Effect of increasing inoculum sizes of pathogenic filamentous fungi on MICs of antifungal agents by broth microdilution method.

Inoculum size is a critical variable in development of methods for antifungal susceptibility testing for filamentous fungi. In order to investigate the influence of different inoculum sizes on MICs of amphotericin B, 5-fluorocytosine, itraconazole, and miconazole, 32 clinical isolates (8 Aspergillus fumigatus, 8 Aspergillus flavus, 5 Rhizopus arrhizus, 8 Pseudallescheria boydii, and 3 Fusarium solani isolates) were studied by the broth microdilution method. Four inoculum sizes were studied: 1 x 10(2) to 5 x 10(2), 1 x 10(3) to 5 x 10(3), 1 x 10(4) to 5 x 10(4), and 1 x 10(5) to 5 x 10(5) CFU/ml. The National Committee for Clinical Laboratory Standards reference method for antifungal susceptibility testing in yeasts was modified and applied to filamentous fungi. The inoculum was spectrophotometrically adjusted, and all tests were performed in buffered medium (RPMI 1640) at pH 7.0 with incubation at 35 degrees C for 72 h. MICs were read at 24, 48, and 72 h. Amphotericin B showed a minimum effect of inoculum size on MICs for all species with the exception of P. boydii (P < 0.05). A significant effect of inoculum size on MICs was observed with 5-fluorocytosine, for which there was an increase of more than 10-fold in MICs against all Aspergillus spp. between inoculum concentrations of 10(2) and 10(4) CFU/ml (P < 0.001). For itraconazole, the results showed a more species-dependent increase of MICs, most strikingly for R. arrhizus and P. boydii. Miconazole, which was tested only with P. boydii, did not demonstrate a significant effect of inoculum size on MICs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multicenter evaluation of proposed standardized procedure for antifungal susceptibility testing of filamentous fungi.

A multicenter study was conducted to expand the generation and analysis of data that supports the proposal of a reference method for the antifungal susceptibility testing of filamentous fungi. Broth microdilution MICs of amphotericin B and itraconazole were determined in 11 centers against 30 coded duplicate pairs of Aspergillus spp., Fusarium spp., Pseudallescheria boydii, and Rhizopus arrhizus. The effect of inoculum density (approximately 10(3) and 10(4) CFU/ml), incubation time (24, 48, and 72 h), and procedure of MIC determination (conventional and colorimetric [Alamar Blue] evaluation of growth inhibition) on intra- and interlaboratory agreement was analyzed. Based on intra- (97 to 100%) and interlaboratory (94 to 95%) agreement for both drugs, the overall optimal testing conditions identified were determination of colorimetric MICs after 48 to 72 h of incubation with an inoculum density of approximately 10(4) CFU/ml. These testing conditions are proposed as guidelines for a reference broth microdilution method.     

Collaborative evaluation of optimal antifungal susceptibility testing conditions for dermatophytes

A multicenter study was conducted to define the most suitable testing conditions for antifungal susceptibility of dermatophytes. Broth microdilution MICs of clotrimazole, itraconazole, and terbinafine were determined in three centers against 60 strains of dermatophytes. The effects of inoculum density (ca. 10(3) and 10(4) CFU/ml), incubation time (3, 7, and 14 days), endpoint criteria for MIC determination (complete [MIC-0] and prominent [MIC-2] growth inhibition), and incubation temperature (28 and 37 degrees C) on intra- and interlaboratory agreement were analyzed. The optimal testing conditions identified were an inoculum of 10(4) CFU/ml, a temperature of incubation of 28 degrees C, an incubation period of 7 days, and MIC-0.     

In vitro fungicidal activities of voriconazole, itraconazole, and amphotericin B against opportunistic moniliaceous and dematiaceous fungi

The NCCLS proposed standard M38-P describes standard parameters for testing the fungistatic antifungal activities (MICs) of established agents against filamentous fungi (molds); however, standard conditions are not available for testing their fungicidal activities (minimum fungicidal or lethal concentrations [MFCs]). This study evaluated the in vitro fungistatic and fungicidal activities of voriconazole, itraconazole, and amphotericin B against 260 common and emerging molds (174 Aspergillus sp. isolates [five species], 23 Fusarium sp. isolates [three species], 6 Paecilomyces lilacinus isolates, 6 Rhizopus arrhizus isolates, 23 Scedosporium sp. isolates, 23 dematiaceous fungi, and 5 Trichoderma longibrachiatum isolates). MICs were determined by following the NCCLS M38-P broth microdilution method. MFCs were the lowest drug dilutions that resulted in fewer than three colonies. Voriconazole showed similar or better fungicidal activity (MFC at which 90% of isolates tested are killed [MFC(90)], 1 to 2 microg/ml) than the reference agents for Aspergillus spp. with the exception of Aspergillus terreus (MFC(90) of voriconazole and amphotericin B, >8 microg/ml). The voriconazole geometric mean (G mean) MFC for Scedosporium apiospermum was lower (2.52 microg/ml) than those of the other two agents (5.75 to 7.5 microg/ml). In contrast, amphotericin B and itraconazole G mean MFCs for R. arrhizus were 2.1 to 2.2 microg/ml, but that for voriconazole was >8 microg/ml. Little or no fungicidal activity was shown for Fusarium spp. (2 to >8 microg/ml) and Scedosporium prolificans (>8 microg/ml) by the three agents, but voriconazole had some activity against P. lilacinus and T. longibrachiatum (G mean MFCs, 1.8 and 4 microg/ml, respectively). The fungicidal activity of the three agents was similar (G mean MFC, 1.83 to 2.36 microg/ml) for the dematiaceous fungi with the exception of the azole MFCs (>8 microg/ml) for some Bipolaris spicifera and Dactylaria constricta var. gallopava. These data extend and corroborate the available fungicidal results for the three agents. The role of the MFC as a predictor of clinical outcome needs to be established in clinical trials by following standardized testing conditions for determination of these in vitro values.     

Modification of rapid susceptibility assay for antifungal susceptibility testing of Aspergillus fumigatus

To improve objectivity and speed of current antifungal mold susceptibility testing, the yeast Rapid Susceptibility Assay (RSA) was adapted for Aspergillus species. The RSA is based on glucose utilization in the presence of an antifungal drug. Aspergillus fumigatus conidia were incubated in 0.2% glucose RPMI 1640 containing 0.03 to 16 micro g of amphotericin B or itraconazole/ml. Drug-related inhibition of glucose utilization correlated with suppression of conidial germination. Following incubation of conidia with various concentrations of antifungal drug, the percentage of residual glucose in the growth medium was determined colorimetrically and plotted against drug concentration to determine the MIC (MIC(RSA)). National Committee for Clinical Laboratory Standards (NCCLS) M38-P testing was also performed to obtain NCCLS MICs (MIC(NCCLS)) for direct comparison with MIC(RSA)s. Conidial inocula of an optical density at 530 nm (OD(530)) of 0.11 facilitated determination of amphotericin B and itraconazole MIC(RSA)s at 16 h equal to or within a single twofold dilution of MIC(NCCLS)s obtained at 48 h. Preliminary testing with a 0.11-OD(530) conidial inoculum of the slower-growing Aspergillus terreus resulted in itraconazole and amphotericin B MIC(RSA)s at 16 h equal to or within a single twofold dilution of MIC(NCCLS)s obtained at 48 h. These data indicate that the mold RSA provides a more objective and rapid method for Aspergillus spp. susceptibility testing than the NCCLS M38-P assay.     

Standardization of a hyphal inoculum of aspergilli for amphotericin B susceptibility testing.

Standardized, evenly dispersed hyphal suspensions served as the inoculum in a microtiter technique for amphotericin B antifungal susceptibility testing. Preliminary testing with six strains of Aspergillus fumigatus and A. flavus produced consistent and reproducible results at 30 degrees C over 24 h. The observed amphotericin B MICs required for hyphae (0.3 to 0.6 microgram/ml) were comparable to MICs required for conidia (0.16 to 0.6 microgram/ml). The results were evaluated and compared with previously published information.     

Susceptibility of 100 filamentous fungi: comparison of two diffusion methods, Neo-Sensitabs and E-test, for amphotericin B, caspofungin, itraconazole, voriconazole and posaconazole

We compared the E-test method to that of the Neo-Sensitabs tablet diffusion assay for evaluating the in vitro susceptibility of 100 clinical isolates of filamentous fungi (Aspergillus spp., Fusarium spp., Scedosporium spp., zygomycetes and other molds) to amphotericin B, itraconazole, voriconazole, caspofungin, and posaconazole. We determined the categorical agreement level between E-test minimum inhibitory concentrations (MIC) and tablet end-points, as opposed to the following disagreement parameters: very major error - resistant parameter (R) in E-test and susceptible (S) in tablet; major error - S by E-test and R by tablet; minor error - shifts between S and susceptible dose-dependent (S-DD) or S-DD and R. We also performed linear regression analyses and computed Pearson's correlation coefficients (R values) between the log transforms of MICs and the inhibition zone diameters of the five studied antifungal agents. For itraconazole we obtained 97% categorical agreement and R = -0.727. Categorical agreement for caspofungin and voriconazole was 96% and R =-0.821 and R = -0.789, respectively. For posaconazole the categorical agreement was 94% and R =-0.743. Amphotericin B exhibited a lower degree of agreement (76%, R = -0.672), especially in studies of Aspergillus spp. Our results suggest a potential value of the Neo-Sensitabs assay for in vitro susceptibility testing of molds to itraconazole, voriconazole, caspofungin and posaconazole, while amphotericin B exhibited an overall lower degree of agreement.     

Sporothrix schenckii sensitivity to voriconazole, itraconazole and amphotericin B.

One hundred clinical isolates of Sporothrix schenckii were tested against voriconazole, itraconazole and amphotericin B using a modification of the NCCLS M27-A in vitro yeast susceptibility testing procedure. NCCLS M38-P for moulds was not used because yeast forms may have been present when the test isolates were incubated at 35 +/- 1 degrees C. The minimum inhibitory concentration (MIC) values were: voriconazole 0.5-8 (geometric mean titer 6.50) microg ml(-1) ; itraconazole 0.03-8 (geometric mean titer 1.56) microg ml(-1); and amphotericin B 0.25-2 (geometric mean titer 1.23) microg ml(-1). The minimum fungicidal concentration (MFC) values were: voriconazole 2-8 (geometric mean titer 7.67) microg ml(-1); itraconazole 0.125-8 (geometric mean titer 7.41) microg ml(-1); and amphotericin B 0.125-2 (geometric mean titer 1.53) microg ml(-1). Based upon MIC values, sensitivity to amphotericin B is strain-dependent. S. schenckii is more sensitive to itraconazole than voriconazole based upon a comparison of MIC geometric means, even though the MIC ranges were essentially the same.     

Influence of glucose supplementation and inoculum size on growth kinetics and antifungal susceptibility testing of Candida spp

The influences of inoculum size and glucose supplementation on the growth kinetics of 60 Candida spp. clinical isolates (Candida albicans, Candida tropicalis, Candida parapsilosis, Candida glabrata, Candida krusei, and Candida lusitaniae [10 isolates each]) are assessed. The combined influence of growth and reading method (visual or spectrophotometric) on the determination of the MICs of amphotericin B, flucytosine, fluconazole, itraconazole, ketoconazole, and voriconazole is also analyzed, and the MICs are compared with those determined by the National Committee for Clinical Laboratory Standards standard microdilution method (NCCLS document M27-A). Glucose supplementation and inoculum size had a significant influence on the growth cycles of these yeasts, and a statistically significant denser growth (optical density at 540 nm) was seen for both incubation periods, 24 and 48 h (P < 0.01). A longer exponential phase and shorter lag phase were also observed. The A540 values at 24 h of incubation with medium containing glucose and an inoculum of 10(5) CFU/ml were >0.4 U for all species, with the exception of that for C. parapsilosis (A540 = 0.26 +/- 0.025). The MICs at 24 h determined by testing with 2% glucose and an inoculum of 10(5) CFU/ml showed the strongest agreement (96.83%) with MICs determined by the reference method. MICs were not falsely elevated, and good correlation indexes were obtained. The reproducibility of results with this medium-inoculum combination was high (intraclass correlation coefficient, 0.955). The best agreement and reproducibility of results for spectrophotometric readings were achieved with endpoints of 50% growth inhibition for flucytosine and azoles and 95% for amphotericin B. Supplementation of test media with glucose and an inoculum size of 10(5) CFU/ml yielded a reproducible technique that shows elevated agreement with the reference procedures and a shorter incubation period for obtaining reliable MIC determinations. The spectrophotometric method offers an advantage over the visual method by providing a more objective and automated MIC determination.     

Susceptibility profile of echinocandins,azoles and amphotericin B against yeast phase of <Emphasis Type="Italic">Talaromyces marneffei</Emphasis> isolated from HIV-infected patients in Guangdong,China

Talaromyces marneffei (T. marneffei) can cause talaromycosis, a fatal systemic mycosis, in patients with AIDS. With the increasing number of talaromycosis cases in Guangdong, China, we aimed to investigate the susceptibility of 189 T. marneffei clinical strains to eight antifungal agents, including three echinocandins (anidulafungin, micafungin, and caspofungin), four azoles (posaconazole, itraconazole, voriconazole, and fluconazole), and amphotericin B, with determining minimal inhibition concentrations (MIC) by Sensititre YeastOne? YO10 assay in the yeast phase. The MICs of anidulafungin, micafungin, caspofungin, posaconazole, itraconazole, voriconazole, fluconazole, and amphotericin B were 2 to >?8 μg/ml, >8 μg/ml, 2 to >?8 μg/ml, ≤?0.008 to 0.06 μg/ml, ≤?0.015 to 0.03 μg/ml, ≤?0.008 to 0.06 μg/ml, 1 to 32 μg/ml, and ≤?0.12 to 1 μg/ml, respectively. The MICs of all echinocandins were very high, while the MICs of posaconazole, itraconazole, and voriconazole, as well as amphotericin B were comparatively low. Notably, fluconazole was found to have a higher MIC than other azoles, and exhibited particularly weak activity against some isolates with MICs over 8 μg/ml. Our data in vitro support the use of amphotericin B, itraconazole, voriconazole, and posaconazole in management of talaromycosis and suggest potential resistance to fluconazole.     

Synergistic interaction of terbinafine with triazoles or amphotericin B against Aspergillus species.

The in vitro activity of terbinafine alone and in combination with other antifungal agents was tested against isolates of Aspergillus fumigatus, A. flavus and A. niger. Testing was performed in a modified National Committee for Clinical Laboratory Standards (NCCLS) macrodilution broth assay, and interactions were examined using a checkerboard design. Terbinafine was highly active against Aspergillus isolates (minimum inhibitory concentration [MIC] 0.01 to 2 microg ml(-1)) with a primary fungicidal action (minimum fungicidal concentration [MFC] 0.02 to 4 microg ml(-1)). Amphotericin B was also highly active and cidal as expected (MIC 1 microg ml(-1), MFC 1 to 4 microg ml(-1)). The triazoles itraconazole and voriconazole were highly active but showed a variable degree of cidal activity against the different strains, voriconazole having the more potent cidal activity. Fluconazole had no significant activity (MIC > 128 microg ml(-1)). Drug combinations were tested in the A. fumigatus and A. niger strains. Terbinafine and amphotericin showed an additive to synergistic interaction depending on the isolate. Combinations of terbinafine with itraconazole or voriconazole displayed a potent synergistic interaction and fungicidal activity against all isolates. Surprisingly, fluconazole also potentiated the activity of terbinafine in an additive to synergistic fashion, despite its lack of activity alone. The results suggest potential clinical application of terbinafine in aspergillosis, either alone or in combination with amphotericin or triazoles.     

Antifungal susceptibility testing method for resource constrained laboratories

Purpose: In resource-constrained laboratories of developing countries determination of antifungal susceptibility testing by NCCLS/CLSI method is not always feasible. We describe herein a simple yet comparable method for antifungal susceptibility testing. Methods: Reference MICs of 72 fungal isolates including two quality control strains were determined by NCCLS/CLSI methods against fluconazole, itraconazole, voriconazole, amphotericin B and cancidas. Dermatophytes were also tested against terbinafine. Subsequently, on selection of optimum conditions, MIC was determined for all the fungal isolates by semisolid antifungal agar susceptibility method in Brain heart infusion broth supplemented with 0.5% agar (BHIA) without oil overlay and results were compared with those obtained by reference NCCLS/CLSI methods. Results: Comparable results were obtained by NCCLS/CLSI and semisolid agar susceptibility (SAAS) methods against quality control strains. MICs for 72 isolates did not differ by more than one dilution for all drugs by SAAS. Conclusions: SAAS using BHIA without oil overlay provides a simple and reproducible method for obtaining MICs against yeast, filamentous fungi and dermatophytes in resource-constrained laboratories.     

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.     

Multicenter evaluation of a new disk agar diffusion method for susceptibility testing of filamentous fungi with voriconazole, posaconazole, itraconazole, amphotericin B, and caspofungin

The purpose of this study was to correlate inhibition zone diameters, in millimeters (agar diffusion disk method), with the broth dilution MICs or minimum effective concentrations (MECs) (CLSI M38-A method) of five antifungal agents to identify optimal testing guidelines for disk mold testing. The following disk diffusion testing parameters were evaluated for 555 isolates of the molds Absidia corymbifera, Aspergillus sp. (five species), Alternaria sp., Bipolaris spicifera, Fusarium sp. (three species), Mucor sp. (two species), Paecilomyces lilacinus, Rhizopus sp. (two species), and Scedosporium sp. (two species): (i) two media (supplemented Mueller-Hinton agar [2% dextrose and 0.5 microg/ml methylene blue] and plain Mueller-Hinton [MH] agar), (ii) three incubation times (16 to 24, 48, and 72 h), and (iii) seven disks (amphotericin B and itraconazole 10-microg disks, voriconazole 1- and 10-microg disks, two sources of caspofungin 5-microg disks [BBL and Oxoid], and posaconazole 5-microg disks). MH agar supported better growth of all of the species tested (24 to 48 h). The reproducibility of zone diameters and their correlation with either MICs or MECs (caspofungin) were superior on MH agar (91 to 100% versus 82 to 100%; R, 0.71 to 0.93 versus 0.53 to 0.96 for four of the five agents). Based on these results, the optimal testing conditions for mold disk diffusion testing were (i) plain MH agar; (ii) incubation times of 16 to 24 h (zygomycetes), 24 h (Aspergillus fumigatus, A. flavus, and A. niger), and 48 h (other species); and (iii) the posaconazole 5-microg disk, voriconazole 1-microg disk, itraconazole 10-microg disk (for all except zygomycetes), BBL caspofungin 5-microg disk, and amphotericin B 10-microg (zygomycetes only).     

Colorimetric assay for antifungal susceptibility testing of Aspergillus species

A colorimetric assay for antifungal susceptibility testing of Aspergillus species (Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Aspergillus nidulans, and Aspergillus ustus) is described based on the reduction of the tetrazolium salt 2,3-bis(2-methoxy-4-nitro-5-[(sulphenylamino)carbonyl]-2H-tetrazolium-hydroxide (XTT) in the presence of menadione as an electron-coupling agent. The combination of 200 microg of XTT/ml with 25 microM menadione resulted in a high production of formazan within 2 h of exposure, allowing the detection of hyphae formed by low inocula of 10(2) CFU/ml after 24 h of incubation. Under these settings, the formazan production correlated linearly with the fungal biomass and less-variable concentration effect curves for amphotericin B and itraconazole were obtained.     

Characterization of clinical strains of Aspergillus terreus complex: molecular identification and antifungal susceptibility to azoles and amphotericin B

We used molecular techniques to analyse 87 (n = 70 patients) Aspergillus terreus complex isolates, all of which were identified as A. terreus sensu stricto. The antifungal susceptibilities determined with CLSI M38-A2 (and Etest for amphotericin B) and expressed as mg/L for range of MIC/MIC(90) /geometric mean were as follows: itraconazole, 0.25-2/2/1.097; voriconazole, 0.125-2/2/1.176; posaconazole, 0.25-1/1/0.836; amphotericin B CLSI, 4-32/16/9.689; and Etest, 0.75-64/6/3.106. The MICs for amphotericin B were significantly higher than those found for the triazoles.     

Antifungal susceptibility and phylogeny of opportunistic members of the order mucorales

The in vitro susceptibilities of 66 molecularly identified strains of the Mucorales to eight antifungals (amphotericin B, terbinafine, itraconazole, posaconazole, voriconazole, caspofungin, micafungin, and 5-fluorocytosine) were tested. Molecular phylogeny was reconstructed based on the nuclear ribosomal large subunit to reveal taxon-specific susceptibility profiles. The impressive phylogenetic diversity of the Mucorales was reflected in susceptibilities differing at family, genus, and species levels. Amphotericin B was the most active drug, though somewhat less against Rhizopus and Cunninghamella species. Posaconazole was the second most effective antifungal agent but showed reduced activity in Mucor and Cunninghamella strains, while voriconazole lacked in vitro activity for most strains. Genera attributed to the Mucoraceae exhibited a wide range of MICs for posaconazole, itraconazole, and terbinafine and included resistant strains. Cunninghamella also comprised strains resistant to all azoles tested but was fully susceptible to terbinafine. In contrast, the Lichtheimiaceae completely lacked strains with reduced susceptibility for these antifungals. Syncephalastrum species exhibited susceptibility profiles similar to those of the Lichtheimiaceae. Mucor species were more resistant to azoles than Rhizopus species. Species-specific responses were obtained for terbinafine where only Rhizopus arrhizus and Mucor circinelloides were resistant. Complete or vast resistance was observed for 5-fluorocytosine, caspofungin, and micafungin. Intraspecific variability of in vitro susceptibility was found in all genera tested but was especially high in Mucor and Rhizopus for azoles and terbinafine. Accurate molecular identification of etiologic agents is compulsory to predict therapy outcome. For species of critical genera such as Mucor and Rhizopus, exhibiting high intraspecific variation, susceptibility testing before the onset of therapy is recommended.     

Microdilution susceptibility testing of amphotericin B, itraconazole, and voriconazole against clinical isolates of Aspergillus and Fusarium species

We compared the activities of amphotericin B, itraconazole, and voriconazole against clinical Aspergillus (n = 82) and Fusarium (n = 22) isolates by a microdilution method adopted from the National Committee for Clinical Laboratory Standards (NCCLS-M27A). RPMI 1640 (RPMI), RPMI 1640 supplemented to 2% glucose (RPMI-2), and antibiotic medium 3 supplemented to 2% glucose (AM3) were used as test media. MICs were determined after 24, 48, and 72 h. A narrow range of amphotericin B MICs was observed for Aspergillus isolates, with minor variations among species. MICs for Fusarium isolates were higher than those for Aspergillus isolates. MICs of itraconazole were prominently high for two previously defined itraconazole-resistant Aspergillus fumigatus isolates and Fusarium solani. Voriconazole showed good in vitro activity against itraconazole-resistant isolates, but the MICs of voriconazole for F. solani were high. RPMI was the most efficient medium for detection of itraconazole-resistant isolates, followed by RPMI-2. While the significance remains unclear, AM3 lowered the MICs, particularly those of amphotericin B.     

In Vitro Antifungal Activity of Clotrimazole (Bay b 5097)

The in vitro antifungal activity of clotrimazole (Bay b 5097) was compared with those of amphotericin B, griseofulvin, nystatin, and pyrrolnitrin. The inhibitory activity of clotrimazole against most systemic pathogens was comparable to that of amphotericin B; minimal inhibitory concentrations of the two drugs for Blastomyces dermatitidis, Histoplasma capsulatum, Sporothrix schenckii, Cryptococcus neoformans, and Coccidioides immitis were in the range of 0.20 to 3.13 and 0.10 to 6.25 mug/ml, respectively. One isolate of Allescheria boydii was resistant to 100 mug of amphotericin B per ml but was inhibited by 6.25 mug of clotrimazole per ml. Clotrimazole was less active than amphotericin B against Candida albicans and Aspergillus fumigatus. The activity of clotrimazole against dermatophytes was comparable to that of pyrrolnitrin; 0.78 mug of either compound per ml was fungicidal for most isolates of Trichophyton sp., Microsporum sp. and Epidermophyton floccosum. Both griseofulvin and nystatin were less active than clotrimazole. The size of inoculum was shown to have a significant effect on the results of in vitro susceptibility testing with clotrimazole.     

Comparison of the E-test with the NCCLS M38-P method for antifungal susceptibility testing of common and emerging pathogenic filamentous fungi

The National Committee for Clinical Laboratory Standards (NCCLS) M38-P method describes standard parameters for testing the fungistatic antifungal activities (MICs) of established agents against filamentous fungi (molds). The present study evaluated the in vitro fungistatic activities of itraconazole and amphotericin B by the E-test and the NCCLS M38-P microdilution method against 186 common and emerging pathogenic molds (123 isolates of Aspergillus spp. [five species], 16 isolates of Fusarium spp. [two species], 4 Paecilomyces lilacinus isolates, 5 Rhizopus arrhizus isolates, 15 Scedosporium spp., 18 dematiaceous fungi, and 5 Trichoderma longibrachiatum isolates). The agreement between the methods for amphotericin B MICs ranged from 70% for Fusarium solani to > or =90% for most of the other species after the first reading; agreement was dependent on both the incubation time and the species being evaluated. Major discrepancies between the amphotericin B MICs determined by the E-test and the NCCLS M38-P method were demonstrated for three of the five species of Aspergillus tested and the two species of Fusarium tested. This discrepancy was more marked after 48 h of incubation; the geometric mean MICs determined by the E-test increased between 24 and 48 h from between 1.39 and 3.3 microg/ml to between 5.2 and >8 microg/ml for Aspergillus flavus, Aspergillus fumigatus, and Aspergillus nidulans. The agreement between the itraconazole MICs determined by the E-test and the NCCLS M38-P method ranged from 83.3% for A. nidulans to > or =90% for all the other species tested; the overall agreement was higher (92.7%) than that for amphotericin B (87.9%). The agreement was less dependent on the incubation time. Clinical trials need to be conducted to establish the role of the results of either the E-test or the NCCLS M38-P method in vitro for molds with the two agents as predictors of clinical outcome.