Journal of Antimicrobial Chemotherapy (2007) 60, 1004– 1009
doi:10.1093/jac/dkm350
Advance Access publication 17 September 2007
Correlation of posaconazole minimum fungicidal concentration
and time – kill test against nine Candida species
G. Sóczó1, G. Kardos1, P. M. McNicholas2, E. Balogh1, L. Gergely1, I. Varga3,
B. Kelentey3 and L. Majoros1*
1
Department of Medical Microbiology, Medical and Health Science Center, University of Debrecen, Hungary;
2
Schering-Plough Research Institute, Kenilworth, NJ, USA; 3Faculty of Dentistry,
Medical and Health Science Center, University of Debrecen, Hungary
Received 13 March 2007; returned 21 May 2007; revised 30 July 2007; accepted 15 August 2007
Objectives: We evaluated the in vitro activity of posaconazole against nine Candida species using
minimum fungicidal concentration (MFC) measurements and time–kill methods.
Methods: MFCs of posaconazole were determined for 209 clinical isolates (32 Candida albicans,
30 Candida glabrata, 21 Candida tropicalis, 29 Candida krusei, 28 Candida parapsilosis sensu stricto,
50 Candida inconspicua, 13 Candida kefyr, 3 Candida lusitaniae and 3 Candida guilliermondii) and
7 ATCC Candida strains. The following strains were tested in time –kill studies: 3 strains each of
C. glabrata, C. kefyr, C. guilliermondii and C. lusitaniae; 2 C. tropicalis; 4 C. albicans; 4 C. inconspicua;
9 C. krusei; 12 C. parapsilosis; and 7 ATCC strains.
Results: Posaconazole was fungicidal in both MFC and time–kill experiments (at 2 mg/L within 48 h in
time –kill assays) against each C. krusei, C. inconspicua and C. lusitaniae strain and was fungistatic
against each C. albicans, C. glabrata, C. tropicalis and C. guilliermondii strain. For the C. parapsilosis
strains, posaconazole MFCs were 2 mg/L for 20 of the 28 isolates tested; however, in the time– kill
tests, even at drug concentrations of 32– 64 3 MIC, posaconazole was fungistatic against all
C. parapsilosis strains tested.
Conclusions: MFC values were good predictors of the fungistatic or fungicidal activity of posaconazole, as determined by time–kill curves, for all tested species except C. parapsilosis. Our results highlight the fungicidal action of posaconazole against a number of clinically relevant Candida species.
Keywords: MFCs, antifungals, triazoles
Introduction
The fungicidal activity of an antifungal agent can be measured
either by determining the minimum fungicidal concentration
(MFC) or using time – kill methodology.1 Although the time –
kill method provides information on the rate of killing of a drug,
it is both cumbersome and time-consuming and is therefore not
suitable for routine laboratory work. Measurement of the MFC
value is a useful alternative; however, the method has not yet
been standardized.1 Cantón et al. 2 observed a good correlation
between the results of time – kill experiments and MFC values
for amphotericin B against a large panel of Candida species.
Similar data for other agents (e.g. triazoles and echinocandins)
do not appear to be available.
Triazoles are commonly regarded as being fungistatic;
however, some triazoles (e.g. voriconazole and posaconazole)
exhibit fungicidal activity against moulds.1 In addition, several
previous studies, using either MFC measurements or time – kill
studies, suggested that triazoles may also show fungicidal
activity against certain Candida species.3 – 6
Posaconazole is an extended-spectrum triazole that is active
in vitro against a wide range of fungal pathogens including most
Candida spp. and the majority of organisms responsible for
causing aspergillosis, cryptococcosis, chromoblastomycosis,
mycetoma and phaeohyphomycosis.7 Posaconazole is also active
against many of the zygomycetes, organisms that were previously regarded as being resistant to most azoles, including the
most widely used fluconazole and voriconazole.7,8 Using a
variety of techniques, posaconazole was shown to be fungicidal
against Aspergillus spp., Cryptococcus neoformans and dermatophytes.9 – 13 This potent in vitro activity has translated into
clinical efficacy against a wide spectrum of mycoses.9,10
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*Corresponding author. Tel: þ36-52-411-717/4501; Fax: þ36-52-414-948; E-mail: [email protected]
.....................................................................................................................................................................................................................................................................................................................................................................................................................................
1004
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Killing activity of posaconazole against Candida species
The aim of this study was to determine whether posaconazole
was fungicidal against both common (Candida albicans,
Candida glabrata, Candida tropicalis, Candida parapsilosis
and Candida krusei) and less common (Candida inconspicua,
Candida kefyr, Candida lusitaniae and Candida guilliermondii)
Candida species using both MFC determinations and time – kill
studies; we also investigated the correlation between the two
techniques.
Materials and methods
diluted 10-fold in sterile saline; four 30 mL aliquots were
subsequently plated onto SDA (if colony counts were suspected to
be ,1000 cfu/mL, undiluted samples were plated). On the basis of
our preliminary results with C. albicans ATCC 14053 and C. krusei
ATCC 6258 strains, the following sampling points were chosen: 0,
6, 18, 24, 36 and 48 h. Colony counts were determined after incubation of the plates at 358C for 48 h. All assays were performed in
duplicate and repeated at least twice. Time –kill curves were prepared using the computer curve-fitting software GraphPad Prism
4.03 for Windows. The limit of exact quantification using this methodology is 50 cfu/mL. Fungicidal activity was defined as a 99.9%
(3 log10) reduction in viable cfu/mL of the starting inoculum.
Clinical isolates
Two hundred and nine yeast isolates (32 C. albicans, 30 C. glabrata,
21 C. tropicalis, 29 C. krusei, 28 C. parapsilosis sensu stricto, 50
C. inconspicua, 13 C. kefyr, 3 C. lusitaniae and 3 C. guilliermondii)
were studied. We also tested seven ATCC Candida strains (C. albicans ATCC 14053, C. tropicalis ATCC 750, C. parapsilosis ATCC
22019, C. krusei ATCC 6258, C. inconspicua ATCC 16783, C. guilliermondii ATCC 6260 and Candida norvegensis ATCC 22977).
Strains were identified using CHROMagar Candida, morphology on
rice extract agar and the API ID32C panel. Species identities of
C. inconspicua and C. parapsilosis isolates were confirmed
by PCR-ribotyping14 and restriction fragment length polymorphism
of the SADH gene,15 respectively.
Drug
Posaconazole was obtained as the reagent grade powder from the
Schering-Plough Research Institute. Stock solutions were prepared
in DMSO as specified by the CLSI (formerly NCCLS) M27-A2
document.16 Final concentration ranges were 0.015–8 mg/L.
Susceptibility testing
Posaconazole MICs for these isolates with the exception of
C. lusitaniae and C. guilliermondii have been published elsewhere.17 For measurement of MFCs, the inoculum recommended in
the CLSI M27-A2 document16 was increased 10-fold to 104 cells/
mL.2 Inoculum densities were confirmed by plating serial dilutions
on Sabouraud dextrose agar (SDA). All other test procedures followed the approved CLSI broth microdilution method. Microdilution
trays were incubated for 48 h and read visually; the MIC was the
lowest concentration of drug that produced a prominent decrease in
turbidity when compared with the drug-free control.
After MICs were read, the entire contents (200 mL) of each well
with drug concentrations above the MIC were plated onto two drugfree SDA plates (100 mL aliquots/plate).2 Aliquots were placed in a
single spot on the agar plate and, after drying, the cells were dispersed by streaking. Plates were incubated at 358C for 48 h; the
MFC was defined as the lowest drug concentration that resulted in a
99.9% reduction in the starting inoculum. All assays were performed
in duplicate and repeated at least twice.
Time– kill studies
In time–kill experiments, the method described by Klepser et al.18
was used. The starting inoculum was 105 cells/mL and drug concentrations ranged from 0.5 to 16 times the MIC; C. parapsilosis strains
were also tested at concentrations 32 and 64 times the MIC. Test
tubes were incubated with agitation in the dark at 358C. At predetermined time points, samples (100 mL) were removed and serially
Results
MFCs
Fungicidal activity is routinely defined as a 99.9% reduction in
cfu over a fixed sampling period. The standard inoculum of
103 cfu/mL (which equates to 102 cells/well when using 100 mL/
well), described in the CLSI document M27-A2,16 is too low to
permit an accurate measurement of a 99.9% reduction in viable
cells. Therefore, as recommended by Cantón et al.,2 in this
study, MIC and MFC determinations were performed using a
10-fold higher inoculum, 104 cfu/mL. The MICs obtained using
the higher inoculum were the same as, or one dilution higher,
those obtained using the standard CLSI inoculum (data not
shown); all MIC values in the following sections were obtained
using the higher inoculum. MFC distributions, geometric mean
values and MFC50/90 values (MFCs for 50% and 90% of the isolates, respectively) are shown in Table 1.
Posaconazole was fungicidal at low concentrations (2 mg/L)
against all of the C. inconspicua and C. lusitaniae strains and
against the majority of C. krusei (24/29, 83%), C. parapsilosis
(20/28, 71%) and C. kefyr (9/13, 69%) strains. In contrast, the
drug was fungistatic (MFC . 8 mg/L) against all of the C. albicans, C. glabrata, C. tropicalis and C. guilliermondii strains
tested. MFC values for the five ATCC strains showed the same
species disposition as the clinical isolates (Table 1).
Time– kill study results
To further characterize the fungicidal activity of posaconazole,
time – kill assays were performed. Strains from a given species
were chosen to represent the entire MFC range of that species;
in those instances where there was a lack of concordance
between the MFC and time – kill experiments, additional strains
were tested. Time – kill curves of all reference strains used were
also determined. The isolates and reference strains tested
together with their MIC and MFC ranges are listed in Table 2;
representative kill curves are shown in Figure 1.
Posaconazole appeared to exhibit the strongest fungicidal
activity against the C. lusitaniae, C. inconspicua and C. kefyr
clinical isolates; at 1 mg/L, the drug resulted in 99.9% killing
within 24 h against two of three C. lusitaniae, three of four
C. inconspicua and two of three C. kefyr isolates (Figure 1). At
48 h, posaconazole was fungicidal against all of the C. lusitaniae, C. inconspicua and C. kefyr isolates (with the exception of
the single C. kefyr isolate with an MFC of .8 mg/L).
Posaconazole also exhibited fungicidal activity against seven out
of nine C. krusei isolates at concentrations 4-fold higher than
1005
Sóczó et al.
Table 1. Posaconazole MFC distributions, geometric means of MFC and MFC50/90 values for test isolates
MFCa (mg/L) distribution
Species (number tested)
0.12
0.25
0.5
1
2
4
8
.8
GMb
MFC range
MFC50/90c
C. albicans (32)
C. glabrata (30)
C. tropicalis (21)
C. krusei (29)
C. parapsilosis (28)
C. inconspicua (50)
C. kefyr (13)
C. lusitaniae (3)
C. guilliermondii (3)
—
—
—
—
—
1
—
—
—
—
—
—
—
—
7
—
—
—
—
—
—
1
3
23
8
—
—
—
—
—
12
10
19
—
—
—
—
—
—
11
7
—
1
3
—
—
—
—
3
—
—
1
—
—
—
—
—
2
8
—
1
—
—
32
30
21
—
—
—
2
—
3
.8
.8
.8
1.69
2
0.57
1.38
2
.8
.8
.8
.8
0.5– 8
0.5– 8
0.12 –1
0.5 to .8
2
.8
.8
.8
.8
2/4
2/8
0.5/1
0.5/.8
2/2
2/2
a
MFC testing conditions were essentially as described in CLSI M27-A2 except that a higher (104 cfu/mL) starting inoculum was used.
GM, geometric mean.
c
MFC50/90, concentrations of drug resulting in a 99.9% reduction in cfu for 50% and 90% of tested isolates, respectively.
b
the MIC after 48 h (but not after 36 h). The remaining two
isolates, with MFC values of 4 and 8 mg/L, exhibited a 99.9%
reduction in cfu at concentrations 8-fold higher than their
respective MICs after 48 h. The only C. krusei strain to exhibit a
99.9% reduction in cfu within 24 h was the ATCC strain 6258;
the drug concentration was 4 mg/L. Finally, against the C. norvegensis ATCC 22977 strain, posaconazole displayed good fungicidal activity after 36 h at concentrations 1– 16-fold higher
than the MIC.
With the exception of the C. albicans strain exhibiting
reduced susceptibility to posaconazole (MIC . 8 mg/L), growth
of all strains of C. albicans, C. tropicalis and C. guilliermondii
was strongly inhibited at drug concentrations ranging from 2- to
Table 2. Candida spp. tested in time–kill assays
Species (number tested)
MIC range of
isolates tested
(mg/L)a
MFC range of
isolates tested
(mg/L)a
C. albicans (4)
C. glabrata (3)
C. tropicalis (2)
C. parapsilosis (12)
C. krusei (9)
C. inconspicua (4)
C. lusitaniae (3)
C. guilliermondii (3)
C. kefyr (3)
C. albicans ATCC 14053
C. tropicalis ATCC 750
C. parapsilosis ATCC 22019
C. krusei ATCC 6258
C. inconspicua ATCC 16783
C. guilliermondii ATCC 6260
C. norvegensis ATCC 22977
0.03 to .8
0.5 to .8
0.06 –0.25
0.06 –0.12
0.25
0.12 –0.25
0.06
0.25
0.06
0.12
0.5
0.12
0.25
0.12
0.25
0.12
.8
.8
.8
0.5 to .8
0.5–8
0.5–1
2
.8
0.5–8
.8
.8
.8
0.5
0.5
.8
0.25
a
MIC/MFC testing followed the CLSI M27-A2 methodology except that a
higher (104 cfu/mL) starting inoculum was used.
16-fold higher than the respective MICs. In contrast, there
was little or no growth inhibition of the C. glabrata strains
at concentrations of posaconazole 16-fold above the MIC.
Twelve C. parapsilosis isolates with MFCs ranging from 0.5 to
.8 mg/L were tested. Although posaconazole did not exhibit
fungicidal activity against any of the strains, growth of all isolates was strongly inhibited at low (0.06 –0.12 mg/L; Figure 1)
drug concentrations.
Discussion
The standard CLSI susceptibility testing method for yeasts recommends an incubation time of 48 h when testing Candida
species.16 As MFC measurements are frequently extensions of
the MIC test—the contents of the optically clear wells in the
MIC test are plated on drug-free medium to quantify changes in
cfu—the same drug exposure time is used when measuring
MFCs. In contrast, time – kill measurements are generally made
over 24 h,1 and this discrepancy makes direct comparisons
between the two methods problematic. To address this problem,
Cantón et al.2 recommended that the incubation time for time –
kill experiments be extended from 24 to 48 h; with the extended
incubation time, they observed a good correlation between
MFCs and time – kill results when examining the activity of
amphotericin B against seven Candida species.
In this study, we also used a 48 h incubation time to evaluate
the fungicidal activity of posaconazole. Posaconazole exhibited
concentration- and time-dependent fungicidal activity against
several Candida species, including fluconazole-resistant
C. krusei isolates, as shown by both MFC measurements and
time –kill curves. Similarly, posaconazole showed excellent and
rapid fungicidal activity against C. inconspicua and C. lusitaniae
strains (with MFCs 2 mg/L); two species that are reported to
exhibit decreased susceptibility to fluconazole and amphotericin
B, respectively.3,14
The correlation between MFCs (read at 48 h) and the
time –kill results was far better at 48 h than at 24 h. This was
most clearly evident for the C. krusei isolates; at 24 h, posaconazole appeared fungistatic at 4 mg/L against 9 of the 10 isolates
1006
Killing activity of posaconazole against Candida species
Figure 1. Representative time–kill plots. Cells were incubated with the indicated amount of posaconazole and samples withdrawn at the indicated time
points and plated on drug-free medium to quantify cfu. The dashed line indicates a 99.9% reduction in the starting inoculum. Each data point represents the
mean + SD (error bar) for two independent experiments, each performed in duplicate. The concentrations of drug tested were as follows: drug-free control,
filled triangles; 0.5 MIC, open squares; 1 MIC, open triangles; 2 MIC, open inverted triangles; 4 MIC, open diamonds; 8 MIC, open circles; and
16 MIC, asterisks.
1007
Sóczó et al.
tested (the exception was the ATCC 6258 strain), but at 48 h, it
(at 2 mg/L) was fungicidal against all isolates tested. The correlation between MFC values and 48 h time –kill results was
excellent for all Candida species, except C. parapsilosis; posaconazole was fungistatic (by time –kill assays) against this
species regardless of the MFC values. Generally, in time – kill
studies, it was fungicidal within 48 h at a lower concentration
than the MFC against all of the C. krusei, C. lusitaniae and
C. inconspicua isolates as well as the two C. kefyr isolates with
MFC values 2 mg/L.
To date, only one other study compared MFCs with time – kill
results when testing the fungicidal action of azoles.19 In this
study, posaconazole exhibited concentration- and timedependent fungicidal activity against an Aspergillus fumigatus
strain at concentrations of 4 mg/L and lower; a 99.9% reduction
in cfu required 8 mg/L posaconazole and 48 h of incubation.
The exact MFC value for this strain was not provided; nonetheless, the time – kill results appear to correlate with the MFC
range of the A. fumigatus strains examined (4.45 + 2.70 mg/L).
For antifungal drugs, there are limited data correlating the
in vitro fungicidal/fungistatic activities of the drugs with clinical
outcomes. The most compelling data, summarized by Pfaller
et al.,1 are from patients treated with amphotericin B; in a series
of studies, microbiological failures (with both yeast and mould
pathogens) appeared to be correlated with elevated in vitro MFC
values. However, in clinical trials comparing a fungicidal drug,
the echinocandins (micafungin and anidulafungin), with a fungistatic drug, fluconazole, for the treatment of oesophageal candidiasis (the predominant pathogen was C. albicans), there were no
significant differences in either clinical or mycological cures.20,21
Furthermore, despite being fungistatic against C. glabrata, in
clinical trials posaconazole was effective in treating oropharyngeal candidiasis in people with human immunodeficiency virus,
including subjects infected with fluconazole-resistant C. glabrata
isolates.22 Clearly, further studies are required to determine
whether there are clinical settings in which a fungicidal drug may
provide an improved response when compared with a fungistatic
drug in the treatment of invasive fungal infection.
In summary, the present study is the first to examine the correlation between MFCs and time – kill results in the determination of the fungicidal activity of an azole antifungal against
Candida species. With the exception of C. parapsilosis, MFC
values proved to be a good predictor of the fungicidal activity of
posaconazole, as measured by time – kill studies, against
Candida species.
Acknowledgements
We would like to thank Cecı́lia Miszti and Erzsébet Falusi for
their valuable help. We are grateful to Dr Rudolf Gesztelyi for
his help with the graphs. We thank Schering-Plough for providing
the posaconazole powder. A part of this work was presented at
the Forty-sixth Interscience Conference on Antimicrobial Agents
and Chemotherapy, San Francisco, CA, 2006 (abstract A-2151).
Funding
The study did not receive any financial support from third
parties.
Transparency declarations
None to declare.
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