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97]

REVIEW ARTICLE

Antifungal Resistance in Dermatology

Varadraj Pai, Ajantha Ganavalli1, Naveen Narayanshetty Kikkeri2

Abstract From the Department of

Cutaneous fungal infections affect more than one‑fourth of world’s population. The pathogenesis Dermatology, Goa Medical College,
and severity of fungal infection depend on various immunological and nonimmunological Bambolim, Goa, Departments of
1
Microbiology and 2Dermatology,
factors. The rampant use of antifungal therapy in immunocompromised individuals marked
SDM College of Medical Sciences
the onset of antifungal drug resistance. Fungal resistance can be microbiological or clinical. and Hospital, Dharwad,
Microbiological resistance depends on various fungal factors which have established due to Karnataka, India
genetic alteration in the fungi. Clinical resistance is due to host‑ or drug‑related factors. All
these factors may cause fungal resistance individually or in tandem. In addition to standardized
susceptibility testing and appropriate drug dosing, one of the ways to avoid resistance is Address for correspondence:
Dr. Varadraj Pai,
the use of combinational antifungal therapy. Combination therapy also offers advantages in
Department of Dermatology,
increased synergistic action with enhanced spectrum activity. Newer insights into mechanisms Goa Medical College,
of drug resistance will help in the development of appropriate antifungal therapy. Bambolim, Goa, India.
E‑mail: docpai@rediffmail.com
Key Words: Antifungal resistance, dermatophytes, mechanisms

Introduction dermatophytes are divided into zoophilic, geophilic,

Fungi have been present for around 1500 million
years with more than 1.5 million species out of which
only about 300 species are known to cause human
infections.[1,2] Fungi were recognized earlier than bacteria
as a pathogenic agent of human disease with David
Gruby describing etiological agent of favus, ectothrix,
and endothrix into three genera, Epidermophyton,
Microsporum, and Trichophyton.[3] Despite their early
discovery, most widespread infectious diseases in the
19th century were attributed to bacterial, parasitic,
and to viral origins.[4] From the mid‑20th century,
the incidence of severe systemic fungal infections
increased significantly, mainly due to increase in the
number of patients with compromised immune system
such as acquired immunodeficiency syndrome (AIDS)
or postorgan transplantation and chemotherapy. The
indiscriminate use of antibiotics added to the worsening
of this picture, leading to the installation of fungal
infections.[5]
Among all fungal infections, superficial mycoses are
the most frequent forms of human infections, affecting
more than 20%–25% of the world’s population.[6] It is
also estimated that 30%–70% of adults are asymptomatic
carriers of these pathogens.[7] In addition, species of
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or anthropophilic, depending on their primary
habitat (animals, soil, or humans, respectively).
Zoophilic species are responsible for about 30% of human
dermatophytoses and usually cause acute inflammatory
features. Anthropophilic species represent about 70% of
infections on these hosts, causing a chronic infection
of slow progression, suggesting that the fungus has
adapted to the human host.[8]
Pathogenesis of Fungal Infections
The successful initiation of infection is a process closely
related to the capability of the infecting dermatophyte
to overcome the host resistance mechanisms.[5]
Factors affecting fungal virulence are:
a. Defect in normal physiological barriers such as
physical and chemical structure of skin, normal
microflora present, exposure to ultraviolet light,
temperature, and humidity[6]
b. Adherence of dermatophytes and hyphae
penetration – adherence of dermatophytes is due
to arthroconidia, which are genetically programmed
disarticulated septate hyphae. It occurs 3–4 hours
This is an open access journal, and articles are distributed under the terms of
the Creative Commons Attribution‑NonCommercial‑ShareAlike 4.0 License,
which allows others to remix, tweak, and build upon the work non‑commercially,
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For reprints contact: reprints@medknow.com

How to cite this article: Pai V, Ganavalli A, Kikkeri NN. Antifungal

DOI: 10.4103/ijd.IJD_131_17 resistance in dermatology. Indian J Dermatol 2018;63:361-8.
Received: February, 2017. Accepted: June, 2018.

© 2018 Indian Journal of Dermatology | Published by Wolters Kluwer - Medknow 361

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Pai, et al.: Antifungal resistance
after contact.[8,9] Hyphae penetration into the stratum
corneum is a result of breakage of disulfide bridges
followed by secretion of variety of enzymes such as
metalloendoproteases (fungalysins) formerly called
keratinases, multiple serine‑subtilisins, proteases,
lipases, elastases, collagenases, phosphatases,
and esterases.[7,8,10] Reduction of disulfide bridges
depends on a sulfite efflux pump encoded by the
TruSSU1 gene, a proposed target for new antifungal
treatment. Sulfite secretion by this transporter
allows the cleavage of the cystine present in keratin,
thereby making it accessible to the action of endo
and exoproteases.[8,10,11]
c. Host response:
• Nonimmunological factors such as inhibitory
effect of sebum, unsaturated transferrin,
α2‑macroglobulin keratin inhibitor, long‑chain
saturated fatty acids in the scalp, serum inhibitory
factors (beta‑globulins, ferritin, and other metal
chelators) binding to iron essential for growth of
dermatophytes inhibit fungal invasion. Macerated
and hydrated skin, altered skin barrier, and
immunodeficiency disorders can promote entry of
dermatophytes[8,10]
• The immunological mechanism is cell‑mediated,
also more effective and protective, and is mediated
by macrophages as effector cells and cytokines
secretion from type 1 T‑helper lymphocytes.[10]
d. Fungal response – dermatophytes have developed
mechanisms that allow them to avoid the host
response such as reduction of inflammation
and phagocytosis by fungal mannans. Mannans
also inhibit the proliferation of keratinocytes,
allowing the establishment of a persistent chronic
infection.[5,8]
The common antifungal agents and their mechanisms of
action depending on the above are given in Figure 1.
Figure 1: The common antifungals and their mechanism of action
362
Antifungal Resistance
The evolution of antimicrobial drug resistance is an
almost inevitable process that is universal in the
microbial world. Although fungal resistance is not at par
with bacterial resistance, the economic facets associated
with fungal infections remains unacceptably high.[1]
Also considering the limited number of antifungal drugs
available, one of the main strategies of improving therapy
in mycoses is overcoming antifungal resistance.[12,18]
In general, clinical resistance is considered to be the
persistence or progression of an infection despite
appropriate antimicrobial therapy.[5,13] Clinical resistance
to antifungals was rare during the early phases of
antifungal therapy.[14] Among the earliest reported case of
drug resistance of dermatophytes was to griseofulvin by
Michaelides et al. in 1961.[15] Antifungal resistance to azoles
was first reported in 1980s to ketoconazole used in patients
of chronic mucocutaneous candidiasis.[13,16] Mukherjee et al.
in 2003 reported primary resistance of Trichophyton rubrum
to terbinafine in patients with onychomycosis.[17] A study
of Candida species in patients with AIDS showed 33% of
late‑stage patients with drug‑resistant strains of Candida
albicans in their oral cavities.[13,18]
Azole resistance in dermatophytes has been reported to
be as high as 19% in certain regions of the world.[19]
Occurrence of antifungal resistance has to be considered
independently for each antifungal class and for each
fungal genus.[14]
Fungal resistance can be:
i. Microbiological resistance or in vitro resistance
ii. Clinical resistance or in vivo resistance.[5,20]
The factors responsible for fungal resistance are given in
Table 1.[5,13,20]
Microbiological resistance refers to nonsusceptibility
of a fungus to an antifungal agent by in vitro
susceptibility testing, in which the minimum inhibitory
concentration (MIC) of the drug exceeds the susceptibility
breakpoint for that organism. Microbiological resistance
can be primary (intrinsic), where the fungi are resistant to
a drug before exposure and secondary (acquired), which
develops in response to exposure to an antimicrobial
agent.[13] Certain fungal species are intrinsically resistant
such as Candida krusei to fluconazole and Cryptococcus
neoformans to echinocandins and nonalbicans Candida to
5‑flucytosine (5FC).[13,20]
Secondary resistance develops among previously
susceptible strains after exposure to the antifungal agent
and is usually dependent on altered gene expression,[20]
for example, terbinafine resistance in T. rubrum,
fluconazole resistance among C. albicans.
Clinical resistance is defined as the failure to eradicate a
fungal infection despite the administration of an antifungal
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Pai, et al.: Antifungal resistance
Table 1: Factors for fungal resistance
Fungal factors
Reducing the accumulation of the drug within the fungal cell
Decreasing the affinity of the drug for its target
Modifications of metabolism to counterbalance the drug effect
Cellular response to stress
Biofilm production
Host factors
State of immunosuppression
Site of infection
Severity of infection
Onset of treatment
Drug related
Fungistatic nature
Dosage
Pharmacokinetics
Drug‑drug interactions
Environmental factors
agent with in vitro activity against the organism. Although
clinical resistance cannot always be predicted, it highlights
the importance of individualizing treatment strategies on
the basis of the clinical situation.[20]
Fungal factors, host factors, and drug pharmacology play
a role in fungal resistance in isolation or in association
with other.
The fungal molecular mechanism can result from gene
amplification, gene transfer, gene deletion, point
mutations, loss of cis‑ and trans‑acting regulatory
elements, and transcriptional activation. All these effects
could be on genes directly involved in the combat
against the cytotoxic compounds and/or could be on
genes involved in their regulation or processing.[5]
The genes responsible for ergosterol synthesis is given in
Figure 2.[13]
Fungal Factors
Decreased accumulation of drug within fungi
Reducing the accumulation of the drug within the fungal
cell is done by increasing the drug efflux mechanism.
Multidrug efflux transporters are membrane proteins
found in all living organisms. These proteins bind
to a variety of structurally and chemically dissimilar
compounds and actively extrude them from the cells.[5]
Mutations (upregulation or overexpression) of the genes
encoding these efflux pumps result in decreased
accumulation of the drug in the cell.[21]
Efflux systems affecting the antifungal drugs vary.
Common drug efflux systems that modulate azole
resistance are the ATP‑binding cassette (ABC) superfamily
and the major facilitator superfamily. Overexpression
of TruMDR1 and TruMDR2 genes in T. rubrum, which
Indian Journal of Dermatology | Volume 63 | Issue 5 | September-October 2018
encoded an ABC transporter was seen following azole
and terbinafine therapy.[5]
Decreasing the affinity of the drug for its
target
A mutation or overexpression of the gene coding for
target enzymes is another mechanism developed by
fungi.
A point mutation in the ERG11 gene that codes for
lanosterol 14α‑demethylase leads to the complete inhibition
of the binding capacity of the azole drug to its target.[14]
Mutation in the squalene epoxide (SE) gene (ERG1) leads
to an amino acid substitution in the SE making the fungi
about 1000‑fold less susceptible to terbinafine.[5]
The overexpression of target enzymes is either by gene
amplification or upregulation of the corresponding
gene.[5] In chromosomal anomalies like isochromosome
formation, an increase in the number of azole‑resistant
genes can occur.[21]
Modifications of metabolism to
counterbalance the drug effect
De novo synthesis of pyrimidines
The antifungal drug 5FC competes with the regular
pyrimidine intermediate metabolites for incorporation
into nucleic acids.[14,22] De Novo increase in pyrimidine
synthesis leads to 5FC resistance as seen in Candida
glabrata.[14]
Paradoxical effect
Few yeasts and filamentous fungi are able to grow in
elevated echinocandin concentrations much higher than
the MICs. This phenomenon, called paradoxical effect
or “eagle effect,” is strain dependent and is due to the
upregulation of the chitin synthesis in the fungal cell
wall after drug administration.[14,20]
Modifications of the ergosterol biosynthetic
pathway
The antifungal activity of azole drugs depends on the
depletion of ergosterol from the fungal membrane and
accumulation of the toxic product 14α‑methyl‑3,6‑diol,
leading to growth arrest. Alteration in the late steps of
the ergosterol biosynthetic pathway through inactivation
of the ERG3 gene can lead to the total inactivation
of C5 sterol desaturase and also can give rise to
cross‑resistance to all azole drugs. This mechanism is
identified in C. albicans.[14,20]
Plasma membrane composition variation
A decrease or total absence of ergosterol in the plasma
membrane through mutations in nonessential genes of
the ergosterol is a rare mechanism of resistance among
polyene drugs, for example, ERG3 mutation in clinical
isolates of C. albicans, ERG6 mutation in C. glabrata.[14]
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Pai, et al.: Antifungal resistance
Figure 2: The genes involved in ergosterol synthesis
Biofilms
Biofilms are sessile microbial communities surrounded
by extracellular polymeric substances with increased
resistance to antimicrobial agents and host defenses.
Both T. rubrum and T. mentagrophytes are capable of
producing biofilms.[23]
Biofilms by yeast and certain molds are frequently
polymicrobial and are resistant to almost all the currently
used antifungals, with the exceptions of echinocandins
and lipid formulations of amphotericin B. Biofilm
resistance is probably the result of multiple factors such as
an increased expression of efflux pumps, a modification of
plasma membrane composition, and the drug sequestration
in biofilm‑produced extracellular matrix.[14,21]
Cellular response to stress or
adaptation
Fungi are remarkably adaptive and have numerous
signal‑transduction pathways to sense and ensure
appropriate physiological mechanisms to adapt to
environmental stress following exposure to an antifungal
agent.[5,21] Hsp90, Hsp104, ubiquitin, calcineurin,
esterases, and oxidative‑stress proteins such as
glutathione synthatase are key cellular regulators critical
for orchestrating cellular responses to drug‑induced
stress, e.g., in T. rubrum following azoles, griseofulvin
and amphotericin B.[5]
Stress adaptation may not induce clinical resistance but
stabilizes the cell in the presence of drug and allows it
to develop more profound resistance mechanisms over
time that are manifested as clinical resistance.[21]
364
Clinical Resistance
Clinical resistance depends on a multiple host‑ and
drug‑related factors which are as follows:[13,20]
a. Patients with severe degree of immunosuppression
with invasive fungal infections may not respond to
antifungals
b. Delay in initiation of adequate dose of antifungal
results in increased chances of treatment failure
c. Fluconazole has better cerebrospinal fluid (CSF)
penetration as compared to itraconazole, therefore,
making it a better choice in treating fungal
meningitis. When the site of infection is necrotic
with poor blood supply, a debulking surgery is
essential to overcome antifungal treatment resistance
d. Compliance in patients requiring long‑term therapies.
stress
Drug‑Related Factors
Various factors such as the fungistatic nature of most
drugs, inappropriate antifungal usage (in cases where
the etiological agent is known), treatment with low
antifungal dosage, long duration of treatment, drug
interactions, and the cost of therapy play a role in
fungal resistance.[21] Combination of polyenes and azoles
with other nephrotoxic drugs can result in treatment
failure.[20]
Environmental resistance
In recent years, the role of environment as a driver
for resistance has become prominent. Fungicidal use
in agriculture differs hugely between different regions,
with the United States using about a tenth of as much
as Europe. Azole‑based fungicides are used in grape and
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cereal production in European countries. A strong link
has been found between countries where azole‑based
fungicides are used and the incidence of antifungal
resistance.[21,24]
Antifungal susceptibility testing
Antifungal susceptibility testing (AFST) has undergone
considerable change from a nonstandardized procedure
that generated results lacking clinical utility to a
standardized procedure. Standardized microdilution‑based
procedures by the Clinical and Laboratory Standards
Institute (CLSI) and the European Committee on
Antibiotic Susceptibility Testing are universally accepted
for performing AFST[25] The CLSI, formerly the National
Committee on Clinical Laboratory Standards has released
four standard methods for antifungal susceptibility
testing, namely, M27‑A3 for macrobroth and microtiter
yeast testing, M38‑A2 for microtiter mold testing, M44‑A
for yeast disc diffusion testing, and M51‑P for mold disc
diffusion testing.[26-31]
AFST patterns in dermatophytes show individual and
regional variation in their outcome. Studies on AFST
showed that T. rubrum and T. mentagrophytes, which
were the common species isolated, showed a higher
MIC to fluconazole. Although generally itraconazole,
terbinafine had lower MICs, few studies demonstrated
a higher MIC to itraconazole and terbinafine. Systemic
griseofulvin and topical amorolfine had lower MIC than
fluconazole. A study done by Koga et  al. showed the
lowest MIC to be with luliconazole.[32‑36] Rex and Pfaller
proposed the “90–60 Rule” which states that infections
caused by isolates that have MICs considered susceptible
respond favorably to appropriate therapy approximately
90% of the time, whereas infections caused by isolates
with MICs considered resistant respond favorably
approximately 60% of the time.[37]
Treatment
Clinically, antifungal resistance may be suspected
in patients with recurrent episodes of infection,
unresponsiveness to the first line of therapy, generalized
involvement, or atypical form of presentation with usual
history of similar lesions in the family.
One of the important treatment measures in recurrent
cutaneous fungal infections is prevention of disease
among family members. Therefore, nonpharmacological
measures like good hygiene play an important adjunct
along with medications.[38]
Good skin hygiene measures include handwash; clipping
of nail; regular bathing and complete drying of the skin;
use of nonocclusive shoes, absorbent socks, and powder;
avoidance of sharing of combs, towels, brushes, bedding,
and hats; and avoidance of barefoot walk in public
bathroom.[39,40]
Indian Journal of Dermatology | Volume 63 | Issue 5 | September-October 2018
Pharmacological measures like prudent use of antifungals
in proper dosing and duration form the mainstay of
therapy. In general, topical agents should be applied
once to twice daily over the lesions and up to at least
2 cm beyond the margins of the lesions for 2–4 weeks
and continued for 1 week after the rash resolves.[41]
Topical allylamines maintain the mycological cure for
longer periods compared to azole drugs.[42] With respect
to systemic antifungals, in addition to a proper
dose, duration, and knowledge of pharmacokinetics,
dosage updosing (pulse or boosted oral antifungal)
and combination antifungals can be tried in recurrent
dermatophytoses. Boosted oral antifungal treatment
is designed to target dormant chlamydospores and
arthroconidia to produce sensitive hyphae which are less
refractory to antifungal treatment.[43,44]
Combination Antifungal Therapy
The availability of new antifungal agents with unique
mechanisms of action and improved tolerability has
widened the possibilities for the use of combination
antifungal therapy for difficult‑to‑treat opportunistic
mycoses.[45] Combining two systemic antifungal agents
has been used in invasive mycoses caused by Candida,
Aspergillus and Cryptococcus.[46] In dermatophytoses,
combination antifungal therapy has been used, wherein
topical and systemic antifungal are combined. Gupta
et  al. used sequential therapy with itraconazole and
terbinafine pulse in toenail onychomycosis.[44,47] The
advantages of combination therapy are increased rate and
extent of fungal killing (synergy), enhanced spectrum
of activity, and decreased likelihood of resistance or
tolerance.[45] Azoles can act in a synergic way when
combined with terbinafine providing good results against
Candida, dimorphic molds, dematiaceous fungi and
yeasts, such as C. glabrata. Azoles with amphotericin B
have mixed response.[46,49]
The disadvantages are increased toxicity, increased
drug interactions, increased cost, and poorly
standardized methods of testing efficacy of the
combination.[45] Itraconazole and voriconazole are potent
inhibitors of cytochrome P450 3A4 and associated
with a greater risk of broader range of potentially
severe drug interactions and hepatotoxicity than
fluconazole.[48] At present, checkerboard dilution assay
is the most common laboratory method for evaluating
antifungal combinations.[45]
Antifungal Drugs Plus Nonantifungal Drugs
Combination of antifungal drugs with nonantimicrobial
agents such as calcineurin inhibitors (cyclosporine A
and tacrolimus), proton‑pump inhibitors, antiarrhythmic
agents, cholesterol‑lowering agents, immunomodulators,
antineoplastic drugs, antiparasitic agents, microbial
metabolites, and human recombinant antibodies has
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Pai, et al.: Antifungal resistance
shown beneficial effects.[46,50] Cyclosporine singularly
is not able to inhibit the fungal growth but increases
susceptibility to fluconazole due to efflux pump deletion
or alteration of stress response caused by calcineurin
during azole therapy.[51]
Studies show that statins are active against Microsporum
canis and T. mentagrophytes. Synergistic interactions
were noticed when simvastatin was combined with
terbinafine against dermatophytes.[52]
Hemopoietic growth factors, such as granulocyte colony
stimulating factor or granulocyte‑macrophage colony
stimulating factor and Th1 cytokines have activity on
the antifungal function of phagocytes and the efficacy
of antifungal agents.[46,53,54]
Fractional carbon‑dioxide laser therapy combined with
topical antifungals such as terbinafine, amolorfine, or
luliconazole was found to be effective in the treatment
of onychomycosis.[55]
Newer Antifungal Drugs
An efficient antifungal should act against a wide range
of fungi having no or low toxicity to the host.[5] One of
the major challenges in developing antifungal drugs lies
in the similarities shared between fungi and their hosts,
thereby restricting the target molecules for antifungal
drugs. Most newer antifungal drugs are designed
considering the following:
i. Drugs having pharmacological similarity with the older
drugs which share the target molecules, with lower
MIC level and specific indications (e.g., isavuconazole,
micafungin, luliconazole)[56,57]
ii. Repurposing of established medications where an old
compound with a known pharmacology is used alone
or in combination with another drug for a newer
indication, for example, calcineurin inhibitors, target
of rapamycin inhibitors, Hsp90 inhibitors, in synergy
with azoles[20,50,58,59]
iii. Recent advances in fungal genomics and proteomics
have revealed target genes, proteins, or virulence
factors required during infection of the host
tissues by dermatophytes, for example, efflux pump
inhibitors (derived compounds of milbemycin), the
transcription factor PacC, a wide‑domain regulatory
protein involved in pathogenicity events or the sulfite
transporters were proposed as interesting targets for
antifungal drugs in dermatology because of their role
in the proteolytic digestion of hard keratin).[5,20,60‑62]
Conclusion
While deeper mycoses are still difficult to treat, common
fungal infections in dermatology were considered easily
treatable. Lately, fungi causing superficial mycoses also
have evolved to develop resistance against commonly
used drugs. Current solutions to this could be good skin
366
hygiene measures, prudent use of antifungals in proper
dosing and duration, appropriate use of susceptibility
testing, usage of older molecules such as griseofulvin or
topical keratolytics in combination with newer drugs and
in appropriate dosages and combination therapy with
two systemic antifungals or systemic antifungal with
topical antifungals and/or topical keratolytics.
The key to managing the widespread nature of this
emerging problem would involve development of drugs
with newer targets, decreased drug interactions, at
affordable price, which at this stage appears distant in
our scenario, knowing the resources required and the
economics involved. However, newer insight on drug
resistance mechanisms could lead to advanced treatment
strategies in managing fungal infections.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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