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Present status of the detection of antifungal resistance: the perspective

from both sides of the ocean

M. Cuenca-Estrella and J . L. Roddguez-Tudela

Servicio de Micologia, Centro Nacional de Microbiologia, Instituto de Salud Carlos 111, Majadahonda, Madrid, Spain

The NCCLS reference methodology for antifungal susceptibility testing is a new milestone of the evolution of
medical mycology. The use of t h s methodology however, is not problem-fi-ee. At present, major limitations
are a trailing phenomenon with azoles, unreliable detection of resistance to amphotericin B, poor growth of
some organisms and unpractical procedures for the clinical laboratory. Herein a overview of NCCLS
guidelines for yeasts and filamentous fungi is presented. Likewise, a review of studies conducted trying to
overcome the limitations of reference procedures is also included. Several alternative approaches are reviewed
as alternative meha, inoculum size and incubation time. Modifications of reading procedure and endpoint
determination are also evaluated. Agar di&sion methods and other methods for susceptibihty testing are cited.
Finally, we discuss the data on correlation of the in vitro results with the in vivo activity.
Clin Microbiol Znfect 2001: 7 (Supplement 2): 46-53

stuhes have been conducted to try to overcome the

limitations. Colorimetric, spectrophotometric and agar d i 6 -
Over the years, many studies on in vitro susceptibility of fungi sion methods have been proposed as modifications of reference
have been reported [l]. Their findings presented basic techniques [3]. The European Committee on Antibiotic
hmitations due to interlaboratory variations and the lack of Susceptibdity Testing (EUCAST) Antifungal Susceptibility
correlation between in vitro and in vivo activity [2,3]. However, Testing Subcommittee has performed several studies and is
significant progress has been made in t h s field. Susceptibility conducting a cooperative study to elaborate a method for
testing of fungi has recently been standardized by the National testing the antifungal susceptibihty of fermentative species of
Committee for Clinical Laboratory Standards (NCCLS), yeasts. Societies of Clinical Microbiology in several European
Subcommittee on Antifungal Susceptibility Tests. At present, countries are also carrying out collective works [8,9]. The aim
these testing methods have only been standardized for Candida of all the studies in progress is to develop a practical reference
spp. and C. neofoovmans (document M27-A) [4], and guidelines methodology for clinical laboratories and a useful tool to
have been proposed for filamentous fungi (document M38-P) predct the outcome of therapy with an antifungal agent.
[5]. The broth dilution tests proposed by the NCCLS feature Here, we present an overview of the NCCLS guidelines for
good interlaboratory reproducibdity. This agreement is yeasts and filamentous fungi, their limitations and some
essential to identify organisms unlikely to respond to certain modifications of these reference methods. We also review
antifungal treatments. the data on the correlation of the in vitro results with in
The use of NCCLS methodology is not problem-free. At vivo activity.
present, the major limitation is the trading phenomenon that
makes visual determination of azole MICs more difficult,
because of partial idubition of fungal growth [6,7]. Some
Antifungal susceptibility testing for yeasts

In 1985, the NCCLS published its first report, in which the

Corresponding author and reprint requests: M. Cuenca-Esaella,
results of a small collaborative study were presented [lo]. Since
Servicio de Micologia, Centro Nacional de Microbiologia, Instituto
de Salud Carlos 111, Ctra Majadahonda-Pozuelo Km 2, 28220 then, a reproducible reference procedure of antifungal
Majadahonda, Madrid, Spain susceptibility testing for yeasts has been developed. Several
Tel.: +34 91 509 7961
cooperative works were conducted to ascertain inoculum
Fax: +34 91 509 7966
E-mail: preparation, inoculum size, choice among several synthetic

0 2001 Copyright by the European Society of Clinical Microbiology and infectious Diseases, CM/, 7 (Suppl. 2). 4653
Cuenca-Estrella and Rodrigez-Tudela Antifungal susceptibility testing 47

media, temperature of incubation, duration of incubation, Obviously, a great deal of effort has gone into the
end-point determination, reference MIC ranges for quality development of the standardized method for antifungal
control strains, and breakpoints for available antifungal agents susceptibility testing and the reference techniques are now
[2-4,11,12]. These studies established a broth macrodilution more reliable and reproducible. However, the use of this
reference procedure. A microdilution method as a modifica- methodology still has some limitations. At present, the main
tion of the macrodilution technique was also developed in problem is the determination of MICs for azoles. This is subject
order to be more practical for the clinical laboratory [13]. to variable interpretation, because of the trailing phenomenon
As a result, the NCCLS document M27-A was published caused by partial inhibition offungal growth [18,20]. As already
[4]. It describes both macrodilution and microdilution stated, visual endpoint determination is a subjective operation,
methods for the antifungal susceptibility testing of Candida particularly with fungistatic agents. Quantification of endpoint
spp. and C. neoformans. Drug stock preparation, storage and determination and solution agitation do not settle the issue. For
dilution techniques are similar to antibacterial testing proce- some isolates, trailing growth is so significant that the MICs after
dures with minor modifications. A completely synthetic 24 h are much lower than after 48 h. Furthermore, a worse
medium is recommended for susceptibility testing. Undefined aspect is that some strains are susceptible to fluconazole and
media can give rise to widely varying results [6]. For this itraconazole (MIC < 8 mg/L and MIC 6 0.12 mg/L
reason, the synthetic medium RPMI-1640 with 1-glutamine respectively) at 24 h, but resistant at 48 h (264 mg/L and > 1
buffered to pH 7.0 with 3-[N-morpholino]-propanesulfonic mg/L) [18]. The relevance of this discordance is unknown but
acid (MOPS) is advocated by the NCCLS. The pH must be evidence suggests that lower MICs correlate most closely with
strictly controlled. Inoculum preparation follows a spectro- the outcome in vioo [21]. MIC determination at 24 h only has
photometric method at 530 nm, and tubes or plates are been proposed to solve the problem [14,16] but numerous
inoculated with a final inoculum of 0.5-2.5 x lo3 CFU/mL. strains show little or no discernible growth after 24 h of
It has been indicated that higher inocula could increase the incubation [22]. Other authoe have addressed inoculum size,
MICs for the drugs tested, and smaller inocula were associated reading method or medium p H [14,15,2>25]. Several studies
with improvements in interlaboratory reproducibihty [14,151. have used spectrophotometric determination of endpoints to
Plates or tubes are incubated at 35 "C for 48 h for Candida spp. eliminate such subjective interpretation [19,20,24].Marr et al.
and for 72 h for C. neofrmanr. reported that adjustment in the pH ofmedia to acidic conditions
The NCCLS method recommends visual reading but this is @H4.5) can reduce trailing in C. albicans without aEecting the
an important source of variability and inaccuracy [16]. For MICs of fluconazole [25]. More comprehensive studies are
amphotericin B, a fungicidal drug, endpoints are easily defined needed to resolve the phenomenon of trailing and to ascertain
and MIC is the lowest drug concentration that prevents any its effects on clinical response.
discernible growth compared with the growth control (drug-free A second major limitation is poor growth of C. neoformans
tube or well). However, fungistatic agents (such as azoles and other nonfermentative yeasts with media recommended
ketoconazole, fluconazole, itraconazole and flucytosine) show by the NCCLS 126,271. Therefore, several authors have
endpoints less clearly defined and introduce sigyficant sub- advised a number of different ways of overcoming the
jectivity in reading results [15,17,18]. The document M27-A problem. Media such as buffered yeast nitrogen base (BYNB)
proposes to quanti+ endpoint determination, so MIC is defined [26] or RPMI supplemented with 2% glucose have been
for the macrodilution procedure as an 80% decrease in turbidity employed [28]. Better rates of growth were obtained with
as compared with growth control. For broth microdilution, BYNB, and MICs were not falsely elevated [29]. Odds et al.
MIC is detem7ined according to the 0-4 scale, as follows: demonstrated that oxygen is a limiting factor for C. neDfomans
growth in liquid media [30] and cultivation under constant
0 0, optically clear;
agitation has been recommended [28].
0 1, slightly hazy;
The other major limitation of the NCCLS methods
0 2, prominent decrease in turbidity;
described for antifungal susceptibility testing is reliable
0 3, slight decrease in turbidity;
detection of resistance to amphotericin B [4]. RPMI medium
0 4, no reduction in turbimty.
yields a range of MICs that span only three or four twofold
MIC is defined as the lowest concentration of drugs with serial dutions [7]. This short range precludes reliable
w h c h the score is < 2. The quantification of endpoint discrimination between susceptible and resistant isolates.
determination and agitation of solutions before reading led to Current reports suggest that testing with Antibiotic Medium
more reproducible endpoints determination [19]. Quality 3 (AM3) instead of RPMI permits us to enhance the ability to
control procedures and reference strains are also included in detect it [31]. However, the reproducibility of this method is
the document [4]. still under study, owing to lot-to-lot variability of AM3 [32].

0 2001 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 7 (Suppl. 2). 46-53
48 Clinical Microbiology and Infection, Volume 7, Supplement 2. 2001

Despite its limitations, the NCCLS reference methodology breakpoints are not avadable but some degree of correlation
for antifungal susceptibility testing is a new d e s t o n e in the has been demonstrated between in uitro test results and
evolution of medical mycology. It is possible, even likely, that response to treatment in animal models p,35].
new antifungal agents require modified methods or that new The limitations in standardized susceptibility testing for
assays will show a better correlation between in vitro and in viuo yeasts are considerable, and developing a reference methodol-
susceptibility results and patients' outcomes. However, at ogy for filamentous fungi represents a major challenge with
present, the NCCLS procedure is the most reliable and unsolved problems. First, the incubation period for obtaining
reproducible methodology, and any progress in this field must the inoculum lasts several days, which is a major limitation for
take it into account. the clinical susceptibdity testing of moulds [2,3]. In addition,
the document M38-P describes guidelines for conidmm-
forming moulds, and conima are small spheres that are easily
Antifungal susceptibility testing for filamentous fungi
quantifiable [5]. However, obtaining conidial suspensions is
The document M38-P describes a method for testing the not possible when the moulds present germinate to hyphal
susceptibility to antifungal agents of filamentous fungi forms. Preparation of hyphal or germinated sporangiospores
(moulds), including Aspergillus spp. Fusarium spp., Rhizopus suspensions is difficult and limited data suggest that t h s is an
arrhizus, Pseudallescheria boydii and Sporothrix schenckii [5]. These important source of discrepancies [36,37]. Collaborative
species are conidium-forming moulds. A working group was studies have not been conducted for nonconidium-forming
formed and charged with the responsibility of carrying out moulds and published results are less consistent in reliabihty
studles to collect data and to propose a way to perform than data obtained with species included in the proposed
susceptibility testing of those fungal species. As a result of two reference method [2,3,38].
collaborative works, the document was published [33,34]. Finally, as mentioned in relation to yeasts, visual reading
The M38-P methodology adopts some of the steps in yeast introduces subjective interpretations of susceptibility results.
testing [4]. The guidelines include both macro- and micro- Using the conventional criterion of complete growth inhlbi-
dilution methods [5]. Drug stock preparation and dilution tion inaccuracy is minimized, but the NCCLS recommends
procedures are similar to the yeast techniques. The synthetic quantification of endpoint determination in order to avoid the
medium RPMI-1640 is also advocated. Fungi are grown on trailing phenomenon with fungistatic agents [5,33]. Data
potato agar slants at 35 "C for 7 days. Inocula are prepared by a recently reported have revealed that 100%inhibition instead of
spectrophotometric procedure at 530 nm, and tubes or plates 50% can be the optimal testing condition to improve
are inoculated with a final suspension of approximately 0.4- reproducibility with azoles and even to detect potential azole
5 x lo4 CFU/mL [7]. It has been indicated that inoculum resistance in moulds. These refined gmdelines and results
preparation as specified here provides the most reproducible obtained by the evaluation of other species of fun@ will be
MIC data. Table 1 displays the optical density range at 530 nm included in a new NCCLS document (M38-T) [7].
correlated with inoculum size for filamentous fungi included
in document M38-P. Plates or tubes are incubated at 35 " C for
72 h and examined daily. Readings are obtained with the aid
of a mirror and endpoint determination adopts criteria The reference method already mentioned is not the best
developed for antifungal testing of yeasts [3]. Interpretative technique for testing all organisms or the most convenient

G a b l e 1 Optical density ranges and inoculum

Species No. Optical density lnoculurn size
sizes for filamentous fungi included in M38-P
observations range mean ( x 10' CFU/mL) dncumsnta

Aspergillus flavus 105 0.09-0.1 1 1.6

Aspergillus fumigatus 104 0 . 0 9 4 . 11 2.7
Fusarium oxysporum 105 0.15-0.1 7 3.0
Fusarium soiani 103 0.15-0.1 7 1.8
Pseudallescherisboydii 99 0.15-0.1 7 1 .o
Rhizopus arrhizus 99 0.1 5-0.1 7 1.3
Sporothrix schenckii NAb 0.09-0.1 1 2.4
Paecilomyces variotii NA 0.11-0.17 1 .1
(ATCC 22315)

aData are from ref. [3].

bNA. not applicable.

Q 2001 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, C M , 7 (Suppl. 2), 46-53
Cuenca-Estrella and Rodrigez-Tudela Antifungal susceptibility testing 49

procedure for routine use in clinical laboratories. As a "C [28]. Some reports have indicated that the maximum rate of
consequence, a number of modifications of the NCCLS growth of C. neojbmw is observed at 30 "C [40]. These reports
methodology have been proposed. Several alternative ap- demonstrate the need for a more reliable and reproducible new
proaches have been evaluated, including techniques used for reference methodology of antihngal susceptibility testing of C.
antibacterial susceptibility testing. In particular, microdilution neofotvnans and other nodermentative yeasts.
procedures are employed in these modifications. Another modification is the supplementation of RPMI with
2% glucose for all organisms [22]. It has been suggested that
glucose supplementation may simplifi endpoint determina-
Alternative media, inoculum size and incubation time
tion, because of higher turbidity of the growth control [14,16].
The document M27-A includes some alternative media for In addition, numerous strains show no discernible growth after
special circumstances but indicates that the utility of these 24 h of incubation when the NCCLS reference method is
modifications remains to be established. These alternative employed, and FWMI-2% glucose has the advantage of
medla are AM3, BYNB and RPMI supplemented with 2% shortening the incubation time [41]. Growth increases when
glucose [4]. Alternative media have been accepted, because of hgher inoculum sizes are employed (lo4 or lo5 CFU/mL)
limitations of the reference techmque [2,6]. and MICs are not falsely elevated [42,43]. Thus, the
The M27 methodology does not permit consistent detec- Antihngal Susceptibility Testing Subcommittee of EUCAST
tion of isolates resistant to amphotericin B. Several works is c q n g out a collaborative study comparing M27-A
suggest that testing with AM3 supplemented with 2% glucose procedures with a new method for the determination of
permits more reliable detection of these isolates, but AM3 is MIC by broth dilution of fermentative species of yeasts. This
not standardized and substantial lot-to-lot variabibty has been methodology includes RPMI-2% glucose and an inoculum
observed [31,32]. Variabihty is a basic limitation for size of lo5 CFU/mL. Figure 1 and Table 2 display preliminary
reproducibility. Thus, other defined synthetic medla could data of the EUCAST cooperative work. The MICs for quality
represent alternatives to detect resistance to amphotericin B. control strains are similar to the NCCLS reference values, and
Preliminary data suggest that Iso-Sensitest could distinguish
susceptible and resistant isolates. Iso-Sensitest is a semidefined
medium for antimicrobial susceptibility testing in which
undefined components are kept to a minimal level [MI.
Ghannoum et ul. reported that the use of BYNB media may
enhance the growth of C. Neofomuns [26]. This yeast exhibits
poor growth with RPMI-1640. Thus, BYNB and an inoculum
of 10" CFU/mL have been defined as the optimal method for
determining the susceptibility of C. neoformans [29]. Inoculum 0 4 8 12.18 20 24 28 92 38 40 44 48

effects in determining MICs of antifungal agents were not IncubanonamelnhQur

observed and improvements in the clinical relevance of Fig 1 Growth kinetics of 24 clinical isolates of Candida spp. (six
antihngal MICs have been found. Other authors have different species). Comparison between NCCLS reference methodol-
recommended the cultivation of the microdilution trays under ogy (RPMI and inoculation of lo3 CFU/mL) and new method,
constant agitation and an inoculum size of lo5 CFU/mL at 35 including 2% glucose and lo5 CFU/mL. OD, optical density.

Table 2 In vitro data on qulaity

control strains Candida krusei Organism Antifungal NCCLS reference RPMI-2% glucose and
ATCC 6258 and Candida agent MIC range (mg/L) lo5 CFU/mL M I C range (rng/L)
parapsilosis ATTC 22019
Candida krusei Amphotericin 6 0.52.0 0.5-1.O
ATCC 6258 Fluconazole 16-64 16-32
ltraconazole 0.12-0.5 0.12-0.5
Ketoconazole 0.1245 0.0601500.25
Flucvtosine 4.0-16 4.0-8.0

Candida parapsilosis Amphotericin B 0.25-1 .O 0.25-1.O

ATCC 2201 9 Fluconazole 2.0-8.0 1.0-4.0
ltraconazole 0.06-0.25 0.12-0.25
Ketoconazole 0.06-0.25 0.06-0.25
Flucytosine 0.12-0.5 0.12-0.5

0 2001 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 7 (Suppl. 2).46-53
50 Clinical Microbio/ogy and Infection, Volume 7. Supplement 2, 2001

the growth kinetics show that 24 h is an optimal incubation The indicator aids in the determimoon of visual MIC
period for MIC determination. These data could confirm that endpoints and could be a more convenient procedure than the
the M27-A technique modified with 2% glucose and higher standard method for use in the clinical laboratory. The agreement
inocula offers the advantages of reducing the incubation time of colorimetricmicrodilution MICs with the NCCLS reference
and simplifying endpoint determination, without sigmficant procedures has been acceptable, with the exception of some
inoculum effects in determining MICs. species (C. albicans for azoles and flucytosine or C.n e o f m n s for
Alternative mema are not described in the document M38- amphotericin B) [49]. For filamentous fungi, colorimetricMICs
P for moulds. However, it has been suggested that inoculum have also been determined and excellent agreement has been
preparation by a spectrophotometric procedure can be a observed. Moreover, colorimetric MICs afier 48-72 h of
source of inaccuracy, owing to the color and size of spores, incubation with an inoculum size of lo4 CFU/mL have been
which are species dependent [36-381. Therefore, individual defined as optimal testing conditions and proposed as guidelines
features of strains could have an influence on the optical for a reference method for testing moulds [34].
density of inoculum suspensions. Reports have indicated that
inoculum size should be adjusted by using a hemacytometer
Agar diffusion and other methods for susceptibility testing
cell-counting chamber [44,45]. Inoculum quantification has to
be performed by plating serial dilutions of suspensions. Other Disk-diffusion tests and E-test strips (As Biodisk, Solna,
authors have reported that the hemacytometer count is less Sweden) have been employed for antifungal susceptibdity
consistent in between-laboratory tests than the spectrophoto- testing. These procedures are straightforward, economical to
metric procedure [6]. RPMI-2% glucose and a higher perform and offer a practical method for the clinical laboratory
inoculum size have also been employed for antifungal [2,3]. However, agreement with reference methods is species
susceptibility testing of moulds, being an acceptable means and medium dependent. Disk-diffusion tests, such as Neo-
of evaluating the in vitro activity of antifungal agents against sensitabs (Rosco Diagnostica, Taastrup, Denmark), have been
these organisms [46,47]. employed in several studies [43,50,51]. Most of these
exclusively evaluated disks of fluconazole and Candida spp.
Work has also been conducted to assess disk-ditrusion
Modifications of reading procedure and endpoint determination
susceptibility testing of dermatophytes [52]. E-test strips have
An alternative to the reference method of visually gradmg been more extensively analyzed [2,3,53,54].This appears to be
turbidity is the determination of endpoints with automated a suitable alternative procedure for testing the susceptibility of
spectrophotometric methods. Microtiter trays and automated yeasts and moulds to azoles and amphotericin B. However,
reading allow the determination of endpoints at hfferent levels poor agreement with the reference methods has been reported
of growth inhibition [14]. Several studies have assessed various for Candida glabrata, C. tropicalis, C. neofomzans and some
levels of growth inhibition, in comparison with that of the filamentous fungi. At present, the role of agar diffusion
drug-free control well [15,19,20,24]. Levels of 50% with the methods is not clearly defined, although it may have promise.
azoles and 80-90% with amphotericin B have provided the Other methodologes for testing the susceptibihty of yeasts
most accurate values [3]. Excellent agreement has been and moulds involve either direct determination of cell mass or
observed between the results of the spectrophotometric and cell viability by fluorescence, or indirect determination of cell
standard methods, but most studies tested exclusively C. mass by calorimetry, analysis of ATP production or radiometry
albicans. Therefore, the collaborative work of the Antifungal [55-571. Expensive equipment is required, some methods are
Susceptibility Testing Subcommittee of EUCAST is assessing very laborious and some involve subjective interpretation.
both automated and visual methods with several yeasts species. Agreement with reference procedures is not evaluated in some
A novel modification is the use of a colorimetric oxidation- of published reports. More comprehensive studies are needed
reduction indicator [2]. This technique is used in bacterial to determine the usefulness of these methodologies.
reference methods and allows the determination of viable cells. Finally, several novel commercial systems for antifungal
Several indicators have been employed, but only the use of susceptibility testing have been developed. They are based on
Alamar Blue (Alamar Biosciences, Inc., Sacramento, USA) has microtitration or colorimetric procedures. The usefulness of
been analyzed in depth. Alamar Blue is an indicator that these techniques remains undetermined [58,59].
changes color from blue to red when reduced in the presence
of microbial growth. It has been incorporated into the
Sensititre Yeast One Antifungal Panel (Accumed Intema-
tional, Ohio, USA), which is a disposable microdilution tray, In vitro susceptibility testing should reliably predict the in uivo
and numerous studies have been carried out [48,49]. response to therapy in human infections. However, numerous

0 2001 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CM/, 7 (Suppl. 2). 4 6 5 3
Cuenca-Estrella and Rodrigez-Tudela Antifungal susceptibility testing 51

factors af3ect the clinical response to antifungal therapy, patients with hematological diseases and Aspgillus spp. infection.
regardless of the MIC endpoint for the infecting organism. Mortality was sigdcantly associated with MICs of amphotericin
Host factors may have more effect on the infection than B 2 2 mg/L. The relationship was more sigdcant for patients
susceptibility to antifungal agents, and a low MIC does not infected by Asperrgillus terreus [62]. In addition, Denning et al.
necessarily predict clinical success [7]. However, an in vitro have identified strains of A. &migatus resistant to itraconazole.
susceptibility test may be able to identify organisms unlikely to These strains showed MICs of itraconazole 2 16 mg/L and were
respond to certain antimicrobial agents. isolated from patients who had fsjled itraconazole treatment [63].
Evidence of a correlation between clinical outcome and The results were confirmed in a neutropenic mouse model of
antifungal susceptibility testing has been limited untd recently. invasive aspergdlosis [64].
Good interlaboratory reproducibility of the NCCLS reference It must be emphasized that breakpoints are tentative and
guidelines for yeasts has led to the establishment of more data are needed. The clinical usefulness of in vitro tests
interpretative breakpoints for fluconazole, itraconazole and must be established in other clinical settings and for other
flucytosine, but for Cundidu species only (Table 3) [4]. organism-drug combinations. More reliable detection of
Breakpoints for fluconazole were established following resistance to amphotericin B is clearly needed. However, the
correlation between azole MICs and the clinical response of currently available in oitro tests can detect both intrinsic and
patients with AIDS, and oropharyngeal candidosis and non- primary resistance (moulds to fluconazole, Trichosporon spp. to
neutropenic patients with candidemia or visceral infection [l- amphotericin B, Cundidu knrsei to fluconazole and others). The
3,7]. Itraconazole breakpoints were defined with clinical data reference methodology is also able to detect multiresistant
on oropharyngeal candidosis only [12]. Breakpoints for organisms (Scedospon'um prol$cam and other species of moulds)
flucytosine were established on the basis of historical data and, moreover, breakpoints for fluconazole and itraconazole
and the pharmacokinetics of the drug, because of the shortage have clinical relevance in oropharyngeal candidal infection in
of available in vitvo results obtained by the NCCLS methods HIV-infected patients [1,7,65,66].
[4]. Breakpoints for amphotericin B and other antifungal
agents have not been proposed. Recent reports point out that
either E-test MICs or minimal hngicidal concentrations are
better predictors of clinical failure to amphotericin B therapy Considerable progress has been achieved in antihngal
than are the reference NCCLS procedures [7,60]. However, susceptibility testing. A reproducible reference methodology
these methodologes have not been standardized. has been developed by the NCCLS. These procedures are not
For the f3amentous fmgi, interpretative breakpoints are not problem-free, but are essential to identify organisms unlikely
available. Historically, it has been suggested that an amphotericin to respond to certain antifungal treatments. At present, major
B MIC > 1 mg/L could correlate with a lack of response of limitations are the trailing phenomenon with azoles, unreliable
treatment in experimental mould infections [1,2,7]. However, resistance to amphotericin B detection, poor growth of some
these data have been reviewed recently. An NCCLS study using organisms and impractical procedures for the clinical labora-
the M38-P methodology has indicated some degree of tory. Several collaborative studies are in progress with the
correlation between in oitm tests and response to treatment in intention to overcome the limitations and to develop a
animal models. Lack of response was observed with R. urrhizus practical reference methodology and a useful clinical tool.
and Aspqillus spp. isolates for whch MICs of amphotericin B Good interlaboratory reproducibility of the the NCCLS
were 2 2 mg/L and MICs of itraconazole >1 mg/L [35]. reference guidelines for yeasts has led to the establishment of
However, other authors report a lack of correlation of in oitro interpretative breakpoints for fluconazole, itraconazole and
amphotericin B susceptibihty testing with outcome in a murine flucytosine. Resistant breakpoints for fluconazole and itraco-
model of Aspergdlus infection [61]. Some data are also available nazole have been well documented, with treatment failure
from clinical stumes. A certain correlation was observed for 29 d y in HIV patients with oropharyngeal candidosis.

Table 3 Interpretative guidelines for

susceptibility testing in vitro of Candida Antifungal Susceptible Susceptible-dose Intermediate Resistant
agent dependent

Fluconazole 68 16-32 - > 64

ltraconazole G0.125 0.25-0.5 - 21
Flucytosine <4 - 8-1 6 > 32
Breakpoints in mg/rnL. Data are from ref. [4].

0 2001 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI,7 (Suppl. Z),46-53
sz Clinical Microbiology and Infection, Volume 7. Supplement 2, 2001

Correlations in other clinical settings are unknown. Further endpoints in antifungal susceptibilitytesting by the National Committee for
Clinical Laboratory Standards meth0d.J Clin Minobiol 1998; 3 6 153-6.
studies are needed to evaluate the clinical usefulness of the in
19. Pfaller MA, Messer SA, Coffmann S. Comparison of visual and spectro-
vitro tests for other species of yeasts and filamentous fun@. photomemc methods of MIC endpoint determinations by using broth
microdilution methods to tests five antifungal agents, including the new
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