ARTICLE
Auteur(s) : Xiao-Fang Li, Yong-Nian Shen, Wei
Chen, Hui Chen, Gui-Xia LV, Wei-Da Liu
Department of Mycology, Institute of Dermatology,
Chinese Academy of Medical Sciences & Peking Union Medical
College, Jiang Wangmiao jie 14, Nanjing 210042, China
accepté le 4 Septembre 2008
Dermatophyte infection is a disease of worldwide distribution
that accounts for the majority of superficial infections.
Trichophyton rubrum is by far the most common pathogen. Less
frequently, non-dermatophytic molds and Candida species give a
similar clinical picture which requires differential diagnosis
[1-3]. The treatment of dermatophyte infection would be most
appropriate when the selection of antimicrobial agent is based on
the identity of the causative agent. In some cases, such as
onychomycosis, the treatment needs to be administered long-term and
enough time must elapse for the nail to grow out completely before
such treatment can be designated as successful. Therefore,
treatment should not be commenced before mycological confirmation
of infection [1, 4].
To date, culturing is the “gold standard” for diagnosis and is
necessary to identify the etiology, which can be helpful for
selecting a therapeutic approach. Unfortunately, all dermatophytes
grow slowly, thus culturing is time-consuming, and difficulties are
sometimes encountered due to phenotypic variations between strains
[5]. In fact, some isolates require the assistance of an
experienced mycologist. Thus, new and reliable methods combined
with easier processes of species identification are needed to
enhance this process.
Dermatophyte test medium (DTM) was developed by Taplin, et al.
in 1969 [6]. Several clinical evaluations have provided further
information about the effectiveness of it in the diagnosis of
dermatophyte infection [7-9]. This culture medium has the similar
nutrients to other fungal culture media (1% soy peptone, 1%
dextrose). Its character is the pH indicator (phenol red) and the
selective inhibitors chlorotetracycline, gentamicin and
cycloheximide, which partly suppress the growth of bacteria, yeasts
and contaminating molds. Dermatophytes induce a color change of DTM
from yellow (the initial pH of the medium is 5.5 ± 0.1) to red, due
to alkaline metabolites. Other organisms may grow, but slowly, and
can be recognized as non-dermatophytes by a lack of color change.
Although DTM does not identify specific organisms, a positive DTM
culture indicated the presence of a dermatophyte.
The advantages of DTM culture are the relative ease for
interpretation of results and faster turn around time than with
other types of fungal culture. However, subsequent studies have
demonstrated the problems associated with the use of DTM, e.g. some
non-dermatophytes exhibit similar characteristics to dermatophytes
in colony morphologies and color alteration [10, 11].
In order to improe the sensitivity, accuracy and feasibility of
DTM, we developed a medium called DBM (trade mark pending) that
also induces a color change in the presence of dermatophytes (from
yellow to blue) and new, related interpretational criteria. The
goal of this study was to compare these two media in the diagnosis
of onychomycosis.
Materials and methods
Media
Sabourauds dextrose agar (Difco), DTM (QUE-BACT). The main
components of DBM were 0.5% soy peptone (ZhongKe, Shanghai, China),
0.5% dextrose (Difco), and 0.05‰ bromothymol blue (BBI), 0.0125%
chloramphenicol (Sigma), 0.05% cycloheximide (Sigma), pH 5.5 ± 0.1.
Specificity of DTM and DBM in axenic
cultures
Reference organisms selected to assay color change specificity of
DTM and DBM were fungi of medical importance in onychomycosis,
including eighteen dermatophytes: T. rubrum (T1c),
Trichophyton schoenleini (T2a), Trichophyton violaceum
(T3d), Trichophyton tonsurans
(T4b),Trichophyton mentagrophytes (T5b),
Trichophyton concentricum (T6a), Trichophyton verrucosum
(T7c), Trichophyton simii (T8a), Trichophyton
meginii (T9d), Microsporum ferrugineum (M1),
Microsporum gypseum (M2b), Microsporum canis
(M3a), Microsporum audouinii (M4a),
Microsporum nanum (M5a), Microsporum fulvum
(M6b), Microsporum distortum (M7),
Microsporum gallinae (M8d), Epidermophyton floccosum
(E1d); fourteen molds: Alternaria alternata
(B1), Rhizopus nigricans (B2), Rhizomucor
variabilis (B3), Scopulariopsis brevicaulis
(B4b), Penicillium islandicum (B27),
Penicillium citreoviride (B30), Fusarium solani
(B24), Curvularia lunata (B25), Aspergillus
fumigatus (A1), Aspergillus flavus (A2),
Aspergillus niger (A3), Sporothrix schenkii (clinical
isolate), Cladosporium (clinical isolate), Acremonium (clinical
isolate). All the reference strains were obtained from China
Culture Collection Center of Medical Mycology (CCCCM). Media were
incubated at 25 °C, and examined daily for growth as well as the
color of the medium around the colonies, over 2 weeks. The test was
repeated at another different time.
Sensitivity of DTM and DBM in axenic
cultures
T. rubrum (T1c) suspension was serially diluted by
10-fold to give concentrations ranging from 102 ~
106 cfu/mL, and was inoculated on DTM and DBM
slants (each with twenty microliters). The lowest concentration
that could make a color change was determined as the lowest limit
of detection (sensitivity). The test was repeated twice.
Clinical evaluation
Patients admitted to our clinic with clinically suspected
onychomycosis from May 2005 to August 2005 were included in this
study. Nail shavings from the suspected portions were used as
clinical specimens after swabbing liberally with alcohol (75%) to
exclude the possibility of the presence of contaminants attached.
After being examined by direct microscopy with 15% KOH, the
scrapings were separated into four parts, and were cultured onto
DTM, DBM, SCCA (Sabourauds dextrose agar contained chloramphenicol
and cycloheximide) and SCA (Sabourauds dextrose agar contained
chloramphenicol), respectively. Each medium was examined every 12
hours for growth and color change (diameters of colonies were
measured when the color of the media began to change). The medical
mycology lab of CCCCM performed strain identification.
Statistical analysis
The results obtained were treated with the SPSS statistical package
(11.0) for ANOVA, T test or Chi-Square test. The statistical level
of significance was 5%.
Results
Specificity of DTM and DBM in axenic
cultures
All tested dermatophytes grew on both DTM and DBM, and caused a
color change of these media when there was very small colonial
growth. An early color shift of DBM from straw yellow to green was
easily observed; for DTM, very careful observation was needed to
find the initial color conversion from orange yellow to salmon
pink. Although most tested molds (11/14) grew and induced a color
change in both DBM and DTM, they made the color alteration at a
slower rate, i.e., no color changes were found even when the
colonies were very obvious (figure 1).
Sensitivity of DBM and DTM in axenic
cultures
Both DBM and DTM could make a color change with as little as
103 cfu/mL (2 × 101 cfu/slant) of
T. rubrum suspension.
Clinical evaluation
Nail sample inoculation and colonial growth
One hundred and two clinical specimens were collected with 71.57%
(73/102) positive rate for microscopy, and 55.88% (57/102) for
culture (with pathogens isolated from at least one of the four
media). The positive rate of culture among SCCA, DTM and DBM was
similar (60.27%, 58.90% and 60.27%, respectively) which was higher
than that of SCA (31.51%). In the culture-positive samples T.
rubrum was identified as the pathogen in 55 cases, C. albicans in 1
case, T. rubrum and C. albicans coexisted in 1 case. Saprophytes
belonged to Aspergillus spp., Penicillium spp., Rhizopus spp.,
Alternaria spp., Cladosporium spp., Acremonium spp., Fusarium spp.,
yeast and bacteria. The initial growth time of pathogens was 5.92 ±
0.37d, 5.03 ± 0.38d and 6.24 ± 0.42d on DTM, DBM and SCCA,
respectively (F = 2.35, P = 0.10). No obvious difference in
colonial appearance was observed among these three media (figure 2).
Color conversion of DTM and DBM
All isolates of dermatophyte (including those coexisting with
nondermatophytic strains) could alter the color of both DTM and
DBM. Similarly, with the axenic culture, it was easier to discover
the early color change in DBM (figure 3). The time for
discoloration was shorter with DBM than with DTM (5.83 ± 0.39 days
vs. 7.32 ± 0.41 days, t = 2.63, P = 0.01).
Thirteen out of twenty five cases (52%) of only
non-dermatophytic growth showed a similar discoloration (10 of
molds, 2 of yeasts, 1 of mold coexisted with yeast) with DTM, and
seven out of eleven (63.64%) with DBM (6 of molds, 1 of bacteria).
However, the colonies were large when the color began to change,
which could be distinguished from dermatophytes (figure 4). Variance of the
colonial diameters when the color began to change between
dermatophytes and nondermatophytes was significant (P <
0.001).
Interpretation of results
According to the conventional interpretational criteria of DTM
(dermatophytes usually made the color change within 7~10 days), 21
cases of 42 (50%) dermatophytic strains isolated from DTM were
mistaken for non-dermatophyte, and 10 cases of 25 non-dermatophytic
cases (40%) were mistaken for dermatophyte, kappa = 0.36; as for
DBM, these false-negative and false-positive percentages were 27.9%
and 54.5%, respectively, kappa = 0.63.
Therefore, we set a new criteria, i.e. a strain with a colonial
diameter ≤ 5 mm when the color began to change would be
identified as a dermatophyte, and > 5 mm would be
identified as non-dermatophytic mold (yeast and bacterial could be
differentiated easily by their colonial morphology). Following
these criteria, for DTM, only 2 dermatophytic cases (4.8%) were
mistaken for non-dermatophytes, and 1 non-dermatophytic case (4.0%)
was mistaken for a dermatophyte, kappa = 0.94; for DBM, these
false-negative and false-positive percentages decreased to 4.7% and
0%, respectively, kappa = 0.94.
The data of sensitivity, specificity, positive predictive value,
negative predictive value, accuracy with the old and new criteria
are shown in table 1.
Table 1 Comparisons for indexes of the conventional and
new criteria differentiate dermatophyte and non-dermatophyte
isolated from DTM and DBM
|
Sensitivity
|
Specificity
|
Positive predictive value
|
Negative predictive value
|
Accuracy
|
|
DTM-i
|
50.0%
|
83.3%
|
67.7%
|
70.4%
|
69.6%
|
|
DTM-i’
|
95.2%
|
98.3%
|
97.6%
|
96.7%
|
97.1%
|
|
DBM-i
|
72.1%
|
89.8%
|
83.8%
|
81.5%
|
82.4%
|
|
DBM-i’
|
95.3%
|
100%
|
100%
|
96.7%
|
98.0%
|
|
u1
|
5.17*
|
3.18*
|
3.77*
|
4.50*
|
5.89*
|
|
u2
|
3.13*
|
3.54*
|
3.66*
|
2.94*
|
4.16*
|
Discussion
The most important character of DBM is the pH indicator.
Bromothymol blue is a widely used pH indicator in culture media of
microorganisms with a pH range of 6.0 ~ 7.6 [12-15]. The
initial pH of DTM or DBM is 5.5 ± 0.1, therefore, bromothymol blue
can reflect the pH shift from acid to alkali quicker than phenol
red (pH range: 6.8 ~ 8.4). Besides, in our experiment, the
order of color alteration of phenol red is orange yellow→salmon
pink→red, and of bromothymol blue is straw yellow→green→blue. This
is may be the reason that it is easier to discover the
discoloration with DBM than with DTM. When applied to clinical
samples, the time for discoloration was shorter in DBM than in DTM
(5.83 ± 0.39 days vs. 7.32 ± 0.41 days, t = 2.63, P = 0.01).
Besides, the original DTM antibiotic formation is replaced by
chloramphenicol in DBM. Chloramphenicol is a broad spectrum
antibiotic that inhibits a wide range of gram-positive and
gram-negative bacteria, and it can be added into media before
autoclaving, thus reducing the procedure. Moreover, DBM contains
less dextrose and soy peptone than DTM, which decreases the
production cost. Although it may affect the growth of some strains
such as T. concentricum and T. verrucosum, no significant effect on
initial color alteration was observed in our study (data not
shown).
Since it is easy to distinguish bacteria and yeasts from
filamentous fungi by their distinctive colonial morphology, the
specificity test of the media was focused on non-dermatophytic
molds and dermatophytes. In our study, more than 50% of
non-dermatophytic isolates made a color alteration, some of which
were as early as dermatophytes. This may be due to the heavy
contamination of nail samples and the strain differences. If we
still follow the conventional criteria which mainly considers the
color alteration, then the percentage of wrong identifications
would be very high. Through careful observation and analysis, we
found dermatophytes could change the color of the media when there
was no obvious or very small colonial growth; in contrast,
non-dermatophytic molds made the color alteration at a slower rate.
Such molds have priority in acid nutrient utilization and grow
faster than dermatophytes, which may account for the above
phenomena. Therefore, we set up new interpretational criteria based
on the colonial diameter when the color began to change
(dermatophyte ≤ 5 mm, non-dermatophytic mold > 5 mm).
Most isolates could be identified correctly by the new criteria,
and the results of differentiation were in good agreement with
those from the professional laboratory of mycology. If the
variations of colonial morphology between dermatophyte and
non-dermatophyte can be recognized (e.g. some of these
non-dermatophytic molds may be recognized by their dark green to
black hyphae; white aerial hyphae are exhibited by dermatophytes),
the accuracy of identification will be further increased.
Our results, obtained from axenic cultures as well as clinical
settings, indicate that the DBM medium is more convenient, rapid,
and economical to use than DTM. Combined with the new
interpretational criteria, it is more accurate in confirming a
diagnosis of onychomycosis. Further research including more types
of dermatophytic infections and a larger number of clinical samples
is required.
Acknowledgments
This work was supported by a grant from Institute of Dermatology,
Chinese Academy of Medical Sciences & Peking Union Medical
College (received by Xiaofang Li). Conflict of interest: none.
References
1 Barry L. Hainer. Dermatophyte infections. Am Fam Physician
2003; 67: 101-8.
2 Ghannoum MA, Hajjeh RA, Scher R, et al. A
large-scale North American study of fungal isolates from nails: the
frequency of onychomycosis, fungal distribution, and antifungal
susceptibility patterns. J Am Acad Dermatol 2000; 43: 641-8.
3 Kemna ME, Elewski BE. A U.S. epidemiologic survey of
superficial fungal diseases. J Am Acad Dermatol 1996; 35:
539-42.
4 Roberts DT, Taylor WD, Boyle J, British
Association of Dermatologists. Guidelines for treatment of
onychomycosis. Br J Dermatol 2003; 148: 402-10.
5 Clayton YM. Clinical and mycological diagnostic aspects
of onychomycoses and dermatomycoses. Clin Exp Dermatol 1992;
17(suppl 1): 37-40.
6 Taplin D, Zaias N, Rebell G, et al.
Isolation and recognition of dermatophytes on a new medium (DTM).
Arch Dermatol 1969; 99: 203-9.
7 Rich P, Harkless LB, Atillasoy ES. Dermatophyte
test medium culture for evaluating toenail infections in patients
with diabetes. Diabetes Care 2003; 26: 1480-4.
8 Elewski BE, Leyden J, Rinaldi MG, et al.
Office practice-based confirmation of onychomycosis: a US
nationwide prospective survey. Arch Intern Med 2002; 162:
2133-8.
9 Singh S, Beena PM. Comparative study of different
microscopic techniques and culture media for the isolation of
dermatophytes. Indian J Med Microbiol 2003; 21: 21-4.
10 Salkin IF. Dermatophyte test medium: evaluation with
nondermatophytic pathogens. Appl Microbiol 1973; 26: 134-7.
11 Carroll HF. Evaluation of dermatophyte test medium for
diagnosis of dermatophytes. J Am Vet Med Assoc 1974; 165:
192-5.
12 Muniesa-Perez M, Jofre J, Blanch AR.
Identification of Vibrio proteolyticus with a differential medium
and a specific probe. Appl Environ Microbiol 1996; 62: 2673-5.
13 Citron DM, Baron EJ, Finegold SM, et al.
Short prereduced anaerobically sterilized (PRAS) biochemical scheme
for identification of clinical isolates of bile-resistant
Bacteroides species. J Clin Microbiol 1990; 28: 2220-3.
14 Nakamura Y, Kano R, Sato HV. Isolates of
Cryptococcus neoformans serotype A and D developed on
canavanine-glycine-bromthymol blue medium. Mycoses 1998; 41:
35-40.
15 Premier RR, Cox JC, Aitken DP, et al. An
evaluation of the use of a pH indicator for the detection of
beta-lactamase in enzyme immunoassay. Immunol Methods 1985; 83:
371-7.
|
Printable version
Version PDF
