The physiological patterns, the sequence polymorphisms of the internal transcriber spacer (ITS), and intergenic spacer regions (IGS) of the rRNA genes and the antifungal susceptibility profile were evaluated for their ability to identify Trichosporon spp. and their specificity for the identification of 49 clinical isolates of Trichosporon spp. Morphological and biochemical methodologies were unable to differentiate among the Trichosporon species. ITS sequencing was also unable to differentiate several species. However, IGS1 sequencing unambiguously identified all Trichosporon isolates. Following the results of DNA-based identification, Trichosporon asahii was the species most frequently isolated from deep sites (15 of 25 strains; 60%). In the main, other Trichosporon species were recovered from cutaneous samples. The majority of T. asahii, T. faecale, and T. coremiiforme clinical isolates exhibited resistance in vitro to amphotericin B, with geometric mean (GM) MICs >4 g/ml. The other species of Trichosporon did not show high MICs of amphotericin B, and GM MICs were <1 g/ml. Azole agents were active in vitro against the majority of clinical strains. The most potent compound in vitro was voriconazole, with a GM MIC <0.14 g/ml. The sequencing of IGS correctly identified Trichosporon isolates; however, this technique is not available in many clinical laboratories, and strains should be dispatched to reference centers where these complex methods are available. Therefore, it seems to be more practical to perform antifungal susceptibility testing of all isolates belonging to Trichosporon spp., since correct identification could take several weeks, delaying the indication of an antifungal agent which exhibits activity against the infectious strain.

Candida dubliniensis is a recently identified chlamydospore-positive yeast species associated with oral candidiasis in human immunodeficiency virus (HIV)-infected (HIV ؉) patients and is closely related to Candida albicans. Several recent reports have described atypical oral Candida isolates with phenotypic and genetic properties similar to those of C. dubliniensis. In this study 10 atypical chlamydospore-positive oral isolates from HIV ؉ patients in Switzerland, the United Kingdom, and Argentina and 1 isolate from an HIV-negative Irish subject were compared to reference strains of C. albicans and Candida stellatoidea and reference strains of C. dubliniensis recovered from Irish and Australian HIV ؉ individuals. All 11 isolates were phenotypically and genetically similar to and phylogenetically identical to C. dubliniensis. These findings demonstrate that the geographical distribution of C. dubliniensis is widespread, and it is likely that it is a significant constituent of the normal oral flora with the potential to cause oral candidiasis, particularly in immunocompromised patients. Recently several independent reports have described the recovery of atypical chlamydospore-positive oral Candida isolates from human immunodeficiency virus (HIV)-infected and AIDS patients in Ireland, the United Kingdom, Switzerland, and Australia which were not readily identifiable as any known Candida species by conventional mycological procedures (1-4,

Five sequential Cryptococcus neoformans isolates recovered from an AIDS patient with recurrent meningitis were analyzed. Four isolates were fluconazole susceptible, while the fifth isolate developed fluconazole resistance. Analysis of the 14-␣ lanosterol demethylase gene (ERG11) showed a point mutation in the resistant strain responsible for the amino acid substitution G484S.Mechanisms of azole resistance already described for yeasts include altered affinity of lanosterol 14-␣ demethylase (ERG11) to azole drugs due to target site mutation or its overexpression and decreased accumulation of drugs due to enhanced energydependent drug efflux (5, 13). Changes in the azole affinity of the lanosterol 14-␣ demethylase have already been related to low-level fluconazole resistance in Cryptococcus neoformans isolates (15). In addition, the decreased affinity of lanosterol 14-␣ demethylase for azole derivatives due to mutations that contributes to the increase in the MICs of fluconazole has been described for sequential clinical isolates of Candida albicans (5, 6). To elucidate if this mechanism could also be implicated in the resistance of C. neoformans to azole, we compared the ERG11 genomic sequence in five sequential isolates recovered from recurrent episodes of cryptococcal meningitis.Clinical case. The five strains of C. neoformans were isolated from a 33-year-old male patient who had been positive for human immunodeficiency virus since 1990 and was presenting advanced AIDS (CD4 ϩ count, Ͻ100 cells/mm 3 ) and recurrent cryptococcosis. The first episode of cryptococcosis was diagnosed in August 1997 at Hospital Fernandez, Buenos Aires, Argentina. During the following 15 months, four more episodes of cryptococcal meningitis were detected and documented by cultures recovered from cerebrospinal fluid. In the first episode the patient was treated with amphotericin B (AMB), and for the rest of the episodes a fluconazole therapy at different doses was always established, reaching a cumulative dose of 336 g at the moment of the fifth episode. The five isolates from each episode were identified as C. neoformans var. grubii by the following parameters: morphology, assimilation and fermentation of carbon and nitrogen compounds, and molecular taxonomy.Susceptibility testing was performed by microdilution and E-test methods. Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 were used as quality control strains throughout the experiments (7). Microdilution method. The susceptibility testing followed the NCCLS recommendations (7) but included some modifications as previously described (11). Briefly, the susceptibility testing included RPMI medium supplemented with 2% glucose as the assay medium (RPMI-2% glucose), an inoculum size of 10 5 CFU/ml, flat-bottomed trays, spectrophotometric reading at 530 nm, incubation at 30°C, and shaking at 350 rpm for 48 h (11). The antifungal agents used in the study were as follows: AMB (Sigma Aldrich Quimica S.A., Madrid, Spain), 5-flucytosine (5FC) (Sigma Aldrich Quimica), fluconazole ...

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A bloodstream infection due to Candida haemulonii afflicting a patient with fever and a medical history of megaloblastic anemia is reported. The clinical isolate was misidentified by the API 20C and VITEK identification systems. The results of susceptibility tests showed that the MIC of amphotericin B for C. haemulonii was 4 g/ml. Additional susceptibility testing procedures based on the use of antibiotic medium 3 and Iso-Sensitest broth were performed, and killing curves were determined. Two collection strains of C. haemulonii were employed as controls. The three isolates exhibited resistance to amphotericin B in vitro regardless of the antifungal susceptibility testing method employed. In addition, the MICs of fluconazole for the three isolates were high. Further studies are needed in order to ascertain whether this species exhibits innate or acquired resistance to amphotericin B and other antifungal agents.