Establishing a method of inoculum preparation for susceptibility testing of Trichophyton rubrum and Trichophyton mentagrophytes

A total of 92 clinical isolates of dermatophytes (52 of Trichophyton rubrum and 40 of Trichophyton mentagrophytes) were selected for testing with six antifungal drugs (terbinafine, griseofulvin, clotrimazole, miconazole, isoconazole, and fluconazole) and two pairs of drug combinations (ketoconazole-cyclopiroxolamine and itraconazole-cyclopiroxolamine). Two methods of inoculum preparation for susceptibility testing were evaluated that used (i) inocula consisting only of microconidia of dermatophytes filtered in Whatman filter model 40 and (ii) unfiltered inocula consisting of hyphae and microconidia. We followed the recommendations of approved document M38-A of CLSI (formerly NCCLS) with some adaptations, including an incubation period of 7 days and an incubation temperature of 28 degrees C. Reference strains of Candida parapsilosis, Candida krusei, Trichophyton rubrum, and Trichophyton mentagrophytes were included as quality-control strains. MICs were consistently higher (usually 1 to 2 dilutions for drugs tested individually) when nonfiltered inocula were tested (P < 0.01) except for terbinafine. Larger MICs were seen when testing drugs with nonfiltered inocula. The curves of drug interaction were used to analyze the reproducibility of the test, and it was shown that high levels of reproducibility were achieved using the methodology that included the filtration step. The standardization of methodologies is the first step to yield reliability of susceptibility testing and to proceed with clinical laboratory studies to correlate MICs with clinical outcomes.     

Evaluation of microdilution and disk diffusion methods for antifungal susceptibility testing of dermatophytes.

A total of 63 dermatophyte strains belonging to 6 species (Trichophyton rubrum (16), T. mentagrophytes (16), T. tonsurans (20), T. violaceum (2), Microsporum canis (7), and Epidermophyton floccosum (2)) were tested for their in vitro susceptibility to a range of antifungal drugs using disk diffusion and Clinical Laboratory Standards Institute M38-A (CLSI, formerly NCCLS) broth microdilution methods. The antifungals used were ciclopirox (CIC), terbinafine (TER), griseofulvin (GRE), fluconazole (FLC), itraconazole (ITR), posaconazole (POS), and ravuconazole (RAV). In the broth microdilution assay terbinafine was found to have the highest activity followed by ravuconazole, posaconazole, ciclopirox, itraconazole, griseofulvin and fluconazole. In the disk diffusion assay terbinafine produced the largest inhibition zone diameters (IZDs) on Dermasel agar media followed by ravuconazole, griseofulvin, posaconazole, and itraconazole. A significant correlation was not observed between the minimum inhibitory concentrations (MICs) and IZDs, but some correlations were observed for POS, RAV, and TER (correlation coefficients r=-0.507, -0.249, -0.267, P<0.05, respectively). MICs obtained by the microdilution method with ITR and GRE did not correlate with IZDs obtained in disk diffusion assays in this study. The ciclopirox (20 microg/disk) and fluconazole (25 microg/disk) did not produce well defined inhibition zone diameters on Dermasel agar medium.     

Interlaboratory study of quality control isolates for a broth microdilution method (modified CLSI M38-A) for testing susceptibilities of dermatophytes to antifungals

The Clinical and Laboratory Standards Institute (CLSI; formerly National Committee for Clinical Laboratory Standards, or NCCLS) M38-A standard for the susceptibility testing of filamentous fungi does not specifically address the testing of dermatophytes. In 2003, a multicenter study investigated the reproducibility of the microdilution method developed at the Center for Medical Mycology, Cleveland, Ohio, for testing the susceptibility of dermatophytes. Data from that study supported the introduction of this method for testing dermatophytes in the future version of the CLSI M38-A standard. In order for the method to be accepted by CLSI, appropriate quality control isolates needed to be identified. To that end, an interlaboratory study, involving the original six laboratories plus two additional sites, was conducted to evaluate potential candidates for quality control isolates. These candidate strains included five Trichophyton rubrum strains known to have elevated MICs to terbinafine and five Trichophyton mentagrophytes strains. Antifungal agents tested included ciclopirox, fluconazole, griseofulvin, itraconazole, posaconazole, terbinafine, and voriconazole. Based on the data generated, two quality control isolates, one T. rubrum isolate and one T. mentagrophytes isolate, were identified and submitted to the American Type Culture Collection (ATCC) for inclusion as reference strains. Ranges encompassing 95.2 to 97.9% of all data points for all seven drugs were established.     

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.     

Antifungal susceptibility testing of dermatophytes: establishing a medium for inducing conidial growth and evaluation of susceptibility of clinical isolates

A standardized reference method for dermatophyte in vitro susceptibility testing is lacking. In a previous study, Norris et al. (H. A. Norris, B. E. Elewski, and M. A. Ghannoum, J. Am. Acad. Dermatol. 40(6, part 2):S9-S13) established the optimal medium and other growth variables. However, the earlier study did not address two issues: (i) selection of an optimal medium for conidial formation by dermatophytes and (ii) validation of the method with a large number of dermatophytes. The present study addresses these two points. To select which agar medium best supported conidial growth, representative isolates of dermatophytes were grown on different agars. Preliminary experiments showed that only oatmeal cereal agar supported the production of conidia by Trichophyton rubrum. We tested the abilities of 251 T. rubrum isolates to form conidia using three different cereal agars and potato dextrose agar. Overall, oatmeal cereal and rice agar media were comparable in their abilities to support T. rubrum conidial growth. Next, we used the oatmeal cereal agar for conidial formation along with the optimal conditions for dermatophyte susceptibility testing proposed by Norris et al. and determined the antifungal susceptibilities of 217 dermatophytes to fluconazole, griseofulvin, itraconazole, and terbinafine. Relative to the other agents tested, terbinafine possessed the highest antifungal activity against all of the dermatophytes. The mean +/- standard error of the mean MICs of fluconazole, itraconazole, terbinafine, and griseofulvin were 2.07 +/- 0.29, 0.13 +/- 0.01, 0.002 +/- 0.0003, and 0.71 +/- 0.05 microgram/ml, respectively. This study is the first step in the identification of optimal conditions that could be used for the standardization of the antifungal susceptibility testing method for dermatophytes. Inter- and intralaboratory agreement as well as clinical correlations need to be established.     

Antifungal drug response in an in vitro model of dermatophyte nail infection.

Despite terbinafine being fungicidal against Trichophyton rubrum in standard NCCLS assays and rapidly accumulating in nails in vivo, onychomycosis patients require prolonged terbinafine treatment to be cured. To investigate this, we developed a more clinically relevant onychomycosis in vitro test model. Human nail powder inoculated with T. rubrum and incubated in liquid RPMI 1640 salt medium, which did not support growth alone, developed extensive and invasive mycelial growth. Antifungal drugs were added at different concentrations and cultures incubated for 1 to 4 weeks. Fungal survival was determined by spreading cultures on PDA plates without drug and measuring CFU after 1 to 4 weeks incubation. Drug activity was expressed as the nail minimum fungicidal concentration (Nail-MFC) required for 99.9% elimination of viable fungus. Terbinafine Nail-MFC was 4 microg/ml after 1 week exposure, decreasing to 1 microg/ml after 4 weeks exposure, much higher than MFCs < or = 0.03 microg/ml determined in standard NCCLS MIC assays. In contrast, other clinically used drugs were unable to kill T. rubrum after 4 weeks incubation in this model. Invasive mycelial growth on nail appears to protect T. rubrum from the cidal action of systemic drugs, thus providing a rationale for the long treatment periods in onychomycosis.     

Selection of candidate quality control isolates and tentative quality control ranges for in vitro susceptibility testing of yeast isolates by National Committee for Clinical Laboratory Standards proposed standard methods.

The National Committee for Clinical Laboratory Standards has developed a proposed standard method for in vitro antifungal susceptibility testing of yeast isolates (National Committee for Clinical Laboratory Standards, document M27-P, 1992). In order for antifungal testing by the M27-P method to be accepted, reliable quality control (QC) performance criteria must be developed. In the present study, five laboratories tested 10 candidate QC strains 20 times each against three antifungal agents: amphotericin B, fluconazole, and 5-fluorocytosine. All sites conformed to the M27-P standards and used a common lot of tube dilution reagents and RPMI 1640 broth medium. Overall, 98% of MIC results with amphotericin B, 95% with fluconazole, and 92% with 5-fluorocytosine fell within the desired 3-log2 dilution range (mode +/- 1 log2 dilution). Excellent performance with all three antifungal agents was observed for six strains: Candida albicans ATCC 90028, Candida parapsilosis ATCC 90018, C. parapsilosis ATCC 22019, Candida krusei ATCC 6258, Candida tropicalis ATCC 750, and Saccharomyces cerevisiae ATCC 9763. With these strains, 3-log2 dilution ranges encompassing 94 to 100% of MICs for all three drugs were established. Additional studies with multiple lots of RPMI 1640 test medium will be required to establish definitive QC ranges.     

Intra- and interlaboratory study of a method for testing the antifungal susceptibilities of dermatophytes

The National Committee for Clinical Laboratory Standards (NCCLS) M38-A standard for the susceptibility testing of conidium-forming filamentous fungi does not explicitly address the testing of dermatophytes. This multicenter study, involving six laboratories, investigated the MIC reproducibility of seven antifungal agents tested against 25 dermatophyte isolates (5 blinded pairs of five dermatophyte species per site for a total of 300 tests), using the method of dermatophyte testing developed at the Center for Medical Mycology, Cleveland, Ohio. The dermatophytes tested included Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton tonsurans, Epidermophyton floccosum, and Microsporum canis. Seven antifungals with activity against dermatophytes were tested, including ciclopirox, fluconazole, griseofulvin, itraconazole, posaconazole, terbinafine, and voriconazole. Interlaboratory MICs for all isolates were in 92 to 100% agreement at a visual endpoint reading of 50% inhibition as compared to the growth control and 88 to 99% agreement at a visual endpoint reading of 80% inhibition as compared to the growth control. Intralaboratory MICs between blinded pairs were in 97% agreement at a visual endpoint reading of 50% inhibition as compared to the growth control and 96% agreement at a visual endpoint reading of 80% inhibition as compared to the growth control. Data from this study support consideration of this method as an amendment to the NCCLS M38-A standard for the testing of dermatophytes.     

Factors affecting griseofulvin susceptibility testing of Trichophyton rubrum in microcultures.

A microculture broth assay system for griseofulvin susceptibility testing of Trichophyton rubrum was further characterized. The effects of mass and number of colony-forming units of a fragmented mycelial inoculum, 5- or 8-day incubation periods, 25 or 32 degrees C incubation temperatures, and the solvents used to dissolve griseofulvin on the minimal inhibitory concentration (MIC) of griseofulvin were determined. An inoculum density with an absorbance of 0.600 at 450 nm ensured successful inoculation of all microcultures. Reduction of the inoculum mass to an absorbance of 0.200 lowered the number of colony-forming units in the inoculum by 60 to 80%. This decreased the efficiency of inoculation but did not alter the resulting MIC. There was no correlation between MIC and the number of colony-forming units used to initiate growth. Neither incubation temperature nor the length of incubation affected the MIC. The use of either acetone or ethanol to solubilize griseofulvin likewise had no effect on the MIC. The mean reproducibility of the MICs determined with the microculture method was 96%.     

Lyophilized microculture susceptibility test for ketoconazole, miconazole, clotrimazole, and griseofulvin against dermatophytes.

A lyophilized microculture antimycotic susceptibility testing system for ketoconazole, miconazole, griseofulvin, and clotrimazole is described. Microculture plates were loaded with 100 microliters of medium and 10 microliters of appropriate concentrations of the four antimycotics and were lyophilized to complete dryness. The lyophilized plates were stored at -70 degrees C or 4 degrees C or in a desiccator at 25 degrees C. Samples from each storage condition were rehydrated at 1, 2, 3, 4, 5, 6, 8, 10, and 12 months and inoculated with Trichophyton mentagrophytes (Robin) Blanchard ATCC 18748. All of the minimal inhibitory concentrations (MICs) generated from the lyophilized microcultures were within one experimental dilution of MICs derived from fresh microcultures. The ability of reconstituted lyophilized microcultures to consistently produce MICs comparable to MICs derived from fresh microcultures was characterized. Nine dermatophyte isolates were tested five times each over a 70-day period. The MICs derived were reproducible and comparable to MICs determined by freshly prepared microculture tests. Lyophilization of freshly prepared antimycotic-containing microcultures does not alter the MIC resolution of the testing system and provides an effective method of storage of prepared antimycotic tests for ketoconazole, miconazole, clotrimazole, and griseofulvin.     

In vitro susceptibility of Microsporum canis and other dermatophyte isolates from veterinary infections during therapy with terbinafine or griseofulvin.

We investigated the in vitro activity of terbinafine against fresh veterinary isolates of Microsporum canis and the potential of this organism to develop resistance in vivo during oral therapy. Dermatophyte cultures (n = 300) were obtained from naturally infected cats and dogs undergoing oral therapy with terbinafine or griseofulvin. M. canis comprised 92% of isolates; other species included Microsporum gypseum and Trichophyton mentagrophytes. Minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) of terbinafine and griseofulvin were determined by broth macrodilution assay. Terbinafine was highly active against all three species with MIC90< or =0.03 microg ml(-1), in agreement with published data. However, terbinafine exhibited primary cidal activity against 66% of Microsporum isolates (n = 275) in contrast to the almost complete cidal effect in Trichophyton (n = 18). Griseofulvin was significantly less active than terbinafine (MIC90 = 4 microg ml(-1)) but had a primary cidal action on about 40% of the isolates. The data were analysed for changes in MIC and MFC during the course of therapy, which could be indicative for development of acquired resistance. Oral treatment of 37 animals with terbinafine for up to 39 weeks caused no increase in MIC or MFC of terbinafine, either in individual patients or in the whole group.     

Dermatophytes growth curve and in vitro susceptibility test: a broth micro-titration method.

The introduction of systemic antifungal drugs which act upon different targets is the main issue of the in vivo antifungal resistance control. Different factors, such as growth curve phase, quality of the specimen, quantity of the inoculum, temperature, pH, culture medium composition, incubation duration and solvent, are believed important factors affecting minimum inhibitory concentration (MIC) value to most of the antifungal agents. We assayed an in vitro susceptibility test with 40 isolates of dermatophytes: Microsporum canis, Trichophyton rubrum, Trichophyton mentagrophytes and Epidermophyton floccosum against griseofulvin, fluconazole, itraconazole and terbinafine, using the guidelines of the M38-P document approved by the NCCLS. We determined the growth curves, to estimate the specific growth rate (mu max) and the generation time (G) of each dermatophyte, using dry weight and spectrophotometry methods. We demonstrate that, at 192 h, all fungi tested had a constant growth curve and we considered this as the optimal time for MIC determination. Terbinafine, griseofulvin and itraconazole possessed the highest antifungal activity against the four groups of dermatophytes studied. Fluconazole demonstrated no efficacy. Our MIC results differ from other authors and this difference is due to the timing of the MIC determination based on the growth curve of each fungi tested.     

Comparative evaluation of Etest and sensititre yeastone panels against the Clinical and Laboratory Standards Institute M27-A2 reference broth microdilution method for testing Candida susceptibility to seven antifungal agents

To assess their utility for antifungal susceptibility testing in our clinical laboratory, the Etest and Sensititre methods were compared with the Clinical and Laboratory Standards Institute (CLSI) M27-A2 reference broth microdilution method. Fluconazole (FL), itraconazole (I), voriconazole (V), posaconazole (P), flucytosine (FC), caspofungin (C), and amphotericin B (A) were tested with 212 Candida isolates. Reference MICs were determined after 48 h of incubation, and Etest and Sensititre MICs were determined after 24 h and 48 h of incubation. Overall, excellent essential agreement (EA) between the reference and test methods was observed for Etest (95%) and Sensititre (91%). Etest showed an >or=92% EA for MICs for all drugs tested; Sensititre showed a >or=92% EA for MICs for I, FC, A, and C but 82% for FL and 85% for V. The overall categorical agreement (CA) was 90% for Etest and 88% for Sensititre; minor errors accounted for the majority of all categorical errors for both systems. Categorical agreement was lowest for Candida glabrata and Candida tropicalis with both test systems. Etest and Sensititre provided better CA at 24 h compared to 48 h for C. glabrata; however, CA for C. glabrata was <80% for FL with both test systems despite MIC determination at 24 h. Agreement between technologists for both methods was >or=98% for each agent against all organisms tested. Overall, Etest and Sensititre methods compared favorably with the CLSI reference method for determining the susceptibility of Candida. However, further evaluation of their performance for determining the MICs of azoles, particularly for C. glabrata, is warranted.     

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.     

In vitro activity of nystatin compared with those of liposomal nystatin, amphotericin B, and fluconazole against clinical Candida isolates

We investigated the in vitro activity of nystatin and liposomal nystatin against 103 Candida isolates to determine the effect of both time and medium on MICs. We also compared the nystatin MICs with those of amphotericin B and fluconazole. Testing was performed in accordance with the National Committee for Clinical Laboratory Standards M27-A microdilution methodology with RPMI 1640, RPMI 1640 supplemented with glucose to 2% (RPMI-2), and antibiotic medium 3 supplemented with glucose to 2% (AM3). While nystatin MICs were similar to or slightly lower than liposomal nystatin MICs in RPMI 1640 and RPMI-2, they were markedly higher than liposomal nystatin MICs in AM3. Use of AM3 and determination of the MIC after 24 h of incubation provided a slightly wider range of liposomal nystatin MICs (0.06 to >16 microg/ml). Under these conditions, the MICs at which 90% of isolates were inhibited of nystatin and liposomal nystatin were 2 and 1 microg/ml, respectively. Nystatin and liposomal nystatin in general showed good activity against all Candida spp. tested. Although the MICs of nystatin and liposomal nystatin tended to rise in parallel with the amphotericin B MICs, nystatin and liposomal nystatin MICs of 1 to 2 and 0.5 to 1 microg/ml, respectively, were obtained for seven and six, respectively, of nine isolates for which amphotericin B MICs were >or=0.25 microg/ml. No correlation between fluconazole and nystatin or liposomal nystatin MICs was observed. As amphotericin B MICs of >or=0.25 microg/ml correlate with in vitro resistance, these results suggest that liposomal nystatin might have activity against some amphotericin B-resistant isolates. In vivo testing in animal models is required for clarification of this issue.     

Quality control guidelines for National Committee for Clinical Laboratory Standards--recommended broth macrodilution testing of ketoconazole and itraconazole.

Ketoconazole and itraconazole were tested in a multilaboratory study to establish quality control (QC) guidelines for yeast antifungal susceptibility testing. Two isolates that had been previously identified as QC isolates for amphotericin B, fluconazole, and flucytosine (Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258) were tested in accordance with the National Committee for Clinical Laboratory Standards M27-P guidelines. Each isolate was tested 20 times with the two antifungal agents in the five laboratories by using a lot of RPMI 1640 unique to each laboratory as well as a lot common to all five laboratories, thus generating 200 MICs per drug per organism. Overall, 96 to 99% of the MICs for each drug fell within the desired 3-log2 dilution range (mode +/- 1 log2 dilution). By using these data, 3-log2 dilution QC ranges encompassing 98% of the observed MICs for three of the organism-drug combinations and 94% of the observed MICs for the fourth combination were established. These QC ranges are 0.064 to 0.25 micrograms/ml for both ketoconazole and itraconazole against C. parapsilosis ATCC 22019 and 0.125 to 0.5 micrograms/ml for both ketoconazole and itraconazole against C. krusei ATCC 6258.     

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.     

Evaluation of Etest Method for Determining Caspofungin (MK-0991) Susceptibilities of 726 Clinical Isolates of Candida Species

The performance of the Etest for testing the susceptibilities to caspofungin (MK-0991) of 726 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 726 isolates with RPMI agar containing 2% glucose (RPG) and were read after incubation for 48 h at 35 degrees C. The Candida isolates included Candida albicans (n = 486), Candida glabrata (n = 96), Candida tropicalis (n = 51), Candida parapsilosis (n = 47), Candida krusei (n = 11), Candida lusitaniae (n = 2), and Candida guilliermondii (n = 33). In addition, a subset of 314 isolates were also tested by Etest using Casitone agar (CAS) and antibiotic medium 3 agar (AM3). The Etest results obtained using RPG correlated well with reference MICs. Overall agreement was 94% with RPG, 82% with CAS, and 79% with AM3. When RPG was used, agreement ranged from 79% for C. parapsilosis to 100% for C. krusei, C. lusitaniae, and C. guilliermondii. When CAS was used, agreement ranged from 0% for C. lusitaniae to 100% for C. glabrata. With AM3, agreement ranged from 0% for C. lusitaniae to 100% for C. guilliermondii. All three media supported growth of each of the Candida species. Etest results were easy to read, with sharp zones of inhibition. In most instances (75%) where a discrepancy was observed between the Etest and the reference method, the Etest MIC was lower. The Etest method using RPG appears to be useful for determining caspofungin susceptibilities of Candida species.     

Further standardization of broth microdilution methodology for in vitro susceptibility testing of caspofungin against Candida species by use of an international collection of more than 3,000 clinical isolates

The influence of test variables on in vitro susceptibility testing of caspofungin was examined with 694 isolates of Candida albicans including seven laboratory-derived glucan synthesis mutants. The conditions providing the greatest separation between the mutant strains and the clinical isolates were RPMI medium, MIC end point criterion of partial inhibition, and incubation for 24 h. These testing conditions were then applied to 3,322 isolates of Candida spp. (3,314 clinical isolates and eight glucan synthesis mutants). Among the 11 isolates for which caspofungin MICs were >/=2 microg/ml, eight were accounted for by the glucan synthesis mutants. The MICs for >99% of isolates were 相似文献   

Activity of TDT 067 (terbinafine in Transfersome) against agents of onychomycosis, as determined by minimum inhibitory and fungicidal concentrations

TDT 067 is a novel carrier-based dosage form (liquid spray) of 15 mg/ml of terbinafine in Transfersome that has been developed to deliver terbinafine to the nail bed to treat onychomycosis. In this study, we report the in vitro activities of TDT 067 against dermatophytes, compared with those of the Transfersome vehicle, naked terbinafine, and commercially available terbinafine (1%) spray. The MICs of TDT 067 and comparators against 25 clinical strains each of Trichophyton rubrum, T. mentagrophytes, and Epidermophyton floccosum were determined according to the CLSI M38-A2 susceptibility method (2008). Minimum fungicidal concentrations (MFCs) were determined by subculturing visibly clear wells from the MIC microtiter plates. TDT 067 demonstrated potent activity against the dermatophyte strains tested, with an MIC range of 0.00003 to 0.015 μg/ml. Overall, TDT 067 MIC(50) values (defined as the lowest concentrations to inhibit 50% of the strains tested) were 8-fold and 60-fold lower than those of naked terbinafine and terbinafine spray, respectively. The Transfersome vehicle showed minimal inhibitory activity. TDT 067 demonstrated lower MFC values for T. rubrum and E. floccosum than naked terbinafine and terbinafine spray. TDT 067 has more potent antifungal activity against dermatophytes that cause nail infection than conventional terbinafine preparations. The Transfersome vehicle appears to potentiate the antifungal activity of terbinafine. Clinical investigation of TDT 067 for the topical treatment of onychomycosis is warranted.