Cryptococcosis is a cosmopolitan infectious disease of humans and animals caused by an encapsulated basidiomycetous yeast-like fungus Cryptococcus neoformans. Clinical manifestations range from asymptomatic carriage in the respiratory tract to chronic pulmonary disease to meningitic disease with dissemination to the skin and other sites also reported. The incidence of infections varies with geographic location and with patient groups; AIDS has now become the most common predisposing factor for infection. Cryptococcal virulence factors include the ability to form a polysaccharide capsule, produce melanin and to grow at 37°C. C. neoformans var. neoformans (serotypes A and D) has a world-wide distribution and been isolated from various sources in nature and is noted for its association with accumulations of avian guano, especially with pigeon excreta. C. neoformans var. gattii (serotypes B and C) has a more restricted global distribution corresponding to a subtropical to tropical climate. Environmental isolations have now established that C. neoformans var. gattii serotype B appears to have a specific ecological association with Eucalyptus camaldulensis and Eucalyptus tereticornis. The global distribution of these two trees appears to correspond to the epidemiologic distribution of cryptococcosis caused by C. neoformans var. gattii. The epidemiology of cryptococcosis can primarily be explained by exposure to an infective aerosolized inoculum, such as basidiospores released from specific host plants and/or desiccated blastoconidia (yeast cells) disseminated from accumulations of dried pigeon dung. The ecology of C. neoformans still remains largely unresolved, studies on the host-parasite interaction between serotype B and E. camaldulensis are still in progress, and extensive environmental searches are now underway to determine the natural habitats of serotypes A, C and D.

View full abstract

The aim of molecular biology studies on medically important fungi is to connect the functions of proteins to their activities through identification of the genes responsible. In this review we focussed on the electrophoresic karyotypes or the banding patterns of chromosomal DNAs separated by pulsed field gel electrophoresis in Candida albicans which are variable among the strains. We also described the isolation and the characterization of a repetitive sequence RPS which might be involved with chromosome length polymorphism via chromosomal rearrangements.

View full abstract

The increasing incidence of fungal infections in immunocompromised individuals has focused attention on the importance of Candida species, particularly Candida albicans as the major opportunistic fungal pathogen. Among several putative virulence factors of C. albicans is an aspartic proteinase produced and excreted by this yeast which has been considered primarily responsible for its pathogenicity. Although this possibility is supported by experimental data reported from several different laboratories, the actual pathogenetic role of C. albicans aspartic proteinase in the development of candidiasis remains unclear. In the course of studies which were attempted to answer this question, we initially undertook the cDNA cloning of this enzyme.
In this paper we report the comparison of the DNA and amino acid sequences among the enzymes secreted by C. albicans including the enzyme cloned by us, C. tropicalis and C. parapsilosis reported previously and describe their functions.

View full abstract

Mitochondrial DNA (mtDNA) analysis was performed in dermatophytes, dematiaceous fungi, and genus Aspergillus. Twenty-three species of genera Microsporum and Arthroderma were divided into 13 groups according to the mtDNA restriction pattern. The results strongly suggested that M. gallinae is an anamorph of A. grubyi, and M. audouinii and M. ferrugineum are anamorphs of A. otae.
Exophiala spinifera showed an extremely heterogeneous restriction pattern, and was considered to be a complex made up of at least 5 species.
Hortaea werneckii was divided into 6 types, but was concluded to be a single species.
From a dendrogram of genus Aspergillus it was suggested that N. fischeri may be a teleomorph of A. neoellipticus.

View full abstract

We studied detection of the Cryptococcus neoformans gene using PCR method. The primer was designed from 20 mer of Cryptococcus neoformans URA 5 gene. Conditions of the PCR method were denaturing at 94°C for 60sec, annealing at 63°C for 90sec, extension 72°C for 60sec, and this was repeated for 36 cycles. DNA extraction was conducted by the glass bead method, the enzyme method or a combination of the two and the glass bead method showed the highest extraction rate in experiments. The series of experiments revealed the high specificity of the adapted PCR method. Sensitivity in nested PCR was 100pg in quantity of DNA and 1×10³ in diluted solution.

View full abstract

A polymerase chain reaction (PCR) for detection of a gene encoding the secretory aspartate proteinase of Candida albicans has been employed to diagnose candidiasis. A 273-base-pair C. albicans DNA fragment was amplified from cerebrospinal fluid with Candida meningitis and 20 paraffin-embedded tissues with candidiasis. Immunohistochemical study revealed the positive staining of anti C. albicans antibody in these 20 cases. This PCR method proved to be practically useful for rapid and sensitive diagnosis of candidiasis.

View full abstract

The PCR (polymerase chain reaction) technique was used to diagnose Candida albicans infection that was common in immunocompromised patients. Oligonucleotide primers were synthesized to amplify a 180bp fragment on the β-tubulin gene of C. albicans.
DNA was amplified by these primers from C. albicans of all the standard strains and the strains isolated from clinical specimens, but not from any other Candida, other fungal, other bacterial or human DNA. The detection limit using nested PCR was 10fg of purified C. albicans DNA. This PCR is more specific, sensitive and rapid than the conventional culture method and is applicable to diagnosis of candidemia.

View full abstract

We developed a new method for detection of Candida albicans cells in human blood by the polymerase chain reaction that amplifies a 125-bp region within a species-specific DNA fragment originating from C. albicans mitochondrial DNA. In preparing template DNA from C. albicans-containing blood, the addition of C. tropicalis cells, together with antibody which reacts with both cells, raised the sensitivity of detection of C. albicans to about 3 cells per 0.1ml.

View full abstract

The effects of storage with programmed freezing on the virulence and morphology of Paracoccidioides brasiliensis were analyzed by using yeast form cells of 3 strains (Bt-9, Pb-18 and B-1183). From each original strain (O: Original), the following three subcultures were prepared: Subculture F, frozen at -135°C for 6 month; subculture OA, original strain through a mouse; and subculture FA, the frozen strain through a mouse. The virulence and morphology of these subcultures were compared with those of the original strains. Amounts of 10⁶ colony forming units of yeast form cells of each strain were inoculated intravenously into young adult male ddY mice. The virulence of the original strain Bt-9 weakened after the storage at -135°C and did not recover even after animal passage. The morphology was also different from that of the original strain. However, the virulence of strains Pb-18 and B-1183 was not weakened by being frozen and stored at that temperature nor was their morphology change. Storage at -135°C with programmed freezing thus seems inappropriate for P. brasiliensis yeast form cells.

View full abstract

The efficacy and safety of a weekly oral dose of 100mg of fluconazole in the treatment of tinea unguium were evaluated in 18 patients, 4 with fingernail tinea and 14 with toenail tinea. After a 24 week follow-up, the clinical effects were excellent in 8 (44.4%), good in 8 (44.4%), fair in 1 (5.6%) and poor in 1 (5.6%). No side effects or abnormal laboratory findings were observed. Both the efficacy rate and the utility rate were 88.9% (16/18). This study suggests that a weekly dose of fluconazole is useful in the treatment of tinea unguium.

View full abstract

To evaluate the fungicidal effects of antifungal agents quickly, we developed a new antimycotic susceptibility test using neutral red (NR). Trichophyton mentagrophytes treated with various concentrations of antifungal agents for 5 days was stained with NR and washed with 4% formol-1% calcium chloride. The NR incorporated into viable cells was extracted with 0.1M acetic acid-50% ethanol. The minimum fungicidal consentrations of antifungal agents were determined from the results of NR absorbencies (A 540), and were compatible with previously described MICs. This newly-developed method will provide a simple, rapid and quantitative procedure for measuring the minimum fungicidal concentrations of antifungal agents.

View full abstract

Two cases of Trichophyton violaceum infection in a family, a 5-year-old girl with tinea corporis and black-dot ringworm, and her 39-year-old mother with tinea unguium were reported. The affected nail showed an uneven and finely wrinkled surface with slight hyperkeratosis, in accordance with reported characteristics of onychomycosis due to T. violaceum.
T. violaceum was also isolated from the asymptomatic scalps of two siblings of case 1 using the tooth-brush method.
The presence of T. violaceum on the skin surface of family members without lesions and the sporadic occurrence of plural patients in a family was thought to be characteristic of an extremely anthropophilic dermatophyte like T. violaceum.

View full abstract

We report a case of primary pulmonary cryptococcosis diagnosed by detection of a cryptococcal antigen.
A 37-year-old female was admitted to Sawara Hospital on August 17, 1993 because of a cough. At the time of admission, physical and laboratory examinations revealed no abnormal findings, but chest X-ray showed an infiltration and consolidated shadows in the right upper lobe. The patient was treated with 200mg/day of minocycline for 8 days, but no clinical improvement was seen either in the cough or by chest X-ray. We then examined the serum cryptococcal antigen and it showed high titer (×64). Our diagnosis was primary pulmonary cryptococcosis.
The patient was then treated with a daily dose of fluconazole 200mg DIV for 7 days and 400mg PO for 22 days. The antigen titer was decreased from ×64 to ×16.
The cryptococcal antigen test was useful in diagnosing of primary pulmonary cryptococcosis and in monitoring the effect of treatment.

View full abstract