Abstract
This study compared nine susceptibility testing methods and 12 endpoints for anidulafungin, caspofungin, and micafungin with the same collection of blinded FKS hot spot mutant (n = 29) and wild-type isolates (n = 94). The susceptibility tests included EUCAST Edef 7.1, agar dilution, Etest, and disk diffusion with RPMI-1640 plus 2% glucose (2G) and IsoSensitest-2G media and CLSI M27A-3. Microdilution plates were read after 24 and 48 h. The following test parameters were evaluated: fks hot spot mutants overlapping the wild-type distribution, distance between the two populations, number of very major errors (VMEs; fks mutants misclassified as susceptible), and major errors (MEs; wild-type isolates classified as resistant) using a wild-type-upper-limit value (WT-UL) (two twofold-dilutions higher than the MIC50) as the susceptibility breakpoint. The methods with the lowest number of errors (given as VMEs/MEs) across the three echinocandins were CLSI (12%/1%), agar dilution with RPMI-2G medium (14%/0%), and Etest with RPMI-2G medium (8%/3%). The fewest errors overall were observed for anidulafungin (4%/1% for EUCAST, 4%/3% for CLSI, and 3%/9% for Etest with RPMI-2G). For micafungin, VME rates of 10 to 71% were observed. For caspofungin, agar dilution with either medium was superior (VMEs/MEs of 0%/1%), while CLSI, EUCAST with IsoSensitest-2G medium, and Etest were less optimal (VMEs of 7%, 10%, and 10%, respectively). Applying the CLSI breakpoint (S ≤ 2 μg/ml) for CLSI results, 89.2% fks hot spot mutants were classified as anidulafungin susceptible, 60.7% as caspofungin susceptible, and 92.9% as micafungin susceptible. In conclusion, no test was perfect, but anidulafungin susceptibility testing using the WT-UL to define susceptibility reliably identified fks hot spot mutants.
Three echinocandin class drugs, anidulafungin, caspofungin, and micafungin, are licensed for the treatment of invasive candidiasis. They are among the preferred agents for invasive candidiasis, as a number of recent fungemia surveys have reported a considerable proportion of cases involving species with reduced susceptibility to fluconazole (3, 4, 24, 28, 31, 37, 44). Additionally, anidulafungin has been associated with an improved success rate, even in cases involving fluconazole-susceptible species (39). Following increased use, sporadic cases of failures associated with elevated MICs have been reported. In the majority of cases, these failures have been associated with mutations in two hot spot regions of FKS genes, which encode the target and major subunit of the 1,3-ß-d-glucan synthase complex (5, 7, 22, 25, 26, 33, 34). Consequently, close monitoring and robust susceptibility testing methods have become increasingly important.
EUCAST and CLSI have developed standard methods based on broth dilution for the susceptibility testing of yeasts (9, 41). Methodological differences include glucose concentration, inoculum size, shape of microtiter wells (flat or round), and end-point reading (visual or spectrophotometric), but the methods are more alike than different and in general generate similar results (11, 42). Recently, CLSI proposed an S value of ≤2 μg/ml as a tentative susceptibility breakpoint for caspofungin, micafungin, and anidulafungin for Candida spp., taking into account analysis of mechanisms of resistance, an epidemiological MIC population distribution, parameters associated with success in pharmacodynamic models, and results of clinical efficacy studies (9, 38). As no significant differences in clinical response were noted among the various species, results for all species were merged, and a susceptibility breakpoint of 2 μg/ml was found to encompass the vast majority of isolates, while not bisecting the population of Candida parapsilosis. The crucial issue is whether current susceptibility testing methods and breakpoints clearly and reliably identify isolates with resistance mechanisms associated with treatment failures (5, 7, 8, 13, 14, 16, 18, 22, 25, 26, 33, 40). Not only have cases involving isolates classified as susceptible using the reference methods been shown to contain resistance mutations (5, 7, 13, 14, 22, 25), but also recent studies suggest that a breakpoint of an S value of ≤2 μg/ml may be too high for anidulafungin and micafungin, considering the 1,3-ß-d-glucan synthase kinetic inhibition data of wild-type and mutant enzymes from resistant strains (17, 18). Finally, we recently reported a resistant Candida albicans isolate that failed to be identified as resistant when the reference methodologies were used, while Etest, agar dilution, and disk diffusion methods correctly identified it (5).
We therefore undertook a comparative study of the two references methods, a modified EUCAST microdilution method using IsoSensitest medium, agar dilution, and disk and Etest diffusion using RPMI-1640 as well as IsoSensitest medium to evaluate their ability to reliably discriminate between a well-characterized panel of wild-type and fks hot spot mutant Candida isolates. The semisynthetic IsoSensitest medium was chosen as an alternative medium due to this medium having previously been shown to be appropriate for amphotericin B MIC testing (10).
MATERIALS AND METHODS
Isolates.
Totals of 94 clinical isolates and two reference strains (C. parapsilosis ATCC 22019 and C. krusei ATCC 6258) were used in this study, including 10 FKS wild-type and 10 fks hot spot mutant C. albicans isolates, 9 FKS wild-type and 11 fks hot spot mutant C. glabrata isolates, 1 FKS wild-type and 1 fks hot spot mutant C. dubliniensis isolate, 13 FKS hot spot wild-type and 3 fks hot spot mutant C. krusei isolates, 19 FKS wild-type C. parapsilosis isolates, and 15 FKS hot spot wild-type and 4 fks hot spot mutant C. tropicalis isolates. Three isolates were found to harbor mutations outside the resistance hot spots and were regarded as wild type concerning echinocandin susceptibility because of their normal kinetic inhibition properties. Thus, a total of 29 isolates with characteristic echinocandin resistance mutations in the hot spot regions were included (Table 1). Species identification was based on colony color and morphology on CHROMagar (CHROMagar Co., Paris, France), microscopic morphology on cornmeal agar (SSI Diagnostica, Hilleroed, Denmark), ability to grow at 37°C and 42°C, and use of a commercial system (ATB ID32C; bioMérieux, Marcy l'Etoile, France). All isolates were coded, and tests performed blinded for susceptibility patterns. Each susceptibility test method was performed in one of the four participating laboratories.
TABLE 1.
Amino acid substitutions within the Fks proteins from the Candida isolates studieda
| Species | GenBank accession no. | Fks protein(s) (amino acid substitution[s]) |
|---|---|---|
| C. albicans | XM_716336 | Fks1p (F641S),c Fks1p (S645Y), Fks1p (S645F), Fks1p (S645P),b Fks1p (S645F and R1361R/H), Fks1p (D648Y), Fks1p (P649H) |
| C. dubliniensis | GQ342611 | Fks1p (S645P) |
| C. glabrata | XM_446406 and XM_448401 | Fks1p (F625S), Fks1p (S629P), Fks1p (D632G), Fks2p (F659V), Fks2p (F659S),c Fks2p (S663P), Fks2p (S663F), Fks2p (D666G), Fks2p (D666E), Fks2p (P667T) |
| C. krusei | EF426563 | Fks1p (R1361G), Fks1p (F655F/C), Fks1p (L658W and L701M), Fks1p (D700M),d Fks1p (L701M)d |
| C. tropicalis | EU676168 | Fks1p (F76S), Fks1p (S80P),b Fks1p (V213I and V265I)d |
Only isolates with mutations in the hot spot regions were regarded as resistant isolates.
n = 3.
n = 2.
Outside hot spots.
Compounds.
A single lot of pure substance for each of the three echinocandins was used throughout the study. Stock solutions were prepared in dimethyl sulfoxide (DMSO; Sigma) for anidulafungin (provided by Pfizer), in water for caspofungin (provided by Merck), and in water or saline for micafungin (provided by Astellas), taking into account the potencies of the powders. MICs obtained by the CLSI M27-A3 method fell within the recommended ranges for both control strains, thus confirming the activity of the powders.
Agar dilution.
Twelve-well multidish plates (1.9 cm2/well) containing a twofold-dilution series of each of the three echinocandins (concentration range of 0.015 to 4 μg/ml, 1-ml volumes) and a drug-free well were prepared using RPMI 1640 2% glucose buffered with MOPS [morpholinepropanesulfonic acid] (RPMI-2G) and IsoSensitest 2% glucose (IsoSensitest-2G), respectively (Balis Laboratorium, Boven Leeuwen, The Netherlands). A 1 to 5 × 106-CFU/ml suspension of each Candida isolate was prepared and diluted 1:6,000, and 20 μl (containing 3 to 16 CFU) were plated on each agar surface. Plates were incubated at 37°C for 48 h. Growth was compared to that in the drug-free control well; wells with tiny microcolonies were ignored.
EUCAST microdilution.
EUCAST microdilution was performed strictly according to the standard (EDef 7.1 [41]) and additionally using IsoSensitest-2G medium (Oxoid S.A., Madrid Spain). Plates were prepared in one batch, sealed in aluminum foil, and stored at −80°C for no longer than 2 months before use. Microtiter plates were read spectrophotometrically at 530 nm after 24 h as well as after 48 h, and the MIC was determined using 50 as well as 90% growth inhibition. C. krusei ATCC 6258 and C. parapsilosis ATCC 22019 were used as quality control strains through all experiments.
CLSI microdilution.
CLSI microdilution was performed in triplicate strictly according to the CLSI M27-A3 standard (9) using three separate batches of plates prepared using the single lots of antifungal compounds. Plates were stored at −86°C for a maximum of 15 days before use. Microtiter plates were read visually, and the MIC was determined using prominent inhibition (corresponding to 50%) as an end point. An additional reading was performed after 48 h. Geometric mean values were determined, and the values between the log2 scale were adjusted to the upper closest log2 value for comparison. C. krusei ATCC 6258 and C. parapsilosis ATCC 22019 were used as quality control strains through all experiments.
Etest and disk diffusion testing.
Susceptibilities of the various isolates to anidulafungin, micafungin, and caspofungin were determined by Etest (AB Biodisk, Solna, Sweden) using RPMI-2G agar (Sigma, Vienna, Austria) according to the manufacturer's recommendations and using IsoSensitest-2G (Difco, Vienna, Austria). For disk diffusion testing, 90-mm plates containing RPMI-2G agar and IsoSensitest-2G (Difco, Vienna, Austria), respectively, were inoculated by swabbing the agar with a swab soaked in a yeast suspension of 1 × 106 to 2.5 × 106 cells/ml. Disks containing 5 μg of drug were prepared by placing 20 μl of the appropriate drug concentration on sterile 6-mm-diameter paper disks and subsequently placing the disks on the inoculated plates. Stock solutions were prepared by dimethyl sulfoxide and sterile water (Sigma, Vienna, Austria) as described above. Etests and disk diffusion were read at 24 h. The Etest MICs were rounded up to the next even log2 concentration for comparison. For disk diffusion, the zone diameters were evaluated in mm.
FKS gene sequence analysis.
Candida sp. genomic DNA was extracted from yeast cells grown overnight in YPD (yeast extract, 2%; BactoPeptone, 4%; dextrose, 4%) broth medium with a Q-Biogene (Irvine, CA) FastDNA kit. PCR and sequencing primers were designed based on the C. albicans FKS1 gene (GenBank accession no. XM_716336), C. glabrata FKS1 and FKS2 (GenBank accession no. XM_446406 and XM_448401, respectively), C. tropicalis FKS1 (GenBank accession no. EU676168), C. krusei FKS1 (GenBank accession no. EF426563), C. dubliniensis FKS1 (GenBank accession no. GQ342611), and C. parapsilosis FKS1 (GenBank accession no. EU221325). The hot spot regions of the FKS genes (35) of all the strains included in this study were sequenced with a CEQ dye terminator cycle sequencing quick start kit (Beckman Coulter, Fullerton, CA) according to the manufacturer's recommendations. Sequence analysis was performed with CEQ 8000 genetic analysis system software (Beckman Coulter, Fullerton, CA) and BioEdit sequence alignment editor (Ibis Therapeutics, Carlsbad, CA). The specific mutations are shown in Table 1.
Evaluation of test performance.
For each of the drug-bug combinations, the following parameters were used to evaluate and compare the test performances: distance between end-point ranges (MIC or zone diameter) for fks hot spot mutant isolates and wild-type isolates calculated as number of twofold-dilution steps (for MIC results) and number of millimeters separating the zones (for disk diffusion) (negative values indicate the degree of overlap expressed as the number of dilution steps or millimeters involved in the overlap); overlap, which is defined as the number of end points for the fks hot spot mutant isolates that overlapped with the end-point range of the wild-type populations; wild-type-upper-limit values (WT-UL), which for MIC tests is defined as two twofold-dilution steps higher than the MIC50 (if the population was truncated with all MICs at or below the lowest concentration tested, the WT-UL was defined as two times the lowest dilution tested); very major errors (VME), which is the number of fks hot spot mutant isolates with a MIC lower or equal to the WT-UL; and major errors (ME), which is the number of wild-type isolates with a MIC above the WT-UL.
RESULTS
For each susceptibility test, 94 isolates were tested against the three candins (282 test results per test). Twenty-nine isolates had FKS mutations in the hot spot regions, leading to 87 test results for such isolates per test.
Reference methods.
Overall wild-type and fks hot spot mutant populations were separated by −1 (the negative value representing an overlap) to 6 twofold-dilution steps using EUCAST (Table 2). The MIC ranges of wild-type and fks hot spot mutant isolates overlapped for 2 of 15 drug-Candida sp. combinations, for anidulafungin and C. krusei and for micafungin and C. glabrata, involving 2/84 test results for fks hot spot mutant isolates (one isolate failed to grow) (Tables 2 and 3). CLSI overlaps between wild-type and fks hot spot mutant populations were seen for four drug-Candida sp. combinations, anidulafungin and C. tropicalis, caspofungin and C. tropicalis, micafungin and C. glabrata, and micafungin and C. tropicalis, involving 5/84 test results for fks hot spot mutant isolates (Tables 3 and 4). Overall, wild-type and fks hot spot mutant isolates were separated by −2 to 5 twofold-dilution steps. Applying the CLSI breakpoint for susceptibility of a MIC of ≤2 μg/ml, 89.2% (25/28) isolates with FKS hot spot mutations were classified as anidulafungin susceptible, 60.7% (17/28) as caspofungin susceptible, and 92.9% (26/28) as micafungin susceptible by the CLSI reference method.
TABLE 2.
MICs obtained using the EUCAST EDef 7.1 (RPMI-2G and 50% inhibition)a
| Drug and species | MIC (μg/ml) |
NG | Total | Distance | Overlap | WT-UL (S ≤ X) | VMEs | MEs | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ≤0.008 | 0.015 | 0.032 | 0.064 | 0.125 | 0.25 | 0.5 | 1 | 2 | 4 | 8 | >8 | ||||||||
| Anidulafungin | |||||||||||||||||||
| C. albicans (R) | 2 | 2 | 2 | 2 | 2 | 10 | |||||||||||||
| C. albicans (S) | 10 | 10 | 3 | 0.015 | |||||||||||||||
| C. dubliniensis (R) | 1 | 1 | |||||||||||||||||
| C. dubliniensis (S) | 1 | 1 | 6 | 0.015 | |||||||||||||||
| C. glabrata (R) | 2 | 3 | 1 | 2 | 2 | 1 | 11 | ||||||||||||
| C. glabrata (S) | 1 | 4 | 4 | 9 | 2 | 0.064 | |||||||||||||
| C. krusei (R) | 1 | 1 | 1 | 3 | 1/3 | 1/3 | |||||||||||||
| C. krusei (S) | 12 | 1 | 13 | −1 | 0.125 | ||||||||||||||
| C. parapsilosis (S) | 5 | 7 | 5 | 1 | 1 | 19 | 4 | 1/19 | |||||||||||
| C. tropicalis (R) | 1 | 1 | 2 | 4 | |||||||||||||||
| C. tropicalis (S) | 2 | 10 | 2 | 1 | 15 | 1 | 0.064 | ||||||||||||
| Caspofungin | |||||||||||||||||||
| C. albicans (R) | 6 | 4 | 10 | 6/10 | |||||||||||||||
| C. albicans (S) | 1 | 9 | 10 | 2 | 2 | ||||||||||||||
| C. dubliniensis (R) | 1 | 1 | |||||||||||||||||
| C. dubliniensis (S) | 1 | 1 | 3 | 2 | |||||||||||||||
| C. glabrata (R) | 1 | 5 | 2 | 2 | 1 | 11 | 6/10 | ||||||||||||
| C. glabrata (S) | 9 | 9 | 1 | 2 | |||||||||||||||
| C. krusei (R) | 1 | 1 | 1 | 3 | 1/3 | ||||||||||||||
| C. krusei (S) | 13 | 13 | 1 | 4 | |||||||||||||||
| C. parapsilosis (S) | 2 | 14 | 3 | 19 | 8 | ||||||||||||||
| C. tropicalis (R) | 1 | 3 | 4 | 1/4 | |||||||||||||||
| C. tropicalis (S) | 11 | 4 | 15 | 1 | 2 | ||||||||||||||
| Micafungin | |||||||||||||||||||
| C. albicans (R) | 1 | 1 | 2 | 5 | 1 | 10 | 1/10 | ||||||||||||
| C. albicans (S) | 4 | 6 | 10 | 2 | 0.064 | ||||||||||||||
| C. dubliniensis (R) | 1 | 1 | |||||||||||||||||
| C. dubliniensis (S) | 1 | 1 | 5 | 0.125 | |||||||||||||||
| C. glabrata (R) | 1 | 2 | 5 | 2 | 1 | 11 | 1/10 | 8/10 | |||||||||||
| C. glabrata (S) | 4 | 5 | 9 | −1 | 0.125 | ||||||||||||||
| C. krusei (R) | 1 | 1 | 1 | 3 | |||||||||||||||
| C. krusei (S) | 12 | 1 | 13 | 2 | 0.5 | ||||||||||||||
| C. parapsilosis (S) | 5 | 10 | 4 | 19 | 4 | ||||||||||||||
| C. tropicalis (R) | 1 | 1 | 2 | 4 | 1/4 | ||||||||||||||
| C. tropicalis (S) | 12 | 3 | 15 | 1 | 0.125 | ||||||||||||||
NG, no growth; distance, number of twofold steps between wild-type (S) and fks hot spot mutant (R) populations; overlap, number of fks R isolates overlapping with the S population/total number of isolates in the group; WT-UL, wild type cut off (for calculation, see Materials and Methods); VMEs, R isolates misclassified as susceptible using the WT-UL as the breakpoint for susceptibility; MEs, S isolates misclassified as resistant. Only isolates with mutations in the hot spot regions were regarded as resistant isolates.
TABLE 3.
Comparison of the following test performance parameters: the proportion of MIC ranges for the 29 fks hot spot mutant isolates overlapping the MIC range for wild-type isolates, the proportion of VMEs in the test results for fks hot spot mutant isolates; and the proportion of MEs in the test results for wild-type isolates when applying the WT-ULs as species-specific susceptibility breakpointsa
| Test | % of MICs for R isolates overlapping with MICs for wild-type population |
% VMEs/% MEs |
||||||
|---|---|---|---|---|---|---|---|---|
| Anidulafungin | Caspofungin | Micafungin | Total | Anidulafungin | Caspofungin | Micafungin | Total | |
| EUCAST (n = 28) | 4 | 0 | 4 | 2 | 4*/1** | 50/0 | 36/0 | 30/0 |
| EUCAST-IsoSensitest (n = 29) | 0 | 3 | 10 | 5 | 7/1 | 10/0 | 48/0 | 22/0 |
| CLSI (n = 28) | 4 | 4 | 11 | 6 | 4/3 | 7/0 | 25/0 | 12/1 |
| CLSI-48h (n = 28) | 7 | 4 | 11 | 7 | 7/0 | 7/3 | 29/0 | 14/1 |
| EUCAST-48h (n = 28) | 4 | 14 | 7 | 8 | 7/1 | 46/4 | 32/0 | 29/2 |
| EUCAST-IsoSensitest-48h (n = 29) | 17 | 52 | 10 | 26 | 0/4 | 17/4 | 41/0 | 20/3 |
| Etest-RPMI (n = 29) | 3 | 3 | 7 | 5 | 3/9 | 10/0 | 10/1 | 8/3 |
| Etest-IsoSensitest (n = 29) | 10 | 17 | 21 | 16 | 7/10 | 3/4 | 24/0 | 11/5 |
| Agardilution-RPMI (n = 28) | 4 | 0 | 4 | 2 | 7/0 | 0/1 | 36/0 | 14/0 |
| Agardilution-Isosensitest (n = 28) | 4 | 4 | 18 | 8 | 7/0 | 0/1 | 71/0 | 26/0 |
| Disk-RPMI (n = 29) | 21 | 10 | 31 | 21 | NA | NA | NA | NA |
| Disk-IsoSensitest (n = 29) | 24 | 3 | 21 | 16 | NA | NA | NA | NA |
VMEs, fks hot spot mutant (R) isolates misclassified as susceptible using the WT-UL as the breakpoint for susceptibility; MEs, wild-type isolates misclassified as resistant using the WT-UL as breakpoint for susceptibility; *, number of VMEs/number of fks mutant test results; **, number of MEs/number of wild-type test results; NA, not appropriate. Only isolates with mutations in the hot spot regions were regarded as resistant isolates.
TABLE 4.
MICs obtained using CLSI M27-A3 (RPMI-2G and 50% inhibition)a
| Drug and species | MIC (μg/ml) |
NG | Total | Distance | Overlap | WT-UL (S ≤ X) | VMEs | MEs | VMEs CLSI-BP | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ≤0.008 | 0.015 | 0.032 | 0.064 | 0.125 | 0.25 | 0.5 | 1 | 2 | 4 | 8 | >8 | |||||||||
| Anidulafungin | ||||||||||||||||||||
| C. albicans (R) | 2 | 6 | 2 | 10 | 10/10 | |||||||||||||||
| C. albicans (S) | 1 | 4 | 3 | 2 | 10 | 2 | 0.125 | |||||||||||||
| C. dubliniensis (R) | 1 | 1 | 1/1 | |||||||||||||||||
| C. dubliniensis (S) | 1 | 1 | 4 | 0.25 | ||||||||||||||||
| C. glabrata (R) | 3 | 5 | 2 | 1 | 11 | 8/10 | ||||||||||||||
| C. glabrata (S) | 5 | 3 | 1 | 9 | 2 | 0.125 | 1/9 | |||||||||||||
| C. krusei (R) | 1 | 1 | 1 | 3 | 2/3 | |||||||||||||||
| C. krusei (S) | 1 | 8 | 3 | 1 | 13 | 2 | 0.25 | |||||||||||||
| C. parapsilosis (S) | 1 | 4 | 9 | 4 | 1 | 19 | 8 | |||||||||||||
| C. tropicalis (R) | 1 | 1 | 2 | 4 | 1/4 | 1/4 | 4/4 | |||||||||||||
| C. tropicalis (S) | 2 | 6 | 6 | 1 | 15 | −2 | 0.125 | 1/15 | ||||||||||||
| Caspofungin | ||||||||||||||||||||
| C. albicans (R) | 2 | 3 | 2 | 3 | 10 | 5/10 | ||||||||||||||
| C. albicans (S) | 1 | 6 | 3 | 10 | 2 | 0.25 | ||||||||||||||
| C. dubliniensis (R) | 1 | 1 | ||||||||||||||||||
| C. dubliniensis (S) | 1 | 1 | 5 | 0.5 | ||||||||||||||||
| C. glabrata (R) | 3 | 4 | 2 | 1 | 1 | 11 | 7/10 | |||||||||||||
| C. glabrata (S) | 1 | 7 | 1 | 9 | 2 | 0.5 | ||||||||||||||
| C. krusei (R) | 1 | 1 | 1 | 3 | 1/3 | 1/3 | ||||||||||||||
| C. krusei (S) | 4 | 7 | 2 | 13 | 1 | 1 | ||||||||||||||
| C. parapsilosis (S) | 4 | 5 | 8 | 1 | 1 | 19 | 4 | |||||||||||||
| C. tropicalis (R) | 1 | 1 | 2 | 4 | 1/4 | 1/4 | 4/4 | |||||||||||||
| C. tropicalis (S) | 10 | 3 | 2 | 15 | −1 | 0.25 | ||||||||||||||
| Micafungin | ||||||||||||||||||||
| C. albicans (R) | 1 | 1 | 2 | 5 | 1 | 10 | 1/10 | 9/10 | ||||||||||||
| C. albicans (S) | 7 | 3 | 10 | 1 | 0.25 | |||||||||||||||
| C. dubliniensis (R) | 1 | 1 | 1/1 | |||||||||||||||||
| C. dubliniensis (S) | 1 | 1 | 4 | 0.5 | ||||||||||||||||
| C. glabrata (R) | 2 | 3 | 2 | 1 | 2 | 1 | 11 | 2/10 | 5/10 | 10/10 | ||||||||||
| C. glabrata (S) | 8 | 1 | 9 | −1 | 0.25 | |||||||||||||||
| C. krusei (R) | 2 | 1 | 3 | 2/3 | ||||||||||||||||
| C. krusei (S) | 2 | 10 | 1 | 13 | 2 | 1 | ||||||||||||||
| C. parapsilosis (S) | 4 | 14 | 1 | 19 | 8 | |||||||||||||||
| C. tropicalis (R) | 1 | 3 | 4 | 1/4 | 1/4 | 4/4 | ||||||||||||||
| C. tropicalis (S) | 5 | 7 | 2 | 1 | 15 | −2 | 0.5 | |||||||||||||
NG, no growth; distance, number of twofold steps between wild-type (S) and fks hot spot mutant (R) populations; overlap, number of R isolates overlapping with S isolates/total number of isolates in the group; WT-UL, wild type cut off (for calculation, see Materials and Methods); VMEs, R isolates misclassified as susceptible using the WT-UL as the breakpoint for susceptibility; MEs, S isolates misclassified as resistant. Only isolates with mutations in the hot spot regions were regarded as resistant isolates.
Modified reference methods.
Microdilution testing was also performed according to EUCAST Edef 7.1 but using IsoSensitest-2G as a test medium (Table 5). Overall wild-type and fks hot spot mutant populations were comparably separated by −1 to 5 dilution steps. The MIC ranges of wild-type and fks hot spot mutant isolates overlapped for two drug-Candida sp. combinations, caspofungin and C. krusei and micafungin and C. glabrata, thus involving 4/87 test results for fks hot spot mutant isolates (Table 3 and 5). Furthermore, EUCAST and CLSI microdilution trays were evaluated after 48 h. However, this led to poorer discrimination between wild-type and fks hot spot mutant isolates (Table 3).
TABLE 5.
MICs obtained using a modified EUCAST methodology (IsoSensitest-2 and 50% inhibition)
| Drug and species | MIC (μg/ml) |
NG | Total | Distance | Overlap | WT-UL (S ≤ X) | VMEs | MEs | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ≤0.008 | 0.015 | 0.032 | 0.064 | 0.125 | 0.25 | 0.5 | 1 | 2 | 4 | 8 | >8 | ||||||||
| Anidulafungin | |||||||||||||||||||
| C. albicans (R) | 2 | 3 | 1 | 2 | 1 | 1 | 10 | ||||||||||||
| C. albicans (S) | 8 | 2 | 10 | 2 | 0.032 | ||||||||||||||
| C. dubliniensis (R) | 1 | 1 | |||||||||||||||||
| C. dubliniensis (S) | 1 | 1 | 5 | 0.032 | |||||||||||||||
| C. glabrata (R) | 1 | 5 | 2 | 2 | 1 | 11 | 1/11 | ||||||||||||
| C. glabrata (S) | 2 | 7 | 9 | 2 | 0.064 | ||||||||||||||
| C. krusei (R) | 1 | 1 | 1 | 3 | 1/3 | ||||||||||||||
| C. krusei (S) | 1 | 9 | 3 | 13 | 1 | 0.064 | |||||||||||||
| C. parapsilosis (S) | 1 | 1 | 6 | 6 | 2 | 2 | 1 | 19 | 2 | 1/19 | |||||||||
| C. tropicalis (R) | 2 | 1 | 1 | 4 | |||||||||||||||
| C. tropicalis (S) | 4 | 9 | 2 | 15 | 2 | 0.064 | |||||||||||||
| Caspofungin | |||||||||||||||||||
| C. albicans (R) | 1 | 1 | 1 | 2 | 5 | 10 | |||||||||||||
| C. albicans (S) | 8 | 2 | 10 | 2 | 0.5 | ||||||||||||||
| C. dubliniensis (R) | 1 | 1 | |||||||||||||||||
| C. dubliniensis (S) | 1 | 1 | 4 | 2 | |||||||||||||||
| C. glabrata (R) | 1 | 5 | 3 | 1 | 1 | 11 | 1/11 | ||||||||||||
| C. glabrata (S) | 9 | 9 | 2 | 1 | |||||||||||||||
| C. krusei (R) | 1 | 1 | 1 | 3 | 1/3 | 1/3 | |||||||||||||
| C. krusei (S) | 3 | 9 | 1 | 13 | −1 | 2 | |||||||||||||
| C. parapsilosis (S) | 1 | 1 | 6 | 9 | 1 | 1 | 19 | 8 | |||||||||||
| C. tropicalis (R) | 1 | 2 | 1 | 4 | 1/4 | ||||||||||||||
| C. tropicalis (S) | 6 | 8 | 1 | 15 | 1 | 1 | |||||||||||||
| Micafungin | |||||||||||||||||||
| C. albicans (R) | 2 | 2 | 3 | 1 | 2 | 10 | 2/10 | ||||||||||||
| C. albicans (S) | 3 | 7 | 10 | 1 | 0.25 | ||||||||||||||
| C. dubliniensis (R) | 1 | 1 | |||||||||||||||||
| C. dubliniensis (S) | 1 | 1 | 3 | 0.25 | |||||||||||||||
| C. glabrata (R) | 3 | 1 | 5 | 1 | 1 | 11 | 3/11 | 9/11 | |||||||||||
| C. glabrata (S) | 9 | 9 | −1 | 0.125 | |||||||||||||||
| C. krusei (R) | 1 | 1 | 1 | 3 | 2/3 | ||||||||||||||
| C. krusei (S) | 1 | 12 | 13 | 1 | 0.5 | ||||||||||||||
| C. parapsilosis (S) | 1 | 2 | 14 | 1 | 1 | 19 | 2 | ||||||||||||
| C. tropicalis (R) | 1 | 3 | 4 | 1/4 | |||||||||||||||
| C. tropicalis (S) | 7 | 8 | 15 | 1 | 0.25 | ||||||||||||||
NG, no growth; distance, number of twofold steps between wild-type (S) and fks hot spot mutant (R) populations; overlap, number of R isolates overlapping with the S population/total number of isolates in the group; WT-UL: wild type cutoff (for calculation, see Materials and Methods); VMEs, R isolates misclassified as susceptible using the WT-UL as the breakpoint for susceptibility; MEs, S isolates misclassified as resistant. Only isolates with mutations in the hot spot regions were regarded as resistant isolates.
Agar diffusion methods.
Susceptibility testing was performed using Etest and 5-μg disks with RPMI-2G medium, as well as IsoSensitest-2G medium as a test agar. By Etest using RPMI-2G (as recommended by the manufacturer), the distances between wild-type and fks hot spot mutant isolates were −4 to 8 dilution steps, and overlapping populations were seen for three drug-Candida sp. combinations, anidulafungin and C. krusei, caspofungin and C. krusei, and micafungin and C. glabrata, involving 4/87 test results for fks hot spot mutant isolates (Tables 3 and 6). When the CLSI breakpoint was applied, 82.8% (24/29) of the isolates with FKS mutations were classified as anidulafungin susceptible, 44.8% (13/29) as caspofungin susceptible, and 96.6% (28/29) as micafungin susceptible. Replacing the medium with IsoSensitest-2G did not improve the performance; thus, overlapping populations were seen for seven drug-Candida sp. combinations involving 14/87 test results for fks hot spot mutant isolates (Tables 3 and 7). By disk test using RPMI-2G agar, overlaps between wild-type and fks hot spot mutant isolates were seen for 9/15 drug-Candida sp. combinations, involving 18/87 test results for fks hot spot mutant isolates (Tables 3 and 8). Using disks and IsoSensitest-2G agar, overlaps between wild-type and fks hot spot mutant isolates were seen for 9/15 drug-Candida sp. combinations, involving 14/87 test results for fks hot spot mutant isolates and 1/195 results for wild-type isolates (Tables 3 and 9).
TABLE 6.
MICs obtained using Etest and RPMI-2G agar
| Drug and species | MIC (μg/ml) |
Total | Distance | Overlap | WT-UL (S ≤ X) | VMEs | MEs | VMEs CLSI-BP | |||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ≤0.002 | 0.004 | 0.008 | 0.015 | 0.032 | 0.064 | 0.125 | 0.25 | 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | >32 | ||||||||
| Anidulafungin | |||||||||||||||||||||||
| C. albicans (R) | 1 | 1 | 3 | 3 | 1 | 1 | 10 | 9/10 | |||||||||||||||
| C. albicans (S) | 6 | 3 | 1 | 10 | 1 | 0.008 | |||||||||||||||||
| C. dubliniensis (R) | 1 | 1 | 1/1 | ||||||||||||||||||||
| C. dubliniensis (S) | 1 | 1 | 8 | 0.015 | |||||||||||||||||||
| C. glabrata (R) | 2 | 4 | 2 | 1 | 1 | 1 | 11 | 9/11 | |||||||||||||||
| C. glabrata (S) | 4 | 3 | 1 | 1 | 9 | 1 | 0.064 | 1/9 | |||||||||||||||
| C. krusei (R) | 1 | 1 | 1 | 3 | 1/3 | 1/3 | 2/3 | ||||||||||||||||
| C. krusei (S) | 2 | 1 | 3 | 2 | 2 | 1 | 2 | 13 | −4 | 0.25 | 2/13 | ||||||||||||
| C. parapsilosis (S) | 2 | 1 | 1 | 1 | 6 | 2 | 5 | 1 | 19 | 2 | 1/19 | ||||||||||||
| C. tropicalis (R) | 2 | 1 | 1 | 4 | 3/4 | ||||||||||||||||||
| C. tropicalis (S) | 5 | 3 | 1 | 4 | 2 | 15 | 2 | 0.015 | 2/15 | ||||||||||||||
| Caspofungin | |||||||||||||||||||||||
| C. albicans (R) | 2 | 2 | 2 | 2 | 1 | 1 | 10 | 2/10 | 4/10 | ||||||||||||||
| C. albicans (S) | 1 | 3 | 5 | 1 | 10 | 1 | 0.25 | ||||||||||||||||
| C. dubliniensis (R) | 1 | 1 | |||||||||||||||||||||
| C. dubliniensis (S) | 1 | 1 | 6 | 2 | |||||||||||||||||||
| C. glabrata (R) | 3 | 3 | 3 | 2 | 11 | 6/11 | |||||||||||||||||
| C. glabrata (S) | 4 | 4 | 1 | 9 | 2 | 0.5 | |||||||||||||||||
| C. krusei (R) | 1 | 1 | 1 | 3 | 1/3 | 1/3 | 1/3 | ||||||||||||||||
| C. krusei (S) | 1 | 7 | 5 | 13 | −1 | 1 | |||||||||||||||||
| C. parapsilosis (S) | 1 | 2 | 3 | 5 | 5 | 3 | 19 | 2 | |||||||||||||||
| C. tropicalis (R) | 1 | 1 | 2 | 4 | 2/4 | ||||||||||||||||||
| C. tropicalis (S) | 4 | 3 | 3 | 5 | 15 | 1 | 0.25 | ||||||||||||||||
| Micafungin | |||||||||||||||||||||||
| C. albicans (R) | 1 | 1 | 4 | 4 | 10 | 10/10 | |||||||||||||||||
| C. albicans (S) | 1 | 7 | 2 | 10 | 2 | 0.032 | |||||||||||||||||
| C. dubliniensis (R) | 1 | 1 | 1/1 | ||||||||||||||||||||
| C. dubliniensis (S) | 1 | 1 | 4 | 0.125 | |||||||||||||||||||
| C. glabrata (R) | 1 | 1 | 1 | 3 | 1 | 3 | 1 | 11 | 2/11 | 3/11 | 11/11 | ||||||||||||
| C. glabrata (S) | 3 | 5 | 1 | 9 | −2 | 0.064 | |||||||||||||||||
| C. krusei (R) | 1 | 1 | 1 | 3 | 2/3 | ||||||||||||||||||
| C. krusei (S) | 1 | 10 | 1 | 1 | 13 | 1 | 0.5 | ||||||||||||||||
| C. parapsilosis (S) | 2 | 2 | 1 | 8 | 3 | 2 | 1 | 19 | 2 | 1/19 | |||||||||||||
| C. tropicalis (R) | 1 | 3 | 4 | 4/4 | |||||||||||||||||||
| C. tropicalis (S) | 9 | 5 | 1 | 15 | 2 | 0.064 | |||||||||||||||||
NG, no growth; distance, number of twofold steps between wild-type (S) and fks hot spot mutant (R) populations; overlap, number of R isolates overlapping with the S population/total number of isolates in the group; WT-UL, wild type cutoff (for calculation, see Materials and Methods); VMEs, R isolates misclassified as susceptible using the WT-UL as the breakpoint for susceptibility; MEs, S isolates misclassified as resistant. Only isolates with mutations in the hot spot regions were regarded as resistant isolates.
TABLE 7.
MICs obtained using Etest and IsoSensitest-2 agar
| Drug and species | MIC (μg/ml) |
Total | Distance | Overlap | WT-UL (S ≤ X) | VMEs | MEs | |||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ≤0.002 | 0.004 | 0.008 | 0.015 | 0.032 | 0.064 | 0.125 | 0.25 | 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | >32 | |||||||
| Anidulafungin | ||||||||||||||||||||||
| C. albicans (R) | 1 | 1 | 1 | 2 | 2 | 3 | 10 | |||||||||||||||
| C. albicans (S) | 4 | 4 | 2 | 10 | 3 | 0.015 | ||||||||||||||||
| C. dubliniensis (R) | 1 | 1 | ||||||||||||||||||||
| C. dubliniensis (S) | 1 | 1 | 5 | 0.015 | ||||||||||||||||||
| C. glabrata (R) | 1 | 1 | 1 | 3 | 2 | 1 | 1 | 1 | 11 | 1/11 | 1/3 | |||||||||||
| C. glabrata (S) | 1 | 1 | 2 | 5 | 9 | 3 | 0.064 | |||||||||||||||
| C. krusei (R) | 1 | 1 | 1 | 3 | 2/3 | 1/3 | ||||||||||||||||
| C. krusei (S) | 1 | 1 | 2 | 3 | 2 | 4 | 13 | −4 | 0.25 | 4/13 | ||||||||||||
| C. parapsilosis (S) | 1 | 2 | 1 | 1 | 6 | 2 | 3 | 2 | 1 | 19 | 2 | 3/19 | ||||||||||
| C. tropicalis (R) | 1 | 1 | 2 | 4 | ||||||||||||||||||
| C. tropicalis (S) | 2 | 1 | 6 | 6 | 15 | 3 | 0.032 | |||||||||||||||
| Caspofungin | ||||||||||||||||||||||
| C. albicans (R) | 2 | 2 | 2 | 1 | 2 | 1 | 10 | |||||||||||||||
| C. albicans (S) | 1 | 1 | 3 | 3 | 2 | 10 | 4 | 0.032 | ||||||||||||||
| C. dubliniensis (R) | 1 | 1 | ||||||||||||||||||||
| C. dubliniensis (S) | 1 | 1 | 7 | 0.125 | ||||||||||||||||||
| C. glabrata (R) | 1 | 4 | 1 | 2 | 1 | 2 | 11 | 1/11 | ||||||||||||||
| C. glabrata (S) | 1 | 1 | 5 | 1 | 1 | 9 | −1 | 0.25 | 1/9 | |||||||||||||
| C. krusei (R) | 1 | 1 | 1 | 3 | 1/3 | 1/3 | ||||||||||||||||
| C. krusei (S) | 3 | 8 | 1 | 1 | 13 | −2 | 1 | |||||||||||||||
| C. parapsilosis (S) | 1 | 1 | 2 | 1 | 4 | 3 | 2 | 5 | 19 | 2 | ||||||||||||
| C. tropicalis (R) | 1 | 2 | 1 | 4 | 3/4 | |||||||||||||||||
| C. tropicalis (S) | 1 | 9 | 2 | 1 | 1 | 1 | 15 | −3 | 0.064 | 2/15 | ||||||||||||
| Micafungin | ||||||||||||||||||||||
| C. albicans (R) | 1 | 1 | 2 | 5 | 1 | 10 | 1/10 | |||||||||||||||
| C. albicans (S) | 3 | 5 | 2 | 10 | 1 | 0.064 | ||||||||||||||||
| C. dubliniensis (R) | 1 | 1 | ||||||||||||||||||||
| C. dubliniensis (S) | 1 | 1 | 6 | 0.064 | ||||||||||||||||||
| C. glabrata (R) | 3 | 2 | 2 | 2 | 1 | 1 | 11 | 5/11 | 5/11 | |||||||||||||
| C. glabrata (S) | 5 | 2 | 2 | 9 | −2 | 0.032 | ||||||||||||||||
| C. krusei (R) | 1 | 1 | 1 | 3 | 1/3 | 1/3 | ||||||||||||||||
| C. krusei (S) | 5 | 4 | 3 | 1 | 13 | −1 | 0.5 | |||||||||||||||
| C. parapsilosis (S) | 1 | 1 | 3 | 2 | 8 | 4 | 19 | 2 | ||||||||||||||
| C. tropicalis (R) | 3 | 1 | 4 | |||||||||||||||||||
| C. tropicalis (S) | 11 | 3 | 1 | 15 | 2 | 0.064 | ||||||||||||||||
NG, no growth; distance, number of twofold steps between wild-type (S) and fks hot spot mutant (R) populations; overlap, number of R overlapping with the S population/total number of isolates in the group; WT-UL, S cutoff (for calculation, see Materials and Methods); VMEs, R isolates misclassified as susceptible using the WT-UL as the breakpoint for susceptibility; MEs, S isolates misclassified as resistant. Only isolates with mutations in the hot spot regions were regarded as resistant isolates.
TABLE 8.
Zone diameters obtained using 5-μg disks and RPMI-2G agara
| Drug and species | Zone diam (mm) |
Total | Distance | Overlap | ||||||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 0 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 | 31 | 32 | 33 | 34 | 35 | 36-39 | 40 | >41 | ||||
| Anidulafungin | ||||||||||||||||||||||||||||||||||||
| C. albicans (R) | 2 | 3 | 1 | 2 | 1 | 1 | 10 | 1/10 | ||||||||||||||||||||||||||||
| C. albicans (S) | 2 | 3 | 1 | 1 | 1 | 1 | 1 | 10 | −1 | |||||||||||||||||||||||||||
| C. dubliniensis (R) | 1 | 1 | 1/1 | |||||||||||||||||||||||||||||||||
| C. dubliniensis (S) | 1 | 1 | −2 | |||||||||||||||||||||||||||||||||
| C. glabrata (R) | 3 | 3 | 2 | 1 | 1 | 1 | 11 | 1/11 | ||||||||||||||||||||||||||||
| C. glabrata (S) | 1 | 2 | 3 | 3 | 9 | −1 | ||||||||||||||||||||||||||||||
| C. krusei (R) | 1 | 1 | 1 | 3 | ||||||||||||||||||||||||||||||||
| C. krusei (S) | 2 | 1 | 3 | 1 | 2 | 4 | 13 | 2 | ||||||||||||||||||||||||||||
| C. parapsilosis (S) | 1 | 1 | 2 | 2 | 4 | 4 | 1 | 1 | 1 | 2 | 19 | |||||||||||||||||||||||||
| C. tropicalis (R) | 1 | 1 | 1 | 1 | 4 | 3/4 | ||||||||||||||||||||||||||||||
| C. tropicalis (S) | 1 | 1 | 2 | 3 | 6 | 2 | 15 | −6 | ||||||||||||||||||||||||||||
| Caspofungin | ||||||||||||||||||||||||||||||||||||
| C. albicans (R) | 3 | 1 | 2 | 2 | 1 | 1 | 10 | |||||||||||||||||||||||||||||
| C. albicans (S) | 1 | 1 | 3 | 1 | 1 | 1 | 1 | 1 | 10 | 2 | ||||||||||||||||||||||||||
| C. dubliniensis (R) | 1 | 1 | ||||||||||||||||||||||||||||||||||
| C. dubliniensis (S) | 1 | 1 | 15 | |||||||||||||||||||||||||||||||||
| C. glabrata (R) | 2 | 1 | 5 | 1 | 1 | 1 | 11 | |||||||||||||||||||||||||||||
| C. glabrata (S) | 7 | 1 | 1 | 9 | 2 | |||||||||||||||||||||||||||||||
| C. krusei (R) | 2 | 1 | 3 | 1/3 | ||||||||||||||||||||||||||||||||
| C. krusei (S) | 1 | 1 | 2 | 1 | 3 | 1 | 3 | 1 | 13 | −3 | ||||||||||||||||||||||||||
| C. parapsilosis (S) | 2 | 3 | 4 | 1 | 3 | 4 | 1 | 1 | 19 | |||||||||||||||||||||||||||
| C. tropicalis (R) | 1 | 1 | 1 | 1 | 4 | 2/4 | ||||||||||||||||||||||||||||||
| C. tropicalis (S) | 1 | 4 | 1 | 5 | 3 | 1 | 15 | −3 | ||||||||||||||||||||||||||||
| Micafungin | ||||||||||||||||||||||||||||||||||||
| C. albicans (R) | 1 | 1 | 1 | 2 | 2 | 2 | 1 | 10 | ||||||||||||||||||||||||||||
| C. albicans (S) | 2 | 4 | 1 | 1 | 1 | 1 | 10 | 1 | ||||||||||||||||||||||||||||
| C. dubliniensis (R) | 1 | 1 | ||||||||||||||||||||||||||||||||||
| C. dubliniensis (S) | 1 | 1 | 2 | |||||||||||||||||||||||||||||||||
| C. glabrata (R) | 1 | 2 | 1 | 1 | 1 | 2 | 1 | 2 | 11 | 5/11 | ||||||||||||||||||||||||||
| C. glabrata (S) | 1 | 1 | 1 | 4 | 1 | 1 | 9 | −6 | ||||||||||||||||||||||||||||
| C. krusei (R) | 1 | 2 | 3 | 2/3 | ||||||||||||||||||||||||||||||||
| C. krusei (S) | 2 | 1 | 3 | 1 | 1 | 3 | 2 | 13 | −1 | |||||||||||||||||||||||||||
| C. parapsilosis (S) | 1 | 2 | 1 | 2 | 4 | 2 | 1 | 1 | 2 | 2 | 1 | 19 | ||||||||||||||||||||||||
| C. tropicalis (R) | 1 | 1 | 1 | 1 | 4 | 2/4 | ||||||||||||||||||||||||||||||
| C. tropicalis (S) | 1 | 2 | 5 | 4 | 1 | 1 | 1 | 15 | −4 | |||||||||||||||||||||||||||
S, wild type; R, fks hot spot mutant; distance, number of twofold steps between S and R population; overlap, number of isolates overlapping/total number of isolates in the group. Only isolates with mutations in the hot spot regions were regarded as resistant isolates.
TABLE 9.
Zone diameters obtained using 5 μg disks and IsoSensitest-2% glucose agara
| Drug and species | Zone diam (mm) |
Total | Distance | Overlap | |||||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 0 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 | 31 | 32 | 33 | 34 | 35-39 | 40 | 41 | ||||
| Anidulafungin | |||||||||||||||||||||||||||||||||||
| C. albicans (R) | 1 | 2 | 2 | 3 | 1 | 1 | 10 | 1/10 | |||||||||||||||||||||||||||
| C. albicans (S) | 1 | 1 | 4 | 1 | 1 | 1 | 1 | 10 | −1 | ||||||||||||||||||||||||||
| C. dubliniensis (R) | 1 | 1 | |||||||||||||||||||||||||||||||||
| C. dubliniensis (S) | 1 | 1 | 6 | ||||||||||||||||||||||||||||||||
| C. glabrata (R) | 1 | 1 | 1 | 3 | 2 | 1 | 1 | 1 | 11 | 3/11 | |||||||||||||||||||||||||
| C. glabrata (S) | 2 | 2 | 2 | 2 | 1 | 9 | −6 | ||||||||||||||||||||||||||||
| C. krusei (R) | 1 | 2 | 3 | 2/3 | |||||||||||||||||||||||||||||||
| C. krusei (S) | 1 | 2 | 2 | 5 | 2 | 1 | 13 | −1 | |||||||||||||||||||||||||||
| C. parapsilosis (S) | 1 | 2 | 3 | 1 | 2 | 5 | 1 | 3 | 1 | 19 | |||||||||||||||||||||||||
| C. tropicalis (R) | 1 | 2 | 1 | 4 | 1/4 | ||||||||||||||||||||||||||||||
| C. tropicalis (S) | 1 | 3 | 7 | 1 | 1 | 1 | 1 | 15 | −3 | ||||||||||||||||||||||||||
| Caspofungin | |||||||||||||||||||||||||||||||||||
| C. albicans (R) | 2 | 4 | 1 | 1 | 2 | 10 | |||||||||||||||||||||||||||||
| C. albicans (S) | 1 | 1 | 3 | 2 | 1 | 1 | 1 | 10 | 2 | ||||||||||||||||||||||||||
| C. dubliniensis (R) | 1 | 1 | |||||||||||||||||||||||||||||||||
| C. dubliniensis (S) | 1 | 1 | 11 | ||||||||||||||||||||||||||||||||
| C. glabrata (R) | 1 | 1 | 5 | 1 | 1 | 1 | 1 | 11 | |||||||||||||||||||||||||||
| C. glabrata (S) | 1 | 4 | 4 | 9 | 4 | ||||||||||||||||||||||||||||||
| C. krusei (R) | 2 | 1 | 3 | 1/3 | |||||||||||||||||||||||||||||||
| C. krusei (S) | 1 | 4 | 3 | 4 | 1 | 13 | −1 | ||||||||||||||||||||||||||||
| C. parapsilosis (S) | 1 | 5 | 3 | 1 | 4 | 4 | 1 | 19 | |||||||||||||||||||||||||||
| C. tropicalis (R) | 1 | 1 | 2 | 4 | |||||||||||||||||||||||||||||||
| C. tropicalis (S) | 1* | 1 | 5 | 7 | 1 | 15 | −3 | 1/15 | |||||||||||||||||||||||||||
| Micafungin | |||||||||||||||||||||||||||||||||||
| C. albicans (R) | 1 | 1 | 2 | 2 | 1 | 1 | 1 | 1 | 10 | 1/10 | |||||||||||||||||||||||||
| C. albicans (S) | 1 | 3 | 2 | 1 | 1 | 1 | 1 | 10 | −1 | ||||||||||||||||||||||||||
| C. dubliniensis (R) | 1 | 1 | |||||||||||||||||||||||||||||||||
| C. dubliniensis (S) | 1 | 1 | 11 | ||||||||||||||||||||||||||||||||
| C. glabrata (R) | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 11 | 4/11 | ||||||||||||||||||||||
| C. glabrata (S) | 2 | 1 | 4 | 1 | 1 | 9 | 6 | ||||||||||||||||||||||||||||
| C. krusei (R) | 1 | 1 | 1 | 3 | |||||||||||||||||||||||||||||||
| C. krusei (S) | 3 | 1 | 1 | 3 | 1 | 1 | 1 | 1 | 1 | 13 | 1 | ||||||||||||||||||||||||
| C. parapsilosis (S) | 1 | 1 | 1 | 3 | 2 | 2 | 2 | 1 | 2 | 1 | 3 | 19 | |||||||||||||||||||||||
| C. tropicalis (R) | 1 | 1 | 1 | 1 | 4 | 1/4 | |||||||||||||||||||||||||||||
| C. tropicalis (S) | 1 | 1 | 2 | 6 | 1 | 1 | 2 | 1 | 15 | −4 | |||||||||||||||||||||||||
S, wild type; R, fks hot spot mutant; distance, number of twofold steps between S and R population; overlap, number of isolates overlapping/total number of isolates in the group; *, regarded as an outlier. Only isolates with mutations in the hot spot regions were regarded as resistant isolates.
Agar dilution.
Agar dilution performed using RPMI-2G resulted in a distance between the wild-type and fks hot spot mutant populations of −1 to ≥5 (Table 10), and overlapping populations were seen for three drug-Candida sp. combinations, anidulafungin and C. tropicalis, caspofungin and C. tropicalis, and micafungin and C. tropicalis, involving 2/87 test results for fks hot spot mutant isolates and 1/195 test results for wild-type isolates (Tables 3 and 10). Performing agar dilution with IsoSensitest-2G did not improve discrimination between wild-type and fks hot spot mutant isolates. Thus, the distances between wild-type and fks hot spot mutant isolates was −1 to >3 dilution steps, and overlaps between wild-type and fks hot spot mutant isolates were seen for five drug-Candida spp. combinations, involving 7/87 test results for fks hot spot mutant isolates (Tables 3 and 11).
TABLE 10.
MICs obtained by agar dilution using RPMI-2G agara
| Drug and species | MIC (μg/ml) |
NG | Total | Distance | Overlap | WT-UL (S ≤ X) | VMEs | MEs | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ≤0.015 | 0.032 | 0.064 | 0.125 | 0.25 | 0.5 | 1 | 2 | 4 | >4 | ||||||||
| Anidulafungin | |||||||||||||||||
| C. albicans (R) | 2 | 3 | 3 | 2 | 10 | ||||||||||||
| C. albicans (S) | 9 | 1 | 10 | 2 | 0.064 | ||||||||||||
| C. dubliniensis (R) | 1 | 1 | |||||||||||||||
| C. dubliniensis (S) | 1 | 1 | >5 | 0.064 | |||||||||||||
| C. glabrata (R) | 2 | 5 | 1 | 2 | 1 | 11 | |||||||||||
| C. glabrata (S) | 9 | 9 | 3 | 0.125 | |||||||||||||
| C. krusei (R) | 1 | 1 | 1 | 3 | 1/3 | ||||||||||||
| C. krusei (S) | 3 | 8 | 2 | 13 | 1 | 0.25 | |||||||||||
| C. parapsilosis (S) | 8 | 4 | 2 | 4 | 1 | 19 | 4 | ||||||||||
| C. tropicalis (R) | 1 | 3 | 4 | 1/4 | 1/4 | ||||||||||||
| C. tropicalis (S) | 3 | 8 | 2 | 2 | 15 | −1 | 0.125 | ||||||||||
| Caspofungin | |||||||||||||||||
| C. albicans (R) | 1 | 9 | 10 | ||||||||||||||
| C. albicans (S) | 1 | 2 | 7 | 10 | 3 | 2 | |||||||||||
| C. dubliniensis (R) | 1 | 1 | |||||||||||||||
| C. dubliniensis (S) | 1 | 1 | >4 | 2 | |||||||||||||
| C. glabrata (R) | 10 | 1 | 11 | ||||||||||||||
| C. glabrata (S) | 9 | 9 | 3 | 4 | |||||||||||||
| C. krusei (R) | 3 | 3 | |||||||||||||||
| C. krusei (S) | 6 | 7 | 13 | 1 | >4 | ||||||||||||
| C. parapsilosis (S) | 13 | 1 | 4 | 1 | 19 | >4 | |||||||||||
| C. tropicalis (R) | 4 | 4 | |||||||||||||||
| C. tropicalis (S) | 7 | 7 | 1 | 15 | −1 | 1/15 | 4 | 1/15 | |||||||||
| Micafungin | |||||||||||||||||
| C. albicans (R) | 2 | 5 | 2 | 1 | 10 | ||||||||||||
| C. albicans (S) | 1 | 7 | 2 | 10 | 2 | 0.25 | |||||||||||
| C. dubliniensis (R) | 1 | 1 | |||||||||||||||
| C. dubliniensis (S) | 1 | 1 | 4 | 0.25 | |||||||||||||
| C. glabrata (R) | 1 | 4 | 3 | 1 | 1 | 1 | 11 | 5/11 | |||||||||
| C. glabrata (S) | 9 | 9 | 1 | 0.25 | |||||||||||||
| C. krusei (R) | 1 | 1 | 1 | 3 | 1/3 | 2/3 | |||||||||||
| C. krusei (S) | 13 | 13 | −1 | 2 | |||||||||||||
| C. parapsilosis (S) | 7 | 10 | 1 | 1 | 19 | 4 | |||||||||||
| C. tropicalis (R) | 3 | 1 | 4 | 3/4 | |||||||||||||
| C. tropicalis (S) | 1 | 10 | 4 | 15 | 1 | 0.5 | |||||||||||
NG, no growth; distance, number of twofold steps between wild-type (S) and fks hot spot mutant (R) populations; overlap, number of fks R isolates overlapping with the S population/total number of isolates in the group; WT-UL, wild-type cutoff (for calculation, see Materials and Methods); VMEs, R isolates misclassified as susceptible using the WT-UL as the breakpoint for susceptibility; MEs, S isolates misclassified as resistant. Only isolates with mutations in the hot spot regions were regarded as resistant isolates.
TABLE 11.
MICs obtained by agar dilution using IsoSensitest-2 agar
| Drug and species | MIC (μg/ml) |
NG | Total | Distance | Overlap | WT-UL (S ≤ X) | VMEs | MEs | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ≤0.015 | 0.032 | 0.064 | 0.125 | 0.25 | 0.5 | 1 | 2 | 4 | >4 | ||||||||
| Anidulafungin | |||||||||||||||||
| C. albicans (R) | 1 | 1 | 2 | 3 | 2 | 1 | 10 | ||||||||||
| C. albicans (S) | 5 | 5 | 10 | 2 | 0.125 | ||||||||||||
| C. dubliniensis (R) | 1 | 1 | |||||||||||||||
| C. dubliniensis (S) | 1 | 1 | 4 | 0.25 | |||||||||||||
| C. glabrata (R) | 3 | 4 | 1 | 1 | 1 | 1 | 11 | ||||||||||
| C. glabrata (S) | 1 | 7 | 1 | 9 | 2 | 0.25 | |||||||||||
| C. krusei (R) | 1 | 1 | 1 | 3 | 1/3 | ||||||||||||
| C. krusei (S) | 12 | 1 | 13 | 1 | 0.25 | ||||||||||||
| C. parapsilosis (S) | 3 | 8 | 6 | 2 | 19 | 4 | |||||||||||
| C. tropicalis (R) | 1 | 1 | 2 | 4 | 1/4 | 1/4 | |||||||||||
| C. tropicalis (S) | 2 | 12 | 1 | 15 | −1 | 0.25 | |||||||||||
| Caspofungin | |||||||||||||||||
| C. albicans (R) | 1 | 9 | 10 | ||||||||||||||
| C. albicans (S) | 2 | 3 | 5 | 10 | 3 | 1 | |||||||||||
| C. dubliniensis (R) | 1 | 1 | |||||||||||||||
| C. dubliniensis (S) | 1 | 1 | >3 | 4 | |||||||||||||
| C. glabrata (R) | 3 | 7 | 1 | 11 | |||||||||||||
| C. glabrata (S) | 3 | 6 | 9 | 3 | 2 | ||||||||||||
| C. krusei (R) | 1 | 2 | 3 | ||||||||||||||
| C. krusei (S) | 8 | 5 | 13 | 2 | 2 | ||||||||||||
| C. parapsilosis (S) | 7 | 8 | 2 | 2 | 19 | >4 | |||||||||||
| C. tropicalis (R) | 1 | 3 | 4 | 1/4 | |||||||||||||
| C. tropicalis (S) | 1 | 12 | 1 | 1 | 15 | −1 | 1 | 1/15 | |||||||||
| Micafungin | |||||||||||||||||
| C. albicans (R) | 2 | 3 | 3 | 1 | 1 | 10 | 2/10 | 5/10 | |||||||||
| C. albicans (S) | 1 | 6 | 3 | 10 | −1 | 1 | |||||||||||
| C. dubliniensis (R) | 1 | 1 | 1/1 | ||||||||||||||
| C. dubliniensis (S) | 1 | 1 | 2 | 1 | |||||||||||||
| C. glabrata (R) | 1 | 2 | 5 | 1 | 1 | 1 | 11 | 1/10 | 8/10 | ||||||||
| C. glabrata (S) | 2 | 7 | 9 | −1 | 0.5 | ||||||||||||
| C. krusei (R) | 2 | 1 | 3 | 2/3 | 2/3 | ||||||||||||
| C. krusei (S) | 13 | 13 | −1 | 2 | |||||||||||||
| C. parapsilosis (S) | 1 | 9 | 8 | 1 | 19 | 2 | |||||||||||
| C. tropicalis (R) | 2 | 2 | 4 | 4/4 | |||||||||||||
| C. tropicalis (S) | 6 | 9 | 15 | 1 | 1 | ||||||||||||
NG, no growth; distance, number of twofold steps between wild-type (S) and fks hot spot mutant (R) populations; overlap, number of fks R isolates overlapping with the S population/total number of isolates in the group; WT-UL: wild-type cutoff (for calculation, see Materials and Methods); VMEs, R isolates misclassified as susceptible using the WT-UL as the breakpoint for susceptibility; MEs, S isolates misclassified as resistant. Only isolates with mutations in the hot spot regions were regarded as resistant isolates.
WT-UL.
Defining susceptibility as a MIC ≤ the WT-UL value, the proportions of fsk hot spot mutants being misclassified as susceptible (VMEs) and MEs (susceptible isolates being classified as resistant) were calculated and presented together with the WT-ULs for each test, compound, and species in Tables 2, 4 to 7, 10, and 11. An overview of VMEs and MEs per test and Candida species is presented in Table 3. The lowest numbers of VMEs and MEs across the three echinocandins were seen for susceptibility testing using CLSI M27A-3 (12% and 0%, respectively), agar dilution with RPMI-2G medium (14% and 0%), and Etest with RPMI-2G medium (8% and 3%). The fewest VMEs and MEs were observed overall for anidulafungin with EUCAST (4% and 1%), CLSI (4% and 3%), and Etest with RPMI-2G (3% and 9%). For micafungin, VME rates of 10 to 71% were observed, and notably both reference tests resulted in a quarter to a third of the fks mutants being misclassified as susceptible (Table 3). For caspofungin, agar dilution with either medium was notably superior, leading to no VMEs and a 1% ME rate, while CLSI, EUCAST with IsoSensitest-2G medium, and Etest were less optimal, leading to VME rates of 7%, 10%, and 10%, respectively, and no MEs.
C. parapsilosis.
The wild-type isolates of C. parapsilosis separated clearly from the other species for anidulafungin and micafungin using microdilution tests and agar dilution, with an epidemiological cut-off value of 2 to 8 μg/ml (Tables 2, 4, 5, 10, and 11). By Etest and disk diffusion, the ranges of MICs/zone diameters were very wide, suggesting low reproducibility and variability of the results (Tables 6 to 9).
DISCUSSION
This study compared the major susceptibility testing methods by evaluating a significant number of fks hot spot mutant and wild-type isolates with nine different tests and 12 different end points on the same collection of blinded isolates. Direct comparison of MICs obtained by the different methods is problematic, as the tests were performed in different laboratories. However, for each test it is possible to evaluate the potential for discriminating fks hot spot mutants from wild-type isolates and then to compare the performances of the individual tests with respect to this crucial parameter, expressed as distance between the populations and number of overlaps, VMEs, and MEs.
When the tests were ranked according to the number of fks hot spot mutant isolates overlapping the susceptible population, agar dilutions using RPMI-2G and EUCAST EDef 7.1 were found to be best at discriminating wild-type isolates from isolates with mutations in the FKS hot spots. EUCAST with IsoSensitest-2G medium, CLSI M27-A3, and Etest using RPMI-2G performed slightly less well, and the remaining tests suffered from a high number of overlaps between isolates with resistance mutations and wild-type isolates. For the agar dilution tests, each wild-type distribution typically spanned 3 log2 dilution steps, in agreement with what we have previously published using one batch of trays in a single lab (6). When results from a large number of isolates, several technicians and batches of media, many laboratories, and other necessarily allowed variations in testing conditions are taken into account, the base of the perceived wild-type distribution broadens to a maximum of five dilution steps, with the MIC50 in the middle and the epidemiological cut-off value (ECOFF) two twofold-dilution steps higher (6). We similarly established species- and drug-specific WT-ULs, applying this principle to the limited number of wild-type isolates included in this study, as a comparison of the numbers of fks hot spot mutants with MICs lower than or equal to the WT-UL value is likely a better predictor of the test's ability to pick up mutant isolates when performed in different laboratories, using different batches of trays, etc.
The lowest numbers of VMEs and MEs across the three echinocandins seen seen for susceptibility testing using CLSI M27A-3, agar dilution, or Etest with RPMI-2G medium, but still ∼10% of the fks hot spot mutants were classified as susceptible. The consequence of this is that even with the best performing assays and a susceptibility breakpoint set as low as the WT-UL, 10% of the fks mutants are misclassified as susceptible. If, on the other hand, the susceptibility breakpoint is defined even lower in order to ensure that fks mutants are classified as nonsusceptible, a number of susceptible isolates will be classified as potentially resistant. The first scenario may not currently affect a great number of cases, as fks mutants are still only sporadically reported, but for individual patients, the consequence may be inappropriate treatment and failure (5, 7, 8, 22, 25, 26, 33, 45). The other scenario will affect a greater number of cases, for which further analyses are needed in order to determine if the isolate is truly resistant or not.
Considerable differences in test performances were seen for the different echinocandins. Caspofungin agar dilution with either medium was superior, in agreement with previous observations (5). For micafungin, in general all micro- or agar dilution tests led to a misclassification of at least a quarter of the fks hot spot mutant isolates. This was due mainly to C. glabrata isolates with resistance mutants not separating very well from the wild-type population (17, 45). Etest with RPMI-2G performed best for micafungin, but still a quarter of the fks mutant C. glabrata isolates were misclassified as susceptible. This raises the question of whether a number of resistant isolates may go unnoticed when current reference methodologies for susceptibility testing are used.
Anidulafungin was the compound for which the fewest VMEs were observed overall. This may suggest that susceptibility testing for this agent could be used as a screening procedure for echinocandin susceptibility. In this study, anidulafungin susceptibility testing by any of the current reference methodologies or Etest with RPMI-2G resulted in misclassifications of 3 to 4% of fks hot spot mutant isolates when the species-specific WT-ULs indicated in Tables 2, 4, and 6 were used as susceptibility breakpoints. These WT-UL values are considerably lower than the currently recommended CLSI breakpoint for susceptibility of an S value of ≤2 μg/ml (9, 38). However, the vast majority of isolates with FKS hot spot mutations were classified as susceptible by CLSI and Etest using the CLSI breakpoint, in agreement with the increasing concern that the breakpoint may be too high (5, 7, 13, 14, 18, 22, 25). This was particularly true for anidulafungin and micafungin, in agreement with the fact that the MICs of these compounds are in general lower than that of caspofungin and much lower than the breakpoint (12, 29, 30). Further studies are needed to establish ECOFFs and breakpoints, reducing the risk of misclassification of resistant isolates.
The MICs of all three candins were considerably higher for C. parapsilosis than for the other species, as previously described (12, 29, 36, 38). C. parapsilosis has a naturally occurring mutation at hot spot 1 in FKS1, accounting for the MIC level being in the same range as those of hot spot mutant isolates of the other Candida species (16). The overall clinical response for invasive C. parapsilosis infections treated with echinocandins is comparable to that for the other species (23, 27, 32, 38), which may in part be due to C. parapsilosis being less virulent (1, 2, 19-21, 46). However, C. parapsilosis was the only species for which the response rate observed with patients with invasive candidiasis receiving anidulafungin was numerically but not statistically significantly poorer than that for fluconazole (response rates of 64% versus 88%) (39), and breakthrough infections with C. parapsilosis in hematological patients have been associated significantly with prior caspofungin use, suggesting that other antifungal drug classes may be more efficacious, although this fungus is not clinically highly resistant (15, 43). These observations suggest that species-specific breakpoints and lower breakpoints than the current CLSI ones are necessary to avoid classifying C. albicans, C. dubliniensis, C. glabrata, and C. tropicalis isolates with resistance mutations as susceptible while not classifying all wild-type C. parapsilosis isolates as resistant.
When the MICs obtained for the three candins were compared, it was noted that the MICs obtained by EUCAST EDef 7.1 were in general higher for caspofungin than those obtained using the CLSI M27-A3 method. And for both tests the MIC50s were higher than those normally obtained (one or two dilutions for CLSI and two dilution steps for EUCAST) (36). The reason for this is not clear, but it indicates a variability associated with caspofungin susceptibility testing, despite performing testing in reference laboratories using a single batch of pure substances and correcting for potency, as we have summarized previously (5). The implication of this for the current study is that WT-UL values and MIC ranges might be lower if repeated with a different batch of pure substance, but it is not likely that it affects the evaluation of the performances of the tests and their ability to differentiate fks hot spot mutants from wild-type isolates. Further studies of caspofungin testing are needed to resolve this issue.
In conclusion, no test was perfect, in the sense that MIC ranges for fks mutants and wild-type isolates were separated by at least three twofold-dilution steps and no overlap existed between wild-type and fks hot spot mutant populations for all three candins and the Candida species included. Until the challenges concerning caspofungin testing reproducibility and micafungin susceptibility testing of C. glabrata are solved, an attractive approach might be to use anidulafungin MICs greater than the species-specific ECOFFs as a marker for possible echinocandin resistance. For this purpose, EUCAST with RPMI-2G or IsoSensitest-2G, CLSI, and Etest are all appropriate methods, while disk testing performed poorly. Possibly resistant isolates should be confirmed and the underlying mechanism investigated by sequence analysis of the FKS hot spot regions. Lack of mutations may represent a falsely elevated MIC during initial testing or resistance due to yet uncharacterized mechanisms.
Acknowledgments
We thank Birgit Brandt for excellent technical assistance. We thank Francoise Dromer, Unité de Mycologie Moléculaire, Institut Pasteur, Paris, France, for providing one of the candin fks hot spot mutant C. krusei isolates (13). We thank Astellas for providing micafungin pure substance, Merck for providing caspofungin pure substance, and Pfizer for providing anidulafungin pure substance.
The study was financially supported in part by research grants from the Investigator-Initiated Study programs of Astellas Pharma, Merck & Co. Inc., and Pfizer Aps. Additionally, it was supported by NIH grant AI069397 to D.S.P.
The opinions expressed in this paper are those of the authors and do not necessarily represent those of the pharmaceutical companies.
Potential conflicts of interest are that M.C.A. has been a consultant for Astellas, Merck, Pfizer, and SpePharm and an invited speaker for Astellas, Cephalon, Merck Sharp & Dohme, Pfizer, Schering-Plough, and Swedish Orphan and has received research funding for this particular study from Astellas, Merck, and Pfizer.
C.L.-F. has been a consultant for Pfizer and Schering Plough and an invited speaker for Pfizer, Gilead, Schering Plough, and Merck Sharp & Dohme and has received research funding from Pfizer, Gilead, Merch Sharp & Dohme, and Schering Plough.
D.S.P. is a shareholder in Merck; has acted as a consultant for Merck, Pfizer, and Astellas; is an advisory board member for Merck, Pfizer, Astellas, and Myconostica; has a patent for assays for resistance to echinocandin-class drugs (application no. 07763-O69WO1); has received research funding (although not for this particular study) from Merck, Pfizer, Astellas, and Myconostica; and has been an invited speaker for Merck, Pfizer, Astellas, and Myconostica.
G.G.-E. has no conflicts of interest.
A.G.L. has no conflicts of interest.
In the past 5 years, J.L.R.T. has received grant support from Astellas Pharma, Gilead Sciences, Merck Sharp & Dohme, Pfizer, Schering Plough, Soria Melguizo SA, the European Union, the Spanish Agency for International Cooperation, the Spanish Ministry of Culture and Education, the Spanish Health Research Fund, the Instituto de Salud Carlos III, the Ramon Areces Foundation, and the Mutua Madrileña Foundation. He has been an advisor/consultant to the Panamerican Health Organization, Gilead Sciences, Merck Sharp & Dohme, Myconostica, Pfizer, and Schering Plough. He has been paid for talks on behalf of Gilead Sciences, Merck Sharp & Dohme, Pfizer, and Schering Plough.
In the past 5 years, M.C.E. has received grant support from Astellas Pharma, BioMerieux, Gilead Sciences, Merck Sharp & Dohme, Pfizer, Schering Plough, Soria Melguizo SA, the European Union, the ALBAN program, the Spanish Agency for International Cooperation, the Spanish Ministry of Culture and Education, the Spanish Health Research Fund, the Instituto de Salud Carlos III, the Ramon Areces Foundation, and the Mutua Madrileña Foundation. He has been an advisor/consultant to the Panamerican Health Organization, Gilead Sciences, Merck Sharp & Dohme, Pfizer, and Schering Plough. He has been paid for talks on behalf of Gilead Sciences, Merck Sharp & Dohme, Pfizer, and Schering Plough.
Footnotes
Published ahead of print on 2 November 2009.
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