Abstract
We performed 24- and 48-h MIC determinations of posaconazole and voriconazole against more than 16,000 clinical isolates of Candida species. By using the 24- and 48-h epidemiological cutoff values (ECVs), the categorical agreement between the 24-h and reference 48-h broth microdilution results ranged from 97.1% (C. parapsilosis and voriconazole) to 99.8% (C. krusei and voriconazole), with 0.0 to 2.9% very major discrepancies (VMD). The essential agreement (within 2 log2 dilutions) between the 24- and 48-h results was 99.6% for both posaconazole and voriconazole. The MIC results obtained for both posaconazole and voriconazole after only 24 h of incubation may be used to determine the susceptibilities of Candida spp. to these important antifungal agents. The applications of ECVs to this large collection of Candida isolates suggests the potential to develop 24-h species-specific clinical breakpoints for both posaconazole and voriconazole.
INTRODUCTION
There now are several lines of evidence that indicate that early and appropriate (correct dose and susceptible organism) antifungal therapy has a positive effect on outcomes and resource utilization for patients with candidemia (2, 14, 15, 19, 22, 24, 28, 39). The choice of antifungal therapy may be optimized by the use of routine onsite antifungal susceptibility testing (4, 5, 8, 13, 16, 18, 23, 27, 37). Specifically, the susceptibility testing of species of Candida such as Candida glabrata results in lower overall treatment costs, based on the de-escalation of therapy from an expensive echinocandin to a triazole antifungal agent for patients with documented candidemia (8).
It is now apparent that the vast majority of Candida species achieve suitable growth to allow the MIC testing of the triazole class of antifungal agents within 24 h of incubation using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution (BMD) method (9–11, 30). Given that a shorter incubation period for antifungal susceptibility testing would avoid the potentially confounding effect of trailing growth on 48-h triazole MICs (3, 30, 35, 36), is more efficient and practical for use in the clinical laboratory (11, 30), and would provide useful information to clinicians sooner rather than later (14, 24), the CLSI Subcommittee for Antifungal Testing has focused attention on the validation of 24-h MIC determinations for the triazoles and Candida spp. (26, 30). The validation of 24-h MIC determinations and the establishment of new 24-h clinical breakpoints (CBPs) has been accomplished for the in vitro testing of fluconazole (32); however, progress has been lacking in this area for the newer triazoles, namely, posaconazole and voriconazole.
A multicenter study conducted by Espinel-Ingroff and colleagues (9) found an excellent essential agreement (EA; percentage of MICs within a 3-dilution range) of 97% for both posaconazole and voriconazole for comparisons of MICs determined at 24 h of incubation with those determined at 48 h for a small collection of 71 isolates of Candida spp. Furthermore, they found a high degree of interlaboratory agreement when posaconazole and voriconazole MICs were read at 24 h in six different laboratories (93 and 94%, respectively) (9). Categorical agreement (CA) between 24- and 48-h readings was assessed for voriconazole using the 48-h CBPs established by the CLSI (7, 29), demonstrating an acceptable overall CA of 93% with no very major errors (VME). Subsequently, Espinel-Ingroff et al. (11) extended the evaluation of 24-h voriconazole MICs using a larger collection of 2,162 isolates of Candida spp. and other yeasts tested in three different laboratories. In this study, they found an 88.6% EA and an overall CA of 93.2%; however, they found unacceptable percentages of VMEs with C. albicans (2.7%), C. glabrata (4.1%), and C. tropicalis (9.7%), leading to the conclusion that the 24-h voriconazole MIC may not be useful in detecting resistance using the existing 48-h CBPs (11).
In the present study, we attempt to expand upon this evaluation using a larger collection of Candida bloodstream infection (BSI) isolates (16,485 isolates) tested against both posaconazole and voriconazole by the CLSI BMD method. In addition to determining the EA between the 24- and 48-h MICs for both agents according to the species of Candida, we use the previously established species-specific epidemiological cutoff values (ECVs) for each agent to examine the CA (wild-type [WT] versus non-WT) between 24- and 48-h readings. Similarly to Espinel-Ingroff et al. (11), we also examine the CA between 24- and 48-h voriconazole MICs using the existing CLSI CBPs.
MATERIALS AND METHODS
Organisms.
A total of 16,485 clinical isolates obtained from more than 100 medical centers worldwide from 2001 through 2009 were tested. The collection included 8,618 isolates of C. albicans, 2,414 of C. glabrata, 2,278 of C. parapsilosis, 1,894 of C. tropicalis, 508 of C. krusei, and 776 of miscellaneous species, including 205 of C. lusitaniae, 177 of C. guilliermondii, 93 of C. kefyr, 50 of C. pelliculosa, 40 of C. famata, 30 of C. metapsilosis, 26 of C. dubliniensis, 19 of C. rugosa, 16 of C. lipolytica, 8 of C. zeylanoides, and 112 of Candida spp. not otherwise identified (Table 1). All isolates were obtained from blood or other normally sterile sites and represented the incident isolate from individual infectious episodes. The isolates were collected at individual study sites and were sent to the University of Iowa (Iowa City, IA) for identification and susceptibility testing as described previously (29, 31, 33). The isolates were identified by standard methods (17) and stored as water suspensions until used in the study. Prior to being tested, each isolate was passaged at least twice onto potato dextrose agar (Remel) and CHROMagar Candida medium (Becton Dickinson and Company, Sparks, MD) to ensure purity and viability.
Table 1.
In vitro susceptibilities of Candida sp. isolates to posaconazole and voriconazole as determined by the CLSI BMD method and read after 24 and 48 h of incubation
| Species | Antifungal agent | No. of strains tested | Incubation time (h) | MICa (μg/ml) |
EA (%) | ||
|---|---|---|---|---|---|---|---|
| Range | 50% | 90% | |||||
| C. albicans | Posaconazole | 8,618 | 24 | 0.007–2 | 0.015 | 0.06 | 99.9 |
| 48 | 0.007–2 | 0.015 | 0.06 | ||||
| Voriconazole | 8,615 | 24 | 0.007–1 | 0.007 | 0.007 | 99.8 | |
| 48 | 0.007–2 | 0.007 | 0.015 | ||||
| C. glabrata | Posaconazole | 2,413 | 24 | 0.007–>8 | 0.5 | 2 | 98.2 |
| 48 | 0.03–>8 | 1 | 2 | ||||
| Voriconazole | 2,414 | 24 | 0.007–8 | 0.06 | 0.5 | 98.7 | |
| 48 | 0.007–>8 | 0.25 | 1 | ||||
| C. parapsilosis | Posaconazole | 2,278 | 24 | 0.007–1 | 0.06 | 0.12 | 99.9 |
| 48 | 0.007–1 | 0.06 | 0.12 | ||||
| Voriconazole | 2,278 | 24 | 0.007–4 | 0.007 | 0.03 | 99.7 | |
| 48 | 0.007–8 | 0.015 | 0.06 | ||||
| C. tropicalis | Posaconazole | 1,894 | 24 | 0.007–2 | 0.03 | 0.12 | 99.8 |
| 48 | 0.007–2 | 0.06 | 0.12 | ||||
| Voriconazole | 1,894 | 24 | 0.007–4 | 0.015 | 0.06 | 99.7 | |
| 48 | 0.007–>8 | 0.03 | 0.06 | ||||
| C. krusei | Posaconazole | 508 | 24 | 0.015–1 | 0.25 | 0.5 | 100.0 |
| 48 | 0.015–4 | 0.25 | 0.5 | ||||
| Voriconazole | 507 | 24 | 0.007–2 | 0.12 | 0.25 | 100.0 | |
| 48 | 0.007–4 | 0.25 | 0.5 | ||||
| Misc.b | Posaconazole | 776 | 24 | 0.007–4 | 0.06 | 0.25 | 99.5 |
| 48 | 0.007–4 | 0.12 | 0.5 | ||||
| Voriconazole | 776 | 24 | 0.007–1 | 0.015 | 0.06 | 99.7 | |
| 48 | 0.007–2 | 0.03 | 0.12 | ||||
| Total | Posaconazole | 16,485 | 24 | 0.007–>8 | 0.03 | 0.25 | 99.6 |
| 48 | 0.007–>8 | 0.03 | 0.5 | ||||
| Voriconazole | 16,484 | 24 | 0.007–>8 | 0.007 | 0.12 | 99.6 | |
| 48 | 0.007–>8 | 0.007 | 0.25 | ||||
50% and 90% indicate the 50% and 90% MICs of isolates tested, respectively.
Miscellaneous species, including C. lusitaniae (205 isolates), C. guilliermondii (177 isolates), C. kefyr (93 isolates), C. pelliculosa (50 isolates), C. famata (40 isolates), C. metapsilosis (30 isolates), C. dubliniensis (26 isolates), C. rugosa (19 isolates), C. lipolytica (16 isolates), C. zeylanoides (8 isolates), and Candida spp. not otherwise identified (112 isolates).
Antifungal agents.
Reference powders of posaconazole and voriconazole were obtained from their respective manufacturers. Stock solutions were prepared in dimethyl sulfoxide, and serial 2-fold dilutions in RPMI 1640 medium (Sigma, St. Louis, MO) buffered to pH 7.0 with 0.165 M MOPS (morpholinepropanesulfonic acid) buffer (Sigma) were made.
Antifungal susceptibility testing.
BMD testing was performed in accordance with the guidelines in CLSI document M27-A3 (6) by using RPMI 1640 medium, an inoculum of 0.5 × 103 to 2.5 × 103 cells/ml, and incubation at 35°C. MICs were determined visually after 24 and 48 h of incubation as the lowest concentration of drug that caused a significant diminution (≥50% inhibition) of growth below control levels (6, 29, 31, 33). The CBPs for voriconazole and all species of Candida were those established by Pfaller et al. (29) and the CLSI (7): susceptible (S), MIC ≤ 1 μg/ml; susceptible dose dependent (SDD), MIC = 2 μg/ml; resistant (R), MIC ≥ 4 μg/ml.
Quality control.
Quality control was performed by testing CLSI-recommended strains C. krusei ATCC 6258 and C. parapsilosis ATCC 22019 (6, 7).
Analysis of results.
The MIC results for each triazole obtained with the CLSI BMD method at 24 h was compared with that obtained at 48 h of incubation. High off-scale MIC results were converted to the next highest concentration, and low off-scale MIC results were left unchanged. Discrepancies of more than 2 dilutions among MIC results were used to calculate the EA. The 48-h CBPs for voriconazole were used to obtain CA percentages between MICs determined at 24 and 48 h for all species. The recently described ECVs for each agent and species also were used to obtain CA percentages between 24- and 48-h readings (33). The ECVs are used to discriminate WT isolates (absence of acquired resistance mechanisms) from non-WT isolates (containing acquired resistance mechanisms) and serve as the most sensitive measure of the emergence of strains with reduced susceptibility to a given agent (20, 21, 33). Using the 48-h CLSI CBPs for voriconazole, very major errors (VME) were identified when the 48-h MIC indicated resistance (R) and the 24-h MIC indicated susceptibility (S). Major errors (ME) were identified when the isolate was classified as R at 24 h and as S at 48 h, and minor errors (M) were identified when one of the readings was SDD and the other was either S or R.
The assessment of CA using the ECVs for each antifungal agent and species was assessed by applying the appropriate ECV to the respective MIC result. Thus, the 24-h ECVs were used to identify the WT and non-WT isolates in each 24-h MIC distribution, and the 48-h ECVs were applied similarly to the 48-h MIC distribution of each species. Very major discrepancies (VMD) were identified when the 48-h MIC was greater than the 48-h ECV for each agent and species (non-WT) and when the 24-h MIC was less than or equal to the 24-h ECV (WT). Major discrepancies (MD) were identified when the isolate's triazole MIC was greater than the ECV (non-WT) at 24 h and less than or equal to the ECV (WT) at the 48-h reading.
RESULTS AND DISCUSSION
Table 1 summarizes the in vitro susceptibilities of 16,485 isolates of Candida spp. to posaconazole and voriconazole, as determined by the CLSI BMD method, read after 24 and 48 h of incubation. The MIC results for each agent were typical of those for each species of Candida (25, 31, 38). The 24-h MIC results tended to be equal to or one 2-fold dilution lower than those determined after 48 h of incubation for both agents and species.
The overall EA between the 24- and 48-h MIC results was 99.6% for both posaconazole and voriconazole. By comparison, Espinel-Ingroff et al. (9) reported an EA of 97% for both agents in the earlier six-laboratory study and only 88.6% for voriconazole in a subsequent three-laboratory study (11).
Of the discrepancies noted between 24- and 48-h readings, the MICs generated at 48 h of incubation were higher than those obtained at 24 h of incubation in 110 of 119 (92.4%) discrepant results (53 of 58 with posaconazole and 57 of 61 with voriconazole). The largest number of discrepancies observed with the comparison of results at 24 versus 48 h occurred with C. glabrata tested against posaconazole (43 discrepant results) and against voriconazole (32 discrepant results).
Regarding the individual species, the EAs between the 24- and 48-h CLSI BMD MIC values were >98.0% for all organism-drug combinations and were >99.0% for all with the exception of C. glabrata and both posaconazole (98.2%) and voriconazole (98.7%) (Table 1). By comparison, the earlier study of Espinel-Ingroff et al. (9) found an overall EA of 97% for both posaconazole and voriconazole and EAs of ≥95% for all organism-drug combinations, with the exception of C. glabrata and voriconazole (89%) and C. tropicalis and voriconazole (93%). In the subsequent study of voriconazole, Espinel-Ingroff et al. (11) reported a lower overall EA of 88.6% for the comparison of results at 24 versus 48 h and EAs of <80% for C. glabrata (73.3%), C. tropicalis (77.2%), and miscellaneous Candida spp. (75%). In each of these instances the discrepancies were due to higher MICs at the 48-h reading, possibly due to trailing growth patterns (11). Although more than 90% of discrepancies in the present study were due to higher MICs after 48 h of incubation, the EA was more in line with the earlier study by Espinel-Ingroff et al. (9), suggesting that the lower level of agreement in the second voriconazole study (11) was due to differences in the culture collections used in the various studies and/or the abilities of the various laboratories to read and interpret trailing growth patterns at 48 h of incubation. It is notable that previous studies have shown trailing growth to be most problematic with C. glabrata and C. tropicalis, with the 24-h MIC deemed to be the correct MIC as determined by sterol inhibition studies and in vivo animal models of invasive candidiasis (3, 35, 36). One possible solution to the problem of trailing MIC endpoints would be the use of a spectrophotometric approach for the reading of MICs, as suggested previously by us and others (3, 9, 32, 34).
Similarly to Espinel-Ingroff et al. (11), we employed the 48-h CLSI CBPs for voriconazole to assess the CA between 24- and 48-h MIC determinations (Table 2). In contrast to Espinel-Ingroff et al. (11), we found the CA to be >95.0% for all species and >99.0% for all species with the exception of C. glabrata (96.0%). While Espinel-Ingroff et al. (11) found an unacceptable VME rate for C. albicans (2.7%), C. glabrata (4.1%), and C. tropicalis (9.7%), the VME rates in the present study ranged from 0.0 to 0.2% for these species (Table 2). Again, these differences could be ascribed to a greater degree of trailing growth in the study of Espinel-Ingroff et al. (11); however, it also should be noted that the proportion of isolates resistant to voriconazole was considerably higher in that study (3.3 and 6.8% at 24 and 48 h, respectively) than in the present study (0.5 and 0.8% at 24 and 48 h, respectively).
Table 2.
Categorical agreement between 24- and 48-h CLSI BMD voriconazole MICs for 16,484 isolates of Candida spp. using CLSI 48-h clinical breakpoints
| Species (no. tested) | Incubation time (h) | % of isolates that testeda: |
CA (%) | % of errors |
||||
|---|---|---|---|---|---|---|---|---|
| S | SDD | R | VME | ME | Minor | |||
| C. albicans (8,615) | 24 | 100.0 | 0.0 | 0.0 | 99.9 | 0.0 | 0.0 | 0.1 |
| 48 | 99.9 | 0.1 | 0.0 | |||||
| C. glabrata (2,414) | 24 | 92.8 | 3.9 | 3.3 | 96.0 | 0.2 | 0.0 | 3.8 |
| 48 | 91.1 | 3.3 | 5.6 | |||||
| C. parapsilosis (2,278) | 24 | 99.9 | <0.1 | <0.1 | 99.7 | 0.1 | 0.0 | 0.2 |
| 48 | 99.5 | 0.3 | 0.2 | |||||
| C. tropicalis (1,894) | 24 | 99.9 | 0.0 | 0.1 | 99.8 | 0.1 | 0.0 | 0.1 |
| 48 | 99.8 | 0.1 | 0.1 | |||||
| C. krusei (507) | 24 | 99.8 | 0.2 | 0.0 | 99.6 | 0.0 | 0.0 | 0.4 |
| 48 | 99.6 | 0.2 | 0.2 | |||||
| Misc.b (776) | 24 | 100.0 | 0.0 | 0.0 | 99.7 | 0.0 | 0.0 | 0.3 |
| 48 | 99.7 | 0.3 | 0.0 | |||||
| Total (16,484) | 24 | 98.9 | 0.6 | 0.5 | 99.3 | <0.1 | 0.0 | 0.6 |
| 48 | 98.6 | 0.6 | 0.8 | |||||
S, susceptible (MIC ≤ 1μg/ml; SDD, susceptible dose dependent (MIC = 2 μg/ml); R, resistant (MIC ≥ 4 μg/ml).
Miscellaneous species, including C. lusitaniae (205 isolates), C. guilliermondii (177 isolates), C. kefyr (93 isolates), C. pelliculosa (50 isolates), C. famata (40 isolates), C. metapsilosis (30 isolates), C. dubliniensis (26 isolates), C. rugosa (19 isolates), C. lipolytica (16 isolates), C. zeylanoides (8 isolates), and Candida spp. not otherwise identified (112 isolates).
The 24- and 48-h ECVs for both triazoles and the five most common species of Candida are shown in Table 3. These ECVs were determined in a previous study (33) and provide another measure of the close agreement between the 24- and 48-h MICs for each agent and these species. It should be noted that the 24-h ECVs for both posaconazole and voriconazole determined using the CLSI method are essentially the same as those obtained using the BMD method of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (12, 34). The application of these ECVs allows an assessment of the CA between the two reading times in terms of their comparability in differentiating WT from non-WT strains within each species.
Table 3.
ECVs for posaconazole and voriconazole and five species of Candidaa
| Species | No. tested | Incubation time (h) | ECV (μg/ml [% of isolates that were ≤ECV]) for: |
|
|---|---|---|---|---|
| Posaconazole | Voriconazole | |||
| C. albicans | 8,619 | 24 | 0.06 (98.4) | 0.03 (99.0) |
| 48 | 0.06 (97.8) | 0.03 (98.4) | ||
| C. glabrata | 2,415 | 24 | 2 (96.1) | 0.5 (90.4) |
| 48 | 4 (94.9) | 1 (91.1) | ||
| C. parapsilosis | 2,278 | 24 | 0.25 (99.3) | 0.12 (97.8) |
| 48 | 0.25 (98.6) | 0.12 (97.8) | ||
| C. tropicalis | 1,895 | 24 | 0.12 (97.8) | 0.06 (97.3) |
| 48 | 0.25 (99.2) | 0.12 (98.0) | ||
| C. krusei | 508 | 24 | 0.5 (99.0) | 0.5 (99.4) |
| 48 | 1 (99.4) | 1 (99.6) | ||
Data were compiled from Pfaller et al. (33).
The CA between the results obtained after 24 h of incubation and those obtained after 48 h of incubation for the two triazoles and each species of Candida was determined by applying the respective 24- and 48-h ECVs for each drug-organism pair (Table 4). Again, excellent CA was observed for all comparisons of results at 24 versus 48 h. The only comparisons with a CA of <99% were C. glabrata and posaconazole (97.2%), C. glabrata and voriconazole (98.6%), C. parapsilosis and voriconazole (97.1%), and C. tropicalis and posaconazole (98.4%). Despite a large number of non-WT isolates (MIC > ECV) identified for most of the species, the VMD rate was <1.0% for all comparisons, with the exception of C. glabrata and posaconazole (2.0%) and C. parapsilosis and voriconazole (2.9%).
Table 4.
Categorical agreement between the results of 24- and 48-h CLSI BMD methods for posaconazole and voriconazole and Candida spp. using ECVs
| Species | Antifungal agent | Incubation time (h) | ECV (μg/ml) | No. of strains tested | No. (%) of isolates with results that were: |
% CA | % of isolates with discrepant results that were: |
||
|---|---|---|---|---|---|---|---|---|---|
| ≤ECV | >ECV | VMD | MD | ||||||
| C. albicans | Posaconazole | 24 | 0.06 | 8,618 | 8,479 (98.4) | 139 (1.6) | 99.3 | 0.6 | 0.1 |
| 48 | 0.06 | 8,426 (97.8) | 192 (2.2) | ||||||
| Voriconazole | 24 | 0.03 | 8,615 | 8,526 (99.0) | 89 (1.0) | 99.4 | 0.5 | 0.1 | |
| 48 | 0.03 | 8,479 (98.4) | 136 (1.6) | ||||||
| C. glabrata | Posaconazole | 24 | 2 | 2,413 | 2,321 (96.2) | 92 (3.8) | 97.2 | 2.0 | 0.8 |
| 48 | 4 | 2,290 (94.9) | 123 (5.1) | ||||||
| Voriconazole | 24 | 0.5 | 2,414 | 2,184 (90.5) | 230 (9.5) | 98.6 | 0.3 | 1.1 | |
| 48 | 1 | 2,201 (91.2) | 213 (8.8) | ||||||
| C. parapsilosis | Posaconazole | 24 | 0.25 | 2,278 | 2,263 (99.3) | 15 (0.7) | 99.2 | 0.7 | 0.1 |
| 48 | 0.25 | 2,246 (98.6) | 32 (1.4) | ||||||
| Voriconazole | 24 | 0.12 | 2,278 | 2,227 (97.8) | 51 (2.2) | 97.1 | 2.9 | 0.0 | |
| 48 | 0.12 | 2,162 (94.9) | 116 (5.1) | ||||||
| C. tropicalis | Posaconazole | 24 | 0.12 | 1,894 | 1,852 (97.8) | 42 (2.2) | 98.4 | 0.1 | 1.5 |
| 48 | 0.25 | 1,878 (99.2) | 16 (0.8) | ||||||
| Voriconazole | 24 | 0.06 | 1,894 | 1,842 (97.3) | 52 (2.7) | 99.1 | 0.1 | 0.8 | |
| 48 | 0.12 | 1,857 (98.0) | 37 (2.0) | ||||||
| C. krusei | Posaconazole | 24 | 0.5 | 508 | 503 (99.0) | 5 (1.0) | 99.2 | 0.2 | 0.6 |
| 48 | 1 | 505 (99.4) | 3 (0.6) | ||||||
| Voriconazole | 24 | 0.5 | 507 | 504 (99.4) | 3 (0.6) | 99.8 | 0.0 | 0.2 | |
| 48 | 1 | 505 (99.6) | 2 (0.4) | ||||||
These results both confirm and extend the previous observations of Espinel-Ingroff et al. (9, 11). First of all, we agree that although the EA between 24- and 48-h MIC results generally is quite good, it must be recognized that for some species the 48-h triazole MICs may be higher than those read after 24 h of incubation, and that this may lead to categorical discrepancies. The results of the present study, however, are in greater agreement with those of the first report by Espinel-Ingroff et al. (9) than with those of the latter (11), suggesting that it should be possible to read MICs for posaconazole and voriconazole after only 24 h of incubation. We have demonstrated the potential for the existing CBPs for voriconazole to be applied to MICs determined after 24 h of incubation with a high level of CA and very acceptable levels of VME (Table 2). Having said this, we agree with Arendrup and Denning (1), who questioned whether a non-species-dependent CBP for a voriconazole susceptibility of ≤1 μg/ml is appropriate given that the vast majority of cases of BSI caused by isolates defined as susceptible are cases of C. albicans, C. tropicalis, and C. parapsilosis, for which MICs are 4 to 5 logs below the suggested CBP (29). The species- and drug-specific ECVs shown in Table 3 begin to address this issue and differentiate WT from non-WT strains at much lower MIC cutoffs than those of the current CBP. In the absence of CBPs for posaconazole, the ECVs allow one to examine the emergence of non-WT strains of each species of Candida.
The application of the species-specific ECVs for posaconazole and voriconazole to the large collection of Candida isolates in the present study allows us to demonstrate the high degree of concordance between the 24- and 48-h MIC readings and their similar ability to identify WT and non-WT strains of Candida. These results indicate that determining the posaconazole and voriconazole MICs for the most common species of Candida after 24 h of incubation is both feasible and representative of the reference MICs determined after 48 h of incubation. Furthermore, we have used the 24- and 48-h ECVs for each triazole and species of Candida to show that the 24-h readings identify the same WT and non-WT populations determined after 48 h of incubation. The latter observation points the way to the development of new 24-h species-specific CBPs for voriconazole and the possibility of similar CBPs for posaconazole and Candida.
ACKNOWLEDGMENTS
Caitlin Howard provided excellent support in the preparation of the manuscript.
This work was supported in part by research grants from Pfizer and Schering-Plough.
Footnotes
Published ahead of print on 2 February 2011.
REFERENCES
- 1. Arendrup M. C., Denning D. W. 2007. Does one voriconazole breakpoint suit all Candida species? J. Clin. Microbiol. 45:2093–2094 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. Arnold H. M., et al. 2010. Hospital resource utilization and costs of inappropriate treatment of candidemia. Pharmacotherapy 30:361–368 [DOI] [PubMed] [Google Scholar]
- 3. Arthington-Skaggs B. A., et al. 2002. Comparison of visual and spectrophotometric methods of broth microdilution MIC end point determination and evaluation of a sterol quantitation method for in vitro susceptibility testing of fluconazole and itraconazole against trailing and nontrailing Candida isolates. Antimicrob. Agents Chemother. 46:2477–2481 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4. Baddley J. W., et al. 2004. Utility of real-time antifungal susceptibility testing for fluconazole in the treatment of candidemia. Diagn. Microbiol. Infect. Dis. 50:119–124 [DOI] [PubMed] [Google Scholar]
- 5. Baddley J. W., Patel M., Bhavnani S. M., Moser S. A., Andes D. R. 2008. Association of fluconazole pharmacodynamics with mortality in patients with candidemia. Antimicrob. Agents Chemother. 52:3022–3028 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6. CLSI 2008. Reference method for broth dilution antifungal susceptibility testing of yeasts, 3rd ed. M27-A3 Clinical and Laboratory Standards Institute, Wayne, PA [Google Scholar]
- 7. CLSI 2008. Reference method for broth dilution antifungal susceptibility testing of yeasts, 3rd informational supplement. M27-S3. Clinical and Laboratory Standards Institute, Wayne, PA [Google Scholar]
- 8. Collins C. D., Eschenauer G. A., Salo S. L., Newton D. W. 2007. To test or not to test: a cost minimization analysis of susceptibility testing for patients with documented Candida glabrata fungemias. J. Clin. Microbiol. 45:1884–1888 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Espinel-Ingroff A., et al. 2005. Comparison of visual 24-hour and spectrophotometric 48-hour MICs to CLSI reference microdilution MICs of fluconazole, itraconazole, posaconazole, and voriconazole for Candida spp.: a collaborative study. J. Clin. Microbiol. 43:4535–4540 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. Espinel-Ingroff A., et al. 2005. International and multicenter comparison of EUCAST and CLSI M27-A2 broth microdilution methods for testing susceptibilities of Candida spp. to fluconazole, itraconazole, posaconazole, and voriconazole. J. Clin. Microbiol. 43:3884–3889 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11. Espinel-Ingroff A., Canton E., Peman J., Rinaldi M. G., Fothergill A. W. 2009. Comparison of 24-hour and 48-hour voriconazole MICs as determined by the Clinical and Laboratory Standards Institute broth microdilution method (M27-A3 document) in three laboratories: results obtained with 2,162 clinical isolates of Candida spp. and other yeasts. J. Clin. Microbiol. 47:2766–2771 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. European Committee on Antimicrobial Susceptibility Testing–Subcommittee on Antifungal Susceptibility Testing (EUCAST-AFST) 2008. EUCAST technical note on voriconazole. Clin. Microbiol. Infect. 14:985–987 [DOI] [PubMed] [Google Scholar]
- 13. Forrest G. 2006. Role of antifungal susceptibility testing in patient management. Curr. Opin. Infect. Dis. 19:538–543 [DOI] [PubMed] [Google Scholar]
- 14. Garey K. W., et al. 2006. Time to initiation of fluconazole therapy impacts mortality in patients with candidemia: a multi-institutional study. Clin. Infect. Dis. 43:25–31 [DOI] [PubMed] [Google Scholar]
- 15. Garey K. W., Turpin R. S., Bearden D. T., Pai M. P., Suda K. J. 2007. Economic analysis of inadequate fluconazole therapy in non-neutropenic patients with candidemia: a multi-institutional study. Int. J. Antimicrob. Agents 29:557–562 [DOI] [PubMed] [Google Scholar]
- 16. Hadley S., Martinez J. A., McDermott L., Rapino B., Snydman D. R. 2002. Real-time antifungal susceptibility screening aids management of invasive yeast infections in immunocompromised patients. J. Antimicrob. Chemother. 49:415–419 [DOI] [PubMed] [Google Scholar]
- 17. Hazen K. C., Howell S. A. 2007. Candida, Cryptococcus, and other yeasts of medical importance, p. 1762–1788 In Murray P. R., Baron E. J., Jorgensen J. H., Landry M. L., Pfaller M. A. (ed.), Manual of clinical microbiology, 9th ed. ASM Press, Washington, DC [Google Scholar]
- 18. Hospenthal D. R., Murray C. K., Rinaldi M. G. 2004. The role of antifungal susceptibility in the therapy of candidiasis. Diagn. Microbiol. Infect. Dis. 48:153–160 [DOI] [PubMed] [Google Scholar]
- 19. Hsu D. I., Nguyen M., Nguyen L., Law A., Wong-Beringer A. 2010. A multicenter study to evaluate the impact of timing of caspofungin administration on outcomes of invasive candidiasis in non-immunocompromised adult patients. J. Antimicrob. Chemother. 65:1765–1770 [DOI] [PubMed] [Google Scholar]
- 20. Kahlmeter G., et al. 2003. European harmonization of MIC breakpoints for antimicrobial susceptibility testing of bacteria. J. Antimicrob. Chemother. 52:145–148 [DOI] [PubMed] [Google Scholar]
- 21. Kahlmeter G., Brown D. F. J. 2004. Harmonization of antimicrobial breakpoints in Europe–can it be achieved? Clin. Microbiol. Newsl. 26:187–192 [Google Scholar]
- 22. Labelle A. J., Micek S. T., Roubinian N., Kollef M. H. 2008. Treatment-related risk factors for hospital mortality in Candida bloodstream infections. Crit. Care Med. 36:2967–2972 [DOI] [PubMed] [Google Scholar]
- 23. Magill S. S., Shields C., Sears C. L., Choti M., Merz W. G. 2006. Triazole cross-resistance among Candida spp.: case-report, occurrence among bloodstream isolates, and implications for antifungal therapy. J. Clin. Microbiol. 44:529–535 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24. Morrell M., Fraser V. J., Kollef M. H. 2005. Delaying empiric treatment of Candida bloodstream infection until positive blood culture results are obtained: a potential risk factor for mortality. Antimicrob. Agents Chemother. 49:3640–3645 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25. Ostrosky-Zeichner L., et al. 2003. Antifungal susceptibility survey of 2,000 bloodstream Candida isolates in the United States. Antimicrob. Agents Chemother. 47:3149–3154 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26. Ostrosky-Zeichner L., et al. 2008. Rationale for reading fluconazole MICs at 24 hours rather than 48 hours when testing Candida spp. by the CLSI M27-A2 standard method. Antimicrob. Agents Chemother. 52:4175–4177 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27. Pappas P. G., et al. 2009. Clinical practice guidelines for the management of candidiasis: 2009 update by the Infectious Diseases Society of America. Clin. Infect. Dis. 48:503–535 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28. Parkins M. D., Sabuda D. M., Elsayed S., Laupland K. B. 2007. Adequacy of empirical antifungal therapy and effect on outcome among patients with invasive Candida species infections. J. Antimicrob. Chemother. 60:613–618 [DOI] [PubMed] [Google Scholar]
- 29. Pfaller M. A., et al. 2006. Correlation of MIC with outcome for Candida species tested against voriconazole: analysis and proposal for interpretive breakpoints. J. Clin. Microbiol. 44:819–826 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30. Pfaller M. A., et al. 2008. Validation of 24-hour fluconazole MIC readings versus the CLSI 48-hour broth microdilution reference method: results from a global Candida antifungal surveillance program. J. Clin. Microbiol. 46:3585–3590 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31. Pfaller M. A., et al. 2008. Selection of a surrogate agent (fluconazole or voriconazole) for initial susceptibility testing of posaconazole against Candida spp.: results from a global antifungal surveillance program. J. Clin. Microbiol. 46:551–559 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32. Pfaller M. A., et al. 2010. Wild-type MIC distributions, epidemiological cutoff values and species-specific clinical breakpoints for fluconazole and Candida: time for harmonization of CLSI and EUCAST broth microdilution methods. Drug Resist. Updates 13:180–195 [DOI] [PubMed] [Google Scholar]
- 33. Pfaller M. A., et al. 2011. Wild-type MIC distributions and epidemiological cutoff values for posaconazole and voriconazole and Candida spp. as determined by 24-h CLSI broth microdilution. J. Clin. Microbiol. 49:630–637 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34. Pfaller M. A., et al. 2011. Comparison of the broth microdilution (BMD) method of the European Committee on Antibacterial Susceptibility Testing (EUCAST) with the 24-h CLSI BMD method for testing susceptibility of Candida species to fluconazole, posaconazole, and voriconazole by use of epidemiological cutoff values. J. Clin. Microbiol. 49:845–850 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35. Revankar S. G., et al. 1998. Interpretation of trailing endpoints in antifungal susceptibility testing by the National Committee for Clinical Laboratory Standards method. J. Clin. Microbiol. 36:153–156 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36. Rex J. H., et al. 1998. Optimizing the correlation between results of testing in vitro and therapeutic outcome in vivo for fluconazole by testing critical isolates in a murine model of invasive candidiasis. Antimicrob. Agents Chemother. 42:129–134 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37. Rex J. H., Pfaller M. A. 2002. Has antifungal susceptibility testing come of age? Clin. Infect. Dis. 35:982–989 [DOI] [PubMed] [Google Scholar]
- 38. Sabatelli F., et al. 2006. In vitro activity of posaconazole, fluconazole, itraconazole, voriconazole, and amphotericin B against a large collection of clinically important molds and yeasts. Antimicrob. Agents Chemother. 50:2009–2015 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39. Zilberberg, et al. 2010. Inappropriate empiric antifungal therapy for candidemia in the ICU and hospital resource utilization: a retrospective cohort study. BMC Infect. Dis. 10:150–156 [DOI] [PMC free article] [PubMed] [Google Scholar]