Journal of Antimicrobial Chemotherapy (2003) 51, 163166

DOI: 10.1093/jac/dkg018

Antifungal activity of the echinocandin anidulafungin (VER002,

LY-303366) against yeast pathogens: a comparative study with M27-A
microdilution method

Mara Pilar Arvalo1*, Alfonso-Javier Carrillo-Muoz2, Javier Salgado1,3, Delia Cardenes1,

Sonia Bri2, Guillermo Quinds4 and Ana Espinel-Ingroff5

1Ctedra
de Medicina Preventiva y Salud Pblica, Facultad de Medicina, Universidad de la Laguna,
38071 La Laguna, Tenerife; 2Departamento de Microbiologa, ACIA, Barcelona;
3Hospital Ntra. Sra. de Candelaria, Tenerife; 4Departamento de Immunologa, Microbiologa y Parasitologa,

Facultad de Medicina, Universidad del Pas Vasco, Bilbao, Spain; 5Medical College of Virginia,
Campus Virginia Commonwealth University, Richmond, VA, USA

Downloaded from http://jac.oxfordjournals.org/ by guest on January 5, 2017

Received 18 December 2001; returned 4 June 2002; revised 15 July 2002; accepted 28 September 2002

This study further evaluated the in vitro activity of anidulafungin (VER002, Versicor Inc.)
(LY303366) against 460 clinical yeast isolates. MICs of anidulafungin, fluconazole and itra-
conazole were determined by following the NCCLS M27-A guidelines. Minimum fungicidal
concentrations (MFCs) of anidulafungin were determined for 230 isolates of Candida spp. The
activity of anidulafungin in vitro was significantly superior (P < 0.05) to those of itraconazole and
fluconazole against Candida albicans, Candida tropicalis, Candida glabrata and Candida krusei,
but anidulafungin was less active for Candida famata and Candida parapsilosis. The differences
were not significant for the other species evaluated.

Introduction azole derivatives fluconazole, itraconazole and voriconazole,

are the only available agents for the management of severe
The emergence of opportunistic fungal pathogens, the in- fungal infections. However, their use is limited by either their
creased variety of mycoses, the recovery of clinical isolates narrow efficacy or safety problems. Although amphotericin B
resistant to conventional antifungal agents and the adverse has been commonly used for the treatment of serious, invasive
side effects of amphotericin B and azoles have promoted the fungal infections, resistance to this agent has been docu-
development of new antifungal agents (triazoles and echino- mented,4 especially in immunocompromised patients infected
candins) as well as of lipid formulations of amphotericin B with emerging yeast or mould pathogens.5,6 The emerging
and nystatin.1,2 fungal pathogens are usually less susceptible to azole com-
The parallel increasing interest in antifungal susceptibility pounds and the management of such infections could be
tests also reflects the need for standardized conditions to problematic.
obtain data in vitro for the prediction of the susceptibility or Anidulafungin (VER002, Versicor Inc., CA, USA)
resistance of clinical isolates to antifungal agents. In the 1990s, (LY-303366) is a semi-synthetic antifungal agent with a
the NCCLS developed a reference method for the suscep- structure related to cilofungin (LY-101216). Its structure cor-
tibility of Candida species and Cryptococcus neoformans.3 responds to a cyclic lipopeptide, which inhibits -1,3-glucan
Although Candida albicans is more often associated with biosynthesis by affecting the glucan-synthase enzyme. The
serious fungal infections than other fungi, other Candida result is fungal cell damage and death. Both the spectrum of
species and yeast-like organisms have emerged as aetiolog- action and the toxicity profile are superior to those of cilofun-
ical agents of serious fungal infections.1 Amphotericin B and gin, being highly active in vitro against fluconazole-resistant
its lipid formulations, the echinocandin caspofungin and the and -susceptible C. albicans, Aspergillus spp. and the cyst
..................................................................................................................................................................................................................................................................

*Corresponding author. Tel: +34-922-319-376; Fax: +34-922-319-378; E-mail: mpareval@ull.es

...................................................................................................................................................................................................................................................................

163
2002 The British Society for Antimicrobial Chemotherapy
 M. P. Arvalo et al.
form of Pneumocystis carinii; it has no activity against C.
neoformans.7 Kill-curves of anidulafungin are similar to
those produced by amphotericin B for C. albicans, Candida
glabrata and Candida krusei.8 The activity in vivo of this
echinocandin has been good in animal models (per os) of
systemic candidiasis and in experimental pneumonia infec-
tions due to P. carinii, as well as in infections produced by
Aspergillus fumigatus (intravenous).5,9 Ongoing Phase II
clinical trials will focus on the safety and efficacy of the intra-
venous treatment with anidulafungin for patients suffering
invasive candidiasis. The present study evaluated the fungi-
static (MICs) antifungal activities in vitro of anidulafungin,
fluconazole and itraconazole for Candida spp. The fungicidal
activity (MFCs) of anidulafungin was evaluated for 230 of the
460 isolates included in the study.
Materials and methods
Antifungals
Anidulafungin, as the standard powder LY-303366, was
kindly provided by Lilly Pharmaceuticals (Indiana, IN, USA),
fluconazole by Pfizer (Sandwich, Kent, UK) and itraconazole
by Janssen Research Foundation (Beerse, Belgium) as stand-
ard powders. Stock solutions of anidulafungin and itra-
conazole (1600 mg/L) were prepared in 100% dimethyl
sulphoxide (DMSO) (Merck, Darmstad, Germany) and
fluconazole stock solutions were prepared in sterile distilled
water. Additive two-fold drug dilutions were prepared in the
corresponding solvents (100% strength) followed by further
dilutions in standard RPMI 1640 broth to twice the strength
required for the final concentrations of 0.00316 mg/L
(anidulafungin, itraconazole) and 0.12564 mg/L (flucon-
azole).
Strains
A total of 460 clinical yeast isolates were evaluated:
C. albicans (n = 317), Candida dubliniensis (n = 38), Candida
tropicalis (n = 27), C. glabrata (n = 25), C. krusei (n = 18),
Candida lusitaniae (n = 15), Candida famata (n = 10) and
Candida parapsilosis (n = 10). The NCCLS quality control
(QC) Candida parapsilosis ATCC 22019 and C. krusei
ATCC 6358 strains3 were included as controls each time a set
of isolates was tested. The 460 isolates were recovered from
blood, vaginal, urine and oropharyngeal clinical specimens;
210 from patients in the USA and 250 from Spain. These iso-
lates were maintained at room temperature in sterile distilled
water and subcultured before testing.
Inoculum preparation
Yeast inoculum suspensions were obtained by taking five
colonies (>1 mm diameter) from 24-h-old cultures grown on
164
Sabouraud dextrose agar. The colonies were suspended in
5 mL of sterile saline (0.85% NaCl). The inoculum suspen-
sions were shaken for 15 s and the inoculum density was
adjusted to the turbidity of a 0.5 McFarland Standard (equiva-
lent to 15 106 cfu/mL) with sterile saline. The suspensions
were diluted 1:1000 in RPMI 1640 to give a final inoculum
suspension equivalent to 0.52.5 103 cfu/mL.
Microdilution tests
The broth microdilution assay was carried out following the
standard conditions described by the NCCLS3 with sterile,
96-well, round-bottomed plates (Corning, New York, NY,
USA) and standard RPMI 1640 buffered to pH 7 with MOPS
as the test medium. On the day of the test, each microdilution
well containing 100 L of the corresponding two-fold drug
dilution was inoculated with 100 L of the two-fold-diluted
cell suspension. Growth and sterility controls were included
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for each isolatedrug combination.
Incubation and scoring of MIC wells
The microdilution trays were incubated at 35C and MICs
were determined visually after 48 h of incubation, using an
inverted mirror. The growth in each MIC well was compared
with the growth in the control (antifungal-free) well. MICs of
anidulafungin were the lowest drug concentration wells that
were optically clear (without any visible growth/turbidity).
MICs of fluconazole and itraconazole were the lowest drug
concentration wells that showed a prominent reduction
(approximately 50%) in growth/turbidity.3
MFCs of anidulafungin
MFCs of anidulafungin were also determined for 215 C. albi-
cans and five isolates each of C. krusei, C. glabrata and
C. tropicalis recovered from a single clinical trial in the USA
from oropharyngeal infections. Briefly, 20 L aliquots were
subcultured from each well that showed complete inhibition
(optically clear) and from the growth control onto Sabouraud
dextrose agar plates. The plates were incubated at 35C until
growth was seen in the growth control subculture (usually
48 h). The MFC was the lowest drug concentration that
resulted in either no growth or fewer than three colonies.
Data analysis
The MIC50 and MIC90 values were calculated as the con-
centrations of antifungal agent that were able to inhibit 50%
and 90% of the isolates, respectively. Geometric mean MICs
were obtained to facilitate comparisons of the activities of the
three drugs. The results were processed statistically using
SPSS PC+ version 9.0 for IBM PC. The criterion for statistical
significance was P < 0.05.
 In vitro activity of anidulafungin against yeast

Results and discussion tors.7,9,10 As previously demonstrated,7 anidulafungin had

Efforts made to standardize methods for antifungal suscep-
tibility testing have resulted in the development of broth
macro- and microdilution standard methods by the NCCLS.3
This methodology has been applied to the evaluation of
antifungal activities of investigational agents, including
anidulafungin,7 which is undergoing clinical evaluations
(Phase II) before FDA approval. Because of that, we further
investigated the antifungal activity of anidulafungin against
the 460 yeasts by following the standard conditions for broth
microdilution testing described in the NCCLS document.
Table 1 summarizes the data for the three agents in vitro for
the 460 clinical isolates evaluated in the present study. The
overall mean MIC of anidulafungin was 1.01 mg/L, which
indicated a higher fungistatic antifungal activity in vitro of
this echinocandin than those of itraconazole (1.97 mg/L) and
fluconazole (19.4 mg/L); these differences were statistically
good fungicidal activity against C. albicans, MFC range
0.030.5 mg/L (MFC50 0.12 mg/L and MFC90 0.5 mg/L). For
other species, the MFC ranges were: C. krusei 0.060.5 mg/L,
C. glabrata 0.22.0 mg/L and C. tropicalis 0.20.5 mg/L.
MFCs were usually the same as MICs or only one to two dilu-
tions higher.
In addition, our data demonstrated that anidulafungin had a
potent activity in vitro against both C. krusei and C. glabrata
(Table 1); these two species are either inherently resistant to
fluconazole (C. krusei) or have lower susceptibility (C. gla-
brata) to this triazole than other Candida species. The MICs
of anidulafungin are relatively high for C. parapsilosis when
they are compared with those of other Candida species, but
the MIC range was broad for this species. The lower suscep-
tibility of C. parapsilosis to both echinocandins, caspofungin
and anidulafungin, has been reported previously.9

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significant (P < 0.05). The antifungal activity in vitro of
anidulafungin was superior to that of itraconazole and
fluconazole for C. albicans, C. tropicalis, C. glabrata and
C. krusei (Table 1). But, anidulafungin was less active against
C. famata and C. parapsilosis. The differences were not
significant for C. dubliniensis and C. lusitaniae. Our results
are in agreement with those reported by other investiga-
In summary, anidulafungin has potential as an active
antifungal agent for the treatment of fungal infections caused
by most Candida species, including those species that have
low susceptibility or are resistant to fluconazole. The relev-
ance of these data in vitro has to be validated in clinical trials.
However, the clinical response can be affected by the
inadequate concentration of the antifungal agent at the site of

Table 1. In vitro susceptibilities (mg/L) of 460 clinical yeast isolates to anidulafungin, fluconazole and itraconazole

Organism (n) Antifungal agent Geometric mean MIC MIC50 MIC90 Range

C. albicans (317) anidulafungin 0.11 0.12 0.25 0.010.5

fluconazole 15.23 0.25 32 0.12>256
itraconazole 1.33 0.06 1 0.01>16
C. dubliniensis (38) anidulafungin 0.96 0.12 4 0.128
fluconazole 5.25 0.5 16 0.1264
itraconazole 3.14 1 >16 0.01>16
C. tropicalis (27) anidulafungin 0.4 0.12 2 0.062
fluconazole 22.05 2 64 0.12128
itraconazole 4.37 0.12 >16 0.01>16
C. glabrata (25) anidulafungin 0.27 0.12 0.5 0.122
fluconazole 38.2 32 64 <0.12>256
itraconazole 7.39 8 >16 0.03>16
C. krusei (18) anidulafungin 0.61 0.12 0.5 0.068
fluconazole 66.67 32 128 0.12>256
itraconazole 2.48 0.5 >16 0.06>16
C. lusitaniae (15) anidulafungin 0.18 0.12 0.12 0.030.25
fluconazole 0.84 0.12 2 0.128
itraconazole 0.07 0.12 0.12 0.121
C. famata (10) anidulafungin 2.03 4 8 0.01>16
fluconazole 34.83 8 16 0.1264
itraconazole 6.41 0.5 1 0.03>16
C. parapsilosis (10) anidulafungin 1.76 1 4 0.124
fluconazole 7.01 1 1 0.0364
itraconazole 0.15 0.06 0.5 0.030.5

165
 M. P. Arvalo et al.
the infection, by impaired host defence mechanisms or by
other hostfungus and antifungal agent interactions.
Acknowledgements
This study was partially supported by Lilly Pharmaceuticals.
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