Comparative sensitivity of yeasts to antifungal agents using a standardized micromethod

The comparative susceptibility of 1 850 yeast strains belonging to 8 species was determined. The standardized micromethods used allows determinations of minimal inhibitory concentrations (MICs) or categorized sensitivities for two different concentrations (AB). Overall results showed that amphotericin B (AMB) is the most active agent, followed by the various imidazoles. 5-fluorocytosine (5FC) was the least effective drug, with 68% susceptible strains. However, results varied widely across species and drugs. For instance, among Candida albicans and Torulopsis glabrata strains, none were resistant to AMB and only 6% were resistant to 5-FC; in contrast, Candida albicans was highly susceptible to imidazoles (0.8 to 2.5% resistant strains) whereas Torulopsis glabrata showed much higher resistance rates (18% of strains for tioconazole and 70% for ketoconazole). Variations in susceptibility were also recorded across imidazoles: clotrimazole, tioconazole and ketoconazole were much more effective against Candida tropicalis and Candida parapsilosis than miconazole and econazole, whereas almost no strains were resistant to AMB and more than 50% of strains were resistant to 5-FC. Results obtained by AB (967 strains) and MIC (455 strains) were consistent for the 1 422 Candida albicans strains. Our results show that standardized micromethods should be used to determine the susceptibility of yeasts to antifungal agents.     

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.     

In vitro antifungal susceptibility testing of Candida blood isolates and evaluation of the E-test method.

Fungal infections have dramatically increased in recent years, along with the increase of drug-resistant isolates in immunocompromised patients. Ninety eight Candida species obtained from blood cultures at the Tri-Service General Hospital, Taiwan, from 1998 to 2000 were studied. These included 50 Candida albicans, 13 Candida glabrata, 24 Candida tropicalis and 11 Candida parapsilosis isolates. To investigate their susceptibility to commonly used antifungal drugs, minimum inhibitory concentrations (MIC) of amphotericin B, fluconazole, flucytosine, and ketoconazole were determined. Both the National Committee for Clinical Laboratory Standards reference broth macrodilution method and E-test were used in parallel. Ninety five isolates (95/98, 96.94%) were susceptible to amphotericin B at a concentration < or = 1 microg/mL. All isolates (100%, 98/98) were susceptible to flucytosine. Approximately 30% of these Candida isolates were resistant to fluconazole. The MIC for 90% of isolates (MIC90) values for both methods for these isolates were 0.5 microg/mL for amphotericin B, 32 microg/mL for fluconazole, 0.25 microg/mL for flucytosine (0.125 microg/mL by E-test method), and 4 microg/mL for ketoconazole. MIC for 50% of isolates (MIC50) values for these agents were 0.25, 2, 0.06, and 0.06 microg/mL, respectively. The essential agreement of MIC values within 2 dilutions for the 2 methods was 99.0% for amphotericin B, 90.8% for ketoconazole, 92.9% for fluconazole, and 91.8% for flucytosine. This study showed that E-test has equivalent performance to the broth macrodilution method and can be used as an alternative MIC technique for antifungal susceptibility testing.     

In vitro susceptibility of Cryptococcus neoformans isolates from patients with acquired immunodeficiency syndrome

Cryptococcus neoformans strains from 26 individual patients with acquired immunodeficiency syndrome (AIDS) and three isolates from patients without AIDS were tested for their susceptibility to amphotericin B, flucytosine, ketoconazole, and miconazole nitrate. Ninety percent of the C neoformans isolates from patients with AIDS were inhibited by drug concentrations within achievable serum levels. The minimum fungicidal concentration of the four tested antifungal agents, however, exceeded obtainable cerebrospinal fluid levels.     

In vitro activities of voriconazole and five licensed antifungal agents against Candida dubliniensis: comparison of CLSI M27-A2, Sensititre YeastOne, disk diffusion, and Etest methods

We compared the in vitro activity of six antifungal agents against 62 isolates of Candida dubliniensis by the Clinical Laboratory Standards Institute (CLSI [formerly National Committee for the Clinical Laboratory Standards]) M27-A2, Sensititre YeastOne, disk diffusion, and Etest methods and we studied the effect of the time of reading. For the azoles, voriconazole was the most potent in vitro followed by fluconazole, ketoconazole, and itraconazole. All the isolates were susceptible to amphotericin B and flucytosine. The highest rate of resistance was obtained against itraconazole with a high number of isolates defined as susceptible dose-dependent. At 24 hr, 100% of the isolates were susceptible to ketoconazole, amphotericin B, and flucytosine, 98% susceptible to voriconazole and fluconazole, and 95% for itraconazole. At 48 hr, 100% of the isolates remained susceptible for flucytosine and amphotericin B, 95% for voriconazole, 93% for fluconazole, 90% for ketoconazole, and 82% for itraconazole. The agreement between the CLSI and the other methods was better at 24 than 48 hr.     

Clinical, microbiological, and experimental animal studies of Candida lipolytica.

Candida lipolytica was recovered from six patients in three different clinical centers. The index isolate caused a persistent fungemia with catheter-associated Candida thrombophlebitis, the second isolate was from a polymicrobial sinusitis, and the remaining four isolates were involved in tissue colonization. These and 20 other isolates were consistent in their morphological and physiological characteristics. All formed true hyphae and blastoconidia on cornmeal-Tween 80 agar and all assimilated glucose, glycerol, and erythritol. In a murine model of disseminated candidiasis, the index isolate that caused clinical fungemia caused no mortality and produced only two lesions on a kidney, as determined at necropsy. The nine isolates selected for in vitro antifungal susceptibility studies had intermediate susceptibilities to amphotericin B but were susceptible to ketoconazole. We conclude that C. lipolytica is a weakly virulent pathogen which may require an intravascular foreign body to cause fungemia.     

Synergic effect of 5-fluorocytosine and imidazole derivatives against yeast strains resistant to 5-fluorocytosine

Antifungal agents associations are widely used in therapy of deep mycotic diseases, particularly amphotericin B-5-fluorocytosine association. Synergistic effect has also been described between 5-fluorocytosine and imidazole derivates. The authors have tested here eventual synergy between 5-fluorocytosine and imidazole derivatives (miconazole, ketoconazole, fluconazole, itraconazole) against 57 years isolates resistant to 5-fluorocytosine by a semi-automated methods in liquid medium (Yeast Nitrogen Base and Brain Heart Infusion). The synergistic effect between 5-fluorocytosine and antifungal imidazoles varies widely with the drug tested. It's more frequent with ketoconazole. Itraconazole and fluconazole present very little synergistic effects in vitro.     

Comparative study of two systems for the determination of the sensitivity of yeasts to antifungal agents]

One hundred yeast strains (including 60 Candida albicans) were tested in two laboratories using two different antifungal susceptibility test kits, ATB Fungus and Mycostandard. Tests were carried out under everyday work conditions. Four antifungal agents were compared: amphotericin B, flucytosine, miconazole, and ketoconazole. Results were discrepant in 19.2% (77/400) of cases. Following retesting of discordant cases with both kits, the agreement rate for strain characterization was 95.5%. Few discrepancies were seen with flucytosine. Conflicting results obtained with amphotericin B were due to poor reproducibility of Mycostandard results, especially for species other than C. albicans. In contrast, reproducibility of the ATB Fungus kit was inadequate for miconazole. The rate of discrepant results was greatest for ketoconazole. Intermediate susceptibility was seen more often with ATB Fungus for C. albicans and with Mycostandard for C. glabrata and C. krusei. The lack of reproducibility under routine working conditions should lead gallery manufacturers to strive to achieve clearer readings.     

In vitro activities of voriconazole,posaconazole, and four licensed systemic antifungal agents against Candida species infrequently isolated from blood

We determined the in vitro susceptibilities of 314 strains of Candida spp., representing 13 species rarely isolated from blood, to posaconazole and voriconazole as well as four licensed systemic antifungal agents (amphotericin B, flucytosine, fluconazole, and itraconazole). The organisms included 153 isolates of C. krusei, 67 isolates of C. lusitaniae, 48 isolates of C. guilliermondii, 10 isolates of C. famata, 10 isolates of C. kefyr, 6 isolates of C. pelliculosa, 5 isolates of C. rugosa, 4 isolates of C. lipolytica, 3 isolates of C. dubliniensis, 3 isolates of C. inconspicua, 2 isolates of C. sake, and 1 isolate each of C. lambica, C. norvegensis, and C. zeylanoides. MIC determinations were made by the National Committee for Clinical Laboratory Standards reference broth microdilution method and Etest (amphotericin B). Resistance to both amphotericin B and fluconazole was observed in strains of C. krusei, C. lusitaniae, C. guilliermondii, C. inconspicua, and C. sake. Resistance to amphotericin B, but not to fluconazole, was also observed among isolates of C. kefyr and C. rugosa. Posaconazole and voriconazole were active (MIC, < or = 1 micro g/ml) against 94 to 100% of these isolates. In contrast to the more common species of Candida causing bloodstream infection, these rare species appear to be less susceptible to the currently licensed systemic antifungal agents, with the exception of voriconazole. Continued surveillance will be necessary to detect the emergence of these species as more prevalent, resistant pathogens. The new triazoles appear to offer acceptable coverage of uncommon Candida sp. bloodstream infections.     

Distribution and antifungal susceptibility of Candida species causing nosocomial candiduria

The aim of the study was to investigate the distribution of Candida species isolated from urine specimens of hospitalized patients in Akdeniz University Hospital, Antalya, Turkey, as well as their susceptibilities to antifungal agents. A total of 100 patients who had nosocomial candiduria between March 2003 and May 2004 at the facility were included in the study. Organisms were identified by conventional methods and the use of API ID 32C strips. Susceptibilities of the isolates to amphotericin B were determined by Etest, whereas the minimum inhibitory concentration (MIC) values of these same strains to fluconazole, voriconazole and caspofungin were assessed using the broth microdilution method. The most common species recovered was C. albicans 44% of all yeasts, followed by C. tropicalis (20%), C. glabrata (18%), C. krusei (6%), C. famata (5%), C. parapsilosis (4%), C. kefyr (2%) and C. guilliermondii (1%). A total of nine (9%) of the isolates, including five C. krusei and four C. glabrata isolates were susceptible dose-dependent (SDD) to fluconazole. In constrast, only two C. glabrata and one C. krusei isolates were resistant to this antifungal. The voriconazole MICs for all Candida isolates were ≤0.5 μg/ml, except for one C. glabrata isolate with a MIC value of 2 μg/ml. Among all isolates, 94% were susceptible to amphotericin B with MIC values of <1 μg/ml and all isolates were susceptible to caspofungin with MIC values of ≤0.5 μg/ml. Future studies are needed to define better treatment regimens for those patients who have fluconazole-resistant Candida urinary tract infections.     

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.     

Antifungal susceptibility of 50 Candida isolates from invasive mycoses in Chile.

We determined the antifungal susceptibility of 50 Candida isolates from invasive mycoses in intensive care unit patients in Chile. Candida albicans (27 isolates) and C. parapsilosis (12 isolates) were the most common etiologic species. Candida glabrata (five isolates) was isolated only from children. Our data are consistent with those of recent Brazilian and Argentinean studies but differ from those of some US, Canadian and Norwegian studies, in which the relatively azole-resistant C. glabrata was the predominant non-C. albicans species seen. All isolates in this study were susceptible to amphotericin B. Itraconazole and fluconazole resistance were observed in 6 and 4% of the isolates, respectively.     

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.     

Isolation and characterization of a polyene-resistant variant of Candida tropicalis.

An atypical variant of Candida tropicalis was recovered from multiple specimens from a patient who had been a recipient of a bone marrow transplant. This yeast variant showed atypical morphology on corn meal agar distinguishable from typical isolates of C. tropicalis by the production of clusters of blastospores. Isolates of the variant produced acid, but no gas, from maltose and sucrose in fermentation tests. Isolates from blood, pleural fluid, respiratory secretions, and stool specimens were susceptible to amphotericin B and nystatin in an agar dilution system. However, eight isolates of the variant C. tropicalis recovered over a period of 4 weeks from the patient's urine after amphotericin B therapy were found to be resistant to amphotericin B and nystatin. The isolate recovered after 7 days of therapy had minimal inhibitory concentrations of 100 micrograms of amphotericin B and 20 micrograms of nystatin per ml, whereas the seven isolates recovered subsequently had minimal inhibitory concentrations of greater than 500 micrograms of amphotericin B and 50 micrograms of nystatin per ml. The resistant isolates concomitantly lost the capacity to utilize amino acids that susceptible isolates could utilize. Ultraviolet absorption spectra of nonsaponifiable fractions of whole cells showed that resistant isolates lacked ergosterol, which susceptible isolates contained.     

Survey of amphotericin B susceptibility of Candida clinical isolates determined by Etest.

BACKGROUND AND PURPOSE: The minimal inhibitory concentrations (MICs) of amphotericin B (AmB) determined by the National Committee for Clinical Laboratory Standards (NCCLS; NCCLS document M27-A) broth dilution method are in a relatively narrow ranges and this may lead to underestimation of the AmB-resistant rate in clinical isolates. We evaluated in vitro susceptibility of clinical isolates of Candida spp. to AmB using Etest and determined the distribution of AmB MICs in different species. METHODS: We used the Etest (AB Biodisk, Solna, Sweden) to evaluate the MICs of Candida isolates randomly collected during 2001-2003 in a teaching hospital. RESULTS: Of the 572 isolates evaluated, Candida albicans (50.7%) was the most common species, followed by Candida tropicalis (23.9%), Candida parapsilosis (13.1%), Candida glabrata (9.4%), Candida krusei (1.9%), and Candida guilliermondii (0.9%). The majority of isolates were from blood (85%). The minimal concentrations of AmB required to inhibit 50%/90% of the isolates (MIC(50)/MIC(90)) were 0.19/0.38 microg/mL for C. krusei, 0.125/0.38 microg/mL for C. glabrata, 0.094/0.25 microg/mL for C. tropicalis, 0.032/0.19 microg/mL for C. albicans, 0.016/0.125 microg/mL for C. parapsilosis, and 0.023/0.032 microg/mL for C. guilliermondii. Only 1 blood isolate of C. glabrata was resistant to AmB (MIC > or =1 microg/mL) [0.17%]. 18.2% of isolates were less susceptible to AmB (MIC > or =0.19 microg/mL) with the highest rates for C. krusei (63.6%), followed by C. glabrata (37.0%), C. tropicalis (29.9%), C. albicans (11.0%), C. parapsilosis (5.3%), and C. guilliermondii (0%). More isolates collected from patients with hematologic malignancy were less susceptible to AmB than those collected from those with other diseases (30.5% vs 15.4%, p<0.05). CONCLUSION: This study demonstrated that AmB resistance remains rare at this hospital in Candida clinical isolates despite increasing use of this agent during the past 4 decades.     

Cross-Resistance of clinical isolates of Candida albicans and Candida glabrata to over-the-counter azoles used in the treatment of vaginitis

Antifungal drug resistance in Candida spp. continues to increase in response to the widespread application of triazole therapeutics among immunosuppressed patients. Azole-based over-the-counter (OTC) antifungal agents used to treat vaginitis have the potential to exacerbate this problem by contributing to the selection of highly resistant strains of Candida in otherwise healthy women. In this study, we show that fluconazole-resistant (MIC > 64 microg/mL) blood stream isolates of Candida albicans and Candida glabrata obtained from cancer patients were cross-resistant to the root drugs miconazole, clotrimazole, and tioconazole (found in several over-the-counter products), but remained susceptible to butoconazole. We also provide evidence that spontaneous mutants of Candida glabrata selected for resistance to clotrimazole were cross-resistant to other azolebased drugs, including fluconazole. Our findings demonstrate cross-resistance of Candida strains to fluconazole and OTC azole antifungals, and support the notion that OTC drugs can promote azole resistance in Candida spp.     

Effects of incubation time and buffer concentration on in vitro activities of antifungal agents against Candida albicans.

Nine selected isolates of Candida albicans were tested for their susceptibilities to amphotericin B and fluconazole by using three methods to assess the effect of incubation time and buffer concentration. By using a microdilution method with 0.0165 M 3-(N-morpholino)propanesulfonic acid (MOPS) and a 24-h incubation time, all of the isolates were found to be susceptible to amphotericin B and fluconazole. After 48 h of incubation, all isolates were still susceptible to amphotericin B. Seven of the nine isolates were resistant to fluconazole, and for the remaining two isolates, MICs increased by fourfold or more but the isolates remained susceptible (MIC, < or = 10 microg/ml). The nine isolates, along with three control strains, were further tested against amphotericin B and fluconazole by a standard broth macrodilution method with both 0.165 and 0.0165 M MOPS. The susceptibility results for fluconazole by the broth macrodilution method with the lower MOPS concentration correlated with the results of the 24-h broth microdilution method for determination of susceptibility or resistance in eight of nine tests and with the results of the 48 h broth microdilution method in three of nine tests. The results of the broth macrodilution method with the standard MOPS concentration did not correlate with any of the results obtained by the 24-h broth microdilution but correlated with results of seven of nine tests by the 48-h broth microdilution method. All nine test strains appeared to be susceptible when they were examined by a flow cytometric method. For clinical yeast susceptibility testing in microdilution panels, the 0.0165 M MOPS concentration combined with 24 h of incubation appeared to be the method of choice. The lower MOPS concentration may also be a useful modification to the tentative broth macrodilution method of the National Committee for Clinical Laboratory Standards. Use of the higher buffer concentration or longer incubation time may lead to false in vitro resistance for agents like fluconazole.     

Wound infection by Prototheca wickerhamii, a saprophytic alga pathogenic for man.

Biopsy of a wound infection of the palmar fascia in a young diabetic woman revealed characteristic periodic acid-Schiff-positive Prototheca species cells with a rosette configuration and internal septation. Prototheca wickerhamii was cultured repeatedly from the wound drainage and the biopsy tissue. Several diagnostic features distinguishing Prototheca species, saprophytic algae, from yeasts are: the formation of endospores by mitosis; greater variation in cell size (2 to 15 mum); the presence of cytoplasmic granules, particularly in old cultures; and the absence of budding forms and pseudomycelia. The organism was resistant to 5-fluorocytosine and the minimal inhibitory concentration of amphotericin B was 12.5 mug/ml. With the exception of the tetracycline group, all other 16 antibacterial agents tested appeared completely ineffective in vitro. A synergism between amphotericin B and tetracycline was clearly demonstrated by the use of the checkerboard method. Infection by Prototheca species may be more common than presently realized due to the common expedient of identifying yeast-like isolates as "yeast--not Candida albicans."     

Pathogenicity and antifungal susceptibility ofChaetomium species

Several reports have been published implicatingChaetomium spp. as opportunistic pathogens. A critical review of these cases was made, and the majority of the responsible strains were studied.Chaetomium globosum was the most common species, being isolated in at least nine clinical cases of infection. Some of these clinical isolates and others from environmental sources were tested against six antifungal agents (5-fluorocytosine, fluconazole, amphotericin B, itraconazole, ketoconazole and miconazole). The 23 strains tested were totally resistant to the first two drugs, and none of the other antifungal agents demonstrated fungicidal activity. There were no significant differences between the susceptibility of the clinical strains and the other strains.     

Physiological and morphological characterization of tert -butylhydroperoxide tolerant Candida albicans mutants

tert -Butylhydroperoxide (t BOOH) tolerant Candida albicans mutants developed from clinical isolates were characterized with increased tolerance of the oxidative stress generating agents t BOOH and H2O2, continuous induction of the antioxidative defence system, reduced pseudohypha and hypha-forming capabilities, decreased phospholipase secretion and delayed growth in Sabouraud dextrose agar and broth media. Changes in antimycotic (fluconazole, voriconazole, amphotericin B, 5-fluorocytosine) tolerances as well as in total and cytochrome c-dependent respirations showed versatile patterns, meanwhile the intensified alternative oxidase-dependent respiration of the mutants indicated that this respiratory pathway was an important element of the antioxidative defence in general. Because the phenotypes of increased oxidative stress tolerance and reduced virulence attribute production always emerged concomitantly in t BOOH-tolerant mutants the natural selection of C. albicans strains more tolerant of oxidative stress is unlikely. Not surprisingly, a screening study failed to detect any C. albicans strains with increased oxidative stress tolerance among 46 randomly selected clinical isolates.