Samaniya Sukroongreung

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 byPenicillium 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 byP. marneffei. Six each ofC. badius (19.4%) andR. pruinosus (75%) yielded cultures of this unique, dimorphicPenicillium species. All of the isolates were readily converted to their unicellular form that multiplies by the process of schizogony by incubating them at 37 °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.

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.

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.

Phenotypic Switching and Genetic Diversity of Cryptococcus neoformans

ABSTRACT 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.

Electrophoretic karyotypes of C. neoformans 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.

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.

Abnormal form of Aspergillus terreus isolated from mycotic abscesses

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

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. 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.