Ben Ami2021 1

R e sis tance t o A n t i f u n ga l
Drugs
a b,
Ronen Ben-Ami, MD , Dimitrios P. Kontoyiannis, MD, ScD *
KEYWORDS
Resistance Fungal Candida Aspergillus Cryptococcus Mechanisms
Animal models
KEY POINTS
Currently, the arsenal of systemic antifungals available for clinical use consists of only 3
major drug classes: triazoles, polyenes (represented by amphotericin B), and the
echinocandins.
Resistance to any one class of drugs can significantly limit treatment options, whereas
multidrug resistance may render fungal infections untreatable.
Antifungal resistance is intrinsic (genetically coded, seen in fungal species that are inher-
ently nonsusceptible to certain antifungals) or acquired (after antifungal exposure, which
can be either genetic or epigenetic).
Genetic changes conferring attenuated susceptibility to antifungals include point muta-
tions and genome rearrangements that result in gene copy number variations and loss
of heterozygosity.
Nongenetic mechanisms of resistance, including tolerance and heteroresistance, are
increasingly recognized as being responsible for antifungal treatment failure associated
with persistent, relapsing and breakthrough infections.
INTRODUCTION
The burden of human disease associated with fungal infections is generally underap-
preciated, with estimates indicating as many as 150 million persons infected annually
with invasive fungi and 1.5 million deaths attributed to these infections.1 Major path-
ogens responsible for invasive fungal diseases (IFD) include Aspergillus species,
which cause sinopulmonary and disseminated disease in patients with hematologic
cancer and stem cell transplantation recipients, Cryptococcus neoformans, a major
cause of meningoencephalitis in immunocompromised persons, and Candida spe-
cies, which frequently cause bloodstream infections and deep-seated infections in
a
Infectious Diseases Department, Sackler School of Medicine, Tel Aviv University, Tel Aviv
Sourasky Medical Center, 6 Weizmann, Tel Aviv 64239, Israel; b Infectious Diseases, University of
Texas M D Anderson Cancer Center, 1515 Holcombe, Houston, TX 77030, USA
* Corresponding author.
E-mail address: dkontoyi@mdanderson.org
Infect Dis Clin N Am 35 (2021) 279–311

https://doi.org/10.1016/j.idc.2021.03.003 id.theclinics.com
0891-5520/21/ª 2021 Elsevier Inc. All rights reserved.
280 Ben-Ami & Kontoyiannis
hospitalized patients.1 Additional disease burden is attributed to chronic fungal respi-

ratory infections, such as chronic pulmonary aspergillosis, chronic fungal sinusitis,
and allergic bronchopulmonary aspergillosis.1 Finally, mucosal and superficial skin in-
fections caused by Candida species and dermatophytes affect a sizable proportion of
the human population, and may have a significant impact on quality of life.1,2
Currently, the arsenal of systemic antifungals available for clinical use consists of
only 3 major drug classes: triazoles, polyenes (represented by amphotericin B), and
the echinocandins. Flucytosine, a nucleotide analogue, is used as an adjunct to
amphotericin B, mainly for the treatment of cryptococcal meningitis. Given the limited
number of therapeutic targets, resistance to antifungals is a serious concern. Resis-
tance to any 1 class of drugs can significantly limit treatment options, whereas multi-
drug resistance may render fungal infections untreatable.
The evolution of drug resistance in pathogenic fungi reflects the selective pressure
of clinical and agricultural antifungal agent use. For a quarter century after its introduc-
tion in 1958, the antifungal landscape was dominated by amphotericin B, a drug
whose use is limited by frequent and sometimes severe adverse reactions, specifically
nephrotoxicity and infusion reactions.3 The first systemic imidazole antifungal, ketoco-
nazole, became available in the early 1980s, followed a decade later by the
first-generation triazoles, fluconazole and itraconazole. Voriconazole, a second-
generation triazole, replaced amphotericin B as the drug of choice for the treatment
of invasive aspergillosis and most other filamentous fungal infections, owing to its
greater efficacy and lower toxicity.4 The introduction of triazoles represented a land-
mark in antifungal treatment, and paved the way for highly potent immunosuppressive
regimens to be given to patients with cancer and to those undergoing stem cell or solid
organ transplantation. Oral formulations and relatively favorable safety profiles make
azoles ideal drugs for chemoprophylaxis and outpatient treatment of IFD, albeit at
the cost of drug–drug interactions and adverse effects. However, the widespread
use of fluconazole in patients with hematologic malignancies was rapidly followed
by an increasing incidence of infection with Candida isolates with either innate or ac-
quired resistance to this drug.5,6 The widespread dissemination of azole-resistant
Aspergillus fumigatus, now detected on 4 continents, is likely associated with the agri-
cultural and industrial use of azoles, and threatens to render these drugs ineffective in
the prevention and treatment of IFD.7,8 A similar, but slower trend followed the intro-
duction of echinocandins as front-line agents for the treatment of invasive candidiasis
in the 2000s.9 More recently, multidrug-resistant Candida auris has emerged as a
pressing global public health concern owing to its ability to cause hospital outbreaks
associated with high mortality rates.10
In this article, we review the mechanisms by which pathogenic fungi survive expo-
sure to antifungal agents and their relevance to therapeutic decisions (Table 1).
DEFINITION OF ANTIFUNGAL RESISTANCE
In vitro measured susceptibility to antifungal drugs, as defined using the minimal inhib-
itory concentration (MIC), has been shown to predict therapeutic response for certain
organism-drug combinations. Candidiasis is the fungal infection where the most data
exist regarding in vitro susceptibility and responses to antifungals. Specifically, in vitro
susceptibility testing is able to predict the clinical response to fluconazole in mucosal
and invasive infections caused by Candida albicans, Candida parapsilosis, and
Candida tropicalis.11–14 Similarly, in vitro nonsusceptibility to the echinocandins corre-
lates with increased failure rates of echinocandin-based therapy in infections caused
by Candida glabrata, Candida krusei, and Candida guilliermondii.15 Clinical
Table 1
Main resistance mechanisms of common fungal pathogens
Mechanism
Amphotericin
Organism Azoles Echinocandins B Refs
Candida species
36–40,44,70,147
C albicans FLC-R: 0.1%–0.4% 0%–0.1% Rare mutations
ERG11 mutations FKS1 hot spot 1/2 mutations with reduced
GOF mutations in transcriptional fitness
regulators TAC1, MRR1, and UCP2
9,47,49–51,70
C glabrata FLC-R: 2.6%–10.6% 0%–2.8% Rare
GOF mutations in transcriptional regulator PDR1 FKS1 and FKS2 hot spot 1/2 mutations
70
C krusei Innately FLC-R 0%–0.7% Rare
ERG11 polymorphism FKS1 hot spot 1/2 mutations
ABC1 upregulation
70–74,76–78
C parapsilosis FLC-R: 0.6%–53% 0%–0.1% Rare
ERG11 mutations Naturally occurring FKS1
GOF mutations in transcriptional polymorphism confers
regulators TAC1 and MRR1 reduced susceptibility
70
C tropicalis 1.1%–9.2% 0%–1.3% Rare
Resistance to Antifungal Drugs

FKS1 hot spot 1 mutations
89,90,171,184
C auris FLC: 90%, VRC: 50% 2%–8% 15%–30%
ERG11 mutations FKS1 hot spot 1 mutations Unresolved
ABC and MFS type efflux activity mechanism
22,126,129,176,177
Cryptococcus neoformans FLC: 0%–50% Innately resistant Rare
Aneuploidy (Chr1)
7,8,122
Aspergillus fumigatus VRC/ITC/PSC: 0%–6% Rare Rare
CYP51 A mutations
Promoter duplications
Abbreviations: ABC, ATP binding cassette; FLC, fluconazole; GOF, gain of function; ITC, itraconazole; MFS, major facilitator superfamily; PSC, posaconazole; VRC,
voriconazole.
281
breakpoints have been defined for these Candida species, taking into account MIC
distributions, pharmacokinetic and pharmacodynamic data, resistance mechanisms
and clinical outcomes related to MIC values.16 Conversely, in the absence of robust
clinical data, the MIC distribution for a fungal species can be used as an alternate
to determine the epidemiologic cutoff values (ECV), defined as the upper MIC limit
for the wild-type population.17 Importantly, the ECV serves mainly to identify fungal
isolates with potential resistance mechanisms and may not necessarily correlate
with clinical response to the drug.
DRIVERS OF ANTIFUNGAL RESISTANCE IN THE ENVIRONMENT AND IN PATIENTS
Resistance to antifungals may be divided into intrinsic (primary) and acquired (second-
ary) forms. Intrinsic resistance is genetically coded and is associated with fungal spe-
cies that are inherently nonsusceptible to certain antifungals, irrespective of previous
exposure to those drugs. The detection of this type of resistance depends on accurate
identification of fungi to the species level, which often requires genomic sequencing to
differentiate among phenotypically similar sibling species.18 Acquired resistance de-
velops as a consequence of exposure to a stressor, often an antifungal drug or its
structural analogue, and may be either genetic or epigenetic. The ability of fungi to
rapidly evolve and adapt to fluctuating environmental conditions facilitates the emer-
gence of resistance during exposure to sublethal drug concentrations.19 Such
changes typically occur when fungi are shielded from both antifungal drugs and the
host immune system within sanctuary sites, such as biofilms formed on mucosal sur-
faces (eg, oropharyngeal candidiasis) or prosthetic material (eg, intravascular cath-
eter), within deep-seated abscesses, cavitary lung lesions, or in the central nervous
system (Table 2). Genetic changes conferring attenuated susceptibility to antifungals
include point mutations and genome rearrangements that result in gene copy number
variations and loss of heterozygosity.20–23
RESISTANCE TO TRIAZOLES
Azoles bind and inhibit the heme group of the cytochrome P-450-dependent enzyme
lanosterol 14-alpha demethylase, impairing a crucial step in ergosterol biosynthesis.
Lanosterol 14-alpha demethylase is encoded by the genes ERG11 in yeasts and
Cyp51 in molds. Depletion of ergosterol and accumulation of toxic methyl-sterols dis-
rupts the normal function of the fungal cytoplasmic membrane, leading to a fungistatic
effect in yeasts and a fungicidal effect in molds.
The azoles are the most widely used antifungal class.24 Voriconazole and isavuco-
nazole are drugs of choice for the treatment of invasive aspergillosis and other invasive
mold infections.25,26 Fluconazole, although currently not a front-line agent for the
treatment of IFD, is still used extensively as step-down treatment after an initial
response to echinocandins for invasive candidiasis and amphotericin B plus flucyto-
sine for cryptococcosis.27–29
Mechanisms of Resistance
Resistance to triazoles occurs by 3 main mechanisms: upregulated lanosterol 14-
alpha demethylase expression, alterations in the triazole binding site, and up-
regulation of multidrug efflux transporters. The predominant mechanism differs among
fungal species, and more than 1 resistance mechanism may be active within a single
isolate.
Resistance to Antifungal Drugs 283
Table 2
Factors driving the emergence of antifungal drug resistance
Factor Example
Drug exposure
Antifungal prophylaxis C krusei in patients with hematologic malignancies
Antifungal drugs used in agriculture A fumigatus with TR34/L98A CYP51A mutation in
patients with no previous exposure to azoles
Insufficient drug concentration
Biofilm Candida FKS mutations and echinocandin resistance
Foreign body Indwelling vascular catheters
Prosthetic heart valves
Urinary bladder catheters
Abscess Candida FKS mutations and echinocandin resistance
Drug–drug interactions P450 3A4 inhibition by triazoles
Bioavailability Posaconazole oral suspension bioavailability
increased by high-fat meal and decreased by PPI.
Voriconazole bioavailability reduced approximately
20% by food.
Food increases the absorption of itraconazole
capsules but reduces the absorption of
itraconazole suspension.
Nonlinear pharmacokinetics Voriconazole and itraconazole have nonlinear
pharmacokinetics, necessitating therapeutic drug
monitoring
Interpatient variation in metabolism CYP2C19 polymorphisms and voriconazole levels
Poor patient compliance
Abbreviation: PPI, proton pump inhibitor.
Candida Species
C albicans is the most frequent Candida species involved in human disease.30 Azole-
resistant C albicans strains were first observed in patients with AIDS and recurring
oropharyngeal candidiasis who were treated for extended periods with fluconazole.31
Relapsing infection and treatment failure were shown to occur with initially susceptible
C albicans strains that had acquired fluconazole resistance.31 Research on these iso-
lates revealed genetic changes that fall into 3 categories: (1) point mutations in the
Erg11p coding sequence, which decrease its affinity to azoles32,33; (2) constitutive
upregulation of Erg11p as a result of gain-of-function (GOF) mutations in the transcrip-
tional regulator Upc2 or ploidy changes involving chromosome 534,35; and (3) GOF mu-
tations in the transcription factors Tac1 and Mrr1 resulting in the upregulation of the
multidrug efflux transporters Cdr1 and Cdr2.36,37 In sequential C albicans isolates
from patients, drug resistance evolves in a stepwise manner, often starting with the
upregulation of transmembrane transporters leading to reduced intracellular accumu-
lation of fluconazole,38 followed by further changes in either expression levels of
Erg11p or the ERG11 coding sequence, resulting in decreased fluconazole binding af-
finity to the target enzyme lanosterol 14-alpha demethylase.31
The upregulation of multidrug efflux transporters seems to be the most rapid adap-
tive response that occurs after exposure to azoles.31 The main efflux transporters in C
albicans are the ABC (ATP binding cassette) type transporters Cdr1 and Cdr2, under
the regulation of Tac1 (transcriptional activator of CDR genes),37,39,40 and the MFS
(major facilitator superfamily) transporter Mdr1 (formerly BENr), which is regulated by

Mrr1 (multidrug resistance regulator).36,38 In drug-susceptible C albicans, efflux trans-
porters are expressed at low or undetectable levels; however, exposure to certain
toxic molecules, such as fluphenazine (for Cdr1/Cdr2) and benamyl (for Mdr1), signif-
icantly increases transporter expression.41,42 Activating mutations in TAC1 and MRR1,
resulting in constitutive overexpression of the associated efflux transporters and multi-
drug resistance, were identified in C albicans clinical isolates from oropharyngeal
candidiasis and in in vitro generated fluconazole-resistant strains.36,37
Upc2 is a homolog of the Saccharomyces cerevisiae transcriptional regulators Upc2
and Ecm22.43 Deletion of Upc2 renders C albicans highly susceptible to azoles, and
activating mutations leading to the constitutive upregulation of Upc2 and Erg11
were found in fluconazole-resistant clinical isolates.44 Activating mutations in UPC2
were found in 20 of 63 clinical fluconazole-resistant isolates, in conjunction with
ERG11 mutations,33 suggesting that this may be an underappreciated mechanism
of azole resistance.
Clinical C albicans isolates with a decreased susceptibility to fluconazole harbor a
large variety of ERG11 mutations, which cluster within 3 highly conserved hot spot re-
gions, corresponding with the predicted catalytic and fluconazole-binding sites
(Y132F, S405F, and G464S), extended external loop (D446E, G448E, F449V, and
G450E), and between the proximal surface and the heme (K143R).32,33 Most of these
substitutions impact sensitivity to fluconazole significantly, but have a much lesser ef-
fect on itraconazole and voriconazole; Y132F is a notable exception because it in-
creases the MIC of all 3 triazoles.33,45
Genome rearrangement is a rapid mechanism of stress adaptation and acquired
drug resistance in asexual yeasts. Aneuploidy was found in 50% of azole-resistant
C albicans clinical isolates, most of which exhibit trisomy or segmental aneuploidy
of chromosome 5.34 An isochromosome composed of 2 left arms of chromosome 5
is associated with a corresponding increased expression of genes carried on the
duplicated chromosomal segment, notably ERG11 and TAC1.34,35 Similarly, the loss
of heterozygosity for a mutated MRR1 allele was found to be a major mechanism of
resistance in C albicans strains overexpressing MDR1.46
C glabrata, the second most frequent cause of invasive candidiasis,30 is a haploid
yeast phylogenetically related to S cerevisiae. C glabrata is innately less susceptible
to azoles than C albicans, with an ECV of 32 mg/L, based on the wild-type MIC distri-
bution according to both the European Committee on Antimicrobial Susceptibility
Testing and the Clinical and Laboratory Standards Institute (CLSI) broth microdilution
methodology.11 However, whereas the European Committee on Antimicrobial Sus-
ceptibility Testing has declined to define clinical breakpoints for C glabrata, the
CLSI has held the position that fluconazole has a place in the treatment of C glabrata
infections, albeit at high doses.47 This view is based on retrospective analyses of pa-
tient data showing an improved outcome of C glabrata bloodstream infection with flu-
conazole dose of greater than 400 mg/d and a dose/MIC ratio of greater than 25.11,48
Thus, the CLSI has dropped the fluconazole susceptible category for C glabrata,
defining isolates as either susceptible dose-dependent or resistant, for a MIC of
32 mg/L or less and greater than 32 mg/L, respectively.11
Azole resistance in C glabrata is attributed primarily to the upregulation of ABC-type
efflux transporters Cdr1, Cdr2 (Pdh1), and, to a lesser extent, Snq2, under the regula-
tion of the zinc finger transcription factor Pdr1.47,49–51 PDR1 activating mutations
resulting in constitutive upregulation of Cdr1 and Cdr2 have been found in most clin-
ical azole-resistant isolates.52,53 GOF mutations cluster in 3 hot spot regions of the
PDR1 coding sequence, and intriguingly, were associated with increased fitness
and virulence in an animal model.53 As is the case with other Candida species, ABC
efflux transporter mediated resistance is associated with extensive cross-resistance
to the azole drugs itraconazole, voriconazole, and posaconazole.47,51 MFS trans-
porters seem to have a minor role in C glabrata azole resistance.54
The isolates of C glabrata carrying a defect in MSH2, which encodes the DNA
mismatch repair protein, have been identified with a high frequency in clinical speci-
mens.55 This so-called mutator phenotype is associated with the rapid acquisition
of resistance to multiple antifungal drug classes in vitro, and promotes breakthrough
infection in a mouse model of gut colonization.55 However, epidemiologic studies have
not identified an association between MSH2 mutations and antifungal resistance in
clinical isolates,56–58 calling into question the importance of this gene for the evolution
of drug resistance in vivo.
Another property of C glabrata underpinning its resilience is its unique ability to
tolerate the inhibition of the ergosterol biosynthesis pathway by acquiring exogenous
membrane sterols in vivo.59 C glabrata uses the sterol transporter Aus1 to take up
cholesterol from serum, thus evading the inhibitory effects of fluconazole.60 In contrast
with C albicans, which imports cholesterol slowly under aerobic conditions, C glabrata
also exhibits rapid cholesterol uptake under anaerobic conditions, and cholesterol
transport is accelerated in the presence of serum and fluconazole.61
C glabrata is able to survive complete loss of its mitochondrial genome while
acquiring azole resistance.62 These petite strains are reminiscent of the long-known
petite cytoplasmic mitochondrial mutant phenotype observed in the phylogenetically
related model yeast S cerevisiae.63,64 Studies of C glabrata petite mutants have found
the upregulation of the transcription factor Pdr1, as well as the efflux transporters un-
der its regulation, Cdr1 and Cdr2.65–67 The role of mitochondrial biogenesis in
response to antifungals is not restricted to Candida; it has been described in other
fungi68 and resistance is not restricted to ergosterol depleted azoles, because it has
also been described with echinocandins.69
C parapsilosis is the third leading cause of candidemia worldwide.30,70 Resistance
to fluconazole, as reported in the SENTRY trial, is infrequent (3.9% overall) but
increasing over time, with greater resistance in Latin America than in other parts of
the world.70 Notably, vast regional differences exist, with resistance rates of greater
than 50% reported in South Africa,71 and multiple countries reporting clonal outbreaks
with fluconazole-resistant strains.72–74 Most fluconazole-resistant C parapsilosis iso-
lates harbor ERG11 mutations, principally Y132F and K143R.72–75 In addition, the
upregulation of Cdr1 and Mdr1, arising from GOF mutations in the zinc cluster regula-
tors, Tac1 and Mrr1, has been observed in some resistant C parapsilosis iso-
lates.74,76–78 Cross-resistance to other azoles occurs in greater than 60% of
fluconazole resistant isolates.79 Interestingly, serial whole genome sequencing of C
parapsilosis isolates revealed divergent effects of prolonged exposure to different
azoles, whereas exposure to fluconazole or voriconazole induced resistance to the
same azole and was caused by MRR1 mutations, exposure to posaconazole induced
cross-azole resistance and attenuated virulence owing to ERG3 mutations.80 Candida
orthopsilosis, a sibling species within the C parapsilosis complex, exhibits a higher
MIC of itraconazole than C parapsilosis sensu stricto,81,82 and fluconazole resistant
Y132F C orthopsilosis isolates have been described.83
C krusei is intrinsically resistant to fluconazole, but remains frequently susceptible to
second-generation triazoles.70 Infection with this species is strongly associated with
the use of fluconazole prophylaxis among patients with hematologic malignancies.5,6
In a large cancer center, C krusei accounted for 24% of Candida bloodstream infec-
tions among patients with hematologic malignancies, but only 2% of infections among
patients with solid tumors; fluconazole prophylaxis was a major risk factor for infection
with C krusei.5 In a national survey of Candida infections in Israel, C krusei bloodstream
infection was limited to tertiary medical centers with hemato-oncology units.84 The
precise mechanism of C krusei fluconazole resistance is incompletely understood,
but is likely a combination of a decreased affinity of Erg11 to fluconazole coupled
with both constitutive and inducible expression of the ABC-type efflux transporter
Abc1.85,86
C auris is a globally emerging pathogen that has been identified as a cause of explo-
sive nosocomial outbreaks.87 C auris presents a therapeutic challenge owing to its
resistance to azoles, and propensity for multidrug resistance.88 Resistance to flucon-
azole, defined using a tentative MIC breakpoint of 32 mg/L or greater, is observed in
approximately 90% of isolates,10,89,90 with the exception of Clade 2 (east Asian Clade),
which is frequently azole susceptible.91 The mechanisms of azole resistance are pleio-
tropic, including ERG11 mutations (Y132F, F126T, and K143R),10 efflux transporters of
both the ABC and MFS types,92,93 and ERG11 gene duplication.94 Bhattacharya and
colleagues94 have shown that, similar to other yeasts, C auris undergoes replicative
aging, where asymmetric cell division generates phenotypic diversity. Interestingly,
older cells exhibited greater tolerance and resistance to azoles associated with
ERG11 and CDR1 gene duplications.94 Although ECVs have not been defined for
extended-spectrum triazoles, posaconazole and isavuconazole exhibit good in vitro
activity against C auris, whereas 50% of isolates have elevated MIC of
voriconazole.10,90,95–97
Candida (Meyerozyma) guillermondii is frequently associated with a high MIC of flu-
conazole and extended-spectrum triazoles.98 Systematic analysis of 164 invasive iso-
lates from China revealed a variety of single and multiple point mutations in the ERG11
gene in non–wild-type isolates.99 Identified point mutations included Y132F, W37C,
P518R, P430Q, Q469K, and I303V.99 Other rare Candida species, such as Pichia nor-
vegensis, Yarrowia lipolytica, and C palmioleophila, frequently have an elevated MIC
for fluconazole and other triazoles.98 However, little is known about the resistance
mechanisms in these species.
Aspergillus Species
Aspergillus species, principally A fumigatus, are the most frequent cause of IFD in
severely immunocompromised individuals.100 The key Aspergillus species involved
in human disease, A fumigatus, Aspergillus flavus, Aspergillus niger, and Aspergillus
terreus are innately sensitive to the triazoles voriconazole, itraconazole, Posacona-
zole, and isavuconazole. However, each of these pathogens resides taxonomically
within a species complex that includes sibling (cryptic) species that are morphologi-
cally highly similar to the sensu stricto species, some of which exhibit intrinsically
high azole MICs. The clinical importance of cryptic Aspergillus species is probably un-
derappreciated, because most cases of invasive aspergillosis in the current era are
based on fungal biomarkers, such as galactomannan and polymerase chain reaction,
and are not confirmed by culture. Furthermore, molecular identification of cultured as-
pergilli to the species level is not undertaken at most clinical microbiology laboratories.
In a population-based survey of mold infections in Spain, 14.5% of Aspergillus isolates
were identified as cryptic species,101 whereas in the Transplant-Associated Infection
Surveillance Network study 6.1% of section Fumigati isolates were cryptic species,
31% of section A niger isolates were reclassified as Aspergillus tubingensis, and all
6 Aspergillus ustus isolates were reclassified as Aspergillus calidoustus.102 ECVs
have not been defined for these uncommon species, but MIC values above the break-
points used for the sensu stricto species have been used tentatively to define
resistance.103 Notable cryptic species with elevated or variable azole MICs include A
calidoustus and Aspergillus pseudodeflectus (section Usti),101,104,105 and Aspergillus
lentulus, Aspergillus felis, Aspergillus viridinutans, Neosartorya pseudofischeri, Asper-
gillus hiratsukae, and Aspergillus tsurutae (section Fumigati).101,102,106–108 A tubingen-
sis, which represents 18% to 51% of clinical Aspergillus isolates belonging to the
section Nigri,101,102,109 has elevated itraconazole MICs, but remains sensitive to other
triazoles.109 Some reports have suggested that A calidoustus is an emerging cause of
invasive aspergillosis among transplant recipients receiving long-term azole prophy-
laxis.105 In a multinational study of invasive aspergillosis caused by section Usti,
47% of cases were breakthrough infections, patients were treated with multiple anti-
fungals, either consecutively or concurrently, and the mortality rate was alarmingly
high at 58%.110 A calidoustus was identified in nearly half the cases.110
A fumigatus possesses 2 CYP51 paralogues, CYP51A and CYP51B, that share 63%
sequence homology.111 Although these genes seem to have a similar substrate and
function, acquired resistance of A fumigatus to azoles is almost always associated
with sequence changes in the CYP51A coding sequence, its promoter, or both, and
only rarely with CYP51B mutations.7 The deletion of CYP51A increases susceptibility
to azoles without changing the ergosterol content or CYP51B expression, suggesting
that CYP51A is responsible for the greater part of 14 alpha demethylase activity
required for growth.111 CYP51A promoter tandem repeats are associated with an
upregulated expression of the CYP51A gene, but are usually not sufficient to induce
outright resistance.112,113 The coding sequence mutations are associated with
decreased affinity of the heme cofactor containing the catalytic domain of the enzyme
to azole drugs.112–114
A limited number of CYP51A mutations, specifically TR34/L98H (34 base pair pro-
moter tandem repeat and a leucine to histidine substitution in position 98)7,115 and
TR46/Y121F/T289A (46 base pair promoter tandem repeat and substitutions tyrosine
to phenylalanine and threonine to alanine in positions 121 and 289, respectively)113
have been described in A fumigatus isolates from patients with no prior exposure to
azoles. These mutations are assumed to occur in the environment, as a result of expo-
sure to azole-like compounds used in agriculture and industry.116 The L98H substitu-
tion alters a highly conserved region of the CYP51 protein, which forms a gate-like
structure leading to the heme cofactor.112 The resulting conformational change re-
stricts the access of azoles to the heme molecule, without affecting the catalytic ac-
tivity of the enzyme.112 The Y121F substitution destabilizes the heme cofactor and
alters a hydrophobic pocket important for azole docking. The T289A substitution
partially restores susceptibility to itraconazole, a characteristic feature of triple mu-
tants.113 Additional CYP51A mutations, specifically amino acid substitutions in posi-
tions G54 and M220, have been identified in A fumigatus isolates from patients with
chronic pulmonary aspergillosis,117,118 and likely emerge in the host during prolonged
or repeated exposure to azole drugs.
Azole-resistant A fumigatus isolates that have wild-type CYP51A are observed un-
commonly. Various functional or genomic changes have been found in such isolates,
including increased expression of CYP51, upregulation of efflux transporters, point
mutations in HMG1 (encoding HMG CoA reductase),119 and in various genes involved
in oxidative stress response.120 Similar to C albicans, A fumigatus imports exogenous
cholesterol under aerobic conditions and is thus able to bypass the antifungal activity
of sterol biosynthesis inhibitors.121
Although reported sporadically since 1997, the emergence of itraconazole-resistant
A fumigatus was first appreciated with the publication by Snelders and colleagues7 of
an epidemiologic survey in the Netherlands. Itraconazole-resistant clinical isolates
were first detected at the Radboud University Nijmegen Medical Center in 1999, and
their prevalence increased over the observation period to 6% in 2007. Strikingly, 94%
of azole-resistant isolates harbored the TR34/L98H genotype.7 Subsequent studies
have identified azole-resistant A fumigatus across Europe, at an average proportion
of 3.2% of clinical isolates.8 Of 497 A fumigatus isolates collected outside of Europe
in the ARTEMIS trial, 29 (5.8%) exhibited elevated MIC of one of the triazoles, 24 of
which originated from Hangzhou, China, and 8 of those had the TR34/L98H
genotype.122
Invasive aspergillosis with azole-resistant A fumigatus has been associated with an
extremely high case fatality rate of 88%.123 The effect of resistance to voriconazole,
defined as a MIC of greater than 2 mg/L, on the outcome of patients with invasive
aspergillosis, was examined in a retrospective multicenter study.124 Voriconazole
resistance, which was mostly associated with environmental CYP51A mutations,
was detected in 19% of A fumigatus clinical isolates and was associated with signif-
icantly greater 42-day mortality compared with infection with voriconazole-
susceptible isolates (49% and 28%, respectively; P 5 .017).124 Treatment of azole-
resistant A fumigatus with voriconazole was associated with significantly greater mor-
tality compared with treatment with an antifungal possessing in vitro activity against
the implicated isolate.124
Cryptococcus neoformans
Although combination treatment with amphotericin B and flucytosine is the recom-
mended induction regimen for the treatment of cryptococcal meningoencephalitis, flu-
conazole is the only drug available for the treatment of this disease in many parts of the
world.125 There are currently no accepted clinical breakpoints for fluconazole and C
neoformans. Pfaller and colleagues126 defined an ECV of 8 mg/L using CLSI microdi-
lution methodology. There has been a constant increase in fluconazole resistance
rates reported in clinical studies, for example, from 0% to 50% in South Africa.127,128
Fluconazole monotherapy is associated with a 10% to 20% relapse rate, with relapse
isolates typically displaying higher fluconazole MIC than incident isolates.22,129,130 An
analysis of serially cultured C neoformans reveals multiple ploidy changes, frequently
involving chromosome 1, with increased copy numbers of ERG11 and AFR1, an efflux
transporter.22 Interestingly, fluconazole resistance did not develop during combination
therapy with fluconazole and flucytosine, an oral combination associated with clinical
success rates equivalent to those of amphotericin B and flucytosine.131 Fluconazole
resistant isolates with ERG11 point mutations have been identified anecdotally.132,133
RESISTANCE TO ECHINOCANDINS
Although echinocandins are used extensively as a front-line treatment for invasive

candidiasis and as salvage therapy for invasive aspergillosis, acquired resistance re-
mains relatively uncommon in Candida species (<3% of isolates).134 The main excep-
tion is C glabrata, where caspofungin resistance is increasing, and many isolates also
display cross-resistance to other echinocandins and triazole antifungals.9,135–138 In
the SENTRY antimicrobial surveillance program, 8% to 9% of C glabrata bloodstream
isolates collected from 2009 to 2010 were found to be resistant to caspofungin.135
Distinct genetic mechanisms contribute to low-level echinocandin resistance or toler-
ance versus bona fide high-level resistance, which is more likely to result in clinical
echinocandin failure.139,140 Decreased echinocandin susceptibility occurs via 3 main
mechanisms: (1) adaptive stress responses, which result in elevated cell wall chitin
content and paradoxic growth in vitro at supra MIC drug concentration; (2) acquired
FKS mutations, which confer decreased glucan synthase sensitivity, elevated MICs,
and are often associated with clinical failure; and (3) intrinsic FKS mutations, which
are naturally occurring mutations in C parapsilosis and C guilliermondii that confer
elevated MIC levels but relatively greater glucan synthase sensitivity compared with
acquired FKS mutations.
Candida Species
Unlike azole antifungals, echinocandins are considered poor substrates for most
multidrug efflux transporters, and only modest MIC changes are observed in C albi-
cans and S cerevisiae strains hyperexpressing fungal ABC or MFS-type efflux trans-
porters.141 Additionally, fluconazole-resistant C albicans strains expressing high
levels of CDR1, CDR2, and/or MDR1 were fully susceptible to caspofungin,142 as
were 351 fluconazole-resistant clinical Candida isolates.143 Therefore, the mechanism
for resistance is expected to reflect direct changes in the glucan synthase enzyme
complex that affect echinocandin binding.144–146
Spontaneous C albicans mutants resistant to caspofungin analogues were selected
in the laboratory and exhibited significantly elevated MICs (>70-fold) and glucan syn-
thase median inhibition concentration values isolated from crude membrane frac-
tions.146 The mutant isolates were found to require 20-fold higher drug doses in a
murine model of invasive candidiasis to achieve a 99% decrease in kidney fungal
burden compared with the wild-type strains. Mutations that conferred resistance to
caspofungin in these laboratory strains (and later clinical strains) were subsequently
mapped to a highly conserved region of the FKS1 gene and a homologous region of
FKS2 in C glabrata. Amino acid substitutions S645F, S645P, and S645Y defined a re-
gion in C albicans (CaFks1 F641-P649) termed hot spot 1, which confers decreased
susceptibility to caspofungin.147 Genetic studies involving an S cerevisiae mutant con-
taining a R1357S substitution helped identify a separate region conferring decreased
susceptibility termed hot spot 2.147 Laboratory and clinical isolates harboring these
FKS mutations exhibit marked changes in the kinetic inhibition profile of glucan syn-
thase, with the hot spot S645 locus having the most pronounced impact, effectively
reducing the sensitivity of the mutant enzyme by 1000-fold to caspofungin with corre-
sponding increases in the MIC. These isolates show a poor response to caspofungin
therapy in animal infection models, even with treatment at high doses.148–150 Similar
resistance patterns were observed with anidulafungin and micafungin, suggesting
that hot spot FKS mutations impart cross-resistance to all of the currently available
echinocandins.
The FKS mechanism of echinocandin resistance extends to non–C albicans species
including isolates of C glabrata, C. guillermondi, C krusei, C parapsilosis, C tropicalis,
and Candida dubliniensis.134 In C glabrata, amino acid modifications at S663 in Fks2,
S629 in Fks1, and P659 in Fks2 are the most prominent amino acid substitu-
tions.139,150 The universality of FKS mutations as a mechanism leading to echinocan-
din resistance was further confirmed with the engineering of S645Y mutation in A
fumigatus resulting in echinocandin MECs of greater than 16 mg/L.151,152 Interestingly,
this resistance pattern has not been detected in clinical strains, despite prolonged
treatment courses in profoundly immunosuppressed patients with documented
aspergillosis.140 Increased accumulation of fluorescently labeled caspofungin was
observed in intracellular vacuoles of echinocandin-resistant Candida, suggesting
this may be a useful assay to monitor the development of resistance.153
Several clinical features have been consistent among case reports of acquired echi-
nocandin resistance in Candida species confirmed by FKS sequencing and altered
glucan synthase kinetic profiles. Patients typically have an untreated medical or
surgical reason contributing to persistent infection in the setting of prolonged or

repeated echinocandin treatment courses,154,155 including persistently low CD41
counts in patients with AIDS and recurrent esophagitis,156–158 retained infected pros-
thetic material or a catheter as the primary source of infection,159 endocarditis,160 or
complicated intra-abdominal infection,161 which may be a reservoir for selection of
echinocandin-resistant strains.162 However, even without such risk factors, rapid
emergence of resistance to echinocandins during therapy has been described anec-
dotally.154,163 Second, renal dysfunction is common in patients who developed ac-
quired echinocandin resistance, although this condition may be a consequence of
the underlying severity of illness for patients predisposed to resistance. Third,
echinocandin-resistant C glabrata may display cross-resistance to polyenes161 and
azoles.9,160
Heat shock protein (Hsp) 90 is a molecular chaperone that regulates the form and
function of diverse client proteins. The pivotal role of Hsp90 in echinocandin resis-
tance, mediating calcineurin-dependent stress responses activated by echinocan-
dins, has been shown for C albicans, C glabrata, and C dubliniensis.164–167 In a
series of elegant experiments, Singh and colleagues164 showed that pharmacologic
inhibition or genetic loss of function of Hsp90 decreased the resistance of clinical iso-
lates to the echinocandins and created a fungicidal combination. Conversely, Hsp90
expression enhanced the efficacy of an echinocandin in a murine model of dissemi-
nated candidiasis. Although not yet validated in the clinic, Hsp90 may prove a prom-
ising target for therapeutics aimed at potentiating the antifungal activity of
echinocandins and reversing echinocandin resistance or tolerance.
There is limited information on the genetic orchestrators of hypersusceptibility to
echinocandins. Gcn5, a lysyl-acetyl transferase that modifies both histone and nonhis-
tone targets, is a master regulator of multiple independent pathways, including adhe-
sion, cell wall–mediated kinase signaling, hypersensitivity to oxidative stress, and
regulation of the Fks1 glucan synthase.168 Deletion of the C albicans Gcn5 resulted
in defects in filamentation, decreased virulence in mice, and hypersensitivity to caspo-
fungin.168 Similarly, the transcription factors Efg1 and Cas5 were shown to orchestrate
the cell wall stress homeostatic response of C albicans to caspofungin.169
Resistance to echinocandins has been observed in 2% to 8% of C auris isolates,
with FKS1 hot spot 1 mutations detected in strains exhibiting a pan-echinocandin
resistant phenotype.89 Breakthrough infection with the emergence of echinocandin
resistance has been observed during treatment of infection with an initially susceptible
C auris isolate.170 Of note, echinocandin resistance has been reported predominantly
in C auris isolates from India, suggesting clade and strain-associated variation in the
propensity for acquiring FKS1 mutations.89,90,171 Shivarathri and colleagues172
showed that the 2-component signal transduction and mitogen-activated protein ki-
nase signaling are important regulators of adherence, antifungal drug resistance to
both amphotericin B and caspofungin, and virulence in C auris clinical strains. Specif-
ically, deletion of the response regulator encoding gene SSK1 and the mitogen-
associated protein kinase HOG1 restored susceptibility to both antifungals, altered
membrane lipid permeability, and cell wall mannan content.172
Aspergillus Species
Similar to Candida, echinocandin resistance in Aspergillus species is known to arise
from amino acid substitutions in b-(1,3)-d-glucan synthase encoded by the FKS1
gene. However, no tall echinocandin nonsusceptible isolates contain mutations in
FKS1. Satish and colleagues173 reported an FKS1-independent mechanism, which
implicated a significant increase in the abundance of dihydrosphingosine and
phytosphingosine in the glucan synthase microenvironment. The authors hypothe-

sized that alterations in the composition of plasma membrane lipids surrounding
glucan synthase results in nonsusceptibility to echinocandins. In their study, it was
shown that caspofungin induces mitochondrial-derived reactive oxygen species and
that dampening reactive oxygen species formation by antimycin A eliminated
caspofungin-induced resistance. These findings are consistent with the prior study
of Chamilos and colleagues,69 which showed that the mitochondrial respiratory
network is implicated in C parapsilosis caspofungin nonsusceptibility.
Interestingly, the innately resistant non-Aspergillus molds such as Fusarium solani
and Mucorales have FKS1 encoding genes that are required for cell wall biosyn-
thesis,174,175 implying that and that the echinocandin resistance in non-Aspergillus
molds is largely FKS1 independent.
Cryptococcus neoformans
C neoformans is innately resistant to the echinocandins, but the underlying mecha-
nisms for this resistance are unclear. Cao and Xue176,177 showed that Cdc50, the
b-subunit of the lipid translocase (flippase), mediates echinocandin resistance in C
neoformans via a dual function of Cdc50 that connects lipid flippase with cytoplasmic
calcium homeostasis and signaling. It has been suggested that fungal melanins pro-
tect C neoformans from echinocandins and amphotericin B, and that this protection
is not apparent on in vitro susceptibility testing.178
RESISTANCE TO AMPHOTERICIN B
Amphotericin B binds ergosterol in fungal membrane and has a rapid fungicidal action
via channel-mediated membrane permeabilization.179 Not surprisingly, resistance of
pathogenic fungi to amphotericin B is rare, as resistance is associated with significant
fitness cost (as discussed elsewhere in this article). Some uncommon fungal patho-
gens have innate resistance, such is C guilliermondii, Scedosporium apiospermum,
A terreus, some members of the Mucorales (eg, Cunnighamella180), and most of the
Fusarium species.179 Rapid acquisition of resistance to amphotericin B has also
been described in some Candida species such as Candida lusitaniae.181
C albicans mutations in the ergosterol biosynthetic pathway leading to replacement
of ergosterol with other sterol intermediates are associated with co-resistance to
azoles and amphotericin B.182,183 Specifically, heterozygous deletion of ERG11
resulted in an azole and amphotericin B–resistant strain whose sterol content was
chiefly lanosterol and eburicol.182 A C albicans strain with mutation in both ERG11
and ERG5 (encoding sterol C22 desaturase), where ergosterol was undetectable
and more than 80% of the sterol content was ergosta 5,7-dienol was similarly resistant
to azoles and amphotericin B.183 C auris is frequently nonsusceptible to amphotericin
B, with 15% to 30% of isolates displaying elevated MIC of amphotericin B (a MIC
of >2 mg/L).10,90,184 Mechanisms of amphotericin B resistance are poorly understood.
A study from Columbia identified regional clustering of amphotericin B resistant
strains, with nonsynonymous mutations identified in the transcription factor FLO8
and a putative transmembrane transporter.184
Studies in the model nonpathogenic yeast S cerevisiae have shown that amphoter-
icin B causes rapidly significant oxidative stress that is accelerated by iron chelation.
Using RNA-seq and network analyses, Pang and colleagues185 reported that ampho-
tericin B induced increased expression of genes involved in iron homeostasis and ATP
synthesis and decreased expression of genes involved in adaptive response to zinc
deficiency and oxidative stress. The clustering of co-expressed genes and network
analysis revealed that many iron and zinc homeostasis genes are targets of transcrip-
tion factors Aft1p and Zap1p. Aft1 and zap1 deletion mutants were hypersensitive to
amphotericin B and oxidative stress (H2O2), further supporting the mechanistic notion
that amphotericin B globally affects the integrity of the cell wall and membrane,
permitting other noxious stimuli, such as iron chelators, to disrupt intracellular pro-
cesses.185 Other studies in C albicans and S cerevisiae implicated the sphingolipid
biosynthetic pathway as a mechanism underlying resistance to amphotericin B,186
In fact, the alteration of the fungal membrane in general, because of mutations or
altered expression of ergosterol biosynthetic genes, results in resistance to amphoter-
icin B in both S cerevisiae and Candida species64 at the expense of hypersensitivity to
other membrane disruptors.187
A terreus, a pathogenic fungus with an intrinsically high MIC of amphotericin B, has
been the focus of research to uncover mechanisms of amphotericin B resistance.
Recent research has shown that heat shock stress response, mediated through
increased expression of the molecular chaperons Hsp90 and Hsp70, reactive oxygen
species scavenging by superoxide dismutase and catalase, and altered mitochondrial
activity all have roles in amphotericin B resistance.188,189
FITNESS CHANGES IN DRUG-RESISTANT FUNGI
Viewed from an evolutionary perspective, the development of drug resistance allows

fungi to survive in the presence of drug, but may compromise fitness, manifesting as a
decreased growth rate and sporulation relative to the isogenic wild-type strain under
nonselective conditions. In practice, dynamic compensatory genomic changes in
fungi harboring resistance-associated mutations result in fitness changes ranging
from attenuation to enhancement.190–194
Azole Resistance and Fitness

The resistance of Candida spp. to azoles has been associated with reduced, un-
changed or increased fitness and virulence.190–192 Sasse and associates194 showed
that this discrepancy can be attributed, at least in part, to the mechanism of resis-
tance. The stepwise acquisition of fluconazole resistance by sequential isogenic iso-
lates of C albicans through Tac1, Mrr1, and Upc2 activating mutations was
accompanied by a concomitant loss of fitness under nonselective growth condi-
tions.194 The fitness cost of such mutations was cumulative and was particularly
robust when combining MRR1 and TAC1 GOF mutations. Their findings suggest
that the energetic burden of constitutive overexpression of both ABC and MFS trans-
porters is disadvantageous, and may explain why these mutations are rarely found
together in fluconazole-resistant clinical strains. Taken together, these studies sug-
gest that limiting azole exposure may facilitate replacement of fitness-impaired flucon-
azole-resistant C albicans by susceptible strains in the population. However, the
dynamics of drug resistance are complicated by the interaction between different
resistance mechanisms and fitness, and the contribution of compensatory mutations
that counterbalance fitness cost.193 In contrast, azole resistance in C glabrata is asso-
ciated with fitness gain and enhanced virulence of azole-resistant isolates.195
Echinocandin Resistance and Fitness

Associations of echinocandin resistance with fitness cost have been described in
some but not all studies. Ben-Ami and colleagues196 compared echinocandin-
resistant C albicans strains with homozygous FKS1 hot spot mutations and FKS1
wild-type strains, and found that the mutants had reduced maximum catalytic
capacity of their glucan synthase complexes and thicker cell walls with higher chitin
contents. FKS1 mutants had reduced growth rates, impaired filamentation capacities,
and were hypovirulent in fly and mouse models of candidiasis, in an inverse correlation
with the cell wall chitin content.196 That decreased fitness of echinocandin-resistant C
albicans was confirmed in a competitive mixed infection model.196 In contrast, loss of
fitness was not found experimentally in echinocandin nonsusceptible C glabrata.197
We conclude that FKS1 mutations that confer echinocandin resistance incur fitness
and virulence costs to C albicans, which may limit their epidemiologic and clinical
impact. However, this relationship may be Candida species or isolate specific.
Amphotericin B Resistance and Fitness

Despite more than 55 years of use, resistance to amphotericin B is rare, suggesting it
is associated with a substantial fitness cost. Vincent and colleagues198 used whole
genome sequencing of several uncommon amphotericin B-resistant clinical and lab-
oratory C albicans strains, and found that mutations associated with amphotericin B
resistance resulted in increased vulnerability to external stresses, including oxidative
stress, high temperature, and neutrophil phagocytosis, requiring high levels of Hsp90
for survival. In addition, these mutants were defective in filamentation and avirulent in a
mouse infection model.198
TOLERANCE AND HETERORESISTANCE IN FUNGI
As noted elsewhere in this article, antifungal resistance has been shown to predict
therapeutic response for specific fungal species–drug combinations.11–14 However,
the correlation between MIC measurement and clinical response to treatment is an
imperfect one. For example, the majority of patients who die of candidemia are
infected with isolates defined as drug susceptible by accepted MIC break-
points.84,199,200 This discrepancy can be ascribed to host, disease, and drug factors
that are unrelated to in vitro susceptibility (Table 3). In addition, it has been hypothe-
sized that treatment outcomes may be affected by drug tolerance, defined as the abil-
ity to grow in the presence of drug concentrations that are greater than the MIC.
Tolerance occurs to reflect phenotypic cell-to-cell heterogeneity within a given fungal
isolate, resulting in subpopulations of cells that are able to grow at high drug concen-
trations.201 Fungal tolerance has been the subject of thoughtful reviews (beyond the
scope of this work) that link the presence of dormant (persister) cells, in the presence
of antifungals to treatment failures.201,202 Biofilms are particular ecological niches
where tolerant fungi exist.203
Persistent infection and emergent fluconazole resistance occur frequently after
treatment of C glabrata infections with this drug.50,51 Hetero-resistance, broadly
defined as significant cell-to-cell variation in drug response, has been found in a large
proportion of susceptible dose-dependent C glabrata clinical isolates, and was hy-
pothesized as a possible mechanism for these phenomena. Hetero-resistance lacks
a standard definition and is not tested for in the routine microbiology laboratory. Using
population analyses, heteroresistance was shown to be a nonbinary continuous
phenotype, with significant hetero-resistance observed in 57% of clinical isolates.
Hetero-resistance was associated with increased efflux transporter activity and
persistent visceral infection despite fluconazole treatment in a mouse model.204 Of in-
terest, functional genomic studies of C glabrata, a frequent MDR or tolerant patho-
genic fungus, identified several novel genes involved in tolerance to azoles and
echinocandins.195,205
Table 3
Causes of antifungal failure in systemic mycoses
Host Drug Pathogen

Prior exposure to antifungals Intermittent vs Species-specific variability in
constant exposure in vitro susceptibility and
virulence
Cavitary lung lesions and Decreased AUC/MIC Isolate-specific variability in vitro
sequestra (eg, abscess)a,b Drug characteristics susceptibility and virulence
Dose
Compliance Volume of distribution Fungal burden
Biofilms
Poor systemic and infection site Lipophilicity
specific immune responsesa
Delayed diagnosisa Sequence of
Obesityb azole use
Drug–drug interactionsb (eg, FLC /
Hypoalbuminemiab PSC, ITC/ PSC)
Liver/renal dysfunctionb
Co-infections
Abbreviations: AUC, area under the curve; FLC, fluconazole; ITC, itraconazole; PSC, posaconazole.
a
Predisposing factors for high fungal burden.
b
Variability/suboptimal pharmacokinetics.
Paradoxic growth, also known as the Eagle effect, where a proportion of the fungal
population (particularly Candida species, but also Aspergillus) is inhibited by low con-
centrations of an echinocandin, yet they are able to grow at much higher drug concen-
tration, is considered a phenotype related to tolerance and has been the subject of
several mechanistic studies that link it with the stress pathways regulated by Hsp90
and calcineurin and is not the result of mutations of the b-glucan synthase. As is the
case with differences in frequency of tolerance in different fungi, paradoxic growth
also seems to be species dependent.171,206 However, the exact mechanisms and clin-
ical implications of paradoxic growth remain unclear.202
TRANSLATIONAL QUESTIONS REGARDING ANTIFUNGAL RESISTANCE
Although tremendous progress has been made in characterizing molecular and epige-
netic mechanisms of resistance, especially in Candida and Aspergillus,207 less is
known about innate or acquired mechanisms of resistance of uncommon, yet
emerging opportunistic yeasts (eg, Trichosporon) and opportunistic molds (eg, Fusa-
rium species). Of note, several of the known resistance mechanisms have been
derived from laboratory-adapted strains, and we know less about the mechanisms
of resistance operating in the clinical setting. Not uncommonly, we miss the opportu-
nity to study the microevolution of resistance in high risk patients that arises from anti-
fungal selection pressure,208 because serial isolates are not available. The exceptions
have historically been patients with HIV/AIDS with oral thrush, women with chronic
recurring vulvovaginal candidiasis, and patients with chronic structural lung disease
with semi-invasive aspergillosis syndromes. The problem is particularly important in
highly immunosuppressed patients with pulmonary mycoses, such as invasive pulmo-
nary aspergillosis.209 It is possible that many of clinical fungal strains isolated from a
breakthrough infection in the setting of prior antifungals, have multiple mechanisms
of resistance or uncharacterized mechanisms of resistance or tolerance.210 This
relationship should be explored further, because the clinical scenario typically is expo-
sure of fungi to repetitive cycles of antifungals, creating a permissive setting for the
acquisition of tolerance, and ultimately, resistance. Furthermore, whether different
mechanisms underlie low-level versus high-level resistance is unclear.209 Fungal ge-
nomes are both complex and dynamic, with frequent insertions, deletions, and rear-
rangements, implying that the contribution of specific polymorphisms to phenotypic
resistance should be interpreted in a wider genomic context.211
In the absence of robust clinical experience, animal models have become an essen-
tial tool for assessing treatment strategies for resistant fungal pathogens.212 However,
these acute animal models rarely simulate clinical scenarios that lead to the emer-
gence of antifungal resistance. Specifically, opportunistic fungal infections typically
occur in patients at the extremes of age or in patients with severe and chronic immu-
nosuppression and multiple comorbidities. In contrast, animal models use relatively
young animals without comorbidities that are exposed acutely to high immunosup-
pression and high fungal inocula to overcome their natural resistance to fungal infec-
tions. Moreover, inbred animal strains do not reflect the genetic heterogeneity one
encounters in real life in humans. In addition, although elegant studies have been
done correlating the pharmacokinetic and pharmacodynamic properties of antifungals
in infection models caused by resistant fungi,213 one needs to be reminded that the
pharmacokinetic and pharmacodynamic behaviors of an antifungal in an animal and
a human might vary significantly depending on the underlying immune status of the
host and differences in anatomy and volume of distribution between mice and human
hosts.8,43 Finally, despite their promise in studying fungal pathogenesis, it is unproven
whether mini host models such as Drosophila melanogaster, Galleria mellonella, and
Caenorhabditis elegans could help to accelerate the study of fungal resistance.
More work is needed to develop these miniature models214 as platforms for high
throughput screening for novel antifungal compounds in the setting of infection by a
resistant fungus.
Few investigators have explored how specific intrinsic and adaptive resistance
mechanisms influence the host immune response to opportunistic fungi. Emerging
work on Candida species could offer insights as to why certain antifungal resistance
mechanisms and Candida species predominate in particular patient populations,
and also suggest immunotherapeutic strategies for combating Candida resistance.215
Although this area of research is still in its infancy, some themes have begun to
emerge. For example, the immune evasion and intracellular persistence of C glabrata
may be key factors in its ability to tolerate and eventually become resistant to multiple
antifungal classes. Furthermore, changes in the cell wall associated with nonsuscept-
ibility to antifungals, coupled with evasion of the host immune response216 and the
immunopharmacologic action of some antifungals, could enhance the immune-
mediated clearance and theoretically avert resistance.217
CLINICAL PERSPECTIVES REGARDING ANTIFUNGAL RESISTANCE
As the use of antifungals is widespread in modern medicine, resistant fungal infections

have been on the rise. This problem of growing antifungal resistance has been com-
pounded sharp focus by the relatively limited pipeline of novel antifungals and the ever
expanding population of immunosuppressed and frail patients where antifungals are
commonly used.
Improvement in fungal diagnostics, including the timely diagnosis of a resistant
fungal infection, is clearly one of the most urgent needs in medical mycology. The
frequency of this problem might be underestimated, given the suboptimal perfor-

mance of current diagnostics and the low frequency of autopsy in modern
hospitals.218
The development of culture-independent diagnostics that are well integrated in the
work flow of the clinical microbiology laboratory could offer a rapid advance to the
field. Importantly, because the susceptibility profile of many different fungi can no
longer be inferred from identification to the species level by conventional culture-
based methods, culture-independent methods that identify both the fungus and its
resistance pattern will be key to exploring the correlations between resistance and
clinical outcomes. Currently, only polymerase chain reaction has shown promise for
both early diagnosis and detection of resistance.219 The sensitivity of polymerase
chain reaction for detection of resistance is limited, because resistance-encoding
genes are frequently single-copy genes, unlike the multicopy genes used for fungal
detection and identification. Matrix-assisted laser desorption ionization–time of flight
may potentially become a useful modality for both identification and detection of resis-
tance, provided comprehensive libraries will be established containing sufficient
numbers of resistant fungi.220
The suboptimal correlation of in vitro methods to evaluate fungal resistance with
outcomes is another problem. In vitro susceptibility testing is performed under con-
ditions aimed at optimizing robustness and reproducibility (eg, high inoculum, aero-
bic growth, glucose-rich medium, planktonic pattern of growth), but these conditions
differ significantly from fungal growth in vivo.221 For example, opportunistic mold in-
fections in the lung are characterized by low infecting inocula, nutrient-poor sub-
strates, biofilm formation, and semianaerobic growth.221 The frequent sequential
exposure of fungi to different antifungals and the widespread use of combination
therapy further complicate the translation of in vitro susceptibility data to the
clinic.222
Not surprisingly, clinical break points have been developed only for Candida spe-
cies, mostly derived from episodes of mucosal or invasive candidiasis in nonneutro-
penic patients. No studies have proven the utility of resistance identified by in vitro
methods for predicting outcomes of candidiasis in critically ill or neutropenic pa-
tients.223,224 These conundrums are even more notable in patients with infections
caused by rare opportunistic yeast or molds, where only ECVs are available to guide
decisions.221,222,225
Critically, the acquisition of a resistant fungal infection is a relatively late event in
most treatment scenarios of immunosuppressed patients,226 where major con-
founders such as activity of underlying disease, comorbidities, drug–drug interactions,
immunosuppression, and source control issues are important confounders.227 Not
surprisingly, current guidelines for the treatment of candidiasis228 and aspergil-
losis211,229 do not provide robust evidence-based recommendations on how to treat
resistant infections because of a lack of good data. There is an urgent need for robust
data on how to treat emerging drug-resistant pathogens, especially C auris,88 C glab-
rata,230 and azole-resistant Aspergillus species.231,232
The introduction of drugs with unique mechanisms of action will be a welcome
addition to our modern armamentarium. However, the market niche and commercial
success of such drugs are uncertain.233 The discovery of new anti-infective agents in
general is biased toward drugs that kill the micro-organism or inhibit its growth. An
interesting direction in antifungal drug development would be the introduction of
drugs that do not inhibit growth, but rather impair key virulence properties, such is
invasion or adherence. Developing drugs that only interfere with virulence might
be beneficial in terms of preservation of a healthy microbiome/mycobiome, a factor
whose role in the proper functioning of the human immune system is increasingly
appreciated.234,235
In conclusion, considerable progress has been made in the field of antifungal resis-
tance, an area of emerging global public health concern. Future research priorities in
this field include mechanistic studies of antifungal stress response, genomic plasticity
in the context of innate and acquired resistance, more suitable animal models to simu-
late specific disease entities and immune deficiencies, novel diagnostics with an
emphasis on rapid point-of-care assays, and refinement of existing in vitro suscepti-
bility testing to produce clinically meaningful endpoints. Pragmatic clinical trials to
study opportunistic mycoses caused by resistant fungi, improved education of pre-
scribers and policy makers, as well as greater efforts toward antifungal stewardship
are also very important.
CLINICS CARE POINTS
Echinocandins are the preferred treatment option for most patients with suspected or
confirmed invasive candidiasis, pending the results of antifungal susceptibility testing.
Amphotericin B–based regimens are preferred as empirical treatment in immunosuppressed
patients with unidentified yeast infections, because the scope of relevant pathogens includes
Cryptococcus species and various yeast-like organisms, such as Trichosporon asahii, that are
intrinsically resistant to echinocandins.
C glabrata has a high propensity to develop resistance to both azoles and echinocandins in
the setting of a high-inoculum infection and poor source control, such as an intra-abdominal
abscess or endovascular infection. The importance of source control in these situations
cannot be overstated.
Voriconazole isavuconazole and posaconazole are adequate treatment options for invasive
pulmonary mold infections other than mucormycosis in immunosuppressed patients. Clinical
risk factors for mucormycosis, such as concurrent sinusitis, the presence of a reversed halo
sign on a computed tomography scan of the chest, and breakthrough infection on azole-
based prophylaxis, are indications for preemptive amphotericin B–based treatment pending
the results of diagnostic procedures.
An institutional antifungal stewardship program is recommended to limit inappropriate
antifungal use, which may be associated with the emergence of resistant fungal species
and excessive costs. Such programs should promote adherence to clinical pathways that
define appropriate indications, dosing regimens, duration of therapy, and de-escalation
based on culture results and biomarker assays.
There is a pressing need for novel diagnostics with an emphasis on rapid point-of-care assays,
and refinement of existing in vitro susceptibility testing to produce clinically meaningful end
points.
ACKNOWLEDGMENTS
D.P. Kontoyiannis acknowledges the Robert C Hickey Chair endowment in Clinical

Care.
DISCLOSURE
R. Ben-Ami has received consulting fees from Merck & Co., Pfizer and Gilead. D.P.
Kontoyiannis has received research support from Gilead and honoraria from Astellas
Pharma US; Gilead Sciences, Inc and Mayne, Inc.; He is member of the Cidara, Inc
Data review Committee.
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Infect Dis Clin N Am 35 (2021) 279–311

hospitalized patients.1 Additional disease burden is attributed to chronic fungal respi-

DEFINITION OF ANTIFUNGAL RESISTANCE

Resistance to Antifungal Drugs

DRIVERS OF ANTIFUNGAL RESISTANCE IN THE ENVIRONMENT AND IN PATIENTS

Abbreviation: PPI, proton pump inhibitor.

(major facilitator superfamily) transporter Mdr1 (formerly BENr), which is regulated by

Although echinocandins are used extensively as a front-line treatment for invasive

surgical reason contributing to persistent infection in the setting of prolonged or

phytosphingosine in the glucan synthase microenvironment. The authors hypothe-

FITNESS CHANGES IN DRUG-RESISTANT FUNGI

Viewed from an evolutionary perspective, the development of drug resistance allows

Azole Resistance and Fitness

Echinocandin Resistance and Fitness

Amphotericin B Resistance and Fitness

TOLERANCE AND HETERORESISTANCE IN FUNGI

Host Drug Pathogen

TRANSLATIONAL QUESTIONS REGARDING ANTIFUNGAL RESISTANCE

CLINICAL PERSPECTIVES REGARDING ANTIFUNGAL RESISTANCE

As the use of antifungals is widespread in modern medicine, resistant fungal infections

frequency of this problem might be underestimated, given the suboptimal perfor-

CLINICS CARE POINTS

D.P. Kontoyiannis acknowledges the Robert C Hickey Chair endowment in Clinical

1. Bongomin F, Gago S, Oladele RO, et al. Global and multi-national prevalence of

35. Selmecki A, Gerami-Nejad M, Paulson C, et al. An isochromosome confers drug

homeostasis and drug resistance in Candida glabrata. Curr Genet 2008;53(5):

82. Arastehfar A, Khodavaisy S, Daneshnia F, et al. Molecular identification, geno-

98. Stavrou AA, Perez-Hansen A, Lackner M, et al. Elevated minimum inhibitory

132. Gago S, Serrano C, Alastruey-Izquierdo A. Molecular identification, antifungal

decreased caspofungin susceptibility. Antimicrob Agents Chemother 2006;

182. Sanglard D, Ischer F, Parkinson T, et al. Candida albicans mutations in the

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