Fungal Genetics and Biology 132 (2019) 103255

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Fungal Genetics and Biology

journal homepage: www.elsevier.com/locate/yfgbi

Review

Antifungal resistance in dermatophytes: Recent trends and therapeutic

implications
⁎
Ananta Khuranaa, , Kabir Sardanaa, Anuradha Chowdharyb
a
Department of Dermatology, Postgraduate Institute of Medical Education and Research, Dr Ram Manohar Lohia Hospital, New Delhi 110001, India
b
Department of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi, New Delhi 110007, India

A RT I CLE INFO AB S T RA CT

Keywords: Dermatophytoses or tinea refers to superficial fungal infection of keratinized tissues. Although generally con-
Tinea sidered easy to treat, recalcitrant infections, presenting as extensive and difficult to treat tinea corporis and
Dermatophyte cruris, are on the rise in some parts of the world. The situation demands an understanding of the pharmaco-
Resistance kinetic and pharmacodynamic properties of the available antifungals against dermatophytes and the possible
Treatment contribution of drug resistance and other factors to the present scenario. In this review, we provide the readers a
Recalcitrant comprehensive account of the available literature on in-vitro and in-vivo resistance to clinically used antifungals
Corporis
among dermatophytes. We have also added, in brief, the relevant skin pharmacokinetics of important systemic
Cruris
drugs. The established and postulated mechanisms of drug resistance are discussed and aspects on lack of in vivo
correlation of in vitro resistance are presented. Finally, the lacunae in our existing knowledge on the topic and
the arenas for future research are highlighted.

1. Introduction treatment of recalcitrant dermatophytoses.

Dermatophytes are a group of primarily pathogenic fungi re-
sponsible for the commonest fungal infection of humans namely der-
matophytoses or tinea. The dermatophytes (Onygenales,
Arthrodermataceae) are a group of closely related filamentous fungi
mainly infecting the keratinized tissues such as skin, hair and nails.
Trichophyton rubrum is the commonest causative species, although in-
cidence of infections caused by T. interdigitale and T. mentagrophytes is
increasing in some parts of the world (Singh et al., 2018; Rudramurthy
et al., 2018; Dabas et al., 2017; Pathania et al., 2018).
The emergence of recalcitrant dermatophytoses, mainly presenting
as tinea corporis and cruris, over the past few years has become a cause
for concern worldwide. Consequently, this emergence has led to re-
newed research interest in dermatophytoses, the commonest fungal
infection of humans (Bishnoi et al., 2018; Pai et al., 2018). In the fol-
lowing sections, we discuss the important drug classes available for
treatment of dermatophytoses and review the existing literature on
antifungal resistance mechanisms specific to each. We further discuss
the clinical utility of individual drugs in the wake of existing data on in-
vitro and in-vivo susceptibility of each. We have primarily focused on
clinically useful systemic antifungals as these form the cornerstone of
2. Antifungals used in dermatophytes and issues of clinical
unresponsiveness
Various systemic antifungals have been used for dermatophytoses
with the initial use of griseofulvin (GRI) introduced in 1950s trans-
cending to ketoconazole (KTZ) introduced in 1980s, and fluconazole
(FLU), terbinafine (TRB) and itraconazole (ITR) which came into use
over the next decade (Sheehan et al., 1999) (Table 1). From the 90s
onwards, the last two have been the mainstay of treatment for tinea
except for tinea capitis, where GRI still remains useful. The treatment of
tinea with systemic antifungals requires that the drug effectively
reaches the most superficial dead layer of the skin i.e., stratum corneum
(SC), to effect its action. Apart from the in vitro efficacy, effective pe-
netration of the systemically administered drug into the SC and its
persistence there (a function of its affinity for keratin or keratin ad-
herence) is an important factor for achieving cure. KTZ, ITR and TRB
have high keratin adherence, while it is low for GRI and FLU
(Cauwenbergh et al., 1988; Sardana et al., 2017) which accounts for the
former three being preferred over the latter two.
In the last decade several new antifungals have been introduced and

Abbreviations: GRI, griseofulvin; KTZ, ketoconazole; FLU, fluconazole; TRB, terbinafine; ITR, itraconazole; MIC, minimum Inhibitory concentration; PK, pharma-
cokinetic; PD, pharmacodynamics; ABC, ATP Binding Cassette; SQLE, squalene epoxidase; once a day, OD; twice a day, BD
⁎
Corresponding author.
E-mail address: drananta2014@gmail.com (A. Khurana).

https://doi.org/10.1016/j.fgb.2019.103255
Received 28 May 2019; Received in revised form 17 July 2019; Accepted 17 July 2019
Available online 19 July 2019
1087-1845/ © 2019 Elsevier Inc. All rights reserved.
A. Khurana, et al. Fungal Genetics and Biology 132 (2019) 103255

Table 1
Antifungals used in dermatophytoses.

Class Drugs Mechanism of action

Azoles Systemic: Fluconazole, Ketoconazole, Itraconazole, Voriconazole, Posaconazole, Inhibition of Lanosterol 14α demethylase
isavuconazole
Topical:Clotimazole, Miconazole, Econazole, Luliconazole, Lanoconazole, Efinaconazole,
Ketoconazole, Sertaconazole, Oxiconazole, Eberconazole, Fenticonazole, Bifonazole
Allylamines Systemic: Terbinafine Inhibition of squalene epoxidase
Topical: Terbinafine, Butenafine, Naftifine
Heterocyclic benzofuran Griseofulvin Inhibition of microtubule aggregation
Polyenes Nystatin, Natamycin, Amphotericin B (topical) Disorganization of the cell membrane by formation of
pores
Morpholine Amorolfine (topical) Inhibition of C-14 reductase and C8 isomerase
Thiocarbamate Tolnaftate (topical) Inhibition of squalene epoxidase
Hydroxypyridones Ciclopirox (topical) Chelation of trivalent metal cations
Inhibition of metal dependent enzymes – catalase,
peroxidase
Inhibition of enzymes involved in mitochondrial electron
transport processes and energy production
Echinocandins Anidulafungin, Caspofungin, Micafungin Glucan synthase inhibition
Others Tavabarole (topical) Cytoplasmic leucyl tRNA synthetase - inhibition of
protein synthesis and termination of cell growth
ME 111 Succinate dehydrogenase inhibitor

some others are under investigation. Efinaconazole and tavabarole are behind that on systemic mycoses. And, in contrast to MIC data which is
approved for use in U.S.A, Europe and many other countries for ony- available from across the globe, data on clinical correlation of anti-
chomycosis and provide modest cure rates (Sahni et al., 2018). Luli- fungal susceptibility testing (AFST) in dermatophytoses is limited
conazole, a topical azole has high in vitro and in vivo activity and the (Khurana et al., 2018; Khurana and Sardana, 2018; Artis et al., 1981;
advantage of once daily application (Sahni et al., 2018). Third gen- Dogra et al., 2019) thus impeding clinical utilization of the in vitro MIC
eration azoles have also shown low MICs but clinical use has been data. It is believed that there is a relationship between in vitro resistance
sparsely reported and is mainly limited in settings of severe infections and clinical failure, but probably not between in vitro susceptibility and
associated with underlying immunodeficiency (Deng et al., 2015; therapeutic success (Espinel-Ingroff, 2000). This is because the in vivo
Jachiet et al., 2015). Echinocandins have also shown in vitro activity response depends on a multitude of factors apart from drug resistance,
against dermatophytes but reports on clinical use are lacking (Bao et al., as mentioned in Table 2. Importantly, the drug works in concert with
2013). There are some in vitro reports on antifungal activity of rapa- the host immunity and any aberrations thereof may lead to a sub op-
mycin and analogs against yeasts but none of activity against derma- timal response. Further, unfavorable PK profile is another important
tophytes as yet (Cruz et al., 2001). determinant especially factors related to the drug’s bioavailability and
ability to reach the site of infection within a specific tissue.
“Resistance” and “recalcitrance” are terms commonly used in con-
text of lack of a clinical response in dermatophytoses. While use of the
term “resistance” is best restricted to the lack of in-vitro inhibition, re- 2.1. Mechanisms of action of antifungals used against dermatophytes
calcitrance is a broader term which is more clinically relevant as it
encompasses both in vitro resistance and all other possible causes of Antifungals used in dermatophytoses largely act on different steps of
therapeutic failure. A few important factors implicated and recognized ergosterol synthesis pathway (Fig. 1). The pathway begins with squa-
in previous reviews are listed in Table 2 (Jones et al., 1990; Sardana lene, which is in turn produced from acetate through acetyl coenzyme
and Khurana, 2018). A, hydroxymethyl glutaryl coenzyme A, and mevalonate (White et al.,
The research on antifungal resistance in dermatophytoses lags 1998). Ergosterol is an essential component of the fungal plasma
membranes and forms the bulk of it. It is not present in mammalian
cells which instead produce cholesterol, thus making this a safe target
Table 2
for drug intervention. Ergosterol also has an essential regulatory or
Factors responsible for recalcitrance in dermatophytoses (Jones et al., 1990;
Sardana and Khurana, 2018 ). “sparking” function where small amounts are essential to “spark” cell
proliferation and for the cells to progress through the cell cycle (White
Factor Implications/scenarios
et al., 1998). This sparking function is independent of the “bulk” or
Fungal factors Host adaptability, structural function, since certain sterols can replace the bulk sterol of
Virulence factors, the membrane without supplying the sparking function for the cell
Probable variation in drug susceptibility cycle (Hitchcock, 1993). The phenomenon by which ergosterol per-
Host factors Immunity related forms these dual roles has been termed “sterol synergism” (Ramgopal
• Iatrogenic and disease related immune defects,
• Impaired local immunity by topical steroid misuse
and Bloch, 1983).
• Predominance of Th2 immune response
Compliance with prolonged treatment
3. Azoles
Drug factors Mechanism of action
MFC/MIC ratio
Suboptimal absorption, Azoles form the largest group of antifungals. Three generations of
Quality concerns,
azoles are currently in clinical use for dermatophytoses. The first gen-
Levels achieved in skin, keratin adherence, eration azoles contain an imidazole in their ring system and are mainly
Resistance used topically (barring KTZ) as they have poor oral bioavailability and
Clinical presentation Onychomycosis (possible role of biofilms)
high toxicity on systemic administration (Mast et al., 2013). The second
Majocchi’s granuloma,
Tinea imbricata and third generation azoles have the triazole ring instead of the imi-
Involvement of palms/soles dazole structure (Mast et al., 2013). The “triazoles” display a broader

2
Fig. 1. Steps of ergosterol synthesis in fungal cell membranes, enzymes involved and genes encoding each enzyme. Also mentioned are the sites of action of
antifungals used against dermatophytes.
spectrum of activity than the imidazoles and have a better safety pro-
file, improved oral bioavailability and pharmacokinetic (PK)/pharma-
codynamics (PD) properties as compared with the imidazoles
(Girmenia, 2009). The second generation azoles include ITR and FLU,
while the third generation include posaconazole, voriconazole, and
isavuconazole (Mast et al., 2013).
The azoles act on ergosterol biosynthesis at the C-14 demethylation
stage, a three step, oxidative reaction catalyzed by the cytochrome P-
450 enzyme – 14α-lanosterol demethylase (P-450DM) (VandenBossche
et al., 1993). The interaction occurs between the nitrogen atom of
azoles with the heme iron of P-450DM. The resultant blockage of the
pathway and accumulation of 14-methylated sterols interferes with the
“bulk” function of ergosterol making the plasma membrane vulnerable
to further damage and altering activity of membrane bound enzymes,
importantly those involved in nutrient transport and chitin synthesis
(Georgopapadakou and Walsh, 1996). Severe ergosterol inhibition
(> 99%) additionally interferes with the sparking functions, affecting
cell growth and proliferation (Barrett-Bee et al., 1991; Nes et al., 1993).
The accumulated sterol intermediates may be diverted to an “alternate”
pathway, resulting in formation of a sterol metabolite –dienol that is
further fungistatic to the cell (Bhattacharya et al., 2018). In addition,
triazoles also affect the reduction of obtusifolione to obtusifoliol, re-
sulting in the accumulation of methylated sterol precursors (Ghannoum
et al., 1994).
Three systemic azoles have been used for treating dermatophytoses
namely KTZ, ITR and FLU. KTZ demonstrated good clinical efficacy and
overcame the problems of long treatment durations, frequent failures,
unfavourable skin PK profile and poor oral bioavailability associated
with the use of GRI, the only other systemic antifungal which was in use
for dermatophytoses at the time (Epstein et al., 1972; Robertson et al.,
1982). It offered the advantages of high keratin adherence and persis-
tent therapeutic levels in the SC till 10-days post treatment cessation
(Haneke, 1987; Jones, 1990). To the best of our knowledge, no clinical
case of resistance to KTZ has been reported till date, although there are
isolated reports of high in vitro MICs (Goh et al., 1994; Gupta and Kohli,
2003a, 2003b; Manzano-Gayosso et al., 2008; Singh et al., 2018).
However, the drug has been withdrawn in some countries, and strict
restrictions and caution advised in some others, in view of its hepato-
toxic adverse effects, although arguments have been raised against this
(Gupta and Lyons, 2015). The drug continues to be used as an effective
topical agent for superficial mycoses (Gupta and Lyons, 2015) and is
occasionally used by clinicians as a reserve drug for recalcitrant der-
matophytoses in a dose of 200–400 mg/day (Robertson et al., 1982).
Fluconazole has good oral bioavailability, accumulates rapidly in SC
and achieves high levels (Wildfeuer et al., 1994). But following treat-
ment discontinuation, there may be a re-diffusion back into the circu-
lation, suggesting low binding avidity. The elimination from SC occurs
with a half life of the order of 60–90 h (slower than elimination from
plasma) (Wildfeuer et al., 1994). Though not specifically indicated for
dermatophyte infection, FLU was documented to be useful in derma-
tophytoses, particularly tinea capitis (Gupta and Cooper, 2008). FLU
was initially used in a dose of 50 mg/day but later in view of the skin PK
characteristics, a weekly dose of 150 mg was tried in trials and reported
to be effective (Stary and Sarnow, 1998; Nozickova, 1998; Suchil et al.,
1992). However, with the SC elimination half life of 60–90 h, the drug
may not persist beyond the inhibitory concentrations for adequate
length of time after a weekly dose. There are reports of clinical non-
responsiveness (Balci and Cetin, 2008) to weekly FLU and several re-
ports of high in vitro MICs (Carrillo-Muñoz et al., 2003; Ghannoum
et al., 2006; Goh et al., 1994; Gupta and Kohli, 2003a, 2003b; Khurana
et al., 2018; Rudramurthy et al., 2018). A striking 48% of isolates were
resistant to FLU in a recent study from India (Singh et al., 2018). It is
not considered a frontline drug for dermatophytoses anymore.
Itraconazole has been effectively used for dermatophytoses over
past three decades. The drug has a favorable skin PK profile, although
oral bioavailability is poor and shows high inter-individual variations
(Allegra et al., 2017). ITR rapidly reaches SC mainly via sebum and
achieves much higher levels in SC than plasma (Cauwenbergh et al.,
Table 3
Standard and modified treatment regimens for tinea corporis/cruris.

Drug Standard recommendations Modifications

Terbinafine + #
250 mg OD (2–3 weeks) 250 mg BD
# γ
Longer durations
&
Itraconazole Short fixed duration regimens : Higher doses
γ

# γ
100 mg OD (15 days) Longer durations
200 mg/day (7 days)
$
Fluconazole 150 mg/week (2–6 weeks)* 50–100 mg OD
Ketoconazole Use restricted in many countries owing to hepatotoxic adverse 200-400 mg/day for recalcitrant infections, with careful monitoring of liver
Λ
effects function tests
+ $
Combination topicals Topical antifungals-azoles and allylamines, for limited disease Salicylic acid – to improve desquamation
Increasing use of ciclopirox and amorolfine as topical antifungals

+
Hay and Ashbee (2016).
&
Parent et al. (1994) and Boonk et al. (1998).
#
Khurana et al. (2018) and Sardana and Gupta (2017).
$
Sardana and Khurana (2018).
* Suchil et al. (1992) and Stary and Sarnow (1998).
γ
Rajagopalan et al. (2018).
Λ
Robertson et al. (1982).
1988). Owing to high keratin adherence, the levels are sustained for
upto 3–4 weeks after treatment discontinuation, depending on the body
site (Cauwenbergh et al., 1988). No definite clinical case of ITR re-
sistance has been reported in dermatophytoses as yet although occa-
sional high MICs have been on record, mainly with T. interdigitale
(Singh et al., 2018). Ususal doses used are mentioned in Table 3. Owing
to the complex manufacturing process of ITR pellet containing capsules,
inferior drug quality may become a reason for poor therapeutic re-
sponse especially in the developing world where several in house pre-
parations are available (Sardana et al., 2018a, 2018b, 2019; Pierard-
Franchimont et al., 1995).
3.1. Mechanisms of resistance to azoles
The mechanisms of azoles resistance in dermatophytes are yet to be
elucidated. However, experimental studies indicate some possible me-
chanisms that are detailed below and depicted in Fig. 2.
3.1.1. Drug efflux
Energy dependent efflux by membrane bound transporters is the
main mechanism of high level azole resistance in pathogenic fungi
(Coleman, 2009). The ATP Binding Cassette (ABC) transporter super-
family is of prime importance here. ABC transporters are primary efflux
transporters which hydrolyze ATP for export of substrates and are re-
sponsible for removal or sequestration of unwanted (toxic) molecules
from cells. These proteins are evolutionarily conserved from
prokaryotes to humans and contain two distinct regions: highly con-
served nucleotide-binding domains (NBD) and variable transmembrane
domains (TMD) (Martinez-Rossi et al., 2018; Wilkens, 2015). Both
importing and exporting ABC transporters are found in bacteria,
whereas the majority of eukaryotic family members function in the
direction of export (Wilkens, 2015). There are 5 families of ABC
transporters namely ABC A, ABC B, ABC C, ABC D and ABC G. Out of
these, three families - ABCB or multi-drug resistance (MDR), ABCC or
multidrug resistance–associated protein (MRP) and ABCG the pleio-
tropic drug resistance (PDR) families have been most extensively stu-
died. Overexpression of ABC transporters occurs in presence of drugs
and these have the potential to cause multi drug resistance (Martinez-
Rossi et al., 2018). PDRs form the largest group among these and confer
resistance to triazoles in Candida albicans (CDR1 and CDR2), C. glabrata
(CgCDR1, CgCDR2 and SNQ2), C. krusei (ABC1) and Cryptococcus neo-
formans (AFR1) among others (Posteraro et al., 2003; Sanglard et al.,
1999; Prasad et al., 1995; Katiyar and Edlind, 2001; Sanglard et al.,
1999; Miyazaki et al., 1998).
Possibility of a multidrug resistance (MDR)-type mechanism was
suggested for dermatophytes with report of clinical T. rubrum isolates
demonstrating resistance to both GRI and tioconazole (Fachin et al.,
1996). Existence of ABC transporters in dermatophytes was demon-
strated in experimental studies much later. Maranhão et al. (2009)
demonstrated sequences similar to genes encoding the multidrug-re-
sistance ABC transporter, copper ATPase, the major facilitator super-
family and a permease in T. rubrum and demonstrated upregulation of
the encoding genes in presence of keratin. A sequence identical to the
TruMDR2 gene, encoding an ABC transporter in T. rubrum, was de-
tected. Further, a DeltaTruMDR2 mutated T. rubrum showed reduced
growth on human nail, suggesting involvement of the transporters in T.
rubrum pathogenecity. Fachin et al (2006) also cloned and sequenced a
single copy gene TruMDR2 from T. rubrum. This showed high homology
with ABC transporters involved in drug efflux in other fungi. Interest-
ingly, there occurred an increased level of transcription of TruMDR2
gene when mycelia were exposed to antifungals acriflvine, KTZ, GRI,
FLU, ITR and tioconazole. TruMDR1 gene, also encoding an ABC
transporter with high homology with ABC transporters involved in drug
efflux in other fungi, was also subsequently isolated from T. rubrum and
a similar increase in expression with exposure to azoles and GRI was
demonstrated (Cervelatti et al., 2006). However, unlike the observa-
tions with TruMDR2, Cervelatti et al (2006) found no basal expression
of TruMDR1 gene in untreated T. rubrum, suggesting that its expression
is only induced by metabolic poisons present in the environment.

Fig. 2. Possible mechanisms of azole resistance in dermatophytes.

pression of the gene products and mutations in Erg11 are reported
3.1.2. Drug target modification mechanisms of azole resistance in yeasts, but these have not been de-
14α-lanosterol demethylase is encoded by Erg11 gene. Over-ex- scribed in dermatophytes as yet (Feng et al., 2017; Xiang et al., 2013).
3.1.3. Stress response
Dermatophytes have been demonstrated to secrete many proteins in
response to environmental and drug exposure stress as an adaptive
mechanism (Peres et al., 2010; Paiãoet al., 2007). Some of these e.g.
heat shock proteins hsp70, hsp90, and PacC are important virulence
factors for dermatophytes (Martinez-Rossi et al., 2017). The relevance
of these to drug resistance has not been clearly described, but it is be-
lieved that stress adaptation stabilizes the cell in the presence of drug
and allows it to develop more profound resistance mechanisms over
time (Perlin et al., 2015). Further, the stress caused by antifungal and
cytotoxic drugs in sub-inhibitory concentrations promotes compensa-
tory stress responses, and likely leads to over-expression of genes in-
volved in cellular detoxification, drug efflux, and signaling pathways
and thus may contribute to drug tolerance (Martinez-Rossi et al., 2018).
Hsp inhibition is a potential target for future research on antifungal
drugs (Martinez-Rossi et al., 2016).
Thus, our understanding of azole resistance in dermatophytes is
limited at present. No particular mechanism of resistance has been Fig. 3. Mechanisms (proven and proposed) of resistance to terbinafine and
thoroughly investigated and proven to be relevant to clinical scenarios. other allylamines.
Further, if there are common mechanisms at play as suggested, the
difference in clinical utility of different azoles for dermatophytoses
substitution in squalene epoxidase (SE) gene (L393F) in a later report
needs further studies.
(Osborne et al., 2005). Interestingly, sequential isolates from the pa-
tient, while on treatment, demonstrated no further increase in MIC
4. Terbinafine and other allylamines
using the microdilution method suggesting a primarily resistant strain
and no tendency of resistance potentiation with continued use
Allylamines work by inhibition of the enzyme squalene epoxidase
(Mukherjee et al., 2003). This observation is further supported by the
(SQLE) in a non-competitive manner, blocking synthesis of 2, 3 oxido-
report on 30 patients of onychomycosis, who remained culture positive
squalene and thus leading to accumulation of squalene and depletion of
despite treatment with TRB for upto 24 weeks (Bradley et al., 1999). All
ergosterol. The affinity of dermatophyte SQLE for TRB is much higher
serial isolates obtained from these patients showed low MICs to TRB
than that of the yeast enzyme or mammalian SQLE (Balfour and Faulds,
and no increase in MICs with continued exposure. The authors con-
1992). In addition, there occurs a possible accumulation of TRB in T.
cluded that drug resistance is unrelated to lack of clinical response in
rubrum, further adding to the potency of the drug (Favre and Ryder,
onychomycosis and other host and organism related factors be con-
1996). A fungicidal effect is achieved in vitro at TRB concentrations
sidered. Subsequently, Osborne et al. (2006) described a new clinical
which do not completely prevent ergosterol biosynthesis suggesting
strain of TRB resistant T. rubrum with an MIC of 64 µg/ml that harbored
that cell death in susceptible organisms may be a consequence of
squalene accumulation rather than ergosterol deficiency (Balfour and a single amino acid substitution (F397L) in the SQLE protein. Later,
Faulds, 1992). The precise effects of intracellular squalene are un- contrary to previous reports, an increase in resistance frequency on
known, but it appears likely that high concentrations cause disruption serial subcultures with sub inhibitory TRB concentrations was demon-
of fungal cell membranes (Ryder, 1990). TRB rapidly reaches SC mainly strated, with mutants having 500–1000 fold increase in MICs to TRB
via secretion into sebum and maintains SC levels beyond treatment (Ghelardi et al., 2014).
cessation, in view of the high keratin adherence (Faergemann et al., Since 2017, emergence of TRB resistance has been reported from
1991, 1993). Its favorable PK profile, good tolerance and lack of drug different geographical locations. Two clinical cases of TRB resistant T.
interactions made it the preferred drug for dermatophytoses since its rubrum infection were recently reported from Denmark in a child with
introduction. congenital icthyosis (Schøsler et al., 2018) and in an adult with Darier’s
disease (Digby et al., 2017). The MICs of these strains were reported as
4.1. Resistance to TRB and other allylamines 4 µg/mL and > 4 µg/mL respectively, but further analysis for mutations
in the SQLE gene was not done. Subsequently, Yamada et al. (2017)
Resistance to TRB in dermatophytes was rarely described until re- reported SQLE gene mutations (leading to aminoacid substitutions at
cently. The various possible mechanisms of terbinafine resistance are Leu393, Phe397, Phe415 and His440positions in the SQLE protein) in
depicted in Fig. 3 and discussed briefly. In early 2000s in-vitro studies
17 isolates including 16 T. rubrum and a single T. interdigitale collected
demonstrated that the frequency of naturally occurring mutants with over a 3-year period from tinea pedis and unguium cases. The MICs
resistance to TRB and of development of resistance during prolonged were reported for 12 of the 17 isolates and ranged between 0.1 to >
−9
exposure to the drug both remain very low (~10 ), compatible with 12.8 µg/ml. Eight of the 17 patients had already been treated with TRB
the reported mechanism of single non-silent nucleotide substitution in at the time of collection of samples, while the treatment data of the
gene encoding SQLE protein (Osborne et al., 2003; Hofbauer et al., other 9 was not available. Rudramurthy et al. (2018) from India re-
2002). Indeed, prior to 2017, there had been only 2 documented cases ported Phe397Leu substitution in SQLE of four T. interdigitale and two
of TRB resistance (Osborne et al., 2006; Mukherjee et al., 2003). T. rubrum isolates from clinical cases of dermatophytoses. TRB MICs
Mukherjee et al. (2003) first described high MICs in 6 T. rubrum isolates were ≥2 µg/ml in 20 of the 127 isolates tested. So far the largest re-
from a single patient of onychomycosis who failed TRB treatment given ported series of TRB resistance in India (Singh et al., 2018) described 20
for 24-weeks. The cause was documented as a single amino acid TRB resistant T. interdigitale isolates, all harboring SQLE mutations,
isolated from tinea corporis and cruris patients from 3 centers in Delhi.
TRB MICs of ≥2 µg/ml were obtained in 32% of the total isolates
tested. A recent study from the same centers (Khurana et al., 2018)
reported clinical correlation of MIC and SQLE mutation data of 30
patients with tinea corporis/cruris. The patients were started treatment
with the standard dose of TRB 250 mg/day, which was increased to
250 mg twice a day if no significant clinical response was obtained after
3 weeks. The response to the higher dose was adjudged after a further
3 weeks period and if still inadequate, the patients were shifted to ITR.
The authors reported a significant difference in the mean MICs of the
three treatment groups and concluded that a TRB strain with MIC <
1 µg/ml is 2.5 times more likely to respond to TRB than one with a
higher MIC. Interestingly, 5 of the 13 patients infected with SQLE
mutation harboring strains responded to prolonged duration/higher
dosage of TRB.
The reported mutations affect the drug binding site of SQLE and
thus may lead to failure of drug enzyme interaction. The isolates re-
sistant to TRB also show cross resistance to other allylamines
(Rudramurthy et al., 2018; Mukherjee et al., 2003). It is a notable
finding that in studies from India, the mutations were present only in
isolates with MICs above 2 µg/ml (Rudramurthy et al., 2018) and 4 µg/
ml (Singh et al., 2018; Khurana et al., 2018), whereas in the isolates
from Switzerland, reported by Yamada et al. (2017), the MICs of mu-
tated isolates varied from 0.1 µg/ml to > 12.8 µg/ml. It is possible that
other mechanisms may also be operating to confer resistance to TRB.
The finding of varying MICs of strains bearing identical substitutions in
amino acid sequence of SQLE further support this possibility. The ob-
servation that disruption of Tru MDR2 gene renders mutants more
sensitive to TRB than control strains (Fachin et al., 2006), suggests that
the alternate mechanism may involve an efflux pump. A terbinafine
resistant isolate grown in presence of 0.14 µg/ml TRB expressed higher
levels of PDR1, MDR1, MDR2 and MDR4 than the TRB susceptible
strains (Kano et al., 2018). On addition of efflux blocker FK-506 to the
media, the MIC of TRB reduced from > 32 µg/ml to 4 µg/ml, demon-
strating an additive effect of the combination and lending further
support to the possible role of efflux pumps in mediating TRB resistance
(Kano et al., 2018). Another possible mechanism may be over expres-
sion of salA gene, encoding salicylate 1-monooxygenase (Santos et al.,
2018). A similar mechanism has also been demonstrated in Aspergillus
nidulans (Graminha et al., 2004). The authors hypothesized that the
protein causes cleavage of the naphthalene nucleus of TRB causing its
degradation and hence resistance.
The experimental evidence of a very low rate of spontaneous and
induced mutations with TRB exposure seems contradictory to the pat-
tern being observed now (Osborne et al., 2003; Bradley, OM). A cidal
drug does not leave many viable fungal elements and further a single
non silent nucleotide substitution is a rare event in itself (Ghelardi
et al., 2014; Osborne et al., 2003). But, the high proportion of resistance
to TRB reported in recent studies (Khurana et al., 2018; Singh et al.,
2018; Rudramurthy et al., 2018) does not corroborate with these. A
recent study on whole genome sequencing of Trichophyton isolates
causing the outbreak of dermatophytoses in India, has demonstrated
that TRB resistant clonal isolates are being transmitted between pa-
tients thus expanding the clonal population (Singh et al., 2019). Omi-
nously, TRB resistance is being increasingly reported from other parts of
the world as well (Digby et al., 2017; Schøsler et al., 2018; Salehi et al.,
2018). Unsupervised topical use of TRB producing sub inhibitory con-
centrations at the site may be one possible factor leading to the chan-
ging scenario, especially in nations where drug regulations are not
strictly enforced. Another more likely factor may be use of TRB for
onychomycosis, an indication for which it has been the worldwide drug
of choice for long. The levels achieved in nail are much lower than
those in SC (Faergemann et al., 1993) and the minimum fungicidal
concentrations (MFC) in experimental models using nail as a substrate
have also been demonstrated to be higher than MFCs determined by
conventional in vitro methods (Osborne et al., 2004). TRB nail-MFC was
4 µg/ml after 1 week exposure to the drug, decreasing to 1 µg/ml after
4-weeks exposure, much higher than MFCs ≤ 0.03 µg/ml determined
in standard MIC assays. The maximum concentration achieved in
nail after 4 weeks of TRB is 0.33 µg/ml (Faergemann et al., 1993).
Further, dense white fungal masses, termed “dermatophytomas” are
often ob-
served on infected nails and are believed to represent biofilm formation
(Burkhart et al., 2002). The physical and metabolic properties of bio-
films protect them from action of antimicrobials and are considered a
factor in promoting drug resistance (Martinez-Rossi et al., 2018). Also,
the ability of Trichophyton spp to form biofilms has been demonstrated
in an experimental model (Costa-Orlandi et al., 2014). Thus nail sites
probably provide the optimum conditions of subinhibitory concentra-
tions for potentiating resistance to TRB.
5. Griseofulvin
GRI was the first systemic antifungal introduced for dermatophy-
toses. Its levels build up rapidly in SC, but also fall rapidly on treatment
discontinuation, with no special binding forces existing to hold GRI in
SC (Epstein et al., 1972). Further, GRI is potentially “washed out” of SC
by the effect of sweating. GRI required prolonged durations of weeks or
months to treat dermatophytoses, likely the result of its mechanism of
action of inhibiting microtubule aggregation requiring a long time to
render dermatophytes in-viable due to their slow growth characteristics
(Epstein et al., 1972). The use of GRI has largely been superseded by
TRB and ITR worldwide, except in tinea capitis (Gupta and Cooper,
2008). Data across different countries documents GRI-resistant isolates
of dermatophytes (Yenişehirli et al., 2013; Chadeganipour et al., 2004;
Ghannoum et al., 2004; Mistik et al., 2006). In a recent study from
India, GRI showed low activity (modal MIC: 4 μg/mL) and 98% isolates
showed MICs ≥ 2 μg/mL (Singh et al., 2018). In another study from
India, GRI was the “most inactive” drug (in vitro) with modal MIC of
32 μg/ml (Rudramurthy et al., 2018). The predominant species in both
these studies was T. interdigitale. Some researchers have reported lower
in vitro activity of GRI for T. mentagrophytes than for T. rubrum (Korting
and Rosenkranz, 1990; Chadeganipour et al., 2004). The low efficacy of
GRI for tinea corporis/cruris was recognized in the early 1980s itself.
Artis et al (1981) compared clinical outcomes in 43 patients prescribed
GRI (250 mg twice daily), with some being on the drug for years.
Thirteen out of 16 patients with tinea corporis failed treatment at this
dose. On comparison with the respective MIC values, the authors in-
ferred that a MIC of ≥3.0 µg/mL or greater indicates relative GRI re-
sistance.
No particular mechanism of action has been described for GRI.
However, it was demonstrated that on challenge with GRI, four
Trichophyton spp, with disrupted MDR2 gene, accumulated high levels
of MDR4 transcripts (Martins et al., 2016). An increased expression of
Tru MDR1 on GRI exposure has also been described (Cervelatti et al.,
2006). Thus as with azoles efflux pumps may be a factor for resistance
to GRI.
6. Resistance potentiation and cross resistance within antifungals
TRB resistant mutants show cross resistance to other SQLE in-
hibitors, as expected from the resistance mechanism involving SQLE
gene mutations (Osborne et al., 2003; Rudramurthy et al., 2018). It is
important to note here that AMF also has a week inhibitory effect on
squalene epoxidase apart from effects on C-14 reductase and C8 iso-
merase (Favre and Ryder, 1996). In a pioneering work done by Ghelardi
et al. (2014) the authors demonstrated an increase in MICs of ITR,
amorolfine (AMF) and TRB on serial sub-culturing with subinhibitory
drug concentrations. The resistance developed with frequencies 100
fold or higher than the frequency of spontaneous drug resistance to
these drugs. However, no ciclopirox (CPX) resistant mutant was isolated
at any point. TRB-resistant mutants showed a 500- or 1000-fold in-
crease in the MIC values of TRB, ITR-resistant mutants a 4- or 8-fold
increase in the MIC values of ITR, and AMF-resistant mutants showed
16- to 64-fold increases in the MIC values of AMF. ITR-resistant mutants
also showed increased MIC values of AMF (8- or 32-fold) and TRB (4- or
8-fold). Further, AMF-resistant mutants showed increased resistance to
TRB (from 4-to 16-fold). However, no variation in the MICs of ITR and
AMF was observed for TRB-resistant mutants. The plausible cause for
such cross resistance may be a mechanism beyond the target gene
modification. Efflux transporters may play a role here. However, re-
versal of TRB and ITR resistance after passage on nonselective media
points against this. In another study, the strains grown with FLU
showed increase in the MICs to both FLU (56.7%) and ITR (63.3%),
while strains propagated with ITR, had increased MICs to both ITR
(80%) and FLU (66.7%) (Hryncewicz-Gwóźdź et al., 2013). Thus, cross
resistance is possible between drugs sharing common targets but in
addition efflux transporters have an ability to produce multi-drug re-
sistance independent of the drug’s mechanism of action.
7. Questions unanswered and directions for future
It is unfortunate that although dermatophytoses is the commonest
fungal infection worldwide, research on it lags behind that on systemic
fungi. While increasing resistance is being described to antifungals in
dermatophytes, new drug development lags far behind. Antifungals for
dermatophytes have one distinct requirement of being able to penetrate
into the SC. Skin PK of most newer antifungals (third generation azoles,
echinocandins) have not been adequately described for any potential
clinical utility (Saunte et al., 2007). A lack of studies on clinical cor-
relation of MICs is also glaring (Dogra et al., 2019). The MIC data of
dermatophytes alone does not offer much to the clinician for effective
patient management as epidemiological cut-offs and clinical break-
points have not been described for dermatophytes. In the absence of
these, comparison of available data on skin PK of antifungals with the
local MIC patterns may be utilized by clinicians to guide treatment
(Khurana et al., 2019b). While combinations of antifungals (different
antifungal classes topically and systemically or two different systemic
antifungals used together) are often prescribed for dermatophytoses,
the practice lacks evidence backing. Checkerboard studies provide the
ideal in vitro approach to determine the utility of such combinations
(providing results as synergy, additivism, indifference or antagonism)
but have rarely been conducted for dermatophytoses especially glab-
rous skin infections (Gupta and Kohli, 2003a, 2003b). These few
checkerboard studies on dermatophytes have all been done in the set-
ting of onychomycosis with an aim to arrive at a useful systemic and
topical antifungal combination. Clinical studies testing combinations of
antifungals are also scarce (Gupta et al., 2001). Most importantly no
synergy study has been performed in patient population who fail oral
antifungals instead data is extrapolated largely from studies on la-
boratory samples that are grown under controlled conditions. Further,
use of topical antifungals may not be economically viable in extensive
disease. Modifications of established treatment regimens are already
being practiced to counter recalcitrance in dermatophytoses especially
in India that is presently facing a disturbing uprise of recalcitrant in-
fections (Table 3). However, many of these practices donot yet have
sceintific evidence backing and thus cautious use in appropriate settings
is advised.
Combinations of conventional systemic antifungals and other drugs
have been tried in experimental and clinical settings. There is in vitro
evidence of antifungal activity of FK506 (Tacrolimus) against a broad
spectrum of fungi (Lee et al., 2018). Also, as mentioned previously,
addition of FK-506 resulted in decrease in MIC of a TRB resistant M.
canis isolate possibly by efflux blockage (Kano et al., 2018). A sy-
nergistic effect of FK-506 and cyclosporine (another calcineurin in-
hibitor) with azoles has been demonstrated on checkerboard studies on
Candida spp. (Zhang et al., 2019). However, clinical studies/reports
supporting their use in dermatophytoses are lacking. Further, the im-
munosuppressive effects of calcineurin inhibitors and pharmacokinetic
interactions of cyclosporine (especially with azoles) preclude clinical
utility of these molecules at present. Topical tacrolimus has also been
reported to cause unusual and deep presentations of dermatophytosis
(Yamamoto and Nishioka, 2003). Newer analogs of FK-506 with lower
immunosuppressive activity are under investigation for potential use in
fungal infections (Lee et al., 2018). Combinations of antifungals with
systemic isotretinoin have also been tried to increase keratinocyte
proliferation with an aim to increase shedding of the fungi (Ardeshna
et al., 2016). But the combination lacks scientific logic as firstly the
reduced sebum secretion can decrease the amount of drug (especially
TRB, ITR) reaching SC (Srivastava and Kothiwala, 2017) and secondly,
there are reports of significant pharmacokinetic interaction of iso-
tretinoin with ITR (von Bernuth and Wahn, 2014). In vitro studies have
demonstrated antifungal effects of statins and synergistic interactions
with TRB and azoles but again clinical utility remains to be established
(Nyilasi et al., 2014).
Dermatophytoses have the unique disadvantage of being amenable
to self treatment leading to unsupervised and often misleading use of
over the counter antifungals that may lead to resistance. Further, to-
pical steroid misuse for cutaneous fungal infections is a common sce-
nario in some countries. The issue has been much discussed in India
especially where combinations of super potent topical steroids and
antifungals are freely available and used by patients for months before
presenting to dermatologists (Khurana et al., 2019a). Steroids have
membrane protective action and thus may diminish the action of an-
tifungals acting at the level of fungal cell membrane which includes
almost all clinically useful antifungals for dermatophytoses (Högl and
Raab, 1980). Further, steroids activate fungal metabolism in low con-
centrations achieved in skin (Erbagci, 2004).
Increasing resistance to TRB, the drug of choice for dermatophy-
toses, necessitates preventive and corrective measures to be taken be-
fore the situation turns worse and spreads globally. An antifungal
stewardship needs to be in place to check antifungal usage patterns in
cutaneous mycoses. Further, it is pertinent to emphasize to restrict use
of combinations of antifungals with steroids. Another useful, though
extreme, step maybe to restrict use of TRB in onychomycosis, the likely
cradle of resistance development to the drug.
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