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Indian Medical Gazette

65

FEBRUARY 2014

Comparative Study

Comparison of the Minimum Fungicidal Concentration of


Clotrimazole, Ketoconazole, Miconazole and Terbinafine
Against Clinical Isolates of Dermatophytes
M. Patankar,
Clinical Medicine Infomatics India, Thane.
A. Bhargava,
A. Ahluwalia
Medical Services, Glenmark Pharmaceuticals Ltd., Mumbai.
Abstract
Superficial fungal infections affect millions of people
worldwide. Earlier most dermatophyte strains had relatively
restricted geographical distribution. But currently,
dermatophytosis has become one of the most common
human infectious diseases worldwide. Fungal infections
are common in hot and humid climate of tropical countries
like India.
Topical and systemic therapies are commonly used to
treat dermatophyte infections.Clotrimazole is one of the
most commonly used topical antifungal drugs. This study
compared the minimum fungicidal concentration (MFC)
of Clotrimazole with Miconazole, Ketoconazole and
Terbinafine in skin dermatophytes.
The study demonstrated that Clotrimazole had lower
MFCs as compared to Ketoconazole and Miconazole against
Trichophyton rubrum, Trichophyton mentagrophytes and
Microsporum canis. Clotrimazole had comparable MFCs
versus Terbinafine against Trichophyton rubrum but it had
lower MFCs against Trichophyton mentagrophytes and
Microsporum canis.
Thus, Clotrimazole is an effective antifungal agent for
dermatophytosis even today.The efficacy of Clotrimazole
even against strains with intermediate resistance or

resistance to the older azole anti fungal drugs reiterate the


current decisions of empirical treatment with topical
Clotrimazole for the management of superficial
dermatophyte infections.
Keywords
dermatophytosis, clotrimazole, minimum fungicidal
concentration (MFC)
Introduction
Superficial fungal infections affect millions of people
worldwide1. Earlier most dermatophyte strains had relatively
restricted geographical distribution. But currently,
dermatophytosis has become one of the most common
human infectious diseases worldwide. Fungal infections
are common in hot and humid climate of tropical countries
like India.These infections have been observed to occur in
both healthy and immunocompromised patients. The chief
etiologic agents implicated include dermatophytes, yeasts
and nondermatophyte moulds. Dermatophytes are
implicated for most superficial fungal infections2 and the
estimated lifetime risk of acquiring a dermatophyte infection
is as high as 1020% as mentioned in a WHO survey3.
Dermatophytes include a group of closely related
keratinophilic fungi. These fungi can invade keratinised

Address for correspondence: Dr A. Ahluwalia, Medical Services, Glenmark Pharmaceuticals Limited, Glenmark House, BDS Marg, Chakala,
Off Western Express Highway, Andheri (E), Mumbai - 400 099. E-mail: amanjita@glenmarkpharma.com

66

tissues such as skin, hair and nails causing


dermatophytosis4. Dermatophytes include three genera
namely Trichophyton, Microsporum, and Epidermophyton5.
Tinea pedis, tinea unguium (onychomycosis), tinea cruris,
tinea mannum, tinea corporis, and tinea faciei are caused
by Trichophyton rubrum6. In addition, other frequently
implicated agents include Trichophyton mentagrophytes,
Microsporum canis, Microsporum gypseum and
Epidermophyton floccosum7.
Dermatophytosis cannot be easily diagnosed based on
the clinical manifestations. It is often confounding and may
mimic the clinical presentation of other dermatological
disorders.The laboratory diagnosis of dermatophytosis is
done by direct microscopic examination of clinical
specimen. This is a rapid diagnostic method8. The in vitro
culture techniques may take up to 8 weeks to yield results9.
The differential diagnosis of dermatophytoses includes
eczema, psoriasis, atopic dermatitis, contact dermatitis,
seborrhoeic dermatitis, etc 10. In immunocompromised
patients, it is more difficult to diagnose dermatophytosis
due to the atypical clinical presentation11.
The treatment of dermatophytoses would be more
effective when the selection of antifungal agent is based on
the identity of the causative agent. Both topical and systemic
therapies are commonly used to treat dermatophyte
infections.
Topical therapy is found to be effective for
uncomplicated cases of dermatophytoses. The key attributes
of a topical agent used for superficial fungal infections
required are: broad-spectrum activity, high mycological cure
rate, low incidence of side effects, convenient dosing, and
low cost. In clinical practice, empirical therapy is adopted
commonly with a broad spectrum topical antifungal drug.
Clotrimazole is an imidazole antifungal agent. It has been
widely used topically for the treatment of superficial
dermatophytosis for over 25 years 12, 13 with a good
satisfaction expressed by both patients and physicians
regarding the efficacy and tolerability profile of this drug.
There is as yet no report of resistance to this drug 14.
There is very little Indian data available regarding the
MFC(Minimum fungicidal concentration) of commonly
reported fungi. Therefore, the current study was undertaken
to evaluate and compare the minimum fungicidal
concentration of Clotrimazole with Miconazole,

Indian Medical Gazette

FEBRUARY 2014

Ketoconazole and Terbinafine in skin dermatophytes.


Materials and Methods
The clinical specimens collected were epidermal scales
which were scraped from near the advancing edges of the
lesions after disinfecting the lesions with 70% alcohol. In
cases where the advancing edges were not evident,
scrapings were collected from areas representing the whole
infected area. A total of 25 dermatophyte strains, including
Trichophyton rubrum (n = 10), T. mentagrophytes (n = 7)
and Microsporum canis (n = 8) were tested.
The broth microdilution assay for antifungal susceptibility
testing of dermatophytes was performed, according to the
CLSI guidelines. The drugs Clotrimazole, Ketoconazole,
Miconazole and Terbinafine were obtained from their
respective manufacturers.The drugs were dissolved in
100% dimethyl sulfoxide. They were subsequently prepared
as stock solution and serial twofold dilutions were
performed. Final concentrations ranged from 0.125 to 64
g/mL for all the antifungal drugs.
Inoculum suspensions of dermatophytes were prepared
from the seven days cultures grown on dermatophyte test
medium and Sabourauds dextrose broth at 28 C. The
fungal colonies were covered with approximately 10mL of
distilled water, and the suspensions were made by scraping
the surface with the tip of a sterile loop. The resulting
mixture of conidia and hyphal fragments was withdrawn
and transferred to sterile tubes and left for 15 to 20 minutes
at room temperature to sediment the heavy particles. The
optical density of the suspensions containing conidia and
hyphal fragments was read at 530 nm, adjusted to
transmittance of 65 to 70% (~2 to 4 106 cells/mL) and
diluted with RPMI 1640 medium to obtain the final
inoculum size of approximately 0.4 to 5 104 cells/mL.
Aliquots of 100 L of these suspensions were inoculated in
well of microtiter plate containing 100 L of specific
antifungal drug concentration and incubated at 28 C. Each
assay was carried out in duplicate.
Endpoint determination
Endpoint determination values were performed visually
every 24 h until the indication of growth in control well
drug-free. For azole agents, the MFC was defined as the
lowest concentration that produced prominent inhibition
of growth (approximately 80% inhibition) while for

Indian Medical Gazette

67

FEBRUARY 2014

Terbinafine, was defined as lowest concentration showing


100% growth inhibition.
Statistical analysis

Fig. 3
Comparison of MFC of Clotrimazole and other antifungal
drugs against Microsporium canis

We applied ANOVA & Tukeys HSD to identify


(1) If 4 treatment groups Clotrimazole, Ketoconazole,
Miconazole and Terbinafine significantly differ with
respect to observed MFC levels for each species
of fungi.
(2) The treatment group that demonstrates lowest MFC
among the antifungal drugs included in study.
Results
Clotrimazole demonstrated lower MFCs (0.01- 0.03
mcg/ml) as compared to Ketoconazole (0.06 mcg/ml) and
Miconazole (0.06-1.25 mcg/ml against Trichophyton rubrum
(Fig. 1) (p<0.01). Clotrimazole demonstrated lower MFCs
(0.03 mcg/ml) as compared to Ketoconazole (0.06 mcg/

ml -0.25 mcg/ml) and Miconazole (1.25 mcg/ml) against


Trichophyton mentagrophytes (Fig. 2) (p<0.01).
Clotrimazole demonstrated lower MFCs (0.01 mcg/ml 0.06 mcg/ml) as compared to Ketoconazole (0.03 mcg/
ml-0.5mcg/ml) and Miconazole (0.6 mcg/ml 1.25mcg/
ml) against Microsporum canis (Fig. 3) (p<0.01).

Fig. 1

Clotrimazole demonstrated comparable MFCs (0.01 0.03 mcg/ml) versus Terbinafine (0.01 mcg/ml - 0.03 mcg/
ml) against Trichophyton rubrum (Fig. 1). Clotrimazole
demonstrated lower MFCs (0.03 mcg/ml) as compared to
Terbinafine (0.03 mcg/ml-0.25 mcg/ml) against
Trichophyton mentagrophytes (Fig. 2). Clotrimazole
demonstrated lower MFCs (0.01 mcg/ml - 0.06 mcg/ml)
as compared to Terbinafine (0.03 mcg/ml - 0.1 mcg/ml)
against Microsporum canis (Fig. 3).

Comparison of MFC of Clotrimazole and other antifungal


drugs against Trichophyton rubrum

Fig. 2
Comparison of MFC of Clotrimazole and other antifungal
drugs against Trichophyton mentagrophytes

We micro monitored the significantly differing pairs


using Tukeys HSD (highest significant difference) method.
After applying ANOVA & Tukeys HSD, evaluation of the
data indicated that 60% of observations were distanced
close to minimum value for Clotrimazole, whereas 40% of
observations are closer to minimum value for Terbinafine.
Thus, Clotrimazole is better anti fungal agent for
Trichophyton rubrum species. In case of Micosporum canis
species, 75% of observations were distanced close to
minimum value for Clotrimazole, whereas 62% of
observations were distanced close to minimum value for
Terbinafine. This indicated that Clotrimazole is better anti
fungal agent for Micosporum canis species. 85% of
observations were distanced close to minimum value for
both Clotrimazole and Terbinafine. Thus, Clotrimazole and
Terbinafine appear to be equally good antifungal agents for
Trichophyton mentagrophytes species.

Indian Medical Gazette

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FEBRUARY 2014

Discussion

3.

The dermatophytes Trichophyton, Epidermophyton and


Microsporum are commonly responsible for most of the
superficial fungal infections. These infections occur in both
healthy and immunocompromised patients. Early
recognition and treatment are vital for reduction of the
morbidity and risk of transmission.

Marques S.A., Robles A.M., Tortorano A.M., Tuculet


M.A., Negroni R, Mendes R.P. Mycoses associated
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4.

Dobrowolska A., Pawe S., Kaszuba C.A., Kozowska M.


PCR-RFLP analysis of the dermatophytes isolated from
patients in Central Poland. Journal of Dermatological
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5.

Weitzman I., Summerbell R.C. The dermatophytes. Clin


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6.

Aly R. Ecology and epidemiology of dermatophyte


infections. J Am Acad Dermatol. 31:S21S25, 1994.

7.

Monod M., Jaccoud S., Zaugg C., Lechenne B., Baudraz


F., Panizzon R. Survey of dermatophyte infectionsin
the Lausanne area Switzerland. Dermatology. 205:201
203, 2002.

8.

Rippon J.W. The Pathogenic Fungi and the Pathogenic


Actinomycetes. 3. Philadelphia: WB Saunders; 1988.
Medical mycology.

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Greenberg R.S., Daniels R.S., Flanders W.D., Eley J.W.,


Boring J.R. Medical epidemiology. 3. McGraw-Hill, New
York, NY; 1996. Diagnostic testing; pp. 7789.

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Barry I. Hainer Dermatophyte Infections. American


family physician. 67: 101108, 2003.

11.

Odom R.B. Common superficial fungal infections in


immunosuppressed patients. J Am Acad Dermatol.
31:5659, 1994.

12.

Calvin O.M., Thomas J.L. Eczema, Psoriasis, cutaneous


infections, acne and other common skin disorders. In:
Fauci, Braunwald, Kasper, Hauser, Longo, Jameson,
Loscalzo, editors. Harrisons Principles of Internal
Medicine. 17th ed. Vol. 1. New York: McGraw-Hill; 2008.
p. 318.

13.

Stary A., Soeltz-Szoets J., Ziegler C., Kinghorn G.R., Roy


R.B. Comparison of the efficacy and safety of oral
fluconazole and topical Clotrimazole in patients with
Candida balanitis. Genitourin Med. 1996;72:98102. Sobel
JD, Brooker D, Stein GE, Thomason JL, Wermeling DP,
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14.

Banerjee M., Ghosh A.K., Basak S., Das K.D.,


Gangopadhyay D.N. Comparative evaluation of
effectivity and safety of topical amorolfine and
Clotrimazole in the treatment of tinea corporis. Indian J
Dermatol. 56(6):657-662, Nov 2011.

It is important to consider the fact that mycoses can


have far reaching consequences in terms of significant
negative impact on the social, psychological and
occupational health. Dermatophytoses are usually treated
with topical antifungal drugs.
Topical agents used in these infections are the imidazoles,
allylamines, tolnafnate and ciclopirox. The imidazole
antifungal agent, Clotrimazole has been widely used topically
in Indian patients for the treatment of superficial
dermatophytosis for over 25 years12, 13. The outcomes of
treatment have been good with satisfaction expressed by
both patients and physicians regarding the efficacy and
tolerability profile of this drug. There is as yet no report of
resistance to this drug.
The current results have demonstrated the continued
efficacy of Clotrimazole against superficial dermatophyte
pathogens. The efficacy of Clotrimazole even against strains
with intermediate resistance or resistance to the older azole
anti fungal drugs reiterate the current decisions of empirical
treatment with topical Clotrimazole for the management of
superficial dermatophyte infections.
The current study corroborates the findings of the earlier
studies and the impression of clinicians in the real life setting
in India. Clotrimazole has been found to be more effective
in this in vitro study than the older imidazoles.
It can thus be concluded that topical Clotrimazole can
be the drug of choice even today in cases of
dermatophytosis.
References
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Pierard G.E., Arrese J.E., Pierard-Franchimont C.


Treatment and prophylaxis of tinea infections. Drugs.
52:209224, 1996.

2.

Aly R. Ecology and epidemiology of dermatophyte


infections. J Am Acad Dermatol. 31:2125, 1994.