JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2005, p. 19171920 Vol. 43, No.

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0095-1137/05/$08.000 doi:10.1128/JCM.43.4.19171920.2005
Copyright 2005, American Society for Microbiology. All Rights Reserved.

Evaluation of Broth Microdilution Antifungal Susceptibility Testing

Conditions for Trichophyton rubrum
D. A. Santos and J. S. Hamdan*
Department of Microbiology, Institute of Biological Sciences, Federal University of Minas Gerais,
Belo Horizonte, Minas Gerais, Brazil
Received 23 September 2004/Returned for modification 5 November 2004/Accepted 29 November 2004

Fifty clinical isolates of Trichophyton rubrum were selected to test with ketoconazole, fluconazole, itracon-
azole, griseofulvin, and terbinafine by following the National Committee for Clinical Laboratory Standards
susceptibility testing guidelines for filamentous fungi (M38-A). In addition, other susceptibility testing con-
ditions were evaluated: (i) three medium formulations including RPMI 1640 (standard medium), McVeigh &
Morton (MVM), and Sabouraud dextrose broth (SDB); (ii) two incubation temperatures (28 and 35C); and
(iii) three incubation periods (4, 7, and 10 days). The strains Candida parapsilosis (ATCC 22019), Candida krusei
(ATCC 6258), T. rubrum (ATCC 40051), and Trichophyton mentagrophytes (ATCC 40004) were included as
quality controls. All isolates produced clearly detectable growth only after 7 days of incubation. MICs were
significantly independent of the incubation temperature (28 or 35C) (P < 0.05). Different incubation periods
resulted in MICs which were consistently different for each medium when azoles and griseofulvin were tested
(P < 0.05). MICs obtained from different media at the same incubation time for the same isolate were
significantly different when azoles and griseofulvin were tested (P < 0.05). MICs were consistently higher
(usually 1 to 2 dilutions) with RPMI than with MVM or SDB (P < 0.05). When terbinafine was tested, no
parameter had any influence on MICs (P < 0.05). RPMI standard medium appears to be a suitable testing
medium for determining the MICs for T. rubrum. MICs obtained at different incubation times need to be
correlated with clinical outcome to demonstrate which time has better reliability.

The dermatophytes are a group of closely related fungi that
have the capacity to invade the keratinized tissue of humans
and other animals and produce dermatophytoses (24). The
incidence of dermatophytoses has increased over recent years,
particularly in immunocompromised patients (7, 22, 23). The
treatment of these cutaneous infections is based on the use of
topical and systemic antifungal agents (19). For localized non-
extensive lesions, topical therapies are generally used. For
tinea unguium, scalp ringworm, extensive lesions, or skin le-
sions with foliculitis, systemic antimycotic agents are necessary
and itraconazole and terbinafine are the oral drugs presently
used most to treat severe conditions (7). Trichophyton rubrum,
among other dermatophytes, is a major causative agent for
superficial dermatomycosis like onychomycosis and tinea pedis
and is known to account for as many as 70% of all dermato-
phyte infections (24). Infections due to T. rubrum are often
associated with frequent relapses following cessation of anti-
fungal therapy (15).
In vitro analysis of the antifungal activity enables the com-
parison between different antimycotics, which in turn may clar-
ify the reasons for the lack of clinical response and assist
clinicians in choosing an effective therapy for their patients (9).
In the in vitro method proposed by the National Committee
for Clinical Laboratory Standards (NCCLS) for testing fila-
mentous fungi, dermatophytes are not included (7). However,
it is important that methodologies used for in vitro testing be
* Corresponding author. Mailing address: Universidade Federal de
Minas Gerais, Departamento de Microbiologia, Av. Anto nio Carlos,
6627, P. O. Box 486, Belo Horizonte, Minas Gerais, Brazil. Phone:
5531 499 2758. Fax: 5531 499 2730. E-mail: handan@mono.icb
.ufmg.br.
standardized to facilitate the establishment of quality control
parameters and interpretative breakpoints (10). In spite of the
lack of a standardized method for testing dermatophytes, sev-
eral authors have published various articles wherein several
species of these fungi have been tested (6, 9, 18, 19). In these
publications, different adaptations or modifications of the
NCCLS method have been made (3, 6).
The purpose of this study was to evaluate the variability of
different microdilution susceptibility testing conditions (me-
dium, incubation time, and temperature) for the determination
of MICs of five antifungal drugs presently available for the
treatment of dermatophytoses for 50 clinical isolates of T.
rubrum.
MATERIALS AND METHODS
Study design. Each isolate was tested with ketoconazole, fluconazole, itracon-
azole, griseofulvin, and terbinafine by following the NCCLS susceptibility testing
guidelines for filamentous fungi (M38-A) (16). In addition, other susceptibility
testing conditions were evaluated: (i) three medium formulations including
RPMI 1640 (standard medium), McVeigh & Morton (MVM), and Sabouraud
dextrose broth (SDB); (ii) two incubation temperatures (28 and 35C); and (iii)
three incubation periods (4, 7, and 10 days).
Isolates. We selected 50 clinical isolates of T. rubrum which were maintained
in sterile saline (0.9%) at 4C until testing was performed. The strains Candida
parapsilosis (ATCC 22019), Candida krusei (ATCC 6258), T. rubrum (ATCC
40051), and Trichophyton mentagrophytes (ATCC 40004) were included as quality
controls.
Media. The standard RPMI 1640 medium was buffered with 0.165 M mor-
pholinepropanesulfunic acid (MOPS) at 34.54 g per liter at pH 7.0. MVM was
prepared as recommended by Shadomy et al. (21), and SDB was prepared at pH
7.0. All drugs were tested in the three media mentioned above for all isolates.
Antifungal drugs. Antifungal drugs were donated as follows: ketoconazole by
Janssen-Cilag, fluconazole by Pfizer, terbinafine by Novartis, and griseofulvin by
Schering Plough. Itraconazole was used in its commercial formulation (Janssen-

1917
1918 SANTOS AND HAMDAN J. CLIN. MICROBIOL.

TABLE 1. Ketoconazole, fluconazole, griseofulvin, itraconazole, and terbinafine in vitro susceptibility data for T. rubrum (ATCC 40004) and
T. mentagrophytes (ATCC 40051) with standard RPMI medium
Geometric mean MIC (g/ml)
Incubation
time Ketoconazole Flucoconazole Griseofulvin Itraconazole Terbinafine
(days)
T. rubrum T. mentagophytes T. rubrum T. mentagophytes T. rubrum T. mentagophytes T. rubrum T. mentagophytes T. rubrum T. mentagophytes

7 0.5 0.5 32.0 16.0 1.0 0.5 0.031 0.125 0.031 0.031
10 0.5 0.5 32.0 32.0 1.0 0.5 0.031 0.125 0.031 0.031

Cilag). All drugs were dissolved in 100% dimethyl sulfoxide (Gibco) following
the protocol of NCCLS and were prepared in stock solutions of 1,000 g/ml.
Drug dilutions. Serial twofold dilutions were prepared according to the
NCCLS approved document (M38-A) at 100 times the strength of the final
concentration, followed by further dilutions (1:50) in RPMI, MVM, or SDB
medium to yield twice the final strength required for the test. Ketoconazole,
itraconazole, and terbinafine were prepared in ranges from 0.031 to 16.0 g/ml.
Fluconazole and griseofulvin were prepared in ranges from 0.125 to 64.0 g/ml.
Preparation of inocula. The isolates were transferred from sterile saline
(0.9%) to potato dextrose agar at 28C for 7 days to produce conidia. The fungal
colonies were covered with 5 ml of sterile saline (0.9%), and the suspensions
were made by gently probing the surface with the tip of a Pasteur pipette. The
mixture of conidia and hyphae fragments was filtered with a Whatman filter
model 40 (pore size, 8 m), which retains hyphae fragments and permits passage
of only T. rubrum microconidia. The densities of these suspensions were adjusted
with a spectrophotometer at a wavelength of 520 nm to a transmittance of 70 to
72%. The inoculum sizes ranged from 2 106 to 4 106 CFU/ml. Inoculum
quantification was made by counting microconidia in a hematocytometer and by
plating 0.01 ml of suspensions in SDA. The plates were incubated at 28C and
were examined daily for the presence of fungal colonies. The inoculum suspen-
sions were diluted (1:50) in RPMI, MVM, or SDB medium to obtain a cell
number ranging from 2 104 to 4 104 CFU.
Test procedure. Flat-bottomed microdilution plates (96 wells) were set up in
accordance with the NCCLS reference method (16). Each microdilution well
containing 100 l of the twofold drug concentration was inoculated with 100 l
of the diluted inoculum suspension. For each test plate, two drug-free controls
were included, one with the medium alone (sterile control) and the other with
100 l of medium plus 100 l of inoculum suspension (growth control). The
microdilution plates were incubated at 28 and 35C and were read visually after
4, 7, and 10 days of incubation.
Reading and interpretation of MICs. Endpoint determination readings were
performed visually based on comparison of the growth in wells containing the
drug with that of the growth control. For azole agents and for griseofulvin, the
MIC was defined as the lowest concentration showing prominent growth inhibi-
tion (a drop in growth corresponding to approximately 80% of the growth
control. For terbinafine, the MIC was defined as the lowest concentration show-
ing 100% growth inhibition. MIC ranges of each drug were obtained to facilitate
comparisons of the activities of tested drugs, as well as readings of the MIC at
which 50% of the isolates were inhibited (MIC50); similarly, MIC90 is the MIC
at which 90% of the isolates were inhibited.
Data analysis. Determinations of all the MICs were repeated twice. Compar-
isons of influence of incubation temperature, incubation time, and tested media
were performed by Wilcoxon (Mann-Whitney) and Kruskal-Wallis tests. A P
value of 0.05 was considered significant.
RESULTS
All isolates produced clearly detectable growth only after 7
days of incubation. Therefore, the first determination of MIC
endpoints was possible only after that time for all testing con-
ditions. Tables 1 and 2 summarize the susceptibility data for
reference dermatophyte isolates and for 50 T. rubrum isolates,
respectively.
Quality control and reference isolates. Fluconazole and itra-
conazole MICs for the strains C. parapsilosis (ATCC 22019)
and C. krusei (ATCC 6258) were within established ranges (2),
and similar values were obtained by using MVM and SDB
media. The MICs for T. rubrum (ATCC 40051) and T. menta-
grophytes (ATCC 40004) are summarized in Table 1.
Effect of the incubation temperature on MICs. The incuba-
tion temperature did not influence MICs significantly (P
0.05). This was true for all tested media and all incubation
times and for all tested drugs (with no dilution interval). MICs
obtained when plates were incubated at 28C (Table 2) were
similar to or even the same as MICs obtained when incubating
plates at 35C.
Effect of the incubation time and media on MICs. Growth
was insufficient at 4 days of incubation for 6 isolates with
RPMI, for 9 isolates with SDB, and for 19 isolates with MVM,
but MICs were obtained with other testing conditions. The
statistical analyses revealed that different incubation periods
resulted in MICs which were consistently different for each
medium when azoles and griseofulvin were tested (P 0.05).
MICs obtained with different media at the same incubation
time for the same isolate were significantly different when
azoles and griseofulvin were tested (P 0.05). MICs were
consistently higher (usually 1 to 2 dilutions) when using RPMI

TABLE 2. Ketoconazole, fluconazole, griseofulvin, itraconazole, and terbinafine in vitro susceptibility data for 50 T. rubrum isolates under
different broth microdilution testing conditions
MIC (g/ml) of:
Incubation
Medium time Ketoconazole Fluconazole Griseofulvin Itraconazole Terbinafine
(days)
Range 50% 90% Range 50% 90% Range 50% 90% Range 50% 90% Range 50% 90%

RPMI 7 0.06252.0 0.5 1.0 1.064.0 64.0 64.0 0.252.0 1.0 1.0 0.0311.0 0.125 0.25 0.031 0.031 0.031
10 0.06252.0 1.0 2.0 8.064.0 64.0 64.0 0.252.0 1.0 1.0 0.0311.0 0.25 0.5 0.031 0.031 0.031

SDB 7 0.0312.0 0.25 1.0 2.032.0 16 32.0 0.252.0 0.5 1.0 0.0310.5 0.0625 0.25 0.031 0.031 0.031
10 0.06252.0 0.5 1.0 4.032.0 32 32.0 0.254.0 1.0 2.0 0.0310.5 0.125 0.25 0.031 0.031 0.031

MVM 7 0.1252.0 0.5 1.0 4.064.0 32 64.0 0.251.0 0.5 1.0 0.0311.0 0.125 0.25 0.031 0.031 0.031
10 0.1252.0 0.5 1.0 4.064.0 64 64.0 0.252.0 1.0 1.0 0.0311.0 0.125 0.5 0.031 0.031 0.031
VOL. 43, 2005 SUSCEPTIBILITY TESTING CONDITIONS FOR T. RUBRUM
than when using MVM or SDB (P 0.05). When terbinafine
was tested, no parameter had any influence on MICs (P
0.05).
DISCUSSION
Although a standard method for the susceptibility testing of
dermatophytes is lacking, there are several reports where the
antimicrobial susceptibility testing of dermatophytes has been
conducted by using either agar macrodilution or broth mac-
rodilution and microdilution tests; these reports have generally
been extensions of either M27-A2 or M38-A methodologies.
Results obtained by some authors (3) have shown great vari-
ability, probably due to the lack of standardization of different
parameters that can influence the MIC determination, such as
method of inoculum preparation (8) and length and tempera-
ture of incubation (4, 7, 14). Ghannoum et al. (9) demon-
strated, in a multicenter study, a high level of intra- and inter-
laboratory agreement for the determination of MICs
undertaken in RPMI medium at a temperature of 35C and
using a 4-day incubation period.
In our study, in general, we followed recommendations of
the NCCLS for testing filamentous fungi with some adapta-
tions. First of all, we used Whatmans filter model 40 to sep-
arate hyphae from conidia, resulting in homogenous inocula
comprised of only microconidia. We think that the separation
of these structures is an important step in MIC determination
because antifungal susceptibilities of hyphae and microconidia
are probably different.
The ideal incubation temperature, 28 or 35C, is still a mat-
ter of debate. It is well known that the majority of dermato-
phyte species show an optimal growth when incubation tem-
perature is between 28 and 30C (7). However, various authors
have proposed higher temperatures, such as 35 or 37C (1, 3, 9,
18, 19). In our experience, this parameter did not significantly
influence (P 0.05) the MICs for a specific incubation time in
all tested media. This result is directly comparable with that
reported by Norris et al. (18) and is different from that re-
ported by Fernandez-Torres et al. (7), who demonstrated bet-
ter growth when fungi were incubated at 28C.
A number of authors have proposed different incubation
times, ranging from 3 to 20 days, for testing dermatophytes (1,
3, 9, 18, 19). After 4 days, T. rubrum was observed to grow
poorly; however, 7 days was found to be sufficient to observe
prominent growth in control wells. Fernandez-Torres et al. (7,
8) obtained similar results. Different findings were obtained by
Jessup et al. (12), Ghannoum et al. (9), and Favre et al. (5),
who found 4 days of incubation sufficient to observe prominent
growth in control wells. Statistical analysis revealed that an
increased incubation time of not only 10 days compared to 7
days increases MICs by 1 to 2 dilutions with the same medium
but also 7 days relative to 4 days (data not shown). The excep-
tion was for terbinafine, where the incubation time did not
influence MICs.
All tested media (standard RPMI broth, MVM, and SDB)
presented statistically different results (P 0.05) with no di-
lution interval, except for terbinafine. Greater MICs were ob-
tained with buffered RPMI relative to other tested media. The
medium proposed by NCCLS allowed adequate growth of all
isolates, confirming reports that this medium produces a suit-
1919
able visible growth of filamentous fungi, including dermato-
phytes (4, 18). Between MVM and SDB, the first was the
medium which was more similar in comparison to RPMI. Al-
though MVM is a chemically defined medium (20) often used
in MIC determination for Paracoccidioides brasiliensis (11) by
the broth macrodilution method, MIC reading in microdilution
plates is harder because of the transparency of this medium
compared to the yellow color of the other media, which con-
fuses during visualization. There is no report about the use of
MVM medium in MIC determination for dermatophytic fungi.
There is a scarcity of reports about the use of SDB for MIC
determination for any fungi, including dermatophytes (17). We
tested SDB medium because its cost is considerably lower than
that of other tested media mentioned above and it is used in all
mycology laboratories; however, SDB presented lower MICs
and had high discrepancy compared to MVM and RPMI (P
0.05).
The evaluation of in vitro activities of tested drugs revealed
that terbinafine was the most potent active drug, confirming
reports by Korting et al. (13) and Fernandez-Torres et al. (6).
Between the azoles, itraconazole was the most active, followed
by ketoconazole and fluconazole. Similar results were obtained
by Korting et al. (13), who tested numerous isolates of T.
rubrum by a RPMI microdilution method. With respect to
griseofulvin, the great majority of tested isolates presented
MICs of 2 g/ml and this result is comparable with that re-
ported by Jessup et al. (12).
In conclusion, this investigation has demonstrated that mi-
crodilution assay for dermatophytes is convenient and repro-
ducible. The results of this study show that the maintenance of
the isolates in sterile saline before transfer to potato dextrose
agar promotes conidial production and enables the use of
inoculum containing only this structure. RPMI standard me-
dium appears to be a suitable testing medium for the determi-
nation MICs for Trichophyton rubrum, strengthening the data
from authors that recommend RPMI for testing dermato-
phytes. In our experiments, temperature did not consistently
influence MICs, revealing that tests could be performed at 28
or 35C. However, MICs obtained at different incubation times
need to be correlated with clinical outcome to demonstrate
which time has better reliability.
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