Phenotypic switching and genetic diversity of Cryptococcus neoformans.

Niger seed agar was used as a primary plating medium for the isolation of Cryptococcus neoformans from cerebrospinal fluid specimens from AIDS patients with untreated primary cryptococcosis. The medium was used as the primary means to detect variations in the colony morphology of the yeast. To search for phenotypic and genetic variations, nine patients individually harboring two or three types of colony morphology were studied. Intraindividual isolates from nine patients had minor variations in the API 20C profile, and the MICs of one or more antifungal agents (amphotericin B, fluconazole, and itraconazole) for isolates from three patients were significantly different. Intraindividual isolates from three patients had minor karyotype differences, and one showed a dramatic chromosomal length polymorphism. In addition, three serial isolates from a patient with two episodes of infection showed similar karyotypes, confirming persistent infection by the same strain. Random amplified polymorphic DNA products were identical for all isolates (including three isolates from a relapse case). Our results provided evidence suggesting that (i) in humans, C. neoformans may undergo phenotypic and genetic changes during early infection prior to antifungal agent administration; (ii) dramatic variations in electrophoretic karyotypes and in phenotypes, as demonstrated during the early infection of one patient, may be due to infection by different strains; and (iii) the use of niger seed agar as a primary plating medium is useful for studying antifungal susceptibility, phenotypic switching, genetic diversity, and multiple-strain infections.

Molecular typing of global isolates of Cryptococcus neoformans var. neoformans by polymerase chain reaction fingerprinting and randomly amplified polymorphic DNA-a pilot study to standardize techniques on which to base a detailed epidemiological survey.

A total of 356 clinical isolates of the encapsulated basidiomycetous fungus Cryptococcus neoformans var. neoformans, obtained from Australia, Argentina, Brazil, India, Italy, New Zealand, Papua New Guinea, South Africa, Thailand and the USA, were analyzed to lay the basis for a comprehensive evaluation of the global genetic structure of C. neoformans. Two polymerase chain reaction (PCR)-based typing techniques were standardized: PCR fingerprinting using a single primer specific to minisatellite or microsatellite DNA, and randomly amplified polymorphic DNA (RAPD) analysis using two combinations of three 20- to 22-mer random primers. Previous studies showed that the resultant profiles are reproducible and stable over time. Identical results were obtained in two different laboratories and by different scientists in the same laboratory. Both typing techniques separated the isolates into four major groups (VNI and VNII, serotype A; VNIII, serotype A/D; and VNIV, serotype D). The majority (78%) of isolates belonged to VNI, compared with 18% VNII, 1% VNIII and 3% VNIV. All US isolates could be differentiated by a unique, strain-specific PCR fingerprint or RAPD pattern in contrast to most of the non-US isolates, which showed a substantially higher degree of genetic homogeneity, with some clonality, in different parts of the world. Isolates obtained from the same patient at different times and from different body sites, had identical banding patterns. Both typing techniques should provide powerful tools for epidemiological studies of medically important fungi.

Pathogenicity of basidiospores of Filobasidiella neoformans var. neoformans.

Basidiospores of Filobasidiella neoformans var. neoformans (progeny of Cryptococcus neoformans MT 100.1 x VR 45980) were able to induce cryptococcosis in Swiss albino mice if inoculated by intraperitoneal injection, nasal instillation or nasal spraying. The latter method, with the aid of a jet nebulizer, was first adopted to imitate the natural entrance of infectious particles. Using this method the small number of basidiospores (7 x 10(3)) could induce cryptococcosis in mice, while the higher number of the parental laboratory-grown yeast cells (1.5 x 10(6)) did not produce infections. By nasal instillation Cyclophosphamide (Cy)-treated mice were more susceptible to the basidiospores, showing 80% cryptococcosis (eight of 10). Seven of the eight infected mice had disseminated cryptococcosis. Immunocompetent mice were more resistant to basidiospore infection than Cy-treated mice, as 40% of that group developed only pulmonary cryptococcosis; none had disseminated infection. Thus, we propose that basidiospores are one form of the infectious propagules of F. neoformans var. neoformans which can cause cryptococcosis, particularly in immunocompromised people.

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.

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 characterized 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 serotype 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.

Occurrence of Penicillium marneffei infections among wild bamboo rats in Thailand.

Penicilliosis marneffei has emerged as an endemic systemic mycosis in Southeast Asia among humans and wild bamboo rats. To gain an insight into the epidemiology of this life-threatening disease, a survey of bamboo rats for natural infections by Penicillium marneffei was carried out in the central plains of Thailand during June-September, 1987. Thirty-one lesser bamboo rats (Cannomys badius) and eight hoary bamboo rats (Rhizomys pruinosus) were trapped. Portions of their internal organs were cultured to determine if they had been infected by P. marneffei. Six each of C. badius (19.4%) and R. pruinosus (75%) yielded cultures of this unique, dimorphic Penicillium species. All of the isolates were readily converted to their unicellular form that multiplies by the process of schizogony by incubating them at 37 degrees C on plates of brain heart infusion agar. Their identity was further confirmed by a specific immunological test. Among the internal organs of the positive rats, the lungs had the highest positivity (83.3%), next in decreased order of frequency were the liver (33.3%) and the pancreas (33.3%). The use and value of domestic and wild animals in locating and demarcating endemic areas of geophilic fungal pathogens are discussed. Penicilliosis marneffei is considered to be a zooanthroponosis--a disease that occurs in lower animals, as well as, humans.

Itraconazole in the treatment of acute vaginal candidosis.

An open non-comparative multicenter study of Itraconazole (Sporal) 400 mg as a single day therapy for vaginal candidosis was carried out at Siriraj Hospital and Chulalongkorn Hospital from 1st November 1988 to 31st August, 1989. Fifty-nine female out patients with vaginal candidosis were included in the study after excluding pregnancy, lactation, mixed vaginal infection and prior antimycotic therapy. Two capsules of 100 mg Itraconazole were given b.i.d. as a single day 400 mg dose. The patients were evaluated at the beginning prior to treatment for physical signs and symptoms, direct microscopic examination, and culture of vaginal fluid. The first and second follow-up were arranged at the end of one week and one month after therapy. The evaluations were the same as in the first visit. The clinical cure rate was 89 and 90 per cent at first and second follow-up respectively. The mycological cure rates were 83.3 and 69.57 per cent at the first and second follow-up respectively. There was one case of Torulopsis glabarta at the second follow-up.

Persistent bacterial endocarditis caused by three isolates of satellite streptococci.

Three isolates of satellite streptococci were cultivated from the blood of a patient affected persistent bacterial endocarditis. They had distinguishable ultrastructural abnormalities. Their cell wall architecture changed from a fuzzy coat (first isolate) to a thick electron transparent layer covered with a rough fuzzy coat (second isolate), and to electron dense globular material which detached from the wall in small patches (third isolate). The antibiotics probably played an important role in changing their architecture. These three isolates were probably derived from the same strain, since they had common biochemical characteristics and they were isolated from the same patient during the course of his endocarditis.

Abnormal form of Aspergillus terreus isolated from mycotic abscesses.

A remarkable outer cell-wall thickening (up to 1.5 muM) 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.

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.