Comparative Evaluation of National Committee for Clinical Laboratory Standards Broth Macrodilution and Agar Dilution Screening Methods for Testing Fluconazole Susceptibility of Cryptococcus neoformans

A simple screening method for fluconazole susceptibility of Cryptococcus neoformans using 2% dextrose Sabouraud dextrose agar (SabDex) with fluconazole was compared to the National Committee for Clinical Laboratory Standards (NCCLS) broth macrodilution method. By this method, fluconazole-susceptible C. neoformans isolates are significantly smaller on medium with fluconazole than on fluconazole-free medium. Isolates with decreased susceptibility have normal-size colonies on medium containing fluconazole. The 48-h NCCLS broth macrodilution MICs (NCCLS MICs) for isolates with normal-size colonies on 8- or 16-μg/ml fluconazole plates were predicted to be ≥8 or ≥16 μg/ml, respectively. On medium with 16 μg of fluconazole per ml, all strains (84 of 84) for which the NCCLS MICs were <16 μg/ml were correctly predicted, as were all isolates (7 of 7) for which the MICs were ≥16 μg/ml. Agar dilution appears to be an effective screening method for fluconazole resistance in C. neoformans.     

Simple method for detecting fluconazole-resistant yeasts with chromogenic agar.

A method for detecting fluconazole-resistant yeasts was developed that uses chromogenic agar containing fluconazole. Yeasts were plated on media with fluconazole at 0, 8, and 16 micrograms/ml. On media without fluconazole, normal growth of susceptible yeasts (defined as those having a fluconazole MIC of < 8 micrograms/ml) was detected, while fluconazole-containing media suppressed susceptible strains and normal colonies of resistant yeasts (fluconazole MICs of > or = 8 micrograms/ml) were detected. This method was used to screen for resistance in oropharyngeal candidiasis. Isolates having fluconazole MICs of > or = 8 micrograms/ml and < 8 micrograms/ml were correctly predicted in 43 of 45 cultures and 115 of 116 cultures, respectively. This screening method appears to be rapid and sensitive for detection of fluconazole-resistant yeasts.     

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

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

Emergence of fluconazole-resistant strains of Candida albicans in patients with recurrent oropharyngeal candidosis and human immunodeficiency virus infection.

After repeated use of fluconazole for therapy of oropharyngeal candidosis, the emergence of in vitro fluconazole-resistant Candida albicans isolates (MIC, > or = 25 micrograms/ml) together with oral candidosis unresponsive to oral dosages of up to 400 mg of fluconazole were observed in patients with human immunodeficiency virus (HIV) infection. Antifungal susceptibility testing was done by broth microdilution and agar dilution techniques on C. albicans isolates recovered from a cohort of patients with symptomatic HIV infection who were treated repeatedly with fluconazole for oropharyngeal candidosis. In vitro findings did show a gradual increase in the MICs for C. albicans isolates recovered from selected patients with repeated episodes of oropharyngeal candidosis. Primary resistance of C. albicans to fluconazole was not seen. Cross-resistance in vitro occurred between fluconazole and other azoles (ketoconazole, itraconazole), but to a lesser extent. The results of the study suggest that the development of clinical resistance to fluconazole could be clearly correlated to in vitro resistance to fluconazole. Itraconazole may still serve as an effective antifungal agent in patients with HIV infection and oropharyngeal candidosis nonresponsive to fluconazole.     

Variations in fluconazole susceptibility and electrophoretic karyotype among oral isolates of Candida albicans from patients with AIDS and oral candidiasis.

DNA subtyping by pulsed-field gel electrophoresis and in vitro susceptibility testing were used to study strain variation and fluconazole resistance in Candida albicans isolates from patients with AIDS undergoing azole (fluconazole and clotrimazole) therapy for oropharyngeal candidiasis. A total of 29 patients suffered 71 episodes of oropharyngeal candidiasis. Overall, 121 isolates of C. albicans recovered throughout the course of treatment of each infection were available for further characterization. DNA subtyping revealed a total of 61 different DNA subtypes. In vitro susceptibility testing of the 121 isolates by using proposed standard methods of the National Committee for Clinical Laboratory Standards revealed MICs of fluconazole ranging from < or = 0.125 to > 64 micrograms/ml. The MIC for 50% of isolates tested was 0.25 microgram/ml, and the MIC for 90% of isolates tested was 8.0 micrograms/ml. MICs were > or = 64 micrograms/ml for only 7.4% of the isolates tested. The majority (62%) of the patients with oropharyngeal candidiasis and undergoing azole therapy were infected or colonized with more than one DNA subtype, and the introduction or selection of strains with a more resistant DNA subtype during the course of fluconazole therapy was not uncommon. With one exception, this did not appear to have an adverse effect on clinical outcome. In contrast, for patients with AIDS and oropharyngeal candidiasis infected with a single DNA subtype of C. albicans, an increase in fluconazole MICs for the infecting strain was rarely demonstrated over the course of therapy.     

Fluconazole disk diffusion procedure for determining susceptibility of Candida species.

A simple disk diffusion test was defined for quick determination of the susceptibility of Candida species to fluconazole. The standard macrotube dilution reference method, the fluconazole E test, and a 25-microgram fluconazole disk test were all performed with each of 250 Candida species selected to provide a broad range of fluconazole MICs. All three methods were in excellent agreement. On RPMI 1640-glucose agar, isolates with inhibition zone diameters of > or = 19 mm were all susceptible (MIC, < or = 8.0 micrograms/ml) by the E test and 94% were susceptible by the macrotube method. Strains with smaller zones were either resistant, intermediate (dose-dependent susceptibility), or susceptible by the reference methods. The disk test did not adequately separate fully resistant strains from those with dose-dependent susceptibility: additional quantitative tests are needed for the few strains that are not unequivocally susceptible by the disk method.     

Interpretation of Trailing Endpoints in Antifungal Susceptibility Testing by the National Committee for Clinical Laboratory Standards Method

Trailing endpoints remain a problem in antifungal susceptibility testing using the National Committee for Clinical Laboratory Standards (NCCLS) method. For isolates for which trailing endpoints are found, MICs of ≤1 μg/ml at 24 h and of >64 μg/ml at 48 h are usually observed. In a study of human immunodeficiency virus (HIV)-infected patients with oropharyngeal candidiasis, we identified three patients with multiple serial isolates for which trailing endpoints were observed with fluconazole. At 24 h, MICs were generally ≤1 μg/ml by both broth macro- and microdilution methods. However, at 48 h, MICs were >64 μg/ml, while the organism remained susceptible by agar dilution testing with fluconazole. Most episodes of oropharyngeal candidiasis with trailing-endpoint isolates responded to doses of fluconazole as low as 100 mg/day. Two patients had both susceptible and trailing-endpoint isolates by NCCLS broth macro- and microdilution testing; these isolates were found to be the same strain by pulsed-field gel electrophoresis using restriction fragment length polymorphisms. Another patient had two different strains, one for which trailing endpoints were observed and one which was susceptible at 48 h. Trailing endpoints may be seen with selected isolates of a strain or may be a characteristic finding for most or all isolates of a strain. In addition, with isolates for which trailing endpoints are observed, reading the endpoint for the NCCLS method at 24 h may be more appropriate.     

Variations in fluconazole susceptibility and DNA subtyping of multiple Candida albicans colonies from patients with AIDS and oral candidiasis suffering one or more episodes of infection.

Five Candida albicans colonies from each infection in AIDS patients receiving fluconazole therapy for oropharyngeal candidiasis over a 2-year period were evaluated by antifungal susceptibility testing and DNA subtyping, and the results were correlated with clinical response to determine the occurrence of clinically significant selection of more-resistant C. albicans over multiple infections. A total of 534 C. albicans isolates were obtained from 38 patients who exhibited 84 episodes of infection. Antifungal susceptibility testing revealed that the MICs for 93% of the isolates were < or = 8.0 microg/ml and the MICs for 7% of the isolates were > or = 64 microg/ml. DNA subtyping revealed 70 different subtypes, with 78% of patients with one infection exhibiting one DNA subtype and 80% of patients with more than one infection exhibiting multiple DNA subtypes. Also, patients who had multiple infections had lower CD4 counts than those with single infections. Differences between the single-infection group and the multiple-infection group regarding the number of DNA subtypes and CD4 counts were both statistically significant. Of the 74 evaluable infections all were successfully treated with regular-dose (100-mg/day) fluconazole, except for three patients who ultimately responded to higher-dose fluconazole. Only one patient may have shown clinically significant selection of a more-resistant C. albicans strain over multiple courses of treatment. Interestingly, MICs reached only 8.0 microg/ml, even though doses of 400 mg of fluconazole were necessary for clinical cure.     

Trends in Antifungal Susceptibility among Swedish Candida Species Bloodstream Isolates from 1994 to 1998: Comparison of the E-test and the Sensititre YeastOne Colorimetric Antifungal Panel with the NCCLS M27-A Reference Method

A comparative evaluation of the NCCLS macrodilution method, the E-test, and the Sensititre YeastOne Colorimetric Antifungal Panel for the susceptibility testing of fluconazole, itraconazole, amphotericin B, and flucytosine was conducted with 233 blood isolates of Candida species collected between 1994 and 1998 in Sweden. Antifungal susceptibility profiles of Candida albicans and non-C. albicans Candida species remained essentially unchanged within the 5-year study period. The overall agreement rates for the E-test and the NCCLS MICs and for the YeastOne and the NCCLS MICs were > or =86 and > or =87%, respectively, within +/-1 dilution for fluconazole, amphotericin B, and flucytosine, and > or =66 and > or =57%, respectively, for itraconazole. The E-test and the YeastOne panels are equivalent, and both are convenient methods for routine use.     

Detection of quinolone-resistant Neisseria gonorrhoeae.

The present National Committee for Clinical Laboratory Standards (NCCLS) guideline for testing Neisseria gonorrhoeae quinolone susceptibility defines only a susceptible category for ciprofloxacin, enoxacin, lomefloxacin, and ofloxacin, while susceptible, intermediate, and resistant categories are defined for fleroxacin. To further define the criteria for detection of quinolone resistance in gonococci, by standard disk diffusion and agar dilution methodologies recommended by the NCCLS, we tested 29 strains of quinolone-resistant N. gonorrhoeae (QRNG) recently isolated from ofloxacin-treated patients who were considered clinical failures. Regression analyses were performed on these results together with those of another 20 strains showing reduced susceptibility and 13 fully susceptible strains (ofloxacin MICs of < or = 0.25 microgram/ml). With 5-micrograms ofloxacin disks, resistance in 27 (93.1%) of the QRNG strains (MICs of > 1 microgram/ml) was detected by the criterion of a zone diameter of < 22 mm, while in the remaining 2 (6.9%), the disks failed to detect resistance. A cluster of 15 highly resistant strains showed ofloxacin MICs of > 4 micrograms/ml and zone diameters of < 13 mm. When tested with 5-micrograms ciprofloxacin disks, the corresponding values for resistance and high-level resistance of these QRNG strains were < 25 mm (MICs of > 0.5 micrograms/ml) and < 15 mm (MICs of > 2 micrograms /ml), respectively. Six strains for which ofloxacin MICs were > or = 8 micrograms/ml showed no zones at all with both 5-micrograms ofloxacin and 5-micrograms ciprofloxacin disks. These QRNG strains are now firmly established in the Southeast Asia region, and it is important for clinical laboratories to recognize these clinically resistant strains and to monitor their spread.     

Preliminary evaluation of a semisolid agar antifungal susceptibility test for yeasts and molds

This report presents a semisolid agar antifungal susceptibility (SAAS) method for the rapid susceptibility screening of yeasts and molds. The reproducibility and accuracy of the SAAS method were assessed by comparing the MICs of amphotericin B and fluconazole obtained for 10 candidate quality control (QC) American Type Culture Collection yeast strains in >/=15 replicates with those found by six independent laboratories using the National Committee for Clinical Laboratory Standards (NCCLS) M27-P broth macrodilution method (M. A. Pfaller et al., J. Clin. Microbiol. 33:1104-1107, 1995). Overall, 96% of MICs for both drugs fell within 1 log(2) dilution of the modal MIC for each strain. The MICs for amphotericin B showed 99% agreement with the NCCLS proposed QC ranges within 1 log(2) dilution. Likewise, the MICs for fluconazole at >/=75% growth reduction showed 99% agreement for seven strains. Three strains, Candida albicans ATCC 24333 and ATCC 76615 and Candida tropicalis ATCC 750, showed a less sharp fluconazole endpoint at >/=75% growth reduction, but at >50% growth reduction, the agreement was 98% within 1 log(2) dilution of the proposed range. The MIC agreement within the proposed range for the suggested QC strains Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 was 100% for fluconazole and 100% within 1 log(2) dilution of the proposed range for amphotericin B. The SAAS method demonstrated the susceptibility or resistance of 25 clinical isolates of filamentous fungi such as Aspergillus fumigatus to amphotericin B, itraconazole, and fluconazole, usually within 48 h. Although the results are preliminary, this SAAS method is promising as a rapid and cost-effective screen and is worthy of concerted investigation.     

Fluconazole and voriconazole multidisk testing of Candida species for disk test calibration and MIC estimation

Fluconazole and voriconazole MICs were determined for 114 clinical Candida isolates, including isolates of Candida albicans, Candida glabrata, Candida krusei, Candida lusitaniae, Candida parapsilosis, and Candida tropicalis. All strains were susceptible to voriconazole, and most strains were also susceptible to fluconazole, with the exception of C. glabrata and C. krusei, the latter being fully fluconazole resistant. Single-strain regression analysis (SRA) was applied to 54 strains, including American Type Culture Collection reference strains. The regression lines obtained were markedly different for the different Candida species. Using an MIC limit of susceptibility to fluconazole of < or =8 microg/ml, according to NCCLS standards, the zone breakpoint for susceptibility for the 25-microg fluconazole disk was calculated to be > or =18 mm for C. albicans and > or =22 mm for C. glabrata and C. krusei. SRA results for voriconazole were used to estimate an optimal disk content according to rational criteria. A 5-microg disk content of voriconazole gave measurable zones for a tentative resistance limit of 4 microg/ml, whereas a 2.5-microg disk gave zones at the same MIC level for only three of the species. A novel SRA modification, multidisk testing, was also applied to the two major species, C. albicans and C. glabrata, and the MIC estimates were compared with the true MICs for the isolates. There was a significant correlation between the two measurements. Our results show that disk diffusion methods might be useful for azole testing of Candida isolates. The method can be calibrated using SRA. Multidisk testing gives direct estimations of the MICs for the isolates.     

Evaluation of in vitro spectra of activity of azithromycin, clarithromycin, and erythromycin tested against strains of Neisseria gonorrhoeae by reference agar dilution, disk diffusion, and Etest methods.

The macrolide-azilide susceptibility testing (agar dilution, disk diffusion, Etest) criteria for 105 Neisseria gonorrhoeae strains were evaluated. In addition, the potencies of azithromycin, clarithromycin, and erythromycin were studied. The most active macrolide-azilide agent was azithromycin (MIC at which 90% of the isolates are inhibited [MIC90], 0.5 microgram/ml) compared with clarithromycin (MIC90, 1.5 to 2 micrograms/ml) and erythromycin (MIC90, 2 to 4 micrograms/ml). The Etest (AB Biodisk, Solna, Sweden) was observed to produce MIC results very similar to those of the reference agar dilution test (GC agar base), with 100% of the results within 1 log2 dilution step of the reference MICs. The disk diffusion test zone diameters for all three drugs correlated at an acceptable level (r = -0.81 to -0.92) with the reference agar dilution MICs. Interpretive criteria for susceptibility were proposed for azithromycin at a MIC of < or = 2 micrograms/ml and a disk diffusion test zone of > or = 25 mm. No category for resistance was proposed because of the paucity of strains for which MICs were > 2 micrograms/ml. These tentative criteria should be further validated by correlations with clinical trial data for gonococcal strains (as they emerge) that have azithromycin MICs above the proposed susceptible category range.     

Two percent sodium chloride is required for susceptibility testing of staphylococci with oxacillin when using agar-based dilution methods.

The need to add NaCl to agar media to ensure accuracy of results when testing staphylococci with oxacillin was investigated. The results of four antimicrobial susceptibility testing methods (agar and broth dilution, E test, and disk diffusion) in which the growth medium contained 0, 2, 4, or 5% NaCl were compared with the results of a hybridization assay using a mec gene probe. We tested 223 strains of staphylococci, 128 of which were mec gene positive. A total of 7 of the 128 positive strains were coagulase-negative staphylococci with 24-h oxacillin MICs of < or = 2 micrograms/ml. Ninety-five isolates were mec gene negative, including seven strains of Staphylococcus aureus with oxacillin MICs of > or = 4 micrograms/ml. The oxacillin MICs for mec gene-positive, oxacillin-resistant strains of staphylococci increased two- to fourfold with the addition of NaCl to the test medium, while the MICs for mec gene-negative strains did not change in the presence of added salt. Very major error rates for the agar dilution and E test methods in the absence of salt ranged from 18.2 to 20.2%. Major error rates for mec gene-negative S. aureus isolates were > 17% for all test methods when 4 or 5% NaCl was added to the test medium. The addition of 2% NaCl to Mueller-Hinton agar for testing of oxacillin resulted in very major error rates of < 1% for the agar dilution and E test methods although the major error rates for the two methods with added NaCl were 8.5 and 6.9%, respectively. The disk diffusion test did not perform well in this study, showing essential error rates of > or = 18.3%. We recommended the addition of 2% NaC1 to Mueller-Hinton agar when testing staphylococci with oxacillin by either the agar dilution or E test method. NaC1 should not be added for the disk diffusion test.     

Metronidazole and clarithromycin resistance in Helicobacter pylori determined by measuring MICs of antimicrobial agents in color indicator egg yolk agar in a miniwell format. The Gastrointestinal Physiology Working Group of Universidad Peruana Cayetano Heredia and the Johns Hopkins University.

Resistance of Helicobacter pylori to metronidazole often causes failure of commonly used combination drug treatment regimens. We determined the MICs of metronidazole and clarithromycin against 18 H. pylori strains from Peru using tetrazolium egg yolk (TEY) agar. The MIC results obtained by agar dilution with petri dishes were compared with the results found through a miniwell format. The results of the two protocols for measuring drug susceptibility differed by no more than 1 dilution in all cases. On TEY agar, bright-red H. pylori colonies were easy to identify against a yellow background. Sixty-one percent (11 of 18) of the strains were resistant to metronidazole (MIC, > or = 4 micrograms/ml) and 50% (9 of 18) were resistant to clarithromycin (MIC, > or = 0.125 micrograms/ml), whereas none (0 of 5) of the strains tested were resistant to tetracycline (MIC, > or = 1 micrograms/ml). Thus, the prevalence of metronidazole and clarithromycin resistance in Peru is higher than that in developed regions of the world. The miniwell plate with TEY agar allows easy H. pylori colony identification, requires about one-third less of the costly medium necessary for petri dish assaying, conserves space, and yields MICs equivalent to those with agar dilution in petri dishes.     

Novel Fluorescent Broth Microdilution Method for Fluconazole Susceptibility Testing of Candida albicans

A comparative evaluation of the reference National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution method with a novel fluorescent carboxyfluorescein diacetate (CFDA)-modified microdilution method for the susceptibility testing of fluconazole was conducted with 68 Candida strains, including 53 Candida albicans, 5 Candida tropicalis, 5 Candida glabrata, and 5 Candida parapsilosis strains. We found trailing endpoints and discordant fluconazole MICs of < 8 microg/ml at 24 h and of > or =64 microg/ml at 48 h for 12 of the C. albicans strains. These strains satisfy the definition of the low-high MIC phenotype. All 12 low-high phenotype strains were correctly shown to be susceptible at 48 h with the CFDA-modified microdilution method. For the 41 non-low-high phenotype C. albicans strains, the CFDA-modified microdilution method yielded 97.6% (40 of 41 strains) agreement within +/-1 dilution at 24 h compared with the reference method and 92.7% (38 of 41 strains) agreement within +/-1 dilution at 48 h compared with the reference method. The five strains each from C. tropicalis, C. glabrata, and C. parapsilosis that were tested showed 100% agreement within +/-2 dilutions for the two methods being evaluated.     

Fluconazole- and itraconazole-resistant Candida albicans strains from AIDS patients: multilocus enzyme electrophoresis analysis and antifungal susceptibilities.

Multilocus enzyme electrophoresis and in vitro susceptibility testing with a broth microdilution method were used to analyze Candida albicans strain diversity in four AIDS patients with recurrent oropharyngeal candidiasis who successively developed clinical resistance to fluconazole (FCZ) and itraconazole (ITZ). One to ten colonies per sample were randomly chosen from oral washings collected before the initial FCZ treatment and just before every other antifungal treatment; a total of 98 isolates were analyzed. Multilocus enzyme electrophoresis analysis revealed 14 different electrophoretic types (ETs). Statistical analysis of genetic distances showed that C. albicans isolates clustered into five subpopulations (I to V). In each subpopulation, isolates are closely related, and genetic distances between subpopulations I to IV are short. In contrast, subpopulation V, which contained isolates typed as ET8 and ET14, is strongly divergent from the others; these isolates may represent atypical C. albicans isolates. Only one patient was infected with a single strain during the course of azole therapy; for the three remaining patients, variants of the same strain and different strains were concurrently isolated. Clinical FCZ resistance was clearly correlated with in vitro data for three patients. Moreover, MICs of ITZ increased during FCZ therapy, and MICs of ITZ which were > or = 1.56 micrograms/ml were found when clinical ITZ resistance occurred; isolates from subpopulation V showed the highest MICs of ITZ. Because of the emergence of clinical ITZ resistance after clinical FCZ resistance, the feasibility of long-term azole therapy for mucosal candidiasis in AIDS patients is questioned.     

Correlation between in vitro and in vivo antifungal activities in experimental fluconazole-resistant oropharyngeal and esophageal candidiasis

Oropharyngeal and esophageal candidiasis (OPEC) is a frequent opportunistic mycosis in immunocompromised patients. Azole-resistant OPEC is a refractory form of this infection occurring particularly in human immunodeficiency virus (HIV)-infected patients. The procedures developed by the Antifungal Subcommittee of the National Committee for Clinical Laboratory Standards (NCCLS) are an important advance in standardization of in vitro antifungal susceptibility methodology. In order to further understand the relationship between NCCLS methodology and antifungal therapeutic response, we studied the potential correlation between in vitro susceptibility to fluconazole and in vivo response in a rabbit model of fluconazole-resistant OPEC. MICs of fluconazole were determined by NCCLS methods. Three fluconazole-susceptible (FS) (MIC, /=64 microgram/ml) isolates of Candida albicans from prospectively monitored HIV-infected children with OPEC were studied. FR isolates were recovered from children with severe OPEC refractory to fluconazole, and FS isolates were recovered from those with mucosal candidiasis responsive to fluconazole. Fluconazole at 2 mg/kg of body weight/day was administered to infected animals for 7 days. The concentrations of fluconazole in plasma were maintained above the MICs for FS isolates throughout the dosing interval. Fluconazole concentrations in the esophagus were greater than or equal to those in plasma. Rabbits infected with FS isolates and treated with fluconazole had significant reductions in oral mucosal quantitative cultures (P < 0.001) and tissue burden of C. albicans in tongue, soft palate, and esophagus (P < 0.001). In comparison, rabbits infected with FR isolates were unresponsive to fluconazole and had no reduction in oral mucosal quantitative cultures or tissue burden of C. albicans versus untreated controls. We conclude that there is a strong correlation between in vitro fluconazole susceptibility by NCCLS methods and in vivo response to fluconazole therapy of OPEC due to C. albicans.     

Rapid identification of fluconazole resistance using Chromagar Candida

Although extremely rare 10 years ago, antifungal drug resistance is becoming a major problem in certain populations, especially in those infected with HIV. This study was undertaken to study the resistance of Candida species isolated in our hospital to Fluconazole using Chrom agar Candida. The Candida strains which were routinely isolated from clinical specimens like blood, urine, sputum, pus, fluid and homograft isolates were included in the study. 142 Candida isolates were tested by using Chrom agar Candida incorporated with fluconazole. 16 strains were found to be resistant to Fluconazole and 126 strains sensitive to Fluconazole. Nine were C tropicalis, 3 C krusei, 2 C guillermondii, 1 Geotrichum candidum and one was an unidentified strain of Candida. The MIC of the 16 strains were done using RPMI 1640 medium by macro broth dilution method. MIC of 9 strains was 64 & > 64 micrograms/ml of 6 strains 32 micrograms/ml and 1 strain 16 micrograms/ml.     

Evaluation of the Etest and disk diffusion methods for determining susceptibilities of 235 bloodstream isolates of Candida glabrata to fluconazole and voriconazole

The performances of the Etest and the disk diffusion methods for testing of the susceptibilities of 235 Candida glabrata isolates to fluconazole and voriconazole were compared with that of the National Committee for Clinical Laboratory Standards (NCCLS) approved standard broth microdilution (BMD) method. The NCCLS method used RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35 degrees C. Etest MICs were determined with RPMI 1640 agar containing 2% glucose (RPG agar) and with Mueller-Hinton agar containing 2% glucose and 0.5 microg of methylene blue per ml (MBE agar) and were read after incubation for 48 h at 35 degrees C. Disk diffusion testing was performed with MBE agar, 25-microg fluconazole disks, and 1- microg voriconazole disks and by incubation at 35 degrees C for 24 h. Overall agreements between the Etest and the BMD MICs obtained with RPG and MBE agars were 91 and 96%, respectively, for fluconazole and 93 and 95%, respectively, for voriconazole. Categorical agreements between the agar-based methods and BMD were 52.3 to 64.7% with fluconazole and 94.8 to 97.4% with voriconazole. The vast majority of the discrepancies by the disk diffusion and Etest methods with fluconazole were minor errors. The agar-based methods performed well in identifying isolates with resistance to fluconazole and decreased susceptibility to voriconazole.