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Journal of Clinical Microbiology, September 2001, p. 3402-3408, Vol. 39, No. 9
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.9.3402-3408.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Colorimetric Assay for Antifungal Susceptibility Testing of
Aspergillus Species
Joseph
Meletiadis,1
Johan W.
Mouton,2
Jacques F. G. M.
Meis,3
Bianca A.
Bouman,1
J. Peter
Donnelly,3
Paul E.
Verweij,1,* and
EUROFUNG Network
Departments of Medical
Microbiology1 and
Hematology,3 University Medical Center
Nijmegen, and Department of Medical Microbiology and
Regional Public Health Laboratory, Canisius Wilhelmina
Hospital,2 Nijmegen, The Netherlands
Received 20 February 2001/Returned for modification 11 April
2001/Accepted 27 June 2001
 |
ABSTRACT |
A colorimetric assay for antifungal susceptibility testing of
Aspergillus species (Aspergillus
fumigatus, Aspergillus flavus, Aspergillus
terreus, Aspergillus nidulans, and Aspergillus
ustus) is described based on the reduction of the tetrazolium
salt
2,3-bis(2-methoxy-4-nitro-5-[(sulphenylamino)carbonyl]-2H-tetrazolium-hydroxide (XTT) in the presence of menadione as an electron-coupling agent. The
combination of 200 µg of XTT/ml with 25 µM menadione resulted in a
high production of formazan within 2 h of exposure, allowing the
detection of hyphae formed by low inocula of 102 CFU/ml
after 24 h of incubation. Under these settings, the formazan production correlated linearly with the fungal biomass and
less-variable concentration effect curves for amphotericin B and
itraconazole were obtained.
| |
TEXT |
Tetrazolium salts are heterocyclic
organic compounds that substitute the natural final acceptor (oxygen)
in the biological redox process and are reduced to formazan derivatives
by receiving electrons enzymically from substances of the hydrogen
transport system or nonenzymically from artificial electron
transporters (phenazine methosulfate and menadione) which enhance the
reaction. Tetrazolium salts can penetrate rapidly into intact cells and directly into subcellular membranes with dehydrogenase activity, where
they are converted to colored formazan derivatives (1, 14). Therefore, they were used as indicators of reducing
systems. The tetrazolium salt MTT has been used for antifungal
susceptibility testing of various yeasts and filamentous fungi, and
testing was in agreement with the corresponding standard methods
recommended by the National Committee for Clinical Laboratory Standards
(NCCLS) (2, 7). The disadvantage of MTT, however, is that
the process includes the solubilization of formazan derivatives. As an
alternative, a new tetrazolium salt,
2,3-bis(2-methoxy-4-nitro-5-[(sulphenylamino)carbonyl]-2H-tetrazolium-hydroxide (XTT) (10), has been employed for antifungal
susceptibility testing of yeasts and resulted in clear-cut
endpoints for various antifungal agents (4, 15). XTT is
converted into a water-soluble formazan, thereby avoiding the
additional steps for the solubilization of formazan derivatives
(8, 12), but needs the presence of an electron-coupling
agent. The nature and the concentration of this agent are critical in
order to obtain a good correlation between the formazan production and
the number of viable fungi and less-variable concentration effect
curves (1, 15).
We developed a colorimetric assay for the quantification of fungal
growth of five different Aspergillus species based on the tetrazolium salt XTT by standardizing various factors that influence XTT conversion. This assay was also tested in the presence of antifungal drugs in order to ascertain its potential for antifungal susceptibility testing of filamentous fungi.
Isolates.
Two clinical isolates of Aspergillus
fumigatus [AZN5161 (S) and AZN 5241 (R)] and one each of
Aspergillus flavus (AZN 510), Aspergillus terreus
(AZN 7320), Aspergillus nidulans (AZN 8933), and
Aspergillus ustus (AZN 9420) from our private collection was selected, and Candida parapsilosis (ATCC 22019) and
Candida krusei (ATCC 6258) were used for quality control.
Isolates were revived by subculturing them twice onto Sabouraud glucose
agar (SAB) plates with chloramphenicol first at 30°C and then at
37°C for 5 to 7 days. Conidia were collected with a swab and
suspended in sterile saline containing 0.05% Tween 20. After the heavy
particles were allowed to settle for 5 to 10 min, the turbidity of
the supernatants were measured spectrophotometrically (Spectronic 20D;
Milton Roy, Rochester, N.Y.) at 530 nm and transmission was adjusted to
80 to 82%, corresponding to an inoculum size of 1 × 106 to 5 × 106 CFU/ml. The inoculum size
was confirmed by plating serial dilutions of conidia suspensions
on SAB plates.
Medium.
RPMI 1640 medium (with L-glutamine;
without bicarbonate) (GIBCO BRL, Life Technologies, Woerden, The
Netherlands) buffered to pH 7.0 with 0.165 M
3-N-morpholinopropanesulfonic acid (MOPS) (Sigma-Aldrich
Chemie GmbH, Steinheim, Germany) was used throughout.
XTT.
XTT (Sigma Chemical, St. Louis, Mo.) was dissolved in
saline at a final concentration of 1 mg/ml. The solution was filtered through a 0.22 µm-pore-size filter.
Electron-coupling agents.
Two electron acceptors were
evaluated, menadione (Sigma-Aldrich Chemie GmbH) and phenazine
methosulfate (PMS) (Sigma-Aldrich Chemie GmbH). Menadione was first
dissolved in acetone at a concentration of 10 mM and was then diluted
1:10 in saline. PMS was directly dissolved in saline at a final
concentration of 1 mM. Further dilutions of both agents were made in saline.
Antifungal drugs.
Itraconazole (Janssen-Cilag, Beerse,
Belgium) and amphotericin B (Bristol-Myers Squibb, Woerden, The
Netherlands) were dissolved in dimethyl sulfoxide (DMSO) at final
concentrations of 3,200 and 1,600 mg/liter, respectively.
XTT assay.
Conidia suspensions of each species were diluted
1:100 in the medium, and 200 µl was inoculated in 96-well flat-bottom
microtitration plates (Costar, Corning, N.Y.). After 24 or 48 h of
incubation at 37°C, 50-µl aliquots of various concentrations of XTT
with either menadione or PMS were added to the wells in order to obtain final concentrations of 200, 100, and 50 µg of XTT/ml and 100, 25, 6.25, 1.56, and 0.39 µM menadione or PMS. The microtitration plates
were incubated further for 6 h at 37°C and the optical density
at 450 nm (OD450) was measured at hourly intervals by spectrophotometer (Rosys Anthos ht3; Anthos Labtec Instruments GmbH,
Salzburg, Austria). The XTT assay was performed for each species in
triplicate and the ODs after 1, 2, and 4 h of exposure to various
concentrations of XTT and the two electron coupling agents of fungi
incubated for 24 and 48 h were plotted.
Quantitative assay of fungal viability.
The relationship
between the number of viable fungi and the amount of XTT reduction was
tested by incubating various inocula of Aspergillus conidia
(102 to 106 CFU/ml) with XTT and various
concentrations of menadione. Conidia suspensions were diluted 1:10
serially in the medium to up to 102 CFU/ml. Then, 96-well
flat-bottom microtitration plates were inoculated with 200 µl of each
conidia dilution. After 24 h of incubation at 37°C, 50-µl
aliquots of XTT and various concentrations of menadione were added to
each well in order to obtain final concentrations of 200 µg/ml for
XTT and 100, 25, 6.25, 1.56, and 0.39 µM for menadione. The
microtitration plates were then incubated for another 2 h, after
which 100 µl of the supernatant of each well was transferred in clean
wells. The OD450 was measured spectrophotometrically. This
experiment was performed in triplicate for each species, and the data
were analyzed by linear regression analysis. Regression lines were
plotted for each concentration of menadione and for each species,
together with the 95% confidence intervals. The slopes and the
r2 of each regression line were
reported as an estimation of steepness of the line and the goodness of
fit, respectively. An r2 value of 1 indicates perfect correlation. The nonlinearity of the curves was
checked by a runs test following the linear regression.
Antifungal susceptibility assay.
Stock solutions of antifungal
drugs were serially diluted twofold in DMSO, and then each drug
concentration was diluted 1:50 in the medium in order to obtain twofold
final concentrations which ranged from 0.015 to 16 mg of amphotericin
B/liter and 0.03 to 32 mg of itraconazole/liter. Wells of 96-well
flat-bottom microtitration plates were filled with 100 µl of each
drug concentration. A drug-free well containing 2% DMSO in the medium
served as the growth control. Each well was inoculated with 100 µl of
conidia suspensions diluted 1:50 in medium in order to obtain a final
inoculum of 1 × 104 to 5 × 104
CFU/ml. The plates were incubated for 24 or 48 h at 37°C, and the MIC-0 was determined visually by four different observers to be the
lowest drug concentration showing no visible growth (NCCLS)
(9). Afterwards, the OD of each well at 405 nm was measured in order to quantify the biomass of fungal growth
(spectrophotometric method). Then, 50 µl of XTT-menadione was
added to each well in order to obtain final concentrations of 200 µg/ml and 25 µM, respectively. After 2 h of further
incubation, the OD of each well at 450 nm was measured in order to
quantify the formazan production (colorimetric method). Background OD
was obtained by spectrophotometric measurements of noninoculated wells
processed in the same way as the inoculated wells. The relative ODs for
each well based on both measurements at 405 and 450 nm were calculated
(in percent) based on the following equation: [(OD of drug containing
well 
background OD)/(OD of drug-free well background OD of
drug-free well)] × 100%. The tests were carried out in triplicate in
three independent experiments for each strain. Results from each
experiment were analyzed by nonlinear regression analysis by using a
four parameter logistic model (sigmoid curve with variable slope) known
as the Emax model, which is described by the
following equation: E = Emax × (D/EC50)m/[1 + (D/EC50)m],
where E is the relative OD (dependent variable),
Emax is the maximum relative OD, D is
the drug concentration (independent variable), EC50 is the
drug concentration producing 50% of the Emax,
and m is the slope that describes the steepness of the curve (3). Since data were normalized by using the relative ODs, the top and the bottom of the Emax model
corresponded to 100 and 0%, respectively. Analysis was carried out
using the GraphPad Prism Software (San Diego, Calif.). Deviation from
the model was tested by the runs test, and goodness of fit was checked
by the r2 values. In order to compare
the concentration effect curves generated by the spectrophotometric and
colorimetric methods, the best-fit values of EC50 and slope
(m) obtained by the regression analysis were used. The
differences between the best-fit values of the concentration effect
curves for all species were analyzed by analysis of variance followed
by a Bonferroni post test. The significance level of 0.05 was chosen.
Results of XTT assay.
The tetrazolium salt XTT was not
metabolized by any of the Aspergillus species until an
electron-coupling agent was added. Conversion of XTT by a certain
amount of hyphae depended on the concentration of XTT and the exposure
time as well as the electron-coupling agent and its concentration
(Fig. 1). Between the three
concentrations of XTT used, 200 µg/ml resulted in
two-times-higher formazan production than 100 and 50 µg/ml, for which
the formazan production was similar. Among the two
electron-coupling agents, menadione was eight times more potent
than PMS for all Aspergillus species. The use of PMS resulted in a background absorbance higher than that of menadione, especially at the concentration of 100 µM, where the OD of the blank
after 6 h of exposure was 0.805 for PMS and 0.347 for menadione. Relatively lower background absorbances (
0.2) were observed at maximal concentrations of 25 µM menadione and 1.56 µM PMS (data not
shown). Any increase of menadione concentration beyond 25 µM did not
have any effect in formazan production when XTT was exposed for longer than 2 h to fungi which were incubated for 48 h.
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FIG. 1.
XTT reduction by Aspergillus species. The
Aspergillus strains were incubated for 24 or 48 h and were
then exposed for 1, 2, and 4 h to various concentrations of XTT,
200 (squares), 100 (triangles) and 50 (circles) µg/ml, combined with
menadione (close symbols) or PMS (open symbols) at various
concentrations (0.39, 1.56, 6.25, 25, and 100 µM). Each data point
represents the average of all strains tested in triplicate.
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Results of quantitative assay of fungal viability.
The
relationship between the XTT conversion and increasing inocula of
Aspergillus species was tested using 200 µg of XTT/ml with
25 µM menadione after 2 h of exposure. The analysis of results showed a linear relationship between the OD and log CFU (Fig. 2). The slopes and the coefficients
obtained by the regression analysis depended on the concentration of
menadione used for each species. A general pattern of concentration
dependence was observed for all five species. At menadione
concentrations lower than 1.56 µM, low XTT conversion rates were
apparent (slopes ranged between 0.01 and 0.13) for all species. For
A. nidulans and A. flavus, low conversion rates
were also observed at a menadione concentration of 6.25 µM (Table
1). The conversion rates increased at
higher concentrations of menadione, generating higher slopes, up to
0.65, as in the case of A. terreus with 100 µM menadione.
However, at this concentration of menadione, larger deviations from
linearity were observed for all species (except A. ustus)
and very low slopes were obtained for A. fumigatus (R) and
A. flavus (0.05 and 0.22, respectively). In addition, at the
concentration of 100 µM menadione, formazan production exceeded
an OD of 3 after 4 h of exposure of XTT, which was the detection
limit of the spectrophotometer, especially when hyphae were exposed to
XTT after 48 h of incubation.
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FIG. 2.
Relationship between the amount of XTT and increasing
inocula of each Aspergillus species. The reduction of 200 µg of XTT/ml in the presence of various concentrations of menadione
(0.39, 1.56, 6.25, 25, and 100 µM) was tested in triplicate for each
strain. The symbols are the means of the triplicates, and the lines
were generated by linear regression analysis. Dotted lines represent
the 95% confidence intervals of the regression lines.
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TABLE 1.
Results of linear regression analysis of the relationship
between the amount of XTT reduction in the presence of different
concentrations of menadione and increasing inocula of each
Aspergillus species after 24 h of
incubationa
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Among the different concentrations of menadione that were evaluated,
relatively higher conversion rates and more linear relationships
between formazan production and viable fungi were generated with
the
concentration of 25 µM for all species (Table
1). In addition,
the
formazan production at this concentration was high within
2 h of
exposure without exceeding the detection limit of the spectrophotometer
(OD of 3) and low background absorbance was
obtained.
Results of antifungal susceptibility assay.
For all
Aspergillus species, the NCCLS MICs of amphotericin B ranged
from 0.5 to 4 mg/liter and those of itraconazole ranged from 0.25 to
0.5 mg/liter except for A. fumigatus (R) and for A. ustus, for which the MICs were higher than 2 mg/liter (Table 2). Concentration effect curves based on
the conversion of XTT by hyphae are shown in Fig.
3 (top). All curves show similar patterns and are characterized by two plateaus connected by a drop in relative OD. In the case of a resistant strain, significant XTT conversion was
observed in higher concentrations [A. ustus and
A. fumigatus (R) against itraconazole; Fig 3]. The
three phases of XTT conversion were clearly distinguishable for
amphotericin B, for which steep concentration effect curves were
obtained. In contrast, for itraconazole, more shallow curves were
generated.
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FIG. 3.
Concentration effect curves for amphotericin B (AB) and
itraconazole (ICZ). Conidia of each Aspergillus sp. tested
were incubated for 24 and 48 h and the OD of hyphal growth at 405 nm was measured for each well. Then, hyphae were exposed to 200 µg of
XTT/ml plus 25 µM of menadione for 2 h and the OD450
was measured. Concentration effect curves based on the colorimetric
method (top) and spectrophotometric method (bottom) were constructed,
and the relative ODs for each drug concentration represent the amount
of formazan produced and the fungal biomass, respectively, for each
method compared with the growth control. A. ustus and
A. fumigatus (R) showed NCCLS MICs higher than 2 mg of
itraconazole/liter. Means with the standard errors of triplicates are
represented for each strain.
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The concentration effect curves obtained by the colorimetric and the
spectrophotometric assessments of fungal growth (Fig.
3) were analyzed
by the
Emax model with variable slope, and the
best-fit values of the two variables EC
50 and slope
(
m) were compared
for each species, drug, and incubation
period. The model fitted
the data very well since the
r2 ranged between 0.85 and 0.99 (median,
0.95) and no statistically
significant deviations from the model were
found (
P > 0.1). No
statistically significant
differences were found by analysis of
variance between the best-fit
values of the two concentration
effect curves for each drug and
incubation period (
P > 0.05) with
the exception of the
concentration effect curves of amphotericin
B after 48 h, where
statistically significantly steeper curves
(higher absolute
m values) were obtained by the colorimetric method
than
by the spectrophotometric method (
P < 0.01). However,
the
slopes and the EC
50s of the fitted model were similar
for the
concentration effect curves obtained by the two methods (Table
2). Overall, the median coefficient variation of the log
EC
50s
and the slope
m of the fitted model for
the six strains among
the replicates was 22 and 25%, respectively, for
the colorimetric
method and 37 and 38%, respectively, for the
spectrophotometric
method. Furthermore, the discrimination between the
in vitro itraconazole-resistant
strains and the other
itraconazole-susceptible strains was clearer
in concentration effect
curves of the colorimetric method than
those of spectrophotometric
method (Fig.
3).
The tetrazolium salt XTT was converted by
Aspergillus
species only in the presence of an electron-coupling agent, as was
found
with yeasts (
14). As opposed to mammalian cells
(
12) but similar
to yeasts (
15), among the
two electron-coupling agents tested
menadione was more potent than PMS.
Since PMS exhibited a background
higher than that found in previous
studies (
12,
13) and inhibitory
effects at elevated
concentrations were reported previously (
1),
menadione was
chosen as the electron-coupling agent for the XTT
assay of
Aspergillus species.
The concentration of XTT is another important factor since it was found
that high concentrations of XTT result in inhibition
of formazan
production while very low concentrations can result
in poor conversion
(
15). No inhibition was observed among the
three
concentrations tested in this study. Preliminary studies
showed that
XTT was not toxic for conidia (unpublished observation).
At higher
concentrations of XTT (200 µg/ml), the rate of formazan
production
increased rapidly. Therefore, a concentration of 200
µg of XTT/ml was
used for further studies. XTT and menadione at
the above concentrations
were stable when they were stored separately
for up to 3 months at room
temperature and 4,
20, and
70°C (unpublished
data).
The linear relationship between the formazan production using 200 µg
of XTT/ml and menadione with increasing inocula of
Aspergillus species indicates that the XTT assay is a
reliable indicator of
fungal biomass. The linearity and the slopes of
this relationship
depended on the concentration of menadione for each
of the
Aspergillus species. At a concentration of 25 µM,
high coefficients and slopes
for all
Aspergillus species
tested were found, unlike with yeasts,
for which lower concentrations
of menadione (1 µM) were required
to generate a linear relation
between XTT conversion and CFU (
15).
For
Aspergillus species, menadione concentrations higher than
25 µM generated a less linear CFU-OD relationship, which explains
the
conclusions of Jahn et al., who found that when XTT was used
with 1,000 µM menadione, a less-well-defined correlation was observed
(
5).
The XTT assay was applied in the presence of antifungal drugs
generating less-variable concentration effect curves. For amphotericin
B, clear-cut endpoints were obtained since XTT conversion was
absent
once the MIC was exceeded, as was found previously (
15).
For itraconazole, shallow concentration effect curves were obtained
due
to a partial inhibition of growth and possible interference
of the drug
in the metabolic status of fungi. Furthermore, as
was found in a
previous study (
14), discrimination between susceptible
and resistant strains may be facilitated by using the XTT assay
since
resistant strains converted XTT even at a high concentration
of
antifungal
drugs.
The need for the use of menadione in the XTT assay described here
offers the possibility of increasing the sensitivity of
this assay by
adjusting the concentration of menadione. In this
way, it would be
possible to detect the metabolic capacity of
slow-growing fungi. Also,
incubation periods proposed by the NCCLS
to determine the MICs may be
shortened if conversion of XTT of
the growth control is sufficient.
However, the development of
a tetrazolium salt which might not require
the addition of an
electron-coupling agent would be a favorable step
since the need
of an electron-coupling agent complicates the assay more
and may
increase variability (
13). Variability may also be
increased
by the lack of a step for the termination of XTT conversion
since
small variation in incubation time would influence the formazan
production.
In conclusion, the XTT-menadione system described in this study
provides an assay which enables the quantification of metabolic
activity of
Aspergillus species and which could be
applied for
other filamentous fungi like it was shown with MTT
(
7).
| |
ACKNOWLEDGMENTS |
This work was supported by the European Commission Training and
Mobility of Researchers Grant FMRX-CT970145 to Joseph Meletiadis and by
the Mycology Research Center of Nijmegen.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Medical Microbiology, University Medical Center Nijmegen, P.O. Box
9101, 6500 HB Nijmegen, The Netherlands. Phone: 31-24-3613514. Fax: 31-24-3540216. E-mail: p.verweij{at}mmb.azn.nl.
EC-TMR-EUROFUNG network (ERBFMXR-CT970145). The Eurofung Network
consists of the following participants: Emmanuel Roilides and Nicos
Maglaveras, Aristotle University, Thessaloniki, Greece; Tore Abrahamsen
and Peter Gaustad, Rikshospitalet National Hospital, Oslo, Norway;
David W. Denning, University of Manchester, Manchester, United Kingdom;
Paul E. Verweij and Jacques F. G. M. Mcis, University of Nijmegen, Nijmegen, The Netherlands; Juan L. Rodriguez-Tudela, Instituto de Salud Carlos III, Madrid, Spain; and George Petrikkos, Athens University, Athens, Greece.
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REFERENCES |
| 1.
|
Altman, F. L.
1976.
Tetrazolium salts and formazans.
Prog. Histochem. Cytochem.
9:1-52[Medline].
|
| 2.
|
Clancy, C. J., and M. H. Nguyen.
1997.
Comparison of a photometric method with standardized methods of antifungal susceptibility testing of yeasts.
J. Clin. Microbiol.
35:2878-2882[Abstract].
|
| 3.
|
Greco, W. R.,
G. Bravo, and J. C. Parsons.
1995.
The search for synergy: a critical review from a response surface perspective.
Pharmacol. Rev.
47:331-385[Medline].
|
| 4.
|
Hawser, S. P.,
H. Norris,
C. J. Jessup, and M. A. Ghannoum.
1998.
Comparison of a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) colorimetric method with the standardized National Committee for Clinical Laboratory Standards method of testing clinical yeast isolates for susceptibility to antifungal agents.
J. Clin. Microbiol.
36:1450-1452[Abstract/Free Full Text].
|
| 5.
|
Jahn, B.,
E. Martin,
A. Stueben, and S. Bhakdi.
1995.
Susceptibility testing of Candida albicans and Aspergillus species by a simple microtiter mena-dione-augmented 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay.
J. Clin. Microbiol.
33:661-667[Abstract].
|
| 6.
|
Levitz, S. M., and R. D. Diamond.
1985.
A rapid colorimetric assay of fungal viability with the tetrazolium salt MTT.
J. Infect. Dis.
152:938-945[Medline].
|
| 7.
|
Meletiadis, J.,
J. F. G. M. Meis,
J. W. Mouton,
J. P. Donnelly, and P. E. Verweij.
2000.
Comparison of NCCLS and 3-(4,5-dimethyl-2-Thiazyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) methods of in vitro susceptibility testing of filamentous fungi and development of a new simplified method.
J. Clin. Microbiol.
38:2949-2954[Abstract/Free Full Text].
|
| 8.
|
Meshulam, T.,
S. M. Levitz,
L. Christin, and R. D. Diamond.
1995.
A simplified new assay for assessment of fungal cell damage with the tetrazolium dye, (2,3)-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carboxanilide (XTT).
J. Infect. Dis.
172:1153-1156[Medline].
|
| 9.
|
National Committee for Clinical Laboratory Standards.
1998.
Reference method for broth dilution antifungal susceptibility testing of conidium forming filamentous fungi. Proposed standard M38-P.
National Committee for Clinical Laboratory Standards, Wayne, Pa.
|
| 10.
|
Paull, D. K.,
H. Shoemaker, and M. R. Boyd.
1998.
The synthesis of XTT: a new tetrazolium reagent that is bioreducible to a water-soluble formazan.
J. Heterocycl. Chem.
25:911-914.
|
| 11.
|
Pfaller, M. A., and M. G. Rinaldi.
1993.
Antifungal susceptibility testing. Current state of technology, limitations, and standardization.
Infect. Dis. Clin. N. Am.
7:435-444[Medline].
|
| 12.
|
Roehm, N. W.,
G. H. Rodgers,
S. M. Hatfield, and A. L. Glasebrook.
1991.
An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT.
J. Immunol. Methods
142:257-265[CrossRef][Medline].
|
| 13.
|
Scudiero, D. A.,
R. H. Shoemaker,
K. D. Paull,
A. Monks,
S. Tierney,
T. H. Nofziger,
M. J. Currens,
D. Seniff, and M. R. Boyd.
1988.
Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines.
Cancer Res.
48:4827-4833[Abstract/Free Full Text].
|
| 14.
|
Seidler, E.
1991.
The tetrazolium-formazn system: design and histochemistry.
Prog. Histochem. Cytochem.
24:1-86[Medline].
|
| 15.
|
Tellier, R.,
M. Krajden,
G. A. Grigoriew, and I. Campbell.
1992.
Innovative endpoint determination system for antifungal susceptibility testing of yeasts.
Antimicrob. Agents Chemother.
36:1619-1625[Abstract/Free Full Text].
|
Journal of Clinical Microbiology, September 2001, p. 3402-3408, Vol. 39, No. 9
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.9.3402-3408.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Shehata, A. S., Mukherjee, P. K., Ghannoum, M. A.
(2008). Comparison between the Standardized Clinical and Laboratory Standards Institute M38-A2 Method and a 2,3-Bis(2-Methoxy-4-Nitro-5-[(Sulphenylamino)Carbonyl]-2H-Tetrazolium Hydroxide- Based Method for Testing Antifungal Susceptibility of Dermatophytes. J. Clin. Microbiol.
46: 3668-3671
[Abstract]
[Full Text]
-
Meletiadis, J., Antachopoulos, C., Stergiopoulou, T., Pournaras, S., Roilides, E., Walsh, T. J.
(2007). Differential Fungicidal Activities of Amphotericin B and Voriconazole against Aspergillus Species Determined by Microbroth Methodology. Antimicrob. Agents Chemother.
51: 3329-3337
[Abstract]
[Full Text]
-
Mowat, E., Butcher, J., Lang, S., Williams, C., Ramage, G.
(2007). Development of a simple model for studying the effects of antifungal agents on multicellular communities of Aspergillus fumigatus. J Med Microbiol
56: 1205-1212
[Abstract]
[Full Text]
-
Meletiadis, J., Stergiopoulou, T., O'Shaughnessy, E. M., Peter, J., Walsh, T. J.
(2007). Concentration-Dependent Synergy and Antagonism within a Triple Antifungal Drug Combination against Aspergillus Species: Analysis by a New Response Surface Model. Antimicrob. Agents Chemother.
51: 2053-2064
[Abstract]
[Full Text]
-
Brun, Y. F., Dennis, C. G., Greco, W. R., Bernacki, R. J., Pera, P. J., Bushey, J. J., Youn, R. C., White, D. B., Segal, B. H.
(2007). Modeling the Combination of Amphotericin B, Micafungin, and Nikkomycin Z against Aspergillus fumigatus In Vitro Using a Novel Response Surface Paradigm. Antimicrob. Agents Chemother.
51: 1804-1812
[Abstract]
[Full Text]
-
Antachopoulos, C., Meletiadis, J., Sein, T., Roilides, E., Walsh, T. J.
(2007). Concentration-Dependent Effects of Caspofungin on the Metabolic Activity of Aspergillus Species. Antimicrob. Agents Chemother.
51: 881-887
[Abstract]
[Full Text]
-
Antachopoulos, C., Meletiadis, J., Sein, T., Roilides, E., Walsh, T. J.
(2007). Use of high inoculum for early metabolic signalling and rapid susceptibility testing of Aspergillus species. J Antimicrob Chemother
59: 230-237
[Abstract]
[Full Text]
-
Knight, S. A. B., Dancis, A.
(2006). Reduction of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) is dependent on CaFRE10 ferric reductase for Candida albicans grown in unbuffered media.. Microbiology
152: 2301-2308
[Abstract]
[Full Text]
-
van Vianen, W., de Marie, S., ten Kate, M. T., Mathot, R. A. A., Bakker-Woudenberg, I. A. J. M.
(2006). Caspofungin: antifungal activity in vitro, pharmacokinetics, and effects on fungal load and animal survival in neutropenic rats with invasive pulmonary aspergillosis. J Antimicrob Chemother
57: 732-740
[Abstract]
[Full Text]
-
Chamilos, G., Lewis, R. E., Kontoyiannis, D. P.
(2006). Inhibition of Candida parapsilosis Mitochondrial Respiratory Pathways Enhances Susceptibility to Caspofungin. Antimicrob. Agents Chemother.
50: 744-747
[Abstract]
[Full Text]
-
Antachopoulos, C., Meletiadis, J., Roilides, E., Sein, T., Walsh, T. J.
(2006). Rapid Susceptibility Testing of Medically Important Zygomycetes by XTT Assay. J. Clin. Microbiol.
44: 553-560
[Abstract]
[Full Text]
-
Chamilos, G., Lewis, R. E., Kontoyiannis, D. P.
(2006). Lovastatin Has Significant Activity against Zygomycetes and Interacts Synergistically with Voriconazole. Antimicrob. Agents Chemother.
50: 96-103
[Abstract]
[Full Text]
-
Wiederhold, N. P., Kontoyiannis, D. P., Prince, R. A., Lewis, R. E.
(2005). Attenuation of the Activity of Caspofungin at High Concentrations against Candida albicans: Possible Role of Cell Wall Integrity and Calcineurin Pathways. Antimicrob. Agents Chemother.
49: 5146-5148
[Abstract]
[Full Text]
-
Heyn, K., Tredup, A., Salvenmoser, S., Muller, F.-M. C.
(2005). Effect of Voriconazole Combined with Micafungin against Candida, Aspergillus, and Scedosporium spp. and Fusarium solani. Antimicrob. Agents Chemother.
49: 5157-5159
[Abstract]
[Full Text]
-
Lewis, R. E., Wiederhold, N. P., Lionakis, M. S., Prince, R. A., Kontoyiannis, D. P.
(2005). Frequency and Species Distribution of Gliotoxin-Producing Aspergillus Isolates Recovered from Patients at a Tertiary-Care Cancer Center. J. Clin. Microbiol.
43: 6120-6122
[Abstract]
[Full Text]
-
Lewis, R. E., Kontoyiannis, D. P.
(2005). Micafungin in combination with voriconazole in Aspergillus species: a pharmacodynamic approach for detection of combined antifungal activity in vitro. J Antimicrob Chemother
56: 887-892
[Abstract]
[Full Text]
-
van de Sande, W. W. J., Luijendijk, A., Ahmed, A. O. A., Bakker-Woudenberg, I. A. J. M., van Belkum, A.
(2005). Testing of the In Vitro Susceptibilities of Madurella mycetomatis to Six Antifungal Agents by Using the Sensititre System in Comparison with a Viability-Based 2,3-Bis(2-Methoxy-4-Nitro-5-Sulfophenyl)-5- [(Phenylamino)Carbonyl]-2H-Tetrazolium Hydroxide (XTT) Assay and a Modified NCCLS Method. Antimicrob. Agents Chemother.
49: 1364-1368
[Abstract]
[Full Text]
-
Lewis, R. E., Wiederhold, N. P., Klepser, M. E.
(2005). In Vitro Pharmacodynamics of Amphotericin B, Itraconazole, and Voriconazole against Aspergillus, Fusarium, and Scedosporium spp.. Antimicrob. Agents Chemother.
49: 945-951
[Abstract]
[Full Text]
-
Chen, J., Li, H., Li, R., Bu, D., Wan, Z.
(2005). Mutations in the cyp51A gene and susceptibility to itraconazole in Aspergillus fumigatus serially isolated from a patient with lung aspergilloma. J Antimicrob Chemother
55: 31-37
[Abstract]
[Full Text]
-
Steinbach, W. J., Perfect, J. R., Schell, W. A., Walsh, T. J., Benjamin, D. K. Jr.
(2004). In Vitro Analyses, Animal Models, and 60 Clinical Cases of Invasive Aspergillus terreus Infection. Antimicrob. Agents Chemother.
48: 3217-3225
[Full Text]
-
Yekutiel, A., Shalit, I., Shadkchan, Y., Osherov, N.
(2004). In Vitro Activity of Caspofungin Combined with Sulfamethoxazole against Clinical Isolates of Aspergillus spp.. Antimicrob. Agents Chemother.
48: 3279-3283
[Abstract]
[Full Text]
-
Tunney, M. M., Ramage, G., Field, T. R., Moriarty, T. F., Storey, D. G.
(2004). Rapid Colorimetric Assay for Antimicrobial Susceptibility Testing of Pseudomonas aeruginosa. Antimicrob. Agents Chemother.
48: 1879-1881
[Abstract]
[Full Text]
-
Balajee, S. A., Weaver, M., Imhof, A., Gribskov, J., Marr, K. A.
(2004). Aspergillus fumigatus Variant with Decreased Susceptibility to Multiple Antifungals. Antimicrob. Agents Chemother.
48: 1197-1203
[Abstract]
[Full Text]
-
Pan, W., Liu, X., Ge, F., Han, J., Zheng, T.
(2004). Perinerin, a Novel Antimicrobial Peptide Purified from the Clamworm Perinereis aibuhitensis Grube and Its Partial Characterization. J Biochem
135: 297-304
[Abstract]
[Full Text]
-
Liu, W., Lionakis, M. S., Lewis, R. E., Wiederhold, N., May, G. S., Kontoyiannis, D. P.
(2003). Attenuation of Itraconazole Fungicidal Activity following Preexposure of Aspergillus fumigatus to Fluconazole. Antimicrob. Agents Chemother.
47: 3592-3597
[Abstract]
[Full Text]
-
Ramani, R., Gangwar, M., Chaturvedi, V.
(2003). Flow Cytometry Antifungal Susceptibility Testing of Aspergillus fumigatus and Comparison of Mode of Action of Voriconazole vis-a-vis Amphotericin B and Itraconazole. Antimicrob. Agents Chemother.
47: 3627-3629
[Abstract]
[Full Text]
-
Lionakis, M. S., Lewis, R. E., Samonis, G., Kontoyiannis, D. P.
(2003). Pentamidine Is Active In Vitro against Fusarium Species. Antimicrob. Agents Chemother.
47: 3252-3259
[Abstract]
[Full Text]
-
Meletiadis, J., te Dorsthorst, D. T. A., Verweij, P. E.
(2003). Use of Turbidimetric Growth Curves for Early Determination of Antifungal Drug Resistance of Filamentous Fungi. J. Clin. Microbiol.
41: 4718-4725
[Abstract]
[Full Text]
-
Meletiadis, J., Mouton, J. W., Meis, J. F. G. M., Verweij, P. E.
(2003). In Vitro Drug Interaction Modeling of Combinations of Azoles with Terbinafine against Clinical Scedosporium prolificans Isolates. Antimicrob. Agents Chemother.
47: 106-117
[Abstract]
[Full Text]
-
Balajee, S. A., Marr, K. A.
(2002). Conidial Viability Assay for Rapid Susceptibility Testing of Aspergillus Species. J. Clin. Microbiol.
40: 2741-2745
[Abstract]
[Full Text]
-
Meletiadis, J., Mouton, J. W., Meis, J. F. G. M., Bouman, B. A., Verweij, P. E.
(2002). Comparison of the Etest and the Sensititre Colorimetric Methods with the NCCLS Proposed Standard for Antifungal Susceptibility Testing of Aspergillus Species. J. Clin. Microbiol.
40: 2876-2885
[Abstract]
[Full Text]
-
Meletiadis, J., Mouton, J. W., Meis, J. F. G. M., Bouman, B. A., Donnelly, P. J., Verweij, P. E., EUROFUNG Network,
(2001). Comparison of Spectrophotometric and Visual Readings of NCCLS Method and Evaluation of a Colorimetric Method Based on Reduction of a Soluble Tetrazolium Salt, 2,3-Bis {2-Methoxy-4-Nitro-5-[(Sulfenylamino) Carbonyl]-2H- Tetrazolium-Hydroxide}, for Antifungal Susceptibility Testing of Aspergillus Species. J. Clin. Microbiol.
39: 4256-4263
[Abstract]
[Full Text]
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