Two cyp51-related genes (cyp51A and cyp51B) encoding 14-␣ sterol demethylase-like enzymes were identified in the opportunistic human pathogen Aspergillus fumigatus. PCR amplification using degenerate oligonucleotides based on conserved areas of cytochrome P450 demethylases of other filamentous fungi and yeasts allowed the cloning and sequencing of two different homologue genes in A. fumigatus. Southern analysis confirmed that both genes hybridized to distinct genomic loci and that both are represented as single copies in the genome. Comparison of the deduced Cyp51A and Cyp51B proteins with the CYP51 proteins from Penicillium italicum, Aspergillus nidulans, Erysiphe graminis, Uncinula necator, Botrytis cinerea, Ustilago maydis, Cryptococcus neoformans, Candida albicans, Saccharomyces cerevisiae, Candida tropicalis, and Candida glabrata showed that the percentages of identity of the amino acid sequences (range, 40 to 70%) were high enough to consider Cyp51A and Cyp51B to be members of the fungal CYP51 family. Fragments from both genes were also cloned from other Aspergillus spp. (A. flavus, A. nidulans, and A. terreus). Phylogenetic analysis showed that, at least in the most pathogenic species of Aspergillus, there are two fungal CYP51 proteins. This is the first report of the existence of two homologue genes coding for 14-␣ sterol demethylase in the fungal kingdom. This finding could provide insights into the azole resistance mechanisms operating in fungi. The primers used here may be useful molecular tools for facilitating the cloning of novel 14-␣ sterol demethylase genes in other filamentous fungi.

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.

The genes encoding 14alpha-sterol demethylases (cyp51A and cyp51B) were analyzed in 12 itraconazole (ITC)-resistant and three ITC-susceptible clinical isolates of Aspergillus fumigatus. Six ITC-resistant strains exhibited a substitution of another amino acid for glycine at position 54, which is located at a very conserved region of the Cyp51A protein. The cyp51A gene from the A. fumigatus wild-type strain (CM-237) was replaced with the mutated cyp51A gene copy of an ITC-resistant strain (AF-72). Two transformants exhibited resistance to ITC, both of which had incorporated the mutated copy of the cyp51A gene.

Seven cases of disseminated infection due toCase 2. The patient in case 2 was a 76-year-old male with a relapse of an acute myeloblastic leukemia. Severe neutropenia developed, the patient became febrile, and blood cultures were obtained. Fever persisted in spite of empirical antibiotic treatment with ceftazidime, amikacin, and vancomycin. Three days after antibacterial treatment, skin nodules appeared and a chest radiograph revealed new bilateral lung infiltrates. Conventional AMB, 1 mg/kg of body weight/day, was added to his treatment regimen. The patient remained febrile and died 5 days later. D. capitatus grew on all blood cultures.Case 3. The patient in case 3 was a 42-year-old male with a relapse of an acute myeloblastic leukemia, type M2, who had been diagnosed 5 years before. Eight days after the chemotherapy was finished, he became febrile and neutropenic. Blood cultures were taken and yielded Escherichia coli and viridans group Streptococcus that responded to treatment with meropenem plus vancomycin. Three days later his fever reappeared, and it was accompanied by tachypnea, diarrhea, and stupor. Blood cultures were drawn, chest radiography revealed new bilateral infiltrates, and conventional AMB was added to his treatment regimen. The patient died of septic shock. Blood cultures yielded D. capitatus.Case 4. The patient in case 4 was a 44-year-old female with a B prolymphocytic leukemia with disease progression in spite of fludarabine and CHOP (cyclophosphamide, hydroxydaunomycin, vincristine [oncovin], and prednisone) treatment. Pentostatin-prednisone was administered but caused severe hematological toxicity. The patient developed pancytopenia, fever, and lung consolidation, and empirical antibacterial treatment and conventional AMB, 1 mg/kg/day, were administered in combination with granulocyte-macrophage colony-stimulating factor. The patient died of septic shock. D. capitatus grew from respiratory samples and blood cultures.Case 5. The patient in case 5 was a 76-year-old female who was diagnosed with acute myeloblastic leukemia. During the administration of chemotherapy, the patient became febrile and responded to treatment with piperacillin-tazobactam plus amikacin. Leptotrichia buccalis was isolated from blood cultures. While she was receiving the antibacterial agents, she became febrile again and a lung consolidation was seen upon chest radiography. Other blood cultures were performed, vancomycin was added to her treatment regimen with no response, and the patient was asked to join a double-blind clinical trial for febrile neutropenia of suspected fungal etiology. The clinical trial compared treatments with caspofungin and liposomal AMB. The patient accepted and was included in the clinical trial. The patient seemed to improve the first 2 days but suddenly worsened, showing jaundice, tachypnea, and pleural ef-* Corresponding author. Mailing address: Unidad de Micología,

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Candida albicans strain delineation by karyotyping, NotI restriction pattern analysis, hybridization with specific probe 27A, and PCR fingerprinting with the phage M13 core sequence were performed with 30 isolates from the oral cavities of 30 human immunodeficiency virus (HIV)-infected patients and 8 reference strains. Within the panel of clinical isolates, 20 were geographically related, although 10 isolates were susceptible to fluconazole and 10 isolates were resistant to fluconazole. The remaining isolates used in this study were fluconazole resistant and geographically unrelated. A composite DNA type was defined for each of the strains as the combination of types obtained by the four molecular methods. By this procedure, a great diversity of DNA types was found among isolates from the oropharynges of HIV-infected individuals with oral candidiasis. This diversity was not reduced when isolates were evaluated on the basis of whether they came from the same geographical locale and whether they were fluconazole resistant. These data refute the idea of a clonal origin for fluconazole-resistant strains among HIV-positive patients. Karyotyping was the least discriminatory method, yielding 19 DNA types among the 38 strains analyzed. Conversely, hybridization with the 27A probe showed a unique DNA pattern for each of the strains examined in this study. Our results demonstrate that at least two different molecular methods are needed for Candida albicans typing and that there is a great deal of strain variation within the species, irrespective of place of origin or antifungal resistance patterns.

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Recently, the methodology that will serve as a basis of the standard for antifungal susceptibility testing of fermentative yeasts of the European Committee on Antibiotic Susceptibility Testing has been described. This procedure employs a spectrophotometric method for both inoculum adjustment and endpoint determination.