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

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

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.     

Quality control guidelines for National Committee for Clinical Laboratory Standards recommended broth macrodilution testing of amphotericin B, fluconazole, and flucytosine.

Amphotericin B, fluconazole, and flucytosine (5FC) were tested in a multilaboratory study to establish quality control (QC) guidelines for yeast antifungal susceptibility testing. Ten candidate QC strains were tested in accordance with National Committee for Clinical Laboratory Standards M27-P guidelines against the three antifungal agents in each of six laboratories. Each laboratory was assigned a unique lot of RPMI 1640 broth medium as well as a lot of RPMI 1640 common to all of the laboratories. The candidate QC strains were tested 20 times each against the three antifungal agents in both unique and common lots of RPMI 1640. A minimum of 220 MICs per drug per organism were generated during the study. Overall, 95% of the MICs of amphotericin B, fluconazole, and 5FC fell within the desired 3 log2-dilution range (mode +/- 1 log2 dilution). Excellent performance with all three drugs was observed for Candida parapsilosis ATCC 22019 and C. krusei ATCC 6258. With these strains, on-scale 3 log2-dilution ranges encompassing 96 to 99% of the MICs of all three drugs were established. These two strains are recommended for QC testing of amphotericin B, fluconazole, and 5FC. Reference ranges were also established for an additional four strains for use in method development and for training. Four strains failed to perform adequately for recommendation as either QC or reference strains.     

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.     

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.     

Quality control limits for broth microdilution susceptibility tests of ten antifungal agents

Broth microdilution susceptibility tests of Candida species have now been standardized by the National Committee for Clinical Laboratory Standards (NCCLS). An eight-laboratory collaborative study was carried out in order to document reproducibility of tests of Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 by the NCCLS method. Replicate broth microdilution tests were used to define control limits for 24- and 48-h MICs of amphotericin B, flucytosine, fluconazole, voriconazole, ketoconazole, itraconazole, caspofungin (MK 0991), ravuconazole (BMS 207147), posaconazole (SCH 56592), and LY 303366.     

Comparison of two microdilution methods for testing susceptibility of Candida spp. to voriconazole

The growing number of fungal infections, coupled with emerging resistance to classical antifungal agents, has led to the development of new agents, among them voriconazole. Susceptibility to voriconazole was tested by using two microdilution techniques: the reference method described in National Committee for Clinical Laboratory Standards document M27-A2 and a colorimetric method, Sensititre YeastOne. A total of 272 Candida isolates (132 of Candida albicans, 62 of C. parapsilosis, 33 of Candida glabrata, 21 of C. krusei, 15 of C. tropicalis, and 9 of C. lusitaniae) and two control strains (C. parapsilosis ATCC 22019 and C. krusei ATCC 6258) were tested. There was a high rate of agreement between the two methods used (97 to 100%).     

Stability of Mueller-Hinton agar supplemented with glucose and methylene blue for disk diffusion testing of fluconazole and voriconazole

The shelf life of Mueller-Hinton agar supplemented with 2% glucose and methylene blue (0.5 micro g/ml) (MH-GMB) prepared in the laboratory to test disk diffusion of voriconazole and fluconazole was assessed using quality control (QC) strains of Candida albicans ATCC 90028, Candida krusei ATCC 6258, and Candida parapsilosis ATCC 22019. MH-GMB agar plates were prepared as described in National Committee for Clinical Laboratory Standards document M44-P, and isolates were tested using 25- micro g fluconazole disks and 1- micro g voriconazole disks over a 36-day period. Zone diameters for fluconazole and voriconazole varied by no more than 4 mm over the study period, and 95 to 100% of results were within the established QC limits for the strains tested. Prepared MH-GMB agar plates provide acceptable performance for disk diffusion testing for at least 30 days when stored at 5 degrees C.     

Rapid flow cytometric susceptibility testing of Candida albicans.

A rapid flow cytometric assay for in vitro antifungal drug susceptibility testing was developed by adapting the proposed reference method for broth macrodilution testing of yeasts. Membrane permeability changes caused by the antifungal agent were measured by flow cytometry using propidium iodide, a nucleic acid-binding fluorochrome largely excluded by the intact cell membrane. We determined the in vitro susceptibility of 31 Candida albicans isolates and two quality control strains (Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258) to amphotericin B and fluconazole. Amphotericin B MICs ranged from 0.03 to 2.0 microg/ml, while fluconazole MICs ranged from 0.125 to 128 microg/ml. This method results in clear-cut endpoints that were reproducible. Four-hour incubation was required for fluconazole, whereas a 2-h incubation was sufficient for amphotericin B to provide MICs comparable to the reference macrodilution method developed by the National Committee for Clinical Laboratory Standards Subcommittee on Antifungal Susceptibility Tests. Results of these studies show that flow cytometry provides a rapid and sensitive in vitro method for antifungal susceptibility testing of C. albicans.     

Quality control guidelines for amphotericin B, Itraconazole, posaconazole, and voriconazole disk diffusion susceptibility tests with nonsupplemented Mueller-Hinton Agar (CLSI M51-A document) for nondermatophyte Filamentous Fungi

Although Clinical and Laboratory Standards Institute (CLSI) disk diffusion assay standard conditions are available for susceptibility testing of filamentous fungi (molds) to antifungal agents, quality control (QC) disk diffusion zone diameter ranges have not been established. This multicenter study documented the reproducibility of tests for one isolate each of five molds (Paecilomyces variotii ATCC MYA-3630, Aspergillus fumigatus ATCC MYA-3626, A. flavus ATCC MYA-3631, A. terreus ATCC MYA-3633, and Fusarium verticillioides [moniliforme] ATCC MYA-3629) and Candida krusei ATCC 6258 by the CLSI disk diffusion method (M51-A document). The zone diameter ranges for selected QC isolates were as follows: P. variotii ATCC MYA-3630, amphotericin B (15 to 24 mm), itraconazole (20 to 31 mm), and posaconazole (33 to 43 mm); A. fumigatus ATCC MYA-3626, amphotericin B (18 to 25 mm), itraconazole (11 to 21 mm), posaconazole (28 to 35 mm), and voriconazole (25 to 33 mm); and C. krusei, amphotericin B (18 to 27 mm), itraconazole (18 to 26 mm), posaconazole (28 to 38 mm), and voriconazole (29 to 39 mm). Due to low testing reproducibility, zone diameter ranges were not proposed for the other three molds.     

Multicenter evaluation of the reproducibility of the proposed antifungal susceptibility testing method for fermentative yeasts of the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antimicrobial Susceptibility Testing (AFST-EUCAST)

Objective   To evaluate the intra- and inter-laboratory reproducibility of a new standard for susceptibility testing of fermentative yeasts. This standard is based on the M27-A procedure of the National Committee for Clinical Laboratory Standards (NCCLS), but incorporates several modifications, including spectrophotometric growth-dependent endpoint reading.
Methods   Nine laboratories participated in the study. Common material lots were used to test six Candida species (one each of C. albicans , C. tropicalis , C. parapsilosis , C. glabrata , C. krusei , and C. lusitaniae ), and two quality control strains ( C. krusei ATCC6258 and C. parapsilosis ATCC22019). Triplicate testing on three separate days was performed in microtiter format with RPMI−2% glucose, pH 7.0. Flucytosine, fluconazole and itraconazole were tested. In total, 3888 MIC values were included in the analyses. Reproducibility was calculated by means of agreement (percentage of MICs within one two-fold dilution of the mode) and intraclass correlation coefficient (ICC, maximum value of 1).
Results   The average intra-laboratory agreements were 99% and 96% after 24 h and 48 h of incubation, respectively, with ICCs of 0.98 and 0.97 ( P  < 0.05). Two strains exhibiting a trailing effect showed intra-laboratory agreement of 92% and ICCs of < 0.91 at 48 h. The inter-laboratory agreement was 94% and 88% after 24 h and 48 h, respectively, with ICCs of 0.93 and 0.91 ( P  < 0.05). Lower values of agreement and ICCs were obtained for strains exhibiting trailing after 48 h of incubation. Itraconazole yielded the lowest values of reproducibility.
Conclusion   The new procedure of EUCAST for antifungal susceptibility testing is a reproducible method within and between laboratories and offers several advantages over the NCCLS approved method.     

Susceptibility of filamentous fungi to voriconazole tested by two microdilution methods

The growing number of fungal infections, coupled with emerging resistance to classical antifungal agents, has led to the development of new agents, among them voriconazole. Susceptibility to voriconazole was tested by two microdilution techniques: the National Committee for Clinical Laboratory Standards reference method M38-A and a colorimetric method, Sensititre YeastOne. The study tested a total of 244 isolates: 223 Aspergillus (136 Aspergillus fumigatus, 37 A. niger, 26 A. terreus, 16 A. flavus, 7 A. nidulans, and 1 A. ustus), 14 Fusarium (8 Fusarium moniliformis, and 6 F. oxysporum), 6 Scedosporium apiospermum, and 1 Rhizomucor pusillus strain and four control strains (Candida parapsilosis ATCC 22019, C. krusei ATCC 6258, A. fumigatus ATCC 204305, and A. flavus ATCC 204304). For all tested species except one F. moniliforme strain and R. pusillus, the MIC, the MIC at which 50% of the isolates are inhibited (MIC(50)), and MIC(90) ranges of 相似文献   

Comparative evaluation of PASCO and national committee for clinical laboratory standards M27-A broth microdilution methods for antifungal drug susceptibility testing of yeasts

The PASCO antifungal susceptibility test system, developed in collaboration with a commercial company, is a broth microdilution assay which is faster and easier to use than the reference broth microdilution test performed according to the National Committee for Clinical Laboratory Standards (NCCLS) document M27-A guidelines. Advantages of the PASCO system include the system's inclusion of quality-controlled, premade antifungal panels containing 10, twofold serial dilutions of drugs and a one-step inoculation system whereby all wells are simultaneously inoculated in a single step. For the prototype panel, we chose eight antifungal agents for in vitro testing (amphotericin B, flucytosine, fluconazole, ketoconazole, itraconazole, clotrimazole, miconazole, and terconazole) and compared the results with those of the NCCLS method for testing 74 yeast isolates (14 Candida albicans, 10 Candida glabrata, 10 Candida tropicalis, 10 Candida krusei, 10 Candida dubliniensis, 10 Candida parapsilosis, and 10 Cryptococcus neoformans isolates). The overall agreements between the methods were 91% for fluconazole, 89% for amphotericin B and ketoconazole, 85% for itraconazole, 80% for flucytosine, 77% for terconazole, 66% for miconazole, and 53% for clotrimazole. In contrast to the M27-A reference method, the PASCO method classified as resistant seven itraconazole-susceptible isolates (9%), two fluconazole-susceptible isolates (3%), and three flucytosine-susceptible isolates (4%), representing 12 major errors. In addition, it classified two fluconazole-resistant isolates (3%) and one flucytosine-resistant isolate (1%) as susceptible, representing three very major errors. Overall, the agreement between the methods was greater than or equal to 80% for four of the seven species tested (C. dubliniensis, C. glabrata, C. krusei, and C. neoformans). The lowest agreement between methods was observed for miconazole and clotrimazole and for C. krusei isolates tested against terconazole. When the data for miconazole and clotrimazole were removed from the analysis, agreement was >/=80% for all seven species tested. Therefore, the PASCO method is a suitable alternative procedure for the testing of the antifungal susceptibilities of the medically important Candida spp. and C. neoformans against a range of antifungal agents with the exceptions only of miconazole and clotrimazole and of terconazole against C. krusei isolates.     

Quality control limits for posaconazole disk susceptibility tests on Mueller-Hinton agar with glucose and methylene blue

A multilaboratory collaborative study was performed in order to propose quality control limits for posaconazole disk diffusion susceptibility tests on Mueller-Hinton agar supplemented with 2% glucose and 0.5 microg of methylene blue per ml. Replicate tests were performed on three lots of prepared media using 5-microg posaconazole disks in each of eight laboratories to generate data to propose quality control zone diameter ranges for tests of Candida albicans ATCC 90028 (24 to 34 mm), C. parapsilosis ATCC 22019 (25 to 36 mm), C. tropicalis ATCC 750 (23 to 33 mm), and C. krusei ATCC 6258 (23 to 31 mm).     

Quality control limits for fluconazole disk susceptibility tests on Mueller-Hinton agar with glucose and methylene blue

An international collaborative study was performed in order to propose quality control limits for fluconazole disk diffusion susceptibility tests on Mueller-Hinton agar with 2% glucose and 0.5 micro g of methylene blue per ml. The supplements may be added before autoclaving the agar, or Mueller-Hinton agar plates may be flooded with a glucose-methylene blue solution. Replicate tests on both types of agar plates generated data to propose zone size limits for tests of Candida parapsilosis ATCC 22019 (22 to 33 mm), C. tropicalis ATCC 750 (26 to 37 mm), and C. albicans ATCC 90028 (28 to 39 mm). C. krusei ATCC 6258 was not useful for this purpose.     

Proposed MIC quality control guidelines for National Committee for Clinical Laboratory Standards susceptibility tests using seven veterinary antimicrobial agents: ceftiofur, enrofloxacin, florfenicol, penicillin G-novobiocin, pirlimycin, premafloxacin, and spectinomycin.

The present multicenter study proposes broth microdilution quality control (QC) ranges for the antimicrobial agents ceftiofur, enrofloxacin, florfenicol, penicillin G-novobiocin, pirlimycin, premafloxacin, and spectinomycin, which are used in veterinary practice. Six separate laboratories tested replicates of National Committee for Clinical Laboratory Standards (NCCLS)-recommended QC organisms (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213, and Enterococcus faecalis ATCC 29212) on medium lots both common and unique to all laboratories. The proposed ranges were within 3 or 4 log2 dilution steps of the modal MICs for all organism-antimicrobial agent pairs, depending on their MIC distributions. With > or = 94.7% of all MIC results being within the proposed QC ranges, all combinations tested comply with NCCLS guidelines and all have been accepted by the NCCLS subcommittee developing susceptibility testing procedures for veterinary laboratories.     

In vitro activity of caspofungin compared to amphotericin B, fluconazole, and itraconazole against Candida strains isolated in a Turkish University Hospital.

We investigated the in vitro activity of caspofungin compared to amphotericin B, fluconazole, and itraconazole against clinical strains of Candida spp. (n =239). Antifungal susceptibility tests were done in accordance with NCCLS M27-A2 microdilution method and the results were read after 24 and 48 h. In general, 24 h MIC readings were similar to those at 48 h for most isolates and all antifungal agents. Caspofungin was active against all species tested. Caspofungin MICs of Candida parapsilosis were slightly higher than those for other Candida spp. Caspofungin MIC (microg/ml) ranges at 24 h for C. albicans, C. glabrata, C tropicalis, C. parapsilosis, C kefyr, C krusei, C. lusitaniae, C. norvegensis, C. guilliermondii and C. lipolytica were 0.06-2, 0.125-2, 0.125-2, 1-4, 0.125-2, 1-2, 0.5-2, 0.5-1, 0.5-2 and 1-2, respectively. Eagle (paradoxical) effect was observed in 31 and 8% of the isolates at highest concentrations of caspofungin and itraconazole, respectively. The activity of caspofungin against fluconazole- and/or itraconazole-resistant isolates was similar to that detected for the susceptible ones. We conclude that caspofungin appears as a promising antifungal agent with enhanced activity against Candida, including the azole-resistant strains.     

Comparison study of broth macrodilution and microdilution antifungal susceptibility tests.

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

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

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

Antifungal susceptibilities of Candida species causing vulvovaginitis and epidemiology of recurrent cases

There are limited data regarding the antifungal susceptibility of yeast causing vulvovaginal candidiasis, since cultures are rarely performed. Susceptibility testing was performed on vaginal yeast isolates collected from January 1998 to March 2001 from 429 patients with suspected vulvovaginal candidiasis. The charts of 84 patients with multiple positive cultures were reviewed. The 593 yeast isolates were Candida albicans (n = 420), Candida glabrata (n = 112), Candida parapsilosis (n = 30), Candida krusei (n = 12), Saccharomyces cerevisiae ( n = 9), Candida tropicalis (n = 8), Candida lusitaniae (n = 1), and Trichosporon sp. (n = 1). Multiple species suggesting mixed infection were isolated from 27 cultures. Resistance to fluconazole and flucytosine was observed infrequently (3.7% and 3.0%); 16.2% of isolates were resistant to itraconazole (MIC > or = 1 microg/ml). The four imidazoles (econazole, clotrimazole, miconazole, and ketoconazole) were active: 94.3 to 98.5% were susceptible at < or =1 microg/ml. Among different species, elevated fluconazole MICs (> or = 16 microg/ml) were only observed in C. glabrata (15.2% resistant [R], 51.8% susceptible-dose dependent [S-DD]), C. parapsilosis (3.3% S-DD), S. cerevisiae (11.1% S-DD), and C. krusei (50% S-DD, 41.7% R, considered intrinsically fluconazole resistant). Resistance to itraconazole was observed among C. glabrata (74.1%), C. krusei (58.3%), S. cerevisiae (55.6%), and C. parapsilosis (3.4%). Among 84 patients with recurrent episodes, non-albicans species were more common (42% versus 20%). A > or = 4-fold rise in fluconazole MIC was observed in only one patient with C. parapsilosis. These results support the use of azoles for empirical therapy of uncomplicated candidal vulvovaginitis. Recurrent episodes are more often caused by non-albicans species, for which azole agents are less likely to be effective.