JAC
Journal of Antimicrobial Chemotherapy (2005) 55, 914–920
doi:10.1093/jac/dki100
Advance Access publication 11 April 2005
A comparative study of fungicidal activities of voriconazole
and amphotericin B against hyphae of Aspergillus fumigatus
Suganthini Krishnan, Elias K. Manavathu* and Pranatharthi H. Chandrasekar
Division of Infectious Diseases, Department of Internal Medicine, 427 Lande Building, Wayne State University,
550 E. Canfield, Detroit, MI 48201, USA
Received 31 January 2005; accepted 20 February 2005
Objectives: To study the in vitro fungicidal activity of voriconazole against hyphae of Aspergillus
fumigatus and compare the results with those obtained for the known fungicidal drug amphotericin B.
Methods: A. fumigatus mycelia were grown on Sabouraud dextrose agar and in peptone yeast extract
glucose broth until the cultures reached a mid-logarithmic growth phase. The fungicidal activities of
voriconazole and amphotericin B against actively growing hyphae of A. fumigatus were examined by a
kill-curve experiment and a fungal cell viability test. For the kill-curve study, the drug-treated hyphae
were washed, homogenized and resuspended in 1 mL of sterile water, diluted 10 –1000 fold and aliquots of 0.1 mL were spread on Sabouraud dextrose agar and allowed to grow for 48 h at 358C. The cfu
were determined and plotted against drug concentrations for each time of exposure to obtain the kill
curve. The viability of drug-treated A. fumigatus hyphae was determined by their ability to reduce tetrazolium compound 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide.
Results: Exposure of A. fumigatus hyphae to several concentrations (1 –16 mg/L) of voriconazole or
amphotericin B for various time intervals killed the hyphae in a time- and drug concentration-dependent manner. Voriconazole at 1 mg/L killed > 95% of the hyphae grown on Sabouraud dextrose agar
after 48 h of exposure, whereas amphotericin B at the same concentration killed 70% of the hyphae
after exposure for the same duration. Approximately 99% killing of hyphae grown in peptone yeast
extract glucose broth was obtained for voriconazole at 1 mg/L after 48 h of exposure, whereas amphotericin B at 1 mg/L yielded 82% killing after 48 h. The fungal cell viability test by tetrazolium reduction
_ 1 mg/L (Sabouraud dextrose agar blocks) and >
_ 2 mg/L (broth
assay showed that mycelia exposed to >
cultures) of voriconazole for 48 h completely failed to reduce 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl2H-tetrazolium bromide. At low concentrations (1 –2 mg/L) amphotericin B had no detectable effect on
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction by drug-treated mycelia,
whereas mycelia treated with 16 mg/L for 48 h showed 50% inhibition of 3-(4,5-dimethyl-2-thiazolyl)2,5-diphenyl-2H-tetrazolium bromide reduction compared with the control.
Conclusions: Voriconazole possesses excellent fungicidal activity against actively growing hyphae of
A. fumigatus. A comparison of results with those obtained for the known fungicidal drug amphotericin
B shows that, in peptone yeast extract glucose broth, voriconazole has superior fungicidal activity
against A. fumigatus hyphae compared with that of amphotericin B.
Keywords: voriconazole, fungicidal activity, Aspergillus fumigatus, fungal hyphae, kill curve, viability test
Introduction
Voriconazole is a second-generation triazole antifungal drug
exhibiting excellent in vitro and in vivo activity against a wide
variety of pathogenic yeasts,1 – 7 and dimorphic8,9 and filamentous
fungi,10 – 28 including Aspergillus species. Recently, it has been
approved by the Food and Drug Administration of the USA for
primary use against infection caused by pathogenic filamentous
fungi, in particular by Aspergillus species.29 Members of the
azole group of antifungal drugs in general are known to have
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*Corresponding author. Tel: +1-313-577-1931; Fax: +1-313-993-0302; E-mail: [email protected]
..........................................................................................................................................................................................................................................................................................................................................................................................................................
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q The Author 2005. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.
All rights reserved. For Permissions, please e-mail: [email protected]
Fungicidal activity of voriconazole
fungistatic qualities, effective in pathogenic yeasts such as
Candida species, resulting in a significant amount of growth
inhibition in the presence of the drug. Removal of the antifungal
drug from exposed fungal cells by washing would release them
from the inhibitory effect of the drug and then they could
resume normal growth under favourable conditions. Thus, prolonged exposure of yeast cells to azoles, including voriconazole,
does not kill the cells but arrests their growth. The lack of fungicidal activity of azoles against the pathogenic yeast Candida
albicans has been previously demonstrated in time–kill experiments.30 On the other hand, voriconazole possesses excellent
fungicidal activity against Aspergillus species when germinated
and ungerminated conidia are used.31 – 35 Although the inhaled
conidia are the primary source of infection in man, such conidia
must germinate producing hyphae for subsequent entry into the
lung alveoli to establish disease. Conidia are generally absent in
infected tissues. Death occurs with hyphal proliferation within
the alveoli, and vascular invasion and dissemination producing
pulmonary and distant infarctions. Thus, it is important that the
antifungal drug treatment should kill the actively growing
hyphae for a successful outcome in invasive aspergillosis.
None of the previous studies on the fungicidal activity of
voriconazole against A. fumigatus included actively growing
hyphae, the biological form of aspergillus found in infected tissues. Therefore, in this study we investigated the in vitro fungicidal activity of voriconazole against actively growing hyphae of
12 clinical isolates of A. fumigatus and the results were
compared with those obtained for the known fungicidal drug
amphotericin B.
Materials and methods
Antifungal drugs
Voriconazole and amphotericin B were obtained as pure powders
from Pfizer Pharmaceuticals, New York, NY, USA and Squibb Institute for Medical Research, Princeton, NJ, USA, respectively. The
stock solutions were prepared by dissolving the powders in dimethyl
sulphoxide at a concentration of 1 mg/mL and were stored as
0.25 mL aliquots at 208C. The frozen stocks were thawed at room
temperature and vortexed gently several times to ensure that any
remaining crystals present were completely dissolved before use.
Since amphotericin B is light-sensitive, the stock solution and the
culture tubes containing amphotericin B were covered with aluminum foil to prevent light exposure. For both antifungal drugs, a
concentration range of 1 –16 mg/L was used for fungicidal testing.
Fungal isolates
A. fumigatus F55064, W73355, H27023, X60141, N31647, H36754,
H50246, F48686, X25419, S66182, W18870 and T45830 (MIC
range: amphotericin B, 0.25– 1 mg/L; voriconazole, 0.062 –
0.25 mg/L) isolated from immunocompromised patients were
obtained from the Microbiology Laboratory of the Detroit Medical
Center, Detroit, MI, USA. The original cultures obtained on Sabouraud dextrose agar slants were subcultured on the same medium to
check the purity and viability of the cultures. For long-term preservation, isolates were stored as conidial suspensions in 25% glycerol
at 708C.
Kill-curve experiment
The fungicidal activity of voriconazole against A. fumigatus hyphae
was examined by kill-curve experiments using mycelia grown on
Sabouraud dextrose agar (VWR Scientific Products, West Chester,
PA, USA) and in peptone yeast extract glucose [Bactopeptone 1 g
(Difco Laboratories, Detroit, MI, USA), yeast extract 1 g (Sigma
Chemical Company, St Louis, MO, USA) and glucose 3 g/L of distilled water] broth. To obtain hyphae grown on solid medium, a
single colony of A. fumigatus was grown on Sabouraud dextrose
agar at 358C for 3 – 4 days from a point source of inoculum. From
the translucent outer zone of the fungal colony consisting of actively
growing non-conidiating hyphae, 5 mm diameter agar blocks carrying thousands of actively growing hyphae were aseptically excised
using a sterile cork borer (Figure 1). These agar blocks were incubated at 358C in 6 mL polystyrene culture tubes in 1 mL of peptone
Figure 1. Photomicrograph of a 3-day-old A. fumigatus colony grown on Sabouraud dextrose agar from a point source of inoculum from which 5 mm diameter agar blocks containing actively growing hyphae were excised. The actively growing region of the colony is represented by the translucent peripheral
region of the colony and consists of unbranched pointed hyphal filaments. Next to the actively growing region is a white zone mainly characterized by
branched hyphae primed to undergo an asexual reproductive phase called conidiation. Hence it is called the pre-conidiation zone. The centre of each A. fumigatus colony is characterized by a bluish yellow colour due to the presence of a massive number of conidiophores bearing asexual spores called conidia, and
this region is called the conidiation zone.
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Krishnan et al.
yeast extract glucose broth (four blocks/tube) containing various
concentrations (1– 16 mg/L) of voriconazole or amphotericin
B. Peptone yeast extract glucose broth with the required amount of
dimethyl sulphoxide was used as a drug-free growth control. After
24 h of incubation, two of the agar blocks were removed from each
drug concentration and washed with 20 mL of sterile distilled water
in a Petri dish for 30 min at room temperature without agitation.
The washed agar blocks containing drug-treated hyphae were homogenized individually for 30 s in distilled water (1 mL/agar block)
using a tissue homogenizer fitted with a sterile miniprobe. The
homogenate was diluted 10 – 1000-fold and 0.1 mL aliquots of the
diluted suspension were spread in duplicate on Sabouraud dextrose
agar. The agar plates were incubated for 48 h at 358C and the number of cfu per agar block was determined. Agar blocks exposed to
peptone yeast extract glucose broth without the drug were used as a
control. The same procedure was repeated for the remaining two
agar blocks in the culture tube after 48 h of drug exposure and the
number of cfu determined. The mean cfu per agar block for each
drug concentration was plotted for 24 and 48 h time intervals to
obtain time – kill curves.
To examine the fungicidal activity of voriconazole and amphotericin B against A. fumigatus hyphae grown in liquid medium by
kill-curve experiment, 1 mL cultures of A. fumigatus isolates were
grown in peptone yeast extract glucose broth for 24 h at 358C from
conidia (1 104 conidia/mL as determined by haemocytometry) in
6 mL polystyrene tubes. To these pre-grown mycelial cultures voriconazole was added to obtain final concentrations of 1 – 16 mg/L and
the cultures were incubated for an additional 24 – 48 h at 358C for
drug treatment. Peptone yeast extract glucose broth containing the
required amounts of dimethyl sulphoxide was used as a control. At
the end of the drug treatment period, 3 mL of fresh peptone yeast
extract glucose broth was added to each tube (total volume 4 mL),
the mycelial suspension was mixed well, diluted 10– 1000-fold by
serial dilution in sterile distilled water and 0.1 mL aliquots of the
diluted mycelial suspension was spread on Sabouraud dextrose agar.
The plates were incubated for 48 h at 358C and the number of cfu
per mL of cultures was determined and plotted against various drug
concentrations for each time interval to obtain kill curves. The minimum fungicidal concentration (MFC) was defined as the lowest con_ 5 cfu/mL of
centration of the antifungal drug that provided <
cultures. The highest possible amount of drug carried over from the
culture tube after the dilution was estimated to be 0.0025 –
0.04 mg/L that had no effect on the growth of A. fumigatus mycelia
on Sabouraud dextrose agar.
centration of the drug that completely prevented the development of
blue colour.
For mycelia grown in liquid medium, cultures of A. fumigatus
were grown in 96-well microtitre plates (0.1 mL/well) in peptone
yeast extract glucose medium for 24 h at 358C from a conidial
suspension (1 106 conidia/mL). Under these conditions, the conidia germinate and produce uniform mycelial growth and the hyphae
adhere tightly to the microtitre plate rendering it highly suitable for
further manipulation with minimal loss of mycelia. After 24 h of
growth, the spent growth medium was removed with a multichannel
pipette and replaced with fresh peptone yeast extract glucose broth
(0.2 mL/well) containing various concentrations (1 –16 mg/L) of
either voriconazole or amphotericin B. The first two vertical columns of the plate (16 wells) were drug-free controls. There were
eight replicate wells for each drug concentration. The plates were
then incubated for either 24 or 48 h, and at the end of the incubation
period the growth medium containing the drug solution was
removed and the fungal cells washed twice with 0.2 mL of sterile
distilled water. Then to each well, 0.1 mL of 100 mM 3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide solution
containing 0.2 mM menadione prepared in peptone yeast extract
Fungal cell viability test
The viability of A. fumigatus hyphae treated with voriconazole or
amphotericin B was evaluated by the 3-(4,5-dimethyl-2-thiazolyl)2,5-diphenyl-2H-tetrazolium bromide reduction assay36,37 using
mycelia grown on Sabouraud dextrose agar and peptone yeast
extract glucose broth. To examine the effect of voriconazole and
amphotericin B on the viability of hyphae grown on solid medium,
Sabouraud dextrose agar blocks containing A. fumigatus hyphae
were prepared and treated with various concentrations of the antifungal drugs as described earlier. After 24 – 48 h of incubation, drugtreated agar blocks were removed and washed with distilled water.
The washed agar blocks (one agar block/well) containing either voriconazole- or amphotericin B-treated hyphae were incubated in fresh
peptone yeast extract glucose broth (0.2 mL/well) containing
100 mM 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide and 0.2 mM menadione for 3 h at 358C for the reduction of
the tetrazolium compound. The MFC was defined as the lowest con-
Figure 2. Effect of voriconazole and amphotericin B on the survival of
A. fumigatus hyphae grown on Sabouraud dextrose agar (a) and peptone
yeast extract glucose broth (b). The data shown here were obtained for A.
fumigatus clinical isolate W73355, and each point represents the mean ± SD
(vertical bar) of two independent time–kill studies.
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Fungicidal activity of voriconazole
broth was added and the plates incubated at 358C for 3 h
for reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium bromide. The MFC was defined as the lowest
concentration of the drug that completely prevented the development of blue colour.
on Sabouraud dextrose agar and peptone yeast extract glucose
broth obtained for voriconazole for 24 and 48 h exposures was
significantly higher than that obtained for amphotericin B.
Viability test
Results
Time –kill study
The fungicidal activities of voriconazole and amphotericin B
against hyphae of a representative A. fumigatus isolate
(W73355) grown on Sabouraud dextrose agar and peptone yeast
extract glucose broth are shown in Figure 2. Exposure of
actively growing hyphae to various concentrations of voriconazole significantly decreased their ability to produce fungal colonies in a time- and drug concentration-dependent manner. For
instance, voriconazole at 4 mg/L showed 96 and 100% killing
of hyphae grown on Sabouraud dextrose agar after 24 and 48 h
of exposure, respectively, whereas amphotericin B at 4 mg/L
showed 50 and 85% killing after 24 and 48 h of exposure,
respectively, compared with the drug-free control (Figure 2a).
Similar results were obtained for hyphae grown in peptone yeast
extract glucose broth, except that almost complete killing was
achieved at 1 mg/L for voriconazole after 48 h of incubation
(Figure 2b). In contrast, amphotericin B at 1 mg/L showed very
poor killing activity ( 62%) against hyphae grown in peptone
yeast extract glucose broth even after 48 h of exposure to the
drug.
A summary of the results of the fungicidal activities of voriconazole and amphotericin B against 12 randomly selected
A. fumigatus clinical isolates from our culture collection is
shown in Table 1. The mean percentage killing of hyphae grown
In addition to kill-curve experiments, we examined the fungicidal activities of voriconazole and amphotericin B against
actively growing A. fumigatus hyphae by a previously described
fungal cell viability test based on the ability of viable fungal
hyphae to reduce the tetrazolium compound 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide. As the fungal
mitochondrial dehydrogenase reduces 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, an insoluble blue
formazan salt will accumulate in the mycelia. The rate of
reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide resulting in the formation of formazan salt is
directly proportional to the amount of viable hyphae, suggesting
that the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide reduction assay can be adapted for qualitative as well as
quantitative measurement of the viability of A. fumigatus hyphae.
Figure 3 shows the effect of voriconazole and amphotericin B
on A. fumigatus mycelia for their ability to reduce the
tetrazolium compound 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl2H-tetrazolium bromide. A. fumigatus hyphae grown on Sabouraud dextrose agar (Figure 3a) and in peptone yeast extract glucose
broth (Figure 3b) were completely killed by voriconazole at 1 and
2 mg/L, respectively, after 48 h of incubation. On the other hand,
amphotericin B at concentrations in the range 1–16 mg/L showed
very poor killing even after 48 h of exposure to the drug, as evaluated by the reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl2H-tetrazolium bromide.
Table 1. Fungicidal activity of voriconazole and amphotericin B against A. fumigatus hyphae grown on solid and liquid growth media
Percentage killing of hyphae grown on
Sabouraud dextrose agar (n = 12)a
Antifungal drug (mg/L)
Voriconazole
0
1
2
4
8
16
Amphotericin B
0
1
2
4
8
16
Peptone yeast extract glucose broth (n = 5)b
24 h exposure
48 h exposure
24 h exposure
48 h exposure
0.0
86.3 ± 6.4
90.5 ± 2.9
96.5 ± 6.7
98.0 ± 2.1
98.8 ± 1.2
0.0
95.1 ± 5.4
96.3 ± 0.5
99.7 ± 1.1
99.9 ± 0.1
99.9 ± 0
0.0
89.5 ± 5.5
93.4 ± 6.6
96.4 ± 3.6
96.9 ± 3.1
98.3 ± 0.2
0.0
99.2 ± 0.8
99.8 ± 0.1
99.9 ± 0
99.8 ± 0
99.9 ± 0
0.0
49.3 ± 9.7
35.5 ± 1.3
39.8 ± 6.0
72.9 ± 3.4
78.7 ± 1.7
0.0
67.4 ± 2.5
81.1 ± 0.8
84.7 ± 1.2
87.9 ± 0.6
88.2 ± 0.5
0.0
64.2 ± 2.2
65.3 ± 0.9
64.0 ± 6.5
68.3 ± 1.8
70.0 ± 3.9
0.0
81.8 ± 4.6
91.0 ± 4.3
91.0 ± 6.5
91.0 ± 2.8
91.8 ± 3.6
a
Each value represents the mean of two independent determinations for 12 A. fumigatus clinical isolates. P values (Student’s t-test) for pairwise comparisons
of the percentage killing obtained for various concentrations of amphotericin B and voriconazole for 24 and 48 h drug exposures were in the range
0.0179–0.00017.
b
Each value represents the mean of two independent determinations each for five A. fumigatus clinical isolates. P values (Student’s t-test) were in the range
0.0287–0.0016.
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Krishnan et al.
Figure 3. Photomicrographs of typical 96-well microtitre plates showing the
effect of voriconazole and amphotericin B on the viability of A. fumigatus
hyphae grown on Sabouraud dextrose agar (a) and peptone yeast extract glucose broth (b). The development of the blue colour due to the reduction of
the tetrazolium compound 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide indicates the presence of viable fungal cells. The data
shown here were obtained for A. fumigatus W73355, and almost identical
results were obtained for all the clinical isolates (n = 12) we examined.
Discussion
Previous studies on the fungicidal activity of triazoles,
including voriconazole, on A. fumigatus used ungerminated
and germinated conidia mainly because of convenience and due
to the absence of a suitable technique for time –kill study using
actively growing hyphae. Since hyphae, not conidia, are found
in the infected tissues of patients, the relevance of using conidia
for kill-curve studies is often questioned. Therefore, using a
novel method, we investigated the fungicidal activity of voriconazole on actively growing A. fumigatus hyphae by kill-curve
experiments, and the results thus obtained were compared with
those obtained for amphotericin B, a known fungicidal agent.
Our results show that voriconazole is a highly effective fungicidal drug against A. fumigatus hyphae in peptone yeast extract
glucose broth and is superior to amphotericin B in its activity.
Unlike unicellular organisms such as bacteria and yeasts that
show uniform growth throughout the cell surface during
vegetative growth phase, filamentous fungi including various
Aspergillus species show polar growth by extending the apices of
each hyphal filament. Thus, the metabolic and biochemical
activity of the fungal cell governing vegetative growth is primarily restricted to the tips of the hyphae. Therefore it is generally
believed that the fungicidal activity of antifungal drugs is directed
to this region of the hyphae and older fungal cultures are less susceptible to the fungicidal activity of the drug. In this study, we
were able to examine the fungicidal effect of voriconazole and
amphotericin B on the actively growing hyphal filaments using
Sabouraud dextrose agar blocks containing growing tips of A.
fumigatus hyphae. The results obtained for the agar block experiments were compared with those obtained for the mycelia grown
for 24 h in peptone yeast extract glucose broth cultures consisting
of actively growing and non-growing hyphal elements. The
hyphal tips on the agar blocks and the mycelia grown in liquid
cultures were killed by voriconazole with almost identical effectiveness, suggesting that A. fumigatus cultures containing growing
and non-growing hyphal elements are equally susceptible to voriconazole as are the actively growing tips of the hyphae.
Previous studies have shown that the fungicidal activity of
amphotericin B against ungerminated and germinated conidia
was superior to that of triazoles, including voriconazole.31 – 33 For
instance, amphotericin B at 1 mg/L killed >99.9% of the conidia
within 6 h of exposure to the drug, whereas almost 24 h of
exposure was required to obtain the same amount of killing with
voriconazole at 1 mg/L. The rapid and efficient fungicidal action
of amphotericin B is thought to be due its ability to form
channels or pores in the cytoplasmic membrane through which
essential nutrients and ions are leaked out of the cell, and the
loss of nutrients and essential ions are thought to be the primary
reason for the lethal effect of amphotericin B. Voriconazole is a
slow-acting fungicidal agent against A. fumigatus, and for voriconazole to be effective as a fungicidal agent, fungal cell growth
equal to several generation times should occur. Therefore, an
extended period of growth in the presence of voriconazole is
required to deplete ergosterol content completely to bring about
a lethal effect on A. fumigatus cells.
The intriguing question is why voriconazole is highly effective against A. fumigatus hyphae as a fungicidal agent compared
with amphotericin B, whereas the reverse is true for ungerminated and germinated conidia of A. fumigatus. It is possible that
a key component(s) that is directly or indirectly involved in the
vegetative growth phase of A. fumigatus is absent or sparsely
present in the conidia and germinating conidia. The function of
this key component is more effectively inhibited by voriconazole, but not by amphotericin B, which results in eventual
fungal cell death. Our recent investigation (E. K. Manavathu and
P. H. Chandrasekar, 2004, unpublished) on the effect of voriconazole on the gene expression profile of actively growing
A. fumigatus mycelia by partial microarray experiments suggests
several possible candidates, including hyp1 and chsE genes,
whose functions are known to be important for the vegetative
and reproductive growth phases of Aspergillus species.
In summary, in contrast to its fungistatic effect against
pathogenic yeasts such as Candida species, voriconazole shows
excellent fungicidal activity against A. fumigatus hyphae as determined by kill-curve experiments and a fungal cell viability test by
the reduction of a tetrazolium compound. A comparison of the
results obtained for the time –kill study and the tetrazolium
reduction assay showed that in peptone yeast extract glucose
broth voriconazole possesses superior fungicidal activity against
A. fumigatus hyphae as compared with that of amphotericin B.
Acknowledgements
We would like to thank Dr William Brown of the Microbiology
Laboratory of the Detroit Medical Center, Detroit, MI, USA, for
providing the A. fumigatus clinical isolates used in this study, and
Pfizer Pharmaceuticals, New York, NY, USA, for financial
support.
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Fungicidal activity of voriconazole
Part of the data described in this manuscript was presented
previously at the Forty-third Interscience Conference on
Antimicrobial Agents and Chemotherapy, 14–17 September
2003, Chicago, USA. Abstract M-1250.
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