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The ribosomal Internal Transcribed Spacer (ITS) regions of the two recognized species of Coccidioides were studied using a reference set of strains that had been previously identified with species defining microsatellite polymorphisms. Unambiguous identification of the two species proved to be possible by amplifying and sequencing the ITS region. PCR-reactions are sensitive to amplification conditions requiring their careful optimization. Stable amplification and sequencing was achieved with primers ITS3 and 4, enabling species diagnosis. Alternatively, Restriction Fragment Length Polymorphism (RFLP) of the entire ITS region using an annealing temperature of 52 degrees C with the restriction enzymes BsrI and XcmI can also distinguish the species. Three strains typifying the species, Glenospora meteuropaea, G. metamericana and Geotrichum louisianoideum, were analyzed and found to be conspecific with C. posadasii. Although these species have nomenclatural priority over C. posadasii, the latter will be proposed for conservation as it has been included in the US select agent list. In addition, Coccidioides immitis is neotypified in this report. Results of antifungal susceptibility testing did not reveal differences between the two species.

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The role of buffering in the determination of the minimal inhibitory concentration (MIC) of fluconazole was studied with Candida species. Agar dilution tests were performed on media (pH 7.25) buffered with either phosphate or morpholinopropane-sulfonic acid (MOPS) or endomethylene-tetrahydrophthalic acid (EMTA), 0.1 mol l-1 each, or on the unbuffered medium. It consisted of casitone and glucose supplemented with FeCl3 and MgSO4. The MICs recorded after 24 h at 37 degrees C extended from 0.1 mg l-1 to > or = 100 mg l-1 on the phosphate and EMTA medium, being concordant on both media. On the MOPS medium and the unbuffered medium the readings were also concordant; the MICs, however, were mostly 25 mg l-1 or higher. This increase of the values--up to six dilution steps--could not be correlated with the amount of acid secreted by the single strains. EMTA proved to be an alternative to phosphate in this system, and because it allows a faster growth of the yeasts it might be superior to phosphate. The concordance of the MIC values in the presence of such different buffer compounds tends to suggest that they indeed indicate the strongest inhibition attainable in vitro by fluconazole. MOPS was confirmed to be of no use in this system.

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The differentiation between a chronic cryptococcal meningitis and a chronic tuberculous meningitis may cause problems for the clinician only if standard microbiological methods are not applied to the diagnosis of both infections. In a male non-AIDS patient (50 y), 11 years after a suggested diagnosis of "tuberculous meningitis", meningoencephalitis with hydrocephalus was diagnosed and treated accordingly without success. Mycobacterium tuberculosis was never found. Because fibrin fibres of a spider web coagulum in the CSF resembled Aspergillus mycelium, the patient was then treated with amphotericin B + flucytosine. Finally, a mycological examination led to the true diagnosis: (1) In the CSF, resembling Aspergillus hyphae were found to be spider web coagulum fibres. (2) Cryptococcal meningoencephalitis based on the detection of Cryptococcus neoformans in CSF and its antigen in serum and CSF. - At post-mortem, cryptococcal meningoencephalitis was established as cause of death. Residual signs of tuberculosis could not be detected in the brain and the meninges. Common clinical similarities of cryptococcal and tuberculous meningitis and the possibility of a double infection are discussed. A comparison of the presence of Cr. neoformans in the meninges of non-AIDS and AIDS patients is made. The formation of spider web coagulum in the CSF is discussed. Proposals for the diagnosis, therapy and prophylaxis of cryptococcal meningitis are made.

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It was demonstrated that the in vitro growth of a mucoid Escherichia coli strain from the urine of an AIDS patient could disturb the concurrent growth of Cryptococcus neoformans and the development of its brown colour effect (BCE) on Staib agar (syn. Guizotia abyssinica creatinine agar, bird seed agar, niger seed agar etc.) supplemented with penicillin + streptomycin. Owing to the supplementation with the triple antibiotic combination of penicillin + streptomycin + gentamicin and the resulting inhibition of E. coli growth, the formation of an intense BCE of the Cr. neoformans colonies after 3 d at 26 degrees C could be observed. On the same medium supplemented with this triple antibiotic combination 40 Cr. neoformans strains tested showed growth with an intense BCE after 3 d at 26 degrees C; but on Emmons' neutral Sabouraud's dextrose agar (NSDA) supplemented with the same triple antibiotic combination, inhibition of growth was found. For the examination of clinical specimens for Cr. neoformans contaminated with gram-negative rod-like bacteria, Staib agar supplemented with this triple antibiotic combination is proposed. Various antibiotic supplements to primary recovery media for fungi are discussed and ecological interrelations of bacteria and fungi are emphasized.

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Cryptococcus neoformans was found as the causative agent of cryptococcosis in 7 (3.6%) out of 195 HIV-positive persons, most of them being hospitalized. These 7 persons included 6 homosexuals and 1 heroin addict. The examinations were performed at the Mycology Unit of the Robert Koch Institute in Berlin (West) between 1984 and 1986. The brown colour effect (BCE) of the C. neoformans colonies on Guizotia abyssinica creatinine agar (with 0.1% glucose) within 2-5 days at 26 degrees C facilitated the diagnosis of disseminated cryptococcosis. In all 7 cryptococcosis cases, the antigen of C. neoformans was detected in serum and CSF by the latex agglutination test. The initial titres ranged from 1:100,000 to 1:160 in the serum and from 1:1280 to 1:10 in the CSF. In comparison to the progressive stage of the infection with the involvement of the various organs and high antigen titres, the fungus may be detected at an early stage in the respiratory tract only where low antigen titres are observed. All the C. neoformans isolates were found to belong to the variety neoformans. Proposals for an effective control of cryptococcosis are made.

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Using the central venous oxygen saturation (SO2v) at rest and during exercise (50 Watt) a new classification of mitral valve disease is proposed, which corresponds with the recommendation of the NHA (1953). Contrary to the mostly subjective classification by clinical features, the new one results from objective hemodynamic parameters. The validity of the new proposed classification is shown using different clinical parameters.

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