Cryptococcus neoformans-Cryptococcus gattii species complex: an international study of wild-type susceptibility endpoint distributions and epidemiological cutoff values for fluconazole, itraconazole, posaconazole, and voriconazole.

Epidemiological cutoff values (ECVs) for the Cryptococcus neoformans-Cryptococcus gattii species complex versus fluconazole, itraconazole, posaconazole, and voriconazole are not available. We established ECVs for these species and agents based on wild-type (WT) MIC distributions. A total of 2,985 to 5,733 CLSI MICs for C. neoformans (including isolates of molecular type VNI [MICs for 759 to 1,137 isolates] and VNII, VNIII, and VNIV [MICs for 24 to 57 isolates]) and 705 to 975 MICs for C. gattii (including 42 to 260 for VGI, VGII, VGIII, and VGIV isolates) were gathered in 15 to 24 laboratories (Europe, United States, Argentina, Australia, Brazil, Canada, Cuba, India, Mexico, and South Africa) and were aggregated for analysis. Additionally, 220 to 359 MICs measured using CLSI yeast nitrogen base (YNB) medium instead of CLSI RPMI medium for C. neoformans were evaluated. CLSI RPMI medium ECVs for distributions originating from at least three laboratories, which included ≥95% of the modeled WT population, were as follows: fluconazole, 8 µg/ml (VNI, C. gattii nontyped, VGI, VGIIa, and VGIII), 16 µg/ml (C. neoformans nontyped, VNIII, and VGIV), and 32 µg/ml (VGII); itraconazole, 0.25 µg/ml (VNI), 0.5 µg/ml (C. neoformans and C. gattii nontyped and VGI to VGIII), and 1 µg/ml (VGIV); posaconazole, 0.25 µg/ml (C. neoformans nontyped and VNI) and 0.5 µg/ml (C. gattii nontyped and VGI); and voriconazole, 0.12 µg/ml (VNIV), 0.25 µg/ml (C. neoformans and C. gattii nontyped, VNI, VNIII, VGII, and VGIIa,), and 0.5 µg/ml (VGI). The number of laboratories contributing data for other molecular types was too low to ascertain that the differences were due to factors other than assay variation. In the absence of clinical breakpoints, our ECVs may aid in the detection of isolates with acquired resistance mechanisms and should be listed in the revised CLSI M27-A3 and CLSI M27-S3 documents.

Clinical and molecular epidemiology of meticillin-resistant Staphylococcus aureus causing bacteraemia in Southern Spain.

BACKGROUND: Some molecular features of meticillin-resistant Staphylococcus aureus (MRSA) isolates causing invasive infections have been shown to have clinical implications. There is a need to monitor the situation using a combination of molecular and clinical data because, although MRSA clones tend to predominate over wide geographical areas, clonal shifts may take place. AIM: To study the epidemiological features and perform molecular characterization of a retrospective cohort of 98 cases of nosocomial and healthcare-associated MRSA bacteraemia in 10 hospitals in Andalusia, Spain. METHODS: Relatedness of isolates was investigated using pulsed-field gel electrophoresis (PFGE), S. aureus protein A (spa) typing and clonal complex (CC) assignment. Staphylococcal chromosomal cassette mec (SCCmec) type and accessory gene regulator (agr) group were studied by polymerase chain reaction. agr function was assessed. RESULTS: Most isolates were CC5, SCCmec type IV and agr group II. The most common spa type was t067. Six major clusters were identified by PFGE. Six small clusters of epidemiologically related cases sharing isolates from the same PFGE subtype were identified. Five percent of isolates had a vancomycin minimum inhibitory concentration (MIC) of 2 µg/mL on broth microdilution, although 44% had an MIC >1 µg/mL on E-test. Variables independently associated with MIC >1 mg/L on E-test were surgery during present admission and Charlson index ≥2. CONCLUSION: A specific CC that has been predominant in Spain over the last decade caused most of the cases in this study. PFGE was more discriminatory than spa typing in showing clusters of epidemiologically related cases. Some patient features were associated with vancomycin MIC >1 mg/L on E-test.

Activity of BAL 4815 against filamentous fungi.

OBJECTIVES: BAL 4815 is a new antifungal drug and it is the active component of the antifungal triazole BAL 8557 (the water-soluble precursor). We studied the in vitro fungistatic and fungicidal activities of BAL 4815 against 103 clinical isolates of filamentous fungi, including 51 isolates of Aspergillus spp. and 52 isolates of non-Aspergillus filamentous fungi. METHODS: We evaluated the in vitro activity of BAL 4815 against 51 isolates of Aspergillus spp., 20 isolates of dematiaceous fungi, 18 isolates of hyaline Hyphomycetes and 14 isolates of Zygomycetes. MICs were determined following the CLSI M38-A broth microdilution method, using RPMI 1640 medium buffered to pH 7.0 with MOPS. Microdilution plates were incubated at 35 degrees C and read at 24 and 48 h (Mucorales were read at 24 h). Minimal fungicidal concentrations were also determined. RESULTS: For all isolates, geometric mean MICs, MIC(50)s, MIC(90)s and MIC ranges (mg/L) were: Aspergillus spp., 1.67, 2, 4 and 0.5-4; dematiaceous fungi, 1.62, 1, >8 and 0.03 to >8; hyaline Hyphomycetes, 2.41, 2, >8 and 0.03 to >8; and Zygomycetes, 6.81, 8, >8 and 0.03 to >8. Differences in susceptibility between genera were noted. Scedosporium prolificans, Fusarium spp., Mucor spp. and Rhizopus spp. (MIC(90) > 8 mg/L) were less susceptible than Aspergillus spp. (MIC(90) = 4 mg/L). CONCLUSIONS: BAL 4815 has excellent in vitro activity against Aspergillus spp. and variable activity against other filamentous fungi.

Antifungal susceptibility of Cryptococcus neoformans isolates in HIV-infected patients to fluconazole, itraconazole and voriconazole in Spain: 1994-1996 and 1997-2005.

The antifungal drug susceptibility of 70 Cryptococcus neoformans isolates obtained in Spain from 1994 to 1996 (23 isolates) and from 1997 to 2005 (47 isolates) was investigated. The MICs of fluconazole, itraconazole and voriconazole were determined by the modified Clinical and Laboratory Standards Institute (CLSI; formerly National Committee for Clinical Laboratory Standards) broth microdilution method. The MIC(50) and MIC(90) for itraconazole and voriconazole did not change significantly from 1994 to 2005. The MIC(50) of fluconazole remained stable and the MIC(90) decreased by 2 log(2) dilution in the isolates collected from 1997 to 2005. We conclude that the in vitro resistance to fluconazole decreased over an 11-year period. In addition, a tendency for the development of possible cross-resistance between the three triazoles was observed.

Evaluation of disk diffusion method for determining posaconazole susceptibility of filamentous fungi: comparison with CLSI broth microdilution method.

The disk diffusion method was evaluated for determining posaconazole susceptibility against 78 strains of molds using two culture media in comparison with the CLSI (Clinical Laboratory Standards Institute) broth microdilution method (M38-A). A significant correlation between disk diffusion and microdilution methods was observed with both culture media.

Comparison of the Etest and microdilution method for antifungal susceptibility testing of Cryptococcus neoformans to four antifungal agents.

We performed a prospective study to compare the Etest and the microdilution method (NCCLS guidelines) for determining the MICs of fluconazole, itraconazole, flucytosine and amphotericin B for 35 strains of Cryptococcus neoformans. For the microdilution method (MDM) RPMI 1640 medium with 2% glucose was used for fluconazole, itraconazole and flucytosine, and Antibiotic Medium 3 for amphotericin B. For the Etest, RPMI 1640 medium with 2% glucose and solidified with 1.5% agar was used for the four antifungal agents. Amphotericin B was also tested on Antibiotic Medium 3 solidified with 1.5% agar. Fluconazole and flucytosine MICs by the Etest showed good correlation with the broth MDM (81.1 and 89.2% agreement within two dilutions, respectively). With the tested population of itraconazole- and amphotericin B-susceptible isolates, the MIC agreement for itraconazole was 54%; amphotericin B showed the lowest agreement (8.1% on Antibiotic Medium 3 and 13.5% on RPMI).

Correlation of fluconazole MICs with clinical outcome in cryptococcal infection.

We have correlated the in vitro results of testing the susceptibility of Cryptococcus neoformans to fluconazole with the clinical outcome after fluconazole maintenance therapy in patients with AIDS-associated cryptococcal disease. A total of 28 isolates of C. neoformans from 25 patients (24 AIDS patients) were tested. The MICs were determined by the broth microdilution technique by following the modified guidelines described in National Committee for Clinical Standards (NCCLS) document M27-A, e.g., use of yeast nitrogen base medium and a final inoculum of 10(4) CFU/ml. The fluconazole MIC at which 50% of isolates are inhibited (MIC(50)) and MIC(90), obtained spectrophotometrically after 48 h of incubation, were 4 and 16 microg/ml, respectively. Of the 25 patients studied, 4 died of active cryptococcal disease and 2 died of other causes. Therapeutic failure was observed in five patients who were infected with isolates for which fluconazole MICs were > or =16 microg/ml. Four of these patients had previously had oropharyngeal candidiasis (OPC); three had previously had episodes of cryptococcal infection, and all five treatment failure patients had high cryptococcal antigen titers in either serum or cerebrospinal fluid (titers, >1:4,000). Although 14 of the 18 patients who responded to fluconazole therapy had previously had OPC infections, they each had only a single episode of cryptococcal infection. It appears that the clinical outcome after fluconazole maintenance therapy may be better when the infecting C. neoformans strain is inhibited by lower concentrations of fluconazole for eradication (MICs, <16 microg/ml) than when the patients are infected with strains that require higher fluconazole concentrations (MICs, > or =16 microg/ml). These findings also suggest that the MICs determined by the modified NCCLS microdilution method can be potential predictors of the clinical response to fluconazole therapy and may aid in the identification of patients who will not respond to fluconazole therapy.

A comparative evaluation of Etest and broth microdilution methods for fluconazole and itraconazole susceptibility testing of Candida spp.

The Etest strip is a promising tool of broad application in clinical microbiology. The method provides MIC readings and is easier to perform than broth microdilution. We carried out a study to compare the MICs of fluconazole and itraconazole obtained by the Etest with those obtained by broth microdilution, performed according to the guidelines of the NCCLS document M27-A, with 402 clinical isolates (360 Candida albicans, 17 Candida tropicalis, nine Candida krusei, nine Candida glabrata and seven Candida parapsilosis) and seven control isolates. The agreement between MICs by the two methods (at +/- 2 dilutions) was 74.5% for fluconazole and 61.4% for itraconazole. These results suggest that further development is necessary to standardize the medium and incubation conditions before introduction of the Etest as a routine method in the clinical microbiology laboratory for fluconazole and itraconazole susceptibility testing.

Response to fluconazole and itraconazole of Candida spp. in denture stomatitis.

The significance of Candida albicans in the development of denture stomatitis (DS), as well as the clinical and microbiological efficacy of treatment with fluconazole and itraconazole was studied in 115 patients affected with DS and 200 controls (100 healthy patients with dental prosthesis and 100 healthy patients without prosthesis). Specimens were taken from all patients; subsequently all patients with positive culture of the DS group were treated with fluconazole. A second specimen was taken after 15 days of treatment with fluconazole, and if the results were positive again, treatment with itraconazole was instituted and the patients were given appointments for taking a third specimen. The incidence of C. albicans was 92% in the group of patients with DS. After treatment with fluconazole, a clinical cure of 97% and a microbiological cure of 78% was obtained in the patients with DS. In 3.2% of the cases strains resistant to fluconazole were found. The cases of microbiological resistance to fluconazole were treated with itraconazole resulting in a clinical cure of 100% and a microbiological cure of 77%. The results show the poor correlation of the clinico-microbiological response after treatment with these antifungal agents in denture stomatitis.

Evaluation of CHROMagar Candida medium for the isolation and presumptive identification of species of Candida of clinical importance.

CHROMagar candida is a new differential culture medium that allows the isolation and presumptive identification of species of yeast of clinical importance. We tested 618 strains of yeast, including 128 direct isolates from clinical specimens. After two days of incubation at 37 degrees C, 339 of 341 Candida albicans, 98 of 99 Candida glabrata, all the Candida tropicalis, and all the Candida krusei were identified correctly. The sensitivity and specificity in these cases were both superior to 99%. Easy to prepare, with low cost, CHROMagar Candida proves to be a useful medium for the identification of species of yeast that are isolated with greater frequency in clinical material and for the identification of mixed cultures.

Antifungal activity of sertaconazole in vitro against clinical isolates of Candida spp..

Sertaconazole is a new azolic derivative containing a benzo(b)-thiophene group, for topical use. It showed in vitro fungistatic and fungicidal activity against yeasts, dermatophytes, opportunistic filamentous fungi and Gram-positive bacteria. In this study, we have evaluated the activity of sertaconazole in vitro against 215 strains of Candida spp. Fluconazole and ketoconazole were used throughout as reference compounds. The minimum inhibitory concentration (MIC) was determined in Casitone medium and the breakpoint was obtained spectrophotometrically and visually. Visual reading of MICs correlated with the spectrophotometric determination, the values of MIC50 and MIC90 showed no difference (+/- 1 dilution) in any of the species tested (range 0.1-16 mg/l). These results show that sertaconazole is an excellent antifungal agent and fungicide in vitro against various species of Candida.

Antifungal activity of LY295337 in vitro against clinical isolates of Candida spp.

LY295337 is a new antifungal agent derived from the echinocandins; it shows in vitro and in vivo activity against a great variety of pathogenic fungi. In this study, we evaluated the in vitro activity of LY295337 against 201 strains of Candida spp. using two culture media: Sabouraud dextrose broth (SDB) and RPMI 1640; fluconazole was used as reference antifungal agent. The minimum inhibitory concentration (MIC) was determined by the broth microdilution method for LY295337 in SDB and RPMI 1640, and for fluconazole by an agar dilution method. The fungicidal activity of LY295337 was also determined. The MIC-50 as well as MIC-90 values obtained for LY295337 in SDB (range 0.025-0.6 mg/l) were lower than those obtained for RPMI 1640 (0.035-1.25 mg/l). All the strains resistant to fluconazole were sensitive to LY295337 (MIC < 0.6 mg/l). These results show that LY295337 is an excellent antifungal agent with a fungicidal activity in vitro against various species of Candida.