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Journal of Clinical Microbiology, May 2008, p. 1793-1803, Vol. 46, No. 5
0095-1137/08/$08.00+0     doi:10.1128/JCM.01883-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Comparison of Neo-Sensitabs Tablet Diffusion Assay with CLSI Broth Microdilution M38-A and Disk Diffusion Methods for Testing Susceptibility of Filamentous Fungi with Amphotericin B, Caspofungin, Itraconazole, Posaconazole, and Voriconazole{triangledown}

A. Espinel-Ingroff1* and E. Canton2

Virginia Commonwealth University Medical Center, Richmond, Virginia,1 Hospital La Fe, Valencia, Spain2

Received 21 September 2007/ Returned for modification 12 November 2007/ Accepted 28 February 2008


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
We compared the Neo-Sensitabs tablet assay to both reference M38-A broth microdilution and disk diffusion methods for testing the susceptibility of 183 filamentous isolates to amphotericin B, caspofungin, itraconazole, posaconazole, and voriconazole. Neo-Sensitabs and disk assay inhibition zone diameters, in millimeters, were obtained on nonsupplemented Mueller-Hinton agar at 16 to 48 h. The reproducibility of zone diameters (i.e., the percentage of replicate zone diameters that were within 2 standard deviations of the means), their correlation with either MICs or minimum effective concentrations (for caspofungin only), and the categorical agreement were similar between tablet and disk assays (93 to 100% [R, >0.70] and 79 to 96%, respectively) with four of the five agents. The exceptions were the results for posaconazole tablets (R, 0.686; disk, 0.757; 84% categorical agreement for tablet and 96% for disk). These data suggest the potential value of the Neo-Sensitabs assay for testing 5-µg caspofungin and 1-µg voriconazole posaconazole tablets against all mold isolates, 8-µg itraconazole and 5-µg tablets against all mold isolates except zygomycetes, and 10-µg amphotericin B tablets against zygomycete isolates only.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS) Subcommittee on Antifungal Susceptibility Tests has developed a reproducible procedure for antifungal susceptibility testing of filamentous fungi (molds) by a broth microdilution format (4, 9, 10). More recently, an agar diffusion method has been developed for testing molds by disk diffusion methodology with five antifungal agents (14). The Neo-Sensitabs assay (A/S Rosco Diagnostica, Taastrup, Denmark) utilizes a 9-mm-diameter (1-mm thickness) tablet for the antifungal susceptibility testing of yeasts and other fungal pathogens with several antifungal agents (Neo-Sensitabs user's guide; A/S Rosco Diagnostica) (2). This assay has been favorably compared with the broth microdilution CLSI M27-A2 method (3) for testing yeasts (1, 20, 23) and, more recently, for mold isolates with posaconazole and amphotericin B (13). Amphotericin B, caspofungin, itraconazole, posaconazole, and voriconazole tablets (A/S Rosco Diagnostica) and 6-mm amphotericin B, itraconazole, and voriconazole disks (Abtek Biologicals Ltd., Liverpool, United Kingdom) are available in Europe but not in the United States.

The purpose of this study was to compare the Neo-Sensitabs tablet assay to both the CLSI reference broth microdilution (document M38-A) assay and the newly developed mold disk diffusion method for testing the susceptibility of 183 molds (Table 1) to five antifungal agents (amphotericin B, caspofungin, itraconazole, posaconazole, and voriconazole). The evaluation included the following determinations: (i) the determination of reference MICs and minimum effective concentrations (MECs; for caspofungin only) of the five agents by the CLSI broth microdilution M38-A method (4, 8, 18), (ii) the determination of inhibition zone diameters (in millimeters) by both disk and commercial Neo-Sensitabs tablet diffusion methods, (iii) the determination of the correlation coefficients between inhibition zone diameters (in millimeters) of both diffusion methods and reference MICs and MECs of the five antifungal agents, and (iv) the determination of the performance of the Neo-Sensitabs method in identifying resistant isolates.


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  		TABLE 1. Set of mold isolates evaluated and in vitro MIC or MEC data

 

   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Isolates. A total of 183 clinical isolates submitted to the Virginia Commonwealth University were evaluated by each method. The set of isolates included strains with different susceptibility patterns for the five agents (Table 1). The CLSI quality control (QC) isolates Candida krusei ATCC 6258 and Paecilomyces variotii ATCC MYA-3630 were tested each time a set of isolates was evaluated with each antifungal agent and by the three procedures. MICs for C. krusei ATCC 6258 were within the established MIC limits of the five antifungal agents (5); the same applied for P. variotii ATCC MYA-3630 with amphotericin B, itraconazole, posaconazole, and voriconazole (12). Caspofungin MEC limits have not been established for molds, but our results were within a three-dilution range (0.015 to 0.06 µg/ml) for the QC P. variotii ATCC MYA-3630 isolate.

(i) CLSI broth microdilution procedure (M38-A). MICs and MECs (for caspofungin only) were determined by the CLSI M38-A broth microdilution method (4, 8, 18). Amphotericin B (Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, CT), caspofungin (Merck Research Laboratories, Rahway, NJ), itraconazole (Janssen, Beerse, Belgium), posaconazole (Schering-Plough Research Institute, Kenilworth, NJ), and voriconazole (Pfizer Central Research, New York, NY) were provided by the manufacturers as standard powders for the preparation of CLSI microdilution trays. Final drug concentrations ranged from 0.01 to 32 µg/ml. Stock inoculum suspensions were prepared from 7-day-old cultures grown on potato dextrose agar (Remel, Lenexa, KS) and were adjusted spectrophometrically to optical densities that ranged from 0.09 to 0.3. The adjusted suspensions were diluted 1:25 (for Scedosporium apiospermum) and 1:50 (for other isolates) in RPMI 1640 broth (containing L-glutamine and 0.165 M morpholinepropanesulfonic acid and without sodium bicarbonate; catalog no. 04-525F; BioWhittaker, Walkersville, MD). Microdilution trays containing 100 µl of the twofold-diluted drugs in RPMI 1640 broth were inoculated with 100 µl of the twofold-diluted inoculum containing between 0.8 x 104 and 5.5 x 104 CFU/ml, as demonstrated by colony counts. Microdilution trays were incubated in ambient air at 35°C and were examined for MIC determinations at 24 (zygomycetes), 72 (Scedosporium spp.), and 48 h (other species) (4). Caspofungin MECs were determined at 48 h (Scedosporium spp.) and 24 h (other species) (8, 14, 18). Reference MICs were defined as the lowest drug concentrations that showed 100% (amphotericin B and triazoles) growth inhibition compared to the growth of the control (4). Caspofungin MECs were defined as the lowest drug concentrations that produced the growth of small, rounded, compact colonies, whereas hyphal growth was seen in the growth control well (8, 14, 18). QC isolates were tested in the same manner.

(ii) Mold disk diffusion procedure. The disk procedure was performed by following the newly identified guidelines for mold testing (14). Briefly, the entire surfaces of nonsupplemented Mueller-Hinton agar plates (150 mm; Hardy Diagnostics, Santa Maria, CA) were inoculated in three directions with the undiluted mold stock inoculum suspensions. The inoculated agar was allowed to dry for 15 to 30 min. Ten-microgram amphotericin B and itraconazole disks (Abtek Biologicals Ltd.) and 5-µg posaconazole, 1-µg voriconazole, and 5-µg caspofungin disks (Becton Dickson and Company, Sparks, MD) were applied to the inoculated agar. The plates were incubated in ambient air at 35°C within 15 min after the disks were applied to the inoculated agar. QC isolates were tested in the same manner.

(iii) Neo-Sensitabs tablet assay. The Neo-Sensitabs tablet assay was performed as described above for the disk methodology on nonsupplemented Mueller-Hinton agar plates (150 mm; Hardy Diagnostics) (13, 14); 9-mm tablets (containing 10 µg amphotericin B, 5 µg caspofungin, 8 µg itraconazole, 5 µg prototype posaconazole, and 1 µg voriconazole), provided by Rosco Laboratory (A/S Rosco Diagnostica), were applied to the inoculated agar. The plates were incubated in ambient air at 35°C. QC isolates were tested in the same manner.

Inhibition zone diameter determination. Zone diameters in both the disk and tablet diffusion assays were measured to the nearest whole millimeter at the point at which there was a prominent reduction of growth (80%) after 16 to 24 h for zygomycetes and after 24 to 72 h for the other species. Microcolonies inside the zone of inhibition and hyphal filaments bending over the inhibition zones were ignored during caspofungin tests, as was slight trailing around the edges during triazole tests, but this was not ignored for amphotericin B (14).

Reproducibility methodology. MICs, MECs, and inhibition zone diameters (for disks and tablets) were obtained with each antifungal agent on three different days for 20 of the 183 isolates evaluated; the selected set of 20 isolates included isolates that were susceptible (MICs or MECs of ≤1 µg/ml) or resistant (MICs or MECs of ≥4 µg/ml) to each of the five antifungal agents. In addition, each of these 20 isolates was tested again when the set of 183 isolates was evaluated by the three methods with each antifungal agent.

Data analysis. Both on-scale and off-scale MICs or MECs, determined by the M38-A reference method (at 24 h for zygomycetes; 48 h for Aspergillus spp., Bipolaris spicifera, Alternaria spp., Fusarium spp., and Paecilomyces spp.; and 72 h for Scedosporium spp.) were correlated with inhibition zone diameters (in millimeters) around disks and tablets obtained at the optimal incubation times (16 to 24 and 48 h). To obtain correlation results (R values), a linear regression analysis by the least-squares method (Pearson's correlation coefficient; MS Excel software) was performed by plotting zone diameters against their respective MIC or MEC endpoints (after log transformation) (Fig. 1 to 5). Diameters obtained on three different days for each of the selected 20 study isolates with each antifungal agent were used to evaluate the reproducibility of both tablet and disk assays. The percentages of reproducibility (i.e., the confidence levels) were obtained based on a selected 12% reproducibility criterion or confidence interval (e.g., 1 mm for 8-mm zones and 5 mm for 42-mm zones); this interval was selected by calculating 2 standard deviations of the relative variation range for each drug-isolate combination (13, 14). Reproducibility values were not obtained as a range of millimeter zone diameter measurements, because millimeter variations in large zones tend to yield larger numbers than the variations in smaller zones. The data generated for both QC isolates (tested more than 10 times) with each antifungal agent also provided zone diameter reproducibility data. Breakpoints are not available for any antifungal agent against molds. However, in a previous study, the following tentative MIC or MEC breakpoints and zone diameter categories, respectively, were assigned using the error-rate bounding method (14): susceptible, ≤1 µg/ml and ≥17 mm (triazoles and caspofungin) and ≥15 mm (amphotericin B); intermediate, 2 µg/ml and 14 to 16 mm (triazoles and caspofungin) and 13 to 14 mm (amphotericin B); and resistant, ≥4 µg/ml and ≤13 mm (triazoles and caspofungin) and ≤12 mm (amphotericin B). In the present study, the performances of both the disk and tablet methods were analyzed using these tentative breakpoints to determine the categorical agreement between the tablet and disk diffusion endpoints and MIC or MEC endpoints of each drug. Major errors were identified as the isolate being resistant by the tablet or disk method but susceptible by the MIC or MEC result, while minor errors were identified by shifts between susceptible and dose-dependent susceptible or between dose-dependent susceptible and resistant. Very major errors were identified as the MIC or MEC showing resistance and the tablet or disk showing susceptibility.


Figure 1
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  		FIG. 1. (a) Correlation of broth microdilution MICs and posaconazole tablet (5 µg) diffusion zone diameters on Mueller-Hinton agar for 183 mold isolates. MICs were determined at the recommended times (24 to 72 h), and zone diameters, in millimeters, were determined at the optimal incubation times (16 to 24 and 48 h). ZD, zone diameter. (b) Correlation of broth microdilution MICs and posaconazole disk (5 µg) diffusion zone diameters on Mueller-Hinton agar for 183 mold isolates. MICs were determined at the recommended times (24 to 72 h), and zone diameters, in millimeters, were determined at the optimal incubation times (16 to 24 and 48 h).

 

Figure 5
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  		FIG. 5. (a) Correlation of broth microdilution MICs and amphotericin B tablet (10 µg) diffusion zone diameters on Mueller-Hinton agar for 183 mold isolates. MICs were determined at the recommended times (24 to 72 h), and zone diameters, in millimeters, were determined at the optimal incubation times (16 to 24 and 48 h). ZD, zone diameter. (b) Correlation of broth microdilution MICs and amphotericin B disk (10 µg) diffusion zone diameters on Mueller-Hinton agar for 183 mold isolates. MICs were determined at the recommended times (24 to 72 h), and zone diameters, in millimeters, were determined at the optimal incubation times (16 to 24 and 48 h).

 

   RESULTS AND DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
We have evaluated for the first time the suitability of the Neo-Sensitabs tablet assay for testing voriconazole, itraconazole, and caspofungin against mold isolates. The suitability of this commercial assay for testing zygomycete and Aspergillus species isolates with amphotericin B and posaconazole has been previously evaluated, but a categorical agreement analysis was not performed in that study (13). Table 1 lists the number of isolates of each species evaluated; the set included the same species that were evaluated in the study that identified the optimal testing guidelines for the disk diffusion testing of molds (14). These optimal testing conditions were used for the evaluation of the Neo-Sensitabs tablet assay in the present study, including the assigned broth dilution and disk diffusion breakpoints. It has been demonstrated that supplemented Mueller-Hinton agar does not support suitable growth for a variety of mold species and that this agar interfered with the correlation between broth dilution and disk diffusion results with either caspofungin for Paecilomyces lilacinus or voriconazole for Aspergillus terreus (14). Because of that, we evaluated the Neo-Sensitabs method with nonsupplemented Mueller-Hinton agar. As previously reported (13, 14), tablet and disk inhibition zone diameters were well defined on this agar and could be determined at 16 to 24 h for zygomycetes, 24 h for most Aspergillus isolates (a few Aspergillus fumigatus and A. terreus isolates needed 48 h of incubation), and at 48 h for the other species. The species evaluated are more frequently associated with severe fungal infections in the immunocompromised host (17), and the antifungal agents are the most important agents licensed for the treatment or prevention of mold infections.

For the 183 isolates and both QC isolates, >95% of the inocula were within the target range of 0.4 x 104 to 5 x 104 CFU/ml. Lower inoculum densities (0.2 x 104 to 0.3 x 104 CFU/ml) were obtained with one to two isolates each of A. terreus, Aspergillus nidulans, and S. apiospermum. Similar reproducibility results for inoculum suspensions have been reported in earlier collaborative studies (92 to 95%) (9, 10). Inoculum suspensions for Alternaria species isolates also were within the target range (optical density, 0.25 to 0.3); however, some of those isolates required a lower incubation temperature (28 to 30°C).

The reproducibility of zone diameters (in millimeters) for the 20 selected resistant and susceptible isolates by the Neo-Sensitabs tablet diffusion assay, which were obtained on three different days with each antifungal agent, was similar to that of the disk diffusion method (confidence level, 93 to 100%). These results also were similar to those obtained for the evaluation of amphotericin B and posaconazole tablets and disks for Aspergillus spp. and the zygomycetes (91 to 100%), as were the results obtained during the recent collaborative disk study for molds (89 to 98%) (13, 14).

Table 2 summarizes the zones of inhibition (in millimeters) obtained with the two QC isolates by the tablet and disk methods. Although reference diameter ranges are not available for either QC isolate against any antifungal agent on nonsupplemented Mueller-Hinton agar, zone diameter data on this agar were compiled for these QC isolates during the collaborative study (14). In general, zone diameters by the disk and tablet assays were within the range of zone diameters obtained in that study (Table 2), which corroborated the reproducibility results obtained by repeated testing for the 20 selected study isolates with each agent. The exceptions were the results with the posaconazole tablet, for which the tablet results were 8 to 11 mm lower than or outside of the zone range obtained with the disk in this and the prior study (13). However, a QC isolate(s) needs to be selected, and standard zone diameter limits established, for mold disk and tablet testing.


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  		TABLE 2. QC strain zone diameters by the Neo-Sensitabs tablet and disk methodsa

 
Table 3 summarizes the correlation coefficients (determined by linear regression analysis) for MIC or MEC results and the corresponding inhibition zone diameters, in millimeters, with the five tablets and disks for the 183 isolates evaluated (Table 1 and Fig. 1 to 5). Although higher R values were observed with the disk method than with the tablet assay, suitable values (R > 0.7 and R2 > 0.5) were obtained with four of the five Neo-Sensitabs tablets. The exception was the R (0.686; R2, 0.47) with the posaconazole tablet, for which the zones were consistently small, especially for certain zygomycete isolates for which the corresponding MICs were low. A similar trend was observed with the amphotericin B tablet, mostly for Aspergillus species and Fusarium species isolates. The correlation results with four of the five Neo-Sensitabs tablets in this study (R, 0.72 to 0.92; R2, 0.518 to 0.846) also were comparable to those reported for Aspergillus spp. (13, 16, 21) and zygomycete (13, 16) isolates with voriconazole (R, 0.79) and posaconazole (R, 0.72 to 0.82) disks as well as in the prior collaborative disk study (R, 0.71 to 0.91) (14).


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  		TABLE 3. Correlation between inhibition zone diameters obtained by the Neo-Sensitabs tablet and disk methods and reference M38-A MICs and MECs (for caspofungin only) of five antifungal agents for 183 mold isolatesa

 
The evaluation of a new method requires the determination of breakpoint category agreement between the new method and the reference method. This type of analysis was performed using the tentative and previously assigned MIC and MEC breakpoints and zone diameter categories (14) as described above. As previously reported, these breakpoints were useful for the purposes of illustration or comparison only. Table 4 depicts the results of the breakpoint category analysis agreement for amphotericin B, caspofungin, itraconazole, posaconazole, and voriconazole tablet and disk inhibition zone diameters. The optimal incubation times for tablet and disk were 16 to 24 h for zygomycetes, A. fumigatus, Aspergillus flavus, and Aspergillus niger and 48 h for the other species on nonsupplemented Mueller-Hinton agar, whereas optimal MICs were obtained at 24 h (zygomycetes), 72 h (Scedosporium spp.), and 48 h (other species). Table 4 also provides the caspofungin MECs from the first reading (16 to 24 [zygomycetes], 48 [Scedosporium spp.], and 24 h [other species]) with tablet and disk zone diameters on the same agar and with the same incubation times.


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  		TABLE 4. Agreement of inhibition zone diameters (by tablet and disk diffusion methods) with MICs or MECs (M38-A method) according to assigned susceptible, intermediate, and resistant categories for 183 mold isolates with five antifungal agents

 
The categorical evaluation of the 5-µg prototype Neo-Sensitabs posaconazole tablet indicated that the proportion of major and very major errors was low (0.5 and 2%) (Table 4 and Fig. 1); four susceptible isolates were categorized as resistant (≤13-mm diameter, major error), and one resistant isolate was categorized as susceptible (≥17-mm diameter, very major error). Of the 25 minor errors, 17 (68%) were among Rhizopus arrhizus and other zygomycete isolates, for which 12 susceptible isolates were categorized as intermediate (14- to 16-mm diameter) and 5 intermediate isolates were categorized as resistant (≤13-mm diameter). In contrast, the 5-µg disk yielded more suitable results (96% overall agreement), and only eight minor errors were observed (mostly for Fusarium species isolates); these results mirrored those obtained in the collaborative study (14). The posaconazole tablet used for the evaluation was the prototype, which could account for its lower level of performance (84% overall agreement) (Table 4).

The 8-µg Neo-Sensitabs itraconazole tablet and the 10-µg disk produced similar numbers of minor errors (tablet, 25; and disk, 24) and percentages of categorical agreement (tablet, 85%; and disk, 87%) (Table 4 and Fig. 2). The only difference was the low percentage of very major (0.5%) and major errors (1%) observed with the tablet. The results also were similar to those of a prior comparison of itraconazole disks and Neo-Sensitabs tablets with a broth dilution methodology for yeast testing despite their different concentrations (15). In this study and as previously reported for the disk test (14), high percentages of minor errors (tablet, 52%; and disk, 63%) were observed among zygomycete isolates by both tablet and disk assays, especially among isolates for which the itraconazole MICs were 2 µg/ml (intermediate category). Therefore, neither itraconazole disks nor tablets are suitable for testing zygomycetes. It is noteworthy that only two to four minor errors were observed among the 87 Aspergillus isolates with both itraconazole and posaconazole tablets and disks.


Figure 2
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  		FIG. 2. (a) Correlation of broth microdilution MICs and itraconazole tablet (8 µg) diffusion zone diameters on Mueller-Hinton agar for 183 mold isolates. MICs were determined at the recommended times (24 to 72 h), and zone diameters, in millimeters, were determined at the optimal incubation times (16 to 24 and 48 h). ZD, zone diameter. (b) Correlation of broth microdilution MICs and itraconazole disk (10 µg) diffusion zone diameters on Mueller-Hinton agar for 183 mold isolates. MICs were determined at the recommended times (24 to 72 h), and zone diameters, in millimeters, were determined at the optimal incubation times (16 to 24 and 48 h).

 
The performance of the 1-µg voriconazole disk was only slightly superior to that of the 1-µg Neo-Sensitabs tablet: 93 and 90% overall categorical agreement, respectively (Table 4). Categorical errors were observed mostly among Fusarium species (six minor errors by disk and tablet tests), Alternaria species isolates, and B. spicifera isolates (four to five minor errors by disk and tablet tests). Two major errors were observed with the disk, and none were observed with the tablet. Some isolates of Fusarium spp. and B. spicifera, for which the voriconazole MICs were 2 µg/ml (intermediate category), were categorized as susceptible (≥17-mm zone diameters) by the tablet and as resistant (≤13-mm zone diameters) by the disk. Both the voriconazole disk and tablet were able to identify as resistant all S. prolificans and zygomycete isolates, as previously reported for the voriconazole disk (14). Therefore, these results suggest that either the tablet or disk assay (R, 0.806 and 0.891, respectively; R2, 0.649 and 0.793, respectively) (Fig. 3) could be the choice for testing the susceptibilities of most mold isolates to voriconazole. Since all zygomycete and Scedosporium prolificans isolates are resistant to voriconazole in this and other studies (6, 11, 14, 19, 22), there is no reason to test these isolates.


Figure 3
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  		FIG. 3. (a) Correlation of broth microdilution MICs and voriconazole tablet (1 µg) diffusion zone diameters on Mueller-Hinton agar for 183 mold isolates. MICs were determined at the recommended times (24 to 72 h), and zone diameters, in millimeters, were determined at the optimal incubation times (16 to 24 and 48 h). ZD, zone diameter. (b) Correlation of broth microdilution MICs and voriconazole disk (1 µg) diffusion zone diameters on Mueller-Hinton agar for 183 mold isolates. MICs were determined at the recommended times (24 to 72 h), and zone diameters, in millimeters, were determined at the optimal incubation times (16 to 24 and 48 h).

 
Table 4 also depicts the categorical agreement between the caspofungin disk and the 5-µg Neo-Sensitabs tablet zone diameters and corresponding MECs. The overall categorical agreement (95 and 96% for tablet and disk, respectively) and R values (0.92 [R2, 0.846] and 0.91 [R2, 0.82], respectively) (Tables 3 and 4 and Fig. 4) were practically the same by both diffusion assays. One very major error (0.5%) was observed with the tablet and none with the disk, while two major errors (1%) were observed with the disk and one with the tablet (0.5%); these errors were among A. nidulans isolates, as previously reported for disk diffusion testing with caspofungin (14). Since both caspofungin tablet and disk assays appear to identify the resistant isolates, they could be the choice for testing the susceptibilities of mold isolates to caspofungin. However, as for voriconazole, there is no reason to test zygomycete, Fusarium species, and S. prolificans isolates (7, 14).


Figure 4
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  		FIG. 4. (a) Correlation of broth microdilution MECs and caspofungin tablet (5 µg) diffusion zone diameters on Mueller-Hinton agar for 183 mold isolates. MECs were determined at the recommended times (24 to 48 h), and zone diameters, in millimeters, were determined at the optimal incubation times (16 to 24 and 48 h). ZD, zone diameter. (b) Correlation of broth microdilution MECs and caspofungin disk (5 µg) diffusion zone diameters on Mueller-Hinton agar for 183 mold isolates. MECs were determined at the recommended times (24 to 48 h), and zone diameters, in millimeters, were determined at the optimal incubation times (16 to 24 and 48 h).

 
The 10-µg Neo-Sensitabs amphotericin B tablet provided the lowest correlation (R, 0.72; R2, 0.518) (Fig. 5) between MICs and inhibition zone diameters compared to those obtained with the five disks and three of the tablets; the posaconazole tablet R value was slightly lower than that with amphotericin B (0.686 versus 0.72 [R2, 0.47 versus 0.518]) (Table 3), but the overall categorical agreement was higher with posaconazole (79 versus 84%) (Table 4). Amphotericin B tablet and disk assays yielded the highest percentage of minor errors despite the suitable R values obtained. In agreement with a previous report (14), both major (false resistant, ≤12-mm zone diameters) and minor errors (34 with the amphotericin B tablet and 30 with the disk) were among Aspergillus species (mostly A. terreus) and Fusarium species (Table 4 and data not shown) isolates; there were either no or a few such conflicting results for Aspergillus spp. with triazole and caspofungin tablets and disks. Five zygomycete isolates were categorized as resistant among the 2-µg/ml MIC group (intermediate category), but as with posaconazole, all other zygomycetes were categorized as susceptible by both tablet and disk assays. Therefore, the amphotericin B tablet and disk appear to be more suitable for testing zygomycetes than for testing other mold species, as previously demonstrated (14).

In conclusion, based on the correlation of inhibition zone diameters with either MICs or MECs, the reproducibility data, and the ability to identify resistant isolates (≥4 µg/ml), the Neo-Sensitabs tablet assay is as suitable as the disk assay for testing molds with caspofungin and voriconazole. Our results also suggest that the itraconazole and prototype posaconazole tablets are not suitable for testing zygomycetes, and neither is the amphotericin B tablet for testing Aspergillus spp., especially A. terreus. Both tablet and disk assays were not always able to differentiate intermediate from susceptible and resistant values for some species and antifungal agent combinations. However, the clinical relevance of these in vitro results has yet to be determined, since breakpoints are not available for mold testing.


   ACKNOWLEDGMENTS
 
Voriconazole disks were provided by Pfizer, posaconazole disks by Schering, and caspofungin disks by Merck. The tablets of the five antifungal agents were provided by Rosco Diagnostica. Mueller-Hinton agar was donated by Hardy Scientific.


   FOOTNOTES
 
* Corresponding author. Present address: 3804 Dover Rd., Richmond, VA 23221. Phone: (804) 358-5895. E-mail: avingrof{at}verizon.net Back

{triangledown} Published ahead of print on 12 March 2008. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

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Journal of Clinical Microbiology, May 2008, p. 1793-1803, Vol. 46, No. 5
0095-1137/08/$08.00+0     doi:10.1128/JCM.01883-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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