In this study, we established an in-house database of yeast internal transcribed spacer (ITS) sequences. This database includes medically important as well as colonizing yeasts that frequently occur in the diagnostic laboratory. In a prospective study, we compared molecular identification with phenotypic identification by using the ID32C system (bioMérieux) for yeast strains that could not be identified by a combination of CHROMagar Candida and morphology on rice agar. In total, 113 yeast strains were included in the study. By sequence analysis, 98% of all strains were identified correctly to the species level. With the ID32C, 87% of all strains were identified correctly to the species or genus level, 7% of the isolates could not be identified, and 6% of the isolates were misidentified, most of them as Candida rugosa or Candida utilis. For a diagnostic algorithm, we suggest a three-step procedure which integrates morphological criteria, biochemical investigation, and sequence analysis of the ITS region.Yeast infections are increasing due to the growing number of immunocompromised and severely ill patients (5, 9). In addition, widespread use of antibiotics and invasive procedures facilitate infections with yeasts (18). Although Candida albicans is still the most frequently encountered yeast species, others have gained increasing importance in the last few years (1). Some species, such as Candida krusei (resistance to fluconazole) or Trichosporon sp. (reduced susceptibility to amphotericin B), may show inherent resistance to antimycotics (13). Rapid and accurate identification is thus essential for proper treatment. Various identification methods have been proposed in the past, including morphology, physiological properties, nucleic acid amplification, restriction fragment length polymorphism analysis, and sequencing (2).For molecular identification, we have chosen sequence analysis since this procedure is simple and can be fully automated. In addition, interpretation of nucleic acid sequences is straightforward and does not depend on too much expertise compared to morphological analyses. As target, we have chosen the internal transcribed spacer (ITS) region, which is located between the highly conserved genes coding for 18S and 28S rRNA. The ITS encompasses the two noncoding regions ITS1 and ITS2, which are separated by the highly conserved 5.8S rRNA gene (20). The ITS1 and ITS2 regions are more variable than the adjacent rRNA gene sequences and thus promise a better separation of closely related species. As the inspection of yeast ITS sequences which are available in the public database GenBank (NCBI) suggested that some entries are incorrect and because certain medically relevant species are not included, we decided to establish an in-house database. Since the number of known yeast species is enormous, we restricted our database to species occurring in the medical diagnostic laboratory.In this study, we compared sequence-based identification with conventional identification. Based on these results, we establish...

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The data raised for Cryptococcus neoformans and cryptococcosis in Austria, Germany and Switzerland during the period from July 1997 to December 1999 are presented and analysed according to the epidemiological criteria. This is part of the ECMM survey in Europe.

We reported previously that Trichophyton mentagrophytes contains a cytoplasmic macromolecule which specifically binds progesterone. Progesterone is also an effective inhibitor of growth of the fungus. We report here studies which characterize more fully the specific binding properties and the functional responses of T. mentagrophytes and taxonomically related fungi to a series of mammalian steroid hormones. Scatchard analysis of [3H]progesterone binding in both the + andmating types of Arthroderma benhamiae and in Microsporum canis revealed a single class of binding sites with approximately the same affinity as that in T. mentagrophytes (Kd, 1 X 10-7 to 2 X 10-7 M). Trichophyton rubrum had a protein with a higher binding affinity (Kd, 1.6 x 10-8 M). Characterization of the [3H]progesterone-binding sites in T. mentagrophytes showed the binder to be a protein which was destroyed by trypsin and heating to 56°C. Previous examination of the steroid-binding specificity in T. mentagrophytes had demonstrated that deoxycorticosterone (DOC) and dihydrotestosterone (DHT) were effective competitors for [3H]progesterone binding. Expansion of this study to include other competitors revealed that R5020 (a synthetic progestin), androstenedione, and dehydroepiandosterone possessed relative binding aflinities which were 20, 11, and 9% of that of progesterone, respectively. Other ligands tested were less effective. Competition studies for the binder in M. canis resulted in similar findings:

Specific binding of [3H]progesterone to cytosol of Trichophyton mentagrophytes was demonstrated. Scatchard analysis of [3H]progesterone binding showed a single class of binding sites with a dissociation constant of 9.5x 108 ± 2.4 x 10-8 M (standard deviation) and a maximal binding capacity of 4,979 ± 3,489 fmol/mg of cytosol protein. Deoxycorticosterone and dihydrotestosterone competitively inhibited binding by 50% at molar ratios of 10:1 and 20:1, respectively. Other steroid hormones that were tested had minimal activity, indicating binding specificity. Steroid hormone actions in T. mentagrophytes were examined in growth studies. Growth was assessed by determination of cellular ATP content. Progesterone inhibited growth in a dose-responsive manner, with a 50% inhibition concentration of 5.5 x 10-6 M. Partial recovery from inhibition occurrred after 24 to 48 h; inhibition could be enhanced by dividing the amount of added progesterone every 24 h. In the same rank order as was their relationship to each other and progesterone in binding studies, deoxycorticosterone and dihydrotestosterone were less effective inhibitors; other steroid hormones that were tested showed no consistent effect. We hypothesize that the binder described, acting as a hormone receptor, is the molecular site of action for the functional effect of the hormone. The functional effect may be related to the observed resistance of females to dermatophytosis. on July 16, 2020 by guest http://iai.asm.org/ Downloaded from on July 16, 2020 by guest http://iai.asm.org/ Downloaded from on July 16, 2020 by guest http://iai.asm.org/ Downloaded from

The antibacterial and antimycotic activity of econazole base, an imidazole derivative, was examined in vitro and in experimental infections of mice. Comparative minimal inhibitory concentration (MIC) determinations indicate econazole as well as miconazole to be of moderate activity against gram-positive bacteria (MICs: 0.78–25 μg/ml) and yeasts (MICs: 1.56–25 μg/ml). Against filamentous fungi, econazole exhibits better in vitro activity than miconazole and – with the exception of Rhizopus oryzae and Absidia corymbifera – MICs are markedly lower than against yeasts. No effect of nutrient media and no effect of the inoculum were observed with the four drugs. A strong influence of bovine serum on MIC values, however, suggested a strong protein binding. In experimental candidiasis of mice, no therapeutic effect with econazole base administered orally or intraperitoneally could be observed (ED₅₀ and ‘minimum life-prolonging dose’: > 200mg/kg). In experimental aspergillosis of mice, a slight effect, as demonstrated by the ‘minimum life-prolonging dose’ of 100 mg/kg, was found. The in vitro and in vivo results are discussed in the light of the available pharmacokinetic and toxicological data. It is concluded that more studies, especially on the pharmacology of econazole and about the clinical efficacy, are needed to come to a definite judgement.

Paracoccidioides brasiliensis, the etiologic agent of paracoccidioidomycosis, causes disease much more frequently in men than it does in women, suggesting that the hormonal milieu of the host might influence P. brasiliensis pathogenicity. We recently demonstrated that cytosol from yeast cultures of P. brasiliensis contains a high-affinity, low-capacity, tritiated 17I-estradiol ([3H]estradiol)-binding protein. Estradiol and, to a lesser degree, diethylstilbestrol (DES), inhibited the transformation of P. brasiliensis cultures from the mycelial to the yeast form, an event critical to the establishment of infection. Our current studies demonstrated a somewhat higher affinity (apparent dissociation constant [Kd],-6 to 12 nM) of the estrogen-binding protein for [3H]estradiol than was previously described for yeast cytosol. The presence of both highand low-affinity estrogen-binding sites in yeast-form P. brasiliensis cytosol was detected after warming the cytosol to 37°C. The high-affinity protein was labile to further heating (56°C), although the low-affinity protein was stable. Additional experiments demonstrated the presence of an estrogen-binding protein in cytosol prepared from mycelial-form P. brasiliensis. This estrogen-binding protein had a slightly lower affinity for [3H]estradiol (Kd-13 nM), and its cytosol contained somewhat fewer binding sites (-78 fmol/mg of protein) than did yeast-form P. brasiliensis cytosol. Of particular interest was the finding that DES, a weak competitor for [3H]estradiol binding in yeast cytosol, displaced [3H]estradiol from the mycelial-form binding moiety. DES had a 50to 100-fold-lower affinity for the [3H]estradiol-binding protein than did estradiol, consistent with its lower bioactivity in the mycelial-to-yeast-form transformation studies. The current results lend further support to our hypothesis that endogenous estrogens in the host, acting through the cytosol binding protein in the fungus, inhibit mycelial-to-yeast-form transformation, thus explaining the resistance of women to paracoccidioidomycosis.