BASIC SCIENCE REVIEW

Azole Resistance in Dermatophytes

Prevalence and Mechanism of Action
Mahmoud Ghannoum, PhD*

Azole antifungal agents (eg, fluconazole and itraconazole) have been widely used to
treat superficial fungal infections caused by dermatophytes and, unlike the allylamines
(such as terbinafine and naftifine), have been associated with resistance development.
Although many published manuscripts describe resistance to azoles among yeast and
molds, reports describing resistance of dermatophytes are starting to appear. In this
review, I discuss the mode of action of azole antifungals and mechanisms underlying
their resistance compared with the allylamine class of compounds. Data from published
and original studies were compared and summarized, and their clinical implications are
discussed. In contrast to the cidal allylamines, static drugs such as azoles permit the
occurrence of mutations in enzymes involved in ergosterol biosynthesis, and the
ergosterol precursors accumulating as a consequence of azole action are not toxic.
Azole antifungals, unlike allylamines, potentiate resistance development in dermato-
phytes. (J Am Podiatr Med Assoc 106(1): 79-86, 2016)

Antifungals Used to Treat Supercial
Fungal Infections
Imidazole and triazole antifungals have been used to
treat dermatophytoses for many decades. In this
review, I discuss their history and use in various
applications and examine the incidence of azole
resistance in dermatophytes. Their mode of action
and the underlying mechanisms of resistance are
discussed and compared with the allylamine class
of compounds.
History of Therapeutic Agents
There are two main classes of antifungal agents for
the treatment of dermatophytosis: azoles and
allylamines, along with griseofulvin, ciclopirox,
and tolnaftate. These agents are available in oral
and topical forms. The azole class of antifungal
drugs includes imidazoles (such as bifonazole,
clotrimazole, econazole, ketoconazole, luliconazole,
miconazole, sertaconazole, and tioconazole) and
triazoles (such as fluconazole, isavuconazole, itra-
conazole, posaconazole, ravuconazole, voricona-
zole, luliconazole, and lanoconazole). Allylamine
antifungals include amorolfine, butenafine, naftifine,
and terbinafine (Fig. 1).
The development of antifungal agents lagged
*Department of Dermatology, Case Western Reserve
University, 11100 Euclid Ave, Cleveland, OH 44106. (E-mail:
mahmoud.Ghannoum@case.edu)
behind that of antibacterial drugs due, in part, to
the relatively low incidence of serious fungal
diseases. Moreover, because fungi are eukaryotic,
similar to human cells, finding agents with selective
toxicity that target the fungus and not the host is
difficult. In 1958, oral griseofulvin was the first
antifungal agent to become available for clinical
use, and it is still used to treat tinea capitis. This
was followed by the introduction of three topical
agentsclotrimazole, econazole, and miconazole
all of which are still mainstays of topical therapy.1
Shortly thereafter, ketoconazole, the first oral drug
with broad-spectrum antifungal activity, was ap-
proved for the treatment of systemic fungal infec-
tions.2,3
In the 1990s, fluconazole and itraconazole were
approved for the treatment of superficial and
systemic infections, followed by terbinafine for the
treatment of onychomycosis and other superficial
dermatophytoses.1,4 Current treatment options for
dermatophyte infections include topical formula-
tions and systemic drugs for more difficult-to-treat
infections. For example, systemic treatment by
griseofulvin, itraconazole, fluconazole, or terbina-
fine is necessary for the treatment of tinea capitis,
and options for the treatment of onychomycosis
include continuous or pulsed oral dosing or
combination topical agents.4,5 Today, newer azoles
and allylamines have increased the size of the
armamentarium available for the treatment of
dermatophyte infections (Fig. 2).

Journal of the American Podiatric Medical Association  Vol 106  No 1  January/February 2016 79
Figure 1. Structures of antifungal agents: econazole (an imidazole) (A), voriconazole (a triazole) (B),
terbinafine (an allylamine) (C), and amorolfine (an allylamine) (D).

Mechanism of Action of the Azole Class
of Antifungals
With some exceptions, including griseofulvin and
ciclopiroxolamine, antifungal agents commonly
used to treat dermatophytoses target the ergosterol
biosynthetic pathway (Fig. 3). Ergosterol is the
major sterol component of the fungal plasma
membrane and is essential for the proper function-
ing of several membrane-bound enzymes. One such
enzyme is chitin synthase, which is crucial for cell
growth and division.6,7 The earlier imidazoles, such
as miconazole, econazole, and ketoconazole, have a
complex mode of action, inhibiting cell membrane
lipid biosynthesis and several membrane-bound
enzymes.1,8 Imidazoles, and, in part, the more recent
triazoles fluconazole, itraconazole, voriconazole,
and luliconazole, share a common mechanism of
ergosterol depletion and accumulation of sterol
precursors.9 The accumulation of these precursors,
including 14a-methylated sterols (lanosterol, 4,14-
dimethylzymosterol, and 24-methylenedihydrola-
nosterol), results in altered plasma membrane
structure and function.10 It is thought that the
primary target of azoles is the heme protein, which
co-catalyzes cytochrome P450dependent 14a-de-
methylation of lanosterol.11 In 2010, Zhang et al12
identified a critical virulence requirement for
ergosterol in vacuolar H -ATPase function. The
authors ability to reverse growth inhibition by
fluconazole with the addition of exogenous ergos-
terol confirms that azole antifungal activity is due to
depletion of ergosterol.12 Mazabrey et al13 used
transmission and scanning electron microscopy to

80 January/February 2016  Vol 106  No 1  Journal of the American Podiatric Medical Association
Figure 2. Timeline of the development of antifungal agents to treat dermatophytoses.
study the effects of imidazoles against dermato-
phytes, from which they suggest that the mitochon-
dria are the first structures to be damaged, with
retraction of cytoplasm and cell wall collapse as
secondary alterations. In addition to inhibition of
the 14a-demethylase, the triazoles also affect the
reduction of obtusifolione to obtusifoliol, resulting
in the accumulation of methylated sterol precur-
sors.14,15
In contrast, the allylamines are potent inhibitors
of squalene epoxidase, an enzyme involved in the
biosynthesis of ergosterol (inhibition of sterol-D14-
reductase and sterol-D7-D8 isomerase).16 This inhi-
bition affects early steps in sterol synthesis and
results in the accumulation of squalene, which is
toxic to the fungal cells. This cell toxicity accounts
for the cidal activity of allylamines compared with
that of azoles, which are static agents.
Incidence of Azole Resistance in
Nondermatophyte Fungal Strains
The increase in Candida-related bloodstream infec-
tions worldwide has been widely publicized, and
although most Candida species remain susceptible
to azoles, Pfaller et al17 identified an emergence of
Journal of the American Podiatric Medical Association  Vol 106  No 1  January/February 2016
azole-resistant Candida glabrata strains from the
SENTRY Antimicrobial Surveillance Program (flu-
conazole, 7.7%; posaconazole, 5.1%; and voricona-
zole, 6.4% of C glabrata strains tested). Other
studies report similar azole resistance in non-
albicans Candida bloodstream infections.18,19 Can-
dida strains isolated from other clinical sources
have also demonstrated increases in azole resis-
tance. For example, Marchaim et al20 reported an
emergence of fluconazole-resistant Candida albi-
cans in patients taking daily doses of fluconazole for
recurrent vaginitis. In addition, Candida strains
from patients with acquired immunodeficiency
syndrome in Ethiopia treated for multiple episodes
of oropharyngeal candidiasis demonstrated resis-
tance to fluconazole (12.2%), ketoconazole (7.7%),
and itraconazole (4.7%).21 Taken together, the
results of these reports suggest that frequent
exposure to azole drugs is a risk factor for the
development of resistance.
Similarly, an increase in the incidence of azole
resistance in molds such as Aspergillus fumigatus
has recently been recognized. Although widespread,
the frequency of itraconazole resistance varies from
7% in the Netherlands to 50% in two US laborato-
ries.22 The exact frequency of azole resistance in
Aspergillus isolates is difficult to determine be-
81
Figure 3. Ergosterol pathway and sites of action. (Adapted from Ghannoum and Rice10; used with permission
from the American Society for Microbiology.)
cause of low culture positivity in patients with
aspergillosis.23 Regardless, molecular assays have
made it possible to trace the geographic spread
throughout Europe and India of voriconazole-
resistant A fumigatus strains by identifying a new
cyp51A-mediated resistance mechanism.24-26 An
alarming discovery from testing soil and other
environmental samples showed cross-resistance to
six widely used triazole fungicides and to voricona-
zole, posaconazole, and itraconazole, raising con-
cerns that exposure to these products will produce
airborne A fumigatus spores that are highly
resistant to the medical triazoles.27
Incidence of Azole Resistance in
Dermatophyte Fungal Strains
Although azole resistance in dermatophyte strains
has been reported in the literature, resistance to
allylamines has been published in only one report,
which showed resistance development in one
patient treated with terbinafine.28 No reports docu-
menting naftifine resistance have been published. In
a retrospective study analyzing in vitro resistance in
Trichophyton rubrum strains from 18 patients with
recalcitrant onychomycosis, Gupta and Kohli29
reported that most sequential isolates were suscep-
tible to ciclopirox, itraconazole, ketoconazole, and
terbinafine. However, a T rubrum strain obtained
from one patient (6% of those studied) whose initial
itraconazole and ketoconazole minimal inhibitory
82 January/February 2016  Vol 106  No 1  Journal of the American Podiatric Medical Association
concentration (MIC) values were 0.125 lg/mL
demonstrated an increase in itraconazole and
ketoconazole MIC values (32 and 4 lg/mL, respec-
tively) after 15 months of antifungal therapy. This
increase in azole MICs, which reached a 256-fold
increase after drug exposure, may be indicative of
the development of azole resistance in dermato-
phytes after repeated exposure.
A similar study in a major Mexican hospital
analyzed in vitro data from 36 patients who failed
treatment for dermatophytoses. Three strains each
of T rubrum and Trichophyton mentagrophytes
and one strain of Trichophyton tonsurans isolated
from these patients demonstrated increased resis-
tance to azoles using the E-test method. All seven
dermatophyte strains (19.4% of those tested) were
resistant to fluconazole (MIC 64 lg/mL), with one
T rubrum strain also resistant to ketoconazole and
itraconazole (MIC 8 lg/mL). The authors in this
study did not analyze sequential isolates obtained
from the same patient at baseline and after
treatment; therefore, it is impossible to determine
whether this represents innate resistance or resis-
tance acquired after repeated drug exposure.30
On the other hand, Goh et al31 studied 100
dermatophyte strains collected from patients in
Singapore who had not previously undergone
systemic or topical antifungal therapy. Patients
presented with various clinical manifestations, with
the most prevalent being tinea corporis (36%), tinea
cruris (22%), tinea pedis (19%), and onychomycosis
(11%). The MICs of ketoconazole and itraconazole
were tested against all dermatophyte strains isolat-
ed using a microdilution assay, resulting in 10% of
ketoconazole and 15% of itraconazole with elevated
MICs (8 lg/mL). This indicates a fairly high
incidence of innate azole resistance in wild-type
dermatophyte strains.
Finally, from a worldwide tinea capitis clinical
trial with participants from the United States,
Central and South America, and India, 817 derma-
tophyte strains were collected at baseline for MIC
testing. Species isolated included T tonsurans (n
718), Trichophyton violaceum (n 13), T menta-
grophytes (n 1), Microsporum canis (n 83), and
Microsporum gypseum (n 2). All strains were
tested against voriconazole and fluconazole using
the Clinical and Laboratory Standards Institutes
M38-A2 microdilution assay. The MIC ranges were
0.002 to 0.06 lg/mL for voriconazole and 0.25 to 32
lg/mL for fluconazole. Of these, 29 strains (3.5%)
had fluconazole MICs of 16 lg/mL or greater.
Although no resistance was found against vorico-
nazole among these isolates, innate resistance to
fluconazole is suggested by these findings.32
In contrast, an increase in the MIC values has not
been reported for drugs in the allylamine class of
antifungals. In a recent study, we evaluated the in
vitro antifungal activity of naftifine against derma-
tophytes as measured by MIC and minimum
fungicidal concentrations and determined whether
exposure to this antifungal agent induces resistance
development. Our data showed that naftifine pos-
sesses potent antifungal activity against dermato-
phytes (including T rubrum, T mentagrophytes, T
tonsurans, Epidermophyton floccosum, and M
canis) and is fungicidal against these fungi. The
MIC range against the dermatophyte isolates tested
was 0.015 to 1.0 lg/mL, with naftifine hydrochloride
being fungicidal against 85% of the Trichophyton
species. Moreover, our data showed that there was
no increase in MIC for any of the six strains tested
after repeated exposure to naftifine hydrochloride.33
Mechanism of Azole Drug Resistance in
Dermatophytes
Resistance to a particular drug can be characterized
by more than one mechanism of action and may be
activated simultaneously. Some of the most fre-
quent resistance mechanisms found in fungal
strains involve a decrease in drug uptake, structural
alterations in the target site, or an increase in drug
efflux.34 Efflux transporters are proteins found in
the cell membrane that bind to compounds,
Journal of the American Podiatric Medical Association  Vol 106  No 1  January/February 2016
including antifungal drugs, and actively extrude
them from the cell.35 Several authors have reported
that increased drug efflux is the resistance mecha-
nism of azoles, including fluconazole, itraconazole,
ketoconazole, and tioconazole.36,37 Although the
mechanism of dermatophyte azole resistance in
vivo has yet to be determined, the T rubrum
TruMDR1 gene was cloned by polymerase chain
reaction using degenerate primers and was identi-
fied as encoding an ATP-binding cassette transport-
er. This gene is overexpressed in the presence of
several different antifungals, including fluconazole,
ketoconazole, and itraconazole, suggesting a role in
drug efflux in this dermatophyte species.36 Se-
quence analysis of the TruMDR1 gene revealed
1511 amino acids with homology to several other
ATP-binding cassette transporters identified in
other fungal strains, including CDR1 and CDR2 of
C albicans. The proteins encoded by these genes,
Cdr1p and Cdr2p, have been shown to confer
resistance to azoles.38,39
Subsequent to the identification of TruMDR1,
Fachin et al 37 described a single-copy gene,
TruMDR2, in T rubrum that also encoded an
ATP-binding cassette transporter. Sequencing of
its amino acids showed high homology with known
ATP-binding cassette transporters present in other
fungi that are responsible for drug efflux. Northern
blot analysis showed an increased level of tran-
scription of this TruMDR2 gene after exposure of
fungal hyphae to antifungals, including fluconazole,
itraconazole, ketoconazole, and tioconazole. Fur-
ther evidence that the transporter encoded by
TruMDR2 is involved in modulating drug suscepti-
bility is the fact that disruption of this gene in T
rubrum increased its sensitivity to terbinafine.
In contrast, modification of the target enzyme
squalene epoxidase by gene mutation (substitution
of a single amino acid in the squalene epoxidase
gene) is considered to be the resistance mechanism
in the allylamine terbinafine.40,41 These mutations
lead to substitutions in amino acids, which are
likely involved in the binding of terbinafine to
squalene epoxidase. For example, single amino acid
exchanges in the region of the protein Erg1 resulted
in high terbinafine resistance in filamentous fungi
and yeast.40-42
Discussion
The incidence of fungal infections has increased
during the past two decades due to the increase in
the number of immunocompromised patients and
the way we practice medicine. Advances such as
83
increased use of medical devices enable patients to
live longer while at the same time increasing their
risk of superficial and systemic fungal infections.
Unfortunately, widespread use of the limited num-
bers of antifungal agents, particularly the azoles, to
treat these infections has led to the development of
drug resistance. Thus, drug resistance in pathogenic
fungi, including the dermatophytes, is of increasing
importance. Although a plethora of published
literature describes the incidence of azole resis-
tance in systemic infections, studies in dermato-
phytes have been limited. However, it is clear that
azoles have a high potential for inducing resistance
in these pathogenic fungi. The prevalence of azole
resistance in dermatophytes has been reported to be
as high as 19% in certain areas worldwide. In
contrast, the potential of allylamines (terbinafine or
naftifine) to potentiate resistance is very low.
The difference in the inability of dermatophyte
strains to adapt to stress exerted by different
classes of antifungals may be due to the differences
in their primary mechanisms of action. For exam-
ple, the primary mode of action of allylamines is the
inhibition of squalene epoxidase. As discussed
previously herein, inhibition of this enzyme leads
to accumulation of the ergosterol precursor squa-
lene (Fig. 3), which is known to be toxic to the
fungal cells and explains the allylamine mechanism
of cidality. Because allylamines are cidal drugs,
their ability to potentiate resistance is greatly
reduced, as shown by our recent study with
naftifine.33 This situation is similar to the cidal
antifungal amphotericin B, a member of the polyene
class of antifungals, in which resistance is rare
despite more than 60 years of use. Another reason
for the lack of resistance to allylamines is the
accumulation of squalene, the first precursor to
ergosterol. This compound has been shown to be
toxic to the fungal cell. In contrast, static drugs such
as azoles inhibit only the growth of the organism,
permitting the occurrence of mutations in enzymes
involved in ergosterol biosynthesis, which serves as
the drug target. In addition, the ergosterol precur-
sors accumulating as a consequence of azole action
are not toxic.
Based on our understanding of the mechanisms of
drug resistance as a template, Alexander and
Perfect43 identified several strategies to overcome
resistance, including improvement of host immune
function, development of new drug delivery systems
for currently available drugs, and development of
new classes of antifungal agents. Furthermore,
molecular studies of the total dermatophyte genome
may guide the future development of new drugs that
84 January/February 2016  Vol 106  No 1  Journal of the American Podiatric Medical Association
have mechanisms of action that will prohibit
resistance development in the wild-type dermato-
phyte population.
In this regard, two new topical agents have
recently been approved by the Food and Drug
Administration for the treatment of onychomycosis.
Efinaconazole may be expected to demonstrate
resistance potential because it shares a mechanism
of action with other triazoles (inhibition of sterol
14a-demethylase).44 Tavaborole, a novel boron-
containing compound, possesses a different mech-
anism of action wherein protein synthesis is
blocked through the inhibition of tRNA synthesis.45
Thus, it is too early to determine whether wild-type
dermatophyte strains will develop resistance to this
topical agent after repeated exposure.
In addition, two novel topical agents are currently
in development that address the challenge of nail
penetration. TDT-067, a carrier-based dosage form
of terbinafine in Transfersome (1.5%) (Celtic Phar-
ma, Hamilton, Bermuda,), is an allylamine and, thus,
most likely will not have the potential for drug
resistance.46 ME1111 (Meiji Seika Pharma, Tokyo
Japan), a small molecular weight antifungal, also
shows low potential for the induction of resistance
in T rubrum and T mentagrophytes strains.47
In conclusion, azole antifungal agents, unlike
terbinafine and naftifine, potentiate resistance de-
velopment in dermatophytes. This resistance affects
not only superficial fungal infections but also
systemic disease. Importantly, azole resistance has
been encountered in strains present in the environ-
ment, which may eventually cause human infec-
tions.
Financial Disclosure: None reported.
Conflict of Interest: Dr. Ghannoum receives
contracts, serves on the advisory boards, and acts
as a consultant for Merz Pharmaceuticals, Meiji
Seika Pharma, Anacor Pharmaceuticals, Novartis
Pharmaceuticals, and Viamet Pharmaceuticals.
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