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The internal transcribed spacer (ITS) region including 5.8S rDNA sequences of 58 isolates of Candida parapsilosis in Brazil and Japan was analyzed. Although most of the C. parapsilosis strains tested were confirmed to belong to three already reported genetically distinct groups (I, II and III) based on their ITS region sequences, 5 strains of the Brazilian isolates showed different sequences from those heretofore reported and suggested a presence of new genotype. For these strains of C. parapsilosis, we proposed a new genetic group (IV). The sequence similarities of this new group of IV to I, II and III were 87.4%, 94.7% and 87.3% in the ITS1 region, respectively. Genetic diversity in ITS regions of the remaining C. parapsilosis strains in Brazil and Japan was also discussed.

One of the main opportunistic fungal infections amongst immunocompromised individuals is oral candidosis, which has been found in up to 90% of human immunodeficiency virus (HIV)-infected patients. This study employed yeasts isolated from the saliva and oral cavities of 114 HIV-infected patients living in Campinas, São Paulo. Of the isolates, 57.8% were identified as Candida albicans and 42.1% as non-C. albicans. The latter isolates were subsequently identified as C. krusei (7.5%), C. lusitaniae (5.2%), C. tropicalis (4.6%), C. parapsilosis (4.6%), C. glabrata (2.8%), C. kefyr (1.7%), C. guilliermondii (1.7%), C. intermedia (1.1%), C. norvegensis (0.5%), and Rhodotorula rubra (1.7%). Susceptibility of the isolates to amphotericin B, fluconazole, miconazole, and itraconazole was also determined by a microdilution method adopted by the National Committee for Clinical Laboratory Standards. The isolates demonstrated various susceptibilities to the antifungal agents. In particular 29 C. albicans and 13 non-C. albicans isolates showed low susceptibility to FLCZ (> 64 micro g/ml). This study revealed huge diversity of Candida species, in particular the increasing emergence of non-C. albicans associated with the oral flora of HIV-infected patients.

One hundred clinical isolates of Cryptococcus neoformans from human immunodeficiency virus (HIV)-infected and non-HIV-infected patients from Brazil, Chile, and Venezuela were separated according to varieties and tested for antifungal susceptibility. A high susceptibility to antifungal agents was observed among all the isolates. The electrophoretic karyotyping of 51 strains revealed good discrimination among Cryptococcus neoformans var. neoformans strains.

Candida dubliniensis is a newly-recognized Candida species and an important infectious pathogen, particularly for HIV-positive patients. >From oral smear samples from the radix linguae of 173 HIV-positive children, we obtained four yeast isolates which took a blue-green color on CHROMagar Candida plate at 37 degrees C for 48 hours from one HIV-positive 3-year-old boy in Brazil. The isolates were difficult to grow on potato dextrose agar plate at 42 degrees C, produced abundant chlamydospores on a cornmeal agar plate with Tween 80, and sprouted germ tubes in saline with horse serum, and the antigenic profile by CANDIDA CHECK test was useless. Carbohydrate assimilation tests by ID32C showed no reference code number in the reference book. The isolates were subjected to molecular biological assay of the DNA sequence of the large-subunit ribosomal DNA region (D1/D2) and randomly amplified polymorphic DNA (RAPD). The DNA sequence agreed with those of standard C. dubliniensis strains, and therefore, the isolates were identified as C. dubliniensis. RAPD band pattern analysis indicated that the clinical isolates might summarize one genotype. Although the child did not present oral lesions, the fungus might be latent for opportunistic infection.

Thirty-three strains of Cryptococcus neoformans were isolated from clinical specimens, including specimens from AIDS patients in Brazil, and were classified into two serotypes; we detected 31 and 2 strains of serotypes A and B, respectively. Random amplified polymorphic DNA (RAPD) fingerprint pattern analyses of these strains of serotypes A and B showed that the patterns were similar for strains of each serotype when three 10-mer primers were used as the RAPD primers. Comparative studies of the fingerprint patterns of the study isolates with those of the reference strains also showed that the RAPD patterns for strains of each serotype were related and that most of the fingerprint bands existed commonly for all strains of each serotype tested. The common RAPD bands (an approximately 700-bp band for serotype A and an approximately 450-bp band for serotype B) were extracted and the DNA sequences were determined. Using this information, we prepared two and one PCR primer pairs which were expected to be specific for C. neoformans serotypes A and B, respectively. Use of each PCR primer combination thus prepared for serotype A or B was 100% successful in identifying the respective C. neoformans serotypes, including the 33 clinical isolates tested in the present study. Among these combinations, one for serotype A was found to amplify DNA from C. neoformans serotype B as well as serotype A. Serotype B-specific PCR primer pairs amplified DNA from not only serotype B strains but also from serotype C strains. The usefulness of other serotype-specific PCR primers for clinical C. neoformans isolates is discussed.

We compared the antigenic characteristics of two thermo-dependent dimorphic fungi isolated from soil in Botucatu, an endemic area of paracoccidioidomycosis (PCM) and Paracoccidioides brasiliensis. The soil isolates grew as cerebriform colonies at 37 degrees C (yeast form) and as cottonous colonies at 25 degrees C (mycelial form). No pathogenicity for ddY mice or hamsters were observed. In immunodiffusion test, there were precipitation bands between the 2 soil isolates and pooled PCM patient sera. There were also common precipitation bands at 21, 50 and 58 kDa between the soil isolates antigens and PCM patient sera by Western-blotting, but no gp43 kDa band. No gene for gp 43 kDa protein was detected in the soil isolates by PCR. The fact that these isolates were obtained from an endemic area of PCM and there were some antigenic similarities between the soil isolates and P. brasiliensis in immunodiffusion test and Western-blotting may have some importance in epidemiological surveys done with paracoccidioidin as well interfering with the immune response of the exposed population.

In an attempt to isolate Paracoccidioides brasiliensis from nature 887 samples of soil from Botucatu, SP, Brazil, were collected cultured in brain heart infusion agar supplemented with dextrose, in potato dextrose agar and in yeast extract starch dextrose agar, all with antibiotics, at 25 degrees and 37 degrees C. Five thermo-dependent dimorphic fungi morphologically resembling P. brasiliensis were isolated; two from armadillo holes; further studies of the biology, antigenicity and genetic features of the five dimorphic fungi are necessary to clarify their taxonomy and their possible relation to P. brasiliensis. In addition, 98 dematiaceous fungi and 581 different species of Aspergillus spp. were also isolated. Our findings emphasize that armadillos and their environment are associated with thermo-dimorphic fungi and confirm the ubiquity of pathogenic dematiaceous fungi and Aspergillus spp.