MYCOSES

ACCEPTED:
JANUARY 4, 1997

40, 291-296 (1997)

Comparison of Etest, microdilution and colorimetric

dilution with reference broth macrodilution method for
antifungal susceptibility testing of clinically significant
Candida species isolated from immunocompromised
patients

Bewertung von Etest, Mikrodilution und kolorimetrischer

Mikrodilution im Vergleich mit der Referenz-Makrodilutionsmethode zur Antimykotika-Empfindlichkeitsprufungklinischer
Candida-Isolate von abwehrgeschwachten Patienten
S. Arikan', D. Cur2 and M. &ova2
Key words. Cudzdu, antifungal susceptibility testing, amphotericin B, fluconazole, Etest, macrodilution, microdilution,
colorimetric microdilution.
Schliisselworter. Cunddu, Empfindlichkeitspriifung,Amphotericin B, Fluconazol, Etest, Makrodilution, Mikrodilution,
kolorimetrischeMikrodilution.

Summary. Amphotericin B and fluconazole

macrodilution minimum inhibitory concentration
values of 101 Candida strains isolated from 91
immunocompromised patients were comparatively
evaluated with the results of Etest, microdilution
and colorimetric microdilution methods. The overall agreement rates of Etest, microdilution and
colorimetric microdilution methods with the reference macrodilution method were found to be
acceptably high after an incubation period of 24
and 48 h (varying from 86 to 93% for
amphotericin B and from 84 to 89% for fluconazole). In addition, the results pointed out relatively
high minimum inhibitory concentration values of
fluconazole for Candida krusei and Candida glabrata
isolates. These methods are not only reliable
alternatives to the present reference macrodilution
method but are also easy-to-perform and less timeconsuming.

'Department of Clinical Microbiology and Microbiology and

'Department of Medicine Section of Infectious Diseases,
Hacettepe University, School of Medicine, Ankara, Turkey.
Correspondence: Dr Murat Akova, Department of Medicine
Section of Infectious Diseases, Hacettepe University School
of Medicine, TR-06100Ankara, Turkey.

Zusammenfassung. Die minimale Hemmkonzentration fur Amphoterin B und Fluconazol

wurde an 101 Candidu-St-men
von 91 Risikopatienten vergleichend im NCCLS-Makrodilutionstest, E-Test, Mikrodilutionstest und
kolorimetrischen Mikrodilutionstest bestimmt. Die
nereinstimmung der drei Teste mit dem Makrodilutions-Referenztest waren bei Ablesung nach
24 und 48 h Inkubation zufriedenstellend (86-93%
fur Amphotericin B und 84489% fur Fluconazol).
Relativ hohe Fluconazol-MHK-Werte fur C. kmei
und C. glabrata wurden bestatigt. Die gepriiften
Methoden enviesen sich als verlaBliche Alternativen zur Makrodilutions-Referenzmethode und
sind leicht durchzufuhren sowie zeitsparend.

Introduction
Fungal infections have emerged as a leading cause
of morbidity and mortality in immunocompromised patients in recent years [l]. Candida
species, particularly Candida albicans is the most
commonly encountered fungal nosocomial pathogen in patients with severe underlying diseases and
immunosuppression [21. After treatment with
broad spectrum antibacterials, immunocom-

promised patients become easily colonized and

infected with Candida [3]. In neutropenic patients
(polymorphonuclear leukocytes <500 mm-3),
repetitive bouts of neutropenic attacks have been
shown to predispose the patient colonized to
become with less virulent but more resistant
Candida species [4].
Emergence of resistance to antifungal agents
among Candida isolates and availability of new
drugs for treatment of invasive fungal infections
have resulted in a great demand for a standardized
in vitro antifungal susceptibility testing procedure
[5, 61. However, diversity of in vitro test results
with respect to the test conditions, such as medium composition, pH, fungal inoculum size and
incubation period displayed difficulties in
standardization.
The National Committee for Clinical
Laboratory Standards (NCCLS) has proposed a
reference broth macrodilution method for antifungal susceptibility testing of yeasts [5]. Although
the development of NCCLS macrodilution methodology is a remarkable progress for in vitro susceptibility testing of yeasts, there are numerous
problems with the procedure. It is time-consuming,
expensive, technically difficult to perform and not
much suited for routine use. The breakpoint minimum inhibitory concentration (MIC) value for
fluconazole has not been well established yet and
the end points are less sharp for azole compounds
and flucytosine, creating a significant source of
variability in interpretation of the results. The
ability to discriminate between amphotericin B
resistant and susceptible isolates is also lacking.
Moreover, in uitro resistance is not always associated with clinical resistance and correlation
between in uitro and in vivo results is not well
established yet [7- lo].
Multicenter trials to develop simpler and more
economical methods are in progress. Among these
are the microdilution method, colorimetric microdilution method and Etest [ l l , 12, 131. The aim
of the present study is to compare the methods
with the NCCLS broth macrodilution reference
method for amphotericin B and fluconazole susceptibility testing of Candida species isolated from
severely immunocompromised patients with
candidosis.

Materials and Methods

Isolates
One hundred and one Candida strains isolated
from clinical specimens of 91 immunocompromised patients who had infection requiring

antifungal therapy were included in the study.

Sabouraud glucose agar (SDA) was used for isolation of the strains. In the case of a positive germ
tube test, the strain was identified as Candida
albicuns. API 20C system (BioMCrieux, Marcy
l'Etoile, France) was used for species identification
if the germ tube test was negative. The isolates
were stored at -70 "C until testing and were
subcultured on SDA at 35 "C prior to antifiungal
susceptibility assay.

Susceptibility testing

I . Macrodilution method. Application of the method

and interpretation of the results were performed
by using the methodology proposed by NCCLS
[5]. (a) Test medium. RPMI-1640 medium with
L-glutamine, without sodium bicarbonate, buffered with 0.165 M morpholiiopropanesulfonic
acid (MOPS, pH 7, Sigma, St. Louis, MO, USA)
was used for dilution of antfingal agents, inoculum preparation and as the test medium.
(b) Antfingal agents. The antifungals were
obtained from the manufacturers as standard powders. Amphotericin B (Squibb) and fluconazole
(Pfizer) were rendered soluble in 100% dimethyl
sulfoxide and further diluted in RPMI-1640
medium. (c) Inoculum preparation. Cundidu isolates
were grown on SDA for 24 h at 35 "C. For each
isolate, five colonies, each at least 1 mm in diameter were picked and suspended in 5 rnl of sterile
0.85% saline, the turbidity of which was later
adjusted spectrophotometrically to that of 0.5
McFarland standard by measuring at 530 nm.
Quantitative colony plate count was performed on
SDA to verify the inoculum size. Adjusted saline
suspension was diluted 1: 100 and then 1 :20 with
RPMI- 1640 medium to yield a final inoculum size
of 0.5-2.5 x lo3 cells per ml to be used in the test.
(d) Procedure. MIC values were determined as
proposed by NCCLS. Briefly, two-fold serial
dilutions of the antifungals were prepared in
"MI-1640
medium resulting in h a l drug concentrations ranging from 16 to 0.03 pgml-' for
amphotericin B and from 64 to 0.125 pg m.-' for
fluconazole. Previously diluted yeast inocula
(0.9 rnl) were added to tubes containing the antifungal dilutions. Test tubes, including the drugfree and yeast-free control tubes were incubated
at 35 "C for 48 h. MIC endpoints were determined
as the concentration of drug producing no visible
turbidity (MIC-O) for amphotericin B and as that
causing greater than 80% inhibition of growth
(MIC-2) for fluconazole.

2. Etest. (a) Test medium. Amphotericin B was

tested on RPh4I 1640 medium supplemented with
mycoses 40, 291-296 (1997)

SUSCEPTIBILITY
TESTING

glucose (2%) and buffered to p H 7 with 0.165 M

MOPS. For fluconazole susceptibility testing, the
only difference was the buffer used, being 0.1 M
potassium phosphate which has been shown to
produce sharper end points for azoles [ 141. Bacto
agar (Difco, Detroit, USA) was added to both
media to produce a h a 1 concentration of 1.5 g
100 ml-'. (b) Etest strips. Amphotericin B and
fluconazole Etest strips were provided by the
manufacturer (AB BIODISK Solna, Sweden). The
concentration gradient ranged from 0.002 to 32
and from 0.016 to 256 pg ml- for amphotericin B
and fluconazole, respectively. The strips were
stored at -20 "C until used. (c) Inoculum preparation. The inoculum concentration prepared in
sterile 0.85% saline was adjusted spectrophotometrically to correspond to the turbidity of 0.5
McFarland standard at 530 nm wavelength.
(d) Procedure. A cotton swab was used to apply
the adjusted inoculum onto the RPMI agar surface
and the plates were left to dry for at least 15 min
before the Etest strips were placed on the agar
surface. The plates were incubated at 35 "C. The
evaluation of the MIC values were performed at
the end of both 24 and 48 h of incubation and
were determined as the lowest concentration at
which the border of the elliptical inhibition zone
intercepted the scale on the Etest strip. The
interpretation of the results in case of the absence
of diffuse end points (presence of microcolonies
inside the inhibition zone or a double halo), was
made according to the criteria documented in the
Etest technical guide for antifungal susceptibility
testing by selecting the MIC value as the point of
approximately 80% growth inhibition [ 141. Since
the Etest scale has a continuous gradient of concentrations rather than two-fold dilutions, the Etest
MIC value was raised to the next two-fold dilution
for the sake of comparison if the obtained MIC
value did not fit one of the dilutions used in the
reference macrodilution method.

'

3. Microdilution and colorimetric microdilution method.

The microdilution test was performed according
to the guidelines proposed for NCCLS macrodilution methodology, except that microdilution trays
were used with a final volume of 100 pl well-'
[12]. MIC values were interpreted after incubation
for 24 and 48 h at 35 "C. The plates were agitated
before reading [ 151. A fluconazole MIC value was
accepted to be the concentration where decrease
in turbidity is prominent compared to the growth
control well (MIC-2) and that of amphotericin B
being the concentration of the we11 which is
optically clear (MIC-0) [lS].
For the colorimetric microdilution test, Alamar
blue (Alamar Biosciences, East Grinstead, UK)
mycoses 40, 291-296 (1997)

OF

CANDIDA
SPECIES

293

which is an oxidation reduction indicator was

added to each well at the time of inoculation
(12.5 pl well-') and MIC values were evaluated
after 24 and 48 h of incubation. An indicator of
growth was the change of colour from blue to
pink. The lowest concentration of the antifungal
which remained blue was accepted as the MIC
value of that strain [ 1 11.

Reference strains
Candida albicms ATCC 90028 and Candida parapsilosis ATCC 90018 strains were included as the
reference strains.
Anabsis of results
Etest, microdilution and colorimetric microdilution MIC values were compared to those of
NCCLS macrodilution method. Discrepancies
among MIC endpoints of no more than f 2
dilutions were used to calculate the percent agreement. The Etest, microdilution or colorimetric
microdilution method for a specific strain was
accepted to be in agreement with the NCCLS
method if the MIC value is within + 2 two-fold
dilutions compared to reference MIC.

Results
In vitro susceptibility test results of amphotericin B
and fluconazole are shown in Tables 1-4. All of
the Candida isolates tested with amphotericin B
Etest strips gave clear end points at the point of
intersection between the zone edge and the strip.
However, for 16 (30%) Candida ahcans and 2
(33%) Candidu tropicalis isolates, instead of sharp
inhibition ellipses, large to minute colonies or a
double halo was observed at the endpoint or
within the whole inhibition ellipse.
Percent agreement rates between the reference
macrodilution and other methods are presented in
Table 5. Evaluation of colorimetric MIC values
after 24 h of incubation was not satisfactory and
the colour change from blue to pink could not be
detected for most of the isolates, therefore, the
agreement rate was assessed only for 48-h incubation results. Both a 24- and 48-h evaluation was
performed for other methods.
Discussion
A standard antifungal susceptibility testing has
long been desired. The reference macrodilution
test [5] recently proposed by the NCCLS has

2%

s. h I K A N ETAL.
~

Table 1. Amphotericin B MIC (pg I&)

Species ( n : 101)

values of 10 1 Candida isolates according to NCCLS macrodilution method and Etest

NCCLS (M27P)

Etest

Range

MIC50

MICSO

Range

MIC50

MICSO

0.06- 1
0.06-4
50.03-2
0.25-2
0.25-1
0.25

0.50
0.50
0.25
1

1
2
1
1
1
-

0.25
0.50
1
0.50
0.50
-

0.50
1
1
1
0.50

0.06-1
<0.03-2
0.06- 1
0.50-1
0.50
0.125-0.25

Candida albicans (n: 53)

Candida krwei (n: 18)
Can& h&r (n : 15)
Candida glubrata (n:7)
Candida tropzcalis (n:6)
Candida parapsilasis (n : 2)

Table 2. Amphotericin B MIC (pg ml-) values of 101 Candida isolates according to microdilution and colorimetric microdilution
method
~

Species ( n : 101)

Candida albicans ( n :53)

Candda ICr?Lsa (n:18)
Candida kefir ( n : 15)
Candida glubrata (n :7)
Candidn tropzcalis (n:6)
Candiah parapsilosis (n :2)

Microdilution

~~

Colorimetric microdilution

Range

MIC50

MICSO

Range

MICSO

mc90

0.06-1
10.03-2
0.06-2
10.03-1
0.25-1
0.125-0.50

0.50
0.50
0.50
1
1

1
1
2
1
1
-

0.25-2
0.1254
0.06-2
10.03-1
1
0.125-1

2
1
1
I

2
2
2
1
1
-

values of 101 Candida isolates according to NCCLS macrodilution method and Etest
Table 3. Fluconazole MIC (pgd-)

Species ( n : 101)

Candtda a1bican.s (n :53)

Candida h e i (n : 18)
Candida k&r ( n: 15)
Candida glabrata (n:7)
Candida tropicalis ( n:6)
Candidaparapsilosis ( n:2)

NCCLS (M27P)

Etest

Range

MIC50

10.125-4
8-64
S 0.125-8
4-64
0.25-8
0.25-0.50

0.50
64
1
8
0.50
-

MICSO
2

> 64
4
64
8
-

Range

MIC50

MICSO

10.125-4
8+64
5 0 . 125-8
0.50-32
1 0 . 125-4
10.125-0.25

0.50
32
0.50
4
0.25
-

1
64
4
16
4
-

Table 4. Fluconazole MIC (pg ml-l) values of 101 Candidu isolates according to microdilution and colorimetric microdilution
Species ( n : 101)

Candida a1bican.s (n:53)

C d i d a kwei (n : 18 )
Candida k& (n : 15)
Candula glabrata (n :7)
Candida tropicalis ( n:6)
Candida pmapsilosis (n : 2)

Microdilution

Colorimetric microdilution

Range

MIC50

10.125-4
8+64
SO.125-8
1-64
1-4
0.25-0.50

0.50
64
0.50
16

2
-

several shortcomings. We compared three dxerent

methods with the reference method in an attempt
to define an easy-to-perform test which has similar
sensitivity and specificity to that of NCCLS. Our
results point out the relatively high MIC values of
fluconazole for C. krusei and C. glabrata isolates.
Similar findings for these two species, together

MICSO
2

>64
8
64
4
-

Raw

MIC50

50.125-4
32-64
0.25-8
4-64
1-8
0.50- 1

0.50
64
1
16
1
-

MICSO
2

=-64
a

64
-

with C. tropicalis were reported in previous

[I, 6, 81. Candida &ofikaZi.~
isolates (n:6) test
a wide range of macrodilution Auconazole MIC
values (0.25-8) as reported previously [8] wherein
the MIC distribution for this species was termed
to be bimodal.
Etest, which is a novel method utilizing a stable
mycoses 40, 291-296 (1997)

SUSCEPTIBILITY
TESTING

OF

CANDIDA
SPECIES

295

Table 5. Percent agreement* of Etest, microdilution and colorimetric microdilution methods with the reference macrodilution
method for susceptibility testing of 101 Cundzdu isolates against amphotericin B and fluconazole after 24 and 48 h of incubation
Antifiigal

Method
Etest

I
Amphotericin B
Fluconazole

Microdilution

Colorimetric microdilutiont
48 h

24 h

48 h

24 h

48 h

90
84

93
87

88

87
87

89

86
87

*agreement within 2 two-fold dilutions; ?percent agreement of colorimetric microdilution method with the NCCLS macrodilution
method was assessed only for 48 h incubation results since the change of colour from blue to pink was not prominent after 24 h
for most of the isolates.

gradient of an antimicrobial agent has proven to

be accurate and simple for in uitro susceptibility
testing of bacteria [171. It provides the determination of MIC value of the related antimicrobial
agent by application of a simple d f i s i o n procedure, Previous studies concerning the application of the method for the antifungals showed
that Etest and NCCLS macrobroth MIC values
are in
good agreement
(89-96%
for
amphotericin B and 80-96% for fluconazole) [ 13,
17, 18, 191. We found the MIC agreement rates
to be 90-93% for amphotericin B and 84-87'10
for fluconazole at 24 and 48 h, suggesting that it
produces comparable results with the reference
method even after 24 h of incubation. In general,
Etest MIC50 and MICSO values of both antifungals tested were either identical or one two-fold
dilution lower than those of the reference method,
as shown in detail in Tables 1 and 3.
Besides being less time-consuming and providing ease of performance, Etest also offers some
advantages for interpretation of the results, especially for azoles. Trailing phenomenon, that is a
result of the partial growth inhibition of azoles,
makes the determination of the exact MIC endpoint difficult [20]. Since fungal growth decreases
very gradually with increasing concentrations of
an azole compound, a MIC value may be overestimated particularly if broth dilution methods
are used. For Etest, trailing effect presents with
the growth of colonies inside the inhibition ellipse.
However, the problem seems less cumbersome
with Etest because it is easier to visualize the
concentration where prominent decrease of
growth starts (MIC-80). We detected such growth
pattern only among C. albicans (30%) and
C. tropicalis (33%) isolates but not in any other
species tested. This seems similar to the results
reported by Colombo et al. [18]. On the other
hand, Eldere et al. [171 detected this growth pattern
in 60% of C. glabrata isolates. Only 7 C. glabrata
isolates were tested in our study and none of them
mycoses 40, 291-296 (1997)

showed growth inside the inhibition ellipse.

Further studies are required to clarify why only
certain species display such a growth pattern.
Broth microdilution method was also comparatively evaluated in the present study and was found
to be in good agreement with the reference method
(88-87% for amphotericin B and 89-87% for
fluconazole at 24 and 48h). Somewhat higher
agreement rates were reported previously (94% at
24 h and 93 at 48 h) [ 111. Agitation of the plates
prior to reading was particularly effective to produce clear-cut visual end points [15, 211.
Alamar blue, which is used for colourimetric
MIC determination as an oxidation-reduction
indicator, is reduced in the presence of actively
metabolizing prokaryotic or eukaryotic cells [22].
It was found to give efficient and reproducible
results when used for in uitro fluconazole susceptibility testing. Pfaller et aL [ 111 reported 94 and
91?Lo agreement with NCCLS macrodilution at 24
and 48 h respectively for fluconazole against
C. albicans. The agreement rate was 95% at 48 h
in another study conducted by Tiballi et al. [22].
Although the agreement rates (86 and 87% for
amphotericin B and fluconazole, respectively)
found in our study are similar to those of the
microdilution method and are acceptably high, in
our experience, the colorimetric method does not
seem to offer any more advantage compared to
the microdilution method and is less satisfactory,
since a prominent change in colour could not be
detected at 24 h.
In conclusion, Etest, microdilution and colorimetric microdilution methods are good and
reliable alternatives for in uitro antifungal susceptibility testing. Etest seems to be relatively more
efficient, particularly for routine use because the
method is easier to perform. However, correlation
of the results with the clinical outcome needs to
be evaluated before antifungal therapy can be
monitorized according to the in uitro susceptibility
test results.

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mycoses 40, 291-296 (1997)