Analysis of the Influence of Tween Concentration, Inoculum Size, Assay Medium, and Reading Time on Susceptibility Testing of Aspergillus spp.
The influence of several test variables on susceptibility testing of Aspergillus spp. was assessed. A collection of 28 clinical isolates was tested against amphotericin B, itraconazole, voriconazole, and terbinafine. Inoculum size (104 CFU/ml versus 105 CFU/ml) and glucose supplementation (0.2% versus 2%) did not have significant effects on antifungal susceptibility testing results and higher inoculum size and glucose concentration did not falsely elevate MICs. In addition, antifungal susceptibility testing procedure with an inoculum size of 105 CFU/ml distinctly differentiated amphotericin B or itraconazole-resistant Aspergillus strains in vivo from the susceptible ones. Time of incubation significantly affected the final values of MICs, showing major increases (two to six twofold dilutions, P < 0.01 by analysis of variance) between MIC readings at 24 and 48 h, but no differences were observed between antifungal susceptibility testing results obtained at 48 h and at 72 h. Significantly higher MICs were uniformly associated with higher concentrations of Tween (P < 0.01), used as a dispersing agent in the preparation of inoculum suspensions. The geometric mean MICs showed increases of between 1.5- and 10-fold when the Tween concentration varied from 0.1% (the geometric means for amphotericin B, itraconazole, voriconazole, and terbinafine were 1.29, 0.69, 1.06, and 0.64 μg/ml, respectively) to 5% (the geometric means for amphotericin B, itraconazole, voriconazole, and terbinafine were 1.97, 5.79, 1.60, and 4.66 μg/ml, respectively). The inhibitory effect of Tween was clearly increased with inoculum sizes of 105 CFU/ml and was particularly dramatic for itraconazole, terbinafine, and Aspergillus terreus. The inoculum effect was not observed when the Tween concentration was below 0.5% (P > 0.01).
JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 2005, p. 1251–1255 Vol. 43, No. 3
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Analysis of the Inﬂuence of Tween Concentration, Inoculum Size,
Assay Medium, and Reading Time on Susceptibility Testing of
Amel Aberkane, Eva Petrikkou, Emilia Mellado,
Juan Luis Rodriguez-Tudela,
and Manuel Cuenca-Estrella*
Servicio de Micologı´a, Centro Nacional de Microbiologı´a, Instituto de Salud Carlos III, Madrid, Spain
Received 13 August 2004/Returned for modiﬁcation 14 September 2004/Accepted 18 November 2004
The inﬂuence of several test variables on susceptibility testing of Aspergillus spp. was assessed. A collection
of 28 clinical isolates was tested against amphotericin B, itraconazole, voriconazole, and terbinaﬁne. Inoculum
CFU/ml versus 10
CFU/ml) and glucose supplementation (0.2% versus 2%) did not have signiﬁcant
effects on antifungal susceptibility testing results and higher inoculum size and glucose concentration did not
falsely elevate MICs. In addition, antifungal susceptibility testing procedure with an inoculum size of 10
CFU/ml distinctly differentiated amphotericin B or itraconazole-resistant Aspergillus strains in vivo from the
susceptible ones. Time of incubation signiﬁcantly affected the ﬁnal values of MICs, showing major increases
(two to six twofold dilutions, P < 0.01 by analysis of variance) between MIC readings at 24 and 48 h, but no
differences were observed between antifungal susceptibility testing results obtained at 48 h and at 72 h.
Signiﬁcantly higher MICs were uniformly associated with higher concentrations of Tween (P < 0.01), used as
a dispersing agent in the preparation of inoculum suspensions. The geometric mean MICs showed increases
of between 1.5- and 10-fold when the Tween concentration varied from 0.1% (the geometric means for
amphotericin B, itraconazole, voriconazole, and terbinaﬁne were 1.29, 0.69, 1.06, and 0.64 g/ml, respectively)
to 5% (the geometric means for amphotericin B, itraconazole, voriconazole, and terbinaﬁne were 1.97, 5.79,
1.60, and 4.66 g/ml, respectively). The inhibitory effect of Tween was clearly increased with inoculum sizes of
CFU/ml and was particularly dramatic for itraconazole, terbinaﬁne, and Aspergillus terreus. The inoculum
effect was not observed when the Tween concentration was below 0.5% (P > 0.01).
The recent increased incidence of fungal infections and the
growing number of new antifungal agents have multiplied the
demand and interest for in vitro antifungal susceptibility test-
ing (12, 20). For determination of MICs of antifungal agents
two approved reference methods have been published by the
National Committee for Clinical Laboratory Standards, the
M27-A2 reference method for testing the susceptibility of yeast
species (16), and the M38-A reference method for ﬁlamentous
fungi (17). The M38-A methodology adopts some of the steps
in yeast testing compiling the results of extensive collaborative
studies on MIC test variables. However, this reference proce-
dure exhibits some limitations such as long incubation periods
for obtaining inoculum (7 to 10 days), problems for obtaining
inoculum when molds present germinate to hyphal forms and
do not form conidia, poor growth of some species with the
assay medium recommended for susceptibility testing, and ﬁ-
nally, the lack of correlation with clinical outcome (6, 7). Some
studies have been conducted trying to overcome the limita-
tions. Alternative assay media and modiﬁcations of inoculum
size, incubation time, reading procedure, and endpoint deter-
mination have been assessed (3).
Taking into account the individuality of the behavior of
fungi, minor variation of test variables can result in signiﬁcant
changes in MICs. Many sources of variation in antifungal MIC
data have been published, such as the nature of the growth
medium (14, 25), size of the inoculum (9), time of incubation
(22), end-point criterion, pH (1, 13), and even the solvent used
to prepare antifungal stock solutions (8) and the inoculum
preparation procedure (21, 24). In addition, polyoxyethylene
sorbitan monolaurate, Tween 20, a nonionic surfactant, has
been widely employed as a dispersing agent in the preparation
of conidial suspensions of hydrophobic fungi, particularly As-
pergillus spp. It makes the dispersion of spores on water easier,
yielding a more reliable procedure for inoculum preparation.
However, surfactants can interact with both organisms and
drugs affecting the activity in vitro of antimicrobial agents (2,
26). The M38-A document recommends the use of a drop of
Tween 20 per ml (approximately 0.01 to 0.02 ml, 0.5 to 1%)
(17) for inoculum preparation, but no standardized amounts of
these agents have been employed in most of the reports pub-
lished until now.
The aim of this study was to investigate the effect of several
test variables on susceptibility testing of Aspergillus spp. The
study included strains from patients who had failed to respond
to itraconazole or amphotericin B therapy and isolates exhib-
iting high MICs whose in vitro data correlated with the results
of animal models of infection (4, 5, 15, 19, 27).
MATERIALS AND METHODS
Fungi. A collection of 28 clinical isolates was tested. They were selected to
represent ranges of susceptibilities in vitro as broad as possible. This collection
included (i) seven isolates of Aspergillus fumigatus kindly provided by David W.
* Corresponding author. Mailing address: Servicio de Micologı´a ,
Centro Nacional de Microbiologı´a, Instituto de Salud Carlos III, Ctra
Majadahonda-Pozuelo, Km 2. 28220, Majadahonda (Madrid), Spain.
Phone: 34-91-5097961. Fax: 34-91-5097966. E-mail: mcuenca-estrella
Denning, CNM-CM-1242 (Mold Collection of the Spanish Center for Microbi-
ology), CNM-CM-1243, CNM-CM-1244, CNM-CM-1245, CNM-CM-1246,
CNM-CM-1247, and CNM-CM-1252; (ii) seven isolates of Aspergillus terreus
(CNM-CM-1572 to CM-1579); (iii) seven isolates of Aspergillus ﬂavus (CNM-
CM-459, CNM-CM-890, CNM-CM-900, CNM-CM-1248, CNM-CM-1264,
CNM-CM-1295, and CNM-CM-1357); and (iv) seven isolates of Aspergillus niger
(CNM CM-152, CNM-CM-519, CNM-CM-794, CNM CM-879, CNM-CM-1524,
CNM-CM-1562, and CNM-CM-1607). A. fumigatus ATCC 9197 and Paecilomy-
ces variotii ATCC 22319 were included as control isolates in each set of exper-
iments. All the strains were stored on slants or in water suspension at ambient
temperature until used.
Antifungal drugs. The antifungal agents utilized were amphotericin B (Sigma-
Aldrich Quı´mica, Madrid, Spain), itraconazole (Janssen Pharmaceutica, Madrid,
Spain), voriconazole (Pﬁzer Ltd, Sandwich, United Kingdom), and terbinaﬁne
(Novartis, Basel, Switzerland). All of them were obtained as reagent-grade pow-
ders from their respective manufacturers. Stock solutions were prepared in 100%
dimethyl sulfoxide (Sigma-Aldrich Quı´mica) at concentrations 100 times the
highest concentration to be tested. All drugs were then diluted in the test
medium and dispensed into 96-well ﬂat-bottom microdilution trays and frozen at
⫺20°C or ⫺70°C (amphotericin B) until needed. The plates contained twofold
serial dilutions of the antifungal drugs with a volume of assay medium of 100 l.
Two drug-free medium wells for sterility and growth controls were used. The
range of concentrations tested was as follow: amphotericin B, from 16 to 0.03
g/ml; itraconazole, from 8 to 0.015 g/ml; voriconazole, from 64 to 0.12 g/ml;
and terbinaﬁne, from 16 to 0.03 g/ml.
Antifungal susceptibility testing. Antifungal susceptibility testing was per-
formed simultaneously with different Tween concentration, assay medium, inoc-
ulum size, and incubation time. Tween 20 (Sigma-Aldrich Quı´mica) was added to
facilitate the preparation of suspensions of spores of Aspergillus spp. Three sterile
water-Tween solutions were used to assess their inﬂuence on MICs (5%, 0.5%,
and 0.1%, corresponding to ﬁnal volumes of 0.05, 0.005, and 0.001 ml, respec-
tively, per ml of inoculum suspension). The surface of the colonies was covered
with a sterile water-Tween solution and scraped with a sterile loop. Conidia were
shaken vigorously for 10 s to break up clumps, transferred to a sterile tube, and
then adjusted by microscopic enumeration with a cell-counting hemacytometer
(Improved Neubauer Chamber; Merck, S. A., Madrid, Spain) to provide a ﬁnal
inoculum of 1 ⫻ 10
to 5 ⫻ 10
In addition, antifungal susceptibility testing was performed simultaneously
with two assay media, (i) standard RPMI 1640, with glutamine and without
bicarbonate, buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid
(MOPS) (Sigma-Aldrich Quı´mica), and (ii) RPMI 1640 supplemented with 18 g
of glucose (Sigma-Aldrich Quı´mica) per liter to reach a ﬁnal concentration of 2%
(RPMI-2% glucose). Both media were prepared as double-strength solutions.
Two different inoculum sizes were also tested, (i) 1 ⫻ 10
to 5 ⫻ 10
(ii) 1 ⫻ 10
to 5 ⫻ 10
On the day of the test, each well was inoculated with 100 l of the corre-
sponding conidial inoculum suspension. All plates were incubated at 35°C. The
MIC endpoint was deﬁned as the lowest drug concentration that showed absence
of growth (100% inhibition). Readings were made after 24, 48, and 72 h of
incubation with the aid of a reading mirror. All the experiments were repeated
three times on different days.
Data analysis. Statistical analysis was done with Statistical Package for the
Social Sciences (version 12.0.) (SPSS S.L, Madrid, Spain). Both on-scale and
off-scale results were included in the analysis. The off-scale MIC values were
converted to the next concentration up or down. The effect of test variables on
the MIC was analyzed with the analysis of variance test. In order to approximate
a normal distribution, the MICs were transformed to log
values. For some cases,
there were clear outliers to the normal distribution after log transformation.
Subsequently, results were also analyzed by the Mann-Whitney U test, a non-
parametric test that permits the inclusion of data treated as outliers for the
analysis of variance. P ⬍ 0.01 was considered signiﬁcant.
Most of the isolates tested grew in the microtiter plates to
detectable endpoints within 48 h. Table 1 summarizes the
range of MICs obtained per isolate regardless of the combi-
nation of variables included in the study. Notably, a wide range
of MICs was found for all the antifungal agents and species,
advancing a great inﬂuence of variables on MICs.
In this sense, the concentration of Tween showed a dramatic
effect on the MICs of antifungal compounds. The analysis of
variance test applied to the data after log transformation pro-
TABLE 1. Range of MIC values obtained for the 28 isolates
Amphotericin B Itraconazole Voriconazole Terbinaﬁne
CNM CM-1242 A. fumigatus 0.06–2.0 0.5 1.0 0.015–8.0 0.5 4.0 0.12–2.0 0.5 1.0 0.25–⬎16.0 4.0 ⬎16.0
CNM CM-1243 A. fumigatus 0.12–2.0 0.5 1.0 0.015–⬎8.0 1.0 8.0 0.12–2.0 0.5 2.0 0.50–⬎16.0 8.0 ⬎16.0
CNM CM-1244 A. fumigatus 0.12–2.0 0.5 1.0 0.015–⬎8.0 0.5 5.2 0.12–4.0 0.5 2.0 0.50–⬎16.0 8.0 ⬎16.0
CNM CM-1245 A. fumigatus 0.06–1.0 0.5 1.0 0.015–⬎8.0 0.75 4.0 0.25–2.0 0.5 2.0 0.50–⬎16.0 8.0 ⬎16.0
CNM CM-1246 A. fumigatus 0.06–2.0 0.5 1.0 0.06–⬎8.0 ⬎8.0 ⬎8.0 0.12–0.50 0.25 0.5 0.50–⬎16.0 4 ⬎16.0
CNM CM-1247 A. fumigatus 0.25–4.0 1.0 2.0 0.50–⬎8.0 ⬎8.0 ⬎8.0 0.25–4.0 1.0 2.0 0.50–⬎16.0 ⬎16.0 ⬎16.0
CNM CM-1252 A. fumigatus 0.25–4.0 1.0 2.0 0.25–⬎8.0 ⬎8.0 ⬎8.0 0.12–4.0 1.0 2.0 0.50–⬎16.0 ⬎16.0 ⬎16.0
CNM CM-1572 A. terreus 0.12–8.0 2.0 8.0 0.03–8.0 0.5 4.0 0.50–8.0 2.0 4.0 0.03–8.0 0.5 4.0
CNM CM-1573 A. terreus 0.12–8.0 2.0 8.0 0.06–8.0 0.5 4.0 0.50–8.0 2.0 4.0 0.03–8.0 0.5 4.0
CNM CM-1574 A. terreus 0.03–8.0 2.0 4.0 0.03–8.0 0.5 4.0 0.50–8.0 2.0 4.0 0.03–8.0 0.5 4.0
CNM CM-1575 A. terreus 0.12–16.0 2.0 4.0 0.015–8.0 0.5 4.0 0.25–8.0 2.0 4.0 0.03–8.0 0.5 4.0
CNM CM-1576 A. terreus 0.12–8.0 2.0 4.0 0.015–8.0 0.5 4.0 0.25–8.0 2.0 4.0 0.03–8.0 0.5 4.0
CNM CM-1578 A. terreus 0.12–⬎16.0 2.0 8.0 0.015–8.0 0.5 4.0 0.25–8.0 1.0 4.0 0.03–8.0 0.5 4.0
CNM CM-1579 A. terreus 0.12–16.0 2.0 8.0 0.03–8.0 0.5 4.0 0.25–8.0 1.0 4.0 0.03–8.0 0.75 4.0
CNM CM-459 A. ﬂavus 1.0–⬎16.0 8 ⬎16.0 0.12–8.0 0.75 4.0 0.25–2.0 1.0 2.0 0.12–4.0 0.37 2.0
CNM CM-890 A. ﬂavus 0.50–8.0 1.0 2.0 0.06–8.0 0.5 4.0 0.12–4.0 1.0 2.0 0.03–4.0 0.25 1.0
CNM CM-900 A. ﬂavus 0.25–8.0 2.0 2.0 0.015–8.0 0.5 4.0 0.25–4.0 1.0 2.0 0.03–2.0 0.25 1.1
CNM CM-1248 A. ﬂavus 1.0–8.0 2.0 4.0 0.03–8.0 0.5 4.0 0.25–4.0 1.0 2.0 0.03–2.0 0.25 1.0
CNM CM-1264 A. ﬂavus 0.25–8.0 1.0 2.0 0.03–8.0 0.5 4.0 0.12–4.0 1.0 2.0 0.03–2.0 0.12 1.0
CNM CM-1295 A. ﬂavus 0.50–8.0 2.0 4.0 0.06–4.0 0.5 4.0 0.25–4.0 1.0 2.0 0.03–4.0 0.25 1.0
CNM CM-1357 A. ﬂavus 0.25–2.0 1.0 2.0 0.03–4.0 0.5 4.0 0.25–4.0 1.0 2.0 0.03–1.0 0.06 0.5
CNM CM-152 A. niger 0.12–4.0 0.5 1.0 0.06–⬎8.0 1.0 4.0 0.12–1.0 0.5 0.5 0.03–2.0 0.25 1.0
CNM CM-519 A. niger 0.06–2.0 0.5 1.0 0.12–⬎8.0 ⬎8.0 ⬎8.0 0.12–2.0 1.0 2.0 0.06–4.0 1.0 2.6
CNM CM-794 A. niger 0.12–8.0 1.0 2.0 0.015–2.0 0.25 1.0 0.12–0.25 0.12 0.25 0.03–2.0 0.25 1.0
CNM CM-879 A. niger 0.03–16.0 2.0 4.0 0.015–⬎8.0 ⬎8.0 ⬎8.0 0.12–4.0 2.0 2.0 0.03–4.0 0.5 2.0
CNM CM-1524 A. niger 0.12–4.0 1.0 1.0 0.06–⬎8.0 1.0 8.0 0.12–1.0 0.5 0.5 0.03–2.0 0.25 1.0
CNM CM-1562 A. niger 0.06–2.0 0.5 1.0 0.06–⬎8.0 1.0 8.0 0.12–1.0 0.5 1.0 0.03–4.0 0.5 2.0
CNM CM-1607 A. niger 0.12–4.0 1.0 2.0 0.06–⬎8.0 1.0 ⬎8.0 0.12–2.0 0.5 1.0 0.06–4.0 0.5 2.0
1252 GOMEZ-LOPEZ ET AL. J. CLIN.MICROBIOL.
vided signiﬁcant differences among MICs obtained for each
Tween concentration regardless of the inoculum size, the assay
medium, and the time of reading.
The highest MICs were uniformly associated with the high-
est concentration of Tween (5%) when all the isolates were
analyzed together (Table 2). This effect was particularly nota-
ble for itraconazole and terbinaﬁne, for which the MICs ob-
tained with the highest Tween concentration were signiﬁcantly
higher (P ⬍ 0.01). In addition, signiﬁcant differences were
found between the MICs of itraconazole and terbinaﬁne
achieved with concentrations of Tween of 0.5% and those
achieved with the lowest concentration of Tween (0.1%, P ⬍
0.01). The results for amphotericin B and voriconazole, how-
ever, were moderately inﬂuenced by the Tween concentration.
The MICs for these agents were signiﬁcantly higher (P ⬍ 0.01)
only when the concentration of Tween used was maximal (5%).
By species, comparisons between the MICs obtained with
5% and 0.1% Tween also resulted in signiﬁcant differences for
the majority of them (P ⬍ 0.01), with major increases between
MICs for itraconazole and terbinaﬁne. A. terreus appeared as
the species most inﬂuenced by Tween concentration, since the
MICs for most of the antifungals tested were signiﬁcantly dif-
ferent (P ⫽ 0.00). Table 2 exempliﬁes the MIC results per
species for each Tween concentration, ﬁxing the medium
(RPMI-2% glucose), the inoculum size (10
CFU/ml), and the
time of reading (48 h) as constants. The geometric mean MICs
showed increases of between 1.5- and 10-fold when the Tween
concentration varied from 0.1% (the geometric means for am-
photericin B, itraconazole, voriconazole, and terbinaﬁne were
1.29, 0.69, 1.06, and 0.64 g/ml, respectively) to 5% (the geo-
metric means for amphotericin B, itraconazole, voriconazole,
and terbinaﬁne were 1.97, 5.79, 1.60, and 4.66 g/ml, respec-
tively). In summary, the concentration of Tween emerged as
having a signiﬁcant inﬂuence on antifungal susceptibility test-
ing of Aspergillus spp. but the magnitude of the effect was
species and antifungal dependent.
Regarding inoculum size, changes in MICs were moderate
when the inoculum size varied from 10
fact, higher inoculum sizes increased the MICs for all the
antifungal agents tested, but these increases were not sta-
tistically signiﬁcant (P ⬎ 0.01) if other test variables were
ﬁxed as constants. Most of the MICs rose one to two twofold
dilutions (data not shown). However, a higher inoculum
effect was observed when MICs were analyzed, taking into
account the combined effect of inoculum size and Tween
concentration. In this sense, signiﬁcantly higher MICs were
observed for antifungal susceptibility tests including both
the highest inoculum size (10
UFC/ml) and the highest
Tween concentration (5%). The combined effect of Tween
concentration and size of inoculum on MICs is exempliﬁed
on Table 3, which exhibits MICs of antifungal agents after
48 h of incubation. The geometric mean MIC increased
from 2- to 14-fold when the inoculum was adjusted to 10
UFC/ml and prepared with the highest concentration of
Tween (5%). Again, the highest increases were observed for
itraconazole and terbinaﬁne. By species, A. terreus was the
most inﬂuenced by the size of the inoculum.
Analyzing MICs by assay medium, antifungal susceptibility
testing results for all the isolates grown in RPMI were identical
to those obtained with RPMI supplemented with glucose. In
addition, glucose supplementation did not affect the MIC val-
ues whatever the ﬁnal Tween concentration used for the inoc-
ulum preparation (P ⬎ 0.01).
Time of incubation signiﬁcantly affected the ﬁnal MICs
(data not shown). A major increase (two to six dilutions) was
observed between 24 and 48 h (P ⬍ 0.01), a difference that
could be explained by the fact that many isolates needed 48 h
for exhibiting detectable growth. However, differences be-
tween MIC readings at 48 and 72 h were minimal (no more
than two dilutions) for most of the antifungals analyzed. A
major dependency on the length of the period of incubation
was noted for amphotericin B, whose MICs were signiﬁcantly
higher for 72 h (P ⬍ 0.01), but the MICs after 48 and 72 h of
TABLE 2. In vitro susceptibilities of Aspergillus spp. obtained with RPMI-2% glucose medium, an inoculum size of 10
UFC/ml, 48 of
incubation, and different Tween 20 concentrations
Species (no. of
Amphotericin B Intraconazole Voriconazole Terbinaﬁne
Range GM Range GM Range GM
A. fumigatus (7) 5 0.50–4.0 1.18 4.0–⬎8.0 8.83 0.25–2.0 1.18 16.0–⬎16.0 27.13
0.5 0.25–2.0 0.69 0.50–⬎8.0 3.28 0.25–4.0 1.22 4.0–⬎16.0 8.27
0.1 0.25–2.0 0.57 0.25–⬎8.0 1.87 0.12–2.0 0.79 2.0–⬎16.0 5.94
A. terreus (7) 5 2.0–8.0 4.56 2.0–8.0 4.88 2.0–8.0 4.56 4.0 4
0.5 2.0–8.0 4.27 0.06–1.0 0.48 1.0–4.0 1.94 0.50–2.0 0.88
0.1 2.0–8.0 3.74 0.25–0.50 0.26 1.0–4.0 1.81 0.25–0.50 0.41
A. ﬂavus (7) 5 1.0–⬎16.0 2.97 2.0–8.0 4.00 1.0–2.0 1.75 1.0–2.0 1.28
0.5 0.50–8.0 1.94 0.50–1.0 0.65 1.0–2.0 1.35 0.12–0.50 0.33
0.1 1.0–8.0 1.70 0.12–0.50 0.35 1.0–4.0 1.26 0.03–0.50 0.14
A. niger (7) 5 0.50–2.0 0.94 1.0–⬎8.0 6.56 0.12–2.0 0.69 1.0–4.0 2.21
0.5 0.25–2.0 0.67 0.12–⬎8.0 1.75 0.12–2.0 0.67 0.25–2.0 0.61
0.1 0.25–2.0 0.77 0.12–⬎8.0 1.34 0.12–2.0 0.69 0.25–1.0 0.41
Total (28) 5 0.50–⬎16.0 1.97 1.0–⬎8.0 5.79 0.12–8.0 1.60 1.0–⬎16.0 4.66
0.5 0.25–8.0 1.40 0.06–⬎8.0 1.58 0.12–4.0 1.21 0.12–⬎16.0 1.15
0.1 0.25–8 1.29 0.12–⬎8 0.69 0.12–4 1.06 0.03–⬎16 0.64
GM, geometric mean.
VOL. 43, 2005 SUSCEPTIBILITY TESTING OF ASPERGILLUS SPP. 1253
incubation were comparable (P ⬎ 0.05) for the rest of the
antifungal agents evaluated.
A great deal of progress has been achieved in antifungal
susceptibility testing of ﬁlamentous fungi. A reproducible ref-
erence methodology has been developed (17). However, the
reference method cannot be the best technique for testing all
organisms and it is possible that new antifungal agents require
modiﬁed methods or new assays show a better correlation
between in vitro and in vivo susceptibility results and patient
outcome. Modiﬁcations have been proposed, but minor varia-
tions can have signiﬁcant inﬂuences on MICs and should be
analyzed in depth before being put into clinical practice. Sig-
niﬁcant inﬂuences on MICs have been described depending on
time of incubation and other test variables (23), and some
reports have pointed out that even large inoculum size and
glucose supplementation could falsely elevate the MICs (18).
However, Denning et al. demonstrated that inoculum sizes
higher than those proposed, 1 ⫻ 10
to 5 ⫻ 10
generate reproducible in vitro susceptibility data for Aspergillus
spp. that can predict clinical outcome (4). They were able to
identify test conditions in vitro that consistently differentiated
resistant Aspergillus strains in vivo from the susceptible ones.
The former had high itraconazole MICs and demonstrated no
beneﬁt over untreated controls in a murine model when
treated with different itraconazole doses. Regarding glucose
supplementation, previous reports suggest no effect on As-
pergillus growth rate when RPMI is supplemented with 2%
glucose, even after incubation for 100 h (14).
Our results agree with previous ﬁndings since no changes in
MICs were found with RPMI or RPMI supplemented with
glucose, and MICs were not falsely elevated when an inoculum
size of 10
UFC/ml was used. In addition, the antifungal sus
ceptibility testing procedure with an inoculum size of 10
UFC/ml distinctly identiﬁed resistant Aspergillus strains in vivo
as the MICs of amphotericin B and itraconazole were ⬎2
g/ml and ⬎8 g/ml, respectively.
A signiﬁcant effect of surfactant on the MIC results of As-
pergillus was observed. We have consistently obtained higher
MICs when major concentrations of Tween were used for the
inoculum preparation. Some reports have described the effect
of nonionic detergent on the activity in vitro of antimicrobial
agents (10, 11). A great inﬂuence was found by Komatsuzawa
et al. even with a concentration of the surfactant as low as
0.015%. Apparently, the effect of Tween is antifungal depen-
dent, and we postulate that it could be related to the solubility
of the antifungal in the medium used. It has been suggested
that some surfactants interfere with antimicrobial activity by
solubilizing molecules of the antimicrobial agent within surfac-
tant micelles, preventing the microorganism-agent interaction.
The surfactant could modify the antifungal solubility index on
RPMI and favor agent precipitation, allowing MICs to be
increased. This study demonstrates that the inhibitory effect of
Tween was dramatic for itraconazole and terbinaﬁne and par-
ticularly signiﬁcant for A. terreus isolates.
The use of Tween for inoculum preparation for antifungal
susceptibility testing of Aspergillus spp. has been universally
accepted. The results of the present study conﬁrm the impor-
tance of standardizing the concentration of surfactant to use
on inoculum preparation. Changes in MICs as great as four
dilutions could change the classiﬁcation of a strain from sus-
ceptible to resistant. This effect is clearly increased with an
inoculum size of 10
UFC/ml. However, signiﬁcant inoculum
effect was not observed when Tween concentration was below
0.5%. Glucose supplementation of assay medium did not have
a signiﬁcant inﬂuence on MICs of the four antifungal agents
tested. We conclude that special attention must be taken to the
addition of Tween 20 for inoculum preparation of Aspergillus
spp., in order to allow reproducible antifungal susceptibility
testing results to be obtained and to avoid falsely elevated
This work was partially supported by a grant from the EC-TMR-
EUROFUNG network (grant ERBFMXR-CT970145), by a grant
from the Fondo de Investigaciones Sanitarias, Instituto de Salud Car-
los III (grant 99/0198), and by research project 99/1199 from the
Instituto de Salud Carlos III.
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TABLE 3. MIC results showing the combined effect of Tween concentration and inoculum size after 48 h of incubation
Species (no. of
Amphotericin B Intraconazole Voriconazole Terbinaﬁne
Range GM Range GM Range GM
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0.50–⬎16.0 1.83 1.0–⬎8.0 5.71 0.12–8.0 1.51 0.50–⬎16.0 4.50
GM, geometric mean.
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