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Journal of Clinical Microbiology, July 2001, p. 2708-2712, Vol. 39, No. 7
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.7.2708-2712.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Novel Fluorescent Broth Microdilution Method for
Fluconazole Susceptibility Testing of Candida
albicans
Robert S.
Liao,1
Robert P.
Rennie,1,2,* and
James A.
Talbot1
Department of Medical Microbiology and
Immunology,1 University of Alberta, and
National Centre for Mycology, University
of Alberta Hospital, Walter C. Mackenzie Health Sciences
Centre,2 Edmonton, Alberta T6G 2J2, Canada
Received 13 October 2000/Returned for modification 5 March
2001/Accepted 19 April 2001
 |
ABSTRACT |
A comparative evaluation of the reference National Committee for
Clinical Laboratory Standards (NCCLS) broth microdilution method with a
novel fluorescent carboxyfluorescein diacetate (CFDA)-modified microdilution method for the susceptibility testing of fluconazole was
conducted with 68 Candida strains, including 53 Candida albicans, 5 Candida tropicalis, 5 Candida glabrata, and 5 Candida
parapsilosis strains. We found trailing endpoints and
discordant fluconazole MICs of <8 µg/ml at 24 h and of 
64
µg/ml at 48 h for 12 of the C. albicans strains.
These strains satisfy the definition of the low-high MIC phenotype. All
12 low-high phenotype strains were correctly shown to be susceptible at
48 h with the CFDA-modified microdilution method. For the 41 non-low-high phenotype C. albicans strains, the
CFDA-modified microdilution method yielded 97.6% (40 of 41 strains)
agreement within ±1 dilution at 24 h compared with the reference
method and 92.7% (38 of 41 strains) agreement within ±1 dilution at
48 h compared with the reference method. The five strains each
from C. tropicalis, C. glabrata, and
C. parapsilosis that were tested showed 100% agreement
within ±2 dilutions for the two methods being evaluated.
| |
TEXT |
Candida is the
fourth most common cause of nosocomial bloodstream infection in the
United States (2, 4), and the rate of primary bloodstream
infections continue to increase (3, 9). In the Americas,
Candida albicans is the species most frequently isolated
from the bloodstream (16). Candidemia is often difficult to diagnose and refractory to therapy. The attributable mortality rate
is 38% in the United States (28) and between 19 and 23% in Canada (9, 25, 29).
Use of the broad-spectrum antifungal fluconazole for the treatment of
serious systemic Candida infections has increased.
Fluconazole is a less toxic alternative to amphotericin B and has been
recommended as primary therapy for candidemia in nonneutropenic and
stable neutropenic patients who have not received prior fluconazole
treatment and in whom Candida krusei is unlikely (5,
24). In vitro susceptibility testing for fluconazole is of
clinical importance, as therapeutic success depends substantially on
achieving plasma fluconazole levels that are sufficiently higher than
MICs (22).
Despite great advances in the standardization of antifungal
susceptibility testing, azole endpoint determination continues to be
problematic and subjective and a major source of interlaboratory variability (6, 18, 23). The trailing-growth phenomenon is
often responsible for these difficulties, whereby partial inhibition of
fungal growth occurs over the range of azole concentrations (6,
13, 14, 23).
Previous work has demonstrated the utility of using fluorescent dyes to
assess the viability of C. albicans exposed to amphotericin B (10). We have investigated the use of the
vitality-specific dye carboxyfluorescein diacetate (CFDA) in the
standardized NCCLS M27-A broth microdilution method (12)
to assess the susceptibility of Candida spp. to fluconazole.
In this study we compared the NCCLS microdilution method with a CFDA
modification in determining fluconazole susceptibility for common
clinical yeast isolates (C. albicans, Candida
tropicalis, Candida glabrata, and Candida parapsilosis).
Antifungal drug.
Fluconazole powder (Pfizer-Roerig, Inc.) was
dissolved in sterile distilled water to make a stock concentration of
10,000 µg/ml and frozen at 
70°C. The stock solution was thawed
once, and fresh dilutions were used.
Yeast isolates.
Yeast isolates were obtained from the National
Centre for Mycology, Division of Microbiology and Public Health,
Edmonton, Alberta, Canada, and two low-high phenotype C. albicans strains were kindly supplied by John H. Rex from the
Center for the Study of Emerging Pathogens and Reemerging Pathogens,
Laboratory of Mycology Research, University of Texas Medical School,
Houston, Tex. The identity of the isolates was confirmed by standard
methods (27); isolates were stored in skim milk at
70°C and were then subcultured twice on Sabouraud dextrose agar
(Difco, Sparks, Md.) before use. These strains included 48 clinical
isolates of C. albicans and homologous control strains ATCC
90028, ATCC 90029, ATCC 24433, ATCC 10231, and ATCC 20408; 4 clinical
isolates of C. tropicalis and ATCC 750; 4 clinical isolates
of C. glabrata and ATCC 90030; and 4 clinical isolates of
C. parapsilosis and ATCC 22019.
Antifungal susceptibility testing.
The reference NCCLS broth
microdilution method was performed as described in the M27-A document
(12). Fluconazole concentrations were diluted in RPMI 1640 medium with L-glutamine. Morpholinepropanesulfonic acid
(MOPS) buffer at 165 mM and pH 7 (Angus Buffers & Biochemicals, Niagara
Falls, N.Y.) and 100-µl aliquots were placed into the wells of
96-well microtiter Linbro plates (Flow Laboratories Inc., McLean, Va.)
with clear, U-shaped well bottoms. The final concentrations of
fluconazole ranged from 0 to 64 µg/ml. Six C. albicans
strains were tested per 96-well plate, which allowed for the empty
outermost wells to be filled with sterile water to minimize evaporation.
Five C. albicans colonies with a diameter of 1 mm were
suspended in sterile 0.85% saline and adjusted to a final
concentration (after inoculation) of 0.5 × 103 to 2.5 × 103
cells per ml in RPMI 1640-MOPS medium. The inoculum was added to the
fluconazole trays, incubated at 35°C, and evaluated at 24 and 48 h.
(i) Reference MIC endpoint.
The reference broth microdilution
was scored by comparing the growth in each well with that in the growth
control (drug-free) well. The MIC was defined as the minimum drug
concentration at which visual growth was determined to be 80% relative
to that of the growth control.
(ii) Fluorescent MIC endpoint.
The CFDA-modified microdilution
method was identical to the method described for broth microdilution
susceptibility testing, except that after determination of visual
endpoints at 24 and 48 h of incubation at 35°C, a fluorescence
assay was also performed. The supernatants were first removed from the
tray wells by using a multichannel pipettor, and the remaining yeasts
were resuspended to 200 µl per well in 35°C-warmed 0.1 M MOPS
buffer at pH 3.5 with 50 mM citric acid. Into each well, 5 µl of a
stock of 5 mg of 5(6)-CFDA (Molecular Probes, Eugene, Oreg.)/ml in
dimethyl sulfoxide was added for a final concentration of 122 µg/ml.
5(6)-CFDA is a lipophilic, nonpolar substrate that traverses the cell
membrane and is hydrolyzed by nonspecific intracellular esterases to
the fluorescent anion carboxyfluorescein (10). A
multichannel pipettor was used to resuspend well contents by pipetting,
alternately filling and emptying the wells 20 times. The tray contents
were then incubated in the dark at 35°C for 1 h. The well
contents were resuspended again as before, and the trays were assayed
for fluorescence with an FL500 microplate fluorescence reader (Bio-Tek Instruments Inc., Winooski, Vt.). 5(6)-CFDA was evaluated using excitation and emission wavelengths of 485 and 530 nm, respectively. The fluorescence of the drug-free control was defined as 100%, and the
fluorescence of the fluconazole-exposed wells was reported proportionally to this value (see Fig. 2). The MIC endpoint was defined
as the lowest fluconazole concentration at which the fluorescence was
reduced to 80% of that of the drug-free growth control. The CFDA
microdilution method was performed in triplicate for each yeast strain assayed.
The technical time required to include the CFDA modification to the
standard microdilution susceptibility test is primarily
dependent on
the 1-h incubation time and on the time required
using a multichannel
pipettor to add the CFDA and resuspend the
yeasts in the
wells.
The comparative evaluation of the reference NCCLS broth microdilution
method and the CFDA-modified microdilution method for
susceptibility to
fluconazole was conducted with 68
Candida strains,
including
53
C. albicans, 5
C. tropicalis, 5
C. glabrata, and
5
C. parapsilosis strains. The
C. albicans isolates chosen covered
a broad range of susceptibility
to fluconazole (Fig.
1). The fluconazole
MICs for control strains were within accepted limits, providing
an
internal control for the NCCLS reference method. Evaluation
by the
reference microdilution method determined that of the
Candida strains tested; 12 strains of
C. albicans
manifested extreme trailing
endpoints producing discordant MICs at
24 h (<8 µg/ml) and 48
h (
64 µg/ml). They were thus
considered to have the low-high
MIC phenotype (
20). All
C. albicans strains with the low-high
phenotype tested by
the CFDA-modified microdilution method were
not discordant between 24 and 48 h. These
C. albicans strains
were all shown to
be susceptible at both 24 and 48 h. In fact,
the MICs were
identical for 11 of the 12 strains.
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FIG. 1.
Range of fluoconazole MICs for 53 isolates of C.
albicans at 24 and 48 h determined with the reference
M27-A broth microdilution method. The fluconazole breakpoints for
Candida species are as follows: susceptible,  8
µg/ml; susceptible-dose dependent, 16 to 32 µg/ml; and resistant,
64 µg/ml (12).
|
|
The CFDA-modified microdilution method allows for the quantification of
fluconazole inhibition and the production of dose-response
curves (Fig.
2). MICs were determined from these
dose-response
curves after plotting the 80% inhibition and rounding up
to the
standardized M27-A endpoint.
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FIG. 2.
Effect of fluconazole on representative strains of
C. albicans, which are low-high phenotype (707-15),
susceptible (Y91), susceptible-dose dependent (Y98), and resistant
(965). Each isolate was tested for susceptibility with the M27-A
microdilution method (MICM27-A) and the CFDA-modified
microdilution method (MICCFDA) at both 24 and 48 h.
Results for the CFDA-modified method are shown graphically as relative
fluorescence units. The fluorescence of the growth in the drug-free
control was defined as 100%, and the fluconazole-exposed wells were
scaled to this value. Error bars indicate standard error. The results
of the reference M27-A method are shown below as a digital image of the
unagitated growth in the 96-well plate at 24 and 48 h.
|
|
Table
1 summarizes the distributions of
the differences in MICs and the percent agreement using the reference
and CFDA-modified
broth microdilution methods for non-low-high
phenotype strains
of
C. albicans. Considering the
non-low-high phenotype
C. albicans strains only, the
CFDA-modified broth microdilution method yielded
97.6% (40 of 41 strains) agreement within ±1 dilution compared
with the NCCLS
reference method. At 48 h the two methods yielded
92.7% (38 of
41) agreement within ±1 dilution and 97.6% (40 of
41) agreement
within ±2 dilutions. Where MIC endpoints differed
between the two
methods, the interpretive susceptibility category
changed for only one
strain. The most common discrepancies between
the different
susceptibilities of
C. albicans strains to fluconazole
were
due to the CFDA-modified microdilution MICs being 1 dilution
lower than
those obtained by the standard method. The five strains
each of
C. tropicalis,
C. glabrata, and
C. parapsilosis showed
100% agreement within ±2 dilutions for the
two methods being evaluated.
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|
TABLE 1.
Distribution of difference between the MICs of
fluconazole for 41 strains of C. albicansa
determined by comparison of the CFDA-modified and reference NCCLS M27-A
broth microdilution methods
|
|
Endpoint determination for fluconazole susceptibility testing is
recognized to be problematic and a significant source of
interlaboratory variability (
6,
18,
23). The
trailing-growth
phenomenon, which describes the partial inhibition of
fungal growth
over most or all of the concentration range (
6,
13,
14,
23), is largely responsible for the difficulties attributed
to endpoint determination, especially with
Candida spp.
(
14).
The impediment that this so-called trailing endpoint
represents
for azole susceptibility testing can with some isolates be
exacerbated
over time, producing discordant MICs of <8 µg/ml at
24 h and of
64 µg/ml at 48 h (
1,
11,
20,
21). Such discordant MICs
place these isolates into susceptible
and resistant MIC interpretive
categories at 24 and 48 h,
respectively, if one uses the NCCLS
M27-A guidelines (
12)
and are conveniently referred to as having
a low-high MIC phenotype
(
20). The present evidence suggests
that the lower MIC
obtained at 24 h using the reference microdilution
method
correlates most closely with the in vivo outcome (
11,
20,
21).
The M27-A method establishes the endpoint for susceptibility testing of
Candida spp. to azoles at 48 h with a criterion of
80%
reduction in growth (
12). Modifications of the M27-A
reference
method are acceptable and expected (
6,
21), and
it has been
suggested that correction can be made for trailing MIC
endpoints
by shortening the incubation time to 24 h and lowering
the MIC
endpoint to the lowest drug concentration producing a 50%
reduction
in growth (
15,
21,
22). The requirement of a
48-h incubation
for optimal testing conditions may represent a barrier
to this
change (
7,
21). In one study (
17) the
results for three
microdilution susceptibility test formats were shown
to be reproducible
and in agreement with one another after 24 h
but required 48 h
to achieve acceptable agreement with the
macrodilution reference
MICs.
Additional studies have proposed the use of a colorimetric endpoint in
a microdilution format by including an oxidation-reduction
indicator
with the yeast and drug prior to incubation (
15).
However,
the colorimetric method also presents trailing azole
endpoints at
48 h (
15,
26), and some species-specific
discrepancies
have been observed (
15).
The CFDA-modified microdilution format does not alter the M27-A
microdilution protocol but instead can be employed at 24 or
48 h
to clarify MIC endpoints. The fluorescent dye CFDA is applied
postincubation to the microdilution tray and thus does not interfere
with the complex interaction between the yeast and antifungal
drug.
The fluorescent dye CFDA is a lipophilic, nonpolar substrate that
diffuses across the cell membrane and is hydrolyzed by nonspecific
intracellular esterases to the fluorescent anion carboxyfluorescein
(
19). Cells with compromised membranes rapidly leak
carboxyfluorescein,
even when residual esterase activity is retained
intracellularly
(
8). The utility of CFDA for assessing the
vitality of
C. albicans exposed to amphotericin B has been
previously demonstrated (
10).
The CFDA-modified microdilution method allowed for the stringent 80%
growth inhibition endpoint to be maintained with fluconazole
susceptibility testing. Furthermore, this method permitted the
evaluation of MICs at 24 or 48 h with clearly demarcated endpoints
despite the trailing-growth phenomenon. The CFDA-modified microdilution
method determined that all 12 low-high phenotype strains of
C. albicans were susceptible to fluconazole and had identical
endpoints
at 24 and 48 h. This result supports the in vivo
evidence suggesting
that the strains of
C. albicans with the
low-high phenotype are
actually susceptible to fluconazole (
1,
20,
21). In fact,
one of the low-high phenotype strains, 707-15, demonstrated to
be susceptible with the CFDA-modified microdilution
method, has
previously been shown to be susceptible in vivo
(
21).
There was excellent agreement between the NCCLS M27-A broth
microdilution method and the CFDA-modified microdilution method
using
an 80% inhibition-of-growth endpoint for strains of
C. albicans without the low-high phenotype. These results demonstrate
that
the CFDA-modified microdilution method for testing fluconazole
is
entirely comparable to the NCCLS reference microdilution method.
The
one strain of
C. albicans for which the fluconazole MIC
differed
in the two methods being compared was shown to be very
resistant
using the M27-A microdilution method and susceptible using
the
CFDA-modified method. This strain has unusual fluorescent staining
properties, and investigations are under way to determine the
nature of
its resistance
mechanism.
A small survey of the other three major bloodstream fungal pathogens
(
16) (
C. tropicalis, C. glabrata, and
C. parapsilosis)
showed excellent agreement between the M27-A
microdilution and
CFDA-modified microdilution methods. Further studies
to evaluate
the applicability of the CFDA-modified method with other
antifungal
agents and other species of yeast are
ongoing.
In summary, the CFDA-modified microdilution method provides objective
and quantifiable endpoints for fluconazole susceptibility
testing at 24 and 48 h which are reproducible and easy to interpret.
It
eliminates the ambiguity of the low-high phenotype while maintaining
the integrity of the NCCLS protocol. This method is simple to
perform
and provides the opportunity for automation and widespread
use.
| |
FOOTNOTES |
*
Corresponding author. Present address: Department of
Microbiology and Public Health, 2B3.08 Walter Mackenzie Centre,
University of Alberta Hospital, 8440-112 St., Edmonton, Alberta T6G
2J2, Canada. Phone: (780) 407-7242. Fax: (780) 407-3864. E-mail:
rpr{at}bugs.uah.ualberta.ca.
| |
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Journal of Clinical Microbiology, July 2001, p. 2708-2712, Vol. 39, No. 7
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.7.2708-2712.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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