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`JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2003, p. 4817–4819
`0095-1137/03/$08.00⫹0 DOI: 10.1128/JCM.41.10.4817–4819.2003
`Copyright © 2003, American Society for Microbiology. All Rights Reserved.
`
`Vol. 41, No. 10
`
`Comparison of In Vitro Activities of 17 Antifungal Drugs against a
`Panel of 20 Dermatophytes by Using a Microdilution Assay
`Bertrand Favre,† Bettina Hofbauer, Kwang-Soo Hildering,‡ and Neil S. Ryder§*
`Novartis Research Institute, Vienna, Austria
`
`Received 11 February 2003/Returned for modification 30 April 2003/Accepted 29 July 2003
`
`The in vitro activities of 17 antifungal drugs against a panel of 20 dermatophytes comprising 6 different
`species were determined using a microdilution assay according to the NCCLS M38-P method with some
`modifications. Terbinafine was the most potent systemic drug while tolnaftate and amorolfine were the most
`active topical agents.
`
`Most superficial infections caused by dermatophytes can be
`rapidly eradicated with topical antifungals. However, two com-
`mon dermatophytoses, tinea capitis and tinea unguium, do not
`respond well to such treatment and require the use of systemic
`antimycotics to be cured (2, 8, 23). Numerous topical agents
`and several systemic ones are available, but comparison of
`their in vitro activity against dermatophytes has been ham-
`pered by the lack of a well accepted MIC assay for these fungi
`(1, 5, 9, 10, 13, 14, 18–20, 25). Recently, several groups have
`adapted the proposed reference method for broth dilution
`antifungal susceptibility testing of conidium-forming filamen-
`tous fungi (17) for developing a more specific assay for der-
`matophytes (6). Since the preparation of conidia inoculum is
`sometimes a challenge with dermatophytes, a microdilution
`assay appears to be the ideal format (5, 6, 13, 20). However,
`assay parameters, such as the temperature, duration, or growth
`inhibition endpoint, are still the subject of debate (11, 12, 21).
`The NCCLS guidelines are primarily aimed toward suscep-
`tibility testing of clinical isolates. The aim of the present study
`was to establish an NCCLS-compatible assay, which was opti-
`mized for our primary purpose of evaluating investigative an-
`tifungal agents.
`Twenty strains of dermatophytes, Trichophyton rubrum (n ⫽
`5), Trichophyton tonsurans (n ⫽ 5), Trichophyton mentagro-
`phytes (n ⫽ 4), Microsporum canis (n ⫽ 4), Microsporum gyp-
`seum (n ⫽ 1), and Epidermophyton floccosum (n ⫽ 1), were
`employed. Five strains were obtained from either the fungal
`biodiversity center (Centralbureau voor Schimmelcultures,
`Utrecht, The Netherlands), T. mentagrophytes strain 560.66
`(Novartis Fungal Index [NFI] 5606), T. tonsurans strains 171.65
`(NFI 5177) and 729.88 (NFI 5178), or American Type Culture
`Collection (Manassas, Va.), T. rubrum strain 18759 (NFI 5182)
`and T. tonsurans strain 10217 (NFI 5176). The others were
`clinical isolates.
`
`* Corresponding author. Mailing address: Infectious Diseases Biol-
`ogy, Room 4210, Novartis Institutes for Biomedical Research, Inc., 100
`Technology Square, Cambridge, MA 02139. Phone: (617) 871-3143.
`Fax: (617) 871-7047. E-mail: neil.ryder@pharma.novartis.com.
`† Present address: Department of Dermatology, University Hospital
`CHUV, Lausanne, Switzerland.
`‡ Present address: Igeneon, Vienna, Austria.
`§ Present address: Novartis Institutes for Biomedical Research, Inc.,
`Cambridge, MA 02139.
`
`RPMI 1640 medium (Invitrogen) with L-glutamine and with-
`out bicarbonate was buffered at pH 7.0 with 0.165 M morpho-
`linepropanesulfonic acid (Sigma). Terbinafine, naftifine, bute-
`nafine, voriconazole, and itraconazole were synthesized at
`Novartis. Fluconazole was extracted and purified from Di-
`flucan tablets (Pfizer). Miconazole, amorolfine, and tolci-
`clate were obtained from Janssen, Roche, and Montedison,
`respectively. Clotrimazole, econazole, ketoconazole, ciclo-
`piroxolamine, tolnaftate, griseofulvine, and undecylenic acid
`were purchased from Sigma, while tioconazole was bought
`from U.S. Pharmacopeia. All drugs were dissolved and two-
`fold serially diluted in dimethyl sulfoxide (DMSO).
`All standard media were purchased from Merck. T. menta-
`grophytes, T. tonsurans, and E. floccosum were grown on Kim-
`mig agar, T. rubrum was grown on potato dextrose agar, and M.
`canis and M. gypseum were grown on malt extract agar at 26°C
`for 2 to 3 weeks. Mycelium and spores were scraped from the
`plates and dispersed in a small volume of Sabouraud 2% dex-
`trose broth (usually 20 ml for 25 plates) using a sterile glass
`homogenizer. After addition of 5% DMSO as a cryopro-
`tectant, the fungal suspension was stored at ⫺80°C (7). The
`viable count was determined by serially diluting the stock in
`0.86% NaCl and spreading 50 ␮l/plate on the same agar me-
`dium as the one used for the inoculum preparation.
`Microdilution plates with flat-bottom well (Greiner) were
`set up in accordance with the NCCLS M38-P reference
`method (17). The final concentration of DMSO was 1%, and
`the inoculum size was 5 ⫻ 103 CFU/ml. Plates were incubated
`for 4 to 5 days, depending on the growth in control wells
`without drug, at 30°C for E. floccosum and M. canis and 35°C
`for the other dermatophytes. Growth inhibition was scored
`visually with the aid of an inverted magnifying mirror from 4 to
`0 according to the NCCLS M38-P reference method, and MIC
`of all tested drugs corresponded to the lowest concentration
`giving a score of 1 (equivalent to about 75% inhibition). After
`MIC determination, the total volume of each well, starting
`from the last well in which growth was observed up to the
`highest drug concentration tested, was transferred into glass
`tubes containing 5 ml of Sabouraud 2% dextrose broth (pH
`6.5). Tubes were incubated for 1 week at 30°C, and growth was
`inspected visually after shaking. The minimal fungicidal con-
`centration (MFC) corresponded to the lowest drug concentra-
`tion (in the assay plate) at which no viable fungus remained.
`
`4817
`
`CFAD v. Anacor, IPR2015-01776
`ANACOR EX. 2023 - 1/3
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`
`4818
`
`NOTES
`
`J. CLIN. MICROBIOL.
`
`NC
`NC
`NC
`NC
`NC
`NC
`NC
`NC
`0.55
`0.44
`0.14
`NC
`NC
`NC
`NC
`NC
`0.07
`
`0.125
`
`⬎32
`⬎128
`⬎2
`⬎8
`⬎4
`
`0.25
`
`0.25
`
`⬎128
`⬎32
`
`⬎4
`⬎16
`⬎1
`⬎4
`⬎0.25
`⬎2
`
`2
`
`MFC
`mean
`Geom.
`
`MFC90
`
`MFC50
`
`MIC
`mean
`Geom.
`
`MIC50MIC90
`
`T.tonsurans
`
`5178
`
`5177
`
`5176
`
`5175
`
`0105
`
`5182
`
`0.125
`
`⬎128
`⬎32
`
`60
`0.7
`0.003
`0.067⬎4
`0.25
`16
`0.032⬎1
`0.083⬎4
`0.003⬎0.25
`0.25
`0.028
`0.029
`0.014
`0.37
`6.3⬎128
`0.033⬎2
`⬎8
`0.22
`⬎4
`0.23
`0.006
`
`0.125
`
`8
`
`1
`
`0.063
`
`Downloaded from
`
`0.008
`0.25
`0.5
`0.063
`0.25
`0.008
`0.063
`0.063
`0.031
`0.5
`32
`0.063
`
`0.5
`0.016
`
`1
`
`64
`
`1
`
`64
`0.5
`0.004
`0.063
`0.25
`0.016
`0.063
`0.004
`0.031
`0.031
`0.008
`0.5
`
`0.031
`0.13
`0.25
`0.004
`
`4
`
`32
`0.5
`0.004
`0.063
`0.25
`0.008
`0.125
`0.004
`0.016
`0.031
`0.008
`0.25
`
`0.031
`0.06
`0.125
`0.008
`
`4
`
`32
`0.5
`0.004
`0.031
`0.50
`0.016
`0.125
`0.004
`0.031
`0.031
`0.016
`0.50
`
`0.008
`0.06
`0.125
`0.008
`
`2
`
`32
`0.5
`0.008
`0.125
`0.50
`0.063
`0.250
`0.001
`0.004
`0.016
`0.008
`0.50
`
`0.063
`0.25
`0.500
`0.008
`
`8
`
`64
`0.5
`0.002
`0.031
`0.13
`0.016
`0.016
`0.002
`0.016
`0.031
`0.008
`0.13
`
`0.016
`0.13
`0.016
`0.002
`
`4
`
`0.5
`0.002
`0.031
`0.13
`0.016
`0.016
`0.001
`0.008
`0.016
`0.008
`0.13
`
`0.031
`0.25
`0.125
`0.002
`
`8
`
`128
`
`0.016
`0.03
`0.500
`0.002
`
`1
`
`http://jcm.asm.org/
`
` on September 29, 2015 by guest
`
`All experiments were repeated at least twice (topical agents) or
`more (systemic drugs). MICs usually did not differ by more
`than one dilution step.
`For our purpose of evaluating drugs against a defined set
`of dermatophytes, large-scale preparation of inoculum with
`well-defined CFU is advantageous. Therefore, we initially
`compared MICs obtained with four drugs, terbinafine, itracon-
`azole, fluconazole, and griseofulvine, against a few dermato-
`phytes using either fresh inocula prepared according to the
`method of Jessup et al. (13) or frozen inocula. The results in-
`dicated that both freezing and the presence of mycelium in the
`inoculum did not significantly affect MICs of antifungals, in
`agreement with results obtained by Manavathu et al. (15).
`The ideal incubation temperature, 28 to 35°C, and time for
`antifungal susceptibility testing of dermatophytes are still a
`matter of debate. In our hands, M. canis and E. floccosum grew
`very poorly at 35°C, so we decreased the temperature to 30°C
`for these two species. Concerning the incubation time, 4 to 5
`days was found to be sufficient to observe prominent growth in
`control wells without drug with our restricted panel of der-
`matophytes, which were selected from a larger panel on the
`basis of
`their abundant conidium production and robust
`growth properties.
`There is no consensus concerning the optimal growth inhi-
`bition endpoint for MICs (5, 6, 21). We uniformly adopted a
`score of 1 as the MIC for all the tested drugs, as recommended
`by Norris et al. (20). The obtained MIC results are presented
`in Table 1. Among the six systemic antifungals tested, flucon-
`azole, griseofulvine, itraconazole, ketoconazole, terbinafine,
`and voriconazole, the allylamine terbinafine was the most po-
`tent agent. In our assay, voriconazole was significantly more
`active than itraconazole, in agreement with the findings of
`Fernandez-Torres et al. (6) but in contrast to the results of
`Perea et al. (21). The reason for these differences is unknown.
`We also measured MFCs with a simple but rigorous method
`requiring complete elimination of viable particles in the cul-
`ture well during the MIC incubation time, while MFC is often
`defined as a ⱖ99% reduction of CFU (3, 4, 16, 22, 24). Amo-
`rolfine and the squalene epoxidase inhibitors, butenafine, naf-
`tifine, and terbinafine, were systematically fungicidal toward
`our panel of dermatophytes within the range of tested concen-
`trations, ⱖ32⫻ the MIC at which 50% of the organisms were
`inhibited (Table 1).
`In summary, the proposed microdilution assay for dermato-
`phytes is convenient and reproducible. While parameters such
`as scoring range and MIC endpoint could be harmonized, it
`appears that the incubation temperature cannot be uniformly
`set at 35°C. The test strains were selected for adequate growth
`and normal susceptibility to standard drugs; we suggest that a
`comparable set of strains could be picked from any dermato-
`phyte collection and used to obtain similar results. Among the
`systemic antifungals tested, terbinafine was the most potent,
`while tolnaftate and amorolfine were the most active topical
`agents.
`
`We thank Ingrid Leitner for the preparation of dermatophyte inoc-
`ula.
`
`andMIC90,MICatwhich50and90%oforganismsareinhibited,respectively.
`ECO,econazole;MCO,miconazole;TIO,tioconazole;AMO,amorolfine;CPX,ciclopiroxolamine;UDA,undecylenicacid;Geom.,geometric;NC,notcalculable.MICsareexpressedinmicrogramspermilliliter.MIC50
`aAbbreviations:TER,terbinafine;ITR,itraconazole;KET,ketoconazole;VOR,voriconazole;FLU,fluconazole;GRI,griseofulvin;BUT,butenafine;NFT,naftifine;TCI,tolciclate;TLN,tolnaftate;CLT,clotrimazole;
`
`bSpeciesandNFIidentificationnumberaregiven.
`
`64
`1.0
`0.002
`0.016
`0.06
`0.016
`0.063
`0.001
`0.008
`0.016
`0.004
`0.50
`
`64
`0.5
`0.004
`0.008
`0.13
`0.016
`0.125
`0.002
`0.031
`0.016
`0.008
`0.50
`
`0.016
`0.06
`0.250
`0.004
`
`1
`
`64
`0.5
`0.004
`0.125
`0.06
`0.031
`0.250
`0.002
`0.031
`0.016
`0.031
`0.50
`
`0.063
`0.13
`0.250
`0.004
`
`4
`
`64
`0.5
`0.004
`0.031
`0.13
`0.016
`0.125
`0.001
`0.016
`0.016
`0.008
`0.50
`
`0.016
`0.13
`0.500
`0.004
`
`2
`
`64
`0.5
`0.004
`0.031
`0.25
`0.016
`0.125
`0.002
`0.016
`0.031
`0.016
`0.50
`
`0.016
`0.06
`0.250
`0.002
`
`1
`
`64
`1.0
`0.008
`0.063
`0.50
`0.063
`0.250
`0.004
`0.063
`0.031
`0.008
`0.50
`
`0.031
`0.13
`0.063
`0.008
`
`4
`
`64
`0.5
`0.002
`0.016
`0.13
`0.008
`0.063
`0.004
`0.031
`0.031
`0.008
`0.50
`
`0.016
`0.13
`0.125
`0.004
`
`2
`
`64
`0.5
`0.004
`0.063
`0.25
`0.031
`0.031
`0.004
`0.063
`0.031
`0.016
`0.25
`16
`0.063
`0.50
`0.250
`0.004
`
`64
`64
`1.0
`1.0
`0.001
`0.008
`0.500
`1.000
`1.00⬎2.00
`1.000
`0.125
`0.500
`0.063
`0.002
`0.004
`0.016
`0.063
`0.016
`0.063
`0.008
`0.031
`1.00
`0.25
`64
`64
`0.063
`0.125
`1.00
`1.00
`1.000
`0.500
`0.004
`0.016
`
`64
`0.5
`0.004
`0.250
`0.50
`0.031
`0.063
`0.008
`0.063
`0.063
`0.031
`0.25
`32
`0.063
`1.00
`0.250
`0.016
`
`64
`1.0
`0.002
`0.250
`0.50
`0.063
`0.031
`0.008
`0.063
`0.063
`0.063
`0.050
`32
`0.063
`1.00
`0.250
`0.016
`
`64
`1.0
`0.002
`0.250
`0.50
`0.063
`0.016
`0.004
`0.063
`0.063
`0.031
`0.25
`16
`0.063
`1.00
`0.250
`0.016
`
`64
`1.0
`0.004
`0.250
`0.50
`0.063
`0.063
`0.063
`0.063
`0.063
`0.031
`0.025
`16
`0.031
`1.00
`0.500
`0.008
`
`UDA64
`CPX
`AMO0.008
`TIO
`0.008
`MCO0.25
`ECO0.016
`0.250
`CLT
`0.008
`TLN
`0.063
`TCI
`0.031
`NFT
`0.031
`BUT
`0.50
`GRI
`FLU
`VOR0.031
`0.13
`KET
`0.250
`ITR
`0.008
`TER
`
`1.0
`
`4
`
`5143
`
`5140
`
`5139
`
`5132
`
`5606
`
`5165
`
`5137
`
`0158
`
`5164
`
`5168
`
`5167
`
`5154
`
`0150
`
`0167
`
`T.rubrum
`
`T.mentagrophytes
`
`seum
`M.gyp-
`
`M.canis
`
`cosum
`E.floc-
`
`Drug
`
`TABLE1.MICof17antifungalsagainstapanelof20dermatophytesa
`
`MICforspeciesb
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`VOL. 41, 2003
`
`NOTES
`
`4819
`
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