The availability of reproducible antifungal susceptibility testing methods now permits analysis of data correlating susceptibility in vitro with outcome in vivo in order to define interpretive breakpoints. In this paper, we have examined the conceptual framework underlying interpretation of antimicrobial susceptibility testing results and then used these ideas to drive analysis of data packages developed by the respective manufacturers that correlate fluconazole and itraconazole MICs with outcome of candidal infections. Tentative fluconazole interpretive breakpoints for MICs determined by the National Committee for Clinical Laboratory Standards' M27-T broth macrodilution methodology are proposed: isolates for which MICs are < or = 8 microg/mL are susceptible to fluconazole, whereas those for which MICs are > or = 64 microg/mL appear resistant. Isolates for which the MIC of fluconazole is 16-32 microg/mL are considered susceptible dependent upon dose (S-DD), on the basis of data indicating clinical response when > 100 mg of fluconazole per day is given. These breakpoints do not, however, apply to Candida krusei, as it is considered inherently resistant to fluconazole. Tentative interpretive MIC breakpoints for itraconazole apply only to mucosal candidal infections and are as follows: susceptible, < or = 0.125 microg/mL; S-DD, 0.25-0.5 microg/mL; and resistant, > or = 1.0 microg/mL. These tentative breakpoints are now open for public commentary.

The antifungal susceptibilities of 232 pathogenic bloodstream Candida isolates collected during a recently completed trial comparing fluconazole (400 mg/day) with amphotericin B (0.5 mg/kg of body weight per day) as treatment for candidemia in the nonneutropenic patient were determined both by the National Committee for Clinical Laboratory Standards M27-P macrobroth methodology and by a less cumbersome broth microdilution methodology. For amphotericin B, M27-P yielded a very narrow range of MICs (0.125 to 1 g/ml) and there were no susceptibility differences among species. For fluconazole, a broad range of MICs were seen (0.125 to >64 g/ml), with characteristic MICs seen for each species in the rank order Candida albicans < C. parapsilosis Х Х C. lusitaniae < C. glabrata Х Х C. krusei Х Х C. lipolytica. The MIC distribution for C. tropicalis was bimodal and could not be ranked. Broth microdilution MICs were within one tube dilution of the M27-P MIC for Ն Ն90% of isolates with amphotericin B and for Ն Ն77% of isolates with fluconazole. For both methods, elevated MICs did not predict treatment failure. In the case of amphotericin B, the MIC range was too narrow to permit identification of resistant isolates. In the case of fluconazole, MICs for isolates associated with failure to clear the bloodstream consistently were equivalent to the median MIC for the given species. Successful courses of therapy were seen with four isolates from four patients despite MICs of Ն Ն32 g/ml. As MICs obtained by M27-P and similar methods correlate with responsiveness to fluconazole therapy in animal models and in AIDS patients with oropharyngeal candidiasis, the lack of correlation in this setting suggests that the MICs for these isolates are at or below the relevant fluconazole breakpoint for this dose of fluconazole and patient setting and that host factors such as failure to exchange intravenous catheters were more important than MIC in predicting outcome.Antifungal susceptibility testing remains in evolution. Recent work by the National Committee for Clinical Laboratory Standards has focused on the development of standard, reproducible methods for testing of yeast. The proposed method M27-P is a broth macrodilution method that has good interand intralaboratory reproducibility (5, 10). As MICs obtained by methods similar to M27-P have generally correlated well with outcome in various animal models of infection, it is anticipated that M27-P (or a method utilizing the principles of M27-P) will prove useful in prediction of the likelihood of response to a given antifungal agent (16). However, no breakpoints have yet been established for M27-P. During our recently completed trial of fluconazole versus amphotericin B for therapy of candidemia in nonneutropenic patients, we collected Candida bloodstream isolates from our patients (15). We now report the distribution of M27-P MICs seen in this collection of pathogenic Candida isolates, the correlation of these MICs with an easily performed broth microdilution variation of M27-P, and the ...

The in vitro activity of daptomycin is affected by the concentration of calcium cations in the test medium. Mueller-Hinton broth is currently adjusted to contain 10 to 12.5 mg of magnesium per liter and 20 to 25 mg of calcium per liter, but for testing of daptomycin, greater concentrations of calcium (50 mg/liter) are recommended to better resemble the normal concentration of ionized calcium in human serum. Two levels of calcium were used for broth microdilution tests of 2,789 recent clinical isolates of gram-positive bacterial pathogens. MICs of daptomycin were two-to fourfold lower when the broth contained additional calcium. For most species, however, the percentages of strains that were inhibited by 2.0 g of daptomycin per ml were essentially identical with the two broth media. Enterococci were the important exception; i.e., 92% were inhibited when tested in calcium-supplemented broth but only 35% were inhibited by 2.0 g/ml without the additional calcium. This type of information should be considered when selecting criteria for defining in vitro susceptibility to daptomycin.Daptomycin is a lipopeptide which is bactericidal against a wide range of gram-positive bacterial pathogens, including the antibiotic-resistant pneumococci, enterococci, and staphylococci that are currently presenting a challenge for the design of empirical chemotherapy. Daptomycin is now being evaluated for possible use in situations in which there may be a high prevalence of antibiotic-resistant gram-positive bacteria (10).The antibacterial activity of daptomycin requires the presence of calcium cations. Previous studies (1-3, 5, 6) have led to the recommendation that when testing daptomycin, the broth medium should contain additional calcium approaching the concentration of ionized calcium that is normally found in human serum (ca. 50 mg/liter). For broth microdilution susceptibility tests of other agents, the National Committee for Clinical Laboratory Standards (NCCLS) recommends cationadjusted Mueller-Hinton broth (CAMHB) that is adjusted to contain 20 to 25 mg of Ca 2ϩ per liter and 10 to 12.5 mg of Mg 2ϩ per liter (7). Those concentrations were selected in order to standardize microdilution tests of the aminoglycosides and tetracyclines, but when testing daptomycin, calcium supplemented CAMHB is recommended (1-3, 6). In the present study, we further assessed the impact of that recommendation by testing more than 2,700 consecutive clinical isolates in both types of broth medium in order to determine which species are likely to be most profoundly affected by the additional calcium. Others have proposed provisional MIC breakpoints of Յ2 g/ml for susceptible, 4 g/ml for intermediate, and Ն8 g/ml for resistant (2, 3, 6, 10). Interpretive criteria have not yet been officially established, but we have focused our attention on MICs ranging from 2.0 to 4.0 g/ml when assessing the effect of adding additional calcium to the broth medium.Consecutive clinical isolates of gram-positive pathogens were collected from 11 medical centers throughout ...

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Interpretive agreements among the results of fluconazole broth microdilution tests, Etests, and disk diffusion tests were documented by evaluating 495 Candida spp. Microdilution reference test results were in agreement with 96% of the Etest results; most discrepancies were minor differences. Fluconazole resistance of Candida krusei strains often required a full 48 h of incubation in order to be observed by the standard method. For the disk diffusion tests that were performed on Mueller-Hinton agar with glucose and methylene blue, 97% of results were in agreement with those of the reference test, especially when zones of inhibition were measured after the first 24 h of incubation. Some Candida glabrata isolates failed to grow satisfactorily until a full 48 h of incubation was completed. Precision was determined by testing 50 selected isolates in triplicate in each of three laboratories. The reproducibility of results of disk diffusion tests was comparable to that of the reference method. With all procedures, determination of test results was particularly challenging with some strains, and new methods are needed in order to improve endpoint definition.Compromised patients, such as those infected with human immunodeficiency virus, are often colonized by and/or infected with fungi, especially Candida spp. Consequently, they frequently receive antifungal agents such as fluconazole for relatively long periods of time during hospitalization. Prolonged exposure to an azole such as fluconazole can select strains with diminished susceptibilities (6, 7), and consequently, increasing doses may be necessary. For this reason, the susceptibilities of isolates recovered from compromised patients receiving prolonged fluconazole treatment should be monitored in order to determine when strains with decreased susceptibilities have been selected (3). In order to do this in a cost-effective way, simple, inexpensive testing procedures are needed, but such tests must be accurate and precise. The purpose of this report is to document the relative levels of accuracy and precision of three procedures for testing fluconazole: broth microdilution, Etest, and disk diffusion. The accuracy of each procedure was assessed by comparing the results to those obtained by the 48-h microdilution reference method of the National Committee for Clinical Laboratory Standards (NCCLS) (8). Precision was measured by performing replicate tests in three separate laboratories. MATERIALS AND METHODSMicroorganisms. For this study, M. A. Pfaller selected 495 clinical isolates of Candida spp. for which there was a broad range of fluconazole MICs. The five species that were represented are shown in Table 1. For testing reproducibility, 50 of the 495 isolates were selected in order to maximize the number of strains for which MICs were near the interpretive breakpoints of Յ8.0 g/ml (susceptible) and Ն64 g/ml (resistant). This subset of strains included 26 strains of Candida albicans and 6 strains each of Candida glabrata, Candida krusei, Candida parapsilosis, and Cand...