestrictive
clinicalbreakpoint[52]. Bytheauthors’opinion[49],interpretingtheMICsobtainedwithcommercial
testsbysuchabreakpointmaygenerateahigherriskofmisclassificationunlessthesusceptibilitytest
isverywellstandardizedagainsttheAFSTreferencemethod. Finally,thediscriminationbetween
WTandTR /L98HmutantisolateswasgreaterfortheMICstriptest,makingitpotentiallysuitable
34
for detecting resistant environmental mutants, provided the full inhibition endpoint is used [49].
Despitebeingpromising,theperformanceoftheMICStripIsavuconazoletestneedstobeconfirmed
in a multicenter study. More recently, Idelevich et al. [50] evaluated Etest (bioMérieux) and MIC
TestStrip(Liofilchem)foritraconazole,posaconazole,andvoriconazole,andonlyMICTestStripfor
isavuconazole,againsttheEUCASTreferencemethodbyusingclinicalconsecutiveisolates(n=24)and
controlstrains(n=15,withdefinedresistantorsusceptiblephenotypesandgenotypes)ofA.fumigatus.
Regarding the clinical isolate collection, both assays had good performance for itraconazole and
voriconazole. However,theMICTestStripshowedlowagreementwiththeEUCASTreferencemethod
duetoahighrateofminorerrors(definedasfalsecategorizationinvolvingintermediateresults)and
majorerrors. WhileMICTestStripperformedwellforisavuconazole,allclinicalisolatesdetermined
to be resistant to a particular azole by the EUCAST reference method were correctly detected as
resistant by both gradient diffusion assays. Regarding the control strain collection, both gradient
diffusiontestshadworseperformance. Inparticular,threemajorerrorsoccurredwithMICTestStrip
forisavuconazole(onestrainwithM220VCYP51AalleleandtwostrainswithWTCYP51Aalleles).
Theauthors[50]concludedthatbothassayscanreasonablybeusedforazolesusceptibilitytesting
withA.fumigatus.
ConcerningtheechinocandinsusceptibilitytestingofCandidaspecieswithEtest(bioMérieux),
Bougnouxetal.[53]recentlyenrolled16Frenchhospitalcenterstoobtain933Candidaisolatesthat
weretestedagainstmicafungin. MICswerealsodeterminedbytheEUCASTreferencemethodata
singlecenter. TheoverallessentialagreementbetweenEUCASTandEtestresultswashigh(98.5%
at ±2 twofold dilutions and 90.2% at ±1 twofold dilutions), whereas a categorical agreement of
98.2%wasobservedforthe742isolatesbelongingtothefivespeciesforwhichclinicalbreakpointsor
ECOFFswereavailable.TheauthorsdemonstratedthattheEtestgavemicafunginsusceptibilityresults
that were very similar to those given by the EUCAST reference method under routine laboratory
testing. Meanwhile, invitro micafungin resistance rates for the Candida species mainly isolated
J.Fungi2018,4,110 6of16
from clinical samples were low (<2% for C. albicans and C. parapsilosis and 3.9% for C. glabrata).
Morerecently,a1-yearsurveyconductedon104Candidaspeciesisolatedfrombloodculturesinan
AustrianhospitalbyAigneretal.[54]showedthatthelevelsofessentialagreementwiththeEUCAST
referencemethodwere97%and92%foranidulafunginandmicafungin. Thecategoricalagreement
oftheEtestwas99%forbothanidulafunginandmicafungin,irrespectiveoftheclinicalbreakpoints
applied(EUCASTversusCLSI).OnlyoneC.glabrataisolatewasclassifiedasechinocandin-resistant
byEtest. Unfortunately,thelackofresistantisolatesinthisstudy’scollectionhinderedmakingany
recommendationsregardingaccurateresistancedetectionbytheEtestmethod.
3. NonconventionalPhenotypicAssaysforTestingFungalSusceptibility
MALDI-TOFMassSpectrometry-BasedAFSTMethods
InmanyEuropeanclinicalmicrobiologylaboratoriessuchasours,theadventofmatrix-assisted
laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has drastically
altered the routine diagnostic workflow [55,56]. As a result, MALDI-TOF MS offers the chance
to identify almost all microbial genera and species with unprecedented reliability, rapidity, and
cost-effectiveness [57]. However, this has occurred quickly for bacteria [58] but not so quickly for
fungi [59]. Difficulties linked to the complexity of the fungal cell hampered early optimization of
sampleanalyticalproceduresand,consequently,large-scaleadoptionofMALDI-TOFMSinmedical
mycology[59]. Todate,MALDI-TOFMSanalysisissuitedformicrobialisolatesculturedfromprimary
samples[60]orpositivebloodcultures[61], significantlyreducingtheturnaroundtimecompared
tobiochemical[62]ornucleicacid–basedtechniques,suchasDNAsequencing[63]. However,high
MALDI-TOF MS performance, particularly for cryptic species within the Candida and Aspergillus
speciescomplexes,canonlybeachievedwiththeappropriatedatabasesprovidedbysomemarketed
MALDI-TOF MS systems [59,60]. While susceptibility testing methods are not directly applicable
to primary samples [64], some recent studies have reported success with phenotype-centered (or
semimolecular)MALDI-TOFMSmethodsforAFST[65].
Inkeepingwithpreviousworkthatintroducedtheminimalprofilechangeconcentration(MPCC)
asanewendpointforAFST[66],ourresearchgroupdevelopedMALDI-TOFMS–basedassaysfor
testingtheechinocandinsusceptibilityoffungalspecies. Inoneofthefirstassays,DeCarolisetal.[67]
obtained mass spectra from fungal cells exposed to different caspofungin concentrations for 15 h,
andthenmatchedthe“intermediate”massspectrawitheachofthe“extreme”massspectrausing
compositecorrelationindex(CCI)analysis. MPCCrepresentstheCCIvalueatwhichaspectrumis
moresimilartothespectrumobservedatthemaximalcaspofunginconcentration(maximumCCI)
thanthespectrumobservedatthenullcaspofunginconcentration(nullCCI).Theauthorsshowedthat
MPCCvaluesapproximatedMIC(orMEC)valuesfor100%ofCandidaandAspergillusisolatestested(in
total,44amongWTandFKS1mutantisolates). Inthesecondassay,whichwasthefirstonesimplified
(herenamedMS-AFST),Vellaetal.[68]provideddiscriminationbetweensusceptibleandresistant
isolatesofC.albicansafter3hofexposureoffungalcellsatthreeantifungaldruglevels: nodrug(null
concentration),intermediate(“breakpoint”),andmaximum(maximalconcentration). Bymeansofthis
“three-point”assay,isolatesweresusceptibleorresistantwhentheCCIvaluesobtainedbymatching
theirbreakpointspectrumwiththeirmaximumspectrumwere,respectively,higherorlowerthanthe
CCIvaluesobtainedbymatchingtheirbreakpointspectrumwiththeirspectrumatnullconcentration.
Usingthiscriterion,100%(51/51)and90.9%(10/11)oftheisolatestestedyieldedMALDIAFSTresults
thatwereinaccordancewiththeWTorFKS1-mutantgenotype,respectively.
Toextendourfindings,Vellaetal.[69]triedtovalidatethe3hMS-AFSTassaywithapanelof80
clinicalisolatesofC.glabratatestedagainstechinocandin(anidulafungin)andtriazole(fluconazole)
antifungal agents. Although acquired azole resistance in Candida species is multifaceted [70],
theinductionofdrugeffluxencodedbyCDRgenesandregulationoftheirexpressionbymutationsin
thetranscriptionfactorCgPdr1(encodedbytheCgPDR1gene)representthemostclinicallyrelevant
J.Fungi2018,4,110 7of16
molecularmechanisms[71,72].Thestudy[69]revealedthat85.0%(68/80)and96.2%(77/80)ofisolates
had classification results for anidulafungin and fluconazole that fully agreed with those obtained
bytheFKS1/FKS2genotypeorCgCDR1/CDR2overexpressionantifungal-resistancemechanisms.
WhenanalyzingtheMS-AFSTresultsaccordingtotheFKS1/FKS2genotype,agreementwas100%
(6/6)forisolateswithamutatedFKS1geneand25.0%(4/16)forisolateswithamutatedFKS2gene.
Thisresultedin15.0%ofincorrectclassificationsforanidulafunginthatinvolvedFKS2HS1mutations.
AccordingtotheCLSIreferencemethod,MS-AFSTassaysyieldedasmanyas11verymajorerrors(i.e.,
aresistantisolatemisclassifiedassusceptible)withanidulafungin,andonlytwoverymajorerrors
withfluconazole. Interestingly,discrepanciescouldberesolvedwithMS-AFSTassaysperformedat
15hofexposuretobothantifungaldrugs;inthiscase,MPCCvalueswerecoincidentwiththeMICsfor
thoseisolatesshowingdiscrepantresults. Takentogether,thesefindingsdemonstratethatMS-AFSTin
the3hformatfailedtodetectC.glabrataisolateswithechinocandin-associatedFKS2mutations.
Contemporarily, but independently from us, Gitman et al. [73] explored the MALDI-TOF
MS–basedmethodtodifferentiateWT(noacquiredresistance)fromnon-WT(acquiredresistance)
isolatesof20Aspergillusspecies(including17A.fumigatus)withrespecttovoriconazole(MIC>ECV;
1µg/mL).Fourof17A.fumigatuswerephenotypicallyandgenotypicallyresistanttovoriconazole,
whereastwoisolatesofAspergillusustusandoneisolateofAspergilluscalidoustusservedascontrols
due to their intrinsic low azole susceptibility. Except for two of four mutant isolates misclassified
asWT,MALDI-TOFMSMPCCsatthe24htimepointwerewithin ±1dilutionoftheBMDMICs
(determinedbytheSYOmethod)forallAspergillusisolates. Inlinewithourstudies[67–69],slightly
longerincubationtimes(30and48h)allowedforaccuratedetectionofisolatesasWTornon-WTby
MS-AFST,incompleteagreementwiththeCYP51Agenesequenceanalysisresults. Remarkably,using
the“three-point”assay(nodrug,16µg/mLvoriconazole,andvoriconazoleECV),theauthors[73]
obtainedthesamefindings,requiring30to48hofincubationwiththedrugpriortoMALDI-TOF
MSanalysis. Itislikelythatsomefungalisolatesrequiringadditionaltimerelatestotheimpossibility
oftheirreachingenoughgrowthand,consequently,levelofexpressedproteinstoallowforcorrect
classificationbytheMS-AFSTassay[69,73].
In the above-mentioned study, Vella et al. [69] obtained reproducibility rates of 98.7% and
97.5%whentestingC.glabrataagainstanidulafunginandfluconazole,respectively,withonlythree
inconsistencies,subsequentlyarbitratedbyathirdrunbeforeincludingthemintheMS-AFSTanalysis.
Inonestudyperformedsimilarlytothatdescribedoriginallybyus[67],Saraclietal.[74]showedthat
thereproducibilityoftheMALDI-TOFMS–basedassayfordiscriminatingsusceptibleandresistant
isolatesofCandidaspecies(35C.albicans,35C.glabrata,and37C.tropicalis)totriazolesvariedbetween
54.3%and82.9%.However,thereproducibilitywashigherforC.glabrataisolates(77.1%forfluconazole)
thanforisolatesfromotherCandidaspecies. Inaddition,applyinga5%toleranceforevaluationofthe
CCIratio(CCI /CCI ratio>1(MS-AFSTassay-classifiedassusceptible);CCI /CCI ratio<1
max null max null
(MS-AFSTassay-classifiedasresistant))didresultinadecreaseinthepercentagesofverymajorand
majorerrorsbyupto33.3%.
4. HowtoBetterUsePhenotypicFungalSusceptibilityResultsintheClinicSetting
Intendedtoreliablyidentifypatientswhoseinfectionislikelytorespondtoagivenantifungal
agent,invitrosusceptibilitytestinghasimprovedourabilitytopredicttheoutcomeoftherapy[9],yet
isconstantlyconfrontedwithincreasingresistancetoantifungalagents[3].Sincethewidespreaduseof
triazolesinearly1990,antifungalresistanceinbothCandidaandAspergillushasbecomeaseriouspublic
healthproblem[75,76]. Theriseinechinocandinresistance,azoleresistance,andcross-resistanceto
twoormoreantifungalclasses(multidrugresistance)inpathogenicfungi[77]hasinvolvedspeciesof
CandidasuchasC.glabrata[78]and,lately,C.auris[79]. Additionally,theincidenceofazole-resistant
A.fumigatushasjeopardizedoutcomesforhigh-riskpatients,becausetheexclusionofazoleantifungal
drugsfromprophylaxisorfirst-linetreatmentofinvasiveaspergillosiswouldlimitdrugchoices[43].
However, we are conscious that various host, drug, and fungal factors contribute to therapeutic
J.Fungi2018,4,110 8of16
failures,andthereisnoabsoluteassociationbetweeninvitroMICandclinicalresponse[75]. Thishas
precludedCLSIorEUCASTfromestablishingCBPsforsomeantifungalagentsandfungalspecies,
J. Fungi 2018, 4, x 8 of 16
eventhoughboththeCLSIandEUCASTmethods,whichresultindifferentCBPs,aregroundedin
pharrmesauclot diny ndaimffeircernets pCoBnPsse, sairne agnriomunadlemdo dine lpshaanrdmpacaotdieynntasm[7ic5 ]r.esponses in animal models and
pCaotinencetsr n[7in5]g. Candida,antifungalsusceptibilityispredictableiftheinfectingorganismisidentified
Concerning Candida, antifungal susceptibility is predictable if the infecting organism is
to the species level, but individual isolates may not follow this course, thus requiring antifungal
identified to the species level, but individual isolates may not follow this course, thus requiring
testing [9]. Importantly, the Infectious Diseases Society of America (IDSA) guidelines for the
antifungal testing [9]. Importantly, the Infectious Diseases Society of America (IDSA) guidelines for
management of candidiasis recommend routinely performing AFST for C. glabrata against azoles
the management of candidiasis recommend routinely performing AFST for C. glabrata against azoles
andechinocandins[5]. ThesameguidelinesmentionthatroutinetestingforCandidaspeciesother
and echinocandins [5]. The same guidelines mention that routine testing for Candida species other
thanC.glabratahaslessvalue[5]. Nevertheless,weagreewiththeopinionbyOstrosky-Zeichnerand
than C. glabrata has less value [5]. Nevertheless, we agree with the opinion by Ostrosky-Zeichner and
AndAesnd[9e]s t[h9]a tthraot urotiunteinlyelyte tsetsitningga annttiiffuunnggaall ssuusscecpeptibtiibliitlyit oyf oalfl abllloobdlosotrdeasmtre aanmd satnerdiles tseitrei liesosliatteesi soof lates
ofCaCnadniddiadas pspeeccieiess mmaayy bbee hheellppffuull ttoo pprroovvididee aann inindedxe xfofro srussucespcetipbtiliibtyil ittryentrdesn adnsda tnhde tehmeeergmenercge eonf ceof
resisrteasniscteanlocec alollcyalalyn danrde greiognioanlalyll.yH. Hoowweevveerr,, iinn aa rreessoouurrccee--rreesstrtircitcetde denevnirvoinromnemnte, nAtF,SATF sShTouslhdo fuolcdusf ocus
on isoonl aisteoslafteros mfrocma sceasseosf otfr etaretamtmenenttf afailiulurree,, bbrreeaakktthhrroouugghh ininfefcetciotino, no,r olrimliimtedit ethderthapeeruatpice uotpitcioonpst, ions,
whicwhhaicreh caoren csoenqsueeqnuceensceosf ouf nudnedrelrylyininggc coommoorrbbiiddiittiieess, ,aaddvveresres eeveevnetsn,t osr, oprrepvrieovuiso aunstiafunntigfauln ugsae l[9u]s. e[9].
Consistent with this, in a scenario of prior echinocandin exposure, Vella et al. [69] proposed that
Consistentwiththis,inascenarioofpriorechinocandinexposure,Vellaetal.[69]proposedthatrapid
rapid detection of C. glabrata isolates as fluconazole-resistant by the MS-AFST assay could alert
detectionofC.glabrataisolatesasfluconazole-resistantbytheMS-AFSTassaycouldalertcliniciansto
clinicians to the potential presence of anidulafungin resistance in these isolates (Figure 1). Despite
thepotentialpresenceofanidulafunginresistanceintheseisolates(Figure1). Despiterepresentinga
representing a personal view of the utility of this approach (and not a recommendation), Figure 1
personalviewoftheutilityofthisapproach(andnotarecommendation),Figure1underlinesthat
underlines that using anidulafungin as a surrogate marker would lead to a scenario in which, if no
using anidulafungin as a surrogate marker would lead to a scenario in which, if no resistance is
resistance is detected, either of the three echinocandins (not only anidulafungin) could be
detecatdemdi,neiistthereerdo. fthethreeechinocandins(notonlyanidulafungin)couldbeadministered.

FiguFrieg1u.reP 1o.t Penotteianltitarle tartematemnetnot foifn ivnvaasisviveeC C.. ggllaabbrraattaa iinnffeecctitoionn babsaesde dono mnamssa sspsescptreocmtreotmrye–t aryn–tifaunntgifaul ngal
suscseupstcibepiltiitbyiltiteys ttienstgin(gM (SM-AS-FASFTS)Tr) erseusultlsts.. IInn aa cclliinniiccaall ccoonntetextx tofo pfrpioriro erchecinhoincaoncdainnd eixnpeoxsuproes, urerseu,lrtes sults
ofsuosfc seupstciebpiltiitbyiliotyr roers riesstiasntacnecteo toa naindiduulalaffuunnggiinn aanndd//oor rflflucuocnoanzaozleo lweiwthiinth 3i nh 3orh, oinr ,ciansecsa osfe sisoolfaitseos lates
with FKS2 HS1 mutations, 6–12 h after testing may guide the appropriate administration of
withFKS2HS1mutations,6–12haftertestingmayguidetheappropriateadministrationofantifungal
antifungal therapy [69].
therapy[69].
J.Fungi2018,4,110 9of16
ConcerningAspergillus,theIDSAguidelinesforthemanagementofaspergillosisrecommend
performingsusceptibilitytestingagainstazolesprimarilyforpatientswhofailtorespondtotherapy
or for epidemiological purposes [6]. However, these guidelines do not specify laboratory-testing
proceduresfortheisolationofAspergillusfromrespiratorytractsamples[46]. Recently,aninternational
expert panel convened to deliberate the management of azole-resistant invasive aspergillosis,
concluding that in culture-positive cases, invitro susceptibility testing is highly indicated when
antifungaltherapyisintended[80]. Uptofivecoloniesneedtobetestedinpatientswhoaretoreceive
antifungal therapy in geographic regions with azole resistance [80], which is also recommended
by the European guidelines for aspergillosis published in early 2018 [81]. Clinically used triazole
antifungalsarederivativesofeitherfluconazole(voriconazoleandisavuconazole)orketoconazole
(itraconazole and posaconazole) as the lead compound. This correlates with the cross-resistance
phenotypesobservedinclinical(andenvironmental)A.fumigatusisolateswiththeCYP51Amutations
thatarewithintheitraconazole/voriconazoleandvoriconazole/isavuconazolecompoundpairs[46].
Therefore, while at least voriconazole and itraconazole are recommended as screening drugs, the
VIPcheck(Nijmegen,TheNetherlands),acommercialagar-basedmethodthatconsistsoffourwells
containingvoriconazole,itraconazole,posaconazole,oragrowthcontrol,wasdevelopedforeasily
discriminatingbetweenazole-susceptibleand-resistantisolatesofA.fumigatus[82]. Clinicalscreening
studiesareencouragedtogenerateepidemiologicaldata,whichinturnmayhelptoreassessclinical
treatmentoptionsonalocalornationalbasis.Inthiscontext,Dudakovaetal.[46]proposedaworkflow
forevaluatingA.fumigatusisolatesfromsuchscreeningstudiesthatidentifiestruepositivesandyields
robust data on the prevalence and phylogenetic relatedness of resistant isolates (Figure 2). In the
authors’ own experience, the species of each isolate can be readily determined via MALDI-TOF
MS using the commercial databases. The isolates with a dark blue-green appearance, which is
common to A. fumigatus and its sibling species, but not a reliable MALDI-TOF MS identification
as A. fumigatus, prove to be cryptic species not yet included in the scheme presented (Figure 2).
Additionally,theauthorscompiledtheMICvaluescorrelatingwithindividualaminoacidsubstitutions
intheCYP51A-encodedenzymeforinterpretationofDNAsequencingdata,especiallyintheabsence
ofculturedA.fumigatusisolates.
AFSTcontinuestobeinstrumentalinidentifyingcasesofinvasivefungalinfectionsassociated
with elevated MICs and in detecting antifungal resistance phenotypes, i.e., determining which
antifungal agents are likely to be clinically inactive [83]. However, for patients with candidemia
who are experiencing clinical response and are infected by seemingly resistant isolates, there is
thepossibilityofMICmisinterpretationduetoartifactssuchastheparadoxicalgrowthseenwith
echinocandinsandthetrailingeffectseenwithazoles[84]. Ontheotherhand,theclinicalmicrobiology
laboratoryisoftenunabletoprovideinformationinaperiodthatcaninforminitialantifungaltreatment
decisions.Forexample,thetotalturnaroundtimeforthecurrentantifungalresistancedetectionprocess
forpatientswithbloodstreaminfectionsis3–5days,sinceitrequiresthreeovernightgrowthsteps
(bloodculture,subculture,andAFSTculture)[63]. Moleculardetectionofresistancegenes,especially
when applied to primary clinical samples [85,86], may be a useful adjunct for fungal surveillance
and disease diagnosis [46]. However, these methods generally do not provide a “comprehensive
assessment of what an organism can be treated with” [87]. Nowadays, growth-based phenotypic
AFSTisstillrequiredtofullydeterminethesusceptibilityprofileoftheinfectingpathogen. Therefore,
recent efforts have led to the development of innovative technologies that promise to shorten the
turnaroundtimeforphenotypicAFSTresults[88]. Designedtoidentifycommoncausesofbacteremia
(~90min)aswellasperformantimicrobialsusceptibilitytesting(~7h)usingpositivebloodcultures,the
FoodandDrugAdministration(FDA)-clearedAcceleratePhenoTestTMBloodCulturekitrepresents
an important breakthrough in the development of rapid phenotypic antimicrobial susceptibility
testing[89]. Furtherapplicationofthisorothergame-changingtechnologiessuchasMALDI-TOF
MS[90]tofungiwouldallowforsimilargainsinAFSTperformance,alongwithimprovementsof
fungaldiagnosticcapability[91].
J.Fungi2018,4,110 10of16
J. Fungi 2018, 4, x 10 of 16

FFiigguurree 22.. PPootteennttiiaall ssccrreeeenniinngg ooff aazzoollee--rreessiissttaanntt AA.. ffuummiiggaattuuss iissoollaatteess.. SShhoowwnn iiss aa sstteeppwwiissee wwoorrkkffllooww
tthhaatt eelliimmiinnaatteess ffaallssee--ppoossiittiivvee rreessuullttss ((iinnddiiccaatteedd bbyy aa rreedd XX)) aanndd ccaann bbee hheellppffuull ttoo bbuuiilldd uupp llooccaall
eeppiiddeemmiioollooggiiccaall ddaattaa oonn tthhee pprreevvaalleennccee aanndd pphhyyllooggeenneettiicc lliinnkkaaggee ooff rreessiissttaanntt iissoollaatteess.. IInn tthhiiss ffiigguurree,,
ttrruuee ppoossiittiivveess aarree ddeerriivveedd ffrroomm tthhee ggrroowwtthh ooff iissoollaatteess oonn sseelleeccttiivvee aaggaarr ppllaatteess ccoonnttaaiinniinngg ddrruugg
ccoonncceennttrraattiioonnss tthhaatt rraannggee ffrroomm 00..55 µgg//mmLL ffoorr ppoossaaccoonnaazzoollee ((PPSSZZ)) ttoo 11 ttoo 44 µgg//mmLL ffoorr iittrraaccoonnaazzoollee
(ITZ) or voriconazole (VRZ). Then, isolates are identified at the species level by multilocus DNA
(ITZ) or voriconazole (VRZ). Then, isolates are identified at the species level by multilocus DNA
sequencing[46](gotpermissiontouse).
sequencing [46] (got permission to use).
5. Conclusions
AFST continues to be instrumental in identifying cases of invasive fungal infections associated
with AeslefvoarteCda nMdiIdCasa nadndA sipne rdgeiltleucst,inthge ainnfteifcutinogna-lr erleastiesdtamncoer tpalhiteynoratytepseas,r ei.set.i,l lduentearcmceipnitnabgl ywhhiigchh,
adnetsipfuitnegarel caegnetntasd avraen lickeeslyin top breo cplhinyilcaaxlilsy, ineaacrltyived [i8a3g]n. oHsoisw, eavnedr, tfroera ptmateiennttso wf iftuhn cgaanlddidiseemasiae sw[h1o].
aArne tiefxupnegraielnpcrionpg hcyllianxicisal wreitshpoannsteim aonldd -aarceti vinefeacztoelde bcyo msepeomunindgsly( proessaisctoannat ziosloelaoters,v othriecroen iasz othlee)
ptoosrseibdiulictey tohf eMinICci dmeniscineteorfprientvaatisoivne dmueo ltdo ianrfteifcatciotsn ssuicnh haisg hth-reis kpapraadtioexnitcsalm garyowbteh asseseonc iwatiethd
ewcihthinobcraenakdtihnrso uagnhd inthfee cttiroanilsincga uesfefdecbt ysereanre wmiuthlt idazruogle-sr es[8is4t]a. nOt mn otlhdes [o9t2h]e.r Thhaenrdef,o rteh,e acccliunriacatel
mdeitcerrombiionlaotgioyn laobfoarnattoifruyn gisa olfstuesnc eupntaibbillei ttyo opfrfouvnidgie, winhfoicrmhamtiaoyn ailns oa inpcelruiodde tnhoant -cAasnp eirngfiollrums minoitlidasl
a(Mntuifcuonraglaels ,tFreuastamrieunmt sdpepci.s,ioornsS.c eFdoors peoxraiummplaep, iothspee rtomtualm tucornmaproleuxn)d, istimmea nfdora ttohrey cautrlreeanstt ainntsifpuencgifiacl
rseitsuisattainocnes dduetreicntgiotnh epcraorceesosf fpoart ipeanttisenwtsit hwiinthv absliovoedfsutnregaamli ninfefecctitoionnss[ 9is]. 3W–5h edtahyesr, tshiencceo nitv reenqtuioinreasl
tahnrteiem oicvreorbniiaglhstu gsrcoewptthib siltietpyst (ebsltosoadr ecustltiullrue,s seufublcuwltilulrde,e apnedn dAFoSnTh couwltufarest) [t6h3e].m Maorclehcutolawr adredtercatpioind
of resistance genes, especially when applied to primary clinical samples [85,86], may be a useful
adjunct for fungal surveillance and disease diagnosis [46]. However, these methods generally do not