We determined the sequence of the intergenic spacer (IGS) 1 region, which is located between the 26S and 5S rRNA genes, in 25 species of the genus Trichosporon. IGS 1 sequences varied in length from 195 to 719 bp. Comparative sequence analysis suggested that the divergence of IGS 1 sequences has been greater than that of the internal transcribed spacer regions. We also identified five genotypes of T. asahii, which is a major causative agent of deep-seated trichosporonosis, based on the IGS 1 sequences of 43 strains. Most of the isolates that originated in Japan were of genotype 1, whereas the American isolates were of genotype 3 or 5. Our results suggest that analysis of IGS regions provides a powerful method to distinguish between phylogenetically closely related species and that a geographic substructure may exist among T. asahii clinical isolates.Fungal rRNA genes are tandemly repeated, with each repeat encoding 18S (small-subunit), 5.8S, and 26S (large-subunit) genes. Two other regions exist in each repeat: the internal transcribed spacer (ITS) region and the intergenic spacer (IGS) region (Fig. 1). Ribosomal DNA (rDNA) has been widely utilized for molecular systematics and the identification of microorganisms. The D1/D2 regions of 26S and ITS sequences have been used mainly to identify pathogenic fungi. At present, the 26S rDNA sequences of almost all yeasts, including nonpathogenic species, have been determined (3,7,8). The analysis of ITS sequences has been carried out mainly for pathogenic yeast species (1,5,9,10,16,19). Peterson and Kurtzman (13) and Sugita et al. (16) demonstrated that a single species showed less than 1% dissimilarity in either the ITS region or D1/D2 26S rDNA. However, these sequence analyses are sometimes incapable of distinguishing between phylogenetically closely related species, such as the three varieties of Cryptococcus neoformans. Although three varieties within a single species can be distinguished for each varietal level by ITS sequence analysis, the distinction is based on differences of only three or four nucleotides (20). Recently, Diaz et al. (2) and Sugita et al. (17) demonstrated that three varieties of C. neoformans were more clearly distinguished by analysis of IGS 1 and IGS 2 sequences than by ITS sequence analysis. Therefore, IGS sequence analysis appears to be a powerful tool for differentiating between phylogenetically very closely related species.

Cryptococcosis is an important fungal disease in Asia with an estimated 140,000 new infections annually the majority of which occurs in patients suffering from HIV/AIDS. Cryptococcus neoformans variety grubii (serotype A) is the major causative agent of this disease. In the present study, multilocus sequence typing (MLST) using the ISHAM MLST consensus scheme for the C. neoformans/C. gattii species complex was used to analyse nucleotide polymorphisms among 476 isolates of this pathogen obtained from 8 Asian countries. Population genetic analysis showed that the Asian C. neoformans var. grubii population shows limited genetic diversity and demonstrates a largely clonal mode of reproduction when compared with the global MLST dataset. HIV-status, sequence types and geography were found to be confounded. However, a correlation between sequence types and isolates from HIV-negative patients was observed among the Asian isolates. Observations of high gene flow between the Middle Eastern and the Southeastern Asian populations suggest that immigrant workers in the Middle East were originally infected in Southeastern Asia.

Antigenic analysis of the four serotypes of Cryptococcus neoformans was carried out by slide agglutination with reciprocal adsorption methods. With this procedure the antigenic patterns of the serotypes were established. Serotypes A and D had antigenic factors 1, 2, 3, 7 and 1, 2, 3, 8, respectively. Serotypes B and C were found to have antigenic factors 1, 2, 4, 5 and 1, 4, 6, respectively. Factor sera, prepared according to the antigenic patterns demonstrated by adsorption studies, proved to be useful for rapidly and accurately identifying C. neoformans serotypes. Some patterns similar to those of the C. neoformans serotypes were observed in five other Cryptococcus species and two Candida species. The proton magnetic resonance spectra of polysaccharides from the C. neoformans serotypes correlated well with their antigenic characteristics. Phenol oxidase test reactions and growth at 37°C were useful criteria for determining which yeasts should be chosen for clinical application of factor sera for serotyping of C. neoformans. Sixty-two Japanese isolates of C. neoformans were serotyped. Fifty-eight of these isolates were serotype A, three were serotype AD , and one was serotype D.

Melanin is a recognized virulence factor in Cryptococcus neoformans; several pathogenetic mechanisms have been suggested. We studied melanin as an antifungal resistance factor. The growth of laccase‐active strains of C. neoformans and C. albidus in L‐DOPA resulted in the production of black pigment. The formal minimal inhibitory concentrations (MICs) of amphotericin B and fluconazole were not changed by melanization. However, when we examined those wells which contained inhibited cells, we found live cells only in wells containing melanized C. neoformans. In contrast, melanization did not protect C. albidus from killing by amphotericin B. In an amphotericin B time‐kill study of C. neoformans, significantly more melanized cells than non‐melanized survived for the first few hours. Fluorescence microscopy and flow cytometry analyses showed that fewer melanized cells were stained with the fluorescent dye MitoRed. Incubation of MitoRed (the model) or amphotericin B with melanin extracted from C. neoformans decreased the free concentrations of these substances. Fluconazole, in contrast, was not removed from solution by melanin. This suggests that neoformans cryptococcal melanin deposited amphotericin B in the cell wall binds, reducing its effective concentrations.

Various modified guanine derivatives were synthesized and introduced into G(4) of d(CGCGCG)(2) to evaluate their capacity to stabilize Z-form DNA. It was found that the incorporation of 8-methylguanosine (m(8)rG) in oligonucleotides stabilizes the Z form more dramatically than does the incorporation of 8-methyl-2'-deoxyguanosine (m(8)G). This enhancement is ascribed to a reduction in the entropic penalty, which arises from the introduction of hydrophilic groups in solvent-exposed regions. The incorporation of m(8)rG into DNA sequences markedly stabilizes the Z form even in the absence of NaCl. The Z-DNA stabilizer allows oligonucleotides with a wide range of sequences to be converted to the Z form. It could be a powerful tool for examining the molecular basis of many types of Z-form-specific reactions at the molecular level under physiological salt conditions.