SUPPLEMENT TO
`
`Journal of the
`
`American
`
`cademy of
`
`DERMATOLOGY
`
`June 1999
`
`V()l.UMI<)40 NUMBICR 6 PART 2
`
`0 Novel treatment strategies for
`
`superficial mycoses
`
`Proceedings of a symposium held at the
`World Congress of Dermatology
`
`Sydney, Australia
`June l997
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`SUPPLEMENT TO
`
`Journal of the
`American Academy of
`DERMATOLOGY
`
`Copyright © 1999 by the American Academy ofDermatology, Inc.
`
`CONTENTS
`
`June, Part 2, 1999
`
`NOVEL TREATMENT STRATEGIES FOR
`SUPERFICIAL MYCOSES
`
`Novel treatment strategies for
`superficial mycoses: introduction
`Boni E. Elewski, MD, and
`Roderick J. Hay, DM, FRCP
`Cleveland, Ohio, and London,
`United Kingdom
`
`Diagnosis of onychomycosis made simple
`David H. Ellis, PhD
`North Adelaide, Australia
`
`Optimal growth conditions for the
`determination of the antifungal susceptibility
`of three species of dermatophytes with the
`use of a microdilution method
`Heather A. Norris, MT (ASCP),
`Boni E. Elewski, MD, and
`Mahmoud A. Ghannoum. PhD
`Cleveland, Ohio
`
`Pharmacokinetics of fluconazole in skin
`and nails
`
`Jan Faergemann, MD, PhD
`Gothenburg, Sweden
`
`Onychomycosis: therapeutic update
`Richard K. Scher, MD
`New York, New York
`
`Treatment of tinea capitis: beyond griseofulvin
`Boni E. Elewski, MD
`Cleveland, Ohio
`
`Oral therapy of common superficial fungal
`infections of the skin
`
`Jack L. Lesher, Jr., MD
`Augusta, Georgia
`
`The management of superficial candidiasis
`Roderick l. Hay, DM, FRCP, FRCPath
`London, United Kingdom
`
`Nfl Mosby
`
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`Jeffrey D. Bernhard. MD
`Associate Editors
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`Founding Editor
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`Editor Emeritus
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`
`the Journal of the
`Vol. 40. No. 6, Part 2, June 1999.
`Aineiican Academy of Dennatology (ISSN 0190-9622)
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`
`Optimal growth conditions for the determination of
`
`the antifungal susceptibility of three species of
`dermatophytes with the use of a microdilution
`method
`
`Heather A. Norris, MT (ASCP), Boni E. Elewski, MD, and Mahmoud A. Ghannoum, PhD
`Cleveland, Ohio
`
`As a prerequisite to standardization of dermatophyte susceptibility testing, conditions that
`support optimal growth of different dermatophyte species must be established. Eighteen
`isolates of Triclwphytun spp. ( T rubrum, T men,tagr0pltytes, T tonsurans) were grown in 4
`different media: RPMI I640 with L—glutamine, without sodium bicarbonate and buffered
`at pH = 7.0; antibiotic medium #3 (Penassay); yeast nitrogen base with 0.5% dextrose
`buffered at pH = 7.0; and Sabouraud dextrose broth. Incubation for 6 days at 35° C pro-
`duced the following results: RPMI and Sabouraud dextrose supported equally sufficient
`growth for all strains tested; Penassay supported growth of only 33% of the isolates test-
`ed, and buffered yeast nitrogen base did not support growth of any isolates. RPMI was
`selected as the optimal medium, and organisms were tested at both 30° C and 35“ C with
`a standardized inoculum density of 103 eonidia/mL. No temperature differences were
`noted in the amount of growth of the dermatophytes tested. With RPMI at an incubation
`temperature of 35° C, 3 inoculum sizes (103. 104, and 105 conidia/mL) were tested against
`4 antifungal agents: griseofulvin, itraconazole, terbinafme, and fluconazole. Inoeulum size
`did not affect minimum inhibitory concentration (MIC) results for itraconazole or
`terbinafme, but a larger inoculum produced a slightly higher MIC for griseofulvin and a
`noticeably higher MIC for fluconazole. Our data support the use of RPMI 1640, 35° C, and
`4 days as an incubation temperature and time, respectively, and an inoculum of 103 eo11i—
`dia/mL as optimal conditions for the determination of the antifungal susceptibility of der-
`matophytes. (J Am Acad Dermatol l999:40:S9-13.)
`
`Data related to susceptibility testing of der-
`matophytes are largely lacking. Efforts by the
`National Committee for Clinical Laboratory
`Standards (NCCLS) have focused primarily on
`testing yeast, and those efforts have resulted in the
`
`From the Center for Medical Mycology, Mycology Reference
`Laboratory, University Hospitals of Cleveland and Case Western
`Reserve University.
`This work was supported by a research grant from Pfizer Inc. Ms.
`Norris has also received research support
`from Janssen
`Pharmaceuticals and Novartis. Dr. Elewski has received research
`grants from Novartis. Janssen, Glaxo, and Westwood, and has
`served as a consultant for Mylan, Janssen. and Novartis. Dr.
`Ghannourn has received research support from Aronex, Janssen,
`Novartis. Nextar, Liposome, Pfizer. and Bristo|»Myers Squibb.
`Reprint requests: Mahmoud A. Ghannoum, PhD, Center for Medical
`Mycology. Mycology Reference Laboratory, University Hospitals
`of Cleveland, 1 l 101 Euclid Ave, LKS 5028. Cleveland, OH 44106.
`Copyright © 1999 by the American Academy of Dermatology, Inc.
`0190-9622/99/$8.00 + 0
`16/0/98106
`
`publication of a Reference Method for Broth
`Dilution Antifungal Susceptibility Testing of
`Yeasts, M27—A.l However, with the increasing
`variety of drugs available to treat dermatophytosis,
`the need for a reference method for dermatophyte
`testing has become apparent?
`Many variables need to be studied when a testing
`method is being developed, because the minimal
`inhibitory concentration (MIC) can be greatly
`affected by changes in inoculum size, length and
`temperature of incubation, media, and endpoint def-
`inition.3 Our aim was therefore to select a group of
`organisms responsible for causing most dermato-
`phytic infections and determine their growth under
`various conditions. Eighteen isolates of Tricke-
`phyton spp. were tested in 4 types of media at both
`30° C and 35° C. Once the media and temperatures
`were selected, we studied the effect of inoculum
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`
`S10 Norris, Elewski, and Ghannomn
`
`Journal of the American Academy of Dermatology
`June 1999
`
`Table I. Components of the 4 culture media used
`
`Component
`
`Ammonium sulfate
`
`p—Aminobenzoic acid
`L—Arginine
`L—Aspa.ragine
`L—Aspartic acid
`Beef extract
`Biotin
`Boric acid
`Calcium chloride
`Calcium nitrate
`
`Calcium pantothenate
`Chlorine chloride
`
`Copper sulfate
`L—Cystine 2HCl
`Dextrose
`Dipotassium phosphate
`Ferric chloride
`Folic acid
`L—Glutamic acid
`L—Glutamine
`
`Glutathione, reduced
`Glycine
`L—I-listidine
`
`L—Histidi11e monohydrochloride
`L-Hydrosyproline
`lnositol
`
`my0—Inositol
`L—Isoleucine
`L-Leucine
`
`L-Lysine HC1
`Magnesium sulfate
`Manganese sulfate
`
`RPMI
`1640*
`
`Yeast nitrogen broth
`with 5% dexti-ose*
`
`Sabouraud
`dextrose broth’?
`
`Penassay
`(antibiotic medium #3)?
`
`1.0 mg
`200 mg
`50 mg
`20 mg
`
`200 ug
`
`l00 mg
`
`3.0 mg
`
`65.2 mg
`2.0 g
`~
`
`I000 ttg
`20 mg
`300 mg
`1.0 mg
`10 mg
`15 mg
`
`20 mg
`
`35 mg
`50 mg
`50 mg
`40 mg
`48.8 mg
`
`5.0 g
`0.2 mg
`
`500 mg
`400 ug
`
`and 105 conidia/mL)
`concentration (10-3,104,
`against 4 antifungal agents: griseofulvin (Sigma
`Chemical Company, St Louis, Mo), itraconazole
`(J anssen Research Foundation, Beerse, Belgium),
`terbinafine (Novartis, East Hanover, NJ), and flu-
`conazole (Pfizer Inc, New York, NY). The results of
`this study serve as the foundation for the develop-
`ment of a standardized susceptibility testing method
`for dermatophytes.
`
`METHODS
`
`Organisms
`
`Two quality control organisms were used, Candida
`parapsilosis ATCC 22019 and Paecilomyces variotii
`ATCC 22319 (American Type Culture Collection,
`Rockville, Md).
`
`Antifungal drugs
`
`Four anti fungal drugs were used: griseofulvin (Sigma
`Chemical Company, St. Louis, Mo), itraconazole (Janssen
`Research Foundation, Beerse, Belgium), terbinafine
`(Novaitis, East Hanover, NJ), and fluconazole (Pfizer
`Inc, New York, NY).
`
`Media
`
`We tested 18 clinical isolates of dcrmatophytes: 6
`strains each of T rubmm, T me/itagrophytes var menta-
`grophyles, and T tonsurans. All strains were isolated
`from nail or hair and were identified at the Center for
`
`Medical Mycology, University Hospitals of Cleveland,
`by conventional methods.4 Isolates were kept frozen at
`—80° C on potato dextrose agar slants until time of use.
`
`Four types of media were used (Table I): RPMI 1640
`(American Biorganics, Niagara Falls, NY) with L—gluta-
`mine, without sodium bicarbonate and buffered at pH =
`7.0; antibiotic medium #3 (Penassay; Difco Labora-
`tories, Detroit, Mich); yeast nitrogen base (Difco
`Laboratories) with 0.5% dextrose buffered at pH = 7.();
`and Sabouraud dextrose broth (Difco Laboratories).
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`

`
`Journal of the American Academy of Dermatology
`Volume 40, Number 6, Part 2
`
`Norris, Elewski, and G/zannoum S11
`
`Yeast nitrogen broth
`with 5% dextrose*
`
`Sabouraud
`dextrose brothi‘
`
`Penassay
`(antibiotic medium #31‘
`
`20 mg
`
`400 ug
`
`1.32 g
`
`Table I. Continued
`
`Component
`
`D,L—Methionine
`L—Methionine
`
`Monopotassium phosphate
`Neopeptone
`Niacin
`Niaeinainide
`D—Pantothenic acid
`
`Phenol red
`
`L—Phenylalanine
`Potassium chloride
`Potassium iodide
`Potassium sulfate
`L—Proline
`
`-Pyridoxine I-lCl
`Riboflavin
`L—Serine
`Sodium chloride
`
`Sodium molybdate
`Sodium phosphate, dibasic
`Thiamine HCI
`L—Threonine
`
`D,L-Tryptophan
`L-Tryptophan
`L—Tyrosine
`L—Valine
`
`Vitamin B12
`Yeast extract
`Zinc sulfate
`
`"Chemically defined media.
`"Chemically nondefined media.
`
`Drug dilutions
`
`Incubation
`
`Serial 2-fold dilutions were prepared according to
`the NCCLS M27—A approved standard} Fluconazole
`and griseofulvin were diluted in water and 100%
`dimethyl sulfoxide, respectively, at 64 ttg/mL to 0.13
`ug/mL. Itraconazole and terbinafine were diluted in
`100% dimethyl sulfoxide at 32 ug/mL to 0.06 pg/1nL.
`
`Inoculum preparation
`
`We prepared a standardized inoeulum by counting
`microconidia. Cultures were grown on a potato dextrose
`agar slant for 6 to l0 days at 30° C. Sterile saline solu-
`tion (O.85%) was added to the slant. and the culture was
`
`gently swabbed with a cotton—tipped applicator to dis-
`lodge conidia from the hyphal mat. The suspension was
`transferred to a sterile tube, and the Volume was adjust-
`ed to 5 mL with sterile saline solution. The resulting
`suspension was counted with a hcmocytomete1' and
`diluted in RPMI 1640 to the desired Concentration.
`
`Microdilution plates were incubated at 30” C and 35°
`C and were read on day 1 through day 6 for growth analy-
`sis and temperature comparison. Microdilution plates for
`testing MIC values were read on days 3, 4, and 5.
`
`Broth mierodilution testing
`
`Microdilution plates were set up in accordance with
`the NCCLS M—27A reference method} with the excep-
`tion of inoculum preparation, described earlier. Briefly,
`column 1 was filled with 200 u,L of RPMI and served
`as a sterility control. Columns 2 through 1 1 were filled
`with 100 uL of the inoculum and 100 uL of the serial-
`ly diluted antifungal agent. Column 12 was filled with
`200 uL of the inoculum and served as a growth control.
`All isolates were run in duplicate and read visually.
`Results of growth in the 4 media at both 30° C and 35°
`C were recorded as follows: (l+) growth barely Visible,
`(2+) moderate growth, and (3+) heavy growth. The
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`
`S12 Norris, Elewski, and Ghannoum
`
`Journal of the American Academy of Dermatology
`June 1999
`
`Table II. Mean growth values in different media at 3 to 5 days incubationl
`
`3 Days
`
`4 Days
`
`Organism
`
`T rubrum
`
`T mentagrophytes
`T tonsurcms
`
`RPM
`
`SAB
`
`PEN
`
`RPM
`
`SAB
`
`PEN
`
`RPM
`
`2.0
`
`1.3
`2.0
`
`2.0
`
`2.5
`1.8
`
`1.5
`
`1.7
`0.0
`
`3.0
`
`2.5
`2.0
`
`2.0
`
`2.5
`1.8
`
`1.5
`
`1.7
`0.0
`
`3.0
`
`2.5
`2.0
`
`PEN
`
`1.7
`
`1.7
`0.0
`
`PEN, Penassay; RPM, RPMI 1640; SAB. Sabouraud dextrose broth.
`*Values were recorded as (l+) barely visible growth, (2+) moderate growth, and (3+) heavy growth.
`
`Table III. Mean MIC (ug/mL) Values for 4 antifungal agents at different inoculum concentrations
`
`Drug
`
`103 Conidia/mL
`
`104 Conidia/mL
`
`105 Conidia/mL
`
`0.20
`0.06
`<0.06
`0.45
`0.1 3
`<0.06
`<0.06
`2.40
`0.63
`<0.06
`<0.06
`7.00
`
`0.35
`0.06
`<0.06
`0.35
`0.15
`0.10
`<0.06
`9.65
`1 .13
`<0.06
`<0.06
`7.00
`
`0.60
`0.06
`<0.06
`2.80
`0.18
`0.14
`<0.06
`9.60
`1.00
`<0.06
`<0.06
`19.0
`
`G l T F G I T F G I T F
`
`Organism
`
`T rubrum
`
`T memagrophytes
`
`T tonsurans
`
`F. Fluconazole; G, griseofulvin; I, itraconazole; T, terbinafine.
`
`Table IV. Antifungal drug concentration in skin
`and nails*
`
`Antifungal agent
`
`Skin level (ug/g)
`
`Nail level (ug/g)
`
`Griseofulvin
`Itraconazole
`Terbinafine
`Fluconazole
`
`16.4
`N/A
`2.5
`23.4
`
`14.61 2
`0.93
`0.39
`11.7
`
`"Drug levels are dependent on the duration of treatment, dosages, and
`host factors.
`llndicates the level in the sole of the foot.
`Adapted froI11 Epstein WL. Shah VP, Riegelman S. Griseofulvin levels
`in stratum corneurn. Arch Dermatol
`1972;l06:344—8;
`from
`Faergemann J, Zehender H, Millerioux L. Levels of terbinafine in plas-
`ma, stratum corneum, dermis—epidermis (without stratum corncum),
`sebum, hair and nails during and after 250 mg terbinafmc orally once
`daily for 7 and 14 days. Clin Exp Dcrmatol 1994; 19: 121-6.
`
`MIC was defined as the point at which the organism
`was inhibited 80% as compared with growth in the
`control well}
`
`RESULTS
`Amount of growth in different media
`
`each isolate and incubated with an equal volume of
`media for 6 days at 35 ° C. The results were as fol-
`lows: No growth was observed in buffered yeast
`nitrogen base for any of the 3 species. Little or no
`growth was observed in Penassay for Tmem‘agr0—
`phyzes or T lonsurcms, although 83% of T mbrum
`isolates demonstrated growth in Penassay. SAB
`and RPMI 1640 supported optimal growth of all 3
`species (Table II).
`
`Comparison of 2 incubation temperatures
`
`An inoculum was prepared as described earlier
`and incubated with an equal volume of RPMI
`1640. Isolates incubated at both 30° C and 35° C
`
`demonstrated equal and adequate growth (22+) by
`the day 4 of incubation for all 3 species.
`
`Effect of inoculum density on the MIC of 4
`antifungal agents
`
`The MIC of each isolate was determined against
`griseofulvin, itraconazole, terbinafine, and flu-
`conazolc with 3 different inocula: 103, 104,and 105
`
`A standardized inoculum density of 103 coni-
`dia/mL was prepared in sterile saline solution for
`
`conidia/mL (Table III). The testing was set up with
`the following variables: RPMI 1640, 35° C incu-
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`
`Journal of the American Academy of Dermatology
`Volume 40, Number 6, Part 2
`
`Norris, Elewski, and Glumnoum S13
`
`bation temperature, and a visual determination of
`the MIC after 4 days of incubation. Inoculum size
`did not affect MIC results for itraconazole or
`
`terbinafine. Slightly higher MIC results were
`observed for griseofulvin at both 104 and 105 as
`compared with 103 conidia/mL for all species test-
`ed. Higher MIC results were observed for flucona-
`zole with 104 and 105 as compared with 103 coni-
`dia/mL for T mentagrophytes, T mbrum, and T
`tonsurans. MICs for fluconazole did not differ
`
`when a 103 inoculum versus a 104 conidia/mL was
`
`used; however, MICs were significantly higher
`when a 105 conidia/mL was used.
`
`DISCUSSION
`
`Choosing a medium that supports adequate
`growth of dermatophytes is the first step in the
`development of a susceptibility testing method.
`The present study demonstrates that RPMI 1640
`and Sabouraud dextrose broth are both suitable
`
`media for microdilution susceptibility testing of
`dermatophytes. For the purpose of standardizing
`the methods, RPMI is the best choice because of its
`
`advantage of being a chemically defined medium
`with no known interference with antifungal agents.
`No difference in the amount of growth was demon-
`strated when isolates were incubated at 30° C and
`
`35° C. A logistical advantage of using 35° C exists
`in that dermatophyte plates can be incubated with
`plates set up for yeast testing, eliminating the need
`for a second incubator for susceptibility testing.
`The length of incubation must also be deter-
`mined, because it will affect the MIC value. The
`
`present data indicate that adequate growth (22+)
`occurs at 4 days for all 3 species tested; thus the
`MIC values recorded reflect a 4-day incubation
`
`period. Selecting an incubation period that is short
`but that can support adequate growth is important
`in providing the clinician with quick and reliable
`laboratory results.
`
`The range and mean MIC values from the pre-
`sent study differed for each of the 4 antifungal
`agents tested (Table III). For each drug evaluated,
`however, the mean MIC fell below the concentra-
`
`tion found in skin or nails after the appropriate
`dosages (Table IV).5'6
`The present study lays a strong foundation on
`which to base future studies. A larger sample of
`dermatophytes needs to be tested, and interlabora-
`tory comparisons must be performed. Addition-
`ally, MIC values need to be correlated with clini-
`cal outcome in future studies to develop interpre-
`tive breakpoints for each isolate/drug combination.
`
`REFERENCES
`
`I
`
`1. National Committee for Clinical Laboratory Standards.
`Reference method for broth dilution antifungal suscepti-
`bility testing of yeasts; approved standard. M27-A, vol.
`17, No. 9. 1996.
`. Lcsher JL. Therapeutic agents for dermatologic fungal
`diseases. In: Elewski BE, editor. Cutaneous fungal infec-
`tions. New York: Igaku—Shoin; 1992. p. 229-41.
`. Rex JH, Pfaller MA, Galgiani .lN, Bartlett MS, Espincl—
`Ingroff A. Ghannoum MA, et al. Development of inter-
`pretive ‘breakpoints for antifungal susceptibility testing:
`conceptual framework and analysis of in vitro-in vivo
`correlation data for fluconazole,
`itracona7.ole, and
`Candida infections. Clin Infect Dis 1997;24:235-47.
`. Larone DH. Medically important fungi: a guide to iden-
`tification. 3rd ed. Washington, DC: ASM Press; 1995.
`. Epstein WL, Shah VP, Riegelman S. Griseofulvin levels
`in stratum corneum. Arch Dermatol 1972;106:344-8.
`. Faergemann .l, Zehender H. Millcrioux L. Levels of
`terbinafine in plasma, stratum corneum, dcrmis-epider-
`mis (without stratum corneum), sebum, hair and nails
`during and after 250 mg terbinafine orally once daily for
`7 and 14 days. Clin Exp Dermatol l994;l9:l2l-6.
`
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