217 DISC

Veterinary Dermatology 2001, 12, 123127

Evaluation of the dermatophyte test medium RapidVet-D

JACQUES GUILLOT,* LAURENCE LATIE ,{ MANJULA DEVILLE,* LENAIG HALOS*
and RENE CHERMETTE*
*UMR 956 INRA-AFSSA-ENVA Biologie Moleculaire et Immunologie Parasitaires et Fongiques, Ecole
Nationale Veterinaire d'Alfort, France, and
{Sano Sante Nutrition Animale, Libourne, France
(Received 6 December 1999; accepted 19 April 2000)

Abstract The performance of the dermatophyte test medium (DTM) RapidVet-D was assessed using hair
samples collected from experimentally infected guinea pigs. Three dermatophyte species were included in the
study: Microsporum canis, Trichophyton mentagrophytes and Trichophyton equinum. DTM substrates were
inoculated with infected hairs and scales, incubated at 18, 21, 24, 27 or 37 8C and examined daily for 15 days.
The rapidity of colour change was clearly related to the incubation temperature and to the number of infected
hairs deposited on the reactive substrates. With the optimum incubation temperature 27 8C, a systematic
colour change could be observed only a few days post-inoculation: 3 days with M. canis infected hairs, 4 days
with T. equinum and 5 days with T. mentagrophytes.
Keywords: dermatophytes, diagnosis, DTM, Microsporum canis, mycology, Trichophyton equinum,
Trichophyton mentagrophytes.

INTRODUCTION
Fungal culture is recognized as the most reliable
method of conrming a diagnosis of dermatophytosis. In 1969, Taplin et al. introduced a specic culture
medium for dermatophyte identication.1,2 The
dermatophyte test medium (DTM) was rst used by
medical practitioners in Vietnam to isolate dermatophytes from soldiers. From that time the use of DTM
has gained wide acceptance in medical mycology and
several media are commercially available in veterinary practice.3,4 Precise formulation may vary
according to the marketed medium but all DTM
substrates contain nutrients to promote the growth of
dermatophytes, selective antibiotics to suppress
growth of most fungal and bacterial contaminants
and the pH indicator phenol red. When a dermatophyte species is cultured on DTM, it preferentially
utilizes the protein components leading to alkaline
pH. The colour change in the medium occurs
simultaneously with the appearance of colony
growth. Saprobic fungi prefer to use carbohydrates
and do not produce alkaline pH before the colony
growth is well established.
In the present study, the performance of RapidVet-D
(dms Laboratories, Inc., Flemington, New Jersey,
USA), a DTM devised for specic diagnosis of animal
dermatophytoses, was evaluated. Hair samples from

Correspondence: Dr J. Guillot, Ecole Nationale Veterinaire

d'Alfort, Parasitologie, 7 avenue du general de Gaulle, 94704
Maisons-Alfort, France. E-mail: j.guillot@vet-alfort.fr
# 2001 Blackwell Science Ltd

experimentally infected guinea pigs were inoculated

onto the DTM. Three dermatophyte species were included in the analysis: Microsporum canis, Trichophyton
mentagrophytes and Trichophyton equinum. Reaction
substrates were incubated at 18, 21, 24, 27 and 37 8C.
MATERIALS AND METHODS
Experimental dermatophytosis
Specic pathogen free (SPF) 6 week-old male guinea
pigs (Hartley) were used. Two weeks before inoculation, the absence of dermatophyte in hair was
conrmed by culture. Experimental cutaneous infection was obtained following Van Cutsem's method.5
Dermatophyte cultures grown for 14 days at 27 8C on
Sabouraud dextrose agar (2% glucose, 1% peptone,
0.5% chloramphenicol, 0.5% cycloheximide and 2%
agar) constituted the inoculum. Aerial sporulated
mycelium was removed and suspended in a mixture
of sterile water and honey (1:1 v:v). The inoculum
was homogenized by crushing. A nal volume of 2
mL was necessary for each animal. The inoculum was
deposited directly on the skin in three areas of about
5 cm2 (one on the back and two on the anks). The
skin had previously been clipped and scaried. The
guinea pigs were examined daily to check the
development of the cutaneous infection. Microscopic
examination of skin scrapings and mycological
cultures were performed on days 7, 14, 21 and 28
post-inoculation. The same procedure was performed
with three dermatophyte species: Microsporum canis,
Trichophyton mentagrophytes and Trichophyton equinum.
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217 DISC
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J. Guillot et al.

The strains had recently been isolated from lesions of

ringworm diagnosed in a Persian cat (M. canis), a dog
(T. mentagrophytes) and a horse (T. equinum).
RapidVet-D characteristics
RapidVet-D is intended for diagnostic use on samples
from dogs, cats, rabbits and horses suspected of being
infected by a dermatophyte species. The reaction
substrate in a glass screw-cap tube should be
inoculated with the hair sample and maintained at
room temperature. The tube should be examined
daily but the test period is deemed terminated at the
end of 3 days. Within this span time, any change in
colour from yellow to red is considered positive for
the presence of a dermatophyte species.
DTM inoculation and incubation
Hair samples from guinea pigs were plucked on days
14 or 21 post-inoculation. In animals infected with
Microsporum canis, hairs that uoresced were selected
under a Wood's light (464 W, Weidman, Germany).
In animals infected with Trichophyton mentagrophytes
or Trichophyton equinum, several hairs within, and
adjacent to, lesions were collected. The performance of
RapidVet-D was assessed using two types of inoculum: a `heavy' inoculum (more than 10 infected hairs)
and a `light' inoculum (one or two infected hairs). Hair
samples were aseptically inoculated on the reaction
substrate. Five incubation temperatures were selected:
18, 21, 24, 27 and 37 8C. Ten reaction tubes were used
for each inoculum, each dermatophyte species and
each incubation temperature. Reaction substrates
were examined daily for two weeks to observe fungal
growth and red colour change. Microscopic examination of a wet mount from fungal colonies, using
lactophenol cotton blue stain, was performed to
conrm the identity between the isolated fungus and
that used for experimental infection.
RESULTS
For each dermatophyte species, experimental infection was obtained in naive guinea pigs. As already
described by Jones6 and Van Cutsem,5 evolution of
the infection was clearly divided in four phases:
incubation, spreading, inammation and spontaneous healing. We systematically observed crusted
and erythematous lesions within 2 weeks after
inoculation. Microscopic and culture examinations
were positive (Fig. 1). Inoculation of Microsporum
canis and Trichophyton mentagrophytes resulted in
very inammatory and extensive lesions. Experimental infection with Trichophyton equinum was obtained
in two out of the three guinea pigs inoculated (Fig. 2).
Exudative lesions resulted in sections of hair matted
together in a scab. In Microsporum canis-infected
guinea pigs, Wood's light positive lesions could be
observed as soon as one week after inoculation.
Natural healing was usually achieved 6 weeks post# 2001 Blackwell Science Ltd, Veterinary Dermatology, 12, 123127

inoculation. However, mycological cultures remained

positive for additional 3 weeks in M. canis-infected
guinea pigs and 2 weeks in T. mentagrophytes and T.
equinum-infected animals.
DTM results with hair samples from experimentally infected guinea pigs are reported in Table 1.
Colour change from yellow to red occurred simultaneously with fungal growth (Fig. 3). The rapidity of
colour change was clearly related to the incubation
temperature and to the number of infected hairs
deposited on the reactive substrates. With a `heavy'
inoculum and a constant incubation temperature of
24 or 27 8C, all reaction tubes were positive within a
few days (3 days for Microsporum canis, 4 days for
Trichophyton equinum and 5 days for Trichophyton
mentagrophytes). On the contrary, low temperatures
(18 and 21 8C) and `light' inoculum resulted in a
delayed colour change: from 6 to 10 days were
usually necessary for 100% positivity. The use of the
elevated temperature of 37 8C did not improve DTM
results for Microsporum canis and Trichophyton
equinum. Trichophyton mentagrophytes was proved
to be more thermotolerant and DTM results at 37 8C
were similar to those obtained at 24 or 27 8C. After
incubation for 10 days, dermatophyte colonies had
systematically developed on the medium and microscopic examination conrmed the identity with the
species used for experimental infection.
DISCUSSION
Dermatophyte test media are widely used for the
diagnosis of human as well as animal dermatophytoses. Interest in DTMs lies on the rapidity of the
result. However, only a few attempts have been made
to evaluate the performance of such media.710
Moreover, the same procedure was systematically
applied in previous investigations: commercially
prepared DTMs were compared with conventional
mycological methods using skin or hair specimens
from humans or animals suspected of having
dermatophytosis. In a study of 1400 human specimens, 97% could be correctly classied as positive or
negative for dermatophytes solely by DTM colour
change.1 Carroll examined specimens from 100
animals and found that DTM was 82% accurate as
a diagnostic test for dermatophytosis.8 The dierence
in accuracy between DTM results with human and
animal specimens was attributed to the greater
abundance of lamentous saprobic fungi on the skin
and hair of animals, especially herbivores.
The originality of the present study lies in the use
of hair samples from experimentally infected guinea
pigs and the comparison of several incubation
temperatures. This procedure allowed comparison
of the size of the inoculum and also the growth
conditions for three dermatophyte species. Whereas
DTM substrates are intended to be maintained at
room temperature for the duration of the test, the

217 DISC
DTM performance

125

Figure 1. Direct examination of

Microsporum canis-infected hairs from
a guinea pig. Small and spherical
arthrospores (24 mm in diameter) are
observed on the surface of the hairs.
Table 1. Delay for colour conversion of RapidVet-D inoculated
with dermatophyte-infected hairs from guinea pigs

Figure 2. Lesions of dermatophytosis caused by Trichophyton

equinum in an experimentally infected guinea pig (21 days post
inoculation).

present study demonstrated that a constant temperature of between 24 and 27 8C was required for a rapid
colour change. Usually the room temperature is not
constant throughout the day. Moreover, great variations may be reported according to local climatic
conditions. One could assume that insucient
incubation temperatures account for false negative
results and variations of DTM performance according to geographical locations and seasons. Another

Inoculum

Delay for DTM colour

change (in days)
minimum
maximum
delayb
delaya

Incubation temperature 18 8C
M. canis low inoculum
M. canis high inoculum
T. mentagrophytes low inoculum
T. mentagrophytes high inoculum
T. equinum low inoculum
T. equinum high inoculum

4
4
5
4
5
5

10
6
10
10
10
10

Incubation temperature 21 8C
M. canis low inoculum
M. canis high inoculum
T. mentagrophytes low inoculum
T. mentagrophytes high inoculum
T. equinum low inoculum
T. equinum high inoculum

4
3
5
3
4
4

6
4
7
7
10
7

Incubation temperature 24 8C
M. canis low inoculum
M. canis high inoculum
T. mentagrophytes low inoculum
T. mentagrophytes high inoculum
T. equinum low inoculum
T. equinum high inoculum

3
2
3
2
4
3

5
3
6
6
6
6

Incubation temperature 27 8C
M. canis low inoculum
M. canis high inoculum
T. mentagrophytes low inoculum
T. mentagrophytes high inoculum
T. equinum low inoculum
T. equinum high inoculum

3
2
4
2
4
3

5
3
5
5
5
4

Incubation temperature 37 8C
M. canis low inoculum
M. canis high inoculum
T. mentagrophytes low inoculum
T. mentagrophytes high inoculum
T. equinum low inoculum
T. equinum high inoculum

5
5
4
2
4
3

7
7
6
6
7
7

colour change observed for the rst time in some reaction substrates.
delay for 100% positivity (10 RapidVet-D tubes inoculated).

limit for DTM sensitivity is the nature and the size of

the inoculum. When only a few infected hairs or
# 2001 Blackwell Science Ltd, Veterinary Dermatology, 12, 123127

217 DISC
126

J. Guillot et al.
DIM (Dermatophyte Identication Medium) was
devised to eliminate the problems associated with
the use of traditional DTMs, especially false positive
results. The medium included antibiotics and the pH
indicator bromocresol purple, and the selected incubation temperature was 37 8C. A study using human
specimens and culture collection isolates demonstrated
very high specicity and sensitivity. The DIM should
rapidly be evaluated for veterinary practice.
REFERENCES

Figure 3. Fungal growth and colour change in RapidVet-D four

days after inoculation of the medium with Microsporum canisinfected hairs (incubation temperature 278C).

scales were collected, cultures took as long as 10 days

to grow. However, the test period of RapidVet-D is
deemed terminated 3 days post-inoculation. This
short time span precludes any positive result with
specimens from mechanical carriers or animals
undergoing antifungal therapy. False negative results
could also be attributed to M. canis strains that do
not produce the red colour change.11
The risk of false positive results constitutes another
problem associated with the use of DTM. Very
rapidly after the original description by Taplin et al.,1
several studies indeed demonstrated that nondermatophytic species, both saprobic or pathogenic, could
cause a colour change as intense and rapid as that
caused by dermatophytes.8,9 However, it seems
commonly admitted that the DTM colour change is
delayed and always occurs after the colony of the
saprobic fungus is well established.
Recently, a new medium was introduced for
isolation and identication of dermatophytes.12 The

1. Taplin, D., Zais, N., Rebell, G., Blank, H. Isolation

and recognition of dermatophytes on a new medium
(DTM). Archives of Dermatology 1969; 99: 2039.
2. Taplin, D., Allen, A.M., Mertz, P.M. Experience with a
new indicator medium for the isolation of dermatophyte
fungi. In: Proceedings of the International Symposium
on Mycoses. Scientic Publication 205. Washington,
DC: Pan American Health Organization, 1970: 558.
3. DeBoer, D.J., Moriello, K.A. Clinical update on feline
dermatophytosis: part I. The Compendium 1995; 17:
1197203.
4. Medleau, L., Ristic, Z. Diagnosing dermatophytosis in
dogs and cats. Veterinary Medicine 1992; 22: 108691.
5. Van Cutsem, J. Animal models for dermatomycotic
infections. In: McGinnis, M.R., Borgers, M. Current
Topics in Medical Mycology. New York: SpringerVerlag, 1989: 135.
6. Jones, H.E. Animal models of human dermatophyte
infections. In: Maibach, H.I. Animal Models in
Dermatology. Edinburgh: Churchill Livingstone, 1975:
16875.
7. Blakemore, J.C. Dermatophyte Test Medium: a
diagnostic aid for practitioners. Veterinary Medicine
1971; 66: 3579.
8. Carroll, H.F. Evaluation of Dermatophyte Test
Medium for diagnosis of dermatophytosis. Journal of
the American Veterinary Medical Association 1974; 165:
1925.
9. Salkin, I.F. Dermatophyte test medium: evaluation with
nondermatophytic pathogens. Applied Microbiology
1973; 26: 1347.
10. Sinski, J.T., Swanson,
J.R., Kelley,
L.M.
Dermatophyte test medium: clinical and quantitative
appraisal. Journal of Investigative Dermatology 1972;
58: 40511.
11. Moriello, K.A., DeBoer, D.J. Fungal ora of the hair
coat of cats with and without dermatophytosis. Journal
of Medical and Veterinary Mycology 1991; 29: 28592.
12. Salkin, I.F., Padhye, A.A., Kemna, M.E. A new
medium for the presumptive identication of
dermatophytes. Journal of Clinical Microbiology 1997;
35: 26602.

Resume La performance du milieu de culture rapide pour dermatophytes (DTM) RapidVet-D a ete evaluee
en utilisant des poils preleves sur des cobayes experimentalement infectes. Trois especes de dermatophytes ont
ete etudiees dans cette etude: Microsporum canis, Trichophyton mentagrophytes et Trichophyton equinum. Les
DTM ont ete inocules avec des poils et des squames infectes, incubes a 18, 21, 24, 27 ou 37 8C et examines
quotidiennement pendant 15 jours. La rapidite de virage de couleur etait clairement liee a temperature
d'incubation et au nombre de poils infectes inocules sur le milieu. A la temperature optimale de 278C, le virage
# 2001 Blackwell Science Ltd, Veterinary Dermatology, 12, 123127

217 DISC
DTM performance

127

de couleur etait systematique, et pouvait etre observe quelques jours seulement apres l'inoculation: 3 jours
avec des poils infectes par M. canis, 4 jours pour T. equinum et 5 jours pour T. mentagrophytes. La specicite
du test a ete evaluee en inoculant sur le milieu des champignons non dermatophytes des genres Acremonium,
Alternaria, Aphanoascus, Aspergillus, Beauveria, Candida, Chrysosporium, Cladosporium, Fusarium, Geotrichum, Malassezia, Mucor, Penicillium, Rhodotorula, Scopulariopsis et Trichoderma. Des dermatophytes non
pathogenes (Microsporum cookei, Trichophyton ajelloi and Trichophyton terrestre) ont egalement ete inocules.
A 27 8C, la plupart des champignons saprophytes ont pousse sur le RapidVet-D en faisant virer le milieu de
couleur. Le virage est apparu en 3 jours pour Aspergillus avus, Geotrichum candidum et pour les especes de
champignons keratinophiles mais non pathogenes Aphanoascus fulvescens, Chrysosporium sp., Microsporum
cookei et Trichophyton ajelloi. [Guillot, J., Latie, L., Deville, M., Halos L., Chermette R. Evaluation of the
dermatophyte test medium RapidVet-D. (Evaluation du milileu de culture rapide pour dermatophytes
RapidVet-D.) Veterinary Dermatology 12: 123127.]
Resumen El rendimiento del medio de prueba de dermatotos (DTM) RapidVet-D fue evaluado utilizando
muestras de pelo obtenidos de cobayas infectados experimentalmente. Se incluyeron tres especies de
dermatotos: Microsporum canis, Trichophyton mentagrophytes y Trichophyton equinum. Los substratos de
DTM fueron inoculados con pelos y escamas infectadas, incubados a 18, 21, 24, 27 o 37 8C y examinados
diariamente durante 15 d as. La rapidez del cambio de coloracion se encontraba claramente relacionado con
la temperatura de incubacion y con el numero de pelos infectados depositados sobre los substratos reactivos.
Con la temperatura optima de incubacion de 27 8C, se pod a observar un cambio de color sistematico solo
unos pocos d as post-inoculacion: 3 d as con pelos infectados con M. canis, 4 d as con T. equinum y 5 d as con
T. mentagrophytes. La especicidad de la prueba fue evaluada por inoculacion sobre el medio de hongos nodermatof ticos (generos Acremonium, Alternaria, Aphanoascus, Aspergillus, Beauveria, Candida, Chrysosporium, Cladosporium, Fusarium, Geotrichum, Malassezia, Mucor, Penicillium, Rhodotorula, Scopulariopsis y
Trichoderma) . Las especies dermatof ticas no-patogenicas (Microsporum cookei, Trichophyton ajelloi y Trichophyton terrestre) fueron tambien examindas. A 27 8C la mayor a de hongos saprotos fueron capaces de crecer
en RapidVet-D y producir un viraje del medio a color rojo. La conversion del color se produjo dentro de los
tres d as en substratos inoculados con Aspergillus avus, Geotrichum candidum y las especies queratinof licas
pero no-patogenicas Aphanoascus fulvescens, Chrysosporium sp., Microsporum cookei y Trichophyton ajelloi.
[Guillot, J., Latie, L., Deville, M., Halos L., Chermette R. Evaluation of the dermatophyte test medium
RapidVet-D. (Evaluacion del medio de prueba para dermatotos RapidVet-D.) Veterinary Dermatology 12:
123127.]
Zusammenfassung Die Leistungsfahigkeit des Dermatophytentestmediums (DTM) RapidVet-D wurde
mittels Haarproben von experimentell inzierten Meerschweinchen bewertet. Drei Dermatophyten wurden
in der Studie verwendet: Microsporum canis, Trichophyton mentagrophytes und Trichophyton equinum. DTMSubstrate wurden mit inzierten Haaren und Schuppen geimpft, bei 18, 21, 24, 27 oder 37 8C inkubiert und fur
15 Tage taglich untersucht. Die Geschwindigkeit der Farbanderung korrelierte eindeutig mit der Inkubationstemperatur und der Zahl der auf dem Substrat deponierten inzierten Haare. Mit der optimalen
Inkubationstemperatur 27 8C war eine systematische Farbanderung nur einige Tage nach der Inokulation
festzustellen: 3 Tage bei mit M. canis inzierten Haaren, 4 Tage bei mit T. equinum und 5 Tage bei mit T.
mentagrophytes inzierten Haaren. Die Spezitat des Tests wurde durch Inokulation des Mediums mit Pilzen,
die nicht zu Dermatophyten zahlten, bewertet (Klassen Acremonium, Alternaria, Aphanoascus, Beauveria,
Candida, Chrysosporium, Cladosporium, Fusarium, Geotrichum, Malassezia, Mucor, Penicillium, Rhodotorula,
Scopulariopsis und Trichoderma). Nicht-pathogene Dermatophytenspezies (Microsporum cookei, Trichophyton
ajelloi und Trichophyton terrestre) wurden auch uberpruft. Bei 27 8C konnten die meisten Saprophyten auf
RapidVet-D wachsen und eine rote Farbanderung hervorrufen. Diese Farbanderung trat innerhalb von 3
Tagen in den Substraten auf, die mit Aspergillus avus, Geotrichum candidum und den keratinophilen, aber
nichtpathogenen Spezies Aphanoascus fulvescens, Chrysosporium sp., Microsporum cookei und Trichophyton
ajelloi geimpft wurden. [Guillot, J., Latie, L., Deville, M., Ha los L., Chermette R. Evaluation of the
dermatophyte test medium RapidVet-D. (Die Bewertung des Dermatophytentestmediums Rapid Vet-D.)
Veterinary Dermatology 12: 123127.]

# 2001 Blackwell Science Ltd, Veterinary Dermatology, 12, 123127