Comparison study of broth macrodilution and microdilution antifungal susceptibility tests.

An evaluation of broth dilution antifungal susceptibility tests was performed by determining both the micro- and macrodilution MICs of amphotericin B, flucytosine, fluconazole, ketoconazole, and cilofungin against 38 isolates of Candida albicans, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Cryptococcus neoformans, and Torulopsis glabrata. The following preliminary antifungal working group recommendations of the National Committee for Clinical Laboratory Standards for broth macrodilution tests with antifungal agents were used: inocula standardized to 1 x 10(4) to 5 x 10(4) CFU/ml with a spectrophotometer, RPMI 1640 medium buffered with morpholinopropanesulfonic acid (pH 7.0), incubation at 35 degrees C for 24 to 48 h, and an additive drug dilution procedure. Broth microdilution MICs were higher (two or more dilutions) than broth macrodilution MICs for all isolates tested with amphotericin B and for most isolates tested with ketoconazole, fluconazole, and cilofungin. MICs of flucytosine were the same by both techniques or lower by the broth microdilution test except in tests with C. neoformans. However, the only statistically significant differences between the two tests were observed with amphotericin B against all isolates (P = 0.01 to 0.07), ketoconazole against C. neoformans (P = 0.01 to 0.02), and cilofungin against C. albicans (P = 0.05 to 0.14). Tests performed with less dense inocula (1 x 10(3) to 5 x 10(3] produced similar results.     

Susceptibility testing of Cryptococcus neoformans: a microdilution technique.

We studied a series of test conditions in a microtiter system to define the optimal method for determining the susceptibility of Cryptococcus neoformans to antifungal agents. Twenty-one isolates of C. neoformans were grown for 24 or 48 h in four chemically defined media: yeast nitrogen base (BYNB 7); RPMI 1640; synthetic amino acid medium--fungal (SAAMF), buffered at pH 7.0 to select the medium that best supported growth of this fastidious yeast; and yeast nitrogen base, pH 5.4 (YNB 5.4). Maximum growth of C. neoformans, at 35 degrees C, was obtained in YNB 5.4, with the next highest growth levels in BYNB 7, SAAMF, and RPMI. Growth at 24 h was uniformly poor in all media and lacked reproducibility. In contrast, incubation for 48 h gave adequate growth with low standard deviations, and 48 h was selected as the optimal incubation period for this study. Comparison of the relationship between growth kinetics and initial inoculum size for eight cryptococcal isolates showed that 10(4) cells per ml yielded optimal growth in BYNB 7 and YNB 5.4, whereas 10(5) cells per ml was optimal in RPMI and SAAMF. Furthermore, variation of inocula from 10(3) to 10(5) cells per ml showed small but significant inoculum effects in determining MICs of fluconazole, amphotericin B, and flucytosine for C. neoformans. Therefore, 10(4) cells per ml was chosen as the optimal inoculum for susceptibility testing in this study. Mean MICs of fluconazole, amphotericin B, and flucytosine for 21 crytococcal isolates in RPMI and BYNB 7 were low (for example, fluconazole had mean MICs of 1.2 and 1.3 micrograms/ml in RPMI and BYNB 7, respectively) and differed significantly from medium to medium. In contrast, the MICs obtained in SAAMF were significantly higher (e.g., fluconazole had a mean MIC of 2.2 micrograms/ml). Variance in MICs was large with fluconazole and flucytosine but small with amphotericin B, irrespective of the medium used. A microtiter system employing BYNB 7 as the medium, 48 h as the incubation period, and 10(4) cells per ml as the final inoculum is a simple, accurate, and reproducible method for the testing of C. neoformans susceptibility to fluconazole, amphotericin B, and flucytosine.     

Comparative sensitivity of yeasts to antifungal agents using a standardized micromethod

The comparative susceptibility of 1 850 yeast strains belonging to 8 species was determined. The standardized micromethods used allows determinations of minimal inhibitory concentrations (MICs) or categorized sensitivities for two different concentrations (AB). Overall results showed that amphotericin B (AMB) is the most active agent, followed by the various imidazoles. 5-fluorocytosine (5FC) was the least effective drug, with 68% susceptible strains. However, results varied widely across species and drugs. For instance, among Candida albicans and Torulopsis glabrata strains, none were resistant to AMB and only 6% were resistant to 5-FC; in contrast, Candida albicans was highly susceptible to imidazoles (0.8 to 2.5% resistant strains) whereas Torulopsis glabrata showed much higher resistance rates (18% of strains for tioconazole and 70% for ketoconazole). Variations in susceptibility were also recorded across imidazoles: clotrimazole, tioconazole and ketoconazole were much more effective against Candida tropicalis and Candida parapsilosis than miconazole and econazole, whereas almost no strains were resistant to AMB and more than 50% of strains were resistant to 5-FC. Results obtained by AB (967 strains) and MIC (455 strains) were consistent for the 1 422 Candida albicans strains. Our results show that standardized micromethods should be used to determine the susceptibility of yeasts to antifungal agents.     

Microtiter broth dilution method for yeast susceptibility testing with validation by clinical outcome.

There is no ideal laboratory procedure or culture medium in current use for susceptibility testing of pathogenic yeasts. Six candidate growth media (RPMI 1640 with L-glutamine, yeast nitrogen base, Casamino Acids medium, Mueller-Hinton broth, Sabouraud dextrose broth, and minimum essential medium-Eagle salts) were screened by spectrophotometric absorbance for nucleic acid and protein. From these, two media were selected: a chemically defined growth medium (RPMI 1640 with L-glutamine) and a chemically complex medium (Casamino Acids). MICs of four antifungal agents (5-fluorocytosine, miconazole, ketoconazole, and amphotericin B) for 84 clinical isolates of various Candida species were then determined with both media in agar dilution and microtiter broth dilution systems. The resultant MICs were correlated with clinical outcome for those isolates obtained from patients treated with single antifungal agents, and susceptibility cut points were calculated. Derived MIC cut points for susceptibility were validated in a murine model of systemic candidiasis. RPMI 1640 with L-glutamine was found to have the lowest absorbance values for both nucleic acid and protein, while Casamino Acids medium was highest in both categories. We found that RPMI 1640 with L-glutamine was superior to Casamino Acids medium in the yield of MICs which correlated with actual clinical and animal outcome data. While there were no significant differences in MICs when RPMI 1640 medium was used, the microtiter broth dilution technique was superior to agar dilution in efficiency and ease of performance. We conclude that a microtiter broth system containing RPMI 1640 medium with L-glutamine is a simple, precise, and economical technique for susceptibility testing of pathogenic Candida species. We also suggest that the validation of susceptibility cut points with patient and animal outcome data make this microtiter broth system a preferential method for yeast susceptibility testing.     

Collaborative investigation of broth microdilution and semisolid agar dilution for in vitro susceptibility testing of Candida albicans.

A study was performed in two laboratories to evaluate the effect of growth medium and test methodology on inter- and intralaboratory variations in the MICs of amphotericin B (AMB), flucytosine (5FC), fluconazole (FLU), itraconazole (ITRA), and the triazole Sch 39304 (SCH) against 14 isolates of Candida albicans. Testing was performed by broth microdilution and semisolid agar dilution with the following media, buffered to pH 7.0 with morpholinepropanesulfonic acid (MOPS): buffered yeast nitrogen base (BYNB), Eagle's minimal essential medium (EMEM), RPMI 1640 medium (RPMI), and synthetic amino acid medium for fungi (SAAMF). Inocula were standardized spectrophotometrically, and endpoints were defined by the complete absence of growth for AMB and by no more than 25% of the growth in the drug-free control for all other agents. Comparative analyses of median MICs, as determined by each test method, were made for all drug-medium combinations. Both methods yielded similar (+/- 1 twofold dilution) median MICs for AMB in EMEM and RPMI, 5FC in all media, and FLU in EMEM, RPMI, and SAAMF. In contrast, substantial between-method variations in median MICs were seen for AMB in BYNB and SAAMF, FLU In BYNB, and ITRA and SCH in all media. Interlaboratory concordance of median MICs was good for AMB, 5FC, and FLU but poor for ITRA and SCH in all media. Endpoint determinations were analyzed by use of kappa statistical analyses for evaluating the strength of observer agreement. Moderate to almost perfect interlaboratory agreement occurred with AMB and 5FC in all media and with FLU in EMEM, RPMI, and SAAMF, irrespective of the test method. Slight to almost perfect interlaboratory agreement occurred with ITRA and SCH in EMEM, RPMI, and SAAMF when tested by semisolid agar dilution but not broth microdilution. Kappa values assessing intralaboratory agreement between methods were high for 5FC in all media, for AMB in BYNB, ENEM, and RPMI, and for FLU in EMEM, RPMI, and SAAMF. One laboratory, but not the other, reported substantial to almost perfect agreement between methods for ITRA, and SCH in EMEM, RPMI, and SAAMF. Both laboratories reported poor agreement between methods for the azoles in BYNB. Discrepancies noted in azole-BYNB combinations were largely due to the greater inhibitory effect of these agents in BYNB than in other media. These results indicate that the semisolid agar dilution and broth microdilution methods with EMEM or RPMI yield equivalent and reproducible MICs for AMB, 5FC, and FLU but not ITRA and SCH.     

Evaluation of Etest method for determining voriconazole and amphotericin B MICs for 162 clinical isolates of Cryptococcus neoformans

The performance of the Etest for voriconazole and amphotericin B susceptibility testing of 162 isolates of Cryptococcus neoformans was assessed against the National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 72 h at 35 degrees C. MICs were determined by Etest for all 162 isolates with RPMI 1640 agar containing 2% glucose (RPG agar) and were read after incubation for 72 h at 35 degrees C. The Etest results for both voriconazole and amphotericin B correlated well with reference MICs. Agreement was 94% for voriconazole and 99% for amphotericin B. When discrepancy was noted between the results obtained by Etest and broth microdilution for voriconazole, the Etest generally provided a higher MIC. The Etest method using RPG agar appears to be a useful method for determining the susceptibility of C. neoformans to voriconazole and amphotericin B.     

Interlaboratory evaluation of Etest method for testing antifungal susceptibilities of pathogenic yeasts to five antifungal agents by using Casitone agar and solidified RPMI 1640 medium with 2% glucose.

An interlaboratory evaluation (two centers) of the Etest method was conducted for testing the antifungal susceptibilities of yeasts. The MICs of amphotericin B, fluconazole, flucytosine, itraconazole, and ketoconazole were determined for 83 isolates of Candida spp., Cryptococcus neoformans, and Torulopsis glabrata. Two buffered (phosphate buffer) culture media were evaluated: solidified RPMI 1640 medium with 2% glucose and Casitone agar. MIC endpoints were determined after both 24 and 48 h of incubation at 35 degrees C. Analysis of 3,420 MICs demonstrated higher interlaboratory agreement (percentage of MIC pairs within a 2-dilution range) with Casitone medium than with RPMI 1640 medium when testing amphotericin B (84 to 90% versus 1 to 4%), itraconazole (87% versus 63 to 74%), and ketoconazole (94 to 96% versus 88 to 90%). In contrast, better interlaboratory reproducibility was determined between fluconazole MIC pairs when RPMI 1640 medium rather than Casitone medium was used (96 to 98% versus 77 to 90%). Comparison of the flucytosine MICs obtained with RPMI 1640 medium revealed greater than 80% reproducibility. The study suggests the potential value of the Etest as a convenient alternative method for testing the susceptibilities of yeasts. It also indicates the need for further optimization of medium formulations and MIC endpoint criteria to improve interlaboratory agreement.     

Comparison of a new colorimetric assay with the NCCLS broth microdilution method (M-27A) for antifungal drug MIC determination

We evaluated a new microtiter assay for antifungal susceptibility testing based on a colorimetric reaction to monitor fungal substrate utilization. This new method (rapid susceptibility assay [RSA]) provides quantitative endpoint readings in less than 8 h compared with visual determination of MIC by the National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution method, which requires a minimum of 48 h of incubation. In this study, we tested clinical isolates from each of the following species: Candida albicans (20 isolates), C. glabrata (20 isolates), C. krusei (19 isolates), C. tropicalis (19 isolates), and C. parapsilosis (28 isolates). RSA and NCCLS broth dilution methods were used to determine the MICs of amphotericin B, fluconazole, itraconazole, and 5-flucytosine for all 106 isolates. RPMI 1640 medium buffered with morpholinopropanesulfonic acid was used for both methods; however, glucose and inoculum concentrations in the RSA were modified. RSA MICs were determined as the lowest drug concentration that prevented glucose consumption by the organism after 6 h of incubation. MICs obtained from the RSA were compared with those obtained from the NCCLS M-27A method read at 24 and 48 h. MIC pairs were considered in agreement when the difference between the pairs was within 2 twofold dilutions. For the 106 isolates tested, amphotericin B and 5-flucytosine demonstrated the highest agreement in MICs between the two methods (100 and 98%, respectively), whereas fluconazole and itraconazole produced less favorable MIC agreement (63.2 and 61.3%, respectively). The azole MIC differences between the two methods were significantly reduced when lower inocula were used with a prolonged incubation time. This preliminary comparison suggests that this rapid procedure may be a reliable tool for the in vitro determination of MICs of amphotericin B and 5-flucytosine and warrants further evaluation.     

Multisite reproducibility of the Etest MIC method for antifungal susceptibility testing of yeast isolates.

A multicenter study was performed to establish the interlaboratory reproducibility of Etest, to provide an additional comparison of Etest MICs with reference broth macrodilution MICs, and to develop some tentative quality control (QC) guidelines for using Etest for antifungal susceptibility testing of Candida spp. Two QC strains, Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258, were tested by Etest against amphotericin B, fluconazole, flucytosine, itraconazole, and ketoconazole in each of four laboratories. The QC strains were tested 20 times each against the five antifungal agents by using a common lot of RPMI agar. A total of 80 MICs per drug per strain were generated during the study. Overall, 98 to 100% of the MICs fell within a 3 log2 dilution range for the respective yeast-antifungal agent combinations. The level of agreement of Etest MICs with broth macrodilution MICs was 86 to 100% with amphotericin B (C. krusei and C. parapsilosis), itraconazole (C. krusei and C. parapsilosis), flucytosine (C. parapsilosis), and fluconazole (C. parapsilosis). A lower level of agreement was observed with ketoconazole (C. krusei and C. parapsilosis). Although all participants reported identical Etest MICs, the MICs of flucytosine and fluconazole when tested against C. krusei fell well above the upper limits of the reference range for this strain. The tentative QC limits for the two QC strains and five antifungal agents when tested by the Etest methodology are the same as the QC limits when tested by the reference broth macrodilution method for amphotericin B and C. krusei, itraconazole and C. krusei, flucytosine and C. parapsilosis, fluconazole and C. parapsilosis, and itraconazole and C. parapsilosis. The Etest QC ranges are 1 dilution broader (4-dilution range) than the reference macrodilution method QC ranges for ketoconazole and C. krusei, amphotericin B and C. parapsilosis, and ketoconazole and C. parapsilosis.     

Evaluation of Etest for direct antifungal susceptibility testing of yeasts in positive blood cultures

The performance of the Etest (AB BIODISK, Solna, Sweden) for direct antifungal susceptibility testing of yeasts in positive blood cultures was compared with that of the macrodilution method for determining the MICs of five antifungal agents. Culture broths with blood from bottles positive for yeasts were inoculated directly onto plates for susceptibility testing with the Etest, and the MICs were read after 24 and 48 h of incubation. A total of 141 positive blood cultures (72 cultures of Candida albicans, 31 of Candida tropicalis, 14 of Candida glabrata, 11 of Candida parapsilosis, 3 of Candida krusei, and 3 of Cryptococcus neoformans, 4 miscellaneous yeast species, and 3 mixed cultures) were tested, and the rates of MIC agreement (+/-1 log(2) dilution) between the direct Etest (at 24 and 48 h, respectively) and macrodilution methods were as follows: amphotericin B, 81.8 and 93.5%; flucytosine, 84.8 and 87.7%; fluconazole, 89.4 and 85.5%; itraconazole, 69.7 and 63.8%; ketoconazole, 87.9 and 79.0%. By a large-sample t test, the difference in log(2) dilution between the direct Etest and the macrodilution method was found to be small (P < 0.05). The lone exceptions were ketoconazole at 48 h of incubation and itraconazole at both 24 and 48 h of incubation (P > 0.05). By Tukey's multiple comparisons, the difference between the direct Etest (48 h) and reference methods among different species was found to be less than 1 log(2) dilution. When the MICs were translated into interpretive susceptibility, the minor errors caused by the direct Etest (at 24 and 48 h, respectively) were as follows: flucytosine, 2.3 and 1.4%; fluconazole, 3.0 and 3.6%; itraconazole, 21.2 and 21.3%. Itraconazole also produced an additional 3.0 and 3.6% major errors as determined by the direct Etest at 24 and 48 h, respectively. It was concluded that, except for itraconazole, the Etest method was feasible for direct susceptibility testing of blood cultures positive for yeasts. The method is simple, and the results could be read between 24 and 48 h after direct inoculation, whenever the inhibition zones were discernible.     

Comparison of the Etest and the sensititre colorimetric methods with the NCCLS proposed standard for antifungal susceptibility testing of Aspergillus species

The susceptibilities of 25 clinical isolates of Aspergillus fumigatus, A. flavus, A. terreus, A. nidulans, and A. ustus to itraconazole and amphotericin B were determined by an agar diffusion-dilution method (the Etest method) and a colorimetric broth microdilution method (the Sensititre method); and the results were compared with those obtained by the NCCLS proposed standard M-38P method for antifungal susceptibility testing of filamentous fungi. Various MIC endpoints for the three methods were determined visually by four different observers in three blinded experiments, and the reproducibilities among the observers (interobserver agreement) and among the replicates (interexperimental agreement) as well as the levels of agreement between the NCCLS, the Etest, and the Sensititre methods were calculated. High levels of reproducibility (within 1 twofold dilution) were found for the NCCLS method (>95%) with the MIC-0 endpoint (complete inhibition of growth) for both drugs and with the MIC-1 endpoint (slight growth) for itraconazole and for the Sensititre method (>90%) with all MIC endpoints, although for the latter the interexperimental agreement for itraconazole was comparatively lower (83 to 93%). The Etest method was less reproducible (67 to 87%) for both drugs. Using the recommended MIC endpoints, high levels of agreement (within one twofold dilution) between the NCCLS and the Sensititre methods for all species were found for amphotericin B (>77%) but not for itraconazole (<66%), for which the MICs by the Sensititre method were up to 3 twofold dilutions lower than the corresponding MICs by the NCCLS method. The use of the first blue well as an endpoint for the Sensititre method and 48 h of incubation improved the levels of agreement with the NCCLS method. Low levels of agreement between the NCCLS and the Etest methods using the recommended MIC endpoints were found for most species, especially after 48 h of incubation (<50%), when the MICs obtained by the Etest method were up to 9 twofold dilutions higher than the corresponding MICs obtained by the NCCLS method. Relatively better agreement was found after 24 h, although it was species dependent, with the highest levels of agreement (>82%) found for A. terreus and A. ustus for amphotericin B and A. fumigatus for both drugs. Overall, better agreement was found when MIC-0 was used as the MIC endpoint for the NCCLS method for both drugs and when the MICs by the Etest method were determined after 48 h of incubation for itraconazole and after 24 h of incubation for amphotericin B.     

Use of fatty acid RPMI 1640 media for testing susceptibilities of eight Malassezia species to the new triazole posaconazole and to six established antifungal agents by a modified NCCLS M27-A2 microdilution method and Etest

A novel formulation of RPMI 1640 medium for susceptibility testing of Malassezia yeasts by broth microdilution (BMD) and Etest is proposed. A modification of the NCCLS M27-A2 BMD method was used to test 53 isolates of Malassezia furfur (12 isolates), M. sympodialis (8 isolates), M. slooffiae (4 isolates), M. globosa (22 isolates), M. obtusa (2 isolates), M. restricta (2 isolates), M. pachydermatis (1 isolates), and M. dermatis (2 isolates) against amphotericin B, ketoconazole, itraconazole, fluconazole, voriconazole, terbinafine, and posaconazole by BMD and Etest. RPMI and antibiotic medium 3 (AM3) were supplemented with glucose, bile salts, a mixture of fatty acids, and n-octadecanoate fatty acids and Tween 20. M. furfur ATCC 14521 and M. globosa ATCC 96807 were used as quality control strains. Depending on the species, MICs were read after 48 or 72 h of incubation at 32 degrees C. Low azole and terbinafine MICs were recorded for all Malassezia species, whereas amphotericin B displayed higher MICs (>/=16 microg/ml) against M. furfur, M. restricta, M. globosa, and M. slooffiae strains, which were AM3 confirmed. Agreement of the two methods was 84 to 97%, and intraclass correlation coefficients were statistically significant (P < 0.001). Because of higher amphotericin B MICs provided by Etest for strains also displaying high BMD MICs (>/=1 microg/ml), agreement was poorer. The proposed media are used for the first time and can support optimum growth of eight Malassezia species for recording concordant BMD and Etest MICs.     

Variability of clarithromycin and erythromycin susceptibility tests with Haemophilus influenzae in four different broth media and correlation with the standard disk diffusion test.

Four separate laboratories performed antimicrobial susceptibility tests with 40 Haemophilus influenzae isolates, each tested in triplicate. Erythromycin and a new macrolide, clarithromycin (A-56268; TE-031), were tested by the disk diffusion method, by the agar dilution procedure in two different media, and by broth microdilution tests in four different media. Erythromycin MICs for 90% of the strains were 16 micrograms/ml in Mueller-Hinton broth with 3% lysed horse blood and NAD, 4.0 micrograms/ml in hemophilus test medium, and 2.0 micrograms/ml in supplemented Schaedler broth or in the fastidious broth medium from Beckman Instruments, Inc. Clarithromycin MICs were generally 1 doubling dilution greater than erythromycin MICs in each of the media. Erythromycin disk tests corresponded best with MICs determined in the fastidious broth medium. In that same medium, clarithromycin MICs were about 1 doubling dilution greater than what would be expected from the results of disk tests. Because there were fewer growth failures, hemophilus test medium is recommended for microdilution tests with H. influenzae. Incubation of all tests for a full 24 h without an increased CO2 atmosphere was needed to achieve maximal precision of the tests. Interlaboratory and intralaboratory reproducibility of all tests was satisfactory.     

Influence of Incubation Time, Inoculum Size, and Glucose Concentrations on Spectrophotometric Endpoint Determinations for Amphotericin B, Fluconazole, and Itraconazole

We addressed the influence of the incubation time (24 h versus 48 h), starting inoculum size (standard inoculum size, ~10³ CFU/ml, versus large inoculum size, ~10⁴ CFU/ml), and supplementation with 2% glucose of RPMI 1640 medium on the spectrophotometric determination of the MICs of amphotericin B, fluconazole, and itraconazole. We compared the MICs determined spectrophotometrically with those determined by the standard broth macrodilution method (National Committee for Clinical Laboratory Standards approved guideline M27-A). The agreement between the results of the spectrophotometric and standard methods for amphotericin B testing was 100%; this agreement was independent of the inoculum size and incubation time. On the other hand, the agreement for the results for fluconazole testing and itraconazole testing was dependent on the inoculum size and incubation time. With large inoculum size, excellent agreement can be achieved at 24 h. With standard inoculum size, acceptable agreement can be achieved only at 48 h. In contrast to previous observations, the addition of 2% glucose did not have any significant impact on the growth density at 24 h, nor did it improve the agreement with the standard method. Furthermore, supplemental glucose might falsely elevate the MIC at 48 h.     

Comparative evaluation of three antifungal susceptibility test methods for Candida albicans isolates and correlation with response to fluconazole therapy.

In vitro susceptibilities were determined for 56 Candida albicans isolates obtained from the oral cavities of 41 patients with human immunodeficiency virus infection. The agents tested included fluconazole, itraconazole, ketoconazole, flucytosine, and amphotericin B. MICs were determined by the broth microdilution technique following National Committee for Clinical Laboratory Standards document M27-P (M27-P micro), a broth microdilution technique using high-resolution medium (HR micro), and the Etest with solidified yeast-nitrogen base agar. The in vitro findings were correlated with in vivo response to fluconazole therapy for oropharyngeal candidiasis. For all C. albicans isolates from patients with oropharyngeal candidiasis not responding to fluconazole MICs were found to be > or = 6.25 micrograms/ml by the M27-P micro method and > or = 25 micrograms/ml by the HR micro method as well as the Etest. However, for several C. albicans isolates from patients who responded to fluconazole therapy MICs found to be above the suggested breakpoints of resistance. The appropriate rank order of best agreement between the M27-P micro method and HR micro method was amphotericin B > fluconazole > flucytosine > ketoconazole > itraconazole. The appropriate rank order with best agreement between the M27-P micro method and the Etest was flucytosine > amphotericin B > fluconazole > ketoconazole > or = itraconazole. It could be concluded that a good correlation between in vitro resistance and clinical failure was found with all methods. However, the test methods used in this study did not necessarily predict clinical response to therapy with fluconazole.     

Evaluation of Etest Method for Determining Posaconazole MICs for 314 Clinical Isolates of Candida Species

The performance of the Etest for posaconazole (SCH 56592) susceptibility testing of 314 isolates of Candida spp. was assessed against the National Committee for Clinical Laboratory Standards (NCCLS) microdilution broth method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35 degrees C. MICs were determined by Etest for all 314 isolates with RPMI agar containing 2% glucose (RPG agar) and were read after incubation for 48 h at 35 degrees C. The Candida isolates included C. albicans (n = 174), C. glabrata (n = 57), C. tropicalis (n = 31), C. parapsilosis (n = 39), C. krusei (n = 5), C. guilliermondii (n = 6), and C. lusitaniae (n = 2). The Etest results correlated well with reference MICs. Overall agreement was 95%, and agreements for individual species were as follows: C. krusei, 100%; C. albicans, 98%; C. tropicalis, 97%; C. glabrata, 93%; C. parapsilosis, 85%; C. guilliermondii, 83%; and C. lusitaniae, 50%. The problem of trailing end points was minimized with RPG agar, and good agreement with broth dilution MICs was obtained when discernible growth within an established ellipse was ignored. The Etest method using RPG agar appears to be a useful method for determining posaconazole susceptibilities of Candida species.     

E-test method for testing susceptibilities of Aspergillus spp. to the new triazoles voriconazole and posaconazole and to established antifungal agents: comparison with NCCLS broth microdilution method

NCCLS document M38-P describes standard parameters for testing the fungistatic activities (MICs) of established agents against filamentous fungi (molds). This study evaluated the in vitro susceptibilities of 15 Aspergillus flavus isolates, 62 A. fumigatus isolates, and 10 isolates each of A. niger, A. nidulans, and A. terreus to voriconazole, posaconazole, itraconazole, and amphotericin B by the E-test and NCCLS M38-P microdilution methods. The agreement (within 3 dilutions) between methods for voriconazole was independent of the E-test incubation time (93.3 to 100% for four of five species at both incubation times). In contrast, with amphotericin B, itraconazole, and posaconazole, E-test results were more dependent on the incubation time for certain species. For A. fumigatus, posaconazole E-test MICs had better concordance with reference values after 48 h (95.2%) than after 24 h (90%), while the highest agreement for itraconazole MICs was after 24 h (90.3 versus 74.2%) of incubation. Better agreement between the methods was also obtained with 24-h E-test amphotericin B MICs for A. flavus (73.3 versus 26.7%) and A. fumigatus (96.7 versus 64.5%). E-test MICs of the four agents had the lowest percentages of agreement with reference values for A. nidulans (60 to 80%). For isolates for which high MICs were obtained for the four agents by the reference method, high MICs were also obtained by E-test at both 24 and 48 h. The utility of in vitro results of either the E-test or the NCCLS broth microdilution (M38-P) method for Aspergillus spp. needs to be established in clinical trials.     

Determination of the range of antibacterial activity by use of viable counts

The activity of three aminoglycosides and six beta-lactam antibiotics on strains of Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus, and enterococci was studied. The minimal inhibitory concentrations (MICs), the minimal bactericidal concentrations (MBCs), and the minimal antibiotic concentrations (MACs) were determined after 5 h of incubation in broth cultures by colony-forming-unit counts. The MICs were also determined by agar dilution after 24 h of incubation. The MICs on agar after 24 h of incubation were higher than those in broth after 5 h of incubation. The differences ranged from 1.1- to 14.2-fold, but in most cases were only three- to fivefold (P less than 0.05 to less than 0.001). The MBCs at 5 and 24 h were comparable in 71% of tests. For current practice, the MBC of enterococci can be determined after 5 h of incubation with antibiotics. The aminoglycosides showed MBCs which were closer to the MICs than were those of the beta-lactam antibiotics, which required a higher multiple of the MIC to show a bactericidal effect. The MBCs of oxacillin and cefamandole for S. aureus after 5 h of incubation were greater than 128 times the respective MICs. The MACs ranged from 1/1.5 to 1/7 of the 5-h MICs. The three endpoints, MIC, MBC, and MAC, indicate the antibacterial range of an antibiotic in terms of inhibition of growth and bacterial survival. The data suggest that the antibacterial range of an antibiotic is similar for most strains of a given species and is, to some extent, a characteristic of similar antibiotics.     

In vitro susceptibilities of sucrose-negative Candida tropicalis, Candida lusitaniae, and Candida norvegensis to amphotericin B, 5-fluorocytosine, miconazole, and ketoconazole.

The MICs and minimal lethal concentrations of four antimycotics, amphotericin B, 5-fluorocytosine, miconazole nitrate, and ketoconazole, were determined for 25 yeast isolates representing species uncommonly implicated in candidiasis. A microdilution procedure was employed with complex and synthetic media. The isolates, in general, were susceptible to the same antimicrobial agents shown to be effective against Candida albicans, but differences between some of the species in relative susceptibilities to the antifungal agents were noted. Isolates of atypical sucrose-negative Candida tropicalis were similar in their susceptibility patterns to typical isolates of the species. Relative resistance to amphotericin B, miconazole nitrate, and ketoconazole was noted for two Candida lusitaniae isolates, but all strains were susceptible to 5-fluorocytosine. Candida norvegensis isolates were more resistant to miconazole and ketoconazole than C. albicans clinical isolates. The microtiter system was satisfactory for determining minimal inhibitory concentrations, but the system is not recommended for detecting finite differences in drug susceptibilities or for detecting drug synergism.     

Effects of incubation time and buffer concentration on in vitro activities of antifungal agents against Candida albicans.

Nine selected isolates of Candida albicans were tested for their susceptibilities to amphotericin B and fluconazole by using three methods to assess the effect of incubation time and buffer concentration. By using a microdilution method with 0.0165 M 3-(N-morpholino)propanesulfonic acid (MOPS) and a 24-h incubation time, all of the isolates were found to be susceptible to amphotericin B and fluconazole. After 48 h of incubation, all isolates were still susceptible to amphotericin B. Seven of the nine isolates were resistant to fluconazole, and for the remaining two isolates, MICs increased by fourfold or more but the isolates remained susceptible (MIC, < or = 10 microg/ml). The nine isolates, along with three control strains, were further tested against amphotericin B and fluconazole by a standard broth macrodilution method with both 0.165 and 0.0165 M MOPS. The susceptibility results for fluconazole by the broth macrodilution method with the lower MOPS concentration correlated with the results of the 24-h broth microdilution method for determination of susceptibility or resistance in eight of nine tests and with the results of the 48 h broth microdilution method in three of nine tests. The results of the broth macrodilution method with the standard MOPS concentration did not correlate with any of the results obtained by the 24-h broth microdilution but correlated with results of seven of nine tests by the 48-h broth microdilution method. All nine test strains appeared to be susceptible when they were examined by a flow cytometric method. For clinical yeast susceptibility testing in microdilution panels, the 0.0165 M MOPS concentration combined with 24 h of incubation appeared to be the method of choice. The lower MOPS concentration may also be a useful modification to the tentative broth macrodilution method of the National Committee for Clinical Laboratory Standards. Use of the higher buffer concentration or longer incubation time may lead to false in vitro resistance for agents like fluconazole.