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Previous Article | Next Article 
Journal of Clinical Microbiology, February 2000, p. 489-491, Vol. 38, No. 2
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Fluorometric Assessment of In Vitro Antidermatophytic Activities
of Antimycotics Based on Their Keratin-Penetrating Power
C. N.
Okeke,
R.
Tsuboi,*
M.
Kawai, and
H.
Ogawa
Department of Dermatology, Juntendo
University School of Medicine, Tokyo, Japan
Received 22 March 1999/Returned for modification 28 June
1999/Accepted 5 November 1999
 |
ABSTRACT |
Keratin particles impregnated with amorolfine or clotrimazole in
serial doubling dilutions (64 to 0.125 µg/ml) were used to evaluate
the activities of these agents against 20 isolates each of
Trichophyton mentagrophytes and Trichophyton
rubrum in a yeast carbon broth medium incorporating Alamar Blue
dye. The proposed MIC with keratin impregnation (MICK) is
defined as the lowest concentration of an agent used to impregnate
keratin particles that effects a fluorescence-based fungal growth
quotient of 0.05 or less. The conventional colorimetric and visual MICs
of amorolfine for the dermatophytes, 
0.03 µg/ml for T. mentagrophytes and 0.063 µg/ml for T. rubrum,
were approximately half of those of clotrimazole for the same isolates.
The superiority of the MICKs of amorolfine for isolates of
T. mentagrophytes (2.0 µg/ml; range, 0.5 to 8.0 µg/ml)
and T. rubrum (4.0 µg/ml; range, 2.0 to 8.0 µg/ml) over those of clotrimazole (32 µg/ml [range, 8.0 to >64 µg/ml] and 64 µg/ml [range, 16 to >64 µg], respectively) may indicate the strong in vivo antidermatophytic activity of amorolfine as a topical agent. The new antidermatophytic susceptibility testing procedure has
potential clinical utility for the in vitro screening of agents for use
in the topical treatment of superficial mycoses.
| |
INTRODUCTION |
Dermatophytoses, which constitute
the majority of superficial fungal infections, are infections of
keratinized tissues such as the stratum corneum, nail, and hair by
dermatophytic fungi. Some agents with high levels of in vitro
antidermatophytic activity show rather poor in vivo effects (5,
16). This difference in the in vivo and in vitro activities of
some agents is due to the dependency of the in vivo action on the
interaction of drug molecules with tissue components (4, 8, 14,
16). Thus, the efficacy of a topically applied antidermatophytic
agent is influenced not only by its antifungal property but also by the ability of the drug molecules to penetrate the keratinized tissue (9).
The effect of the MIC of an agent as determined under in vitro
conditions that approximate the situation in vivo would be a better
predictor of in vivo efficacy. Merten and Lippold (9) estimated the relative therapeutic potencies of antimycotics against onychomycoses using an efficacy coefficient based on the rate of
maximum flux through hoof membrane and an independently determined MIC.
Polak (15), using keratin into which solubilized
antimycotics were impregnated and placement of the keratin into agar
wells, assessed the antifungal activities of agents by measuring the zones of inhibition surrounding the wells in cultures.
In this study, Alamar Blue, a dye that exhibits both fluorescence and
colorimetric changes caused by cellular metabolic reduction (12,
18, 19), was used in a quantitative fluorometric assay to
determine the activities of keratin-bound amorolfine and clotrimazole against isolates of Trichophyton mentagrophytes and
Trichophyton rubrum.
| |
MATERIALS AND METHODS |
Antifungal agents.
Amorolfine powder was a gift from Kyorin
Pharmaceutical Co. Ltd. (Tokyo, Japan), and clotrimazole powder was
purchased from Sigma Chemical Co. (St. Louis, Mo.). Stock solutions
(100 mg/ml) of the agents were prepared by dissolving the powders in
100% dimethyl sulfoxide. The stock solutions were frozen at 
20°C
until use.
Fungal isolates and inoculum preparation.
Twenty isolates
each of T. mentagrophytes and T. rubrum were
obtained from the culture collection of the Research Institute for
Chemobiodynamics, Chiba University, Chiba, Japan. Homogeneous suspensions of conidia and hyphal fragments in 0.85% saline were prepared from 7-day cultures on potato dextrose agar slants (Difco Laboratories, Detroit, Mich.). The optical densities of the suspensions were read at 530 nm and were adjusted to 0.15 to 0.17 to yield 0.6 × 106 to 1.4 × 106 and 0.7 × 106 to 1.2 × 106 CFU of T. mentagrophytes and T. rubrum per ml, respectively
(3).
Broth microdilution antifungal susceptibility testing.
The
MICs of the antidermatophytic agents were determined by modifying the
broth microdilution antifungal susceptibility test procedure
recommended by a subcommittee of the National Committee for Clinical
Laboratory Standards for antifungal susceptibility testing of
filamentous fungi (3). Briefly, the test was performed with
RPMI 1640 medium supplemented with L-glutamine but without sodium bicarbonate (Life Technologies, Grand Island, N.Y.). The medium
was buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid
(MOPS) buffer (Sigma).
A series of doubling dilutions (128 to 0.002 µg/ml) of the stock
solutions of the agents were prepared in 2× RPMI 1640 medium (20.8 g/liter). One hundred microliters of each dilution was then mixed with
an equal volume of a 1:50 dilution of the fungal suspension (approximately 2 × 104 CFU/ml) in 20% (vol/vol)
Alamar Blue dye (Sensititre/Alamar, Westlake, Ohio) in sterile
distilled water. A final volume of 200 µl of the reaction mixture
contained 104 CFU of fungus per ml and the agents at
concentrations ranging from 64 to 0.001 µg/ml. A drug-free medium was
inoculated and was used as a growth control. The blank medium was free
of drug and fungus.
The microdilution plates were incubated at 30°C for 96 h, and
the endpoints were read as the MICVIS and
MICCOL. The MICVIS is defined as the lowest
concentration of an agent at which there is no visually observable
growth in broth, and the MICCOL is defined as the lowest
concentration of an agent that prevents the development of a red color
in broth. Fungal growth activity was also measured by fluorescence
determination at excitation and emission wavelengths of 544 and 590 nm,
respectively, by using a microplate fluorescence reader
(Fluoroskan Ascent Spectrofluorometer; Labsystems, Helsinki, Finland). Growth was expressed as a quotient calculated in the following manner: (fluorescence intensity in dilution broth fluorescence intensity in blank medium)/(fluorescence intensity in
growth control broth fluorescence intensity in blank medium).
Keratin impregnation antidermatophytic susceptibility test.
A quantitative susceptibility test method based on the
keratin-penetrating powers of agents was developed by using amorolfine and clotrimazole as the test drugs against 20 isolates each of T. mentagrophytes and T. rubrum.
Keratin particles were prepared as described by Negi et al.
(10). Briefly, scrapings of the stratum corneum from healthy human soles were cut into pieces, suspended in water, and homogenized with Polytron (Brinkmann, Lucerne, Switzerland). The keratin particles, which were repeatedly washed in distilled water until the optical density of the wash solution was less than 0.01 at 280 nm, were used
after lyophilization. One hundred milligrams of the particles was
suspended in 1.0 ml of a series of doubling dilutions (64 to 0.125 µg/ml) of the stock solutions of amorolfine or clotrimazole in water,
and the suspensions were kept for 1 h at 32°C to allow penetration of the keratin by the drug molecules. The keratin particles
were then precipitated, washed twice in 1 ml of distilled water, and
dried in vacuo. The keratin was dispensed in 5-mg amounts into wells of
a flat-bottom and lidded 24-well plate (Iwaki Glass, Tokyo, Japan) and
were sterilized overnight in ethylene oxide gas at 37°C.
Four hundred microliters of filter-sterilized yeast carbon base medium
(14.625 g/liter; Difco Laboratories) and 50 µl each of fungal
suspension (approximately 106 CFU/ml) and Alamar Blue
indicator were added to each well. The final volume of the 500-µl
reaction mixture contained approximately 104 CFU of fungus
per ml. Broths incorporating drug-free keratin served as growth
controls. Blanks were inoculated with 50 µl of sterile 0.85% saline
in place of the fungal suspension. The plates were incubated at 30°C
for 96 h, during which time the cultures were agitated
continually. One hundred microliters of culture medium was then
transferred to a 96-well microtiter plate (Becton Dickinson and Co.,
Lincoln Park, N.J.), and the fluorescence was measured as described
above. Fungal growth was expressed as a quotient: (fluorescence
intensity in broth containing drug-treated keratin particles fluorescence intensity in blank medium)/(fluorescence intensity in
broth containing drug-free keratin particles fluorescence intensity in blank medium). The MIC with keratin impregnation (MICK) is defined as the lowest concentration of an agent
used to impregnate keratin particles that effects a fluorescence-based fungal growth quotient of 0.05 or less in assay broth.
In the preliminary studies, the effect on the fungal growth quotient of
varying the impregnation time of the keratin particles in the agent
solution, the fungal inoculum size, and the cultural incubation
period was investigated by using keratin particles impregnated in
4 µg of amorolfine or clotrimazole per ml. An isolate of T. mentagrophytes (IFM 45794) was used as the test fungus. The time
of impregnation of the keratin in the agents was set at 1 h, the
inoculum size was set at 104 CFU/ml, and the incubation
period was set at 96 h. When any one of these
parameters was varied, the other two parameters were fixed at the
following settings: keratin drug impregnation periods of 10, 60, and 120 min; fungal inoculum sizes of 103, 104,
and 105 CFU/ml; and cultural incubation times of 72, 96, and 120 h.
| |
RESULTS |
Determination of MICCOLs and
MICVISs.
The MICCOLs and
MICVISs of amorolfine and clotrimazole for the isolates of
T. mentagrophytes and T. rubrum are presented in Table 1. Intraspecific variations within
narrow ranges of drug concentrations were observed in the
MICCOLs and MICVISs of both agents. The
MICCOL was generally lower than the corresponding MICVIS by 1 to 3 dilutions for each drug-fungus pair. The
MICCOLs and MICVISs of amorolfine were lower
than those of clotrimazole for the same test isolates.
The fungal growth quotients of the MICVISs by the broth
dilution method were within the range of 0.030 to 0.049 and 0.028 to
0.042 for amorolfine and clotrimazole, respectively, indicating a more
than 95% reduction in fungal growth activity relative to that in the
growth control broth. On this basis, a fungal growth inhibition
quotient of 0.05 was adopted as the inhibition threshold in the
subsequent keratin impregnation antidermatophytic susceptibility test.
Figure 1 shows the curves relating the
fungal growth quotients for an isolate of T. mentagrophytes
(IFM 45828) to the corresponding drug concentrations. The dotted line,
indicating the 0.05 threshold of inhibition, intersects the curves at
the MICVISs of amorolfine and clotrimazole, 0.008 and 0.125 µg/ml, respectively.
View larger version (17K):
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FIG. 1.
Curves relating the fluorescence-based fungal growth
quotient for an isolate of T. mentagrophytes (IFM
45828) to concentrations of amorolfine ( ) and clotrimazole ( ) in
the conventional broth microdilution antifungal susceptibility test.
The dotted line indicates the 0.05 fungal growth quotient inhibition
threshold.
|
|
Determination of MICK.
Preliminary studies
showed that the growth quotient of T. mentagrophytes
IFM 45794 in cultures with amorolfine-impregnated keratin
particles decreased in correlation to an increase in the keratin impregnation period (0.071, 0.047, and 0.042 for 10, 60, and 120 min, respectively), the fungal inoculum size (0.037, 0.028, and 0.019 for 103, 104, and
105 CFU/ml, respectively), and the incubation time (0.046, 0.013, and 0.004 for 72, 96, and 120 h, respectively). The data
for clotrimazole are not shown. The MICKs of
the agents, derived from the growth quotients under the
conditions of 1 h of keratin impregnation, an inoculum
size of 104 CFU/ml, and a 96-h incubation period are
presented in Table 1. The most frequent MICKs of
amorolfine (modal MICK) for isolates of T. mentagrophytes and T. rubrum (2.0 and 4.0 µg/ml,
respectively) were considerably lower than the corresponding modal
MICKs of clotrimazole (32 and 64 µg/ml,
respectively) (Table 1). The MICKs of clotrimazole
for two and five isolates of T. mentagrophytes and
T. rubrum, respectively, were higher than 64 µg/ml,
the maximum concentration of the agents used for keratin impregnation.
Figure 2 shows the curves relating the
fungal growth quotients of an isolate of T. mentagrophytes
(IFM 45828) to the corresponding drug concentrations used for the
impregnation of the keratin particles. The inhibition threshold line
intersects the curve at the MICK of amorolfine (8 µg/ml)
for the isolate. Keratin particles impregnated with 64 µg of
clotrimazole per ml effected a growth quotient of 0.173 for the
isolate.
View larger version (16K):
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[in a new window]
|
FIG. 2.
Curves relating the fluorescence-based fungal growth
quotient of an isolate of T. mentagrophytes (IFM 45828) to
the concentrations of amorolfine () and clotrimazole ( ) in the
keratin-impregnation antifungal susceptibility test. The dotted line
indicates the 0.05 fungal growth quotient inhibition threshold.
|
|
| |
DISCUSSION |
In this study, a novel susceptibility testing method was
developed and compared with the broth microdilution method for
the evaluation of the antifungal activities of amorolfine and
clotrimazole against isolates of T. mentagrophytes and
T. rubrum.
The MICCOLs of both agents for the dermatophytes were lower
than the corresponding MICVISs. The fungal growth at
96 h in the presence of 1 or 2 higher dilutions compared with the
MICVIS, although visible as a mycelial mat at the bottom of
the wells, failed to effect a color change in the broth. Similar
variable results in colorimetric and visual MICs have been reported by other workers (3, 6, 13, 19). In our study, the discrepancy between the MICCOL and MICVIS for each
drug-fungus pair was not greater than 3 dilutions and thus was not
considered significant, in line with the study conducted by the
National Committee for Clinical Laboratory Standards (3).
In view of the dose-response relationship between the fungal growth
quotient and the corresponding drug concentration (Fig. 1), we adopted
a standard quotient of 0.05 as the threshold of inhibition for the
determination of MICK. Tellier et al. (17) had
earlier proposed a 90% reduction in the optical density of the reduced
tetrazolium salt as a colorimetric MIC for antifungal susceptibility
testing of yeasts.
The fungal growth quotient, and consequently the MICK, was
dependent on experimental parameters such as the impregnation time of
the keratin particles, the inoculum size, and the incubation period.
These parameters, in addition to the composition of the medium, have
been known to affect the intensities of the in vitro antifungal actions
of agents by the conventional broth dilution method (3, 11,
14). The reduction in the growth quotient that correlated with an
increased inoculum size or incubation time resulted from more rapid
fungal growth in the control broth than in the test broth. The
MICK of an agent would also depend on the type of the
keratin used in the test, with the values obtained with
drug-impregnated hard keratin (hair) differing from those obtained with
soft keratin (stratum corneum) according to their different porosities
for the agent molecules. The maximum flux of an agent into keratin is
also known to depend on the surface area of the particle exposed to
impregnation (9).
The considerably superior MICK of amorolfine for the
dermatophytes over that of clotrimazole, relative to the closer
MICCOLs and MICVISs of the two agents, was due
as much to the potent antifungal activity of the agent as to its
ability to penetrate and bind to keratin. In a related study, an
efficacy coefficient calculated from independently determined values of
the maximum flux of the agent in bovine hoof membrane and the MICs also
pointed to amorolfine as the most effective of 10 antimycotics against
onychomycoses (9). On the basis of the conventional MICs
obtained in our study, the in vivo efficacies of both agents in the
topical treatment of dermatophytosis would be equal, which is in
opposition to the considerable superiority of amorolfine, as indicated
by the MICK. The increasing numbers of reports on the
successful treatment of refractile fungal nail infections with
amorolfine-containing lacquer are in keeping with the MICK
index (1, 2, 7).
In conclusion, the keratin impregnation antidermatophytic
susceptibility testing method is quantitative and objective, and the
MICK may be a useful index of the in vivo potency of a
topical agent relative to those of other antimycotics.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department
of Dermatology, Juntendo University School of Medicine, 2-1-1, Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Phone: 81-3-5802-1089. Fax: 81-3-3813-9443. E-mail:
tsuboi{at}med.juntendo.ac.jp.
| |
REFERENCES |
| 1.
|
Cohen, P. R., and R. K. Scher.
1994.
Topical and surgical treatment of onychomycosis.
J. Am. Acad. Dermatol.
31:S74-S77[Medline].
|
| 2.
|
Downs, A. M.,
J. T. Lear, and C. B. Archer.
1999.
Scytalidium hyalinum onychomycosis successfully treated with 5% amorolfine nail lacquer.
Br. J. Dermatol.
40:555.
|
| 3.
|
Espinel-Ingrof, A.,
M. Bartlett,
R. Bowden,
N. X. Chin,
C. Cooper, Jr.,
A. Fothergill,
M. R. McGinnis,
P. Menezes,
S. A. Messer,
P. W. Nelson,
F. C. Odds,
L. Pasarell,
J. Peter,
M. A. Pfaller,
J. H. Rex,
M. G. Rinaldi,
G. S. Shankland,
T. J. Walsh, and I. Weitzman.
1997.
Multicenter evaluation of proposed standardized procedure for antifungal susceptibility testing of filamentous fungi.
J. Clin. Microbiol.
35:139-143[Abstract].
|
| 4.
|
Espinell-Ingroff, A.,
T. M. Kerkering,
P. R. Goldson, and S. Shadomy.
1991.
Comparison of broth macrodilution and microdilution antifungal susceptibility.
J. Clin. Microbiol.
29:1089-1094[Abstract/Free Full Text].
|
| 5.
|
Galgiani, J. N.
1987.
Antifungal susceptibility tests.
Antimicrob. Agents Chemother.
31:1867-1870[Free Full Text].
|
| 6.
|
Ghannoum, M. A.,
A. S. Ibrahim,
Y. Fu,
M. C. Shafiq,
J. E. Edwards, and R. S. Criddle.
1992.
Susceptibility testing of Cryptococcus neoformans: a microdilution technique.
J. Clin. Microbiol.
30:2881-2886[Abstract/Free Full Text].
|
| 7.
|
Haria, M., and H. M. Bryson.
1995.
Amorolfine. A review of its pharmacological properties and therapeutic potential in the treatment of onychomycosis and other superficial fungal infection.
Drugs
49:103-120[Medline].
|
| 8.
|
Hawser, S. P.,
H. Norris,
C. J. Jessup, and M. A. Ghannoum.
1998.
Comparison of a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) colorimetric method with the standardized National Committee for Clinical Laboratory Standards method of testing clinical yeast isolates for susceptibility to antifungal agents.
J. Clin. Microbiol.
36:1450-1452[Abstract/Free Full Text].
|
| 9.
|
Mertin, D., and B. C. Lippold.
1997.
In vitro permeability of the human nail and of a keratin membrane from bovine hooves: prediction of the penetration rate of antimycotics through the nail plate and their efficacy.
J. Pharm. Pharmacol.
49:866-872[Medline].
|
| 10.
|
Negi, M.,
R. Tsuboi,
T. Matsui, and H. Ogawa.
1984.
Isolation and characterization of proteinase from Candida albicans: substrate specificity.
J. Invest. Dermatol.
83:32-36[CrossRef][Medline].
|
| 11.
|
Odds, F. C.
1980.
Laboratory evaluation of antifungal agents: a comparative study of five imidazole derivatives of clinical importance.
J. Antimicrob. Chemother.
6:749-761[Abstract/Free Full Text].
|
| 12.
|
Pfaller, M. A., and A. L. Barry.
1994.
Evaluation of a novel colorimetric broth microdilution method for antifungal susceptibility testing of yeast isolates.
J. Clin. Microbiol.
32:1992-1996[Abstract/Free Full Text].
|
| 13.
|
Pfaller, M. A.,
B. Bushelman,
M. J. Bale,
M. Lancaster,
A. Espinel-Ingroff,
J. H. Rex, and M. G. Rinaldi.
1994.
Multicenter comparison of a colorimetric microdilution broth method with the reference macrodilution method for in vitro susceptibility testing of yeast isolates.
Diagn. Microbiol. Infect. Disease
19:9-13.
|
| 14.
|
Polak, A.
1983.
Antifungal activity in vitro of Ro 14-4767/002, a phenylpropyl-morpholine.
Sabouraudia
21:205-213[Medline].
|
| 15.
|
Polak, A.
1993.
Kinetics of amorolfine in human nails.
Mycoses
36:101-103[Medline].
|
| 16.
|
Rex, J. H.,
M. A. Pfaller,
M. G. Rinaldi,
A. Polak, and J. N. Galgiani.
1993.
Antifungal susceptibility testing.
Clin. Microbiol. Rev.
6:367-381[Abstract/Free Full Text].
|
| 17.
|
Tellier, R.,
M. Krajden,
G. A. Grigoriew, and I. Campbell.
1992.
Innovative endpoint determination system for antifungal susceptibility testing of yeasts.
Antimicrob. Agents Chemother.
36:1619-1625[Abstract/Free Full Text].
|
| 18.
|
Tiballi, R. N.,
X. He,
L. T. Zarins,
S. G. Revanka, and C. A. Kauffman.
1995.
Use of a colorimetric system for yeast susceptibility testing.
J. Clin. Microbiol.
33:915-917[Abstract].
|
| 19.
|
To, W. K.,
A. W. Fothergill, and M. G. Rinaldi.
1995.
Comparative evaluation of macrodilution and Alamar colorimetric microdilution broth methods for antifungal susceptibility testing for yeast isolates.
J. Clin. Microbiol.
33:2660-2664[Abstract].
|
Journal of Clinical Microbiology, February 2000, p. 489-491, Vol. 38, No. 2
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
