Molecular Methods for the Diagnosis of Invasive Candidiasis

Abstract

Invasive infections caused by members of the genus Candida are on the rise. Especially patients in intensive care units, immunocompromised patients, and those recovering from abdominal surgery are at risk for the development of candidemia or deep-seated candidiasis. Rapid initiation of appropriate antifungal therapy can increase survival rates significantly. In the past, most of these infections were caused by C. albicans, a species that typically is very susceptible to antifungals. However, in recent years a shift towards infections caused by non-albicans species displaying various susceptibly patterns has been observed and the prompt diagnosis of the underlying species has become an essential factor determining the therapeutic outcome. The gold standard for diagnosing invasive candidiasis is blood culture, even though its sensitivity is low and the time required for species identification usually exceeds 48 h. To overcome these issues, blood culture can be combined with other methods, and a large number of tests have been developed for this purpose. The aim of this review was to give an overview on strengths and limitations of currently available molecular methods for the diagnosis of invasive candidiasis.

Full text

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J.Fungi2020,6,101;doi:10.3390/jof6030101www.mdpi.com/journal/jof
Review
MolecularMethodsfortheDiagnosis
ofInvasiveCandidiasis
IrisCamp,KathrinSpettelandBirgitWillinger*

DivisionofClinicalMicrobiology,DepartmentofLaboratoryMedicine,MedicalUniversityofVienna,
1090Vienna,Austria;iris.camp@meduniwien.ac.at(I.C.);kathrin.spettel@meduniwien.ac.at(K.S.)
*Correspondence:birgit.willinger@meduniwien.ac.at
Received:25May2020;Accepted:4July2020;Published:6July2020
Abstract:InvasiveinfectionscausedbymembersofthegenusCandidaareontherise.Especially
patientsinintensivecareunits,immunocompromisedpatients,andthoserecoveringfrom
abdominalsurgeryareatriskforthedevelopmentofcandidemiaordeep‐seatedcandidiasis.Rapid
initiationofappropriateantifungaltherapycanincreasesurvivalratessignificantly.Inthepast,
mostoftheseinfectionswerecausedbyC.albicans,aspeciesthattypicallyisverysusceptibleto
antifungals.However,inrecentyearsashifttowardsinfectionscausedbynon‐albicansspecies
displayingvarioussusceptiblypatternshasbeenobservedandthepromptdiagnosisofthe
underlyingspecieshasbecomeanessentialfactordeterminingthetherapeuticoutcome.Thegold
standardfordiagnosinginvasivecandidiasisisbloodculture,eventhoughitssensitivityislowand
thetimerequiredforspeciesidentificationusuallyexceeds48h.Toovercometheseissues,blood
culturecanbecombinedwithothermethods,andalargenumberoftestshavebeendevelopedfor
thispurpose.Theaimofthisreviewwastogiveanoverviewonstrengthsandlimitationsof
currentlyavailablemolecularmethodsforthediagnosisofinvasivecandidiasis.
Keywords:Candida;invasive;diagnosis;molecular;candidemia;T2
1.Introduction
ThegenusCandidacomprisesadiversegroupofdimorphicfungithatarecommensalinhabitants
ofmucousmembranes[1]somespecies,likeC.parapsilosis,additionallycanbefoundascolonizers
onthehumanskin[2].Candidaspecies,therefore,areoftenisolatedfromnon‐sterileclinicalsamples,
suchasswabsfromthegastrointestinalorurogenitaltract.Eventhoughthesefindingsusuallydo
notholdanypathologicvalueinasymptomaticimmunocompetentpatients,Candidacancause
invasiveinfectionsthataremarkedbyhighmortalityrates[3,4].Especiallypatientsonintensivecare
units(ICU),aswellasimmunosuppressedorneutropenicpatients,areathigherriskforthe
developmentofaninvasivecandidiasis(IC)[5],andimprovedtreatmentstrategiesandsurvivalrates
foranumberofseverediseaseslikehematologicmalignancieshaveledtoanevergrowingpoolof
patientssusceptibletoandaffectedbyinvasivefungaldiseases[6].Thepopulation‐basedincidence
ofIChasbeenreportedtobebetween1.9and24cases/100,000/peryear[7–16].Worldwide,over
250,000casesofICandmorethan50,000deathsperyearareduetotheseinfections[17].ICoften,but
notalways,goesalongwithcandidemia,andindeedCandidaspp.havebeenreferredtobeingthe
fourthmostcommoncauseofnosocomialbloodstreaminfections[18].
Inmostcases,ICoriginatesfromthepatient’sownflora,andtheriskforthedevelopmentof
suchaninfectionincreaseswiththenumberofbodysitescolonizedbyCandida[19–21].Eventhough
C.albicansisstillthespeciesresponsibleformostcasesofIC,moreandmoreinvasiveinfectionsdue
tonon‐albicansspecieshavebeennotedinrecentyears[22–27].Thisisrelevantasnotonlyvirulence
andpathogenicitybutalsoresistanceprofilesvarybetweenspecies.WhileC.albicansusuallyis
susceptibletoallmajorgroupsofantifungals,C.glabratacanacquireresistancetoazoles[28],C.

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parapsilosisandC.guilliermondiitoechinocandins[29,30],C.lusitaniaemaybelesssusceptibleto
amphotericinB[31],andC.kruseiisolatesareintrinsicallyresistanttofluconazole.Thus,theobserved
shifttowardsnon‐albicansspeciesmakesitmoredifficulttochoosetheappropriateempirictherapy.
AnotherconcernistheemergenceofC.auris.Thisoftenmulti‐resistantpathogenwasfirstdescribed
inJapanin2009[32].Sincethen,severaloutbreakshavebeenreported[33–35].
ThespectrumofclinicalsignsandsymptomsofICiswideandcanbeunspecific,butaninvasive
fungaldiseaseshouldalwaysbeconsideredifthepatient’sconditiondoesnotimproveunder
antibiotictherapy,especiallyifcolonizationwithCandidaspp.hasbeenobservedinahigh‐risk
patient.ToimprovetheoutcomeofIC,thepromptinitiationofanappropriateantimycotictherapy
isessential[36].Giventhedifferencesinresistancepatterns,fastspecies‐levelidentificationis
requiredforchoosingthecorrectantimycoticagentwhenresultsofantimicrobialsusceptibility
testingarenotyetavailable.
Cultureremainsoneofthekeymethodsfordiagnosingafungalinfection.However,the
definitivetreatmentofICisoftendelayedbytheinsensitivityofculture,andthisdelaymayleadto
highmortalityrates(35–75%)[37].Eventhoughbloodcultures(BC)aresensitiveatdetectingviable
Candidacells,withalimitofdetectionofonecolonyformingunit(CFU)/mL,theiroverallsensitivity
acrossthespectrumofICisonly50%,andtheyhavealagtimeforidentificationofupto5days
[38,39].Nevertheless,BCiscurrentlyconsideredthe“goldstandard”intheeventofanysuspected
caseofinvasivefungalinfection,butthecombinationofculturewithothermethodscanfacilitatea
timelierdiagnosis.Molecularamplificationtechniquesenablefastandsensitivedetectionand
identificationbydirectlydetectingandanalyzingtinyamountsoffungalDNApresentinaclinical
samplewithouttheneedforpriorcultivation,whichmakesthesetestsappealingfortheearly
diagnosisofIC,particularlyforcasesofICthataremissedbyculture.MultiplePCRassaystargeting
variousgeneticsequences(18SrDNA,28SrDNA,5.8SrDNA,internaltranscribedspacerregionsand
mitochondrialDNA)havebeendevelopedforthedetectionofabroadrangeoffungiindifferent
specimenssuchasblood,serum,plasma,bronchoalveolarlavage(BAL),sterilefluidsandtissues.
Dependingontheprimersused(i.e.primerstargetingeitherconservedorspecies‐specificregions),
fungalpathogenscanbedetectedinapanfungaloramorespecificmanner.Thesensitivityand
specificityofthevarioustechniquesarevariable,butmostlyanimprovedsensitivityisobserved
whencomparedtoclassicalculturalbasedmethods[40].Apartfromthedetectionandanalysisof
nucleicacids,molecularassayscanalsobebasedonproteomicprofiling.Inthisreview,wewillfocus
oncommerciallyavailabletests(Figure1).

Figure1.OverviewofavailablemoleculartestsforthediagnosisofinvasiveCandidainfections.BDG:
beta‐D‐glucan;
a
wholeblood,
b
wholeblood,plasmaandserum,
c
plasmaandsyntheticBAL,
d
various
clinicalsamples,
e
extractedDNA

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Table1.Listofbloodculture‐dependenttests.
ProductManufacturerCandidaspp.
Detected
Assay
TimeMethodApproval
noGramstainrequired
SepsiTyper®Bruker
Daltonicspan‐Candida15–20
min
protein
extraction
followed
by
MALDI‐
TOFMS
CE/IVD
FilmArray®BCID
PanelBiomerieux
C.albicans,
C.glabrata,
C.parapsilosis,
C.tropicalis,
C.krusei
60minMultiplex
PCRCE/IVD
Accelerate
PhenoTestTMBCKit
Accelerate
Diagnostics
C.albicans,
C.glabrata90minautomated
FISHCE/IVD
SepsisFlowChipMaster
DiagnosticaC.albicans3hMultiplex
PCRCE/IVD
Gramstainrequired
CandidaQuickFISH®OpGen
C.albicans
C.parapsilosis,
C.glabrata
20minFISHCE/IVD
YeastTrafficLight
PNAFISH®OpGen
C.albicans/C.
parapsilosis,
C.tropicalis,
C.glabrata/C.
krusei
90minFISHCE/IVD
eplex®BCIDFPPanelGenMarkDx
C.albicans,
C.auris,
C.dubliniensis,
C.famata,
C.glabrata,
C.guilliermondii,
C.kefyr,
C.krusei,
C.lusitaniae,
C.parapsilosis,
C.tropicalis
90minMultiplex
PCRCE/IVD
MALDI‐TOFMS:matrix‐assistedlaserdesorption/ionizationtimeofflightmassspectrometry;
CE/IVD:ConformitèEuropëenne/invitrodiagnostic;FISH:fluorescenceinsituhybridization.
2.BloodCulture‐DependentMolecularDiagnostics
Thesetestsystems(Table1)aredesignedforusewithaliquotsofpositivebloodculturesamples.
Thus,thetimerequiredforapositivebloodculturecannotbeeliminated.Asthepathogenloadis
highinpositivebloodculturebottles,sensitivityisnotachallengefortheseassays.
TheMALDISepsityper®IVDKit(BrukerDaltonics,Bremen,Germany)allowsforpathogen
identificationviaMALDI‐TOFMSanalysisfromaliquotsofpositivebloodculturebottlesafterashort
proteinextraction.Inarecentlypublishedstudy[41],62.5%ofallCandidaisolatescouldbeidentified
withtheMALDISepsityper®IVDKitdirectlyfrompositivebloodculturebottles.IfaBruker
Biotyperinstrumentisavailable,thistestisaratherinexpensivealternativetotestsbasedonnucleic
aciddetection.Anotherbenefitisthepotentialtoidentifyallpathogensincludedinthedatabase;
thus,rareyeastsincludingC.auriscanalsobeidentified.

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TheFilmArray®BCIDPanel(Biomerieux,Marcyl’Etoile,France)isaConformitèEuropëenne/in
vitrodiagnostic(CE/IVD)‐certifiednestedmultiplexPCRsystem.Twenty‐fourpathogens,including
thefivemostcommonCandidaspp(C.albicans,C.glabrata,C.parapsilosis,C.tropicalis,andC.krusei),
canbedetectedbytheassaywithminimalhands‐ontimeandaturn‐aroundtimeof1h.Inastudy
withbothclinicalandspikedsamples,asensitivityof99.2%andaspecificityof99.9%wereobserved
forCandidaspp.whenresultsoftheBCIDpanelwerecomparedwithconventionalculture[42].In
anotherrecentlypublishedstudy,theFilmArray®BCIDpanelwasperformedon85positiveblood
cultureswithyeastsvisibleintheGramstain.Atotalof91yeaststrainswereisolatedbyculture,and
84oftheisolatesbelongedtooneofthefiveCandidaspeciescontainedinthepanel.Allofthosewere
identified.Sevenisolatesbelongedtospeciesnottargetedbythetest;thosewerenotdetected.Ten
bloodculturescontainedmorethanonepathogen,andallpathogensincludedinthepanelwere
identifiedcorrectly[43].SincethetestpanelincludesyeastsaswellasGram‐positiveandGram‐
negativebacteria,itisnotnecessarytoperformaGram‐stainpriortotheassay.
TheAcceleratePhenoTestTMBCKit(AccelerateDiagnostics,Tuscon,AZ,USA)isanother
CE/IVDapprovedtestsystemthatallowstheidentificationandrapidphenotypicantimicrobial
susceptibilitytestingofseveralGram‐positiveandGram‐negativebacteriainpositivebloodcultures.
Inadditiontobacterialpathogens,thesystemcandetectC.albicansandC.glabrata.However,rapid
susceptibilitytestingisnotavailableforfungi.ThetestwasrecentlyevaluatedinastudybyBurnham
etal.[44];10ofthe125bloodculturespositiveforpathogensincludedinthetest’spanelcontained
C.glabrata,and5culturescontainedC.albicans.TheassayfailedtodetectC.glabrataintwoofthese
samples,whilethreefalsepositiveresultswithC.glabrata,aswellasonefalsepositiveC.albicans
result,werereported.Thus,forC.albicans,asensitivityof100%andaspecificityof99.3%were
reported,whilethesensitivityandspecificityforC.glabratawere80.0%and97.9%respectively.Five
falsepositiveresultsforC.glabratawerealsoreportedinastudypublishedin2017[45].Atthetime,
thisunfavorableoutcomewasexplainedbytheuseoftheoldersoftwareversion(v1.0),anditwas
discussedthatsoftwareupdatesmightresolvetheissue.
TheSepsisFlowChip(MasterDiagnostica,Granada,Spain)isaCEandIVDapprovedmultiplex
PCRtestwhichisabletodetect22resistancegenesinadditiontomorethan40pathogensincluding
C.albicansfrompositiveBCinthreehours.Thisisachievedbytheuseofbiotinylatedprimers,
automatedreversehybridizationtoachipmembraneandsubsequentimmunoenzymaticdetection
ofpositivesignals.Inthefirstevaluationonclinicalsamples[46],sixyeast‐positiveBCwereincluded.
FiveofthesecontainedC.albicans,andallweredetectedbythetest.OneBCcontainedC.parapsilosis;
forthissample,theassaydidnotyieldapositiveresult.Thus,forC.albicans,asensitivityof100%
wasreported.
TheCandidaQuickFISH®andtheYeastTrafficLightPNAFISH®(OpGen,Gaithersburg,MA,
USA)areCE/IVDcertifiedtestsusingpeptidenucleicacidprobesforfluorescenceinsitu
hybridization.Theycanbeperformeddirectlyonaliquotsfromyeastpositivebloodculturebottles
(i.e.whenyeastcellshavebeenobservedintheGramstain).WiththeYeastTrafficLightPNAFISH®,
identificationofC.albicans/C.parapsilosis,C.tropicalis,andC.glabrata/C.kruseiispossiblewithin90
min,whiletheCandidaQuickFISH®candetectC.albicans,C.parapsilosis,andC.glabratawithin20
min.Inalargeevaluationstudywith216bloodculturesamples[47],theYeastTrafficLightPNA
FISH®yieldedthecorrectresultin96%ofcases.OneisolateofC.parapsilosiswasmisidentifiedasC.
tropicalis,andafalsenegativeresultwasobtainedforonecaseofC.parapsilosisandonecaseofC.
tropicalis.Additionally,crossreactivitywithC.bracarensis,C.nivariensis,C.orthopsilosis,N.delphensis,
andR.mucilaginosawasobserved.Drawbacksmightincludethelimitedspectrum,aswellasthe
requirementforafluorescencemicroscope.
FortheePlex®BCID(GenMarkDX,Carlsbad,CA,USA),arecentlyCE/IVDcertifiedsystem,
thereisachoiceofthreepanels.Thus,dependingontheresultsoftheGram‐stainperformedon
positivebloodcultures,therespectivepanelwillbeused.Thefungalpathogen(FP)paneltargets11
Candidaspp.(C.albicans,C.auris,C.dubliniensis,C.famata,C.glabrata,C.guilliermondii,C.kefyr,C.
krusei,C.lusitaniae,C.parapsilosis,andC.tropicalis)aswellasCryptococcusgattii,Cryptococcus
neoformans,FusariumandRhodotorula,andprovidesresultsin1.5h.Huangetal.tested210positive

J.Fungi2020,6,1015of14
bloodculturesfrompatientswithbloodstreaminfectionswiththeappropriatepanel.Yeastswere
onlyobservedintheGramstainofsevensamples;sixofthesesamplescontainedCandidaspp.
includedinthepanel,andallofthosewereidentifiedbythetest.OnesamplecontainedC.
inconspicua,whichisnotincludedinthepanelandthuswasnotidentified[48].
Table2.Listofbloodcultureindependenttests.
ProductManufacture
r
Candidaspp.
Detected
Assay
TimeMethodApproval
DNAextractionsteprequired
Singletarget: 
AurisID®Olm
DiagnosticsC.auris45minaqPCRCE/IVD
Fungiplex®
Candidaauris
Bruker
DaltonicsC.auris<2haReal‐time
PCRRUO
Multiplextests: 
CandID®Olm
Diagnostics
C.albicans
C.glabrata
C.parapsilosis
C.krusei
C.dubliniensis
C.tropicalis
45minaMultiplex
qPCRCE/IVD
Fungiplex®
Candida
Bruker
Daltonics
C.krusei
C.glabrata
Candidaspp.
(including:C.
albicans,

C.parapsilosis,

C.tropicalis,

C.dubliniensis)
<2ha
Multiplex
real‐time
PCR
CE/IVD
Fungiplex®
Universal
Bruker
DaltonicsCandidaspp.<2ha
Multiplex
real‐time
PCR
RUO
MycoRealCandida Ingenetix
C.albicans,
C.dubliniensis,
C.glabrata,
C.krusei,
C.lusitaniae,
C.parapsilosis,
C.tropicalis
2ha Multiplex
PCR RUO
MagicplexTM
SepsisSeegene
C.albicans
C.tropicalis
C.parapsilosis
C.glabrata
C.krusei
6hbMultiplex
PCRCE/IVD
Broad‐spectrumtests: 
HybcellPathogens
DNAxBCubeDx
Microarray:
C.albicans
C.dubliniensis
C.parapsilosis
C.tropicalis
C.glabrata
+1panfungal
target
6hc
Panfungal
PCR(28s)&
Microarray
CE/IVD

J.Fungi2020,6,1016of14
Sequencing:
pan‐Candida
SepsiTestTM‐UMD
Molzym
Molecular
Diagnostics
pan‐Candida24h
Broad‐
spectrum
PCR(18S)
CE/IVD
MycoRealFungiIngenetixpan‐Candida24h
Broad‐
spectrum
PCR(ITS2)
RUO
Fullyautomated: 
T2CandidaPanelT2
Biosystems
C.albicans/tropicalis
C.glabrata/krusei
C.parapsilosis
3–5h
Multiplex
PCR
followedby
automated
T2MRbased
detection
CE/IVD
aexcludingDNAextraction;bincludingDNAextraction;cifsequencingisnotrequired.CE/IVD:
ConformitèEuropëenne/invitrodiagnostic;RUO:researchuseonly;ITS:internaltranscribedspacer
2region.
3.BloodCultureIndependentMolecularDiagnostics
Bloodcultureindependentmolecularassays(Table2)canbeperformeddirectlyonwholeblood,
serum,orplasmasampleswithouttheneedtowaitforpositivebloodcultures.Thus,thetimesaving
potentialishigherthanwithbloodculturedependenttestsystems.Sincethepathogenloadinthe
bloodislow,thesensitivityofthesetestsystemscanbeanissue.Alargenumberofassaysisavailable
todayforthemoleculardiagnosisofIC;however,manyarein‐houseassaysorcommercially
availableresearch‐use‐onlytests.Broadspectrumtests,aswellastargetedmultiplexassays,are
available.Theprincipleofabroadspectrum/panfungaltestistoamplifyconservedtargetregions
thattheoreticallycanbefoundacrossallfungalspecies.Forspeciesidentification,obtainedamplicons
havetobeanalyzedfurther.Panfungalassaysgenerallyarelesssensitivethanassaystargeting
certainpathogenswithspecies‐specificprimers,buthavetheabilitytodetectallfungalpathogens,
notjustthemostfrequentones.AnissuewiththeuseofmultiplexPCRcanarisefromthefactthat
cliniciansarenotusuallyfamiliarwiththetestpanelsandthuscouldassumethatanegative
multiplexresultissufficientforrulingoutaninvasivefungalinfection.Therefore,itisimportantto
specifywhichpathogensarecoveredonthereportscreatedbytheclinicalmicrobiology/mycology
laboratory.
SomeofthetestsdescribedherecomewiththeirownDNAextractionkit,whileanumberof
differentextractionkits/protocolsarerecommendedforothertests.Asfungalcellsaredifficultto
lyse,theprotocolusedfortheextractionofnucleicacidsmighthavealargeeffectontheassay’s
outcome.Therefore,assayslackingtheirownDNAextractionmethodcanbemoredifficultto
standardize.
CandID®undAurisID®(OlmDiagnostics,NewcastleuponTyne,England)aretwonew
CE/IVDcertifiedqPCRteststhatcanbeperformedwithvariousreal‐timePCRinstruments.The
CandIDkitdetectsC.albicans,C.glabrata,C.parapsilosis,C.krusei,C.dubliniensis,andC.tropicalis;the
AurisID®kitdetectsC.aurisonly.Resultsareavailablewithin45minfromnucleicacidextraction;
noextractionprotocol/kitsarerecommended.Accordingtothemanufacturer,bothkitshavebeen
validatedwithfungalcultures,theCandID®kithasadditionallybeenvalidatedwithplasmaand
syntheticBALsamples,theAurisID®kitwithbloodsamples.Tothebestofourknowledge,no
studiesevaluatingtheclinicalperformanceoftheassayshavebeenpublishedsofar.
TheFungiplex®CandidaIVDPCRKit(BrukerDaltonik,Bremen,Germany)detectsC.krusei,C.
glabrataandCandidaspp.(including:C.albicans,C.parapsilosis,C.tropicalis,andC.dubliniensis)in
wholeblood,plasmaandserum.ForDNAextraction,kitsfromQiagenandBiomerieuxare
recommended,andtheassaymanualprovidesinstrumentsettingsforanumberofdifferent
thermocyclers.InasmallprospectivestudyonICUpatientswithsuspectedIC,theFungiplex®

J.Fungi2020,6,1017of14
CandidadetectedeightoutofeightpatientswithICandreachedasensitivityof100%anda
specificityof94.1%[49].BrukeralsooffersthepanfungalFungiplex®UniversalRUOPCRKitand
theFungiplex®CandidaAurisRUOPCRKitforusewithextractedDNA.
TheMagicplexTMSepsisReal‐timeTest(Seegene,Seoul,SouthKorea)isaCE/IVDapproved
multiplexrealtimePCRdetecting90pathogensatgenuslevel,and27pathogens,includingfive
Candidaspp.(C.albicans,C.tropicalis,C.parapsilosis,C.glabrata,C.krusei),atspecieslevelwithinsix
hoursfromwholeblood.TheSeegeneBloodPathogenKitTMisusedforthepre‐treatmentand
extractionofDNA,andthisstepisfollowedbyaconventionalPCR(onetubeforGram‐positive
bacteriaandresistancemarkersandonetubeforGram‐negativebacteriaandfungi)foramplicon
generation.Ifampliconsaredetected,theconventionalPCRisfollowedbytworeal‐timePCRsfor
screeningandspecieslevelidentification.Seegeneofferssoftware(SeegeneViewer)forthe
interpretationofresults.DeninaetalcomparedtheMagicplexTMtesttobloodculturein150samples
from89patients.Candidaspp.weredetectedbytheMagicplexTMinfoursamples;onlyoneofthese
sampleswasaccompaniedbyapositivebloodculture[50].Inarecentlypublishedstudy,14patients
withICwereincluded.Innineofthesepatients,Candidawasonlydetectedbybloodculture,intwo
patientsonlybytheMagicplexTMassay,andinthreepatientsbybothmethods[51].Importantly,the
twoisolatesdetectedonlybytheMagiplexTMassaybelongedtoC.parapsilosis,whichiswellknown
tobeacolonizingspecies.Thus,thedetectionofC.parapsilosishastobeinterpretedwithcaution.
Moreover,theauthorsdescribethatthetest’slowsensitivitymakesitsimplementationasaroutine
testinclinicalmicrobiologylaboratoriesdifficult.
TheMycoRealCandida(Ingenetix,Vienna,Austria)isaresearch‐use‐onlymultiplexPCRfor
thedetectionofC.albicans,C.dubliniensis,C.glabrata,C.krusei,C.lusitaniae,C.parapsilosis,andC.
tropicalis.Inthisassay,species‐specificbiprobesareused.Thetestwasevaluatedinastudyusing
bothspikedandclinicalsamples[52].Resultsoftheanalyticalandclinicalevaluationshowedthat
thisassaywashighlysensitiveandcanbeusedinclinicallaboratoriesasasimplescreeningtestfor
thementionedCandidaspecies.IngenetixalsoofferstheMycoRealFungi,aresearch‐use‐only
panfungaltesttargetingtheinternaltranscribedspacer(ITS)2region.Theassaykitcontainsprimers,
probes,andapositivecontrol,whilethereactionmixhastobeprovidedbytheuser.ForDNA
extractionfromsamples(blood,sterilefluids,tissue,paraffinembeddedtissue,andBAL),amodified
protocolforusewiththeHighPurePCRTemplatePreparationKitfromRocheDiagnosticsis
recommended.ThesystemhasbeenvalidatedfortheLightCycler®2.0instrument(Roche
Diagnostics),andaLoD95%of15CFU/PCRisreportedbythemanufacturer.Obtainedamplicons
havetobesequencedforspeciesidentification.Thistestwasbasedonanin‐housetest[53,54].
TheSepsiTestTM–UMD(MolzymMolecularDiagnostics,Bremen,Germany)isasystemforthe
CE/IVDcertifiedbroad‐rangedetectionofintactbacterialandfungalpathogenswithananalytical
sensitivityrangingfrom10to80CFU/mL.Inadditiontowholebloodsamples,thistestisvalidated
forsterilefluids,tissuesamples,andswabs.ForpathogenenrichmentandDNAextraction,Molzym
offersanautomatedsolution,inwhichthesestepsareperformed,fullyautomatedbytheSelectNATM
plusrobot(Micro‐DxTMCEIVD).Asemi‐automatedsolution(UMD‐SelectNATMCEIVD)with
manualpathogenenrichment,followedbyautomatedDNAisolationononeofthefollowing
instruments—Liaison®Ixt(Diasorin),Arrow®(Nordiag),Seeprep12TM(Seegene),orGenoXtract®
(HainLifescience)—aswellasmanualextractionarepossible.Afterwards,16Sand18SrRNAgenes
areamplifiedintwoseparatereactions.Obtainedampliconshavetobesequenced,andsequences
arethenanalyzedwiththefreeonlineSepsiTestTM‐BLASTtool.Themajoradvantageofthisbroad‐
rangetestisitswidespectrumwhichincludesfastidiousorganismsthatarenotdetectablebyculture.
IncaseofapositivePCRresult,theneedforsequencingincreasesthetimetogaintheresult.Even
thoughseveralstudieshaveevaluatedtheperformanceofthetest[55–59],fewcasesofICwere
includedinthesestudies.Schreiberetal.reportedonecaseofC.albicansdetectedinbloodcultures
ofapatientwithanegativePCRresult[56],andNiemanetal.foundC.albicansintwopatients,the
yeastwasonlydetectedbybloodculturesinone,andonlyinthePCRassayinthesecondpatient
[58].

J.Fungi2020,6,1018of14
TheHybcellPathogensDNAxB(CubeDx,St.Valentin,Austria)isarecentlyCE/IVDapproved
testforthedetectionofbacteria,resistancegenes,andfungi.DNAisisolatedfrom500mLwhole
bloodwiththeGINApathogenenrichmentandDNApurificationkit(CubeDx).Subsequently,four
separatePCRreactionsarecarriedout(positivecontrol,bacterialpanel:16SrDNA,fungalpanel:28s
rDNA,andpanelfortheresistancemarkersvanA,vanB,mecA,andmecC)andafluorescentdyeis
incorporatedintoampliconsduringthePCR.UponcompletionofthePCR,PCRproductsare
transferredtocylindricalmicroarrays—so‐calledhybcells—andampliconsareidentifiedinthe
hyborgdevicebybindingtoimmobilizedprobesviaelongationanddetectionoffluorescencesignals.
Thesystemcanidentify1panbacterialtarget,4bacterialgeneraand28bacterialspecies,aswellas1
panfungaltarget,2fungalgenera,and13fungalspecies.Thus,sequencingofthePCRproductsisnot
necessaryifthepathogenisincludedinthetestpanel,andresultscanbeavailablewithin3h.Should
specieslevelidentificationyieldnoresultinasamplepositiveforthepanbacterialorthepanfungal
target,leftoverPCRproductscanbesubjectedtoSangersequencing.Thistestiscurrentlyunder
evaluation.Sofar,nopeer‐reviewedstudyresultsareavailable.
TheT2CandidaPanel(T2Biosystems)isaCE/IVDapprovedtestforuseontheT2Dxinstrument,
whichutilizesT2MagneticResonance(T2MR).TheCandidapanelcandetectthreegroupsofCandida
(C.albicans/C.tropicalis,C.glabrata/C.krusei(whichalsoincludesS.cerevisiaeandC.bracarensis),and
C.parapsilosis(whichincludesC.orthopsilosisandC.metapsilosis))inEDTAbloodsampleswith
minimalhands‐on‐time.Afterin‐cartridgeDNAextraction,theITS2regionisamplified.Amplicons
aredetectedviahybridizationwithspecificcaptureprobescarryingsuperparamagneticparticles.The
resultingagglomerationoftheseparticlesinducesashiftinthesample’smagneticresonance.This
methodisabletodetectminimalamountsofintacttargetcells(1CFU/mL)—butnotfreeDNA—with
atimetoresultof3–5h.Neelyetal.[60]evaluatedthemethodforthedetectionofCandidain2013.
Intheirstudy,wholebloodsampleswerespikedwithdifferentconcentrationsofCandidaspp.
includedinthepanel;highagreementratesbetweentheT2MRandBC(97.8%positiveand100%
negativeagreement)wereobserved.Mylonakisetal.laterevaluatedthetestinalargeprospective
studywithsamplesfrom1801patients;250ofthesesampleswerespikedwithCandidaspp.[61].The
overallanalyticalsensitivityoftheT2MRwas91.6%anditsspecificitywas99.4%.In31cases,T2MR
andBCdidnotyieldthesameresults;2patientswerepositiveinBCbutnotwiththeT2MR,while
samplesfrom29patientswerepositivewithT2MRbutnegativeinBC.Arendrupetal.recently
conductedanotherprospectivestudywith126ICUpatientswhichwereclassifiedintogroupsof
proven,likely,possible,orunlikelyICbasedontheresultsofBC,culturefromsterilesites,
colonization,aCandidaantigenassay,andclinicalfindings.ComparedwithBCandtheantigentest,
theT2CandidaPanelhadthehighestsensitivityforthedetectionofIC[62],eventhoughthe
sensitivitywaslowerthanobservedinthe2015Mylonakisetal.study[61].Thebestsensitivitywas
achievedbyacombinationofBCandtheT2CandidaPanel[62].Asthetestalsodetectsnon‐viable
cells,T2MRmightbeausefultoolforthemonitoringofcandidemiauponinitiationofantifungal
therapy.ThiswasdemonstratedinastudypublishedbyMylonakisetalin2018[63].Follow‐ up
bloodsamples(bloodculturesandwholeblood)from31patientswithcandidemiawereincluded.
ThirteenpatientshadatleastonepositiveT2MRresult,whileBConlydetectedthepresenceof
Candidain4ofthese13patients.Clancyetal.[64]comparedthepositivityoffollow‐upsamplesfrom
152patientswithcandidemia.Duringasecondblooddraw,samplesforT2MRandacompanionBC
(cBC)wereobtained.SamplesfortheT2werefrozenandanalyzedinbatches.Inpatientsunder
antifungaltherapy,theT2MRassaywasmoreoftenpositivethanthecBC(50%vs.21%),whereasno
differencewasobservedinuntreatedpatients.Invalidreportswerereportedin9%ofT2samples;
thismightbeduetotheanalysisoffrozensamples.Thetestonlydetectsintactorganisms(notfree
DNA),andthereforeshouldnotbeperformedonfrozensamples.Thus,studiesworkingwithfrozen
samplesmightnotreflecttrueperformancecharacteristics.Zurletal.analyzedfrozensamplesfrom
32patientswithcandidemiaandfrom22patientswithdeep‐ seatedcandidiasis[65].Samplesfor
T2MRtestingwerecollectedatvarioustimepointsrangingfrom2daysbeforeuntil5daysafterthe
indexculture(CandidapositiveBCorsterilesiteculture).Severalinvalidsamples/instrumenterrors
wereobserved.Furthermore,eightsampleswhichwerecollectedconcurrentlywiththepositive

J.Fungi2020,6,1019of14
indexBCyieldednegativeT2MRresults.Intwocases,theT2detectedaCandidaspeciesdifferent
fromthespeciesfoundintheBC.Inthegroupofpatientswithdeep‐seatedcandidiasis,the
T2Candidapanelgaveatleastonepositiveresultinsixpatients(27.3%).Remarkably,allBCs
collectedfrompatientswithdeep‐seatedcandidiasisremainednegative.Thus,eventhoughthe
percentageofpositiveT2resultsdoesnotseemveryhigh,thisisaninterestingobservation,sincethe
diagnosisofdeep‐seatedcandidiasisisverychallenging.InadditiontotheT2CandidaPanel,
T2Biosystemsalsooffersaresearch‐use‐onlypanelforthedetectionofC.auris[66]inskinswab
samples.InadditiontothediagnosticperformancetheroleofT2Candidaasaprognosticandpatient
managementtoolshouldalsobeevaluated.Munozetal.showed,inaprospectiveobservational
multicenterstudyofpatientsreceivingdefinitiveantifungaltreatmentforcandidemia,thatapositive
T2MRwasassociatedwithahigherriskofpooroutcome,whilethedetectionofbeta‐D‐Glucandid
notcorrelatewiththeoutcome.ApositiveT2Candidaresultwithinthefirst5daysafterthereportof
apositiveBCwasanindependentriskfactorforcomplicatedcandidemia,definedbyattributable
mortalityordevelopmentofmetastatic,deep‐seatedinfection[67].Inanothermulti‐center
investigation,Munozetal.showedthatT2Candidaperformedinpatientswithprovencandidemia
maybeabettermarkerofcomplicatedinfectionthanfollow‐upbloodculturesordetectionofbeta‐
D‐glucan(BDG).TheseresultsindicatethatT2Candidamayinfluencethelengthandtypeof
antifungaltherapyinthispopulationandmightbeusedinthesenseofantimicrobialstewardship
[68].Asaconsequence,testresultscouldbeusedtoexpediteantifungaltreatmentofcandidemia,
andreduceoverallantifungalusagewithoutanegativeeffectonpatientoutcomes.UsingT2Candida
incombinationwithculturesislikelytooffergreatestvalue.However,antifungaltherapymay
influencethesensitivityoftheperformanceoftheT2CandidaPanel.AsdescribedbyClancyetal.
T2Candidashowedlimitedsensitivity(36%)/negativepredictivevalue(NPV)(80%)intheMADRID
prospectiveobservationalstudyundertheinfluenceofempiricalantifungaltherapy,whereas
specificity/positivepredictivevalue(PPV)wasexcellent(100%),indicatingarolebettersuitedto
confirmingadiagnosisorpersistentinfection[69].Ashasbeennoted,stratificationofhigh‐risk
patientsthroughrisk‐predictionmodelingisessentialtoachieveasufficientpre‐testprobability.
Irrespectiveoftheprevalenceofdisease,theNPVoftheT2testis>98%,butaprevalenceofaround
10%maybeoptimal,providingaPPVandNPVofapproximately82%and99%,respectively[69,70].
4.Summary
Diagnosingfungalinfectionshasalwaysbeenchallenging.Advancesinmoleculardiagnostic
technologieshavegeneratedarangeoftestswithrapidturnaroundtimesforthediagnosisand/or
screeningofpatientsatriskforinvasivefungalinfections.IncreasingexperiencewithPCRassaysfor
thedirectdetectionoffungiinclinicalspecimensandavailableclinicalvalidationstudieshave
positionedtheseassaytypeswellonthewaytobecomingroutineinclinicallaboratories.
SeveralPCRassays,includingcommerciallyavailablekits,havebeendevelopedforthe
detectionofCandidaspp.inpatientswithcandidemiaandIC.Thehighsensitivitymakestheseassays
appealingtoolsfortheearlydiagnosisofIC.DependingonthemethodusedforDNAextraction,free
DNAorintactpathogencellsaredetected.Thisdifferencecanberelevantforpatientsunder
antifungaltherapyasthismayspecificallyinfluencetheoutcomeofmolecularteststhatdetectonly
intactcells.Ontheotherhand,theinterpretationofpositiveresultsfromassaysdetectingfreeDNA
canbechallenging.Thus,thepositionofCandidaPCRassaysinthediagnosticalgorithmofICisnot
easytoestablish.Aspublisheddatashow,CandidaPCRhasahighersensitivitythanbloodculture
butshowsthebestefficacywhenusedinconjunctionwithbloodculturesand/oradditionaltestssuch
asthedetectionofBDG.Inaddition,theuseofmoleculartechniquesforpositivebloodcultures
allowsamorerapididentificationofCandidaspp.
Asearlyinitiationofeffectiveantifungaltherapyisassociatedwithimprovedoutcomes[71],it
iscrucialtostartatargetedtherapyasearlyaspossible.Directmoleculardetectionorrapid
identificationofCandidaspp.frombloodculturesbyuseofmolecularassaysshowsthepotentialfor
earlyadministrationofanoptimalantifungaltherapy.Inaddition,theseassaysmayallowforthe

J.Fungi2020,6,10110of14
correctchoiceoflengthandtypeofantifungaltherapy,andmaythusbeusedinthesenseof
antimicrobialstewardship.
However,manyoftheseassaysremainunderinvestigationastheyhavenotbeenvalidatedfor
diagnosingICinmulti‐centerstudies.Thechoiceofadoptinganin‐houseratherthancommercial
assayisdependentuponcosts,aswellasworkflowandcapacityinindividuallaboratories[72],and
resultsofmolecularassaysshouldalwaysbeinterpretedwithcaution[73].Therefore,moredatafrom
multi‐centerstudiesisneededforafinalassessmentofcommercialassays.
Authorcontributions:I.C.wrotethefirstdraftandadaptedthemanuscriptfollowingreviews,K.S.was
involvedinthereviewofexistingliterature,designedthefigureandtables,andreviewedthemanuscript,and
B.W.wasinvolvedinconceptualizing,writingandreviewingthemanuscriptbeforesubmission.Allauthors
havereadandagreedtothepublishedversionofthemanuscript.
ConflictsofInterest:TheauthorsarecurrentlyconductingastudyontheclinicalperformanceoftheT2Candida
Panel.Forthestudyperiod,theT2DxinstrumentandnecessarykitsaresuppliedbyT2Biosystems/Biomedica.
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