Electrophoretic karyotypes of <Emphasis Type="Italic">C. neoformans</Emphasis> serotype A recovered from Thai Patients with AIDS

Thirty-seven clinical isolates of C. neoformans were recovered from AIDS patients and all were serotype A according to standard typing tests. They were further analyzed using RAPD, PCR fingerprinting, and PFGE along with 2 additional reference isolates ATCC 34871 (serotype A) and RV 45981 (serotype D). Using 2 different RAPD primers, all of the clinical isolates and the reference serotype A (ATCC 34871) gave similar RAPD patterns while serotype D (RV 45981) gave distinctive pattern. Corresponding result was also obtained upon PCR by using a primer for microsatellite (GACA)₄. However, using a primer specific to minisatellite M13 + 1, all PCR fingerprinting gave similar gel patterns (M1) for 35/37 of the clinical isolates and the reference serotype A while two clinical isolates generated different patterns called M2 and M3. The reference serotype D gave distinctive pattern called M4. PFGE gave 17 different karyotypes that could be categorized into 4 groups named EKA (1–6), EKB (1–5), EKC (1– 5) and EKD (1). The reference serotype A fell into group EKA as EKA6 while the reference serotype D fell into group EKC as EKC5. Among the clinical isolates, EKA group (20/37 isolates) and type EKA1 (16/20) dominated with only one isolate each for types EKA2 to EKA5. The next most prevalent was group EKB (12/37 isolates) which dominately fell in type EKB1 (8/12) and only one isolate each for types EKB2 to EKB5. Group EKC (4/37 isolates) and group EKD (1/37) had only one isolate for each type (EKC1 to EKC 4 and EKD1). The 2 predominant karyotypes (EKA1, 16/37 and EKB1, 8/37) may represent two originally common clones of C. neoformans expose among the patients. The high discriminatory power of PFGE infers the benefit of subtyping which lead to better understanding on the epidemiology and pathogenic potential of C. neoformans subtypes. Moreover, PCR fingerprinting and RAPD infer the feasibility of detail analysis between serotypes A and D for unencapsulated C. neoformans.     

A new aspect of the life cycle ofCandida tropicalis

The life cycle ofCandida tropicalis has been elucidated by nuclear staining. We were able to distinguish 5 stages: (1) the sexually active unstable haplophase which either can mutate directly to (2) the sexually inactive haplophase, or is converted to (3) the diplophase by “autoploidization”. The unstable diplophase changes into the sexually inactive haplophase, which is stable, by “budding meiosis’. Chlamydospores (4), which are possibly formed by sexually inactive haploid cells, are true resting spores, and during their germination only mitosis takes place. The buds formed during germination may develop into either the sexually active, or the inactive haplophase. During budding meiosis in the diplophase, multinucleate giant cells (5) may occur. The authors do not consider the latter to be metabasidia. Instead, they might in some way be homologus with asci, and their daughter nuclei might be comparable with the nuclei of ascospore initials. These new nuclei migrate successively into new buds which process results in a short chain of haploid cells. True ascospores have not been found. From these findings it is likely thatC. tropicalis is closely related to the ascomycetes rather than to the Hemibasidiomycetes.     

Serotypes of Cryptococcus neoformans isolated from patients prior to and during the AIDS era in Thailand

One hundred and eighty-seven strains of Cryptococcus neoformans isolated from patients in Thailand were charcterized by biochemical varieties relating to serogroups. Canavanine-glycine-bromothymol blue (CGB) agar was used for differentiating the varieties of C. neoformans. Slide agglutination tests were performed with Crypto Check (Iatron, Inc., Tokyo) to determine their serotypes. Fifty-five percent (10 out of 18) of the pre-AIDS isolates were serotype B, 28% were serotype A, 5% were serotype D, and an unexpected 11% (2 out of 18) were serotype C. These are the first to be recorded in Asia. In contrast, among the 169 clinical isolates obtained between January 1993 and March 1995 (AIDS epidemic), serotype A was outstandingly predominant-93% (157 out of 169), serotype B was relatively low (3.6%) and both serotypes D and AD were 1.8%. The pattern of serotypes of the 59 isolates from known HIV-positive patients was closely similar to the total isolates during the AIDS epidemic. In determining the varieties of C. neoformans by CGB, only 1 of the 187 isolates gave a false reaction. On the basis of our findings, we believe that in the pre-AIDS era either C. neoformans var. gattii serorype B or serotype C were the common causative agents of cryptococcosis in Thailand. The advent of AIDS changed the pattern of serotypes with serotype A becoming predominant as has been reported world wide.     

Survey of sensitivity of twelve yeast genera toward T-2 toxin.

A survey was made to detect the sensitivity of 12 yeast genera to T-2 toxin. Seventy-five yeasts isolated from various sources were tested for their susceptibility to T-2 toxin. The MIC of T-2 for these yeasts varied from 1.0 to greater than 8.0 micrograms/ml. Of the yeasts studied, Kluyveromyces fragilis showed the greatest sensitivity, which ranged between 0.5 and 2.5 micrograms of T-2 toxin per ml of culture medium. The roles of incubation temperature, size of the inoculum, and incubation time on the MICs were determined. The results suggest that in comparison with other yeasts, K. fragilis is very sensitive to T-2 toxin.     

Abnormal form of Aspergillus terreus isolated from mycotic abscesses

A remarkable outer cell-wall thickening (up to 1.5 m) was observed on septate hyphae obtained from pus collected from multiple abscesses of a 25-year-old female patient. Ultrastructural examination of the hyphae showed a thick electron dense layer of microfibrillar material surrounding the electron transparent cell wall. The organism was able to grow only on hypertonic media upon initial isolation but on later subculture it grew on normal isotonic media. The thick microfibrillar material diminished progressively upon subculture but could be demonstrated in 7 day secondary cultures in isotonic liquid medium. There, microfibrillar bridges appeared to bind hyphae together. The observations suggested that this microfibrillar material was a true extracellular component. The immunological status of the patient was not examined, but her 10 year history of multiple mycotic abscesses and dermatophytoses suggested some abnormalities.     

Recovery of Cryptococcus neoformans from the nasopharynx of AIDS patients

Nasopharyngeal swabbings, obtained from AIDS patients, were plated onto Niger seed agar containing antibiotics. Cryptococcus neoformans was isolated from 35 out of 84 patients (41.7%) diagnosed as primary cryptococcal cases before antifungal administration, and 8 out of 86 (9.3%) cryptococcosis patients on antifungal therapy. The fungus could not be isolated from any of 447 samples from 194 AIDS patients not diagnosed with cryptococcosis. These findings are novel in that the presence of C. neoformans in AIDS patients at this site has never been looked at previously. This revised version was published online in June 2006 with corrections to the Cover Date.     

A glutinous rice culture medium for demonstration of chlamydospores of candida albicans

Thirty-four recent isolates ofCandida albicans from clinical material were cultured on glutinous rice agar at 21 pH values ranging from 2.2 to 11.9. After incubation at 25°C all isolates produced chlamydospores on this medium at pH values from 6.6 to 8.0 with an optimum pH of 7.1. Nineteen stock cultures and all recent isolates ofCandida albicans were used to compare the new glutinous rice agar with 9 other culture media recommended for chlamydospore formation. The results indicated that the new medium was superior in terms of (1) economy, (2) rapid production of chlamydospores, (3) transparency and (4) ease of investigation by direct microscopic examination.

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Zusammenfassung Vierunddreißig jüngst isolierte Stämme vonCandida albicans aus klinischem Material sind auf Glutin-Reisagar innerhalb 21 pH-Werte vom 2.2 bis 11.9 gezüchtet worden. Nach Inkubation bei 25°C haben alle Stämme auf diesem Medium bei den Werten von pH 6.6 bis 8.0 Chlamydosporen produziert mit dem Optimum bei pH 7.1. Neunzehn Stammkulturen und alle jüngst isolierten Stämme vonC. llbicans sind verwendet worden um den neuen Glutin-Reisnährboden mit neun anderen, empfohlenen Nährböden fur Chlamydosporen-Produktion zu vergleichen. Die Ergebnisse zeigten, daß der neue Nährboden in folgenden Beziehungen vortrefflicher war: 1) Wirtschaftlichkeit; 2) rasche Chlamydosporen-Produktion; 3) Durchsichtigkeit; 4) Leichtigkeit bei direkter mikroskopischer Untersuchung.

     

Survey of sensitivity of twelve yeast genera toward T-2 toxin

A survey was made to detect the sensitivity of 12 yeast genera to T-2 toxin. Seventy-five yeasts isolated from various sources were tested for their susceptibility to T-2 toxin. The MIC of T-2 for these yeasts varied from 1.0 to greater than 8.0 micrograms/ml. Of the yeasts studied, Kluyveromyces fragilis showed the greatest sensitivity, which ranged between 0.5 and 2.5 micrograms of T-2 toxin per ml of culture medium. The roles of incubation temperature, size of the inoculum, and incubation time on the MICs were determined. The results suggest that in comparison with other yeasts, K. fragilis is very sensitive to T-2 toxin.