Comparative evaluation of ATB Fungus 2 and Sensititre YeastOne panels for testing in vitro Candida antifungal susceptibility.

Abstract

ATB Fungus 2 and SensititreYeastOne are commercial methods for antifungal susceptibility testing of yeasts. The agreement between these two methods was assessed with a total of 133 Candida strains (60 Candida albicans, 18 Candida dubliniensis, 29 Candida glabrata, and 26 Candida krusei). MIC endpoints were established after 24 h of incubation at 36-/+1 degrees C by each method. Intra-laboratory reproducibility of both methods was excellent (=or>99%). Overall agreement between ATB Fungus 2 and Sensititre YeastOne 3 MICs (within 2 dilutions) was 91.2-97.7% for amphotericin B, 5-fluorocytosine and itraconazole, and 82.7% for fluconazole. The categorical agreement when ATB Fungus 2 results were compared to those by SensititreYeastOne 3 was 93.2-98.5% for 5-fluorocytosine and amphotericin B, but lower for the triazoles (72.9-75.9%). This easy to perform method could be an alternative for routine use in the clinical microbiology laboratory for susceptibility testing of common Candida spp.

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Rev Iberoam Micol 2008; 25: 3-6
Comparative evaluation of ATB
Fungus 2 and Sensititre YeastOne
panels for testing in vitro Candida
antifungal susceptibility
Elena Eraso1, Maite Ruesga1, María Villar-Vidal1,
Alfonso Javier Carrillo-Muñoz2, Ana Espinel-Ingroff3and
Guillermo Quindós1
1Laboratorio de Micología Médica, Departamento de Inmunología, Microbiología y Parasitología, Facultad de
Medicina y Odontología, Universidad del País Vasco, Apartado 699, E-48080 Bilbao, Spain; 2Departamento de
Microbiología, ACIA, P.O. Box 10178, E-08010 Barcelona, Spain; 3Virginia Commonwealth University Medical
Center, 1101 E. Marshall Street, Sanger Hall, Room 7-049, P.O. Box 980049, Richmond, VA 23298-0049, USA
ATB Fungus 2 and SensititreYeastOne are commercial methods for antifungal
susceptibility testing of yeasts. The agreement between these two methods
was assessed with a total of 133 Candida strains (60 Candida albicans,
18 Candida dubliniensis, 29 Candida glabrata, and 26 Candida krusei).
MIC endpoints were established after 24 h of incubation at 36 ± 1 ºC by each
method. Intra-laboratory reproducibility of both methods was excellent
(? 99%). Overall agreement between ATB Fungus 2 and Sensititre YeastOne 3
MICs (within 2 dilutions) was 91.2-97.7% for amphotericin B, 5-fluorocytosine
and itraconazole, and 82.7% for fluconazole. The categorical agreement when
ATB Fungus 2 results were compared to those by SensititreYeastOne 3 was
93.2-98.5% for 5-fluorocytosine and amphotericin B, but lower for the triazoles
(72.9-75.9%). This easy to perform method could be an alternative for routine
use in the clinical microbiology laboratory for susceptibility testing of common
Candida spp.
ATB Fungus 2, Sensititre YeastOne, In vitro antifungal susceptibility, Candida
Evaluación comparativa de ATB Fungus 2 y
Sensititre YeastOne en el estudio de la sensibilidad in
vitro de Candida a los antifúngicos
ATB Fungus 2 y SensititreYeastOne son métodos comerciales para el estudio
de la sensibilidad in vitro de levaduras a los antifúngicos. La concordancia
entre estos dos métodos fue evaluada con un total de 133 aislamientos de
Candida (60 Candida albicans, 18 Candida dubliniensis, 29 Candida glabrata y
26 Candida krusei). La lectura de las CMIs se realizó para cada método
después de 24 h de incubación a 36 ± 1 ºC. La reproducibilidad
intralaboratorio de ambos métodos fue excelente (? 99%). La concordancia
global entre las CMIs de ATB Fungus 2 y Sensititre YeastOne 3 (en un rango
± 2 diluciones) fue de 91,2-97,7% para anfotericina B, 5-fluorocitosina e
itraconazol, y 82,7% para fluconazol. La concordancia por categorías cuando
los resultados de ATB Fungus 2 fueron comparados con los de
SensititreYeastOne 3 fue de 93,2-98,5% para 5-fluorocitosina y anfotericina B,
pero más baja para los triazoles (72,9-75,9%). Este método sencillo de realizar
puede ser una alternativa para uso de rutina en el laboratorio de microbiología
clínica en el estudio de la sensibilidad de Candida spp.
ATB Fungus 2, Sensititre YeastOne, Sensibilidad in vitro a los antifúngicos,
Candida
Corresponding author:
Dr. Guillermo Quindós
Laboratorio de Micología médica
Departamento de Inmunología, Microbiología y Parasitología
Facultad de Medicina y Odontología, Universidad del País Vasco
Apartado 699
E-48080 Bilbao, Spain
Tel.: +34 946012854
Fax:+34 946013495
E-mail: guillermo.quindos@ehu.es
©2008 Revista Iberoamericana de Micología
Apdo. 699, E-48080 Bilbao (Spain)
1130-1406/01/10.00 €
Summary
Key words
Resumen
Palabras clave
Special article

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Candida albicans is the most commonly species
isolated from patients with candidiasis. Other species such
as Candida glabrata, Candida parapsilosis, Candida kru-
sei, or Candida dubliniensis are being frequently isolated
from immunocompromised or surgically treated patients.
A high variability in the susceptibility of clinical isolates
to antifungal agents has been reported among these Can-
dida spp. [7,8,10,16], emphasizing the importance of per-
forming species identification and antifungal susceptibility
testing [8,16].
In 2002, the Clinical and Laboratory Standards Ins-
titute (CLSI, formerly NCCLS) and the European Commit-
tee on Antibiotic Susceptibility (EUCAST) proposed two
broth dilution reference methods [2,4,9] for antifungal sus-
ceptibility testing of yeasts of clinical importance, but these
standardized methods are time-consuming and cumber-
some for routine use in the clinical laboratory. However, in
recent years, a number of commercial methods have been
introduced as easy-to-use, rapid alternatives [2,3,5,14,15].
One of these commercial method is the ATB Fungus 2,
which allows the determination of the susceptibility of
Candida spp. and Cryptococcus neoformans to antifungal
agents in a semi-solid medium under conditions similar to
those described in both EUCAST and CLSI reference
methods [2,12,13,15].
The aim of this study was to evaluate the performan-
ce of the ATB Fungus 2 panel (bioMérieux, Marcy l’Étoile,
France) in comparison to the Sensititre YeastOne 3 (Trek
Diagnostic Systems, East Grinstead, UK) for the in vitro
antifungal susceptibility testing of clinically important
Candida isolates.
Materials and Methods
Yeast isolates. We tested eight reference strains
(C. albicans, ATCC 76615, ATCC 90028, ATCC 90029,
and NCPF 3153; C. dubliniensis, NCPF 3949, and CECT
11473; and C. glabrata, ATCC 90030, and NCPF 3240)
and 125 clinical isolates including 56 C. albicans, 16 C. du-
bliniensis, 27 C. glabrata, and 26 C. krusei; susceptible (S),
susceptible dose dependent (SDD), intermediate (I) and or
resistant (R) isolates to the antifungal agents evaluated
were included in the set. The isolates were identified by
conventional mycological methods such as, the germ tube
induction test in serum, microscopical morphology and
chlamydospore formation in corn meal agar with Tween
80, and carbon source assimilation by the API ID 32C
(bioMérieux) [1]. CLSI QC isolates C. krusei ATCC 6258
and C. parapsilosis ATCC 22019 were used as control
isolates. Prior to testing, each strain was subcultured for
24-48 h at 36 ± 1 ºC on Sabouraud glucose agar (Difco,
St. Louis, MO) and Candida ID2 (bioMérieux) to ensure
viability and purity.
Susceptibility testing. The antifungal susceptibility
of the study and QC isolates was evaluated by both ATB
Fungus 2 (bioMérieux) and Sensititre YeastOne 3 (Trek
Diagnostic Systems) methods. MIC endpoints of each iso-
late were determined after 24 h of incubation at 36 ± 1 ºC
by both methods.
The ATB Fungus 2 panel (bioMérieux) consists of
16 pairs of wells. The first pair does not contain any anti-
fungal agent and is used as growth control. The next 15
pairs contain four antifungal agents at the following drug
concentrations: 5-fluorocytosine 0.5-64 µg/ml, amphoteri-
cin B 0.5-16 µg/ml, fluconazole 0.25-128 µg/ml, and itra-
conazole 0.125-4 µg/ml. The ATB Fungus 2 panel was eva-
luated by following the manufacturer’s instructions [2,15].
Briefly, 20 µl of a 2 McFarland standard yeast suspension
was added to the specific growth medium (ATB Fungus 2
medium). After the homogenization step, each well was
inoculated with 135 µl of the homogenized inoculum.
Following 24h of incubation at 36 ± 1 ºC, growth in the
strips was read visually by two independent readers.
According to the manufacturer’s instructions, the amphote-
ricin B MIC corresponded to the lowest concentration ena-
bling complete growth inhibition. A certain amount of trai-
ling growth was disregarded for fluconazole, itraconazole
and 5-fluorocytosine and the MICs were the lowest drug
concentrations that showed a prominent reduction in
growth [2,15].
Sensititre YeastOne 3 (Trek Diagnostic Systems)
contains several antifungal drugs, but only the four present
in the ATB Fungus 2 strip were evaluated at the following
drug concentrations: 5-fluorocytosine 0.03-64 µg/ml, am-
photericin B 0.008-16 µg/ml, fluconazole 0.125-256 µg/ml,
and itraconazole 0.008-16 µg/ml. The Sensititre YeastOne
3 antifungal panel was also evaluated according to the
manufacturer’s instructions. Briefly, 20 µl of a 0.5 McFar-
land standard yeast suspension was transferred into 11 ml
of the RPMI broth tube and each well of the panel was
inoculated with 100 µl of the diluted inoculum. The plates
were visually read after 24 h of incubation at 36 ± 1 ºC.
Growth in each well was indicated by a color change from
blue (no growth) to red (growth). For amphotericin B, the
MIC corresponded to the lowest concentration enabling
complete growth inhibition (first blue well). For the other
agents, the MIC was defined as the lowest drug concentra-
tion preventing the development of the red color (or first
blue or purple well) corresponding to a significant inhibi-
tion of fungal growth.
Data analysis. Both on-scale and off-scale MICs by
each method were included in the analysis. The evaluation
of the reproducibility was based on the comparison of
MICs of each antifungal agent obtained for each strain and
by each method, as well as both types of reading by the
ATB Fungus 2 panel. MICs were considered in agreement
when the values were within no more than 2 dilutions. The
performance of the ATB Fungus 2 panel in identifying
resistant isolates was evaluated by determining the catego-
rical agreement between the two methods by using CLSI
M27-A2 MIC breakpoint categories [4-6,8-10]. A strain
was considered -R- to 5-fluorocytosine, fluconazole, and
itraconazole, if the MICs were ? 32, ? 64 and ? 1 µg/ml,
respectively; -SDD- or -I- (only for 5-fluorocytosine) if the
MICs were between 8-16, 16-32, and 0.25-0.5 µg/ml,
respectively; and -S-, if the MICs were ? 4, ? 8 and
? 0.12 µg/ml, respectively. Breakpoints are not available
for amphotericin B against any fungal species; however,
based on serum concentrations, an isolate was considered
-R- if the MIC was ? 1 µg/ml and -S- if the MIC was
< 1 µg/ml. Discrepancies of MIC endpoints between the
two methods were considered major errors when the iso-
late was R by the ATB Fungus 2 but -S- by the Sensititre
YeastOne 3, while minor errors were identified when there
were categorical shifts between -S- and -SDD- or between
R and -SDD-. Very major errors were identified when the
ATB Fungus 2 categorized an isolate as -S- and the Sensi-
titre YeastOne 3 as -R-.
Results and Discussion
Different ready-to-use kits have been commerciali-
zed for antifungal susceptibility testing of clinically impor-
tant yeasts [5,6,14]. The ATB Fungus 2 panel is an impro-
ved version of the original panel, because it includes
fluconazole and itraconazole, two of the most widely used
4
Rev Iberoam Micol 2008; 25: 3-6

Page 3

antifungal drugs in therapy. Although both ATB Fungus 2
and Sensititre YeastOne 3 have demonstrated good to exce-
llent agreements with the CLSI M27 method in different
studies [2,5,13,15], limited data are available regarding the
agreement of results obtained by these methods. Because
of that we have evaluated the reproducibility of MICs
results obtained by both methods.
In the present study, MIC readings for the two QC
strains were within the reference limits described in the
CLSI M27-A2 document [9] and in the guides for both
commercial methods. Replicate testing of the QC strains
also demonstrated excellent agreement between both
methods with each antifungal agent (? 99%). The repro-
ducibility of MIC results for 8 of the 133 study strains that
were tested in triplicate in each sequential test was exce-
llent by each method (? 99% global reproducibility). The
agreement (no more than two dilutions) of MIC results
obtained by ATB Fungus 2 and Sensititre YeastOne 3 for
the 133 strains was good to excellent (91.7-97.7%) for
three of the four agents (Table). The agreement was lower
for fluconazole (82.7%).
The categorical agreement was good to excellent
(93.2 to 98.5%) with 5-fluorocytosine and amphoteri-
cin B, but lower with both triazoles (72.9-75.9%) (Table).
Although the majority of discrepant results were due to
minor errors, the categorical evaluation indicated that the
ATB Fungus 2 failed to identify 3 of the 12 itraconazole
and 4 of the 21 fluconazole resistant isolates; no very
major errors were observed with amphotericin B. Most
minor errors were also due to higher fluconazole and itra-
conazole MIC results by Sensititre YeastOne 3 than by the
ATB Fungus 2 (18 and 11 fluconazole and itraconazole
SDDresults,respectively,wereS).Minorerrorswerebelow
7% with 5-fluorocytosine. In two recent reports [2,15], the
comparison between CLSI or EUCAST reference methods
with either Sensititre YeastOne 3 or ATB Fungus 2 have
demonstrated excellent agreement (> 90%) between these
methods for all antifungal agents tested, except for itraco-
nazole (88%).
The present study mirrors these results for ampho-
tericin B and 5-fluorocytosine, but in contrast the agree-
ment was lower with fluconazole (82.7%) and higher with
itraconazole (91.7%) (Table).
In summary, ATB Fungus 2 is a simple, effective
and reproducible method and may be a valuable alternative
for testing the antifungal activity of 5-fluorocytosine and
amphotericin B against commonly isolated Candida spp.
The results were less favorable for testing fluconazole and
itraconazole due the false susceptible results for 3 of the 6
fluconazole -R- and 3 of the 12 itraconazole -R- isolates.
Further comparisons with one of the two reference
methods (CLSI or EUCAST) should clarify this issue
since false-susceptible (very major errors) have been
reported when the CLSI method has been compared to the
Sensititre YeastOne when testing fluconazole [11].
The authors were financed in part with grants
PI030662, and PI061895 from Fondo de Investigación
Sanitaria and and IT-222-07 from Gobierno Vasco-
Eusko Jaurlaritza. We are grateful to Ignacio
Urrechaga and José Manuel Torres, from bioMérieux
España, for the donation of the ATB Fungus 2 panels
used in this study.
5
ATB Fungus 2 for in vitro Candida antifungal susceptibility
Eraso E, et al.
Table. Essential agreement between ATB Fungus 2 and Sensititre
YeastOne panels.
Antifungal agents
5FCAMBFLCITC
MIC agreement
Equivalent MIC
127/133
(95.5%)
130/133
(97.7%)
110/133
(82.7%)
122/133
(91.7%)
Different MICs
6/133
(4.5%)
3/133
(2.3%)
23/133
(17.3%)
11/133
(8.3%)
Category agreement
Total agreement
124/133
(93.2%)
131/133
(98.5%)
97/133
(72.9%)
101/133
(75.9%)
Minor errors
9/133
(6.8%)
0/133
(NA)
30/133
(22.6%)
23/133
(17.3%)
Major errors
0/133
(NA)
2/133
(1.5%)
2/133
(1.5%)
6/133
(4.5%)
Very major errors
0/133
(0%)
0/133
(0%)
4/133
(3%)
3/133
(2.3%)
5FC= 5-fluorocytosine, AMB= amphotericin B, FLC= fluconazole, and ITC= itraconazole.
NA=not applicable.

Page 4

1. Baires-Varguez L, Cruz-García A,
Villa-Tanaka L, Sánchez-García S,
Gaitán-Cepeda LA, Sánchez-Vargas LO,
Quindós G, Hernández-Rodríguez C.
Comparison of a randomly amplified
polymorphic DNA (RAPD) analysis and ATB
ID 32C system for identification of clinical
isolates of different Candida species. Rev
Iberoam Micol 2007; 24: 148-151.
2. Durussel C, Parreno D, Nougier L,
Monnin V, Zambardi G, Bille J. Comparative
study of various methods, NCCL M27-A2,
EUCAST and ATB Fungus 2 (bioMérieux)
for the in vitro antifungal susceptibility
testing of Candida sp. Clin Microbiol Infect
2004; 10 (Suppl. 3): 112-113.
3. Espinel-Ingroff A. Comparison of three
commercial assays and a modified disk
diffusion assay with two broth
microdilution reference assays for testing
zygomycetes, Aspergillus spp., Candida
spp., and Cryptococcus neoformans with
posaconazole and amphotericin B. J Clin
Microbiol 2006; 44: 3616-3622.
4. Espinel-Ingroff A, Barchiesi F,
Cuenca-Estrella M, Pfaller MA, Rinaldi M,
Rodríguez-Tudela JL, Verweij PE.
International and multicenter comparison
of EUCAST and CLSI M27-A2 broth
microdilution methods for testing
susceptibilities of Candida spp. to
fluconazole, itraconazole, posaconazole,
and voriconazole. J Clin Microbiol 2005;
43: 3884-3889.
5. Espinel-Ingroff A, Canton E, Gibbs D,
Wang A. Correlation of Neo-Sensitabs
tablet diffusion assay results on three
different agar media with CLSI broth
microdilution M27-A2 and disk diffusion
M44-A results for testing susceptibilities of
Candida spp. and Cryptococcus
neoformans to amphotericin B,
caspofungin, fluconazole, itraconazole, and
voriconazole. J Clin Microbiol 2007; 45:
858-864.
6. Espinel-Ingroff A, Pfaller M, Messer SA,
Knapp CC, Holliday N, Killian SB.
Multicenter comparison of the Sensititre
YeastOne colorimetric antifungal panel
with the NCCLS M27-A2 reference
method for testing new antifungal agents
against clinical isolates of Candida spp.
J Clin Microbiol 2004; 42: 718-721.
7. Gutiérrez J, Morales P, González MA,
Quindós G. Candida dubliniensis, a new
fungal pathogen. J Basic Microbiol 2002;
42: 207-227.
8. Hajjeh RA, Sofair AN, Harrison LH,
Lyon GM, Arthington-Skaggs BA, Mirza
SA, Phelan M, Morgan J, Lee-Yang W,
Ciblak MA, Benjamin LE, Sanza LT,
Huie S, Yeo SF, Brandt ME, Warnock DW.
Incidence of bloodstream infections due
to Candida species and in vitro
susceptibilities of isolates collected from
1998 to 2000 in a population-based
active surveillance program. J Clin
Microbiol 2004; 42: 1519-1527.
9. National Committee for Clinical
Laboratory Standards. Reference method
for broth dilution antifungal susceptibility
testing of yeasts. Approved standard.
2nd Ed. NCCLS document M27-A2.
Wayne, PA: National Committee for
Clinical Laboratory Standards; 2002.
10. Pfaller MA, Diekema DJ. Epidemiology of
invasive candidiasis: a persistent public
health problem. Clin. Microbiol. Rev.
2007; 20: 133-163.
11. Pfaller MA, Espinel-Ingroff A, Jones RN.
Clinical evaluation of the Sensititre
YeastOne colorimetric antifungal plate for
antifungal susceptibility testing of the
new triazoles voriconazole, posaconazole,
and ravuconazole. J Clin Microbiol 2004;
42: 4577-4580.
12. Quindós G, Ruesga MT,
Martín-Mazuelos E, Salesa R,
Alonso-Vargas R, Carrillo-Muñoz AJ,
Brena S, San MR, Pontón J. In-vitro
activity of 5-fluorocytosine against 1,021
Spanish clinical isolates of Candida and
other medically important yeasts. Rev
Iberoam Micol 2004; 21: 63-69.
13. Quindós G, Salesa R, Carrillo-Muñoz AJ,
Lipperheide V, Jaudenes L, San-Millan R,
Torres-Rodriguez JM, Pontón J.
Multicenter evaluation of ATB fungus: a
standardized micromethod for yeast
susceptibility testing. Chemotherapy
1994; 40: 245-251.
14. Rementeria A, Sánchez-Vargas LO,
Villar M, Casals JB, Carrillo-Muñoz AJ,
Rodríguez-Andrés C., Eraso E,
Quindós G. Comparison of tablet and
disk diffusion methods for fluconazole
and voriconazole in vitro activity testing
against clinical yeast isolates.
J Chemother 2007; 19: 172-177.
15. Torres-Rodríguez JM, Alvarado-Ramírez E.
In vitro susceptibilities to yeasts using the
ATB(R) FUNGUS 2 method, compared
with Sensititre Yeast One(R) and standard
CLSI (NCCLS) M27-A2 methods.
J Antimicrob Chemother 2007; 60:
658-661.
16. Tortorano AM, Kibbler C, Peman J,
Bernhardt H, Klingspor L, Grillot R.
Candidaemia in Europe: epidemiology
and resistance. Int J Antimicrob Agents
2006; 27: 359-366.
6
References
Rev Iberoam Micol 2008; 25: 3-6