In Vitro Interactions of Micafungin with Amphotericin B against Clinical Isolates of Candida spp.
The in vitro activity of amphotericin B in combination with micafungin was evaluated against 115 isolates representing seven species of Candida. Overall, the percentages of synergistic interactions were 50% and 20% when the MIC-2 (lowest drug concentration to cause a prominent reduction in growth) and MIC-0 (lowest drug concentration to cause 100% growth inhibition) end point criteria, respectively, were used. Antagonism was not observed. Some of the interactions were confirmed by time-kill assays.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 2008, p. 1529–1532 Vol. 52, No. 4
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
In Vitro Interactions of Micafungin with Amphotericin B against
Clinical Isolates of Candida spp.
Alfonso J. Carrillo,
J. F. Cano,
F. Javier Pastor,
and Josep Guarro
Unitat de Microbiologia, Facultat de Medicina i Cie`ncies de la Salut, Universitat Rovira i Virgili, Reus, Spain
Departamento de Inmunologı´a, Microbiologı´a y Parasitologı´a, Facultad de Medicina y Odontologı´a,
Universidad del Paı´s Vasco, Bilbao, Spain
; and Departamento de Microbiologı´a,
Asesorı´a Cientı´ﬁca y de Investigacio´n Aplicada, Barcelona, Spain
Received 21 August 2007/Returned for modiﬁcation 10 November 2007/Accepted 20 January 2008
The in vitro activity of amphotericin B in combination with micafungin was evaluated against 115
isolates representing seven species of Candida. Overall, the percentages of synergistic interactions were
50% and 20% when the MIC-2 (lowest drug concentration to cause a prominent reduction in growth) and
MIC-0 (lowest drug concentration to cause 100% growth inhibition) end point criteria, respectively, were
used. Antagonism was not observed. Some of the interactions were conﬁrmed by time-kill assays.
Candida spp. are an important cause of nosocomial infec-
tions with high morbidity and mortality (13). The spectrum
of invasive candidiasis is changing, and the incidence of
infections due to non-C. albicans species is rising (13). Am-
photericin B (AMB) is one of the antifungal agents most
commonly used to treat most invasive mycoses, but its tox-
icity limits its use. Fluconazole is commonly used for the
treatment of candidiasis, but its activity against non-C. al-
bicans species, especially C. glabrata, is variable, and it has
no activity against C. krusei (18, 19). Echinocandins consti-
tute a new and unique class, in that they inhibit synthesis of
a distinct major cell wall component, ␤-(1,3)-
sulting in morphological changes to the cell wall. Micafun-
gin (MFG) is a promising echinocandin that was recently
approved by the FDA and has demonstrated activity against
Candida species, although it has shown high MICs (⬎8
g/ml) against isolates of some species, such as C. parapsi-
losis (1, 6). A combination of MFG with AMB, two drugs
with different targets, could be of interest for improving
clinical results, shortening the duration of treatment, and
reducing toxic drug doses, which is especially important for
AMB. This combination has been synergistic in vitro against
Cryptococcus spp., Rhodotorula glutinis, Trichosporon asahii,
and Scedosporium spp. (15, 16, 21) and showed efﬁcacy for
the treatment of murine disseminated infection with Tricho-
sporon asahii, C. glabrata, and Aspergillus spp. (3, 8, 12, 17).
Moreover, the combination of other echinocandins, e.g.,
caspofungin, with AMB has shown positive in vitro and in
vivo interactions against C. parapsilosis and C. glabrata (2,
12). We considered it interesting to evaluate whether this
combination could also be beneﬁcial for the treatment of
infections by Candida spp., and we tested its activity against
isolates representing seven of the most common species.
We tested a total of 145 clinical isolates (C. albicans [n ⫽
35], C. dubliniensis [n ⫽ 20], C. glabrata [n ⫽ 15], C. krusei
[n ⫽ 35], C. lusitaniae [n ⫽ 10], C. parapsilosis [n ⫽ 15], and
C. tropicalis [n ⫽ 15]). In order to ﬁnd out which species all
the strains received as C. parapsilosis complex belonged to,
the internal transcribed spacer 1 (ITS1) adjacent to the 5.8S
rRNA gene was ampliﬁed and sequenced using primers
ITS5 and ITS2 (20). All the sequences obtained showed
100% similarity with the sequence of the type strain of
C. parapsilosis (CBS 604; GenBank accession number
AJ635316). Drug interactions were assessed by a checker-
board microdilution method after 48 h of incubation at 35°C
(4, 11). The drugs were obtained as pure powders. AMB
(USP, Rockville, MD) was diluted in dimethyl sulfoxide and
MFG (Astellas Pharma Inc., Tokyo, Japan) in sterile dis-
tilled water. The ﬁnal dimethyl sulfoxide concentration was
1%. Antifungal agents were placed in rows or in columns in
the trays, with concentrations ranging from 4 to 0.06 g/ml
for AMB and from 16 to 0.03 g/ml for MFG. One of the
limitations of the checkerboard method for some antifungal
combinations is deciding which end point has to be used to
evaluate interactions between the drugs (10). The recom-
mended end points for AMB and MFG are different, i.e.,
the lowest drug concentration to show 100% growth inhibi-
tion (MIC-0) for the former (11) and a prominent reduction
in growth (MIC-2) for the latter (14); however, the end
points used in the checkerboard procedure should be the
same for the two drugs tested. Since it is difﬁcult to decide
which of the two end points should be used, we used both for
the two drugs when tested alone and in combination. We
used the fractional inhibitory concentration index to quan-
tify and classify drug interaction (4). Interaction was con-
sidered synergistic if the fractional inhibitory concentration
index was ⱕ0.5, indifferent if it was ⬎0.5 and ⱕ4, and
antagonistic if it was ⬎4. The procedure, conservation of the
strains, and quality controls have all been detailed previ-
ously (15). Approximately 80% of the tests were repeated,
and the results showed the same tendencies (data not
shown). However, when the results did not coincide, the test
* Corresponding author. Mailing address: Unitat de Microbiologia,
Facultat de Medicina, Universitat Rovira i Virgili, Carrer Sant
Llorenc¸, 21.43201 Reus, Spain. Phone: 977-759359. Fax: 977-759322.
Published ahead of print on 28 January 2008.
was repeated and the mode of the three MICs was consid-
ered. The MICs were compared using the Mann-Whitney U
test. Calculations were made using Graph Pad 4.0 and SPSS
version 14.0 for Windows.
In order to conﬁrm the results obtained with the check-
erboard method, time-kill studies were performed using two
strains (one that showed indifference and one that showed
synergism by the ﬁrst methodology at the MIC-2 end point)
belonging to the three most common species (C. albicans
FMR 9600 and FMR 9542, C. glabrata FMR 8498 and FMR
8489, and C. parapsilosis FMR 9601 and FMR 9544). Both
AMB and MFG were used at 1⫻ MIC and 2⫻ MIC. The
numbers of CFU were determined at 0, 2, 4, 6, and 24 h. The
limit of detection was 20 CFU/ml. Synergism and antago-
nism were deﬁned, respectively, as an increase or decrease
of ⱖ2 log
CFU/ml in antifungal activity compared with the
most active single agent, while a change of ⬍2 log
was considered indifferent (4).
Table 1 shows the in vitro results using the MIC-0 or
MIC-2 as the end point for both drugs. The MIC-0 and
MIC-2 of MFG were signiﬁcantly different for all the species
(P ⬍ 0.05) except C. albicans (P ⫽ 0.09), C. glabrata (P ⫽
0.12), and C. lusitaniae (P ⫽ 0.19). The geometric mean
MIC (MIC-2) of MFG was lower than 1 g/ml against all
species tested with the exception of C. parapsilosis, which
agrees with results reported by other authors, who also
obtained high MFG MICs for this species (1, 6). Differences
between MIC-0 and MIC-2 were lower for AMB, being
signiﬁcant only for C. parapsilosis, C. tropicalis, and C. dub-
liniensis (P ⬍ 0.0001, P ⫽ 0.0078, and P ⫽ 0.009, respec-
tively). For every species there was more synergism when
MIC-2 values rather than MIC-0 values were used as end
points. Using the MIC-2, synergistic interactions were ob-
tained for 52% (75/145) of the isolates, while using the
MIC-0, synergistic interactions were obtained for only 22%
(32/145) of them. This combination could potentially be
more useful in the case of those species with strains that are
resistant to both antifungals, such as C. parapsilosis. Al-
though this species mainly showed very high MFG MICs,
these were reduced signiﬁcantly when the two drugs were
combined (P ⬍ 0.0001), showing 40 to 60% synergistic in-
teractions. The efﬁcacy of the combination of AMB with one
echinocandin, caspofungin, has already been proved in a
murine model of disseminated infection with C. parapsilosis
One hundred percent agreement was obtained between
the checkerboard and time-kill (at both concentrations) pro-
cedures for the six strains tested. Figure 1 shows the time-
kill curves for the three strains that showed synergism or
indifference at 1⫻ MIC.
In summary, we have demonstrated that the combination
MFG and AMB has some synergistic effect against Candida
species, although with important differences between the two
reading criteria used. Further studies with animal models are
warranted to determine the efﬁcacy of this combination and to
determine which reading criterion is more predictive for clin-
TABLE 1. In vitro activities of MFG and AMB, alone and in combination, against clinical isolates of Candida spp. as determined using the MIC-0 or MIC-2 end point
(no. of isolates)
MIC-0 end point MIC-2 end point
Geometric mean (range) MIC, g/ml % of isolates showing: Geometric mean (range) MIC, g/ml % of isolates showing:
AMB MFG AMB/MFG
Synergism Indifference AMB MFG AMB/MFG Synergism Indifference
C. krusei (35) 2.11 (0.5–8) 0.96 (0.25–4) 0.81/0.15 (0.06–2/0.03–2) 26 74 1.25 (0.5–2) 0.65 (0.12–4) 0.49/0.10 (0.06–2/0.03–1) 37 63
C. albicans (35) 0.73 (0.5–2) 0.31 (0.25–16) 0.36/0.08 (0.06–2/0.03–8) 8.5 91.5 0.70 (0.5–1) 0.23 (0.25–8) 0.16/0.04 (0.06–0.25/0.03–0.5) 71 29
C. parapsilosis (15) 2 (2) 14.49 (8–⬎16) 0.67/1.15 (0.25–1/0.03–8) 40 60 0.74 (0.5–2) 9.28 (8–16) 0.22/0.16 (0.12–0.25/0.03–4) 60 40
C. tropicalis (15) 1.66 (1–4) 1.44 (0.5–32) 0.49/0.12 (0.06–1/0.03–1) 47 53 0.87 (0.25–4) 0.47 (0.25–1) 0.17/0.06 (0.06–0.25/0.03–0.5) 53 47
C. dubliniensis (20) 0.41 (0.12–1) 1.46 (0.25–⬎16) 0.14/0.29 (0.06–0.5/0.06–1) 35 65 0.14 (⬍0.06–0.5) 0.32 (0.06–⬎16) 0.08/0.07 (0.06–0.25/0.03–0.5) 50 50
C. glabrata (15) 0.75 (0.25–1) 0.27 (0.12–0.5) 0.31/0.11 (0.06–1/0.03–0.5) 0 100 0.69 (0.25–1) 0.24 (0.12–0.5) 0.23/0.06 (0.06–1/0.03–0.5) 53 47
C. lusitaniae (10) 0.44 (0.12–1) 1.18 (0.12–2) 0.24/0.60 (0.06–0.5/0.03–1) 0 100 0.35 (0.06–1) 0.86 (0.12–2) 0.19/0.31 (0.06–0.25/0.03–1) 20 80
AMB and MFG in combination.
1530 NOTES ANTIMICROB.AGENTS CHEMOTHER.
This work was supported by a grant from Fondo de Investigaciones
Sanitarias from the Ministerio de Sanidad y Consumo of Spain (PI
1. Andes, D., and N. Safdar. 2005. Efﬁcacy of micafungin for the treatment of
candidemia. Eur. J. Clin. Microbiol. 24:662–664.
2. Barchiesi, F., E. Spreghini, S. Tomassetti, D. Giannini, and G. Scalise. 2007.
Caspofungin in combination with amphotericin B against Candida parapsilo-
sis. Antimicrob. Agents Chemother. 51:941–945.
3. Clemons, K. V., M. Espiritu, R. Parmar, and D. A. Stevens. 2005. Compar-
ative efﬁcacies of conventional amphotericin B, liposomal amphotericin B
(AmBisome), caspofungin, micafungin, and voriconazole alone and in com-
bination against experimental murine central nervous system aspergillosis.
Antimicrob. Agents Chemother. 49:4867–4875.
4. Eliopoulos, G. M., and R. C. Moellering. 1991. Antimicrobial combinations,
p. 432–492. In V. Lorian (ed.), Antibiotics in laboratory medicine, 3rd ed.
The Williams & Wilkins Co., Baltimore, MD.
5. Reference deleted.
6. Laverdiere, M., D. Hoban, C. Restieri, and F. Habel. 2002. In vitro
activity of three new triazoles and one echinocandin against Candida
bloodstream isolates from cancer patients. J. Antimicrob. Chemother.
7. Reference deleted.
8. Marine´, M., C. Serena, F. J. Pastor, and J. Guarro. 2006. Combined anti-
fungal therapy in a murine infection by Candida glabrata. J. Antimicrob.
9. Reference deleted.
10. Mukherjee, P. K., D. J. Sheehan, C. A. Hitchcock, and M. A. Ghannoum.
2005. Combination treatment of invasive fungal infections. Clin. Microbiol.
11. National Committee for Clinical Laboratory Standards. 2002. Reference
method for broth dilution antifungal susceptibility testing of yeasts. Ap-
proved standard M27–A2, 2nd ed. National Committee for Clinical Labo-
ratory Standards, Wayne, PA.
12. Olson, J. A., J. P. Adler-Moore, P. J. Smith, and R. T. Profﬁtt. 2005. Treat-
ment of Candida glabrata infection in immunosuppressed mice by using a
combination of liposomal amphotericin B with caspofungin or micafungin.
Antimicrob. Agents Chemother. 49:4895–4902.
13. Patterson, T. F. 2005. Advances and challenges in management of invasive
mycoses. Lancet 366:1013–1025.
14. Pfaller, M. A., L. Boyken, R. J. Hollis, S. A. Messer, S. Tendolkar, and D. J.
Diekema. 2006. Global surveillance of in vitro activity of micafungin against
Candida: a comparison with caspofungin by CLSI-recommended methods.
J. Clin. Microbiol. 44:3533–3538.
15. Serena, C., B. Fernandez-Torres, F. J. Pastor, L. Trilles, M. S. Lazera, N.
Nolard, and J. Guarro. 2005. In vitro interactions of micafungin with other
antifungal drugs against clinical isolates of four species of Cryptococcus.
Antimicrob. Agents Chemother. 49:2994–2996.
FIG. 1. Time-kill studies conducted at 1⫻ MIC for C. albicans, C. parapsilosis, and C. glabrata. Synergistic interactions were observed for C.
albicans FMR 9542 (A), C. parapsilosis FMR 9544 (C), and C. glabrata FMR 8489 (E), and indifferent interactions were observed for C. albicans
FMR 9600 (B), C. parapsilosis FMR 9601 (D), and C. glabrata FMR 8498 (F).
OL. 52, 2008 NOTES 1531
16. Serena, C., M. Marine, F. J. Pastor, N. Nolard, and J. Guarro. 2005. In vitro
interaction of micafungin with conventional and new antifungals against
clinical isolates of Trichosporon, Sporobolomyces and Rhodotorula. J. Anti-
microb. Chemother. 55:1020–1023.
17. Serena, C., F. J. Pastor, F. Gilgado, E. Mayayo, and J. Guarro. 2005. Efﬁcacy
of micafungin in combination in a murine model of disseminated tricho-
sporonosis Antimicrob. Agents Chemother. 49:497–502.
18. Spellberg, B. J., S. G. Filler, and J. E. Edwards. 2006. Current treatment
strategies for disseminated candidiasis. Clin. Infect. Dis. 42:244–251.
19. Swinne, D., M. Watelle, and N. Nolard. 2005. In vitro activities of voricon-
azole, ﬂuconazole, itraconazole and amphotericin B against non Candida
albicans yeast isolates. Rev. Iberoam. Micol. 22:24–28.
20. Tavanti, A., A. D. Davidson, N. A. R. Gow, M. C. J. Maiden, and F. C. Odds.
2005. Candida orthopsilosis and Candida metapsilosis spp. nov. to replace
Candida parapsilosis groups II and III. J. Clin. Microbiol. 43:284–292.
21. Yustes, C., and J. Guarro. 2005. In vitro synergistic interaction between
amphotericin B and micafungin against Scedosporium spp. Antimicrob.
Agents Chemother. 49:3498–3500.
1532 NOTES ANTIMICROB.AGENTS CHEMOTHER.