What Could Be the Role of Antifungal Lock-Solutions?

15 Pages·2017·0.23 MB·English

pathogens
Review
What Could Be the Role of Antifungal
Lock-Solutions? From Bench to Bedside
ChristineImbert1,2,*andBlandineRammaert2,3,4
1 LaboratoryofEcologyandBiologyofInteractions,UniversityofPoitiers,UMRCNRS7267,
F-86073Poitiers,France
2 FacultyofMedicineandPharmacy,UniversityofPoitiers,F-86073Poitiers,France;
[email protected]
3 CHUPoitiers,InfectiousandTropicalDiseaseUnit,F-86073Poitiers,France
4 INSERMU1070,F-86073Poitiers,France
* Correspondence:[email protected]
Received:28November2017;Accepted:3January2018;Published:6January2018
Abstract: Candidemiarelatedtothepresenceofabiofilmareoftenreportedinpatientswithvascular
catheters. Oncetheyaremature,biofilmsarepersistentinfectiousreservoirs,andtheyeastsdispersed
frombiofilmscancauseinfections. Sessileyeaststypicallydisplayincreasedlevelsofresistanceto
mostantimicrobialagentsandsystemictreatmentsusuallyfailtoeradicatepreviouslyformedfungal
biofilms. Inacurativestrategy,antifungallocktherapymayhelptosterilizecatheters,withveryhigh
concentrationsofantifungalagents,whicharenotcompatiblewithsystemicuse.Thisstrategyhas
beenstudiedbyseveralauthorsininvitroandinvivostudies,andmorerarely,inclinicalsettings
foradultandpaediatricpatients. Ourstudyaimstoassesstheefficacyoftheantifungalsolutions
usedforlocktherapyanddemonstratedbythedifferentteams.
Keywords: biofilm;antifungalagents;candidemia;cathetercarrierpatient
1. Introduction
Candida albicans belongs to human microflora and is a major opportunistic human pathogen.
Thisspeciesisresponsibleforavarietyofdiseases,rangingfromoralthrushtocandidemia,depending
onpatientstatusandfragilityand/orimmunosuppressionlevel. Differentfactorscontributetothe
pathogenicpotentialofC.albicans.Althoughadherenceabilityandbiofilmlifestyleareamongthe
mostimportantfactors,wemayalsomentionthigmotropism,yeast-to-hyphatransition,secretionof
hydrolases,andsoon[1–3].Adherencetoaninertorlivingsurfaceistheearliestphaserequiredduring
themulti-stepmechanismleadingtoformationofabiofilm.Biofilmhasbeendefinedasastructured
communityofmicrobialcellsenclosedinaself-producedpolymericmatrix[4]. Inbiofilms,sessile
cellstypicallydisplayincreasedlevelsofresistancetomostantimicrobialagentsandtohostdefence
mechanisms,aswell[5,6].TheabilityofC.albicanstoformbiofilmsassociatedwithbioticsurfaces,
such as oral mucosa, and abiotic surfaces, such as implanted devices, can strongly impact human
health. Importantly, biofilms are persistent infectious reservoirs of yeast cells dispersed from the
biofilmthatcauselocalandsystemicinfections[7]. Yeastcellsdispersedfrombiofilmsarenotsimilar
toregularplanktonicyeastcells. Inarecentstudy,Uppuluriandcollaboratorsdemonstratedthatcells
arelikelytobedispersedprimarilyasyeastandthatdispersiontendstooccurcontinuouslythroughout
biofilmdevelopmentratherthanbeingamassiveevent[8]. Theyalsoconcludethatcomparedtotheir
planktoniccounterparts,dispersedcellsdisplaydistinctphenotypicproperties,includingenhanced
adherence,filamentationandpathogenicityinamurinemodel.
Ideally, a fully effective antimicrobial candidate treatment would kill the free microbial cells
dispersedfromthebiofilmatthesametimeastheeradicationofthebiofilmitself. Itwouldalsobe
Pathogens2018,7,6;doi:10.3390/pathogens7010006 www.mdpi.com/journal/pathogens
Pathogens2018,7,6 2of15
reallyadvantageousifthisidealtreatmenttotallyremovedthebiofilmandmadethecathetersurface
totallysmoothsoastoavoidoratleastdelaytheformationofanewbiofilm. Itcouldbebasedon
onlyoneoracombinationofmolecules. Inthisway,theacuteinfectionwouldbecuredandrelapse
certainlydelayed. Unfortunately,thisidealtreatmentcandidatehasyettobediscovered.
Candidemiaareassociatedwithreportedmortalityratesrangingfrom30%to60%andattributable
mortalityratesbetween25%and40%,C.albicansbeingthemainspeciesinvolved[9–14]. Outerand
innersurfacesofvascularcatheters,especiallylong-termones,areparticularlygoodsubstratesfor
C.albicansbiofilmdevelopment,andareconsequentlyathighriskofbiofilm-relatedcandidemiafor
patients. Incaseofacatheter-relatedbloodstreaminfection(CRBSI)causedbyCandidaspp.,catheter
removaliscurrentlyrecommended. Ithasbeenwidelyshownthatnon-removalisassociatedwith
increased mortality and more prolonged candidemia [15–24]. This recommendation has certainly
beengivenduetotheinsufficientactivityofcurrentlyavailablesystemicantifungalagentsagainst
Candida spp. biofilms. Many invitro and invivo studies have evaluated the ability of azoles,
echinocandinsandamphotericinBtoeradicateapreviouslyformedbiofilm. Theygenerallyshowed
thatazoleshadpooractivityagainstC.albicansbiofilms. Onthecontrary,echinocandinsandlipid
formulationsofamphotericinBgenerallydemonstratedhigheractivity,eveniftotaleradicationwas
not obtained. Lipid formulations of amphotericin B appeared more effective than amphotericin B
deoxycholate[5,6,9,25–29].
However, catheter replacement is not possible for all patients. For instance, in neonatology
unitsextremelylowbirthweightinfantsareparticularlyatriskforCRBSI[30].Candidemiaoccurs
in approximately 10% of reported bloodstream infections in neonates [31]. The risk of CRBSI in
neonatologyunithasbeenshowntoincreaseduringthe2weeksofcatheterinsertion[32]. Limitedor
noalternativeintravenousaccess,particularlyinchildren,leadstodevelopmentofnewapproaches
whencathetersareinfectedwithCandida. So,antifungallocktherapymaybehelpfulforthesepatients,
in combination with conventional antifungal systemic therapy. Lock therapy involves instilling
highconcentrationsofantimicrobialagents(usuallyfrom100-to1000-foldtheminimalinhibitory
concentration(MIC))intothecatheterlumenforextendedperiodsoftime[33,34].Thisstrategyis
ratherwell-documentedanddefinedforantisepticandantibioticapproaches. However,theroleof
antifungallocktherapyagainstCandidaisstillnotwell-defined[15]. Biofilmsmaybelocatedonboth
the outer and inner surfaces of vascular catheters. Of course, in case of an extra-luminal biofilm,
antifungallockwouldfailtoeradicatebiofilm.
Theaimofthecurrentarticleistofocusonthedataavailableintheliteraturegivinginformation
aboutantifungallocktherapyapproachinordertohavebetterknowledgeofthisstrategy. Wewill
describe its efficacy and the conditions that would be the most appropriate in terms of antifungal
agent,concentration,contactduration,andtreatmentfrequency. Therewillbethreepartssuccessively
focusingoninvitro,invivoandclinicaldata. Onlyconventionalantifungalagentswillbeincludedin
thisstudy. Manyteamshavedevelopedinvitromodelsmimickinglockstrategytostudytheinterest
ofthemainavailableantifungalagentsusedaslocksolutions. Themainissueisthevariabilityofthe
developedmodelsthatcomplicatecomparativeanalysisoftheresultsavailableinliterature. Indeed,
lockconcentration,timeandnumberofrepeats,aswellasbiofilmsubstrates,maturationstatusof
treated biofilms, and the method used to evaluate lock activity differ frequently according to the
studies(Table1). However,publishedresultsaregenerallyinagreementandwewilltrybelowto
summarizethemainideasregardingtheefficacyofantifungallocksagainstCandidaspp.biofilms.
Pathogens2018,7,6 3of15
Table1. Methodstoevaluatetheactivityofantifungallocksolutionsaccordingtoselectedinvitrostudies. d-AmB=AmphotericinBdeoxycholate; L-AmB=
liposomalAmphotericinB;ALT:antifungallocktreatment;XTT:2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide.
Investigation Methodfor
CandidaSpeciesand Ageofthe Antifungal
SurfaceNature ALTDuration ofTreatment Antibiofilm MajorConclusions Reference
NumberofStrains TreatedBiofilm Solutions
Persistence Evaluation
48h-lockofcaspofunginat2µg/mL
Caspofungin; 24,48and72h
C.albicans(2) 12hand5days 100%silicone 12h XTT ormicafunginat5µg/mLreduced Cateauetal.,2008[35]
micafungin post-lock
biofilmsby47%
d-AmB;
Azolesat1mg/mLeradicatedall
C.albicans(1); fluconazole; 1,3,5,7,10and14days
5days(with XTT+CFU biofilmswithin7to14days;azoles
C.glabrata(1); polyurethane itraconazole; (locksolutionsreplaced none Koetal.,2010[36]
shaking) counts weresuperiortod-AmBand
C.tropicalis(1) voriconazole; every2days)
caspofungintoeradicatebiofilms
caspofungin
48h-lockofposaconazolereduced
C.albicansbiofilmsby<50%compared
to≥60%withechinocandins.
Posaconazole;
C.albicans(10); 24,48and72h Greatersustainedefficacyof
12hand5days 100%silicone Caspofungin; 12h XTT Cateauetal.,2011[26]
C.glabrata(6) post-lock micafungincomparedwith
micafungin
caspofunginagainstallbiofilmsof
C.glabrata,albeitnoobviousdifference
forC.albicansbiofilms
C.albicans(20); inefficacyofazolesagainstallspecies
d-AmB;
C.parapsilosis(40); ofC.albicansbiofilms;d-AmBactivity
voriconazole;
C.tropicalis(20); 24h polystyrene 24h none XTT waslimitedandstraindependent. Fiorietal.,2011[37]
anidulafungin;
C.glabrata(20); Anidulafunginmoreactive
caspofungin
C. krusei(20) thancaspofungin
d-AmB;
Noazolecansterilizecatheters;
fluconazole; 1,3,5,7days Determination
C.albicans(1); catheterstreatedwithd-AmBor
5days silicone itraconazole; (locksolutionsreplaced none ofviablecount Öncüetal.,2011[38]
C.parapsilosis(1) caspofunginwerecompletelysterileat
voriconazole; every2days) (nodetails)
thefifthday
caspofungin
Highandpersistentinhibitoryactivity
C.albicans(2); ofL-AmBusedat1000µg/mLfor
C.glabrata(2); 12hand5days 100%silicone L-AmB; 4,12and24h 24and48h XTT shortlockbutnofullbiofilm Touletetal.,2012[27]
C.parapsilosis(2) eradication;C.parapsilosisbiofilmless
susceptiblethanthatofotherspecies.
L-AmBat256to2048µg/mLinhibited
80to90%ofC.albicansbiofilm;
L-AmB; comparableinvitroefficacyofL-AmB
C.albicans(1);
anidulafungin; 24,48and72h XTT+CFU andcaspofunginagainstC.albicans Simitsopoulouetal.,
C.lusitaniae(6); 48h polystyrene 24h
caspofungin; post-lock counts maturebiofilms;anidulafunginand 2014[39]
C.guillermondii(5)
micafungin micafunginlessactivethan
caspofunginagainstC.albicans,
C.guilliermondiiandC.lusitaniae.
Pathogens2018,7,6 4of15
Table1.Cont.
Investigation Methodfor
CandidaSpeciesand Ageofthe Antifungal
SurfaceNature ALTDuration ofTreatment Antibiofilm MajorConclusions Reference
NumberofStrains TreatedBiofilm Solutions
Persistence Evaluation
50%biofilmreductionwasobtained
with4-foldlessanidulafungin
(≤0.25µg/mL)thanL-AmB
silicone; L-AmB;
C.parapsilosis(2) 48h 48h none XTT (1µg/mL);90%biofilmreductionwas Basasetal.,2016[40]
polystyrene anidulafungin
obtainedusinganidulafunginat
1µg/mLcomparedtoL-AmBat
>1024µg/mL
Highefficacyofthecombination:20%
Silicone; XTTandCFU ethanol,0.01565µg/mLmicafungin
C.albicans(5) 24h micafungin 24h none Lownetal.,2016[41]
polystyrene counts and800g/mLdoxycyclineagainst
formingandmaturebiofilms
Pathogens2018,7,6 5of15
2. AzoleLockSolutions
In most instances, all available data suggested that azole lock solutions fail to eradicate
preformedC.albicansbiofilms,regardlessoftestconditions.Öncüstudiedtheefficacyofthreeazole
(fluconazole,itraconazoleandvoriconazole)lockstoeradicateamatureC.albicansbiofilm,5daysold,
previously formed on silicone catheters, using only one strain. Fungal eradication was evaluated
by determining the viable count. The author showed that no azole sterilized catheters, with more
than 105 CFU after the 7-day lock period compared to more than 106 CFU on the non-treated
catheters. Very high concentrations of major azole solutions failed to eradicate biofilms despite
extendedlockduration(24heach)andrepetition(for7days)[38].Fiorietal.alsotestedfluconazole
and voriconazole lock solutions, and included 20 clinical strains of C. albicans in their study;
biofilmswereformedonpolystyrenesurfaces(96-wellmicrotiter-basedmethod)buttheirmaturation
status was unclear [37]. The lowest azole concentrations at which a 50% decrease in biofilm
metabolicactivity(2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide[XTT]
method)wasobservedwerealways>128µg/mL(fluconazoleandvoriconazole),confirmingtheir
inefficiencyagainstC.albicansbiofilms[37].Theselocksolutionswerealsotestedagainstnumerous
C. non-albicans biofilms (C. glabrata, C. parapsilosis, C. tropicalis and C. krusei). Again, the minimal
concentrationsreducingbiofilmsbyhalfalwaysexceeded>128µg/mL[37].Ourteamevaluatedthe
efficacy of posaconazole lock solutions to eradicate 12 h-young and 5-day-old mature biofilms of
C.albicanspreviouslyformedonsiliconecatheters.Inthisstudyposaconazolewastestedat10mg/L
corresponding to concentrations approximately 100–200 MIC and 16 strains of Candida sp. (ten of
C.albicansandsixofC.glabrata)werestudied[26].PosaconazoleinducedC.albicansbiofilminhibition
rangingfrom54.2%(youngbiofilms)to57.6%(mature)24hafterlocksolutionremoval. Inhibition
persistedovera48hpost-lockperiod(inhibitionrangingfrom49.7%to48.4%respectively).Inthis
study posaconazole halved C. albicans biofilms regardless of their maturity level with a persistent
effect. However,underthesametestconditionsposaconazoledidnotinhibitC.glabratabiofilms[26].
These results concurred with those reported by other teams who obtained no more than 40%
inhibitionofC.albicansbiofilmtestedatdifferentmaturationphases(24hto72h-oldbiofilms)[42,43].
IntheirstudyKatragkouetal. testedvoriconazoleandposaconazoleconcentrationsupto256µg/mL
and prepared biofilms on silicone elastomer surfaces [43]. Their results showed that these highly
concentratedlocksolutionswerenotabletoreduce72h-oldC.parapsilosisbiofilmmetabolicactivity
bymorethanabout60%(voriconazole)or40%(posaconazole). Onlyonestudygavedivergentresults
showingthatfluconazole,itraconazole,andvoriconazoleatconcentrationsof1mg/mLeliminated
detectableviabilityinbiofilms(5-day-old)madebyC.albicans,C.glabrataandC.tropicaliswithin7,10
and14daysonpolyurethanesurfaces,respectively[36]. Inthistreatment,locksolutionswerereplaced
every2days. Fourteendaysisaverylongtimeandtestedlockperiodsareusuallyshorter,which
mayhelptoexplainthedivergentdata. Inaddition,theseresultsmaybestrain-dependantasauthors
studiedonlyonestrainofeachspeciesofCandida.Toourknowledge,thisstudyfromKoetal. isthe
onlyonedemonstratingthatitraconazole, fluconazole, andvoriconazolearegenerallysuperiorto
caspofunginandamphotericinB[36].
Finally, invitro literature data primarily underscores the inability of available azoles, such as
fluconazole,itraconazole,voriconazoleandposaconazoletoreducethemetabolicactivityinC.albicans
biofilmsbymorethanhalf,regardlessofthetestedbiofilmmaturationstatus,azoleconcentrationand
contactduration. Giventhefailureofinvitrodata,noclinicaldataexistonazolelock. Oneinvivo
study using a rabbit model of C. albicans CRBSI was consistent with invitro models. This study
assessedtheefficacyofliposomalamphotericinB(L-AmB)comparedtofluconazolelock-therapies[44].
Theantifungalsolutionswerelockedinthelumenofeachcatheterfor8hperdayfor7days. L-AmB
wasmoreefficientincathetersterilizationthanfluconazole.
Pathogens2018,7,6 6of15
3. AmphotericinBLockSolutions
Available data regarding the efficacy of antifungal lock solutions based on amphotericin B
deoxycholate (d-AmB), and lipid formulations of amphotericin B, such as amphotericin B lipid
complex (ABLC) and L-AmB will be reported in this part. Öncü evaluated invitro the capacity
ofamphotericinBlocksolutions(36µg/mLto120µg/mL)toeradicatemature(5-dayold)biofilms
formed on silicone catheters using a viability approach (CFU method) as previously mentioned
for azole testing [38]. Results showed that d-AmB significantly decreased the number of yeasts
insidecatheters,whichwerecompletelysterileatthefifthday.Furthermore,d-AmBefficiencywas
observedasearlyasthefirstdaywhateverthetestedconcentration.Similarresultswereobtained
on C. parapsilosis biofilms using d-AmB at 75 µg/mL to 250 µg/mL. Ko et al. also investigated
invitrotheinterestofd-AmBlocksolutionsininhibitingmature(5-dayold)biofilms[36]. C.albicans,
C. glabrata and C. tropicalis species (one strain of each) were investigated and d-AmB minimal
inhibitory concentrations for biofilm cells (CFU method, polyurethane surfaces) were rather low:
16mg/mL,32mg/mLand4mg/mL,respectively. However,aspreviouslymentioned,theseauthors
reportedratherdivergentresults,andfoundthattestedazolecompoundsweregenerallysuperior
tod-AmBineradicationofCandidabiofilms. Finally,Fiorietal. reportedontheinvitroanti-biofilm
activity of d-AmB lock solutions against C. albicans, C. glabrata, C. tropicalis, C. krusei (20 strains of
each)andC.parapsilosis(40strains)assessingtheinhibitionofsessileyeastmetabolismcausedby
the drug (XTT method) [37]. They reported that the lowest d-AmB concentrations at which 50%
decrease in biofilm was observed ranged from 0.5 µg/mL to >32 µg/mL for all tested strains of
C. albicans, C. glabrata, C. parapsilosis, and C. tropicalis, and from 1 µg/mL to >32 µg/mL for those
ofC.krusei. Actually,theconcentrationresponsiblefor50%biofilminhibitionwas ≤ 1µg/mLfor
45%(C.parapsilosis),40%(C.tropicalis),30%(C.albicansandC.glabrata)andonly1%(C.krusei)ofthe
studiedstrains.Furthermore,theconcentrationscausing90%decreasewere>32µg/mLforC.albicans,
C.parapsilosisandC.kruseistrains,whereastheywere32µg/mLand16µg/mLforC.tropicalisand
C.glabratastrains. Finally,evenifd-AmBlocksolutionsdisplayedlittleactivityagainstsessileCandida
yeasts,theseresultsbasedontheinvestigationofnumerousstrainsofeachstudiedCandidaspecies
clearlydemonstratedthatthisactivitywasbothlimitedandstrain-dependent.
However, some early invitro experiments demonstrated the anti-biofilm activity of lipid
formulations of amphotericin B, such as ABLC, and their significantly higher anti-biofilm activity
againstC.albicansandC.parapsilosiscomparedtod-AmB[6]. Threestudiespublishedbetween2012
and 2016 studying different Candida species focused on the efficiency of L-AMB lock solutions in
fightingCandidabiofilms[27,39,44]. Touletetal. investigatedinvitrotheactivityofL-AmBsolutions
at200µg/mLand1000µg/mLagainstbiofilmsofC.albicans,C.parapsilosisandC.glabrataonsilicone
catheters(2strainsofeachspecies,XTTmethod)andaged12hor5days. Theirefficacywasanalysed
depending on lock duration (4 h, 12 h or 24 h) and in each case the authors evaluated post-lock
remanence[27]. TheyshowedthatL-AmBsolutionsusedat1000µg/mLreduced12hand5-day-old
Candidaspp. biofilmsregardlessoflockduration(inhibitionalways≥80%forC.albicansandC.glabrata
and≥62%forC.glabrata),butcouldnevertotallyeradicatebiofilms. Loweractivitieswereobtained
usingL-AmBat200g/mL,withinhibitionpercentagesforC.albicans,C.glabrataandC.parapsilosis
ranging from 73.5% to 91.5%, 77.5% to 93% and 41.5% to 88.5%, respectively. This study showed
that L-AmB solutions at 1000 µL/mL used for short lock times (≤4 h) could significantly reduce
biofilmsofC.albicansandC.glabrataforupto48h(post-lockremanence)butwerelessefficientagainst
C.parapsilosisbiofilms. Basasetal. alsoevaluatedtheefficacyofL-AmBinreducingmaturebiofilms
(48h-old)ofC.parapsilosisonsiliconesurfaces,studyingtwostrains[40]. Theyreportedthatthelowest
L-AmBconcentrationscausinga50%anda90%decreaseinC.parapsilosisbiofilmwere0.5or1µg/mL
(depending on the strain) and >1024 µg/mL, respectively, confirming how difficult it is to totally
eradicatebiofilms,evenifthedrugremainseffective. Biofilmreductionwasevaluatedaccordingto
decreasesinyeastviability(Live/deadstaining)andmetabolism(XTTmethod). Interestingly,these
authorscomparedL-AmBactivitiesdependingonthebiofilmsubstrateandshowedthat48h-old
Pathogens2018,7,6 7of15
biofilms formed on polystyrene surfaces were much easier to reduce compared to those prepared
onsilicone: 50%biofilmreductionat0.125µg/mLor0.25µg/mLdependingonthestrain,and90%
reductionat8µg/mLor16µg/mL.Thisfindinghighlightstheinfluenceofsubstratesandtheneed
tousematerialsmimickingthoseusedtomanufacturemedicaldevices. Finally,Simitsopolousetal.
investigatedinvitrotheinterestofL-AmBagainstmaturebiofilms(48h-old)ofC.albicans(1strain),
C. lusitaniae (6 strains) and C. guilliermondii (5 strains) using both metabolic (XTT) and viability
(CFU counts) approaches, considering locks for 24 h [39]. In these conditions, the lowest L-AmB
concentrationscausing50%decreaseinbiofilmswere0.25µg/mL,2µg/mLand0.125µg/mLfor
C.albicans,C.guilliermondiiandC.lusitaniae,respectively,whichseemsclosetotheresultspresented
by Fiori et al. [37]. At 256 µg/mL to 2048 µg/mL, L-AmB induced strong biofilm damage against
C.albicans(about80%to90%decrease),whichisconsistentwiththeresultsofTouletetal.[27].
Fewpatientshavebeentreatedwithd-AmBantifungallock[25,45–50](Table2).d-AmBantifungal
lockhasbeenshowntobeeffectivein6/10patients.However,failuresoftherapyareoftenunpublished.
In addition, the patients received different systemic antifungal therapies that were likely to bias
analysisofthesedata[25].Nevertheless,d-AmBasanantifungallockfailedtofindaplaceinaclinical
setting, and was not reinforced by animal models. Indeed, the only experimental invivo model
comparingd-AmBandcaspofunginwasfavouredofcaspofungin[51].Shufordetal.usedarabbit
modelofC.albicanscatheter-relatedbloodstreaminfection(CRBSI).Asiliconecatheterwasinserted
intotheexternaljugularveinoftherabbit. Theinfectedcatheterwasuseddailytoadministeredan
antifungaldrugandwaslockedafterinjectionwithasolutioncontainingheparinandtheantifungal
drug. Using d-AmB, 13/16 catheters were sterile at day 7 compared to all catheters locked with
caspofungin[51].
TheotherinvivomodelsfocusedonlipidformsofAmphotericinB.Mukherjeeetal.usedarabbit
modelofC.albicansCRBSItotestantifungallockwithABLC[52]. Asiliconecatheterwaslockedfor
4or8hperdaywithasolutioncontainingABLCandheparinizedsaline. Atday11ofantifungallock,
ABLC-lockedcatheterswereallsterilizedcomparedtocontrol,whateverthedurationoflocktherapy.
Inanothermodel,L-AmBwassuperiortofluconazoleineradicatingC.albicansbiofilm[44]. Allthese
animalstudiesusedC.albicansandsiliconecatheters.
L-AmBhasbeenusedaslocktherapyforfewpatients[53–55](Table2). L-AmBlockwasused
inapatientonhemodialysiswithpersistentCRBSIduetoC.albicansonapolyurethanecatheter[53].
The catheter could not be removed due to lack of other available sites for vascular access. It was
exchangedoverawire.Alocksolutioncontaining2.67mg/mLofL-AmBin5%dextroseplus200UI
(66UI/mL)ofheparinwaspreparedanddwelledfor12hinthelumenofthenewcatheter. Antifungal
lock with L-AmB was continued for 6 days concomitantly with systemic micafungin. Systemic
antifungal treatment was prolonged 6 months without any relapse of fungal infection. The same
protocolofL-AmBlockwasusedfortheothercases,leadingtosuccessin7/8episodes[54,55].
The only clinical prospective study of antifungal lock came from a pediatric context [56].
ThisstudyinvolvedL-AmBandvariouspolyurethaneandsiliconecentralvenouscatheters(CVC)
fromdifferentmanufacturers. L-AmBsolutionat2mg/mLwasinstilleddailyintothecatheterin
12childrenhaving13episodesofCandidaCRBSI[56]. Theantifungalsolutiondwelledfor8–12hin
thecatheterlumen. Allchildrenreceivedanadditionalsystemicantifungaldrug,eithercaspofungin
orL-AmB.BloodculturesfromeachlineoftheCVCwerecollectedeveryday. Thespeciesinvolved
wereC.albicans,C.glabrata,C.lusitaniae,C.parapsilosis,andRhodotorularubra. Theprimaryendpoint
wastwoconsecutivenegativeCVC-bloodcultureswithin5daysofinitiatingantifungallock. Atotalof
10infectionsin10patients(77%)mettheprimaryendpoint,andcurewithoutrelapsewasobtained
in5of13(38%)infections. Culturebecamenegativewithin2.4days(range1–5days). Althoughthe
numberofpatientsofthispilotstudywasinsufficienttoconcludethatL-AmBantifungallockwas
fullyeffective,itprovidedencouragingresults.
Pathogens2018,7,6 8of15
Table2.Antifungallockinclinicalsetting.Onlycaseswithsufficientdatawereincludedinthetable.Forallcases,seeref[25].Thereisnodataforazoleantifungal
lockorothertypeofechinocandins.d-AmB=AmphotericinBdeoxycholate;L-AmB=liposomalAmphotericinB;ALT:antifungallocktreatment;BC:bloodculture.
Episode SystemicAntifungal
PientAge ALTDuration AntifungalLockSolution Fungus MajorConclusion Reference
Number Treatment
deoxycholateAmB
Success;BCnegativeafter7daysofALT;
35y.o. 1 Nosystemictherapy 12h/dayfor21days d-AmB2.5mg/mL Malasseziafurfur Arnowetal.[46]
feverresolutionafter2daysofALT
d-AmBfor3daysthen
30y.o. 1 8–12h/dayfor15days d-AmB2.5mg/mL C.glabrata Initialsuccessbutrelapse6weekslater;nodetailsonBC
fluconazolefor4days
Benoitetal.[47]
EradiactionofC.albicansbutfailureforC.glabratathat
1 d-Ambfor1day 6h/dayfor7months d-AmB2.5mg/mL C.albicans+C.glabrata
40y.o. recurred5daysafterALTdiscontinuation
Successafter8monthsofATL;BCnegativefor8months;
2 Fluconazolefor3days 6h/dayfor8months d-AmB2.5mg/mL C.glabrata
nodataafterALTwithdrawal
d-AmBfor6daysthen Success;ALTstartedafter6daysofsystemic
13y.o. 1 24h/dayfor20days d-AmB2.5mg/mL C.parapsilosis Wuetal.[48]
fluconazole(noduration) antifungaltherapy;
Success;feverresolutionafter3daysofALT;BC
2y.o. 1 d-AmBfor7days 12h/dayfor14days d-AmB2.5mg/mL C.albicans
negativeattheendofALT Vialeetal.[49]
Success;feverresolutionafter4daysofALT;BC
65y.o. 1 Fluconazolefor7days 12h/dayfor14days d-AmB2.5mg/mL C.albicans
negativeattheendofALT
Success;ALTstarted2daysafterantifungalsystemic; Angel-Morenoetal.
40y.o. 1 Fluconazole(noduration) 6h/dayfor14days d-AmB5mg/mL C.glabrata
surveillanceBCnegative;nofurtherdetails [50]
liposomalAmB
Castagnolaetal.
infant 1 L-AmBfor14days 8h/dayfor14days 2.67mg/mL+heparin100UI C.parapsilosis Success;BCnegativeafter2daysofALT
[53]
1 L-AmBfor8days 8h/dayfor7days 2.67mg/mL+heparin200UI C.glabrata+C.albicans Success;ALTstartedat24hofsystemictreatment
17
month-old 2 L-AmBfor10days 8h/dayfor10days 2.67mg/mL+heparin200UI C.albicans+C.glabrata Failure;nodetails
3 L-AmBfor16days 8h/dayfor16days 2.67mg/mL+heparin200UI C.glabrata Success;BCnegativeafter6daysofALT Buckleretal.[54]
Success;ALTstartedat24h;BCnegativeafter
7y.o. 1 L-AmBfor21days 8h/dayfor17days 2.67mg/mL+heparin200UI C.albicans
24hofALT
6
1 L-AmBfor15days 8h/dayfor15days 2.67mg/mL+heparin200UI C.parapsilosis Success;BCnegativeafter8daysofALT
month-old
1y.o. 1 L-AmBfor14days 8h/dayfor14days 2.67mg/mL+heparin200UI C.guillermondii Success;BCnegativeafter3daysofALT
Success;BCnegativebeforeinitiationofALT;ALT
24h/day,changeevery PaulDimondietal.
64y.o. 1 Micafunginfor14days 2.67mg/mL+heparin200UI C.albicans startedaftercatheterexchangeoverawireandafter
12h,for6days [55]
9daysofsystemicantifungal
Caspofungin
9y.o. 1 caspofungin 12h/dayfor14days 3.33mg/mL+heparin200UI C.lipolytica Success;BCnegativeafter4daysofALT Özdemiretal.[52]
Failure;ALTstarted7daysaftersystemiccaspofungin:
1.5y.o. 1 caspofungin 12h/dayfor14days 3.33mg/mL+heparin200UI C.parapsilosis Isgüderetal.[51]
BCstillpositiveat14days
Pathogens2018,7,6 9of15
Finally,availabledataobtainedwithinvitromodelsgenerallysuggestbetteractivityofL-AmB
locksolutionscomparedtod-AmBlock-solutions;theysuggestthatL-AmBlocksolutionscouldhelpto
significantlyreduceCandidaspp. maturebiofilmswithoutbeingabletototallyeradicatesessileyeasts.
Theyhighlighttheinfluenceofboththenatureofthesubstrate(polystyrene,silicone,polyurethane,
andsoon),lockduration,andageofthetreatedbiofilm.
4. EchinocandinLockSolutions
Echinocandinsactbyinhibitingthebiosynthesisof1.3-beta-glucans,whicharekeyconstituentsof
thefungalcellwall,andthismodeofactionhasencouragedresearcherstoinvestigatetheiranti-biofilm
activity. Theanti-adherentandanti-biofilminterestofechinocandinswasrapidlyshown,evenusing
lowconcentrations,closetoMIC[6,9]. Morerecently,astudydoneonmorethan200Candidaisolates
helped to show that whereas fluconazole belonged to non-active anti-biofilm agents, caspofungin
belongedtohighlyactiveanti-biofilmagents[24]. Anidulafungin,caspofunginandmicafunginall
belongtoechinocandins,andyetmoststudiesinvestigatingtheechinocandinpossiblyusedinlock
therapyfocusoncaspofungin,andtoalesserextentonmicafunginandanidulafungin.
Cateauetal.havedevelopedaninvitromodeltoevaluatetheeffectofa12hlockwithcaspofungin
(2µg/mL)ormicafungin(5µg/mL)solutionsagainstyoung(12hold)ormature(5daysold)C.albicans
on silicone surfaces; tested concentrations corresponded to about 100 times MIC and anti-biofilm
activitywasevaluatedbasedonreductioninthemetabolicactivityofsessileyeasts(XTTmethod)[35].
Their results showed that even 48 h after the end of the lock, 12 h and 5-day-old biofilms treated
by caspofungin and micafungin were still inhibitedby at least 55% (caspofungin: ≈60% to ≈90%)
(micafungin: ≈55%to≈92%)and47%(caspofungin: ≈47%to≈88%)(micafungin: ≈54%to≈91%),
respectively. The weakness of this study is that only two C. albicans strains were studied and that
they were collection instead of clinical strains. However, the same lock-model was then applied
to biofilms prepared in the same manner, with all 8 C. albicans and 6 C. glabrata clinical strains
isolatedfrominfectedcatheters,andthisconfirmedtheinterestofechinocandinlock-solutions[26].
C.glabrataclinicalstrainswereallisolatedfrominfectedcatheters,andtherebyconfirmedtheinterest
of echinocandin lock-solutions. Irrespective of antifungal concentration and biofilm age (12 h or
5daysold),echinocandinsstillinhibitedC.albicansbiofilmsbyatleast65%(caspofungin: inhibition
≥≈77%,micafungin: ≥≈65%)48haftertheendofthelock. Thestraindependenceofresultswas
mentionedinthecaseofmature5-day-oldbiofilmbutnotfor12hones. RegardingC.glabratabiofilms,
inhibitorypercentagescausedbycaspofunginat5g/mLwerestillatleast55.8%(12hbiofilms)or
44.4%(5daysold)48haftertheendofthelockcomparedtothoseofmicafunginat5g/mLwhichwere
stillatleast92.4%(12holdbiofilms)or90.3%(5daysold). So,micafunginshowedgreatersustained
efficacythancaspofunginagainstboth12hand5-day-oldbiofilmsofC.glabrata,whereastherewasno
obviousdifferenceinthecaseofC.albicansbiofilms. Inanyevent,noneofthetestedconditionstotally
eradicatedbiofilms,aswasreportedbythesameteamregardingL-AmB[27]. Öncüstudiedthekilling
activity(CFUcounts)ofcaspofunginsolutions(300to1000timesMIC)against5-day-oldbiofilmson
siliconecatheters;thelockperiodswere1,3,5and7days,caspofunginsolutionsbeingreplacedevery
2days)[38]. Theweaknessofthisstudyisthatonlytwostrainswerestudied,onefromeachspecies.
Theresultsshowedthat,forbothC.albicansandC.parapsilosisbiofilms,caspofunginlocksolutions
significantlydecreasedthenumberofyeastsinsidecatheters,startingonthefirstdayofthetreatment,
andthecatheterswerecompletelysterileatthefifthday,whichcorrespondstoefficacycomparableto
thatreportedbythisauthorusingamphotericinB,asmentionedinthepreviouspart.
Unlikeothers,Koetal. reportedquitelowactivity(CFUmethod)ofcaspofunginlocksolutions
against5-day-oldbiofilmsofC.albicansandC.glabrataonpolyurethanesurfaces,thelowestcaspofungin
concentrationsatwhicha50%decreaseinbiofilmwasobservedremaining>256µg/mL[36].
Only the study of Simitsopoulou et al. compared the activity on anidulafungin, caspofungin
andmicafunginlocksolutionsfor24hagainstmetabolismandcultivabilityofsessileyeastsfrom
mature (48 h old) biofilms of C. albicans; however, they considered only one collection strain [39].
Pathogens2018,7,6 10of15
The lowest average concentrations at which a 50% decrease in C. albicans biofilm was observed
were very close to each other, whatever the tested drug: 0.25 µg/mL for both micafungin and
anidulafungin and 0.5 µg/mL for caspofungin. However, the highest inhibition of sessile yeast
metabolism reached 100% and was only obtained by caspofungin, the other echinocandins being
less active. This was similar to results reported by the team using L-AmB, as mentioned in the
previous part; taken as a whole these results suggested the high and comparable invitro efficacy
of L-AmB and caspofungin against mature biofilms of C. albicans. Simitsopoulou et al. extended
the study to 6 and 5 clinical strains of C. lusitaniae and C. guilliermondii, respectively and showed
that anidulafungin and micafungin had reduced activity against C. lusitaniae and C. guilliermondii
biofilmscomparedwithcaspofungin: thelowestconcentrationofanidulafunginandmicafunginat
whicha50%decreaseinbiofilmwasobservedrangedfrom32µg/mLto>2,048µg/mL,biofilmsof
C.lusitaniaebeingthemostresistant[39]. Interestingly,caspofunginwasthemostactiveagentagainst
C.lusitaniaeandC.guilliermondiibiofilms,achievingcompleteandpersistent(upto72hpost-lock)
biofilmeradicationatlockconcentrationsrangingfrombetween512µg/mLto2048µg/mLdepending
onthetestedstrains. Fiorietal. comparedtheanti-biofilmactivityofanidulafunginandcaspofungin
locksolutionsinthepresenceorabsenceof50%serum,studyingnumerousclinicalstrainsof5Candida
species[37].Overall,thepresenceofserumincreasedconcentrationsatwhicha50%decreaseinbiofilm
wasobserved,whateverthetestedechinocandin,andtheseconcentrationsweregenerallylowerfor
anidulafungincomparedtocaspofungin,suggestingthesuperiorityofanidulafunginagainstsessile
yeasts. Moreprecisely,inpresenceofserum,concentrationswererelativelycloseforC.albicansand
C.tropicalisbiofilms,rangingfrom0.5µg/mLto2µg/mL(anidulafungin)or2µg/mLto8µg/mL
(caspofungin) and 1 µg/mL to 4 µg/mL (anidulafungin) or 2 µg/mL to 8 µg/mL (caspofungin),
respectively.AnidulafunginwasclearlymoreactiveagainstC.glabrata(between1µg/mLand2µg/mL
(anidulafungin)orbetween2µg/mLand16µg/mL(caspofungin))andC.krusei(between4µg/mL
and 16 µg/mL (anidulafungin) or ≥8 µg/mL (caspofungin)). However, both were only weakly
activeagainstC.parapsilosis(≥4µg/mL).Finally,theresultsofBasasetal. werebasedonmetabolic
(XTTmethod)andviabilitytests(staining)andunderscoredthehighefficacyofanidulafunginagainst
C.parapsilosismaturebiofilms(48h-old)onsiliconesurfaces: theyreportedaconcentrationatwhich
a50%or90%decreaseinbiofilmwasobserved≤0.25µg/mLorequalto1µg/mL.Underthetest
conditions,anidulafunginthusappearedmuchmoreeffectivethanL-AmB[40]. Lownetal. recently
studied the efficacy of a 24 h lock treatment based on micafungin used alone or combined with
ethanoland/ordoxycyclineineradicatinga24h-oldbiofilmdevelopedinpolystyrenemicroplate
wells under static conditions [41]. Five C. albicans strains were studied, two clinical ones, derived
echinocandin-resistantisolatesandthreecollectionones,andanti-biofilmactivitywasevaluatedwith
metabolic(XTT)andcultivability(CFUcounts)assays.Theresultsdemonstratedthatthecombination
comprising20%ethanol,0.01565µg/mLmicafunginand800g/mLdoxycyclinewashighlyeffective
against both forming and mature C. albicans. This combined lock-solution reduced the metabolic
activityoftreatedbiofilmsto≤2%,andpreventedtheregrowthfrombothformingandmaturebiofilms;
however,thissolutionwasnotmoreactivethan20%ethanolusedalone. Byusingathree-druglock
therapyapproach,authorswishedtomaximizethebroad-spectrumactivityofthesolutiontoinclude
bothyeastsandbacteria,whichwouldalsoprobablyreducetheriskforresistancedevelopment.
Lownetal.havementionedthatonlylimitedinvestigationshavebeenperformedontheeffect
of high concentrations of ethanol on catheter integrity, depending on the material. Use of such
acomplexthree-druglocksolutionmayconsequentlyhelptominimizetheethanolconcentration
used. Anapproachcombininga1:1mixtureof70%ethanolandmicafungin5mg/Lwasappliedas
antifungallocktherapytotreatapreterminfantforC.albicansCRBSI[57]. Thepatientalsoreceived
systemicL-AmBat5mg/kg/day. Thelocksolutiondwelledfor12h. After48hacombinationtherapy
ofL-AmBandmicafunginforpersistentpositivebloodcultureswasadministered,andthecatheter
waslockedagainforanother12hwithsuccess. Systemicantifungaltherapywasdiscontinuedafter