The Journal of Infectious Diseases

SUPPLEMENT ARTICLE

The Role of In Vitro Susceptibility Testing in the

Management of Candida and Aspergillus
Luis Ostrosky-Zeichner1 and David Andes2
1Division of Infectious Diseases, McGovern Medical School, University of Texas Health Science Center at Houston; and 2Division of Infectious Diseases, University of Wisconsin,
Madison

Antifungal susceptibility testing has evolved from a research technique to a standardized and well-validated tool for the clinical man-
agement of fungal infections and for epidemiological studies. Genetic mutations and phenotypic resistance in vitro have been shown
to correlate with clinical outcomes and treatment failures, and this in turn has led to the creation of clinical breakpoints and, more
recently, epidemiological cutoff values for clinically relevant fungal pathogens. Resistance mechanisms for Candida and Aspergillus
species have been extensively described and their corresponding genetic mutations can now be readily detected. Epidemiological
studies have been able to detect the emergence of regional clonal and nonclonal resistance in several countries. The clinical micro-
biology laboratory is expected to transition from culture and traditional susceptibility testing to molecular methods for detection,
identification, and resistance profiling over the next 5–10 years.
Keywords.  antifungal susceptibility testing; Candida; Aspergillus.

At its core, the minimum expectation for susceptibility testing is
to reliably identify patients whose infection is likely to respond
to or fail a given antifungal agent. This is a very important prem-
ise as it has the implication to drive both the use of a particular
antifungal over another or, in some cases, preclude the use of a
potentially lifesaving drug.
The relative recent development of new antifungal classes
and novel antifungals has prompted the parallel develop-
ment and standardization of antifungal susceptibility testing
(AST) in the past few decades as a tool to optimize antifungal
therapy [1]. Early attempts to standardize AST were crippled
by different groups working with divergent techniques and
media, as well as by the relative low frequency of truly resistant
organisms. It wasn’t until the early 1990s that the Clinical and
Laboratory Standards Institute and the European Committee
on Antimicrobial Susceptibility Testing reference methods
emerged, bringing standardization and reproducibility to the
field; although still significantly behind antibacterial suscep-
tibility testing availability and standardization, AST has ulti-
mately “come of age” [2] and is now a common tool available
to clinicians worldwide for clinical management of highly com-
plex fungal infections and to researchers to track the emergence
and spread of antifungal resistance. The recent expansion of
commercial and automated methods has ultimately brought a
new level of sophistication to the management of patients at the
Correspondence: L. Ostrosky-Zeichner, MD, Division of Infectious Diseases, McGovern
Medical School, University of Texas Health Science Center at Houston, 6431 Fannin, MSB
2.112, Houston 77030, TX (luis.ostrosky-zeichner@uth.tmc.edu).
The Journal of Infectious Diseases®  2017;216(S3):S452–7
© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society
of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.
DOI: 10.1093/infdis/jix239
bedside in near–real time. Collections of isolates bundled with
clinical outcomes and pharmacokinetic/pharmacodynamic
(PK/PD) data led to the creation of breakpoints that, at least for
azoles, have now been validated with a decade or 2 of clinical
correlation [2–4]. Finally, the availability of massive multina-
tional isolate surveys has started to fill the gap for organism/
drug combinations for which clinical correlates and PK/PD
data are lacking by pointing us in the direction of epidemio-
logical cutoff values (ECVs) to try to distinguish wild-type iso-
lates from those that may harbor a genetic mutation and are less
likely to respond to a given antifungal [5, 6].
GENOTYPIC VERSUS PHENOTYPIC RESISTANCE:
THE GREAT DISCONNECT?
In vitro susceptibility testing in the clinical laboratory setting
is intended to inform the clinician if an antifungal would be
expected to result in effective therapy [7, 8]. Thus, in general,
if a minimum inhibitory concentration (MIC) is elevated and
categorized as resistance for a given bug–drug combination, one
would predict treatment failure. The in vitro phenotype of an
elevated MIC is commonly explained by 1 or more resistance
mechanisms. These genotypic changes are typically either muta-
tions in a drug target or epigenetic changes such as upregulation
of a drug efflux pump. For the clinician, the advantage of a quan-
titative MIC result is the ability to decide if a drug and dosing
regimen may provide the drug exposure needed for efficacy. This
is typically based on predictions from PK/PD analyses [9–12].
One limitation of susceptibility testing is the relatively long time
required for the assay result. This is particularly important for
fungi, as the period of time between organism isolation to MIC
can be several days. Given the demonstrated importance of early
initiation of optimal antifungal therapy, this test performance

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 shortcoming is less than optimal [13–16]. In fact, mortality has
been observed to increase 3-fold with delay of appropriate anti-
fungal therapy for invasive candidiasis by as little as 12 hours.
One approach to provide an earlier indication that a ther-
apy would be expected to fail is to assess for genotypic changes
responsible for reduced antifungal effect and the elevated MIC
phenotype [17–20]. These nucleic acid–based assays have been
developed for several of the more common resistance mecha-
nisms due to drug target mutations. For example, investigators
have developed assays to detect polymorphisms in the genes
encoding the echinocandin glucan synthase enzyme in Candida
and the triazole target in Aspergillus, encoded by FKS1/FKS2
and CYP51, respectively [19, 21, 22]. The assays have demon-
strated the ability to detect the great majority of isolates
exhibiting reduced susceptibility to the respective antifungals.
Although these are not yet commercially available, academic
laboratory studies suggest that results indicating the presence
of a mutation linked to antifungal resistance may be possible
within a few hours after organism identification. This advance
could lead to earlier appropriate antifungal therapy. However,
there are potential limitations even for these genotypic studies.
Most often, the identification of a defined resistance mecha-
nism via genotypic studies correlates with an elevated MIC and
subsequently a lower likelihood of clinical success with a given
antifungal therapy [23–25]. However, there are factors that
impact the predictive value of the genotypic result. First, there
may be >1 mechanism that accounts for the resistant phenotype.
For example, triazole resistance in Candida albicans has been
linked to drug target mutations, upregulation of several efflux
pumps, and overexpression of the drug target [26]. Even in the
case of echinocandin resistance in Candida species, where the
majority of the phenotype seems accounted for by mutations in
the glucan synthase genes, resistant strains lacking these muta-
tions have been identified [27]. Second, the quantitative pheno-
type for certain genotypic mutants can be variable. For example,
mutants in both the FKS and CYP51 genes can result in MIC
values that can vary up to 100-fold. Animal model PK/PD anti-
fungal studies with these mutants demonstrate treatment effi-
cacy against some strains for which the mutation results in only
a modest MIC change [28]. Furthermore, these studies predict
the ability for successful therapy with additional dose escalation
for certain antifungals with a relatively wide therapeutic win-
dow [28–30]. These considerations are potentially important
for patients as there are limited second-line antifungal options.
For instance, the only viable option for echinocandin and tri-
azole resistance in patients infected with Candida glabrata is a
polyene. Unfortunately, several studies have demonstrated the
toxicity and associated mortality linked to polyene therapy for
invasive fungal infections [31, 32].
Additionally, there is growing evidence that fitness of the organ-
ism and the immune state of the host are of equal, if not greater,
importance for predicting the outcome of fungal infections. The
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relevance of the host immune state in modulating treatment effi-
cacy has been widely accepted. For example, searches for inde-
pendent predictors of outcome in numerous patient populations
with invasive fungal infections have identified host immune
defects such as neutropenia or immunosuppressive therapies as
conferring negative impact [33–35]. The influence of virulence
changes in the organism has also been associated with a variety of
resistance mechanisms. Specifically, several studies have reported
a fitness cost linked to the acquired resistance mechanisms. For
example, mutations in glucan synthase in C. albicans have been
shown to reduce fitness in animal models [36, 37]. In both the
Drosophila and mouse infection models, survival was nearly
twice as great in animals infected with FKS mutants compared to
wild-type. The impact of these observations in patients remains
less clear. Despite these caveats, phenotypic assays have been of
clear clinical relevance for clinicians and patients. The availabil-
ity of more rapidly available genotypic assays that are most often
congruent would be a welcome tool for physicians.
An additional issue that is not addressed by either the clini-
cally available in vitro testing or the emerging genotypic assays
is the treatment hurdle incorporated during biofilm growth.
Biofilm growth typically incurs up to a 1000-fold increase in
drug resistance that is not accounted for in the commonly used
planktonic assays [38, 39]. Studies in both invasive aspergillosis
and Candida device infections have suggested the importance
of these biofilm-dependent research mechanisms on patient
outcome [31, 40, 41]. Future studies should explore incorpora-
tion of phenotypic and genotypic biofilm assays in management
of relevant invasive fungal infections.
CASE STUDY: ANTIFUNGAL SUSCEPTIBILITY
TESTING IN CANDIDA AND DETECTION OF
RESISTANCE
Antifungal resistance emergence is, in general, less common
than that reported with antibacterials. However, there have been
several notable examples of relatively rapid and impactful resis-
tance emergence in the setting of antifungal therapy. Perhaps
the largest historically is triazole-resistant Candida in the set-
ting of mucosal candidiasis in patients with human immunode-
ficiency virus/AIDS [4, 26, 42]. In retrospect, this epidemic was
the perfect storm that combined prolonged antifungal exposure
with profound and persistent host immunodeficiency. In large
part, this crisis was averted with effective antiretroviral ther-
apy, which reduced patient immunosuppression and the need
for prolonged triazole exposure. Over time, multiple genetic
mutations have been described and are known know to account
for the majority of strains that exhibit azole resistance. These
mutations include upregulation of CDR- and MDR-encoded
efflux pumps, alteration or upregulation of the ERG11 gene, and
mutations of the ERG3 gene as well [43].
A similar but less common scenario has arisen for C.  gla-
brata and echinocandins. Candida glabrata has emerged in
 North America and several other regions as the second most
common Candida species in the setting of invasive candidia-
sis [44, 45]. The similarities to the earlier epidemic include the
relative importance of prior echinocandin exposure [23, 46].
Additionally, the potential importance of the host immune
system is also suggested as many patients have had underly-
ing malignancy or organ transplantation [23, 47]. Additionally,
research rapidly identified the mechanism underlying the echi-
nocandin treatment failures [48, 49]. As with the triazole resis-
tance scenario, there was robust debate regarding the exact in
vitro susceptibility change linked to poor patient outcome [50,
51]. Differences from the prior include the relatively short period
of echinocandin exposure needed for resistance compared to
that associated with triazole resistance. Treatment durations
as short as 7–30 days have been correlated with echinocandin
resistance emergence as opposed to the often many months
of therapy noted in the setting of triazole resistance. Another
distinct feature of the Candida isolates that have emerged in
this outbreak is the relatively high rate of resistance to both
echinocandins and triazoles despite the lack of a mechanistic
or physical link between the involved genes [52, 53]. Recently
reported mechanistic studies may help explain this difference.
Specifically, the identification of a hypermutable genotype in
C.  glabrata is very likely responsible for the relatively rapid
genetic changes in this species following echinocandin expo-
sures [54]. Initial reports of resistance initially emanated from
tertiary care centers, presumably due to their compromised
patient populations and the relatively high rate of echinocan-
din resistance. In some of these medical centers, resistance rates
have approached 20%. Conversely, rates have been lower in
other centers, presumably due to less drug use and perhaps also
the rarity of testing for resistance [55]. However, the emergence
of echinocandins at the first-line therapy for invasive candidi-
asis and the recommendation for routine testing from recent
guidelines has uncovered a growing rate of resistance across
the United States [31, 56]. The continued development of both
phenotypic and genotypic susceptibility assays will be critical
for the optimal management of infections with this increasingly
important Candida species [57].
CASE STUDY: ANTIFUNGAL SUSCEPTIBILITY
TESTING IN ASPERGILLUS AND DETECTION OF
RESISTANCE
Azole-resistant Aspergillus was practically considered to be
nonexistent. Resistance did not emerge until the 1990s, when
azoles started to be used more extensively for both prophylaxis
and therapy of invasive aspergillosis, and AST was more stan-
dardized. Some centers started to investigate prophylaxis break-
throughs and therapeutic failures as possible cases of resistance
with elevated MICs [58].
As with Candida, the first mechanisms of resistance were
found to be those related to point mutations in the CYP51A gene,
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interfering with ergosterol biosynthesis [59]. Depending on the
specific mutations, individual isolates can be found to be single
or pan–azole resistant [60]. For many years, mutations at this site
were believed to be the primary drivers of azole resistance, with
a prevalence of approximately 5% within Aspergillus isolates in
large collections. However, despite being frequently found and
described, mutations in the CYP51A gene do not account for the
vast majority of azole-resistant strains. Over time, more cryp-
tic Aspergillus species with intrinsic antifungal resistance, such
as Aspergillus terreus to amphotericin B and Aspergillus ustus to
azoles, have also been increasingly reported [61].
More recently, and perhaps more disturbing, cases of azole-re-
sistant invasive aspergillosis started to be reported in patients
without prior azole exposure [62]. AST was instrumental in
identifying these cases with elevated MICs, and very elegant
epidemiology and molecular genetics studies have pinpointed
the source of these organisms: azole-related agricultural pesti-
cides and fungicides. These agents promote the newly described
TR34/L98H and TR46/Y121F/T289A mutations, which are
being found both in environmental and patient isolates from
Europe, Asia, and, more recently, in the United States [63–66].
PRACTICAL ADVICE AND COMMON PITFALLS IN
ANTIFUNGAL SUSCEPTIBILITY TESTING
The Infectious Diseases Society of America (IDSA) guidelines
for the management of invasive candidiasis mention that while
antifungal susceptibility is predictable if the causative agent is
identified to the species level, individual isolates may not fol-
low this logic [56]. Therefore, AST is recommended routinely
for C.  glabrata against azoles and echinocandins, mention-
ing that there is less value in routine testing for other species.
Nevertheless, we think there is value in routine susceptibility
testing of all bloodstream and sterile site isolates of Candida
to monitor susceptibility trends and emergence of resistance,
locally and regionally. In a resource-constrained environment,
susceptibility testing should focus on all bloodstream or ster-
ile site isolates of C. glabrata and other Candida species isolates
in the settings of treatment failure, breakthrough infection, or
limited therapeutic options due to underlying comorbidities,
adverse events, allergies, or previous exposures. While clini-
cal correlation between fluconazole and flucytosine MICs and
outcomes is robust, interpretation of MICs and making clini-
cal decisions regarding echinocandins, second-generation tri-
azoles, and amphotericin B is more problematic. An important
point to consider for patients who are experiencing clinical
response and have seemingly resistant isolates is the possibil-
ity of MIC misinterpretation due to artifacts such as 24-hour
vs 48-hour reading discrepancies, trailing seen with azoles, and
the “paradoxical” effect seen with echinocandins [67]. Finally,
as discussed above, PK/PD, host immune status, and site pene-
tration need to be taken into account together with drug MICs
when making therapeutic decisions.
 For aspergillosis, the IDSA guidelines discourage routine
susceptibility testing against azoles during primary therapy,
recommending it primarily for patients failing therapy or for epi-
demiological purposes [68]. This is further compounded by the
fact that breakpoints and ECVs have not clearly shown clinical cor-
relation. However, the general thinking is that second-generation
triazole MICs <4 μg/mL can be considered amenable to treatment.
THE FUTURE: ANTIFUNGAL SUSCEPTIBILITY
TESTING VERSUS EMERGING MOLECULAR
METHODS
As discussed above, after many decades, AST is finally stan-
dardized and available both as a reference method and in
commercial forms/automated systems that have taken it out of
reference and research laboratories, and into clinical microbiol-
ogy laboratories. Current clinical guidelines now recommend
and incorporate AST for specific situations during the care of
patients with invasive fungal infections, such as for all sterile
site isolates of Candida [56] or when a resistant isolate is sus-
pected in aspergillosis [68].
Nevertheless, clinical microbiology is moving away from
traditional cultures and susceptibility testing into the realm of
rapid molecular diagnostics [69–71]. As research continues to
accumulate showing that time to optimal/targeted therapy has a
strong correlation with patient outcomes such as mortality rates
and length of stay, the probes, panels, and arrays are starting
to permeate into clinical laboratories. The next 3–5  years will
be characterized by punctual /targeted rapid diagnostics such
as polymerase chain reaction for multiple organisms or “syn-
dromic panels” such as those currently available for central ner-
vous system, gastrointestinal, and respiratory infections or for
bacteremia/fungemia [72, 73]. The next 5–10 years will, in turn,
focus on rapid whole-genome sequencing to directly detect
microorganisms in blood or other body fluids or tissues.
As the tide turns to direct genetic material detection of
microorganisms for rapid diagnosis, the gap will be suscepti-
bility testing as the organisms will not be cultured or available
for traditional susceptibility testing methodologies. However,
as detection systems evolve, so will the capability of resistance
detection by identifying relevant mutations that are likely to
convey phenotypic resistance, so that not only is the organism
detected but a resistance profile is produced for the clinician
based on known mutations that are likely to translate into clin-
ical resistance. Early examples of such applications include the
description of matrix-assisted laser desorption/ionization–time
of flight (MALDI-TOF) signatures that not only are species
specific but also correlate with resistance, such as has been
shown for C. glabrata [74, 75], automated systems that detect
FKS mutations in multiple Candida species [20], or detection
of Aspergillus mutations directly from respiratory samples [76].
We estimate that as systems automate and gain portability, the
next 10 years will bring the transition of these rapid molecular
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diagnostics from the research laboratory to the reference center,
then the clinical microbiology laboratory, and ultimately to the
point of care.
Notes
Financial support.  L. O.-Z. has received grants and personal fees from
Astellas, Pfizer, Scynexis, and Cidara; personal fees from Merck and Gilead;
and grants from Meiji and Janssen. D.  A.  has received grants and other
funding from Astellas and Scynexis.
Supplement sponsorship.  This work is part of a supplement sponsored by
grants from Astellas Pharma Global Development, Inc. and Merck & Co., Inc.
Potential conflicts of interest.  Both authors: No reported conflicts of
interest. Both authors have submitted the ICMJE Form for Disclosure of
Potential Conflicts of Interest. Conflicts that the editors consider relevant to
the content of the manuscript have been disclosed.
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