Table Of Content235
Development of Interpretive Breakpoints for Antifungal Susceptibility Testing:
Conceptual Framework and Analysis of In Vitro—In Vivo Correlation Data for
Fluconazole, Itraconazole, and Candida Infections
John H. Rex, Michael A. Pfaller, John N. Galgiani, From the Division of Infectious Diseases, Department of Internal
Marilyn S. Bartlett, Ana Espinel-Ingroff, Medicine, Center for the Study of Emerging and Reemerging Pathogens,
Mahmoud A. Ghannoum, Michael Lancaster, University of Texas Medical School, Houston, Texas; Department of
Pathology, University of Iowa College of Medicine, Iowa City, Iowa;
Frank C. Odds, Michael G. Rinaldi, Thomas J. Walsh,
Veterans Affairs Medical Center and University of Arizona College of
and Arthur L. Barry for the Subcommittee on Antifungal
Medicine, Tucson, Arizona; Department of Pathology, Indiana
Susceptibility Testing of the National Committee for University Medical Center, Indianapolis, Indiana; Division of Infectious D
o
Clinical Laboratory Standards Diseases, Medical College of Virginia, Richmond, Virginia; University w
n
Center for Medical Mycology, Department of Dermatology, University lo
Hospitals of Cleveland and Case Western Reserve University, School of ad
e
Medicine, Cleveland, Ohio; AccuMed International, Inc., Westlake, d
Ohio; Janssen Research Foundation, Beerse, Belgium; Laboratory fro
m
Service, University of Texas Health Science Center, Audie L. Murphy
h
Memorial Veterans Hospital, San Antonio, Texas; Infectious Diseases ttp
MSaercytliaonnd, ;P aenddia Ctrliicn iBcraaln Mchic, rNoabtiioolnoagly C Inasntcieturt eIn, sTtiutaultae,t iBn,e tOhreesgdoa,n s://a
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The availability of reproducible antifungal susceptibility testing methods now permits analysis of m
ic
data correlating susceptibility in vitro with outcome in vivo in order to define interpretive .o
u
breakpoints. In this paper, we have examined the conceptual framework underlying interpretation p
.c
of antimicrobial susceptibility testing results and then used these ideas to drive analysis of data o
m
packages developed by the respective manufacturers that correlate fluconazole and itraconazole /c
id
MICs with outcome of candidal infections. Tentative fluconazole interpretive breakpoints for MICs /a
determined by the National Committee for Clinical Laboratory Standards' M27-T broth macrodilti- rtic
tion methodology are proposed: isolates for which MICs are 8 .tg/mL are susceptible to flucona- le-a
zole, whereas those for which MICs are ,64 itg/mL appear resistant. Isolates for which the MIC bs
of fluconazole is 16-32 tcg/mL are considered susceptible dependent upon dose (S-DD), on the basis tra
c
of data indicating clinical response when > 100 mg of fluconazole per day is given. These breakpoints t/2
4
do not, however, apply to Candida krusei, as it is considered inherently resistant to fluconazole. /2
Tentative interpretive MIC breakpoints for itraconazole apply only to mucosal candidal infections /23
5
and are as follows: susceptible, -_0.125 tcg/mL; S-DD, 0.25 —0.5 ttg/mL; and resistant, .- 1.0 iug/mL. /4
2
These tentative breakpoints are now open for public commentary. 4
4
4
4
b
y
Because of advances in therapeutic technology as well as of antifungal agents are under active development. With this g
u
the AIDS epidemic, rates of fungal infections have increased multiplicity of therapeutic options comes a growing need for es
manyfold in recent years [1-3]. While amphotericin B and clinically relevant susceptibility testing methodologies that can t o
n
flucytosine were long the only available systemic antifungal be used to assist in selection of the best treatment strategy for 06
agents, both of these drugs are somewhat difficult to use, and a given patient. M
a
y
the pharmaceutical industry has responded to the demand for 2
0
new therapies for fungal infections with the introduction of See pages 248-9 for editorial response. 1
9
ketoconazole, itraconazole, fluconazole, and the lipid formula-
tions of amphotericin B.
As recently as a decade ago, antifungal susceptibility testing
These drugs are only the beginning otherazoles, lipid for-
was only occasionally employed and had not been carefully
mulations of other polyenes, and even completely new classes
studied. Studies at that time demonstrated that interlaboratory
.1. reproducibility was problematic. For example, one study found
up to 50,000-fold interlaboratory variations in absolute MIC
Received 7 June 1996; revised 19 August 1996. values when laboratories were allowed to employ any arbitrary
Financial support: This work was supported in part by the U.S. Department testing method [4], and another study found that up to 512-fold
of Veterans Affairs and by grants from Pfizer, Inc., U.S. Pharmaceuticals
variations in MIC values still remained when the collaborating
Group, and Janssen Pharmaceutica.
Reprints or correspondence: Dr. John H. Rex, 6431 Fannin, 1728 JFB, laboratories employed a single published, but unstandardized,
Houston, Texas 77030. methodology [5].
Clinical Infectious Diseases 1997; 24:235-47 In response to these problems, the National Committee for
© 1997 by The University of Chicago. All rights reserved.
1058-4838/97/2402 — 0024$02.00 Clinical Laboratory Standards (NCCLS) established the Sub-
236 Rex et al. CID 1997;24 (February)
Table 1. Summary of the M27 methodology developed by the Now that the procedures required to reduce interlaboratory
NCCLS [15, 16]. variability have been identified, the next step is development
of interpretive breakpoints for various organism-drug combina-
Implementation in the M27
tions [26]. This process, however, is far from straightforward.
Factor methodology
As a catalyst for this step, the manufacturers of fluconazole
Methodology Broth macrodilution, 1 mL final (Pfizer, New York, NY) and itraconazole (Janssen Pharmaceu-
volume; or broth microdilution, tica, Titusville, NJ) recently presented the first antifungal sus-
0.2 mL final volume
ceptibility testing data packages ever received by the NCCLS
Medium RPMI-1640 containing 0.165 M
Subcommittee for Antifungal Susceptibility Testing. In this
MOPS* (pH, 7.0)
Fungal inoculum 0.5 —2.5 X 103 organisms article, we, the members of the subcommittee, have examined
Incubation temperature 35°C the conceptual framework underlying interpretation of such D
o
Incubation time 48 h (Candida species) or 72 h antimicrobial susceptibility testing results by reviewing the les- w
n
(Cryptococcus neoformans) sons learned during development of interpretive breakpoints loa
Endpoint Amphotericin B: optically clear tube; d
for antibacterial susceptibility testing. We have employed this e
azoles and 5-fluorocytosine: 80% d
reduction in turbidity by framework to analyze the data packages and propose tentative fro
m
comparison with growth control breakpoints for susceptibility testing of fluconazole and itraco- h
Drugs and quality control Two QC isolates and corresponding nazole against Candida species. ttp
(QC) isolates pQrCoc readnugrees a erest aspbelicsihfieedd v fioar the M23 s://ac
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amphotericin B, flucytosine, Conceptual Framework d
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ketoconazole, itraconazole, and m
fluconazole [17, 18] On the basis of all available information, and especially ic.o
considering the insights generated during the development of up
* Morpholinepropanesulfonic acid. .c
susceptibility testing methods and interpretive breakpoints for o
m
antibacterial testing, we believe that four key principles of /c
id
interpretation of antimicrobial susceptibility testing can be elu- /a
committee for Antifungal Susceptibility Testing in 1982. While cidated. These principles are (1) an MIC is not a physical or rtic
le
many approaches were possible [6], the subcommittee initially chemical measurement; (2) host factors are often more im- -a
b
elected to pursue standardization of broth-based methodologies portant than susceptibility test results in determining clinical s
tra
for determination of the susceptibility of yeasts in the genera outcome; and (3) susceptibility in vitro does not predict suc- c
t/2
Candida and Cryptococcus. Working both independently cessful therapy but (4) resistance in vitro should often predict 4
/2
[7-10] and in cooperation with the NCCLS subcommittee therapeutic failure. We would like to illustrate each of these /2
3
[11-14], a number of investigators collaborated in studies that principles in turn. 5
/4
examined the role of variables such as inoculum size, inoculum 24
4
preparation, medium composition, incubation time, incubation 4
4
temperature, incubation volume, and endpoint definition in in- An MIC Is Not a Physical or Chemical Measurement b
y
terlaboratory variability. If an MIC is not a physical or chemical measurement like gu
e
Collectively, these studies led to the development of NCCLS a drug level or a blood cell count, what then does it measure? st o
document M27, titled "Reference Method for Broth Dilution The answer is that any given MIC method measures an MIC n
0
Antifungal Susceptibility Testing of Yeasts." This document not the MIC. This principle arises owing to the method depen- 6 M
was first published as M27-(P)roposed in 1992 [15] and has dence of all MIC measurement techniques, and similar logic a
y
recently been updated as M27-(T)entative [16]. The M27 meth- applies to any other such measurement (e.g., minimum fungi- 2
0
1
odology is summarized in table 1 and has been reviewed in cidal concentrations or time kill curves). 9
detail elsewhere [6, 19]. During the development process, the Even though standardized, reproducible, and yielding values
major objective of the subcommittee was achieved: testing that expressed in ug/mL, all MIC methodologies produce MICs
follows the M27 procedure will demonstrate interlaboratory that are substantially a function of the conditions selected by
reproducibility that is comparable to that obtained for antibacte- the tester. Changes in test format, inoculum size, time, tempera-
rial testing [17, 18]. ture, and endpoint definition can all produce variations in the
While the initial specification of M27 employed a cumber- measured MIC that can be quite dramatic [4, 5, 23]. In short, an
some broth macrodilution technology, simplifications of the MIC should be expected to have meaning only in relationship to
methodology by conversion to a broth microdilution format MICs obtained under standardized conditions for strains that
[11], by use of novel agar-based technologies [20-23], and by are known to be susceptible or resistant on the basis of clinical
use of colorimetric indicators [24, 25] are promising and sug- information.
gest that more convenient implementations of M27 will be This arbitrary nature of MIC values has several important
feasible. Indeed, a broth microdilution adaptation of M27 has consequences. First, the very magnitude of the variation sug-
been included in the recent M27-T document [16]. gests that approaches to interpretive breakpoint specification
CID 1997;24 (February) Interpretive Breakpoints for Antifungal Susceptibility Testing 237
that depend substantially on examining the ratio of the MIC Table 2. Factors other than susceptibility in vitro that influence the
to achievable serum (or tissue) levels may be problematic. outcome of an infection.
Second, even though absolute MICs vary, isolates will typically
Factor Specific examples
preserve their relative rank order across testing methods [4, 5].
That is, if the MIC for isolate A exceeds the MIC for isolate Drug pharmacokinetics Inadequate drug dosing; inadequate penetration
B when both are tested by one method, the same relationship of the drug to the site of infection; chemical
will often hold for other methods. instability of the drug; reduction of free
drug by protein binding; interactions
This preservation of relative rank order is not absolute, how-
between drugs
ever, and MIC variability may even make interpretation of such
General host factors Inflammatory response; antibody response;
relative MIC differences (or similarities) somewhat treacher- phagocyte function; underlying disease
ous. This latter concept has been well illustrated in recent Site of infection Abscess drainage; sequestrum formation; D
o
work with antifungal agents. For example, in a situation where presence of a foreign body (e.g., an w
n
relative MIC differences were easily seen, studies with cilofun- intravascular catheter or prosthetic cardiac lo
a
valve) d
gin demonstrated a 16-fold range in MIC values for a collection e
Pathogen Toxin production or other virulence factors; d
of Candida isolates by a broth macrodilution assay in a buffered evasion of host inflammatory response or of fro
m
synthetic medium at a pH of 7.4, but only a 2-fold range at a antibiotic by entry into a protected site (e.g., h
pH of 3. When examined in an animal model, the broad MIC an intracellular location) ttp
s
range at a pH of 7.4 did not translate into an outcome difference NOTE. This information is summarized from [32-36]. ://a
in vivo [27]. ca
d
Conversely, susceptibility testing by M27-T of Candida iso- em
lates that had been proven amphotericin B — susceptible or of authors have discussed such factors [32-36], and the major ic
.o
-resistant in an animal model showed that the MICs for the factors are listed in table 2. The importance of this issue cannot up
resistant isolates were at best 2-fold to 4-fold higher than those be overemphasized, especially for immunocompromised pa- .co
m
for the susceptible isolates [28]. When the assay was modified tients. As so eloquently discussed by Verbist [37], antibiotics /c
id
by use of antibiotic medium 3 broth and a reading at 24 hours, rarely cure infections outright. More often, they only buy time /a
this difference was increased to 8-fold to 16-fold. Finally, when during which the patient's host defenses can recover. rtic
le
the assay was fundamentally changed by shifting from a broth Looking first at factors related to pharmacokinetics of the thera- -a
b
microdilution format to an agar-based format with use of the peutic agent, the potential importance of inadequate drug dosing s
tra
Etest (AB BIODISK, Solna, Sweden), the difference in suscep- is obvious. If the drug is not taken by the patient, not absorbed, c
tibilities broadened to as much as 64-fold [23]. Conceptually or given at a dose and frequency that result in trivial drug concen- t/24
/2
similar results have been shown for amphotericin B susceptibil- trations, successful therapy would seem unlikely. Along these /2
3
ity testing of Cryptococcus neoformans [29]. same lines, drug stability is also important. For example, in a 5
/4
Demonstrations of the arbitrary nature of MIC measurements study of a large number of variously substituted azole derivatives 2
4
4
are not limited to antifungal agents. In one study, a retrospec- both in vivo and in vitro, Boyle et al. [33] found that those classes 4
4
tive correlation was performed between cefoxitin MICs and of molecules that would be expected to be relatively resistant to b
y
outcome of Bacteroides fragilis infection by measuring the metabolic degradation by the host had a good in vivo—in vitro gu
e
MICs with use of five different types of agar and two different correlation, whereas compounds that were predicted to be readily s
t o
broths. Five of the methods showed a nonsignificant trend to- degraded did not demonstrate such a correlation. n
0
ward a higher geometric mean MIC among isolates from the The effect of protein binding has recently been studied and 6
M
patients for whom therapy failed, one method produced sig- reviewed [34, 38, 39], and it is generally accepted that only a
y
nificantly higher MICs for the failure isolates, and the final free drug can readily exert its antimicrobial effect. The clinical 2
0
1
method produced lower MICs for the failure isolates [30]. In relevance of this principle was vividly illustrated during recent 9
another study, the addition of serum to a standard assay proce- therapy trials with daptomycin. In these studies, strong activity
dure increased the measured MIC of a series of quinolones in vitro did not translate into efficacy in vivo, and the major
against Staphylococcus aureus (presumably because of protein confounding factor appeared to be protein binding [40-44].
binding) while simultaneously prolonging the postantibiotic The relationship between protein binding and antifungal activ-
effect due to the quinolones [31]. ity, however, is not well understood. For example, both itraco-
nazole and amphotericin B are highly protein-bound, yet they
exhibit potent antifungal activity.
Host Factors Are Often More Important Than Susceptibility
Finally, coadministered agents can influence an antimicro-
Test Results in Determining Clinical Outcome
bial agent's activity. Such effects include interference with the
Further compounding the difficulty with predicting outcome absorption of the antimicrobial agent (e.g., sucralfate binds
from an MIC measurement are numerous host factors that, at ketoconazole and blocks its absorption [45]); speeding up the
least at times, appear to have more impact on the outcome of degradation of the antimicrobial agent (e.g., rifampin induces
an infection than does relative susceptibility in vitro. A number hepatic microsomal enzymes and can dramatically lower azole
238 Rex et al. CID 1997;24 (February)
blood levels [46-50]); and antagonism of the action of the vide an estimate of drug activity, the MIC is only part of the
antimicrobial agent (e.g., although not consistently borne out picture, and many other factors can intervene to produce failure.
by studies in vivo, depletion of cell-membrane ergosterol by Thus, it is apparent that a patient might be infected with an
prior azole therapy has the theoretical potential to reduce the exquisitely susceptible organism, yet fail to respond or relapse
efficacy of amphotericin B [51]). after apparent response because of something as simple as a
Next, the importance of an adequate host immune response retained foreign body. In short, susceptibility in vitro is only
is clear. Inadequate neutrophil function or number, inadequate the beginning of appropriate therapy and does not by itself
humoral responses, and lack of an appropriate cell-mediated guarantee success; all other relevant factors must also be ad-
immune response can both predispose to and result in perpetua- dressed.
tion of an infection. An example that integrates these ideas is Conversely, a patient infected with an extremely resistant
the augmentation of host immune response by IFN--y that has organism might have a self-limited infection and improve de- D
o
recently been shown to be useful in clearance of otherwise spite therapy. If, however, effective therapy was truly needed w
n
refractory nontuberculous mycobacterial infections [52]. More to resolve the infection, the presence of antimicrobial resistance loa
d
subtle interactions between antimicrobial agents and host im- should lead to therapeutic failure. In short, appropriately mea- e
d
munity are also possible. For example, microbial adherence sured resistance in vitro will often (but not always) correlate fro
is a critical prerequisite for many infections, and quite low with failure. These critical concepts were emphasized by Sand- m h
concentrations of antimicrobial agents have been shown to alter ers and Sanders [35] and led to a call for the emphasis of ttp
s
both bacterial and fungal surfaces so that adherence was less- testing in vitro to be changed from susceptibility testing to ://a
ened [53, 54]. Alternatively, low concentrations of an antimi- resistance testing [62]. ca
d
crobial agent might also partially damage the organism and e
m
enhance the ability of host defenses to completely eradicate ic
the infection [55, 56]. Last, while not strictly related to control How, Then, Can We Use MICs to Derive Interpretive .ou
p
of the infection, underlying disease has a tremendous effect on Breakpoints? .co
m
global outcome, and a patient with a rapidly fatal underlying The challenge, therefore, is to describe the rough relationship /c
illness may die of or with an infection despite appropriate and between MIC and outcome and to identify an MIC methodol- id/a
effective therapy. ogy that, at some MIC, predicts an increased probability of rtic
The importance of proper management of a local site of infec- treatment failure. On the basis of the just-discussed concepts, le-a
tsiioten ains da lrseom ionvcaol notfr oavneyr ftoibreleig. nA bdoedqiueas taer ed rwaienlla geset aobfl itshhee din pfericntceid- iat nisu mpobsesr iobfl ea ptop raonacahlyezs et ot hinet eardpvraetnivtaeg berse aaknpdo dinist asdpvecainfitcaagteiosn o.f bstrac
ples that, if ignored, almost always lead to failure of even the t/2
Ratio of MIC to blood or tissue levels. The principal advan- 4
most appropriate antimicrobial therapy. Microbes adhere to the /2
tage of this method is its simplicity. During early drug develop- /2
surface of foreign bodies, and this site appears to afford them a ment, measurements in vitro are the easiest way to estimate 35
relative degree of protection from the action of antimicrobial drug activity and can also be used to estimate concentration- /424
agents [57, 58]. If therapy is to be attempted without removal of 4
dependent drug effects, time-dependent drug effects, and post- 4
4
the foreign body, recent work suggests that radically different antibiotic effects. Unfortunately, a simple correlation of achiev- b
y
testing methodologies may be required before susceptibility mea- able blood or tissue levels with MIC (or any other arbitrarily gu
surFeimnaelnlyts, wthoeu blde hbaev rieolre ovaf ntht eto p caltihnoicgaeln o iutstceolfm cea n[5 i7n]f.luence the selected measurement in vitro) would be expected to correctly est o
identify the optimal breakpoint only occasionally. These con- n
outcome of antimicrobial therapy. On one hand, pathogens can cepts are well illustrated above and also by a recently suggested 06
produce factors that, once released, are beyond the ability of M
approach to breakpoint specification [63]. a
y
any antimicrobial to influence. One example of this would be Using a formula based on maximum blood concentration, 2
0
the neurotoxin produced by Clostridium tetani [59]. Alterna- 1
protein binding, and half-life, the Working Party of the British 9
tively, the organism can evade destruction by entering a site
Society for Antimicrobial Chemotherapy suggested a method
where it is relatively protected both from the host inflammatory
for computing breakpoints. However, the authors point out that
response and from the antimicrobial agent (e.g., Listeria mono-
"its rigid universal application is impossible," and the formula
cytogenes can escape from the- phagolysosome and replicate
also includes two components that are varied arbitrarily to
freely in the cytoplasm [60]). Finally, the anti-Candida activity
accommodate specific features of a given organism-antimicro-
of at least one form of lipid-associated amphotericin B has
bial combination. Even with these shortcomings, such an ap-
been shown to be dependent on production of phospholipase
proach might be useful as a starting point for interpretive
by the target microorganism [61] .
breakpoint specification, provided that the likely need for sub-
Susceptibility In Vitro Does Not Predict Successful Therapy, sequent adjustments is kept in mind.
but Resistance In Vitro Should Often Predict Therapeutic Analogy to closely related drugs. If a new drug is known
Failure to have a similar mode of action and similar pharmacokinetics
These last two intertwined principles are a natural conse- to a previously studied agent, one might also expect that the
quence of the first two principles. Even though MICs can pro- breakpoints would be similar. This logic is frequently used
CID 1997;24 (February) Interpretive Breakpoints for Antifungal Susceptibility Testing 239
within the multiple classes of cephalosporins [64, 65]. This Summary. It is clear that no one method for specification
method, however, is only useful if well-studied analogs of the of interpretive breakpoints is entirely suitable—or, as stated by
drug in question exist. In the case of the antifungal agents, this one author, "criteria for susceptibility testing have frequently
is only partially true for one pair of drugs: ketoconazole and resulted from a blend of science, faith, and business" [73].
itraconazole. However, the analogy even in this situation is loose. However, if breakpoints are deemed desirable, then a decision
Examining MIC population distributions. When testing of must be made, and this decision is ultimately a consensus
an agent demonstrates a bimodal distribution of MICs, the two interpretation of the available data. In the case of antibacterial
groups seem natural candidates for defining susceptibility and agents, breakpoints have been specified by many different
resistance [66]. Unfortunately, such a division is not necessarily groups over the years, and Baquero has performed an interest-
correct, and the situation with cilofungin (see above) is a good ing comparative survey of these breakpoints [73].
example of this problem. Conversely, a unimodal population When the susceptible (S) and resistant (R) breakpoints of D
o
may be observed, with all organisms appearing susceptible and six methods were compared for 11 antibiotics, substantial dis- w
n
similarly responsive in vivo. In such an instance, a tentative cordance for the S breakpoint was apparent, but equally inter- lo
a
d
"susceptible" breakpoint will often be proposed that is based esting was the substantial concordance for the R breakpoint. e
d
on population distribution and pharmacokinetic parameters, As it is detection of resistance that is actually paramount [62], fro
m
and the categories "moderately susceptible" and "resistant" it is indeed encouraging that the results for variant methods
h
will be reserved for use when less-responsive isolates are de- would relate so closely for the R breakpoint. We can only hope ttp
s
tected clinically [67-69]. for a similar degree of correlation for consensus breakpoints ://a
Studies of efficacy in animal models. Animal studies offer for antifungal agents, although consensus does not in any way ca
d
one method for fully integrating the effects of both the antimi- guarantee that the breakpoints are truly correct. em
crobial and host factors. By the performance of parallel studies ic
.o
of organisms that differ only in their susceptibility in vitro, the up
effect of MIC can be studied in detail. This technique is power- Case Study Number 1: Fluconazole and Candida .co
m
ful, and studies with antifungal agents have often demonstrated The data package on fluconazole contained MIC and out- /c
id
general correlations with MIC and outcome [6]. come data for 528 Candida isolates (77% C. albicans, 13% C. /a
As a specific example, recent work has shown clear relation- glabrata, 5% C. tropicalis, 3% C. krusei, and 2% other species) rtic
le
ships between MIC and minimum effective dose for some from 460 episodes of infection in 316 patients enrolled in six -a
b
antifungals [70-72]. Unfortunately, animal models of infection trials of fluconazole as therapy for oropharyngeal candidiasis s
tra
are not necessarily good mimics of the human infection. In in patients with AIDS (420 isolates from 361 episodes of infec- c
addition, drug kinetics often differ substantially between man tion in 219 patients) and three trials of fluconazole as therapy t/24
/2
and other animals. However, these types of results can provide for nonneutropenic patients with bloodstream or visceral can- /2
3
a very useful starting point. dida infection (108 isolates from 99 episodes of infection in 97 5
/4
Clinical trials in humans. Paradoxically, clinical trials in patients). Of these, 9 isolates from 8 episodes of oropharyngeal 24
4
humans are often less than satisfactory as a method for de- candidiasis in 8 patients were excluded from the analysis be- 4
4
termining interpretive breakpoints. While the relevance of cause of lack of information on either the MIC for the isolate or b
y
studying infections in humans is obvious, studies in humans the dosage of fluconazole used to treat the episode of infection. gu
e
suffer from the substantial heterogeneity that is present in most Descriptions of the six studies of oropharyngeal candidiasis st o
patient populations. While this problem can be partially ad- have been published [74-79], as have details on one of the n
0
dressed by study of certain infections that frequently occur in three trials of therapy of systemic candidiasis [80]. The MIC 6
M
certain well-defined populations (e.g., urinary tract infections of fluconazole for the isolates was measured according to the a
y
in healthy women), there is usually no easy way to eliminate NCCLS M27-T broth macrodilution methodology [16]. The 2
0
1
the effects of host factors in the study of seriously ill patients. clinical outcomes of the patients were assessed by generally 9
Furthermore, because it is conventional to use susceptibility accepted clinical criteria. Successful therapy of oropharyngeal
with respect to tentative breakpoints as a screening criterion candidiasis required resolution of all clinical symptoms as well
for a clinical trial, results from clinical trials will usually only as complete resolution of visible lesions, while successful out-
be helpful if a tentative breakpoint for resistance has been set come of therapy for systemic infection required resolution of
too high. fever and clearance of the infected site or organ(s).
On the other hand, proving that the tentative breakpoint is The package also contained data on outcome of fluconazole
too low and that potentially susceptible organisms are being therapy in 47 episodes of infection with C. neoformans in 43
labeled resistant is much more difficult. This, along with the patients, but this set of data was not analyzed because of its
smaller number of high-MIC isolates normally found in most small size as well as recent data suggesting that growth of
studies, means that clinical trials provide a great deal of data on C. neoformans in RPMI-1640 medium is at times too slow to
the efficacy of the antimicrobial against putatively susceptible yield reliable results [29, 81]. The data for candida infections
isolates but very little data regarding isolates for which MICs have been analyzed by means of the concepts outlined above,
are higher. and the subcommittee has used these analyses, along with a
240 Rex et al. CID 1997;24 (February)
Table 3. Success rate of treatment with fluconazole, as related to C. albicans in a murine model of hematogeneous candidiasis
the M27-T-determined MIC of the drug. [71]. Overall, a good correlation existed between MICs of
fluconazole and response to therapy, as assessed by prolonged
Dosage of fluconazole used for each
survival and clearance of Candida species organisms from the
patient-episode-isolate event
kidneys of infected mice. Higher doses were required to pro-
100 mg/d >100 mg/d long survival of mice infected with strains with higher flucona-
zole MICs.
MIC Success, % Success, % Similarly, Graybill [70] demonstrated a good general correla-
(p,g/mL) Success/n* (95% CIt) Success/n* (95% CIt)
tion between response in vivo and fluconazole MICs in a mu-
.-.0.0625 8/8 100 (63-100) 1/1 100 (2 —100) rine model of candidiasis. Mice infected with C. albicans
0.125 5/5 100 (48-100) strains for which MICs were x0.25 iig/mL responded well to D
o
0.25 78/78 100 (95-100) 30/38 79 (63-90) modest doses of fluconazole, whereas long-term survival of w
n
0.5 60/61 98 (91-100) 32/40 80 (64-91) mice infected with strains for which MICs were 16-64 loa
1 49/49 100 (93-100) 21/26 81 (61-93) d
pg/mL required larger doses of fluconazole. Finally, Walsh et e
2 27/28 96 (82-100) 12/14 86 (57-98) d
4 17/18 94 (73-100) 8/10 80 (44-97) al. recently demonstrated a strong correlation between high fro
m
8 10/11 91 (59-100) 13/16 81 (54-96) MICs and a requirement for higher doses of fluconazole in a h
16 12/15 80 (52-96) 15/18 83 (59-96) rabbit model of oropharyngeal candidiasis [95]. Whether anti- ttp
3n264 196//1222 7753 ((4530--9859)) 198//1390 4960 ((3505--61030)) fourn wgaitlh r eas pcoondseep econrdreenlatt evsa rbieasbtl we istuhc phe aask aprleaasm uan dcoern cthene tcrautrivone s://aca
Total n 302 217 d
remains to be determined. em
* No. of treatment successes/total no. of patient-episode-isolate events (n) . ic.o
t The 95% CIs for the percentages of events successfully treated were calcu- u
p
lated with use of a binomial approximation technique [96]. .c
Clinical Data Correlating Fluconazole MIC with Outcome o
m
/c
Analyses of rates of success vs. MIC are given in table 3, id
/a
number of additional pieces of information, to assign tentative along with a summary analysis in table 4. In these analyses, rtic
breakpoints for fluconazole when used to treat candida infec- each isolate from each episode of infection from each patient le
-a
tions. was analyzed as a separate patient-episode-isolate event. That b
s
is, if 3 different species of Candida were obtained from a tra
c
patient during 1 episode of infection, and 2 different species t/2
Pharmacology of Fluconazole were obtained during a second episode of infection, then 5 4/2
/2
Fluconazole is a bistriazole antifungal with a half-life of patient-episode-isolate events would be available for analysis. 35
—30 hours in adults and —15 hours in children and 11%-12% The problem of multiple isolates and multiple episodes was /42
4
protein binding [82-84]. Fluconazole is widely distributed in resolved by considering alternative analyses of (1) only one 44
body tissues and is somewhat concentrated in the liver, spleen, isolate per patient-episode (the one associated with the highest 4 b
y
and kidney [85]. Currently used dosages span the range of 50 MIC) or (2) only one isolate per patient (the first one associated g
u
mg/d to 800 mg/d. Dosages of 100 mg/d (1.5 mg/[kg • d]) pro- with the highest MIC). These alternative analyses produced es
duce peak serum levels of —6.7 ktg/mL; 400 mg/d (6 results that were essentially identical to those of the analysis t on
mg/[kg • d]) produces peak levels of 20-30 Ag/mL; and the in table 4 (data not shown). In addition, analyses limited to 06
linear pharmacokinetics of fluconazole would predict peak se- just the patients with oropharyngeal candidiasis and just the Ma
y
rum levels of 40-60 ug/mL at a dosage of 800 mg/d (12 mg/ 2
0
[kg • d]) [86, 87]. With daily dosing, trough levels would be 19
approximately half the peak level. ° Table 4. Summary analysis of critical fluconazole MIC ranges.
Daily fluconazole dose
Fluconazole MICs and Results of Animal Studies
100 mg >100 mg
When measured by the NCCLS M27-T methodology, flu-
conazole MICs exhibit predictable patterns that are characteris- MIC No. of Success, % No. of Success, %
tic for each species of Candida, with MICs being lowest for (//g/mL) events* (95% CIt) events* (95% CIt)
C. albicans (MIC —1 tig/mL) and highest for C. krusei
90, 253 98 (96-100) 150 81 (74-87)
(MIC90, —64 pg/mL) [88-92]. These MIC differences corre- 16-32 27 78 (58-91) 28 86 (67-96)
late well with the dosage of fluconazole required for effective 64 22 73 (50-89) 39 46 (30-63)
therapy [70, 71, 93, 94].
* Total no. of patient-episode-isolate events.
For example, Anaissie et al. studied the correlation between
t The 95% CIs for the percentages of events successfully treated were calcu-
fluconazole MIC and median effective dose for 16 strains of lated with use of a binomial approximation technique [96].
CID 1997;24 (February) Interpretive Breakpoints for Antifungal Susceptibility Testing 241
patients with invasive candida infection also produced similar 96%), and 41% (22%-61%), respectively. It is again notewor-
results. thy that the change in response rate above 32 ktg/mL corre-
As there is strong information to support the concept that sponds closely to the —25 ig/mL peak serum level of flucona-
response to fluconazole is dependent on dose of fluconazole, zole that would be expected for the average dose of fluconazole
the subcommittee elected to divide the data by fluconazole given these patients.
dose prior to analysis. Minor episodes of mucosal infection are It is also interesting to observe in these data on patients
usually treated with 100 mg/d, whereas invasive disease and treated with > 100 mg/d, the consequences of the principles
other more refractory forms of infection are often treated with discussed in the first half of this paper: therapeutic failures
higher daily doses. Shown on the left in table 3 and table 4 were noted even with isolates for which MICs were very low,
are the outcomes in 302 patient-episode-isolate events treated while success was obtained at times even when the MICs were
with 100 mg/d. Of these, 293 were from patients with oropha- very high. The higher overall failure rate is a consequence of D
o
ryngeal candidiasis and nine were from patients with invasive increasing severity of illness in these patients; failure in this w
n
disease. patient population is often due to a mixture of clinical events loa
d
As can be seen, the success rate for the patients treated with (i.e., events related to factors such as those listed in table 2) e
d
100 mg/d is >90% up to an MIC of 8 µg/mL. While the as well as microbiological events such as persistent infection. fro
m
success rate at an MIC of 8 µg/mL appears to be slightly less Overall, however, the data support the concept that response h
than the success rate for lower MICs, the small number of to fluconazole is a function of both MIC and drug dose. Finally, ttp
s
observations at this MIC limits the strength of this observation, while the data package included a small number of C. krusei ://a
and the 95% confidence interval for the success rates includes isolates, it is now generally thought that C. krusei is intrinsi- ca
d
100% for all MIC values up to and including 8 µg/mL. Stated cally resistant to fluconazole no matter what its measured MIC em
differently, the 95% confidence interval for the success rate [97], and the committee thus believes that an MIC determina- ic.o
for all isolates for which MICs are a4 µg/mL is 96%-100% tion for this species is not required. up
.c
and is unchanged by inclusion of the isolates associated with The data in the fluconazole package are quite consistent with o
m
an MIC of 8 µg/mL. Conversely, the success rate for isolates data from other authors. While this topic has recently been /c
id
for which MICs are 11,g/ mL is definitely lower, with an reviewed for data generated by a variety of methods [26], /a
overall success rate of 76%. The 95% confidence limits on this looking only at reports which used the NCCLS methodology rtic
le
estimate are 61% and 87% and also distinctly lower than the reveals a consistent pattern. Four papers have described consis- -a
b
success rate for isolates associated with an MIC of ktg/ tent failure of 100-200 mg/d when the MIC exceeded 8-25 stra
mL. It is noteworthy that the change in response rate above 8 µg/mL [98-101], and four other papers found consistent failure c
µg/mL corresponds closely to the 6.7 ,ug/mL peak serum level of 400-800 mg/d at an MIC of _.64 ,ug/mL [77, 79, 102, t/24
/2
of fluconazole that would be expected for the average dose of 103]. Unfortunately, all of these data are from patients with /2
3
fluconazole given these patients. oropharyngeal candidiasis in the setting of HIV infection. Addi- 5
/4
Shown on the right in table 3 and table 4 are the outcomes tional MIC data for large numbers of patients with deep-seated 24
4
in 217 patient-episode-isolate events treated with > 100 mg/d or hematogenous candida infections have not been reported. 4
4
(range, 150-850 mg/d; median dose, 400 mg/d). Of these, by
118 involved patients with oropharyngeal candidiasis and 99 gu
Tentative Fluconazole Breakpoints e
involved patients with invasive disease. The success rate is st o
strikingly uniform at 80%-90% for all isolates with MICs of Considering both the overall pattern of the clinical data and n
0
32 µg/mL. The higher success rates at the two very lowest its correlation with the pharmacokinetic behavior of flucona- 6 M
MIC values are not considered reliable estimates because of zole, it seems reasonable to conclude that (1) response to flu- a
y
the very small number of observations at these MICs. conazole does vary with MIC, as determined by the M27-T 20
1
Taken together, these data document an 82% success rate methodology; (2) higher doses of fluconazole can be used to 9
(95% CI, 76%-87%) in these episodes'of infection. A substan- treat patients infected with isolates for which MICs are higher;
tial decrease in success rate is noted, however, with MICs of and (3) failure of fluconazole therapy becomes likely when the
64 ,cg/mL. While less precise because of the progressively MIC determined by the NCCLS M27-T methodology substan-
smaller numbers of patients involved, this analysis can be fur- tially exceeds the predicted peak serum levels of fluconazole
ther subdivided by disease group (oropharyngeal candidiasis expected for a given dosing regimen.
-
vs. invasive infection), and the general dose-response relation- The data presented herein, coupled with what is known of
ship was present for both groups but somewhat more obvious the pharmacokinetics of fluconazole, provide an opportunity to
for the patients with oropharyngeal candidiasis. propose tentative interpretive breakpoints for fluconazole and
For patients with invasive disease, the response rates (95% Candida isolates in much the same manner as is done with
CI) were 71% (60%-81%), 91% (59%-100%), and 58% certain bacterial pathogens and newly introduced antibacterial
(28%-85%) at MICs of 16-32, and µg/mL, respec- agents [68, 69]. It must be clearly understood, however, that
tively. The corresponding success rates for patients with oro- these conclusions are strongest for patients with oropharyngeal
pharyngeal candidiasis were 92% (83%-97%), 82% (57%— candidiasis and C. albicans infection and that the available
242 Rex et al. CID 1997;24 (February)
Table 5. Tentative interpretive guidelines for susceptibility testing mostly mucosal infection (table 3). Second, inclusion of this
in vitro of Candida isolates. value in either the S or S-DD range is unlikely to ever affect
patient care in a meaningful way: patients with invasive infec-
Range of MICs (pg/mL) per category
tions are treated with larger doses of fluconazole (thus render-
Antifungal Susceptible– ing the distinction between S and S-DD moot), whereas MICs
agent Susceptible (S) dose dependent (S-DD)* Resistant (R) are normally used in a post-hoc fashion for patients with muco-
sal infection. It should be noted, however, that the inclusion
Fluconazole ._8 16-32 64
of the value of 8 pg/mL in the S-DD range is otherwise just
Itraconazole -.0.125 0.25-0.5 -1
as reasonable as its inclusion in the S group.
NOTE. Shown are the tentative interpretive breakpoints (pg/mL) for flu-
conazole and itraconazole when the MIC is determined by M27-T. For fluco- D
nazole, these guidelines are based substantially on experience with mucosal Case Study Number 2: Itraconazole and Candida o
w
infections but are consistent with the limited information for invasive candida n
infections in nonneutropenic patients. Isolates of C. krusei are assumed to be The data package on itraconazole contained MIC and out- loa
d
intrinsically resistant to fluconazole, and their MICs should not be interpreted come data for 355 Candida species isolates (87% C. albicans, e
d
wsuistche tphtiibs lsec raalneg. eI to ifs f laulcsoon paezrotlien eisn nt otht aktn tohwen 8 w pi,tgh/ cmeLrta uinptpye—r tbhoeu dnadta f owro tuhled 9% C. glabrata, 2% C. krusei, and 2% other species) from 316 fro
m
permit selection of either 4 pg/mL or 8 pg/mL for this cutoff (see text). For episodes of infection in 316 different patients enrolled in four h
itraconazole, the data are based entirely on experience with mucosal infections, trials of itraconazole solution as therapy for oropharyngeal can- ttp
aabnlde .data supporting breakpoints for invasive candida infections are not avail- didiasis in patients with AIDS. While it is not currently indi- s://a
* Susceptibility is dependent on achieving the maximal possible blood level. cated for treatment of oropharyngeal candidiasis, such approval ca
d
For fluconazole, doses of 400 mg/d or more may be required for adults with seems likely, and the committee has analyzed the data package em
normal renal function and habitus. For itraconazole, measures to ensure ade- on this basis. Details on one of these trials have been presented ic
quate drug absorption and plasma itraconazole concentrations of ...--0.5 kig/mL .o
may be required for optimal response. If MICs are measured with use of a in abstract form [104], while publication of the other three up
scale that yields results falling between categories, the next higher category is trials is pending. The MIC of itraconazole for the isolates was .co
implied. Thus, an isolate for which the MIC of fluconazole is 12.5 pg/mL m
would be placed in the S-DD category. These breakpoints were adopted at an measured according to the NCCLS M27-T broth macrodilution /cid
open meeting of the subcommittee held 1 June 1996 in Reston, Virginia. methodology [16]. The clinical outcomes for the patients were /a
Pursuant to NCCLS policy, these breakpoints are open for public commentary assessed by generally accepted clinical criteria. As with the rtic
for a period of 1 year. le
data for fluconazole, the itraconazole data have been analyzed -a
b
following the precepts outlined above. s
tra
c
t/2
data for correlating MIC with outcome are much more limited 4
Pharmacology of Itraconazole /2
for non-albicans candida infections and for invasive candida /2
3
infections. Itraconazole is a triazole antifungal with complex, nonlinear 5/4
In proposing breakpoints, the subcommittee also considered pharmacological behavior. A single 100-mg dose of itracona- 24
4
the settings in which MIC testing could and should be used. zole produces peak plasma levels measured by HPLC of —0.35 4
4
Routine testing of mucosal isolates is an unnecessary expense, pg/mL. Increasing the dose to 200 mg increases the peak level by
g
and most microbiology laboratories will likely institute policies to —0.55 p,g/mL. At steady state, 200 mg/d of itraconazole u
e
that specify susceptibility testing only for selected mucosal produces peak plasma levels of 1.4-2 p,g/mL with a terminal st o
isolates and isolates from normally sterile body sites. In both half-life of —60 hours (Janssen Pharmaceutica, data on file). n
0
of these circumstances, the purpose of testing will generally Increasing the dose generally results in increased plasma levels, 6 M
be to determine if a large dose of fluconazole (400-800 but both oral bioavailability and hepatic clearance are unpre- a
y
mg/d or 6-12 mg/[kg • d] for adults with normal habitus and dictable, and determination of plasma levels may be necessary 20
1
renal function) is likely to fail. . to optimize therapy [105, 106]. The recently developed itracon- 9
Within this context, tentative fluconazole breakpoints of .-._ 8 azole solution in cyclodextrin appears to resolve this problem
p,g/mL as susceptible and _.--64 p,g/mL as resistant appear rea- at least partially [107].
sonable (table 5). Isolates inhibited by fluconazole at concentra- Itraconazole has an active metabolite, hydroxy-itraconazole,
tions of 16-32 p,g/mL are clearly in a range that responds that results in blood levels measured by bioassay that are ap-
to increased doses of fluconazole, and the subcommittee thus proximately triple those measured by HPLC [108]. Itraconazole
proposed the novel category S-DD (susceptibility—dose depen- is 99.8% protein-bound [108]. Because of its lipophilicity, tis-
dent) to describe these isolates. Isolates for which MICs are 8 sue distribution of itraconazole is related to lipid content, and
.tg/mL present a problem due to the relative paucity of data body fluids with high water content (e.g., CSF, saliva, and
for this MIC, and it was elected to include the value of 8 vitreous fluid) contain insignificant amounts of itraconazole
kig/mL in the S range for two reasons. [109, 110]. Skin concentrations may be 2-10 times higher than
First, all MICs up to and including 8 p,g/mL do include plasma concentrations [111]. Currently used doses span the
100% success in their 95% confidence interval for success range of 200-400 mg/d, and the maximum readily tolerated
when 100 mg/d is used as therapy in a group of patients with dose is —600 mg/d [106, 112].
CID 1997;24 (February) Interpretive Breakpoints for Antifungal Susceptibility Testing 243
Table 6. Success rate of treatment with itraconazole, as related to A limited amount of published data suggest that patients
the M27-T-determined MIC of the drug. with AIDS who are infected with strains of C. albicans with
decreased susceptibility to fluconazole may respond to elevated
Itraconazole blood level
doses of itraconazole [107, 120, 121]. These data, plus what
µg/mL >0.5 µg/mL is known about the pharmacokinetics of itraconazole, made it
reasonable to stratify the analysis of outcome by both MIC and
MIC (mg! Success, % Success, % plasma itraconazole level, and the committee elected to focus
mL) Success/n* (95% Cr) Success/n* (95% CP) on this analysis. Plasma levels were available for 264 patient-
episode-isolate events and ranged from zero to 3 µg/mL. The
-...0.015 23/25 92 (74-99) 10/11 91 (59-100)
0.03 22/24 92 (73-99) 19/22 86 (65-97) average plasma level was 0.55 µg/mL.
0.06 34/40 85 (70-94) 25/27 93 (76-99) Analyses stratifying outcome by plasma level and using cut- D
o
0.125 13/16 81 (54-96) 16/19 84 (60-97) offs of 0.2 µg/mL, 0.5 .tg/mL, and 0.8 ug/mL were considered, w
n
0.25 7/11 64 (31-89) 10/12 83 (52-98) but all demonstrated a similar pattern that is well represented loa
0.5 6/15 40 (16-68) 6/9 67 (30-93) d
by the detailed analysis in table 6 and the summary analysis e
u12 25//610 2803 (3(36--51060)) 47//710 7507 ((3158--9930)) in table 7, using a cutoff of 0.5 µg/mL. The success rate of d fro
m
Total (n) 147 117 itraconazole is 81% for those patients infected with isolates h
inhibited by -_0.125 µg/mL, and the outcome appears indepen- ttp
t* T Nhoe. 9o5f% tr eCaItsm feonr tt hsue cpceerscseenst/atogteasl onfo e. voefn ptsa tsiuencct-eespsifsuolldye t-riesoatleadte w eevreen ctsa lc(un-). dent of plasma itraconazole level. Among patients with plasma s://a
c
lated with use of a binomial approximation technique [96]. levels µg/mL, the success rates decrease for those patients ad
infected with isolates for which MICs are 0.25-0.5 .tg/mL em
(50%) and 1.0 µg/mL (44%). ic.o
u
Itraconazole MICs and the Results of Animal Studies In contrast, the association of decreased success and in- p
.c
creased MIC, although apparent, is less striking among patients o
When measured by the NCCLS M27-T methodology, itra- m
with plasma levels >0.5 pg/mL. Similar results were seen if /c
conazole MICs exhibit predictable patterns that are characteris- id
the analysis was limited to the C. albicans strain present in all /a
tic for each species of Candida. The vast majority of isolates but two of the patients (data not shown). This suggests that rtic
are inhibited by <1 .tg/mL. Itraconazole MICs are lowest for le
although infections due to isolates for which itraconazole MICs -a
bCr.a atlab aicnadn Cs (.M krIuCs9e0i, —(M0I.C19205, —ktg1/-m2 La)g a/mndL h) i[g8h8e-s9t2 f]o. rI sCo.l agtleas- are µg/mL are less likely to respond than those due to bstra
more susceptible isolates, the therapeutic effect may be en- c
for which the MICs of fluconazole are elevated usually also t/2
hanced by ensuring that plasma levels exceed 0.5 µg/mL. It is 4
require higher concentrations of itraconazole for inhibition /2
worth emphasizing that these data apply only to oropharyngeal /2
[113-116]. In the few instances where isolates for which MICs 3
of both fluconazole and itraconazole were elevated have been candidiasis, and there are as yet no data from patients with 5/4
tested in animal models, treatment with itraconazole has failed invasive candida infections. 244
4
to influence the infection [117, 118]. 4 b
When determining itraconazole MICs, it is important for the Tentative Itraconazole Breakpoints y gu
operator to be aware that suitable solvents must be used when e
s
preparing serial dilutions of water-insoluble compounds such as As with fluconazole, consideration of the overall clinical data t o
n
itraconazole. In one well-studied example, use of aqueous media and their correlation with the pharmacokinetics of itraconazole 0
6
during serial dilution of a water-insoluble compound led to deliv- allows us to conclude that response of oropharyngeal candidia- M
a
ery of < 10% of calculated amount of drug [119]. Overestimation y 2
of the MIC would result from such an error, and one procedure for 01
9
properly preparing dilutions of itraconazole and similar insoluble
Table 7. Summary analysis of critical itraconazole MIC ranges.
drugs is given in the published methodology [16].
Itraconazole plasma concentration
Clinical Data Correlating Itraconazole MIC with Outcome
0.5 µg/mL >0.5 µg/mL
Analyses of rates of treatment success vs. MICs are given
in table 6. As with the analysis for fluconazole, each separate MIC No. of Success, % No. of Success, %
(µg/mL) events* (95% CV) events* (95% CIt )
isolation from each patient was considered as a separate event.
Unlike the fluconazole analysis, however, 98% of the patient- 105 88 (80-93) 79 89 (79-95)
isolate events in the database were treated with itraconazole at 0.25-0.5 26 50 (30-70) 21 76 (53-92)
a dosage of 200 mg/d. Although this precluded an analysis of 16 44 (20-70) 17 65 (38-86)
outcome by dosage, itraconazole plasma levels determined by
* Total no. of patient-episode-isolate events.
HPLC were available for many of the patients, and this allowed t The 95% CIs for the percentages of events successfully treated were calcu-
an analysis of outcome stratified by plasma concentration. lated with use of a binomial approximation technique [96].
244 Rex et al. CID 1997;24 (February)
sis to itraconazole does vary with MIC as determined by the data for systemic infections in neutropenic patients) is notewor-
M27-T methodology. Based on the available data, tentative thy, and collection of additional data is certainly warranted.
itraconazole interpretive breakpoints of -_ 0.125 pg/mL as sus- Third, the data are at present inadequate to allow interpreta-
ceptible and ._- 1.0 pg/mL as resistant are reasonable (table 5). tion of fluconazole and itraconazole MICs for fungi other than
Because infections due to isolates for which the itraconazole Candida. Recent data have demonstrated that this may be possi-
MICs are 0.25-0.5 fig/mL respond more often if higher itraco- ble for fluconazole against C. neoformans and Histoplasma
nazole plasma levels are ensured, the subcommittee proposes capsulatum [81, 123], but more work is needed in this area.
that these isolates be placed in the S-DD category. Fourth, interpretive criteria for the other principal antifungal
agents (flucytosine and amphotericin B) are also needed. All
of these areas are currently under intensive study.
Conclusions and Future Directions
D
o
Evidence to support the clinical relevance of antifungal sus- w
n
ceptibility testing is accumulating at a slow but steady pace. Acknowledgments loa
d
With the advent of standardized antifungal susceptibility testing e
d
methods, it is now possible to perform large-scale surveys of The authors thank Drs. C. Douglas Webb and Daniel J. Sheehan fro
m
clinical isolates [122]. Such surveys may be used to develop of Pfizer, Inc., U.S. Pharmaceuticals Group, and Drs. Bruce h
population-distribution profiles of MIC values for antifungal Moskovitz and Jane Wu of Janssen Pharmaceutica for assistance ttp
s
agents against various species of fungi and to begin to correlate with collection and analysis of secondary information supporting ://a
MIC data with clinical response. interpretation of the data packages. ca
d
Ideally, the selection of interpretive breakpoints for antimi- em
crobial agents is based on a combination of clinical outcomes, ic
.o
pharmacokinetic data, and the susceptibility profiles in vitro of References up
.c
the organisms of interest. However, as reviewed in detail above, o
1.Anaissie EJ, Bodey GP, Rinaldi MG. Emerging fungal pathogens. Eur J m
no method of breakpoint determination is perfect, and integra- Clin Microbiol Infect Dis 1989;8:323-30. /cid
tion of multiple pieces of information is required. The pre- 2. Banerjee SN, Emori TG, Culver DH, et al. Secular trends in nosocomial /a
dictive power of even the best interpretive breakpoints should primary bloodstream infections in the United States, 1980-1989. Am rtic
not be overestimated, and it is imperative that continued efforts J Med 1991; 91(suppl 3B):865 -9S. le-a
3. Fisher-Hoch SP, Hutwagner L. Opportunistic candidiasis: an epidemic b
be made to develop additional clinical data to support any s
of the 1980s. Clin Infect Dis 1995;21:897-904. tra
proposed interpretive breakpoints for antifungal susceptibility c
4. Galgiani JN, Reiser J, Brass C, Espinel-Ingroff A, Gordon MA, Kerkering t/2
testing. TM. Comparison of relative susceptibilities of Candida species to three 4
/2
These caveats understood, the tentative interpretive antifungal agents as determined by unstandardized methods. Antimi- /2
breakpoints proposed herein for fluconazole and itraconazole crob Agents Chemother 1987;31:1343-7. 35
versus Candida isolates are a step toward the application of 5. Caalnhtoifuunn gDaLl ,s uRsocbepetritbsi lGitDy t, eGstasl igni athnei JUNn,i teetd a Sl.t aRteess uanltds ionft ear lsaubrovreayto oryf /4244
antifungal susceptibility testing in the clinical arena. Selective 4
comparison of broth dilution testing of flucytosine and amphotericin 4
application of testing in vitro may be useful clinically as an B. J Clin Microbiol 1986;23:298-301. by
adjunct to other clinical and experimental data in determining 6. Rex JH, Pfaller MA, Rinaldi MG, Polak A, Galgiani JN. Antifungal gu
e
the course of action in a difficult case; however, we do not susceptibility testing. Clin Microbiol Rev 1993; 6:367-81. s
7. Espinel-Ingroff A, Kerkering TM, Goldson PR, Shadomy S. Comparison t o
recommend that antifungal susceptibility testing be performed n
study of broth macrodilution and microdilution antifungal susceptibil- 0
as a matter of routine. 6
ity tests. J Clin Microbiol 1991;29:1089-94. M
A number of areas for future work are immediately evident. 8. Guinet R, Nerson D, De Closets F, et al. Collaborative evaluation in ay
First, the committee is currently considering preliminary infor- seven laboratories of a standardized micromethod for yeast susceptibil- 2
0
mation that suggests that reading MICs after 24 hours rather ity testing. J Clin Microbiol 1988;26:2307-12. 19
9. Shawar R, Paetznick V, Witte Z, Ensign LG, Anaissie E, LaRocco M.
than 48 hours of incubation may improve the degree of correla-
Collaborative investigation of broth microdilution and semisolid agar
tion with outcome in vivo. If this observation is confirmed,
dilution for in vitro susceptibility testing of Candida albicans. J Clin
then shorter incubation times will be strongly preferred by both Microbiol 1992; 30:1976 -81.
clinicians and clinical laboratories. Such a change to the 10.Polak A. Multicenter-Studie zur Standardisierung der Empfindlich-
M27-T methodology will require development and validation keitsbestimmung von Pilzen gegen 5-Fluorcytosin and Amphotericin
B. Mykosen 1987; 30:306-314.
of appropriate quality control information, however.
11.Espinel-Ingroff A, Kish CW Jr, Kerkering TM, et al. Collaborative com-
Second, the strength of the in vivo-in vitro correlation is
parison of broth macrodilution and microdilution antifungal suscepti-
poor for isolates at the higher MIC values, for non-albicans bility tests. J Clin Microbiol 1992;30:3138-45.
Candida isolates, and for systemic infections. While the pro- 12.Fromtling RA, Galgiani JN, Pfaller MA, et al. Multicenter evaluation of
posed fluconazole breakpoints are based on reasonable extrapo- a broth macrodilution antifungal susceptibility test for yeasts. Antimi-
crob Agents Chemother 1993; 37:39-45.
lations from the outcomes of patients with oropharyngeal candi-
13. Pfaller MA, Burmeister L, Bartlett MS, Rinaldi MG. Multicenter evalua-
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of data for systemic infections (and especially the absence of 1988;26:1437-41.
Description:University of Texas Medical School, Houston, Texas; Department of. Pathology, University new therapies for fungal infections with the introduction of.
