Pharmacology and therapeutics

In vitro activities of antifungal drugs against dermatophytes

isolated in Tokat, Turkey

G€ulg€un Yenis�ehirli1, MD, Ebru Tunc�o�glu1, MD, Aydan Yenis�ehirli2, MD, PhD, andYunus Bulut1, MD, PhD

1Departments of Microbiology and Clinical

Microbiology, Faculty of Medicine,

Gaziosmanpas�a University, Tokat, Turkey,

and 2Departments of Pharmacology,

Faculty of Medicine, Gaziosmanpas�aUniversity, Tokat, Turkey

Correspondence

G€ulg€un Yenis�ehirli, MD

Department of Microbiology and Clinical

Microbiology

Faculty of Medicine, Gaziosmanpas�aUniversity

Tokat 60100

Turkey

E-mail: gulgun.yenisehirli@gop.edu.tr

Conflict of interest: none

Abstract

Background In this study, we aimed to establish the in vitro antifungal susceptibilities of

terbinafine, miconazole, itraconazole, ketoconazole, griseofulvin, and amphotericin B

against dermatophyte isolates.

Methods One hundred and seventy-seven clinical isolates were tested: Trichophyton rubrum

(n = 78), Trichophyton mentagrophytes (n = 49), Epidermophyton floccosum (n = 30),

Trichophyton verrucosum (n = 16), and Trichophyton tonsurans (n = 4). The broth

microdilution assay for antifungal susceptibility testing of dermatophytes was performed

according to Clinical and Laboratory Standards Institute guidelines in the M38-A2 document.

Results Our minimum inhibitory concentration (MIC) results showed that the values for

terbinafine for all dermatophyte isolates were significantly lower than the values for

amphotericin B, miconazole, itraconazole, ketoconazole, and griseofulvin. For T. rubrum

isolates, amphotericin B was more active than miconazole, itraconazole, and ketoconazole.

Among the antifungal drugs tested, griseofulvin had the highest minimum inhibitory

concentration values for T. mentagrophytes isolates.

Conclusion Terbinafine was found to be the most effective antifungal drug against all

tested dermatophyte isolates. Griseofulvin was the less active antifungal drug against

T. mentagrophytes isolates. Performing antifungal susceptibility testing is especially

important for screening the development of antifungal resistance.

Introduction

Dermatophytes are a group of fungi that infect kerati-nized tissues such as hair, skin, and nail.1 In recent years,the incidence of infections caused by dermatophytes hasincreased considerably, especially in immunocompromisedpatients. These fungal infections establish an importantpublic health problem because of prolonged treatment ofthe disease and its refractivity to therapy.2 The selectionof proper therapeutic options is usually decided by loca-tion and extent of infection. Topical therapies with azolesare used for localized dermatophyte infections. For thetreatment of extensive or resistant dermatophytosis, sys-temic drugs are required.3,4 Although many topical andoral antifungal agents are available for the treatment ofdermatophytosis, some patients have a poor clinicalresponse to these drugs. As antifungal resistance in fungalisolates has been reported both in vitro and in vivo,in vitro antifungal susceptibility testing has becomeimportant for choosing an effective antifungal therapy.5–8

In vitro susceptibility testing also provides monitoring of

the development of antifungal resistance. Recently, theClinical and Laboratory Standards Institute (CLSI) hasdeveloped an approved standard method for testing anti-fungal susceptibility of filamentous fungi, including thedermatophytes.9

In this study, we aimed to establish the in vitro antifun-gal susceptibilities of terbinafine, miconazole, itracona-zole, ketoconazole, griseofulvin, and amphotericin Bagainst dermatophyte isolates.

Materials and methods

All dermatophyte isolates were obtained from the culture

collection at the mycology laboratory of the Gaziosmanpasa

University Hospital. The identification of dermatophyte

isolates was performed by the macroscopic and

microscopic examination of colonies, urease test, in vitro

hair perforation test, and temperature tolerance test. One

hundred and seventy-seven clinical isolates were tested:

Trichophyton rubrum (n = 78), Trichophyton mentagrophytes

(n = 49), Epidermophyton floccosum (n = 30),

ª 2013 The International Society of Dermatology International Journal of Dermatology 2013, 52, 1557–1560

1557

Trichophyton verrucosum (n = 16), and

Trichophyton tonsurans (n = 4).

For in vitro antifungal testing, miconazole, itraconazole,

ketoconazole, griseofulvin, and amphotericin B (Sigma–Aldrich,

St. Louis, MO, USA) were used as standard powders.

Terbinafine hydrochloride (batch no: ACEH004493) was

provided by Bilim Pharmaceuticals (Istanbul, Turkey).

The broth microdilution assay for antifungal susceptibility

testing of dermatophytes was performed according to CLSI

guidelines in the M38-A2 document.9 Isolates were subcultured

onto oatmeal agar (Difco, Detroit, MI, USA) for T. rubrum and

potato dextrose agar (Oxoid, Basingstoke, Hampshire, UK) for

T. mentagrophytes, E. floccosum, T. verrucosum, and

T. tonsurans. Plates were incubated at 30 °C for 4–5 days.

Stock inoculum suspensions were obtained from each strain by

covering the fungal colonies with 1 ml of sterile saline and

gently probing the surface with the tip of a transfer pipette. The

resulting mixture of conidia was transferred to sterile tubes and

allowed to settle for 10–15 minutes. Then, conidia were counted

with a hemocytometer and diluted with RPMI 1640 medium

(Sigma) buffered with MOPS (3-[N-morpholino] propanesulfonic

acid) (Sigma) to obtain the final inoculum size of approximately

1–3 9 103 conidia/ml.

Drug dilutions and inoculum were added in 96-well round

bottom microtiter plates and incubated at 35 °C for 4–7 days.

Growth and sterility controls were included with each isolate

tested. Each organism was tested in duplicate. Minimum

inhibitory concentrations (MICs) were determined visually using

an inverted reading mirror and were defined as the lowest drug

concentration that caused 80% inhibition of the growth as

compared with growth in the growth control well. The quality

control was performed by using Candida parapsilosis ATCC

22019 as a standard strain. As the interpretive breakpoints for

dermatophytes have not yet been set up, we used the

parameters established in the CLSI M38-A2 document for

filamentous fungi.9

Statistical analysis

n represents the number of studied species, and mean MIC and

geometric mean (GM MIC) values were calculated using

GraphPad Prism 5 demo version (GraphPad Software Inc., San

Diego, CA, USA). MIC values were compared with the Mann–

Whitney U-test, and P < 0.05 was considered significant.

Results

The MIC ranges, MIC50, MIC90, mean MIC, and GMMIC values of terbinafine, amphotericin B, miconazole,itraconazole, ketoconazole, and griseofulvin for all der-matophyte isolates are summarized in Table 1.Terbinafine was found to be the most effective drug

against all tested dermatophyte isolates (P < 0.05). ForT. rubrum isolates, the MIC ranges and MIC90 values of

miconazole, itraconazole, and ketoconazole were foundto be similar. The statistical analysis of MIC resultsshowed that amphotericin B was more active than mico-nazole, itraconazole, and ketoconazole (P < 0.05).Against T. mentagrophytes isolates, griseofulvin wasfound to be less active than other tested antifungal drugs(P < 0.05). The griseofulvin MIC was � 4 lg/ml for fiveT. mentagrophytes isolates. The MIC results obtained forE. floccosum showed that amphotericin B was moreactive than miconazole, ketoconazole, and griseofulvin(P < 0.05). Itraconazole was found to be more activethan other azoles against E. floccosum isolates(P < 0.05). On the other hand, no significant differenceswere observed between the MIC values of miconazole,itraconazole, and ketoconazole for other dermatophytespecies (P > 0.05).Statistical analysis of MIC values of all tested drugs for

T. tonsurans isolates showed no significant differencebetween the drugs (P > 0.05). Griseofulvin MIC valuesfor two isolates of T. verrucosum and one isolate ofT. tonsurans were 16 lg/ml and 4 lg/ml, respectively.

Discussion

In recent years, numerous studies on the in vitro antifun-gal susceptibility testing of dermatophytes have beendone.4–8, 10–16 However, the reference method for theantifungal susceptibility testing of dermatophytes was notavailable until 2008. In 2008, the CLSI published theM38-A2 document. This document describes testing con-ditions for dermatophytes.9 In our study, we used this ref-erence method for in vitro susceptibility testing ofdermatophyte isolates.Antifungal susceptibility testing results obtained in this

study showed that terbinafine was the most effective drugagainst all dermatophyte isolates (P < 0.05). Terbinafinewas effective against dermatophyte isolates with a MICrange of 0.007–0.5 lg/ml. Similar findings were reportedby other researchers.10,12,14–17 For T rubrum isolates,amphotericin B was more active than miconazole, itraco-nazole, ketoconazole, and griseofulvin (P < 0.05). GMMIC values of miconazole, itraconazole, and ketoconazolewere 0.10 lg/ml, 0.11 lg/ml, and 0.12 lg/ml, respectively.These values were similar to those reported by Fernandez-Torres et al.,10 Ara�ujo et al.,11 and Gupta and Kohli.12

GM MIC values of itraconazole and ketoconazole forT mentagrophytes were found to be 0.13 lg/ml and0.2 lg/ml, respectively. These MIC values were higherthan those reported by €Ozk€ut€uk et al.13 and similar tothose reported by Fernandez-Torres et al.,10 Ara�ujoet al.,11 and Gupta and Kohli.12 On the other hand, theGM MIC values of amphotericin B (0.14 lg/ml) andmiconazole (0.16 lg/ml) in our study were lower than in

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Pharmacology and therapeutics Antifungal susceptibility of dermatophytes Yenis�ehirli et al.1558

the study of Fernandez-Torres et al.10 In comparison withother tested drugs, griseofulvin was the drug that pre-sented the highest GM MIC and mean MIC values forT. mentagrophytes isolates (P < 0.05). Griseofulvin isonly effective on dermatophytes.18 It has been the first-line antifungal agent for the treatment of dermatophytosisfor many years, but today it is not widely used.19 Manystudies have documented griseofulvin-resistant isolates ofdermatophytes and existence of strains with elevated MIClevels to griseofulvin.5,6,8,20 Some researchers havereported lower in vitro activity of griseofulvin forT. mentagrophytes than for T. rubrum.6,8

In the current study, miconazole, itraconazole, andketoconazole showed similar activity against E. floccosumisolates. The GM MIC values of amphotericin B, mico-nazole, itraconazole, and ketoconazole for E. floccosum

isolates were 0.14 lg/ml, 0.29 lg/ml, 0.13 lg/ml, and0.43 lg/ml, respectively. These values have been foundhigher than those obtained in the study of Fernandez-

Torres et al.10 The MIC values of griseofulvin obtainedfor E. floccosum have been found similar to those docu-mented by Chadeganipour et al.8 and Perea et al.16

In our study, the MIC values of griseofulvin for twoisolates of T. verrucosum were 16 lg/ml. Similar resultswere reported by Chadeganipour et al.8 who found threegriseofulvin-resistant T. verrucosum isolates from Iran.The in vitro antifungal activity of amphotericin B,

miconazole, itraconazole, ketoconazole, and griseofulvinagainst tested T. tonsurans was found to be similar(P > 0.05). This situation could be attributed to thesmall sample size of isolates. GM MIC values of ampho-tericin B, miconazole, itraconazole, ketoconazole, andgriseofulvin for T. tonsurans isolates were 0.14 lg/ml,0.20 lg/ml, 0.24 lg/ml, 0.24 lg/ml, and 0.70 lg/ml,respectively. Similar results have been documented byother researchers.10,21

The results of this study indicate that terbinafine wasthe most effective antifungal drug against all tested

Table 1 In vitro antifungal activities of terbinafine, amphotericin B, miconazole, itraconazole, ketoconazole, and griseofulvinagainst all dermatophyte isolates

Species Antifungal drugs

MIC range

(lg/ml)

MIC50

(lg/ml)

MIC90

(lg/ml)

GM

(lg/ml) Mean � SEM MIC (lg/ml)

T. rubrum (n = 78) Terbinafine 0.007–0.5 0.03 0.125 0.04 0.06 � 0.009

Amphotericin B 0.03–0.5 0.06 0.25 0.07 0.10 � 0.01

Miconazole 0.03–1 0.125 0.5 0.10 0.16 � 0.02

Itraconazole 0.03–1 0.06 0.5 0.10 0.20 � 0.03

Ketoconazole 0.03–1 0.125 0.5 0.12 0.23 � 0.02

Griseofulvin 0.03–2 0.06 0.5 0.08 0.19 � 0.04

T. mentagrophytes

(n = 49)

Terbinafine 0.015–0.125 0.06 0.125 0.06 0.07 � 0.005

Amphotericin B 0.03–2 0.125 0.5 0.14 0.34 � 0.07

Miconazole 0.03–2 0.125 2 0.16 0.47 � 0.09

Itraconazole 0.03–0.5 0.25 0.5 0.13 0.24 � 0.02

Ketoconazole 0.03–4 0.25 1 0.20 0.67 � 0.15

Griseofulvin 0.03–16 0.5 2 0.49 1.55 � 0.45

E. floccosum

(n = 30)

Terbinafine 0.007–0.06 0.03 0.06 0.02 0.03 � 0.003

Amphotericin B 0.03–1 0.125 1 0.14 0.32 � 0.06

Miconazole 0.03–2 0.5 2 0.29 0.64 � 0.11

Itraconazole 0.03–1 0.06 1 0.13 0.25 � 0.06

Ketoconazole 0.125–2 0.25 2 0.43 0.67 � 0.12

Griseofulvin 0.03–2 0.25 2 0.35 0.92 � 0.16

T. verrucosum

(n = 16)

Terbinafine 0.007–0.125 0.015 0.06 0.01 0.02 � 0.008

Amphotericin B 0.06–1 0.25 1 0.29 0.47 � 0.09

Miconazole 0.125–2 0.5 2 0.56 0.96 � 0.21

Itraconazole 0.125–2 0.5 2 0.59 0.92 � 0.19

Ketoconazole 0.25–4 1 2 0.91 1.37 � 0.30

Griseofulvin 0.06–16 0.25 2 0.41 2.38 � 1.33

T. tonsurans

(n = 4)

Terbinafine 0.007–0.125 0.06 0.125 0.04 0.02 � 0.008

Amphotericin B 0.03–0.5 0.125 0.5 0.14 0.22 � 0.10

Miconazole 0.03–1 0.25 1 0.20 0.38 � 0.21

Itraconazole 0.06–1 0.125 1 0.24 0.42 � 0.21

Ketoconazole 0.06–2 0.125 2 0.24 0.60 � 0.46

Griseofulvin 0.125–4 0.5 4 0.70 1.40 � 0.88

MIC50, MIC for 50% of the isolates; MIC90, MIC for 90% of the isolates; GM, geometric mean; SEM, standard error ofmean.

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Yenis�ehirli et al. Antifungal susceptibility of dermatophytes Pharmacology and therapeutics 1559

dermatophyte isolates. Griseofulvin was the less activeantifungal drug against T. mentagrophytes isolates. Per-forming the antifungal susceptibility testing is particularlyimportant for screening the development of antifungalresistance. Besides, to establish interpretive breakpoints,in vitro susceptibility tests results must be supported byclinical outcome data.

Acknowledgments

This study was supported by Gaziosmanpasa UniversityResearch Fund. (Project no: 2006/15).

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