In Vitro Activity of Two Echinocandin Derivatives, LY303366 and MK-0991 (L-743,792), Against Clinical Isolates of Aspergillus, Fusarium, Rhizopus, and Other Filamentous Fungi

LY303366 and MK-0991 (previously L-743,792) are new echinocandin derivatives with excellent broad-spectrum antifungal activity. We investigated the in vitro activity of LY303366, MK-0991, itraconazole, amphotericin B, and 5-flucytosine against 51 clinical isolates of filamentous fungi, including Aspergillus flavus (10), A. fumigatus (12), Fusarium spp. (13), Rhizopus spp. (6), Pseudallescheria boydii (5), and one isolate each of Acremonium spp., A. niger, A. terreus, Paecilomyces spp., and Trichoderma spp. In vitro susceptibility testing was performed using a microdilution broth method performed according to National Committee for Clinical Laboratory Standards guidelines. LY303366 was two- to fourfold more active than MK-0991 against A. flavus, A. fumigatus, and Trichoderma spp. Both LY303366 and MK-0991 were considerably more active (MIC₉₀ of 0.03–0.12 μg/mL) than itraconazole, amphotericin B, and 5-flucytosine against Aspergillus spp., but were less active than itraconazole and amphotericin B against Rhizopus spp. MK-0991 was more active than either LY303366 or itraconazole against Acremonium spp., Paecilomyces spp., and P. boydii. These data demonstrate promising activity of both LY303366 and MK-0991 against Aspergillus spp. and other species of filamentous fungi that are likely to be encountered clinically. Further in vitro and in vivo investigation is indicated.     

Postantifungal effects of echinocandin, azole, and polyene antifungal agents against Candida albicans and Cryptococcus neoformans

The postantifungal effect (PAFE) of fluconazole, MK-0991, LY303366, and amphotericin B was determined against isolates of Candida albicans and Cryptococcus neoformans. Concentrations ranging from 0. 125 to 4 times the MIC were tested following exposure to the antifungal for 0.25 to 1 h. Combinations of azole and echinocandin antifungals (MK-0991 and LY303366) were tested against C. neoformans. Fluconazole displayed no measurable PAFE against Candida albicans or Cryptococcus neoformans, either alone or in combination with either echinocandin antifungal. MK-0991, LY303366, and amphotericin B displayed a prolonged PAFE of greater than 12 h against Candida spp. when tested at concentrations above the MIC for the organism and 0 to 2 h when tested at concentrations below the MIC for the organism.     

In Vitro Activity of Voriconazole Against Candida Species

The in vitro activity of voriconazole was compared with that of itraconazole and fluconazole against 181 isolates of Candida albicans, 124 isolates of Candida glabrata, and 20 isolates of Candida krusei obtained from the early 1980s through the mid-1990s. Voriconazole had greater intrinsic activity than fluconazole or itraconazole against all three Candida species. For C. glabrata, C. krusei, and C. albicans, the MIC₅₀ values for voriconazole were 1 μg/mL, 0.5 μg/mL, and 0.01 μg/mL, respectively, compared with fluconazole MIC₅₀ values of 8 μg/mL, 64 μg/mL, and 0.25 μg/mL, respectively. If isolates from AIDS patients were excluded, MIC values for isolates from the 1990s were no higher than those noted for isolates from the 1980s. Voriconazole, a new triazole antifungal agent, appears to have enhanced activity against these three species of Candida; the clinical relevance of these findings should be studied in treatment trials.     

In vitro antifungal activity of BMS-181184 against systemic isolates of Candida,Cryptococcus, and Blastomyces species

BMS-181184 is a water-soluble derivative of the pradimicin group of antifungal compounds. We determined the in vitro activities of BMS-181184 and comparator agents amphotericin B, 5-fluorocytosine, fluconazole, and ketoconazole against 184 systemic fungal isolates collected at the Health Sciences Centre in Winnipeg, Canada, between 1987 and 1995. BMS-181184 demonstrated MICs of between 1 and 8 μg/mL for all Candida albicans, Candida glabrata, Candida tropicalis, Candida krusei, Candida lusitaniae, and Cryptococcus neoformans isolates tested. BMS-181184 was less active against Candida parapsilosis (MIC₉₀ = 16 μg/mL) and Blastomyces dermatitidis (MIC₉₀ = 32 μg/mL). Isolates of Candida species with fluconazole MICs of ≥16 μg/mL and those with fluconazole MICs of ≤8 μg/mL demonstrated similar BMS-181184 sensitivities.     

Comparison of the in vitro activities of the echinocandin LY303366, the pneumocandin MK-0991, and fluconazole against Candida species and Cryptococcus neoformans.

Two new glucan synthesis inhibitors, the echinocandin LY303366 and the pneumocandin MK-0991 (formerly L-743,872), were studied for their antifungal activities in vitro in relation to each other and in relation to the activity of the triazole fluconazole. Systematic analysis of broth macrodilution testing by varying the starting inoculum size, medium composition, medium pH, temperature of incubation, length of incubation, or selection of endpoints failed to identify significant differences in antifungal activity for either LY303366 or MK-0991 in comparison to the activity under standard test conditions specified for other antifungal agents in National Committee for Clinical Laboratory Standards (NCCLS) document M27A. Under standardized conditions, both drugs exhibited prominent activity against Candida species including Candida glabrata and Candida krusei but showed little activity against Cryptococcus neoformans. This spectrum of activity differed from that of fluconazole, which exhibited marginal activity against C. glabrata and C. krusei but prominent activity against other Candida species and C. neoformans. For individual strains, broth microdilution MICs of LY303366 and MK-0991 were similar to but frequently higher than broth macrodilution results. In contrast, fluconazole broth microdilution MICs were often lower than broth microdilution results. We conclude that the test conditions specified in NCCLS document M27A are applicable to these two new glucan synthesis inhibitors and that systematic differences between broth microdilution procedures and the broth macrodilution reference standard will need to be addressed before the two test methods can be used interchangeably.     

In Vitro Susceptibilities of Candida Bloodstream Isolates to the New Triazole Antifungal Agents BMS-207147, Sch 56592, and Voriconazole

BMS-207147, Sch 56592, and voriconazole are three new investigational triazoles with broad-spectrum antifungal activity. The in vitro activities of these three agents were compared with those of itraconazole and fluconazole against 1,300 bloodstream isolates of Candida species obtained from over 50 different medical centers in the United States. The MICs of all of the antifungal drugs were determined by broth microdilution tests performed according to the National Committee for Clinical Laboratory Standards method using RPMI 1640 as a test medium. BMS-207147, Sch 56592, and voriconazole were all quite active against all Candida sp. isolates (MICs for 90% of the isolates tested [MIC₉₀s], 0.5, 1.0, and 0.5 μg/ml, respectively). Candida albicans was the most susceptible species (MIC₉₀s, 0.03, 0.06, and 0.06 μg/ml, respectively), and C. glabrata was the least susceptible (MIC₉₀s, 4.0, 4.0, and 2.0 μg/ml, respectively). BMS-207147, Sch 56592, and voriconazole were all more active than itraconazole and fluconazole against C. albicans, C. parapsilosis, C. tropicalis, and C. krusei. There existed a clear rank order of in vitro activity of the five azoles examined in this study when they were tested versus C. glabrata: voriconazole > BMS-207147 = Sch 56592 = itraconazole > fluconazole (MIC₉₀s, 2.0, 4.0, 4.0, 4.0, and 64 μg/ml, respectively). For isolates of Candida spp. with decreased susceptibility to both itraconazole and fluconazole, the MICs of BMS-207147, Sch 56592, and voriconazole were also elevated. These results suggest that BMS-207147, Sch 56592, and voriconazole all possess promising antifungal activity and that further in vitro and in vivo investigations are warranted to establish the clinical value of this improved potency.     

National Epidemiology of Mycoses Survey: A Multicenter Study of Strain Variation and Antifungal Susceptibility Among Isolates of Candida Species

The National Epidemiology of Mycoses Survey (NEMIS) involves six academic centers studying fungal infections in surgical and neonatal intensive care unit (ICU) patients. We studied variation in species and strain distribution and antifungal susceptibility of 408 isolates of Candida spp. Candida spp. were isolated from blood, other normally sterile site cultures, abscesses, wounds, catheters, and tissue biopsies of 141 patients hospitalized in the surgical (107 patients) and neonatal (34 patients) ICUs of medical centers located in Oregon, Iowa, California, Texas, Georgia, and New York. Isolates were also obtained from selected colonized patients (16 patients) and the hands of health care workers (27 individuals). DNA typing was performed using pulsed field gel electrophoresis, and antifungal susceptibility to amphotericin B, 5-fluorocytosine, fluconazole, and itraconazole was determined using National Committee for Clinical Laboratory Standards (NCCLS) methods. Important variation in susceptibility to itraconazole and fluconazole was noted: MICs of itraconazole ranged from 0.25 μg/mL (MIC₉₀) in Texas to 2.0 μg/mL (MIC₉₀) in New York. Similarly, the MIC₉₀ for fluconazole was higher for isolates from New York (64 μg/mL) compared to the other sites (8–16 μg/mL). In general, DNA typing revealed patient-unique strains; however, there were 13 instances of possible cross-infection noted in 5 of the medical centers. Notably, 9 of the 13 clusters involved species of Candida other than C. albicans. Potential transmission from patient-to-patient (C. albicans, C. glabrata, C. tropicalis, C. parapsilosis) and health care worker-to-patient (C. albicans, C. parapsilosis, C. krusei) was noted in both surgical ICU and neonatal ICU settings. These data provide further insight into the epidemiology of nosocomial candidiasis in the ICU setting.     

In Vitro Activities of Voriconazole (UK-109,496) and Four Other Antifungal Agents against 394 Clinical Isolates of Candida spp.

Voriconazole (formerly UK-109,496) is a new monotriazole antifungal agent which has potent activity against Candida, Cryptococcus, and Aspergillus species. We investigated the in vitro activity of voriconazole compared to those of fluconazole, itraconazole, amphotericin B, and flucytosine (5FC) against 394 bloodstream isolates of Candida (five species) obtained from more than 30 different medical centers. MICs of all antifungal drugs were determined by the method recommended by the National Committee for Clinical Laboratory Standards using RPMI 1640 test medium. Overall, voriconazole was quite active against all the yeast isolates (MIC at which 90% of the isolates are inhibited [MIC₉₀], ≤0.5 μg/ml). Candida albicans was the most susceptible species (MIC₉₀, 0.06 μg/ml) and Candida glabrata and Candida krusei were the least (MIC₉₀, 1 μg/ml). Voriconazole was more active than amphotericin B and 5FC against all species except C. glabrata and was also more active than itraconazole and fluconazole. For isolates of Candida spp. with decreased susceptibility to fluconazole and itraconazole MICs of voriconazole were also higher. Based on these results, voriconazole has promising antifungal activity and further in vitro and in vivo investigations are warranted.     

In vitro susceptibilities of clinical yeast isolates to a new echinocandin derivative, LY303366, and other antifungal agents.

LY303366 is a new semisynthetic echinocandin derivative with potent, broad-spectrum fungicidal activity. We investigated the in vitro activity of LY303366, amphotericin B, flucytosine (5FC), fluconazole, and itraconazole against 435 clinical yeast isolates (413 Candida and 22 Saccharomyces cerevisiae isolates) obtained from over 30 different medical centers. MICs for all five antifungal agents were determined by the National Committee for Clinical Laboratory Standards method with RPMI 1640 test medium. LY303366 was also tested in antibiotic medium 3 as specified by the manufacturer. Overall, LY303366 was quite active against all of the yeast isolates when tested in RPMI 1640 (MIC at which 90% of the isolates are inhibited [MIC90], 1.0 microg/ml) but appeared to be considerably more potent when tested in antibiotic medium 3 (MIC90, 0.03 microg/ml). When tested in antibiotic medium 3, LY303366 was 16- to >2,000-fold more active than itraconazole, fluconazole, amphotericin B, or 5FC against all species except Candida parapsilosis. When tested in RPMI 1640, LY303366 was comparable to amphotericin B and itraconazole and more active than fluconazole and 5FC. All of the isolates for which fluconazole and itraconazole had elevated MICs (> or = 128 and > or = 2.0 microg/ml, respectively) were inhibited by < or = 0.007 microg of LY303366/ml when tested in antibiotic medium 3 and < or = 0.5 microg/ml when tested in RPMI 1640. Based on these studies, LY303366 has promising antifungal activity and warrants further in vitro and in vivo investigation.     

Variations in DNA subtype,antifungal susceptibility,and slime production among clinical isolates of Candida parapsilosis

Candida parapsilosis is an important nosocomial pathogen that can proliferate in high concentrations of glucose and form biofilms on prosthetic materials. We investigated the genotypic diversity, slime production, and antifungal susceptibility among 60 isolates of C. parapsilosis from 44 patients and 10 patient care providers from five different medical centers. Molecular typing was performed using macrorestriction digest profiles with BssHII followed by pulsed-field gel electrophoresis (REAG) and by electrophoretic karyotyping (EK). Slime production was evaluated by growing the organisms in Sabouraud broth with 8% glucose and examining the walls of the tubes for the presence of an adherent slime layer. Antifungal susceptibility to amphotericin B, 5-fluorocytosine, fluconazole, and itraconazole was determined using National Committee for Clinical Laboratory Standards proposed standard methods. Overall 28 different DNA types were identified by REAG and EK methods. MIC₉₀ values ranged from 0.12 μg/ml for itraconazole to 1.0 μg/ml for fluconazole and amphotericin B. Sixty-five percent of the isolates produced slime: 37% were moderately to strongly positive, 28% were weakly positive, and 35% were negative. Overall, 83% of blood and catheter isolates were slime positive versus 53% of isolates from all other sites (P < 0.05). These data underscore the genetic diversity and susceptibility of C. parapsilosis to antifungal agents. Slime production may be important in enabling C. parapsilosis to cause catheter-related bloodstream infections.     

In vitro activity of a novel broad-spectrum antifungal, E1210, tested against Candida spp. as determined by CLSI broth microdilution method

The in vitro activity of the novel antifungal agent E1210 and four comparators (caspofungin, fluconazole, posaconazole, and voriconazole) was determined against 90 clinical isolates of Candida using Clinical and Laboratory Standards Institute methods. The collection was composed of 21 Candida albicans, 20 C. glabrata, 25 C. parapsilosis, and 24 C. tropicals, and also included 21 fluconazole-resistant and 15 caspofungin-resistant strains. E1210 was highly active against all the species tested and was more potent than all comparators. The MIC₉₀ results (μg/mL) for E1210, caspofungin, fluconazole, posaconazole, and voriconazole, respectively, were as follows by species: C. albicans (0.06, 4, ≥64, 0.5, 0.5), C. glabrata (0.06, 2, 32, 1, 1), C. parapsilosis (0.06, 4, 16, 0.12, 0.25), and C. tropicalis (0.06, 4, ≥64, 0.5, 2). E1210 was also the most active agent against fluconazole-resistant strains of C. albicans (MIC range, 0.015–0.12 μg/mL), C. glabrata (0.06 μg/mL), C. parapsilosis (MIC range, 0.06–0.05 μg/mL), and C. tropicalis (MIC range, 0.008–0.06 μg/mL), and was the most potent agent tested against caspofungin-resistant strains of C. albicans (MIC range, 0.008–0.12 μg/mL), C. glabrata (MIC range, 0.03–0.06 μg/mL), and C. tropicalis (MIC range, 0.015–0.06 μg/mL).     

In Vitro Activities of Terbinafine against Cutaneous Isolates of Candida albicans and Other Pathogenic Yeasts

Terbinafine is active in vitro against a wide range of pathogenic fungi, including dermatophytes, molds, dimorphic fungi, and some yeasts, but earlier studies indicated that the drug had little activity against Candida albicans. In contrast, clinical studies have shown topical and oral terbinafine to be active in cutaneous candidiasis and Candida nail infections. In order to define the anti-Candida activity of terbinafine, we tested the drug against 350 fresh clinical isolates and additional strains by using a broth dilution assay standardized according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS) M27-A assay. Terbinafine was found to have an MIC of 1 μg/ml for reference C. albicans strains. For 259 clinical isolates, the MIC at which 50% of the isolates are inhibited (MIC₅₀) of terbinafine was 1 μg/ml (fluconazole, 0.5 μg/ml), and the MIC₉₀ was 4 μg/ml (fluconazole, 1 μg/ml). Terbinafine was highly active against Candida parapsilosis (MIC₉₀, 0.125 μg/ml) and showed potentially interesting activity against isolates of Candida dubliniensis, Candida guilliermondii, Candida humicola, and Candida lusitaniae. It was not active against the Candida glabrata, Candida krusei, and Candida tropicalis isolates in this assay. Cryptococcus laurentii and Cryptococcus neoformans were highly susceptible to terbinafine, with MICs of 0.06 to 0.25 μg/ml. The NCCLS macrodilution assay provides reproducible in vitro data for terbinafine against Candida and other yeasts. The MICs for C. albicans and C. parapsilosis are compatible with the known clinical efficacy of terbinafine in cutaneous infections, while the clinical relevance of its activities against the other species has yet to be determined.     

Evaluation of Endpoints for Antifungal Susceptibility Determinations with LY303366

We have previously reported poor correlation between the in vitro fungicidal activity of LY303366 and MIC results in RPMI medium based upon the manufacturer’s suggested susceptibility endpoint, lack of visual growth. Additionally, we have noted a significant trailing effect with LY303366 when MICs are determined in RPMI medium. These observations have led us to evaluate an alternative susceptibility endpoint for LY303366, an 80% reduction in growth compared with control (similar to that utilized for azoles). Two isolates each of Candida albicans, Candida glabrata, and Candida tropicalis were selected for testing. MICs were determined for LY303366 in RPMI 1640 medium buffered with morpholinepropanesulfonic acid. MICs were determined with suggested (MIC₁₀₀) and experimental (MIC₈₀) endpoints. The minimal fungicidal concentration (MFC) of LY303366 for each isolate was also determined. Time-kill curves were determined in RPMI medium with each isolate at concentrations of LY303366 ranging from 0.125 to 16× MIC₈₀ to assess the correlation between MIC₈₀ and fungicidal activity. Lastly, fungi exposed to LY303366 were examined via scanning electron microscope (SEM) for evidence of drug-induced ultrastructure change. MIC₈₀s for test isolates ranged from 0.015 to 0.12 μg/ml and were consistently three to five wells less than MIC₁₀₀s. Good correlation was observed between fungicidal activity, as assessed by kill curves, and the MIC₈₀. SEM data revealed significant ultrastructure changes induced by LY303366 even at sub-MIC₈₀s. Based on our results demonstrating better correlation between MIC₈₀ and fungicidal activity, i.e., time-kill curves and MFCs, we suggest that 80% reduction in visible growth be utilized as the endpoint for susceptibility determinations with LY303366 in RPMI medium.     

Antifungal Susceptibility Profiles of Bloodstream Yeast Isolates by Sensititre YeastOne over Nine Years at a Large Italian Teaching Hospital

Sensititre YeastOne (SYO) is an affordable alternative to the Clinical and Laboratory Standards Institute (CLSI) reference method for antifungal susceptibility testing. In this study, the MICs of yeast isolates from 1,214 bloodstream infection episodes, generated by SYO during hospital laboratory activity (January 2005 to December 2013), were reanalyzed using current CLSI clinical breakpoints/epidemiological cutoff values to assign susceptibility (or the wild-type [WT] phenotype) to systemic antifungal agents. Excluding Candida albicans (57.4% of all isolates [n = 1,250]), the most predominant species were Candida parapsilosis complex (20.9%), Candida tropicalis (8.2%), Candida glabrata (6.4%), Candida guilliermondii (1.6%), and Candida krusei (1.3%). Among the non-Candida species (1.9%), 7 were Cryptococcus neoformans and 17 were other species, mainly Rhodotorula species. Over 97% of Candida isolates were susceptible (WT phenotype) to amphotericin B and flucytosine. Rates of susceptibility (WT phenotype) to fluconazole, itraconazole, and voriconazole were 98.7% in C. albicans, 92.3% in the C. parapsilosis complex, 96.1% in C. tropicalis, 92.5% in C. glabrata, 100% in C. guilliermondii, and 100% (excluding fluconazole) in C. krusei. The fluconazole-resistant isolates consisted of 6 C. parapsilosis complex isolates, 3 C. glabrata isolates, 2 C. albicans isolates, 2 C. tropicalis isolates, and 1 Candida lusitaniae isolate. Of the non-Candida isolates, 2 C. neoformans isolates had the non-WT phenotype for susceptibility to fluconazole, whereas Rhodotorula isolates had elevated azole MICs. Overall, 99.7% to 99.8% of Candida isolates were susceptible (WT phenotype) to echinocandins, but 3 isolates were nonsusceptible (either intermediate or resistant) to caspofungin (C. albicans, C. guilliermondii, and C. krusei), anidulafungin (C. albicans and C. guilliermondii), and micafungin (C. albicans). However, when the intrinsically resistant non-Candida isolates were included, the rate of echinocandin nonsusceptibility reached 1.8%. In summary, the SYO method proved to be able to detect yeast species showing antifungal resistance or reduced susceptibility.     

Antifungal susceptibility trend and analysis of resistance mechanism for Candida species isolated from bloodstream at a Japanese university hospital

We compared the susceptibility of six commercially available antifungal agents (fluconazole, itraconazole, voriconazole, caspofungin, micafungin, and amphotericin B) against 133 Candida bloodstream isolates between 2008 and 2013 at Aichi Medical University Hospital. C. albicans was the most common isolate, followed by C. parapsilosis, C. glabrata, and C. tropicalis. MIC₉₀s of voriconazole against C. albicans, C. parapsilosis, and C. tropicalis were the lowest and that of micafungin against C. glabrata was the lowest among the agents tested. Of the 133 isolates, two strains were identified as drug-resistant. One was a fluconazole-resistant C. glabrata strain, in which the ATP-binding cassette (ABC) transporter gene expression was upregulated. The other was a micafungin-resistant C. glabrata strain, that had 13 amino acid substitutions in FKS1 and FKS2, including a novel substitution V1342I in FKS1 hotspot 2. We also evaluated the susceptibility of T-2307, a novel class of antifungal agents used in clinical trials, against the fluconazole- and micafungin-resistant C. glabrata strain; the MICs of T-2307 were 0.0039 and 0.0078 μg/mL, respectively. In conclusion, the incidence of bloodstream infection caused by drug-resistant Candida spp. was rare from 2008 to 2013 at our hospital. Of 133 isolates, only two strains of C. glabrata were resistant to azoles or echinocandins, that upregulated the ABC transporter genes or had novel FKS mutations, respectively.     

Comparison of EUCAST and CLSI broth microdilution methods for the susceptibility testing of 10 Systemically active antifungal agents when tested against Candida spp.

The antifungal broth microdilution (BMD) method of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) was compared with Clinical and Laboratory Standards Institute (CLSI) BMD method M27-A3 for amphotericin B, flucytosine, anidulafungin, caspofungin, micafungin, fluconazole, isavuconazole, itraconazole, posaconazole, and voriconazole susceptibility testing of 357 isolates of Candida. The isolates were selected from global surveillance collections to represent both wild-type (WT) and non-WT MIC results for the azoles (12% of fluconazole and voriconazole results were non-WT) and the echinocandins (6% of anidulafungin and micafungin results were non-WT). The study collection included 114 isolates of Candida albicans, 73 of C. glabrata, 76 of C. parapsilosis, 60 of C. tropicalis, and 34 of C. krusei. The overall essential agreement (EA) between EUCAST and CLSI results ranged from 78.9% (posaconazole) to 99.6% (flucytosine). The categorical agreement (CA) between methods and species of Candida was assessed using previously determined CLSI epidemiological cutoff values. The overall CA between methods was 95.0% with 2.5% very major (VM) and major (M) discrepancies. The CA was >93% for all antifungal agents with the exception of caspofungin (84.6%), where 10% of the results were categorized as non-WT by the EUCAST method and WT by the CLSI method. Problem areas with low EA or CA include testing of amphotericin B, anidulafungin, and isavuconazole against C. glabrata, itraconazole, and posaconazole against most species, and caspofungin against C. parapsilosis, C. tropicalis, and C. krusei. We confirm high level EA and CA (>90%) between the 2 methods for testing fluconazole, voriconazole, and micafungin against all 5 species. The results indicate that the EUCAST and CLSI methods produce comparable results for testing the systemically active antifungal agents against the 5 most common species of Candida; however, there are several areas where additional steps toward harmonization are warranted.     

In vitro efficacy of 5 antifungal agents against Candida parapsilosis,Candida orthopsilosis, and Candida metapsilosis as determined by time–kill methodology

Killing activity of amphotericin B, fluconazole, voriconazole, posaconazole, and 5-fluorocytosine was determined against 6 Candida parapsilosis, 3 Candida orthopsilosis, and 4 Candida metapsilosis clinical isolates. After 24 h, 1 of 6 C. parapsilosis, 1 of 3 C. orthopsilosis, and 3 of 4 C. metapsilosis isolates were killed at 1 to 4 μg/mL (1–8× MIC) amphotericin B. The remaining isolates were killed by 2 to 4 μg/mL amphotericin B after 48 h. Fluconazole was fungistatic at ≥1× MIC (0.5–2 μg/mL) against C. parapsilosis and at ≥2× MIC (4–8 μg/mL) against C. orthopsilosis and C. metapsilosis isolates. Voriconazole inhibited C. parapsilosis at ≥1× MIC (0.015–0.12 μg/mL), but the other 2 species were inhibited only at 4 to 8× MIC (0.25–0.5 μg/mL). Against C. orthopsilosis and C. metapsilosis, posaconazole was fungistatic close to the MIC (0.03–0.06 and 0.015–0.03 μg/mL, respectively). Against C. orthopsilosis and C. metapsilosis, fluconazole and voriconazole, but not posaconazole, seem to be less active in vitro than against C. parapsilosis.     

Susceptibility of fluconazole-resistant clinical isolates of Candida spp. to echinocandin LY303366, itraconazole and amphotericin B

The in vitro activity of LY303366 was compared with those of itraconazole and amphotericin B against 156 fluconazole-resistant (MIC > or = 16 mg/L) clinical isolates of CANDIDA: spp. An adaptation of the NCCLS reference method was employed for determination of MICs. LY303366 was more potent than either itraconazole or amphotericin B against Candida albicans, Candida glabrata, Candida krusei and Candida tropicalis, even against isolates with itraconazole MICs > or = 1 mg/L. LY303366 was less potent in vitro against Candida parapsilosis and Candida guilliermondii isolates. LY303366 has promising antifungal activity and warrants further investigation.     

Saccharomyces cerevisiae infections and antifungal susceptibility studies by colorimetric and broth macrodilution methods

Saccharomyces cerevisiae was isolated in large numbers from operative specimens from two patients with perforated bowel and peritonitis and from the blood of another patient treated with extracorporeal membrane oxygenation. Susceptibility studies were performed on these three isolates and another 29 isolates that colonized or caused infection in a total of 19 patients seen over the last decade. All isolates had low minimum inhibitory concentration (MIC) values for amphotericin B (MIC₉₀ of ≤0.02 μg/ml) and flucytosine (MIC₉₀ of 0.2 μg/ ml), and a broader range of MIC values for itraconazole (MIC₉₀ of 0.8 μg/ml) and fluconazole (MIC₉₀ of 4 μg/ml). A colorimetric method using Alamar blue reagent showed good concordance with the standard broth macrodilution method for amphotericin B, flucytosine, and fluconazole, but less good concordance for itraconazole. Serious infections with S. cerevisiae probably should be treated with amphotericin B, with or without the addition of flucytosine.     

Comparison of the Vitek 2 yeast susceptibility system with CLSI microdilution for antifungal susceptibility testing of fluconazole and voriconazole against Candida spp., using new clinical breakpoints and epidemiological cutoff values

A commercially available, fully automated yeast susceptibility test system (Vitek 2; bioMérieux, Marcy d’Etoile, France) was compared in 3 different laboratories with the Clinical and Laboratory Standards Institute (CLSI) reference microdilution (BMD) method by testing 2 quality control strains, 10 reproducibility strains, and 425 isolates of Candida spp. against fluconazole and voriconazole. Reference CLSI BMD MIC endpoints and Vitek 2 MIC endpoints were read after 24 hours and 9.1–27.1 hours incubation, respectively. Excellent essential agreement (within 2 dilutions) between the reference and Vitek 2 MICs was observed for fluconazole (97.9%) and voriconazole (96.7%). Categorical agreement (CA) between the 2 methods was assessed using the new species-specific clinical breakpoints (CBPs): susceptible (S) ≤2 μg/mL, susceptible dose-dependent (SDD) 4 μg/mL, and resistant (R) ≥8 μg/mL for fluconazole and Candida albicans, Candida tropicalis, and Candida parapsilosis and ≤32 μg/mL (SDD), ≥64 μg/mL (R) for Candida glabrata; S ≤0.12 μg/mL, SDD 0.25–0.5 μg/mL, R ≥1 μg/mL for voriconazole and C. albicans, C. tropicalis, and C. parapsilosis, and ≤0.5 μg/mL (S), 1 μg/mL (SDD), ≥2 μg/mL (R) for Candida krusei. The epidemiological cutoff value (ECV) of 0.5 μg/mL for voriconazole and C. glabrata was used to differentiate wild-type (WT; MIC ≤ ECV) from non-WT (MIC > ECV) strains of this species. Due to the lack of CBPs for the less common species, the ECVs for fluconazole and voriconazole, respectively, were used for Candida lusitaniae (2 μg/mL and 0.03 μg/mL), Candida dubliniensis (0.5 μg/mL and 0.03 μg/mL), Candida guilliermondii (8 μg/mL and 0.25 μg/mL), and Candida pelliculosa (4 μg/mL and 0.25 μg/mL) to categorize isolates of these species as WT and non-WT. CA between the 2 methods was 96.8% for fluconazole and 96.5% for voriconazole with less than 1% very major errors and 1.3–3.0% major errors. The Vitek 2 yeast susceptibility system remains comparable to the CLSI BMD reference method for testing the susceptibility of Candida spp. when using the new (lower) CBPs and ECVs.