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SHORT COMMUNICATION/COURTE COMMUNICATION

Determination of antifungal susceptibility ofAspergillus spp. responsible for otomycosis byE-test methodDetermination de la sensibilite a divers antifongiques dAspergillus sp.responsable dotomycose par la methode du E-test

E. Aktas a, N. Yigit b,*

a Microbiology and Clinical Microbiology Department, Ataturk University Medical Faculties, 25070 Erzurum, Turkeyb Medical Laboratory Department, Health Services Vocational Training School, Ataturk University, 25070 Erzurum, Turkey

Received 28 December 2008; received in revised form 26 February 2009; accepted 13 March 2009

Available online 28 April 2009

KEYWORDSAspergillus spp.;Otomycosis;Ketoconazole;Itraconazole;E-test

SummaryObjective. In this study,we investigate in vitro activities of twoazolederivates, ketoconazoleand itraconazole, against Aspergillusspp. isolates causing otomycosis.Materials and methods. Mycological analysis of samples from ear canals of patients wasperformed by culturing onto Sabourauds dextrose agar and by evaluating microscopically.

Aspergillusspp. was identified with colony morphology and microscopic appearance, and testedfor susceptibility to ketoconazole and itraconazole by E-test method. A total of 52 isolatescomprising 32 A. niger, 12 A. fumigatus and eight A. flavus were tested.Results. Minimal Inhibitory Concentration (MIC) ranges of itraconazole and ketoconazole havebeen found as follows: 0.53 and 0.254 mg/ml for A. niger, 14 and 1.53.0 mg/ml for

A. fumigatusand 0.751.5 and 0.250.75 mg/ml for A. flavus at 24 h.Conclusion. It has been observed that both of the antifungal agents showed an in vitro activityagainst all Aspergillusspp. tested.# 2009 Elsevier Masson SAS. All rights reserved.

MOTS CLSAspergillus sp. ;Otomycose ;Ktoconazole ;

RsumObjectif. Dans cette tude, nous avons test la sensibilit in vitro au ktoconazole et litraconazole de souches dAspergillussp. isoles dotomycoses.Materiels et methodes. Les analyses mycologiques des prlvements du conduit auditif despatients ontt faites par culture sur Sabourauddextrose agar. Les souches dAspergillussp. ontt identifies par examen macroscopique et microscopique et leur sensibilit au ktoconazole

Journal de Mycologie Mdicale (2009) 19, 122125

* Corresponding author.E-mail address: nimyigit@hotmail.com (N. Yigit).

1156-5233/$ see front matter # 2009 Elsevier Masson SAS. All rights reserved.doi:10.1016/j.mycmed.2009.03.004

mailto:nimyigit@hotmail.comhttp://dx.doi.org/10.1016/j.mycmed.2009.03.004http://dx.doi.org/10.1016/j.mycmed.2009.03.004mailto:nimyigit@hotmail.com

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Itraconazole ;E-test

et litraconazole a t teste par la mthode du E-test. Au total, 52 souches ont t isolesdont 32 A. niger, 12 A. fumigatuset huit A. flavus. Les concentration minimale inhibitrice (CMI)de litraconazole et du ktoconazole sont respectivement comprises entre 0,53 et 0,254 mg/ml pour A. niger, 14 et 1,53,0 mg/ml pour A. fumigatuset 0,751,5 et 0,250,75 mg/ml pour

A. flavusaprs 24 heures dincubation.Conclusion. Les deux antifongiques tests ont une bonne activit in vitro contre toutes lessouches dAspergillussp. testes.# 2009 Elsevier Masson SAS. Tous droits rservs.

Introduction

Moulds of the genus Aspergillus are widespread in the envi-ronment, being found in the soil, in the air, on plants and ondecomposing organic matter. In the home, these moulds areoften found in dust and on food. Moulds of the genus Asper-

gillus are opportunistic pathogens, especially in immunecompromised patients [20].

Otomycosis is described as fungal infection of external earcanal. This infection is worldwide in distribution but it is

more common in tropical and subtropical regions and occursin adults of all ages and both sexes: children are lesscommonly affected. Main fungi causing this condition are

Aspergillus spp., particularly A. fumigatus, A. niger andA. flavus [11,13,15].

Currently, amphotericin B, itraconazole, variconazole,ketoconazole and caspofungin have been approved for thetreatment of aspergillosis [10,13,18]. However, not all spe-cies have the same susceptibility pattern and it may benecessary to perform in vitro susceptibility testing for selec-tion and monitoring of antifungal therapy. Various techni-ques have been used to test filamentous fungi, includingbroth macro- and microdilution methods, agar dilution anddisc diffusion. A reference method for filamentous fungi, the

NCCLS M38-P protocol was published in 1998. However, thisstandardized method is cumbersome and time-consuming toperform in the busy diagnostic laboratory and interpretationof results is not always easy with azole antifungal agents dueto trailing endpoints. This has lead to the development ofseveral simpler methods of susceptibility testing [14].

E-test is a simple, agar based, quantitative minimal inhi-bitory concentration (MIC) method. The reagent consists of athin, calibrated plastic strip with a predefined, exponentialand continuous gradient of antifungal across 15 twofolddilutions. E-test has been satisfactorily used to test bacteria,yeasts and moulds [12,14,19]. Good correlation has beenreported between E-test and CLSI reference methods when

pathogenic fungi were tested against a number of azoleantifungal agents [13,6,8,16].In this study, we aimed at investigating in vitro activities

of ketoconazole and itraconazole against Aspergillus spp.isolates causing otomycosis by E-test method.

Materials and methods

Aspergillus isolates

We performed mycological analysis on debris and scarpingsamples from the ear canals of 52 patients who had beenclinically diagnosed with otomycosis. Fungal elements with

septate hyphaewere seen in the microscopicevaluationof earspecimens. These specimens were inoculated onto Sabou-rauds dextrose agar (SDA) (Difco, USA) slants and incubatedat 26 8C and 35 8C until the growth was obtained. The plateswere not discarded before four weeks. Aspergillus spp. wereidentified with colony morphology and microscopic appea-rance. A total of 52 isolates from 52 patients, each from adifferent patient, were identified. A. fumigatus ATCC 204305were used as reference strains for susceptibility testing.

E-test procedure for Aspergillus spp.Aspergillus spp. were obtained from 7-day-old cultures onSDA. One milliliter of sterile 0.85%NaCI was added to thecultures and mixed gently with Pasteur pipette to suspendconidia and hyphal particles. The suspension was transferredto a 13 mm 100 mm sterile tube, vortexed gently, andallowed to stand for 15 min. The supernatant was thenremoved and adjusted with sterile NaCI to the turbidity ofa 0.5 McFarland standard (corresponding to approximately106 colony forming units/mL). Test were performed in RPMI1640 medium (Gibco, New York, USA), pH 7.0, supplementedwith 2% glucose, buffered with morpholinepropanesulfonicacid (MOPS) (FisherBiotech, New Jersey, USA) and 1.8% agar

(Difco, USA). E-test strips containing a continuous concen-tration gradient of itraconazole (0.00232 mg/ml), ketoco-nazole (0.00232 mg/ml) were obtained from AB Biodisk(Stockholm, Sweden). All strips were stored at 20 8C untiluse and left at room temperature for 20 min. The E-test wasperformed according to manufacturers instruction. Forty-five milliliters of the RPMI 1640 agar was dispensed into150 mm diameter Petri dishes. The plates were inoculatedby dipping a sterile swab into the inoculum suspension andstreaking it across the entire surface of agar in three direc-tions. The plates were dried at room temperature for 15 minbefore E-test strips were applied, and were incubated at35 8C for 2448 h. MIC values were the drug concentration atwhich the border of the elliptical inhibition zone intersectedthe scale on the antifungal strip. MICs were determined asthe lowest concentration of drug that gave approximately80% inhibition of the growth control for the tested drugs.

Results

Aspergillus strains

Overall, 52 Aspergillus strains were isolated 52 patients withotomycosis. A. niger(61.5%) was the most frequent speciesrecovered, followed by A. fumigatus (23.0%) and A. flavus(15.3%).

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Aspergillus MIC results

The MIC data of the itraconazole and ketoconazole againstthe Aspergillus spp. are shown in Table 1. MIC ranges ofitraconazole and ketoconazole have been found as follows:0.53 and 0.254 mg/ml for A. niger (32 strains), 14 and1.53.0 mg/ml for A. fumigatus (12 strains) and 0.751.5and 0.250.75 mg/ml for A. flavus (eight strains) accordingto 24 h results. The MIC range of two antifungal agents for

A. fumigatus and A. flavus strains tested after 24 and 48 hincubation was similar while MIC range of two azole for

A. niger was 0.56.0 and 0.756 mg/ml at 48 h. The MICrange of itraconazole and ketoconazole for the referencestrain A. fumigatus ATCC 204305 was 0.1251 and 0.751.5respectively (Table 1).

Discussion

Otomycosis or fungal otitis externa has typically been des-cribed as fungal infection of the external auditory canal withinfrequent complications involving the middle ear. Althoughrarely life-threatening, the disease process presents a chal-lenging and frustrating entity for both patients and otola-ryngologys, for it frequently requires long term treatmentand follow-up, yet the recurrence rate remains high [10].

The detection and identification of the Aspergillus spp.which may cause such disease, and determination of theirantifungal susceptibilities will conduct the effective the-

rapy. Recently, the number of studies about the resistanceto antifungal drugs has increased and primary and secondaryresistance among strains causing human mycosis has beenreported [11,13].

The CLSI M38-A document describes both macro andmicrodilution methods for the antifungal susceptibility test-ing of opportunistic filamentous fungi, which has improvedthe interlaboratory agreement of MICs. These methods arecumbersome and time-consuming; other approaches havebeen evaluated for fungal testing in recent years. Amongthese approaches, E-test has been suggested as an alterna-tive procedure for antifungal susceptibility testing ofmoulds. The E-test used in the present study has been

demonstrated in many studies that it has potential value

for testing the susceptibilities of Aspergillus spp.[4,5,7,9,12,14,17,19].In this study, we tested in vitro activities of itraconazole

and ketoconazole, against Aspergillus spp., which wereidentified as the causative agents of otomycosis by usingE-test. The strains of 32 A. niger, 12 A. fumigatus and eight

A. flavus had itraconazole MICs in the range 0.53, 14, and0.751.5 mg/ml at 24 h, respectively. MIC ranges this speciesat 48 h were 0.56.0, 1.56.0 and 1.01.5 mg/ml, respec-tively (Table 1).

Itraconazole,available as oral capsule,oral suspension andintravenous forms is a triazole antifungal known as having invitro and in vivo activities against Aspergillus species [18]. Invitro activity of this agent for Aspergillus spp. has previously

been tested using E-test by several investigators. The authorsreported the itraconazole MIC ranges from 0.19 to 32.0 mg/mlfor A. niger, 0.033.0 mg/ml for A. flavus and 0.02332.0 for

A. fumigatus [4,5,7,9,11,13,14,16].The other azole derivate ketoconazole, especially topical

form was for its efficacy against Aspergillus spp. causingotomycosis. Topical ketoconazole had a higher resolutionrate and lower rate of disease recurrence [10]. The MICvalues of ketoconazole obtained in our study for A. niger,

A. fumigatus and A. flavus have been found 0.254.0, 1.53.0 and 0.250.75 mg/ml at 24 h and 0.756.0, 1.54.0 and0.51.0 mg/ml at 48 h, respectively (Table 1).

As a result, in vitro data obtained in this study suggest that

both of the antifungal agents showed an in vitro activityagainst all Aspergillus spp. tested and two azole derivatesseems to be as antifungal agents in the therapy of otomycosisdue toAspergillus spp. Studies with large numbers of isolatesneed to be performed to learn more about the antifungalresistance of Aspergillus spp. causing otomycosis to diffe-rent agents and preferably finding testing system that canidentify the resistance documented in vivo.

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Table 1 Minimal Inhibitory Concentration (MIC) data of itraconazole and ketoconazole for Aspergillusspp.Valeurs des CMI de litraconazole et du ketoconazole pour Aspergillus sp.

Species (n) Antifungal agent Incubation time (h) MIC range (mg/ml) MIC50 (mg/ml) MIC90 (mg/ml)

A. niger (32) Itraconazole 24 0.53 2.0 2.048 0.56.0 2.0 4.0

Ketoconazole 24 0.254 1.0 3.048 0.756 1.0 4.0

A. fumigatus (12) Itraconazole 24 14 1.5 4.048 1.56 1.5 4.0

Ketoconazole 24 1.53.0 1.5 3.048 1.54.0 3.0 3.0

A. flavus (8) Itraconazole 24 0.751.5 0.75 1.548 1.01.5 1.0 1.5

Ketoconazole 24 0.250.75 0.25 0.7548 0.501.0 0.5 1.0

A. fumigatus ATCC 204305 Itraconazole 48 0.1251 Ketoconazole 48 0.751.5

124 E. Aktas, N. Yigit

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