In vitro activities of the new antifungal drug eberconazole and three other topical agents against 200 strains of dermatophytes

We have compared the in vitro activity of the new antifungal drug eberconazole with those of three other topical antifungal agents, clotrimazole, ketoconazole, and miconazole, against 200 strains of dermatophytes. MICs were determined by a microdilution method with optimal conditions determined in a previous study (an inoculum of 10(4) CFU/ml, an incubation temperature of 28 degrees C, an incubation period of 7 days, and a MIC endpoint of 100% inhibition of growth). In general, the four drugs tested showed low MICs. However, eberconazole was more active (P < 0.05) than the other three drugs against the majority of the species tested. Eberconazole represents an advantageous alternative for dermatophytoses where a topical therapy is required.     

Evaluation of broth microdilution testing parameters and agar diffusion Etest procedure for testing susceptibilities of Aspergillus spp. to caspofungin acetate (MK-0991)

The NCCLS M38-A document does not describe guidelines for testing caspofungin acetate (MK-0991) and other echinocandins against molds. This study evaluated the susceptibilities of 200 isolates of Aspergillus fumigatus, A. flavus, A. nidulans, A. niger, and A. terreus to caspofungin (MICs and minimum effective concentrations [MECs]) by using standard RPMI 1640 (RPMI) and antibiotic medium 3 (M3), two inoculum sizes (10(3) and 10(4) CFU/ml), and two MIC determination criteria (complete [MICs-0] and prominent growth inhibition [MICs-2]) at 24 and 48 h. Etest MICs were also determined. In general, caspofungin MIC-2 and MEC pairs were comparable with both media and inocula (geometric mean ranges of MECs and MICs, respectively, with larger inoculum: 0.12 to 0.64 microg/ml and 0.12 to 0.44 microg/ml with RPMI versus 0.04 to 0.51 microg/ml and 0.03 to 0.21 microg/ml with M3); however, MEC results were less influenced by testing conditions than MICs, especially with the larger inoculum. Overall, the agreement between caspofungin Etest MICs and broth dilution values was higher with MECs obtained with M3 (>90%) and the large inoculum than under the other testing conditions. Because RPMI is a more stable and chemically defined medium than M3, the determination at 24 h of the easier visual MECs with RPMI and the inoculum recommended in the M38-A document appears to be a suitable procedure at present for in vitro testing of caspofungin against Aspergillus spp. Future in vitro correlations with in vivo outcome of both microdilution and Etest procedures may detect more-relevant testing conditions.     

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.     

Analysis of the influence of Tween concentration, inoculum size, assay medium, and reading time on susceptibility testing of Aspergillus spp

The influence of several test variables on susceptibility testing of Aspergillus spp. was assessed. A collection of 28 clinical isolates was tested against amphotericin B, itraconazole, voriconazole, and terbinafine. Inoculum size (10(4) CFU/ml versus 10(5) CFU/ml) and glucose supplementation (0.2% versus 2%) did not have significant effects on antifungal susceptibility testing results and higher inoculum size and glucose concentration did not falsely elevate MICs. In addition, antifungal susceptibility testing procedure with an inoculum size of 10(5) CFU/ml distinctly differentiated amphotericin B or itraconazole-resistant Aspergillus strains in vivo from the susceptible ones. Time of incubation significantly affected the final values of MICs, showing major increases (two to six twofold dilutions, P < 0.01 by analysis of variance) between MIC readings at 24 and 48 h, but no differences were observed between antifungal susceptibility testing results obtained at 48 h and at 72 h. Significantly higher MICs were uniformly associated with higher concentrations of Tween (P < 0.01), used as a dispersing agent in the preparation of inoculum suspensions. The geometric mean MICs showed increases of between 1.5- and 10-fold when the Tween concentration varied from 0.1% (the geometric means for amphotericin B, itraconazole, voriconazole, and terbinafine were 1.29, 0.69, 1.06, and 0.64 mug/ml, respectively) to 5% (the geometric means for amphotericin B, itraconazole, voriconazole, and terbinafine were 1.97, 5.79, 1.60, and 4.66 mug/ml, respectively). The inhibitory effect of Tween was clearly increased with inoculum sizes of 10(5) CFU/ml and was particularly dramatic for itraconazole, terbinafine, and Aspergillus terreus. The inoculum effect was not observed when the Tween concentration was below 0.5% (P > 0.01).     

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.     

Comparison of Spectrophotometric and Visual Readings of NCCLS Method and Evaluation of a Colorimetric Method Based on Reduction of a Soluble Tetrazolium Salt, 2,3-Bis {2-Methoxy-4-Nitro-5-[(Sulfenylamino) Carbonyl]-2H- Tetrazolium-Hydroxide}, for Antifungal Susceptibility Testing of Aspergillus Species

The susceptibilities of 25 clinical isolates of various Aspergillus species (Aspergillus fumigatus, A. flavus, A. terreus, A. ustus, and A. nidulans) to itraconazole (ITC) and amphotericin B (AMB) were determined using the standard proposed by NCCLS for antifungal susceptibility testing of[filamentous fungi, a modification of this method using spectrophotometric readings, and a colorimetric method using the tetrazolium salt 2,3-bis [2-methoxy-4-nitro-5-[(sulfenylamino) carbonyl]-2H-tetrazolium-hydroxide] (XTT). Five MIC end points for ITC (MIC-0, no visible growth or 93%. Between visual and spectrophotometric readings, high levels of agreement were found for AMB (approximately 97%) and MIC-1 (approximately 92%) and MIC-2 (approximately 88%) of ITC. Poor agreement was found for MIC-0 of ITC (51% after 24 h), since the spectrophotometric readings resulted in higher MIC-0 values than the visual readings. The agreement was increased to 98% by shifting the threshold level of MIC-0 from 5 to 10% relative optical density and by establishing an optical density of greater than 0.1 for the GC as the validation criterion. No statistically significant differences were found between the NCCLS method and the XTT method, with the levels of agreement exceeding 97% for MIC-0 of AMB and 83% for MIC-0, MIC-1, and MIC-2 of ITC. The XTT method and spectrophotometric readings can increase the sensitivity and the precision, respectively, of in vitro susceptibility testing of Aspergillus species.     

Rapid inoculum standardization system: a novel device for standardization of inocula in antimicrobial susceptibility testing.

A rapid inoculum standardization system for antimicrobial susceptibility testing without incubation or the conventional turbidity adjustment has been developed. The rapid inoculum standardization system consists of a plastic rod with cross-hatched grooves on one end and a specific nutrient medium in a vial. The crosshatched grooves are designed to pick up and release a known number of viable microorganisms. In use, the end of the rod is touched to five colonies 1 to 2 mm in diameter from a primary agar plate, thus filling the grooves with bacteria. The rod is placed into the vial, and the bacteria are suspended in the medium by agitation with a Vortex Genie Mixer. The resulting suspension contains 5 X 10(7) to 5 X 10(8) CFU/ml for most gram-negative bacilli and gram-positive cocci. Microorganisms such as streptococci that have colonies less than 1 mm in diameter require as many as 10 colonies for an adequate inoculum suspension. Ninety-five commonly encountered bacterial isolates were tested in triplicate by agar plate counts. The resulting overall geometric mean of the agar plate counts was 1.52 X 10(8) CFU/ml for the species tested. We have found that the rapid inoculum standardization system provides a consistent and reproducible method for the standardization of inoculum for antimicrobial susceptibility testing without the incubation period and turbidity adjustment.     

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.     

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.     

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.     

Multilaboratory Evaluation of In Vitro Antifungal Susceptibility Testing of Dermatophytes for ME1111

ME1111 is a novel small molecule antifungal agent under development for the topical treatment of onychomycosis. Standardization of the susceptibility testing method for this candidate antifungal is needed. Toward this end, 8 independent laboratories determined the interlaboratory reproducibility of ME1111 susceptibility testing. In addition, we subsequently identified 2 strains as quality control (QC) isolates for the method. In the reproducibility study, 5 blinded clinical strains each of Trichophyton rubrum, Trichophyton mentagrophytes, and Epidermophyton floccosum were tested, while the QC study tested 6 blinded T. rubrum or T. mentagrophytes ATCC strains. Testing was performed in frozen microtiter panels according to the Clinical and Laboratory Standards Institute (CLSI) M38-A2 methodology. In the reproducibility study, 9 of 15 clinical strains showed interlaboratory agreement of >90% at the 80% inhibition endpoint, with a range of agreement of 76.2% to 100%. In the QC study, 4 of the 6 ATCC strains showed interlaboratory agreement of >90%. ME1111 demonstrated excellent interlaboratory agreement when tested against dermatophytes. Based on this data, the CLSI Subcommittee on Antifungal Susceptibility Tests approved the susceptibility testing of ME1111 against dermatophytes according to M38-A2 methodology, which stipulates RPMI 1640 as the test medium, an inoculum size of 1 to 3 × 10³ CFU/ml, and an incubation time and temperature of 96 h at 35°C. The MIC endpoint should be 80% inhibition compared with the growth control. T. rubrum ATCC MYA-4438 and T. mentagrophytes ATCC 28185 were selected as QC isolates, with an acceptable range of 0.12 to 1 μg/ml for the two strains.     

Evaluation of blood culture media for isolation of pyridoxal-dependent Streptococcus mitior (mitis).

Nutritional variant streptococci identified as pyridoxal-dependent Streptococcus mitior (mitis) account for 5 to 6% of streptococcal endocarditis and may be a cause of "culture-negative" endocarditis. Hence, growth of three variant strains in 11 commercial blood culture broths was compared to that in fresh heart infusion broth. For simulation of clinical specimens, culture bottles were injected with 5 ml of human blood, inoculated with approximately 500 colony-forming units (CFU) per bottle, and monitored for 7 days with Gram stains and viable counts. Only Thiol broth (Difco Laboratories, Detroit, Mich.) supported growth without blood at this low inoculum. In media containing blood, maximal growth of 10(9) CFU/ml was reached within 2 days of incubation, and heavy turbidity was consistently observed in only heart infusion broth, Thiol broth, and media supplemented with pyridoxal hydrochloride. Columbia broth (BBL Microbiology Systems, Cockeysville, Md.) with increased cysteine, thioglycollate broth, and one brain heart infusion broth produced moderate growth (1 x 10(8) to 5 x 10(8) CFU/ml), whereas Columbia broth, another brain heart infusion broth, and two brands of tryptic soy broth showed fair growth (1 x 10(7) to 4 x 10(7) CFU/ml). The poor growth (1 x 10(6) to 3 x 10(6) CFU/ml) observed in three other brands of tryptic soy broth was often not apparent macroscopically or by Gram stain. Furthermore, on growth occurred in 40% of tryptic soy broth cultures inoculated with 50 CFU. Therefore, to ensure isolation of these variant streptococci from clinical blood cultures, a medium containing thiol compounds or supplemented with pyridoxal should be used. Subcultures should be made within 2 days of incubation to blood agar enriched with pyridoxal or containing a Staphylococcus sp. streak for satellitism.     

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)     

Growth of Campylobacter pylori in liquid media.

Until recently, broth cultivation techniques for Campylobacter pylori were unavailable. We developed a method to cultivate bacterial cells within 24 h in liquid media. Cultivation in broth depended on the adequate dispersion of appropriate gases. A static broth at 37 degrees C in a GasPak jar (BBL Microbiology Systems, Cockeysville, Md.) with a CampyPak (BBL) envelope did not support growth after 5 days of incubation. A broth placed in a flask on a Gyrotory water bath shaker (150 rpm; New Brunswick Scientific Co., Inc., Edison, N.J.) fitted with a gassing hood connected to a gas mixture of 10% CO2, 5% O2, and 85% N2 supported good growth. An initial inoculum of 10(5), 10(3) to 10(4), or 10(2) CFU/ml resulted in greater than or equal to 10(8) CFU/ml after incubation for 24, 48, or 72 h, respectively. Under these conditions, the bacteria grew as motile, spiral bacilli rather than the oval and coccal bacilli occasionally reported. Several bases supported good growth when supplemented with serum. For the determination of basal growth conditions, brucella broth base was used. Fetal calf serum (1%) provided maximum growth. Vitox was not necessary for growth and did not augment growth. C. pylori grew over a wide optimal pH range of 5.5 to 8.5.     

Improved growth of Campylobacter pylori in a biphasic system.

The recovery of Campylobacter pylori from clinical specimens is difficult, even when done with an optimal medium, atmosphere, and temperature. The growth of this organism was investigated by comparing a biphasic system with broth culture. The effects of gyration, inoculum, and pH were studied. Brucella agar and broth supplemented with 2.5% fetal bovine serum were used. Growth in the biphasic system was an average of 2 log units (7 X 10(8) versus 5 X 10(6) CFU/ml) greater than that in the broth system (P less than 0.01), and this occurred 12 to 24 h sooner in the biphasic system. When gyration was added, an average of 1 log unit of growth improvement was seen in comparable systems. Improved growth was also seen with low inoculum levels, in which stationary-phase cells in the broth system reached 10(5) CFU/ml compared with 10(7) CFU/ml in the biphasic system. At the three pH ranges studied, growth was best at pH 8 to 9 (6 X 10(9) CFU/ml), averaging 2 log units greater growth than that at pH 6 to 7 and 4 log units greater growth than that at pH 4.5 to 5.5 (P less than 0.01). The improved recovery of the organism for low inoculum levels in a biphasic system may be important for long-term storage and clinical isolation.     

Sequential parametric optimization of lipase production by a mutant strain Rhizopus sp. BTNT-2

Lipase production by the mutant strain Rhizopus sp. BTNT-2 was optimized in submerged fermentation. Different chemical and physical parameters such as carbon sources, nitrogen sources, oils, inoculum level, pH, incubation time, incubation temperature and aeration have been extensively studied to increase lipase productivity. Potato starch (1.25% w/v) as a carbon source, corn steep liquor (1.5% w/v) as a nitrogen source and olive oil (0.5% v/v) as lipid source were found to be optimal for lipase production. The optimal levels of other parameters are 4 ml of inoculum (2.6x10(8) spores/ml), initial pH of 5.5, incubation time of 48 hours, incubation temperature of 28 degrees C and aeration rate of 120 rpm. With the optimized parameters, the highest production of lipase was 59.2 U/ml while an yield of only 28.7 U/ml was obtained before optimization resulting in 206% increase in the productivity.     

Evaluation of broth microdilution antifungal susceptibility testing conditions for Trichophyton rubrum

Fifty clinical isolates of Trichophyton rubrum were selected to test with ketoconazole, fluconazole, itraconazole, griseofulvin, and terbinafine by following the National Committee for Clinical Laboratory Standards susceptibility testing guidelines for filamentous fungi (M38-A). In addition, other susceptibility testing conditions were evaluated: (i) three medium formulations including RPMI 1640 (standard medium), McVeigh & Morton (MVM), and Sabouraud dextrose broth (SDB); (ii) two incubation temperatures (28 and 35 degrees C); and (iii) three incubation periods (4, 7, and 10 days). The strains Candida parapsilosis (ATCC 22019), Candida krusei (ATCC 6258), T. rubrum (ATCC 40051), and Trichophyton mentagrophytes (ATCC 40004) were included as quality controls. All isolates produced clearly detectable growth only after 7 days of incubation. MICs were significantly independent of the incubation temperature (28 or 35 degrees C) (P < 0.05). Different incubation periods resulted in MICs which were consistently different for each medium when azoles and griseofulvin were tested (P < 0.05). MICs obtained from different media at the same incubation time for the same isolate were significantly different when azoles and griseofulvin were tested (P < 0.05). MICs were consistently higher (usually 1 to 2 dilutions) with RPMI than with MVM or SDB (P < 0.05). When terbinafine was tested, no parameter had any influence on MICs (P < 0.05). RPMI standard medium appears to be a suitable testing medium for determining the MICs for T. rubrum. MICs obtained at different incubation times need to be correlated with clinical outcome to demonstrate which time has better reliability.     

Evaluation of differential inoculum disk diffusion method and Vitek GPS-SA card for detection of oxacillin-resistant staphylococci.

This study was conducted in order to compare the accuracy of detection of oxacillin-resistant staphylococci, defined by microdilution MICs, population analyses, and mec gene hybridization, with the Vitek GPS-SA Susceptibility Card with that of the standard inoculum (10(7) CFU) and high-inoculum (10(9) CFU) disk diffusion tests. By the standard inoculum disk diffusion test, 10 of 67 (15%) isolates of oxacillin-resistant Staphylococcus aureus and 3 of 47 (6%) isolates of Staphylococcus epidermidis were falsely susceptible after 24 h of incubation at 35 degrees C. By the high-inoculum disk diffusion test (10(9) CFU), 4 of the 10 isolates of S. aureus remained falsely susceptible, whereas none of the isolates of S. epidermidis was falsely susceptible. Of the 10 isolates of S. aureus falsely susceptible by the standard disk test, only one remained falsely susceptible after an additional 24 h of incubation at 22 degrees C. All four isolates of S. aureus that were falsely susceptible by the high-inoculum disk diffusion test after 24 h of incubation at 35 degrees C became resistant after an additional 24 h of incubation at 22 degrees C. Thus, extended incubation of both the standard and high-inoculum disk diffusion tests increased their accuracy in detecting oxacillin resistance. All isolates of oxacillin-resistant staphylococci were accurately detected with the Vitek software upgrades (6.1 and 7.1) of the GPS-SA card.     

Effect of delays in processing on the survival of Mycobacterium avium-M. intracellulare in the isolator blood culture system.

Concentrations of Mycobacterium avium-M. intracellulare ranging from 10(-1) to 10(3) CFU/ml were added to blood, placed in Isolator tubes, and held at room temperature for intervals ranging from 4 h to 56 days before being processed (centrifugation and culture on Middlebrook 7H10 agar). At all concentrations tested, M. avium-M. intracellulare was recovered after hold times ranging from 4 h to 7 days; the number of final CFU actually increased progressively for hold times of 8 h or more. Hold times of up to 7 days did not increase the time from processing to the first appearance of visible colonies. At an inoculum of 10(2) CFU/ml, M. avium-M. intracellulare was recovered from Isolator tubes processed 56 days after inoculation. Two Isolator blood cultures were drawn from a patient with AIDS; M. avium-M. intracellulare was recovered from the sample processed immediately and from the sample processed after a hold time of 7 days. Since M. avium-M. intracellulare survives for prolonged periods in Isolator tubes, blood cultures may be collected in outpatient settings or in hospitals without mycobacterial culture facilities and shipped to reference laboratories for processing without loss of viability.     

Spectrophotometric method of inoculum preparation for the in vitro susceptibility testing of filamentous fungi.

Homogeneous inoculum suspensions of 29 isolates of clinically important filamentous fungi were adjusted with a spectrophotometer (530 nm) to obtain standardized preparations containing 1 x 10(6) to 5 x 10(6) CFU/ml. Colony counts (CFU per milliliter) of 1 x 10(6) to 5 x 10(6) were achieved on three different days for isolates of Aspergillus spp., Pseudallescheria boydii, and Sporothrix schenckii (80% +/- 2% transmission), and colony counts of 7 x 10(5) to 2.9 x 10(6) (70% +/- 2% transmission) were achieved for Mucor spp. and Rhizopus spp.