In vitro susceptibility of filamentous fungi from mycotic keratitis to azole drugs

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ORIGINAL ARTICLE/ARTICLE ORIGINAL

In vitro susceptibility of filamentous fungi from mycotic keratitis to azole drugs

Sensibilite ´ in vitro aux azole ´ s de champignons filamenteux, agents de ke ´ ratite fongique

C.S. Shobana

^a,

* , A. Mythili

^a

, M. Homa

^b

, L. Galgóczy

^b

, R. Priya

^a

, Y.R. Babu Singh

^a

, K. Panneerselvam

^c

,

C. Vágvölgyi

^b

, L. Kredics

^b

, V. Narendran

^d

, P. Manikandan

^d,e

aDepartmentofMicrobiology,Dr.G.R.DamodaranCollegeofScience,AvanashiRoad,CivilAerodromePost, Coimbatore,641014,Tamilnadu,India

bFacultyofScienceandInformatics,DepartmentofMicrobiology,UniversityofSzeged,Szeged,Hungary

cDepartmentofMicrobiology,MRGovernmentArtsCollege,Mannargudi,614001,Tamilnadu,India

dDepartment of Microbiology, Aravind Eye Hospital and Post-Graduate institute of Ophthalmology, Coimbatore,641014,Tamilnadu,India

eDepartmentofMedicalLaboratory,AppliedMedicalSciencesCollege,MajmaahUniversity,SaudiArabia

Received25June2014;receivedinrevisedform29September2014;accepted20October2014

KEYWORDS Mycotickeratitis;

Fungalisolates;

Antifungalsusceptibility andazoledrugs

Summary

Objective.— Theinvitroantifungalactivitiesofazoledrugsviz.,itraconazole,voriconazole, ketoconazole,econazoleandclotrimazolewereinvestigatedinordertoevaluatetheirefficacy againstfilamentousfungiisolatedfrommycotickeratitis.

Methods.— Thespecimencollectionwascarriedoutfromfungalkeratitispatientsattending AravindeyehospitalandPost-graduateinstituteofophthalmology,Coimbatore,Indiaandwas subsequently processed for the isolation of fungi. The dilutions of antifungal drugs were preparedin RPMI1640 medium. Minimuminhibitory concentrations(MICs) were determined andMIC50andMIC90werecalculatedforeachdrugtested.

Results.— Atotalof60fungalisolateswereidentifiedasFusariumspp.(n=30),non-sporulating moulds (n=9), Aspergillus flavus (n=6), Bipolaris spp. (n=6), Exserohilum spp. (n=4), Curvulariaspp.(n=3),Alternariaspp.(n=1)andExophialaspp.(n=1).TheMICsofketocona- zole,clotrimazole,voriconazole, econazoleanditraconazoleforallthefungalisolatesranged between 16mg/mLand0.03mg/mL,4mg/mLand0.015mg/mL,8mg/mL and0.015mg/mL,8mg/mL

* Correspondingauthor.

E-mailaddress:shobanasenthilkumar@gmail.com(C.S.Shobana).

Availableonlineat

ScienceDirect

www.sciencedirect.com

http://dx.doi.org/10.1016/j.mycmed.2014.10.024 1156-5233/#2014ElsevierMassonSAS.Allrightsreserved.

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and0.015mg/mLand32mg/mLand0.06mg/mLrespectively.FromtheMIC50andMIC90values,it couldbedecipheredthatinthepresentstudy,clotrimazolewasmoreactiveagainstthetestisolates atlowerconcentrations(0.12—5mg/mL)whencomparedtootherdrugstested.

Conclusion.— Theresultssuggestthatamongstthetestedazoledrugs,clotrimazolefollowedby voriconazoleandeconazolehadlowerMICsagainstmouldsisolatedfrommycotickeratitis.

#2014ElsevierMassonSAS.Allrightsreserved.

MOTSCLÉS Kératitemycosique; Sensibilitéaux antifongique; Médicamentsazolés

Re´sume´

Objectif.— L’activitéantifongiqueinvitrodesazolésàsavoir,l’itraconazole,levoriconazole, le kétoconazole,l’éconazole et leclotrimazole aétéétudiée afin d’évaluerleur efficacité vis-à-visdeschampignonsfilamenteuxisolésdekératitemycosique.

Me´thodes.— Les échantillons provenant de patients consultant pour kératite fongique au Aravind EyeHospital et au Post-Graduate Institute ofOphthalmology, Coimbatore, enInde ontétémisenculturepourrecherchedechampignons.Lesdilutionsdesantifongiquesontété réaliséesenRPMI1640.Lesconcentrationsminimalesinhibitrices(CMI)ontétédéterminéeset lesCMI50etCMI90ontétécalculéespourchaqueantifongiqueétudié.

Re´sultats.— Soixantesouches dechampignonsontétéisolées:Fusariumspp.(n=30), moi- sissuresnefructifiantpas(n=9),Aspergillusflavus(n=6),Bipolarisspp.(n=6),Exserohilum spp.(n=4),Curvulariaspp.(n=3),Alternariaspp.(n=1)etExophialaspp.(n=1).LesCMIdu kétoconazole,duclotrimazole,duvoriconazole,del’itraconazoleetdel’éconazolevis-à-visde l’ensembledesisolatsfongiquesvariaientrespectivemententre16mg/mLet0,03mg/mL,4mg/

mLet0,015mg/mL,8mg/mLet0,015mg/mL,8mg/mLet0,015mg/mLet32mg/mLet0,06mg/

mL.ÀpartirdesvaleursdesCMI₅₀etCMI₉₀quenousavonsobtenues,leclotrimazoleseraitla moléculelaplusactivevis-à-visdesisolatsétudiés,avecdesconcentrations(0,12à5mg/mL) plusfaiblesquecellesdesautresantifongiquestestés.

Conclusion.— Lesrésultatssuggèrentque,parmilesantifongiquesazoléstestés,leclotrimazole suivi par le voriconazole et l’éconazole avaient les CMI les plus basses vis-à-vis des moisissuresisoléesdekératitesmycosiques.

#2014ElsevierMassonSAS.Tousdroitsréservés.

Introduction

Microbialkeratitisisthemostcommonsevereocularinfec- tion and may be caused by a variety of bacteria, fungi (yeasts,mouldsandmicrosporidia)andprotists(e.g.Acan- thamoeba). It is characterized by an acute or sub-acute onsetofpain,conjunctivalinjectionandcornealulceration withastromalinflammatoryinfiltrate[42,16,6,36].Keratitis duetofilamentousfungiisbelievedtousuallyoccurfollow- ingtrauma, the key-predisposingfactor, in healthyyoung malesengagedinagriculturalorotheroutdoorwork[8].The traumatizingagentscanbeof plantor animalorigin(even dustparticles),thateitherdirectlyimplantfungalconidiain the corneal stroma, or abrade the epithelium-permitting invasion by exogenous fungi [41]. The etiologic agents of mycotic keratitis show a varying pattern with respect to geographic locality andclimatic conditions[7].More than 105speciesoffungispanning70generahavebeenreported tocausemycotickeratitis[1].Ofthese,Fusariumspp.and Aspergillusspp.arethemostcommonetiologicalagentsof cornealulcerations[2,5,28].

Pujol etal.[32]reported that amphotericin B(AMB)is probablythemosteffectivedruginvivo,althoughtherehave been many clinical treatment failures. Natamycin, a tetraene polyene,has long been considered the mainstay oftreatmentforfilamentousfungalkeratitis.Althoughthese drugshavepoorocularpenetration,theyhaveprimarilybeen usefulincaseswithsuperficialcornealinfection[29].Azoles

(imidazoles and triazoles)viz.,ketoconazole (KTZ),mico- nazole(MCZ),fluconazole(FLZ),itraconazole(ITC),econa- zole(ECN)andclotrimazole(CLT),inhibitfungalergosterol biosynthesisatlowconcentrations,whileathigherconcen- trationstheyappear tocausedirectdamagetothefungal cellwalls[40].AccordingtoSrinivasan[35],ongoingresearch towards rapid diagnosis and specific drug therapy could minimizethemorbiditycausedbythispreventabledisease.

The current knowledge on antifungal susceptibilities is mainlybasedonWesternliteratureandlocaldataavailable inIndiapertainingtofilamentousfungiotherthanFusarium and Aspergillus are inadequate. The present study was undertakentoisolateandidentifyfilamentousfungiinvolved in mycotickeratitis fromthepatientsattending atertiary care eyehospital inCoimbatore,Tamilnadu,India,andto determine their in vitro susceptibility against five azole antifungal drugs by employing the Clinical and laboratory standardsinstitute(CLSI)brothmicrodilutionmethodM38- A2document[9].

Materials and methods

Samples andfungalisolates

This non-randomizedstudy was carriedout atAravind eye hospitalandPost-graduateinstituteofophthalmology,Coim- batore,India.Thespecimencollectionwascarriedoutbet- weenOctober2012andAugust2013.Cornealscrapingswere

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performed under aseptic conditionson each ulcer using a flame sterilized Kimura’s spatula, after instillation of 4%

preservativefreelignocaine(lidocaine)[18].Materialobtai- nedfromscrapingtheleadingedgeandthebaseoftheulcer was inoculateddirectly onto 5% sheep’sblood agar(SBA), chocolateagar(CA)andpotatodextroseagar(PDA)(250gof potatoslices,15gagar,10gdextroseand1000mLdistilled water),aswellasintobrainheartinfusion(BHI)brothwithout gentamicinsulphate.SBAbase,CAplatesandBHIbrothwere purchasedfromHimediaLaboratories,Mumbai,India.Plates wereincubatedunderaerobicconditionsat378C,whilethe PDAbottleswere incubatedat278Cforfungalgrowth,for 72h.TheobtainedmaterialwassubjectedtoGramstaining and 10% potassium hydroxide mount. Any positive fungal isolate was identified to the genus level and Aspergillus spp. were identified to the species level based on colony morphologyandlactophenolcottonbluemountpreparation of the fungal cultures employing thecellophane tape flag method[3,14,24].

Invitroazole susceptibilitytesting

AspertheCLSIguidelines,ineverybatchofMIC,A.flavus ATCC204304 was includedas referencestrain[9]. Allthe fungalisolatesweresubculturedonSDAplatesandincubated at 308C for 7 to 15 days. The inoculum suspension was preparedbyharvestingthesporesfrommatureplatesinto steriledistilledwater.Thesporesuspensionwasthenadjus- tedspectrophotometricallytotherequiredoptical density for eachspecies asoutlinedinCLSIM38-A2 document[9], providinganinoculumconcentrationof0.410⁴to510⁴ CFU/mL,whichwas verifiedbycolonycount.Furtherdilu- tions (1:50) were carried out using RPMI 1640 medium (Sigma-Aldrich,St.Louis,MO,USA).

The clinically used and commercially available azole drugsviz.,ITC(Sigma-Aldrich,St.Louis,MO,USA),voriconazole(VRC)(Aurolab,Madurai,India),KTZ(Himedia,Mum- bai,India),ECN(Aurolab,Madurai,India)andCLT(Aurolab, Madurai,India)werechosenforthepresentstudy.Thedrugs were dissolved in dimethyl sulfoxide and the dilutions werepreparedinRPMI1640inordertoachievearangeof 8—0.015mg/mL(ECN, VRCandCLT), 32—0.06mg/mL(ITC) and16—0.03mg/mL(KTZ).

Forthebrothmicrodilutionmethod,100mLofeachdrug dilutionand100mLof theprepared sporesuspensionwere addedintoU-bottomedmicrotiterplatewells.Twoseparate wellsweremaintained,oneasgrowthcontrol(100mLmedia and100mLinoculum)andanotherassterilitycontrol(100mL mediaand100mLwater).Theplateswereincubatedat288C untilgrowthwasvisibleingrowthcontrol.MICsweredeter- mined visually with the aid of a reading mirrorand were definedas thelowest drugconcentrationthat caused 80%

inhibitionofthegrowthincomparisontothegrowthcontrol.

TheMIC50wastakenastheMICthatwasthemedianvalueand similarly,theMIC90wasthe90thpercentilevalueandrepre- sentedtheconcentrationofdrugthatwouldinhibit90%of theisolatestested[10].

Results and discussion

Of 108 ocular samplesthat wereprocessed, a total of 48 isolatesofbacteriaand60isolatesoffilamentousfungiwere

obtained.Eachofthepositivesamples(n=60)grewonlyone moldandmixedinfectionswerenotobtained.Thesefilamen- tousfungalisolateswereidentifiedasFusariumspp.(n=30), non-sporulating moulds (n=9), Aspergillus flavus (n=6), Bipolaris spp. (n=6),Exserohilum spp. (n=4), Curvularia spp. (n=3), Alternaria spp. (n=1), and Exophiala spp.

(n=1).Inthepresentstudy,thecommonaetiologicalagents ofcornealulcerswereidentifiedasfilamentousfungirather thanbacteria.SimilarresultswereobtainedbyBharathietal.

[6],Manikandanetal.[24]andHomaetal.[15]inSouthIndia.

Lecketal.[22]alsoreportedsimilarresultsfromSouth,North andEastIndia.ButotherstudiesfromThailandandMalaysia reportedthatthemostfrequentcausativeagentsofmicrobial keratitiswerebacteria[34,27].

Manystudiesreportedthatamongthefilamentousfungi, Fusarium and Aspergillus species were identified as most commoncornealpathogens[23,39,37,33].Thepresentstudy showedthat50%ofcornealinfectionswerecausedbyFusa- rium spp., whereas Aspergillus spp. were responsible for only10% of fungal cornealinfections. Dematiaceousfungi suchas Bipolarisspp., Exserohilum spp.,Curvularia spp., Alternariaspp.andExophialaspp.werealsoisolatedinthe present study. Similar incidences of dematiaceous fungal keratitis have been reported previously [23,39,11]. Nine (15%)oftheobtained60fungalisolateswerenotidentifiable owingtolackof sporulation.Similartoourfindings,Srini- vasan et al.[37] revealed that out of 155 fungal isolates culturedfrom154cornealulcers,47.1%wereFusariumspp., 16.1%wereAspergillus spp.,and theremaining organisms wereadiversemixtureofunusualfungalpathogensincluding a large number of unidentified dematiaceous (13.5%) and hyaline(9.6%)fungalspecies.

Thecommonlyusedantifungalmedicationsinthetreat- ment of fungal keratitis include polyenes (natamycin and AMB) andazoles (KTZ, FLZ, ITCand ECN). However, it is difficulttowidelyusetopicalnatamycinduetoitshighprice [43].FusariumandAspergillusstrainsarequitesensitiveto AMB,butpoorpenetration intocorneasandobvious simu- lativesymptomsmakeitstopicalpreparationunsuitableto beadministeredwithalargedosageandforalong-time[43].

Azoles,mostcommonlyFLZandITC[4,21]areoftenchosen asthecombinedmedicationstoreducethetoxicityandside effectsofAMB.

TheMIC50andMIC90oftheazoledrugstestedforvarious fungal isolates are shown in Table 1. CLT was the most effective antifungal drug against all the isolates tested exceptfortheNSM,forwhichITCwas notablyactive.CLT wastheonlydrugthatwasmostactiveagainst(1—4mg/mL) fusaria.VRC,ECNandCLT(0.015—0.5mg/mL)followedby ITC(0.25—0.5mg/mL)weremorepromisingagainstA.flavus isolates. Bipolaris isolates were inhibited effectively by certain concentrations of CLT and ECN in the range of 0.125—1mg/mL. Exserohilum isolates were inhibited by CLTandVRCintheconcentrationrangeof 0.03—2mg/mL.

Incomparison withother drugs, VRCand CLTshowedthe lowestMICrange (0.125—0.25mg/mL)for Curvulariaisola- tes.

Theabilitytoinhibitfungalisolatesatsuchlowconcen- trationshowsthatCLTcouldbeusedinthefirst-linetherapy ofmycotickeratitis.However,Manikandanetal.[24]repor- tedtherequirementofahigherconcentrationofCLTagainst Aspergillusspp.comparedtothepresentstudy.Itisnotable

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thatFusariumstrainsweresusceptibletoECNatalowerMIC similar tothereports of Galarretaetal.[12].The present findingsrevealedthat,Fusariumspp.andExophialaspp.were resistanttoVRC andhence higherconcentration ofdrug is required for the effective treatment. However, there are studies thathave suggested thatVRC mayhave a broader antifungalspectrum[19,20].Similartoourfindings,Lalitha etal.[17] andIqbal etal. [31] stated thatFusarium spp.

had highest MICs to VRC. Prajna et al. [25] reported that monotherapywithtopicalvoriconazolecannotberecommen- dedforfilamentousfungalkeratitis.Additionally,thereare reportsofintraocularVRCtobesafeinvitroandinvivoandless toxictotheretinathanAMB[26,13].Pfalleretal.[30]reported thatITCexertsnegligibleactivityagainstFusariumspp.andit hasalsobeenstatedthatITChasseldombeenadministeredfor Fusariuminfectionswithnon-univocalresults[38].

Table1 Invitrosusceptibilityoffilamentousfungiisolatedfromkeratomycosistoazoledrugs.

Sensibilite´ invitrodeschampignonsfilamenteuxisole´ sdeke´ ratomycoseauxme´ dicamentsazole´ s.

Agents Groups MICrange^a

(mg/mL)

MIC50b

(mg/mL)

MIC90c

(mg/mL)

GM^d (mg/mL)

CLT Fusariumspp. 1—4 4 4 3.134

Non-sporulatingmolds 0.25—4 1 2 1.166

Aspergillusflavus 0.015—0.5 0.125 0.5 0.156

Bipolarisspp. 0.125—1 0.125 0.5 0.197

Exserohilumspp. 0.03—2 0.12 2 0.205

Curvulariaspp. 0—0.25 0.25 0.25 0.25

Alternariaspp. — — — —

Exophialaspp. — — — —

ECN Fusariumspp. 2—8 4 8 5.401

Bipolarisspp. 0.25—1 0.5 1 0.5

Curvulariaspp. 0—0.5 0.5 0.5 0.5

KTZ Fusariumspp. 8—16 16 16 13.928

Aspergillusflavus 0.25—2 2 2 1.259

Bipolarisspp. 0.125—4 0.5 1 0.629

Exserohilumspp. 0.03—8 0.125 8 0.416

Curvulariaspp. 1—2 1 2 1.259

ITC Fusariumspp. 16—32 32 32 31.269

Bipolarisspp. 0.25—8 0.25 0.5 0.561

Exserohilumspp. 0.06—32 0.06 32 0.471

Curvulariaspp. 0.25—0.5 0.5 0.5 0.396

VRC Fusariumspp. 1—8 4 8 4

Bipolarisspp. 0.06—4 0.25 0.25 0.278

Curvulariaspp. 0.125—0.25 0.25 0.25 0.198

MIC:minimuminhibitoryconcentration;Antifungalagents:CLT:clotrimazole;ECN:econalole;KCN:ketoconazole;ITC:itraconazole;VRC:

voriconazole.

aIntervalbetweenthelowestandhighestMICs.

bMinimuminhibitoryconcentrationmedianoftheantifungalagent.

c Minimumconcentrationoftheantifungalagent90thpercentile.

dGeometricmeanofMICs.

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OurresultsindicatedthathigherconcentrationofITCand KTZwasrequiredfortheinhibitionoftheinvolvedfilamentous fungalisolates,andbasedonthedataobtained,CLTfollowed byVRCandECNarethesuggestedantifungalagentsforthe first-linetherapyofhumankeratomycosescausedbyfilamen- tousspecies.Inaddition,thisstudyhasgeneratedMICdatafor sparinglytested filamentousfungi suchas Alternaria spp., Bipolaris spp.,Curvularia spp.,Exserohilum spp. andExo- phialaspp.

Conclusion

Overall,thedeterminationofMICsoftheinvestigatedfive- azoleantifungaldrugsagainstthefilamentousfungicausing keratitiswasusefulinunderstandingtheefficacyofincrea- sedconcentrationsofthedrugsininhibitingfungalgrowth.

Thepresentstudyobservedavariationintheoverallactivity of the azole drugs depending on the type of the fungal species and the drug concentration.Due to the fact that thepracticeofsubjectingfungalisolatestoantifungalsus- ceptibility testsisuncommonacrossthe diagnosticmicro- biology laboratories in India, and that the susceptibility pattern is depending from the involved fungus as well as thenatureandconcentrationoftheapplieddrug,itisfurther emphasizedthattheisolatesshouldcompulsorilybeexami- nedfor theirsusceptibilitytoensureanaccuratetherapy.

Disclosure of interest

Theauthorsdeclarethattheyhavenoconflictsofinterest concerningthisarticle.

Acknowledgment

The research of M.H., L.G., Cs.V. was supported by the European Union andthe State of Hungary,co-financed by the European Social Fund in the framework of TÁMOP 4.2.4.A/2-11-1-2012-0001‘‘NationalExcellence Program’’.

The relating research groups were also supported by the University Grants Commission (UGC), Bahadur Shah Zafar Marg,NewDelhi-110002,India[F.No.42—469/2013(SR)]

andtheIndo-Hungarianbilateralexchangeprogramno.IA/

INSA-HAS Project/2013-2015/189 providing infrastructure andresearchequipment.

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