Diagnostic Microbiology and Infectious Disease

48 (2004) 153–160 www.elsevier.com/locate/diagmicrobio

Review

The role of antifungal susceptibility testing in the therapy of candidiasis

Duane R. Hospenthala,*, Clinton K. Murraya, Michael G. Rinaldib
a
Infectious Disease Service, Department of Medicine, Brooke Army Medical Center, Fort Sam Houston, TX 78234-6200, USA
b
Fungus Testing Laboratory, University of Texas Health Sciences Center at San Antonio, San Antonio, TX 78229-3900, USA
Received 10 August 2003; accepted 23 September 2003

Abstract
Prior to the introduction of azoles, no real need for antifungal susceptibility testing (AFST) existed, as amphotericin B was the only agent
available to treat systemic candidiasis. Introduction of fluconazole and itraconazole provided alternate, less toxic antifungal therapies.
Intrinsic resistance of Candida krusei, decreased susceptibility of Candida glabrata, and development of resistance by Candida albicans
(in mucosal disease in AIDS) to azoles led to development of our current AFST methodologies. The goal of AFST, like that of antibacterial
susceptibility testing, is to predict clinical response, or at least to forecast failure. Although the ability of AFST to predict clinical outcome
(clinical correlation) is still being fully elucidated, current methodologies do appear to reliably predict clinical resistance to azoles. Ready
access to AFST is currently limited, affecting its timely use, but even with this lack of timeliness, AFST can still play an important role
in patient care. Important potential roles include: 1) use in the development of local antibiograms to aid empiric selection of antifungals;
2) testing of isolates from candidemia or deep infection to aid in selection of long-term therapies; and, 3) the testing of isolates from recurrent
mucosal disease to aid in selection of alternative regimens. © 2004 Elsevier Inc. All rights reserved.

Keywords: Antifungal; Susceptibility testing; Candidiasis

1. Introduction patients who progressed to AIDS. Reports of OPC clinically

Prior to the introduction of the triazole antifungal agents
in the 1980s, the need for antifungal susceptibility testing
was limited. At that time, the only agent available to treat
non-mucosal fungal infections was i.v. amphotericin B. The
toxicity of this agent was well known, but without alternate
effective drugs, selection was limited to treatment with
amphotericin B or no treatment at all. The introduction of
the systemic triazole antifungal agents, fluconazole and itra-
conazole, allowed clinicians the ability to select alternate
antifungal therapy for candidiasis. This introduction oc-
curred at the beginning of the acquired immune deficiency
syndrome (AIDS) epidemic, where the association of hu-
man immunodeficiency virus (HIV) infection and oropha-
ryngeal candidiasis (OPC or thrush) became quickly appar-
ent, as did the severity of thrush and candidal esophagitis in
* Corresponding author: Tel.: ⫹1-210-916-4355; fax: ⫹1-210-916-
0388.
E-mail address: Duane.Hospenthal@amedd.army.mil (D. Hospenthal).
The views expressed in this abstract are those of the authors and do not
reflect the official policy or position of the Department of the Army,
Department of Defense, or the U.S. Government.
unresponsive to fluconazole or requiring higher doses for
response rapidly began to appear in the literature. The in-
trinsic resistance of C. krusei to azole antifungals and de-
creased susceptibility of C. glabrata also became apparent
during the early study and use of the systemic azole anti-
fungals, although most of the isolates recovered in azole-
resistant OPC in AIDS were C. albicans. This clinical
experience with azole-resistant OPC led to interest in and
development of our current antifungal susceptibility testing
methodologies. With the introduction of highly active anti-
retroviral therapy (HAART) for HIV infection, the predict-
able stepwise development of azole-resistant oropharyngeal
candidiasis has become uncommon. Unfortunately, over
this same period candidemia and other non-mucosal candi-
diasis in the non-HIV-infected population has increased in
incidence in association with our use of an ever broadening
spectrum of antibacterial agents, our ability to support very
sick patients in intensive care units, and our increased use of
immunocompromising therapeutic regimens. In addition to
amphotericin B, fluconazole, and itraconazole, today’s cli-
nician can now choose caspofungin from the new echino-
candin class of antifungal agents, and the new broad spec-
trum azole, voriconazole. Other azole and echinocandin

0732-8893/04/$ – see front matter © 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.diagmicrobio.2003.10.003
154 D.R. Hospenthal et al. / Diagnostic Microbiology and Infectious Disease 48 (2004) 153–160
drugs for use in treating fungal infections are currently
being evaluated, and thus, antifungal susceptibility testing
will play an ever-increasing role in the selection of which
antifungal drug to use.
Antimicrobial susceptibility testing provides the clini-
cian data to predict the clinical response to specific antibi-
otic therapy. Susceptibility testing of bacteria has become a
routine, commonly automated part of the modern clinical
microbiology laboratory. The goal of antifungal suscepti-
bility testing is the same as that of antibacterial susceptibil-
ity testing, to predict clinical response, or at least, to forecast
failure. In the development of successful antimicrobial sus-
ceptibility testing, the first step is to develop methodologies
that are reproducible between laboratories. Only by follow-
ing this standardization can testing be studied on a large
scale to assess its ability to predict treatment outcome.
Although testing for bacteria has become commonplace,
antifungal susceptibility testing has only recently become
standardized and available on a more widespread basis. An
approved National Committee for Clinical Laboratory Stan-
dards (NCCLS) antifungal susceptibility testing method,
NCCLS document M27-A, was published in 1997, with a
second edition released in 2002 (NCCLS document M27-
A2). This method has allowed standardization of testing to
produce interlaboratory results that can be reliably com-
pared and examined in patient care. The ability of antifungal
susceptibility testing to predict clinical outcome, or produce
clinical correlation, is still being fully elucidated. The
M27-A2 methodology currently provides interpretive
criteria for yeasts of the Candida species and three antifun-
gals, fluconazole, itraconazole, and flucytosine. Breakpoints
for amphotericin B, the newer azoles and echinocandins
are not currently established, although the document sug-
gests a minimal inhibitory concentration (MIC) to predict
amphotericin B resistance. Although limited, the current
methodology can provide data to answer one of the most
common questions of antifungal therapy, “Can fluconazole
or another azole be used in place of the more toxic ampho-
tericin B?”
Access to “in-house” antifungal susceptibility testing is
not widely available. Most physicians are limited in their
use of antifungal testing by delays in receiving results in
a timely manner. Even if results are not available within
the timeframe that we now expect for antibacterial sus-
ceptibility testing, antifungal susceptibility testing can still
play an important role in patient care. Three important
potential roles include: 1) development of a local anti-
biogram to aid clinicians in their selection of empirically
used antifungal agents, 2) testing of isolates from candi-
demia or deep infection to allow follow on data should
patients need prolonged therapy and in cases where they are
failing empiric therapy; and, 3) testing of isolates from
recurrent mucosal disease to aid in selection of alternative
regimens.
2. Antifungal susceptibility testing
2.1. NCCLS methodology
Beginning in the 1980s, extensive planning, interlabora-
tory cooperation, and study led to the first NCCLS-ap-
proved methodology for testing antifungal susceptibility in
1997 (NCCLS, 1997, Pfaller and Yu, 2001; Rex et al.,
1993). This first big step has allowed investigators to study
and produce results that can be readily compared to each
other. The second edition, NCCLS document M27-A2, de-
scribes methodology to produce testing results that are re-
producible between institutions (NCCLS, 2002). This doc-
ument specifies standardization of media, inoculum,
laboratory conditions (time and temperature), and drug so-
lutions. The methodologies include set up instruction for
both macrodilution and microdilution testing. Quality con-
trol limits at both 24 and 48 h of incubation have been
standardized for amphotericin B, fluconazole, flucytosine,
itraconazole, ketoconazole, voriconazole, ravuconazole, and
posaconazole. Apart from the NCCLS document, study has
led to proposed quality control limits for anidulafungin and
caspofungin as well (Barry et al., 2000).
2.2. EUCAST methodology
The Antifungal Susceptibility Testing Subcommittee of
the European Committee on Antimicrobial Susceptibility
Testing (AFST-EUCAST) has also developed a standard-
ized methodology for testing antifungal susceptibility. This
methodology is based on the NCCLS M27-A, with modi-
fications that attempt to produce better objective endpoint
data. These modifications include a different inoculum size,
use of 2% dextrose supplemented medium and flat-bot-
tomed wells, spectrophotometric readings, and 50% inhibi-
tion endpoints. This method has been evaluated for intra-
and interlaboratory agreement (Cuenca-Estrella et al., 2003)
and with the newer agents, voriconazole and caspofungin
(Chryssanthou and Cuenca-Estrella, 2002). This methodol-
ogy compares favorably with the NCCLS methodology and
theoretically should produce more reliable results.
2.3. Interpretive breakpoints
Interpretive breakpoints for candidal yeast have been
established in the M27-A2 document for fluconazole, itra-
conazole, and flucytosine (Table 1). The breakpoints for the
azole antifungals were agreed upon from evaluation of data
sets containing mostly clinical response of mucosal candi-
diasis in HIV-infected individuals. Most of the fluconazole
data (420 of 528 isolates) and all of the itraconazole data
come from this group of infections. Additional fluconazole
data were obtained from invasive infections due to Candida
spp. in nonneutropenic hosts. Because of this limitation, the
clinical relevance of even these breakpoints is unclear in
other clinical settings. Data provided by the manufacturers
 D.R. Hospenthal et al. / Diagnostic Microbiology and Infectious Disease 48 (2004) 153–160 155

Table 1 duces MIC with a limited range for Candida isolates. This
NCCLS, interpretive breakpoints for Candida isolates* makes determination of breakpoints, and thus assessment of
Drug Minimal inhibitory concentration (␮g/mL) clinical correlation, difficult, although it was agreed that
Susceptible Susceptible-dose Intermediate Resistant
isolates with amphotericin B MIC ⬎ 1 ␮g/mL would likely
dependent be resistant to therapy with that agent. Use of antibiotic
medium #3 (AM3) in place of RPMI-1640 increased the
Fluconazole ⱕ8 16–32 - ⱖ64
Itraconazole ⱕ0.125 0.25–0.5 - ⱖ1
range of MIC values produced with amphotericin B, but the
Flucytosine ⱕ4 - 8–16 ⱖ32 clinical correlation of these numbers have even less data to
support usable breakpoints. In addition to the lack of a
* Candida krusei is presumed intrinsically azole resistant, thus break-
points do not apply to this species.
reliable breakpoint for conventional (deoxycholate) ampho-
tericin B, no breakpoints have been established for any of
the newer lipid-based formulations of this drug. With in-
of fluconazole and itraconazole have been analyzed based creasing call for the replacement of conventional amphoter-
on clinical response as well as dose of drug administered icin B with lipid-based formulations in routine use of this
(Rex et al., 1997). From these data evaluation arose the drug (Ostrosky-Zeichner et al., 2003), more data are needed.
category of susceptible-dose dependent (S-DD). This In vitro comparison of liposomal amphotericin B (AmBi-
unique category was proposed to identify yeast isolates with some; Fujisawa Healthcare, Deerfield, IL), amphotericin B
intermediate susceptibilities, between fully susceptible and lipid complex (Ablecet; Elan Pharmaceuticals, Princeton,
fully resistant, recovered from individuals who responded to NJ), and conventional amphotericin B has been reported. In
doses of fluconazole or itraconazole that were higher than a large multicenter trial, MICs were lower in conventional
those that were at that time considered the standard. This drug, followed by lipid complex, and then liposomal drug
designation was given to isolates with fluconazole MIC ⬎ 8 (Jessup et al., 2000). Two smaller studies found MICs
␮g/mL and ⬍ 64 ␮g/mL, as persons with disease caused by among these three compounds to be much closer (Gottfreds-
these Candida appeared to respond if therapeutic doses of son et al., 2001; Kuhn et al., 2002). This issue is compli-
⬎100 mg daily were used. This observation was virtually cated further by the fact that lipid-based drugs are given at
limited to HIV-infected individuals with oropharyngeal can- doses 3-10 times those of conventional amphotericin B, and
didiasis and its application in other forms of candidiasis was that the level of available drug at various sites of infections
not as well established. The existence of S-DD isolates is not well understood.
raises the issue of effective dosing of the azoles, especially For the new broad spectrum triazole antifungals,
fluconazole. In early studies and on at initial FDA approval, posaconazole, ravuconazole, and voriconazole, MICs can
daily fluconazole doses of 50 mg and 100 mg were com- be determined between 0.03 and 16 ␮g/mL using the stan-
monly used. Currently, daily fluconazole doses are gener- dard described in M27-A2. Although most isolates are in-
ally 400 mg or higher. For fluconazole, the prescribed daily hibited by ⱕ1 ␮g/mL of the agents, correlation between
dose in milligrams approximates its area under the curve MIC and outcome of treatment with these agents is limited.
(AUC). An AUC/MIC (or dose/MIC) ratio greater than 25 Caspofungin, an echinocandin, is the first of its class to be
has been correlated with 97% cure rate in the original FDA approved for clinical use. NCCLS M27-A2 has not
fluconazole data set used to establish NCCLS breakpoints defined breakpoints and MICs for this antifungal agent. The
(Rex et al., 1993). This supports the use of higher doses of MIC90 for Candida isolates is 1.0 ␮g/mL and 99% of
fluconazole in those individuals with S-DD isolates (Table isolates with high-level resistance to both fluconazole and
2). itraconazole were inhibited by ⱕ1 ␮g/mL (Pfaller et al.,
Breakpoints for agents other than fluconazole, itracon- 2003). MICs are consistent across most Candida species
azole, and flucytosine are not provided in the M27-A2 with the exception of Candida parapsilosis and Candida
document. Use of this method with amphotericin B pro- guilliermondii. These two species commonly produce
higher MICs that often are associated with trailing phenom-
enon, the clinical significance of which is unclear (Deres-
Table 2 inski and Stevens, 2003).
Daily dose of fluconazole likely to be effective against Candida based Currently, end point readings are recommended to be
on MIC* performed at 48 h for MICs by the NCCLS method and at
Daily dose MIC 24 h by the EUCAST. Approximately 5% of isolates will
have differing readings, from susceptible to resistant, be-
100 mg ⱕ4 ␮g/mL
200 mg ⱕ8 ␮g/mL tween the 24 and 48 hour reading due to trailing growth. It
400 mg ⱕ16 ␮g/mL appears based upon in vivo evaluation in a murine model of
800 mg ⱕ32 ␮g/mL disseminated candidiasis that those determined to be resis-
* Assumptions: AUC/MIC ⱖ25 should be effective. Daily dose of tant at 48 h and not 24 h should be categorized as “suscep-
fluconazole approximates AUC in average size patient with normal renal tible”. Because of this controversy, MIC ranges for the
function. NCCLS microdilution method quality control strains and
156 D.R. Hospenthal et al. / Diagnostic Microbiology and Infectious Disease 48 (2004) 153–160
the eight systemic antifungal agents listed in that document
are provided for both 24 and 48 hour incubations.
2.4. Clinical correlation
The ultimate goal of susceptibility testing is to predict
how patients will respond to therapy. Unfortunately, multi-
ple factors in addition to in vitro susceptibility testing re-
sults influence the clinical response of patients to antifungal
therapy. These include site of infection, host immune status,
drug pharmacokinetics, and patient compliance. Responses
at mucosal sites are almost certainly much easier to produce
then say, on the heart valves, at other deep sites, or in
association with prosthetic material. Whether a catheter is
removed or not can affect the response to therapy. Patient
immune status certainly plays a critical role in response to
fungal infection and therapy. Response to the treatment of
mucosal infection in the setting of AIDS appear directly
related to the degree of immunocompromise present. Poor
response to therapy of fungal infections is also seen in other
immunocompromised states including other immune defi-
ciency syndromes and in persons receiving cancer chemo-
therapy. Pharmacokinetics also effect therapeutic outcome.
Issues of absorption and drug levels seen with drug-drug
interactions are important factors to consider, especially
with the use of azole antifungal agents.
These factors aside, direct clinical correlation of MIC
data and therapeutic outcome is currently somewhat limited
in antifungal therapy. Again, the bulk of data and most
studies with large numbers of patients are those involving
treatment of oropharyngeal candidiasis in HIV-infected per-
sons with fluconazole. Correlative data for deeper infec-
tions, usually denoted by positive blood culture results, have
not always supported antifungal susceptibility testing re-
sults. Initial data from the largest prospective trial of flu-
conazole versus amphotericin B treatment of candidemia in
non-neutropenic patients found an inverse relationship be-
tween outcome and fluconazole MIC (Rex et al., 1995). A
narrow range of amphotericin B MICs were noted among
these isolates and no correlation of these MICs with out-
comes were identified. A more recent study employing the
NCCLS method did find correlation between MIC and re-
sponse to fluconazole treatment in deep infections (Lee et
al., 2000). In a report of the outcomes of 32 patients without
HIV infection, 79% of persons with sensitive yeasts and
66% of those with S-DD isolates responded to therapy.
Neither of two subjects with resistant isolates responded to
therapy. Another recent study of 48 severely ill subjects
with oropharyngeal candidiasis chiefly in association with
malignancy and cancer therapies documented an association
between therapeutic failure independently with persistent
neutropenia and in vitro resistance (Arikan et al., 1998). In
that study 6 of 13 (46%) subjects with resistant isolates
responded to antifungal therapy compared to 28 of 35 (80%)
subjects with sensitive isolates. Work using non-NCCLS
methodology also supports the role of antifungal suscepti-
bility testing and clinical outcome. Kovacicova et al. (2000)
found correlation between MICs produced by a disk diffu-
sion method and attributable mortality in 161 patients with
candidemia. Attributable mortality was reported to be 19%
(4 of 21) in those patients with resistant isolates compared
with 9% (12 of 140) in those with susceptible isolates.
Correlation of response and MIC in patients with anti-
fungal drugs other than fluconazole is much more sparse.
An early report examining correlation of amphotericin B
susceptibility and outcome, using methodology similar to
the final NCCLS standard, found that none of ten patients
with candidemia and MIC ⬎ 0.8 ␮g/mL survived, whereas
eight of seventeen (47%) with MIC ⱕ0.8 ␮g/mL did sur-
vive (Powderly et al., 1988). Application of the NCCLS
methodology to test isolates against amphotericin B has not
produced as encouraging results. Nguyen et al. (1998) stud-
ied the response of 105 patients with candidemia treated
with amphotericin B. They found a narrow range of MIC
among the isolates, but noted an improved range and out-
come predictability if results from minimal lethal concen-
trations were examined. This work did support a MIC re-
sistance breakpoint of ⬎ 1 ␮g/mL for amphotericin B. A
more contemporary study, using the Etest method (not the
NCCLS M27-A) found that 44% (11 of 25) of patients with
isolates having MIC ⱖ0.38 ␮g/mL survived versus an ob-
served 84% (62 of 74) survival in those whose isolates had
MIC of ⬍0.38 ␮g/mL (Clancy and Nguyen, 1999).
2.5. Other methodologies and technologies
In addition to providing a reproducible backbone to co-
ordinate investigation of antifungal susceptibility testing
and clinical correlation, the methodology included in the
M27-A2 document also provides a standard to compare
newer testing methods and technologies. The M27-A2 has
important limitations that include problems of trailing end-
points (and their interpretation) produced by the azole an-
tifungal agents and the narrow range of MIC results ob-
served with amphotericin B testing. This method is also
inherently labor intensive for the small numbers of isolates
recovered daily at most institutions. Use of NCCLS broth
microdilution method is less cumbersome than the NCCLS
broth macrodilution method, and microtiter plates can be
prepared in advance and frozen for later use. Many other
methodologies and technologies have or are being examined
to determine antifungal susceptibility. Many of these are
broth-based methods that include colorimetric or fluorescent
indicators that allow improved ease of reading or allow
automation. Virtually all new methodologies are now tested
against the approved NCCLS broth microdilution standard
to allow others to better judge their merits. In addition to
microdilution modifications, other methodologies include
calibrated strips incorporating a gradient of drug (Etest),
disk diffusion, and more recently “real-time” flow cytom-
etry methods (Pfaller and Yu, 2001).
 D.R. Hospenthal et al. / Diagnostic Microbiology and Infectious Disease 48 (2004) 153–160
2.5.1. Commercially available products
Several products that employ variations of broth mi-
crodilution methodologies are available commercially.
These include Candifast (International Microbio/Stago
Group, Diagnostic International Distribution, Milan, Italy),
Fungitest panel (Sanofi Diagnostics Pasteur, Paris, France),
Integral System Yeasts (Liofilchem Diagnostics, L’Aquila,
Italy), and Sensititre YeastOne (Trek Diagnostic Systems,
Inc., Westlake, OH/Trek Diagnostic Systems Ltd., East
Grinstead, England). Three recent studies have compared
one or more of these products to the NCCLS methodology
(Chryssanthou, 2001; Morace et al., 2002; Posteraro et al.,
2000). The Sensititre YeastOne product was studied in all of
these, comparing favorably with the NCCLS M27-A meth-
odology. Both the Candifast and Integral System Yeasts
products compared poorly to the NCCLS methodology in
the studies reported by Morace et al. and Posteraro et al. The
Fungifast product was included only in the largest of these
three studies (Morace et al., 2002), but showed good agree-
ment with the M27-A method. In that study, which com-
pared six commercial products to the reference standard
using 800 clinical Candida isolates and fluconazole, agree-
ment was seen with 81.4% of isolates employing the Sen-
sititre YeastOne, 77.7% with Fungitest, 37.6% with Integral
System Yeasts, and 22.1% with Candifast. The Sensititre
YeastOne has received FDA approval and is currently avail-
able commercially in the United States. The Sensititre Yea-
stOne is a pre-filled microtiter plate system modeled on the
M27-A method, which includes a colorimetric growth indi-
cator (AlamarBlue), is stable at room temperature for up to
two years, and allows production of fluconazole, itracon-
azole, and flucytosine MIC results.
Another highly studied and popular methodology is the
Etest (AB Biodisk, Slovna, Sweden). Fluconazole, ketocon-
azole, itraconazole, voriconazole, flucytosine, and ampho-
tericin B Etest strips are available commercially from AB
Biodisk. This antimicrobial gradient embedded strip tech-
nology is approved in the US to determine bacterial MIC,
and although approved in Europe, Etest for antifungal
agents is not approved currently for clinical use in the
United States. The Etest and NCCLS microdilution methods
have been compared extensively and shown to produce
good agreement (Arendrup et al., 2001; Arikan et al., 1997;
Barry et al., 2002; Chryssanthou, 2001; Koc et al., 2000;
Martı́n-Mazuelos et al., 1999; Matar et al., 2003; Maxwell
et al., 2003; Morace et al., 2002; Pfaller et al., 1998a; Pfaller
et al., 2000; Pfaller et al., 1998b; Vandenbossche et al.,
2002).
Another methodology that has been examined in multi-
ple studies and is currently available is disk diffusion test-
ing, a method which uses drug-embedded disks on solid
agar lawns of yeast. Study of this methodology has chiefly
examined susceptibility of Candida to fluconazole, although
amphotericin B (Arendrup et al., 2001) and voriconazole
(Matar et al., 2003) have each been evaluated in comparison
to the NCCLS method in at least one study. Disks contain-
157
ing 25 ␮g (Barry et al., 2002; Matar et al., 2003; Morace et
al., 2002) or 15 ␮g of fluconazole (Arendrup et al., 2001;
Vandenbossche et al., 2002) have been examined. Results
obtained with this methodology have been shown to com-
pare favorably with the NCCLS M27-A microdilution stan-
dard as well as to the Etest methodology (Arendrup et al.,
2001; Barry et al., 2002; Matar et al., 2003; Morace et al.,
2002; Vandenbossche et al., 2002). Recently, a NCCLS
method for antifungal disk diffusion susceptibility testing
has been proposed (NCCLS, 2003).
2.5.2. Newer technologies
Two recent studies have examined the use of viability
markers and flow cytometry to rapidly detect antifungal
susceptibility or resistance (Pina-Vaz et al., 2001; Wenisch
et al., 2001). In one of these studies (Wenisch et al., 2001),
this technology compared favorably with NCCLS method-
ology and showed correlation with clinical outcome. If
proven practical, this technology could potentially reduce
the wait time for results by days.
3. Suggested roles for antifungal susceptibility testing
The role for antifungal susceptibility testing currently is
dependent on local availability and often cost issues. Anti-
bacterial susceptibility testing is widely available and ex-
pected by clinicians. With the large number of bacterial
isolates recovered daily at most larger institutions, and the
availability of automated testing technologies, costs for an-
tibacterial susceptibility testing are much lower per isolate
than those needed to produce antifungal susceptibility re-
sults. Not only do fungi make up only small numbers of
isolates recovered in clinical microbiology laboratories, but
the cumbersomeness of the currently approved methodol-
ogy has resulted in antifungal susceptibility testing not be-
ing available on premises at most institutions. Current use of
this resource is usually dependent on the cost to the insti-
tution and the time it takes to receive results from a referral
laboratory.
Ensuring that the local clinical laboratory identifies yeast
isolates to species is still the best first step that can be done
to help in selecting antifungal agents. Candida species do
not all share similar susceptibility to currently available
antifungal agents. Candida krusei is usually resistant to
fluconazole and the other azoles, Candida lusitaniae has
been associated with amphotericin B resistance, and Can-
dida glabrata commonly produces higher MIC than other
species of Candida to azole agents. Knowing the particular
species causing disease is often of great value to the clini-
cian, as well as knowing what the local prevalence of these
more resistant species is.
Whether testing is available locally or at a reference
laboratory, antifungal susceptibility testing can be incorpo-
rated into the antifungal decision making process in benefit
of the patient (Table 3). The following applications can be
158 D.R. Hospenthal et al. / Diagnostic Microbiology and Infectious Disease 48 (2004) 153–160
Table 3
Suggested use of antifungal susceptibility testing
Setting
Isolated mucosal candidiasis*
Recurrent mucosal candidiasis
Colonization in high risk patient, empirical
antifungal therapy being considered
Candidemia and deep infection
Deep infections requiring prolonged therapy
(osteomyelitis, endocarditis, etc.)
* Including oropharyngeal candidiasis (thrush), esophagitis, vaginitis, intertrigo, and onychomycosis.
employed to maximize the benefit of antifungal susceptibil-
ity testing: 1) batching of key isolates for antifungal sus-
ceptibility testing can be performed to study local patterns
of susceptibility and establish an antibiogram for the local
area or institution; 2) testing of isolates from patients with
candidemia or deep infection to provide follow on data
should the individual patient need prolonged therapy or if
disease progresses on empirically initiated antifungal ther-
apy; and, 3) testing of isolates from persons with recurrent
mucosal disease to detect development of resistance and to
select alternate antifungal therapy if needed.
3.1. Local epidemiology/antibiogram
Knowledge of local susceptibility patterns, in conjunc-
tion with knowledge of local prevalence or identification of
more resistant species (Candida krusei, C. lusitaniae, and C.
glabrata), can greatly aid in selection of effective antifungal
agents for empiric use. As an example, although Candida
krusei is generally resistant to azole antifungal agents, this
organism often accounts for only rare isolates at most hos-
pitals (Krcmery and Barnes, 2002; Pfaller et al., 2001). This
may not be true at large cancer centers where fluconazole
prophylaxis is widely used (Safdar et al., 2001). Candida
glabrata commonly is less susceptible to azoles than other
Candida species. Often falling into the S-DD category,
identifying this yeast often results in treatment with ampho-
tericin B or other non-azole antifungals or with higher doses
of azole drug. Recent study of the susceptibility of candi-
demia isolates has found decreasing prevalence of C. gla-
brata resistance and decreasing MICs noted for these yeasts
(Pfaller et al., 2001). If local susceptibilities to this and other
common Candida species are known, this information can
allow intelligent decisions on antifungals selection, espe-
cially that used empirically.
3.2. Candidemia and deep infection
In addition to identifying yeast isolates to species and
determining local antibiograms, specific antifungal suscep-
tibility testing can be quite helpful in the setting of candi-
Suggestion
Empiric therapy, no testing necessary
Determine species to help guide therapy, consider susceptibility testing if treatment
failure or progressive disease
Determine species to help guide potential future therapy, batch test isolates
periodically to establish local antibiogram
Determine species to guide therapy, consider susceptibility testing if azole therapy
is to be used, batch test isolates periodically to establish local antibiogram
Perform susceptibility testing on all isolates
demia and deep infection. In candidemia associated with
line infections, susceptibility testing is unlikely to be of
great importance unless the subject does not respond
promptly to initial therapy. Testing results may take many
days to become available, but even in this setting, they may
be of great benefit to allow adjustment of therapy once the
results are known. In candidal infections that require pro-
tracted therapy, susceptibility testing can help document
sensitivity and thus support use of less toxic agents such as
an azole. It would seem prudent to always perform such
testing in this setting. Another setting in which this testing
can contribute to patient care is in those infections seen in
patients previously treated with fluconazole either as pro-
phylaxis or in recurring disease. Candidemia in a bone
marrow transplant patient receiving fluconazole prophylaxis
should always raise concerns about the development of
resistance. Antifungal susceptibility testing can be helpful
in determining whether azole therapy would be effective in
this setting.
3.3. Recurrent mucosal disease
Initial episodes of mucosal candidiasis, including OPC,
esophagitis, vaginitis, intertrigo, and onychomycosis, virtu-
ally never need identification of the causative yeast to spe-
cies and are not an indication for susceptibility testing. Most
of these candidal infections can be diagnosed clinically and
treated empirically with topical or systemic antifungal
agents. In persons with recurrent mucosal candidiasis, ac-
quisition of more resistant Candida species and develop-
ment of resistance has been well documented. In this set-
ting, species identification and susceptibility testing may
enhance antifungal selection and patient response to ther-
apy.
4. Conclusions
Currently, antifungal susceptibility testing is limited by
delays in obtaining results, cost, and lack of well-estab-
lished correlation between MIC and clinical response for all
 D.R. Hospenthal et al. / Diagnostic Microbiology and Infectious Disease 48 (2004) 153–160
available agents in the various candidal infections. More
rapid, cheaper, locally available testing is desirable and
large studies to prove clinical correlation of MIC and out-
come are required. Regardless of these limitations, antifun-
gal susceptibility does currently offer the clinician a valu-
able tool to assist him or her in important treatment
decisions for patients infected with candidal yeasts.
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