Strains of Cryptococcus neoformans expressing heteroresistance to fluconazole have been described previously. The present study was conducted to investigate the prevalence of heteroresistance among clinical isolates of C. neoformans and to characterize the heteroresistant phenotypes. A total of 107 clinical isolates of C. neoformans for which the MICs of fluconazole ranged from 0.25 to 32 g/ml were selected. The isolates were chosen to represent a broad geographic distribution. Of the 107 C. neoformans isolates tested, 4 grew on medium containing fluconazole at concentrations that were four to eight times higher than the MICs for each strain. A fifth isolate, for which the fluconazole MIC was 32 g/ml, grew on agar with 64 g of fluconazole per ml. These five isolates (4.7% of the total number) were confirmed to exhibit heteroresistant compositions by population analysis. The degree and frequency of resistance varied among the isolates. Stepwise selection by exposure to fluconazole resulted in subclones of all five strains for which the fluconazole MIC was >64 g/ml. Subclones of three strains demonstrated a homogenous population of resistant cells on medium containing 64 g of fluconazole/ml. The resistance was sensitive to incubation temperature, that is, heteroresistance was demonstrable only at 30°C by agar-based tests, and was reversible through serial transfers on fluconazole-free medium over a period of 8 days. These results suggest that the fluconazole-heteroresistant phenotype of C. neoformans exists in a significant proportion of clinical isolates and that fluconazole resistance can be developed by selection from heteroresistant clones and induction by exposure to fluconazole.

The in vitro activities of the new triazole, ravuconazole (BMS-207147), were compared to those of fluconazole and itraconazole against 541 clinical isolates of Cryptococcus neoformans. Isolates were obtained from cerebrospinal fluid (396), blood (116), and miscellaneous clinical specimens (29). Overall, ravuconazole (MIC at which 90% of the isolates are inhibited [MIC 90 ], 0.25 g/ml) was more active than either itraconazole (MIC 90 , 0.5 g/ml) or fluconazole (MIC 90 , 8 g/ml). Among the isolates inhibited by >16 g of fluconazole/ml, 90.2% were inhibited by <1 g of ravuconazole/ml. On the basis of our findings and the favorable pharmacokinetic properties of ravuconazole, we suggest that ravuconazole may be useful for the treatment of infectious diseases due to C. neoformans and that further clinical studies to confirm these promising in vitro results are warranted.Cryptococcus neoformans has a worldwide distribution and is one of the most important agents of life-threatening infection among the community-acquired opportunistic fungal pathogens (4). Since the 1980s the incidence of Cryptococcus infections in some countries has increased dramatically as a result of AIDS (4, 7). In the United States, the majority of studies report a prevalence of C. neoformans infection among human immunodeficiency virus (HIV)-infected patients in the 5 to 10% range (4, 11). For the treatment of cryptococcal meningitis, fluconazole has been primarily used for maintenance therapy or prophylaxis (15). However, concerns regarding fluconazole-resistant strains of C. neoformans have been expressed by several investigators (2, 3). Itraconazole has been found to be less effective than fluconazole in the treatment of cryptococcal meningitis in HIV-infected patients (17). For these reasons, investigation of the activities of newer antifungal agents against C. neoformans is desired.Ravuconazole (BMS-207147) is a novel triazole antifungal agent (1, 18) with a broad antifungal spectrum and potent activities against major pathogenic fungi such as Aspergillus fumigatus, C. neoformans, Candida spp., and dermatophytes (5,6,8,9). Although its activity against C. neoformans is promising, earlier investigations included limited numbers of clinical isolates, and there is a lack of comparative data with other azole agents. This study provides comparative in vitro susceptibility data for three triazole antifungal agents against a large number of clinical isolates of C. neoformans.A total of 541 clinical isolates of C. neoformans from geographically diverse locations were selected for this study. The collection included 396 isolates from cerebrospinal fluid cultures, 116 from blood cultures, and 29 from miscellaneous clinical specimens (pleural fluid, urine, etc.). All isolates were stored as suspensions in sterile distilled water at room temperature until the study was performed. Prior to testing, each isolate was subcultured at least twice on potato dextrose agar plates (Remel, Lenexa, Kans.) to ensure purity and optimal growth.Standard antifungal pow...

The Vitek automated susceptibility testing system with a modified Gram-Positive Susceptibility (GPS) 106 Card (bioMerieux Vitek, Inc., Hazelwood, Mo.) and a rapid slide latex agglutination test (MRSA-Screen; Denka Seiken Co., Ltd., Tokyo, Japan) were evaluated for their ability to detect oxacillin resistance in Staphylococcus aureus. The oxacillin-salt agar screen (OS) test, the reference broth microdilution method, and the detection of the mecA gene by PCR were compared with the commercial products. A total of 200 contemporary (1999) bloodstream infection isolates were collected from the SENTRY Antimicrobial Surveillance Program, representing diverse geographic areas throughout the world. Among the 99 mecA-positive isolates, 3 isolates were found negative by the MRSA-Screen. Another two isolates did not grow on OS plates and had MICs of 0.5 and 2 g/ml with the Vitek GPS card. All 101 mecA-negative isolates were also found negative by the MRSA-Screen and were categorized as susceptible by the GPS card. Overall, the MRSA-Screen, GPS card, and OS test had sensitivities of 96.9, 98.0, and 98.0% and specificities of 100.0, 100.0, and 98.0%, respectively. MRSA-Screen was a rapid (<15 min) and simple test to perform, and the GPS card provided results in <8 h. Both methods were sensitive and specific for detecting staphylococcal oxacillin resistance in the clinical microbiology laboratory.Methicillin-resistant Staphylococcus aureus (MRSA) is a significant pathogen that has steadily emerged over the last four decades to now cause both nosocomial and community-acquired infections (2). The optimal phenotypic method for detecting (methicillin) oxacillin resistance in S. aureus remains controversial. Moreover, errors in the detection of oxacillin resistance can lead to important adverse clinical consequences. False-susceptible results may contribute to treatment failure as well as to the unchecked spread of MRSA due to a failure to apply appropriate infection control measures. False-resistant results contribute to increased health care costs related to unnecessary isolation precautions, in addition to the consequences of overuse of glycopeptide antimicrobials agents, with associated resistance risk (8). The definitive genotypic test for oxacillin resistance in S. aureus (mecA gene detection by PCR) is not practical for routine performance in clinical laboratories. Therefore, there is an urgent need for a rapid, sensitive, and specific test for MRSA that can be easily performed in clinical microbiology laboratories.This study was performed to compare two newly available tests: (i) the Vitek automated susceptibility testing system with the recently modified Gram-Positive Susceptibility (GPS) 106 Card (bioMerieux Vitek, Inc., Hazelwood, Mo.) and (ii) a rapid slide latex agglutination test (16) for the detection of oxacillin resistance in recent (1999) blood culture isolates of S. aureus obtained from patients worldwide.

The Vitek automated susceptibility testing system with a modified gram-positive susceptibility (GPS) 106 card (bioMerieux Vitek, Inc., Hazelwood. Mo.) and a rapid slide latex agglutination test (MRSA-Screen test; Denka Seiken Co., Ltd., Tokyo, Japan) were evaluated for their abilities to detect oxacillin resistance in coagulase-negative staphylococci (CoNS). The reference broth microdilution method and the detection of the mecA gene by PCR ("gold standard" reference result) were used to compare the results obtained with the commercial products. A total of 123 clinical isolates consisting of eight species were selected from U.S. surveillance collections. Among the mecA-positive isolates (95 strains), 30 isolates were initially negative on the MRSA-Screen test read at 3 min. When the agglutination reaction was extended for 10 min, 26 of the 30 isolates became positive. For a different four isolates, the oxacillin MIC was <0.25 g/ml on the Vitek GPS 106 card. Among the mecA-negative isolates (28 strains), for two Staphylococcus warneri, two S. lugdunensis, and two S. saprophyticus strains MICs were >0.5 g/ml by the reference broth microdilution method. Four of these isolates were also categorized as resistant with the Vitek GPS 106 card and two isolates were positive by the MRSA-Screen test. Overall, the MRSA-Screen test, GPS 106 card, and reference broth microdilution method had sensitivities of 95.7 (result at 10 min), 95.7, and 100%, respectively, and specificities of 92.8, 85.7, and 78.5%, respectively. Although the MRSA-Screen test required a slight procedural modification, both commercial methods achieved a sensitivity and specificity at detecting oxacillin resistance in CoNS at a level that was acceptable for clinical laboratory use.