INTRODUCTION

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In the fourth edition of The Yeasts: A Taxonomic Study, published in 1998, the number of species belonging to the genus Cryptococcus was increased from 19 to 34 (2). Basidiomycetous Candida species, which contain xylose in cell hydrolysates, were incorporated into Cryptococcus. For example, Candida curvata and Candida humicola were reclassified as Cryptococcus and named Cryptococcus curvatus and Cryptococcus humicolus, respectively.

Serological characteristics have been used in yeast taxonomy since the antigenic analyses by Tsuchiya et al. (21) and have been applied to the rapid and accurate identification of medically important yeast species (5, 15). Previously, we analyzed the antigens of C. neoformans serotypes in adsorption experiments and prepared eight factor sera (6). Using these sera, we reported the antigenic patterns of 13 species and 5 varieties of Cryptococcus and Candida curvata and Candida humicola. Ten Cryptococcus species other than C. neoformans shared the eight antigens. Using slide agglutination patterns, the Cryptococcus species and the two species of Candida studied were grouped into seven groups. Groups 1, 2, 3, and 5 corresponded to C. neoformans serotypes A, D, A-D, and C, respectively. Candida humicola belonged to group 2, and Candida curvata belonged to group 3.

Recently, the number of reports of the isolation of non-neoformans Cryptococcus species from clinical specimens and opportunistic infection by this species have been increasing (9, 11, 12). More taxonomic information and methods for identifying Cryptococcus species are required. This study investigated the antigenic patterns of the accepted 34 Cryptococcus species using type strains and compared the results to the phylogenies reported by Takashima and Nakase (20) and Fonseca et al. (4) based on 18S ribosomal DNA (rDNA) and the D1-D2 regions of the large-subunit (LSU) rDNA sequences, respectively. Furthermore, we demonstrated serological heterogeneity within species using strains of C. curvatus, C. humicolus, and C. laurentii and compared the results to the intraspecies diversity reported by Sugita et al. based on the sequences of internal transcribed spacer (ITS) regions and 28S or 18S rDNA (18, 19).

	MATERIALS AND METHODS

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Strains used. The type strains of 34 Cryptococcus species were used (Table 1). All the strains were obtained from the Centraalbureau voor Schimmelcultures, Baarn, The Netherlands. The strains of C. curvatus, C. humicolus, and C. laurentii from the Centraalbureau voor Schimmelcultures were compared with strains from the Japan Collection of Microorganisms. C. neoformans serotypes A CDC551, B NIH112, C NIH18, and D NIH52 were also used for preparation of factor sera.

Preparation of antigen for slide agglutination test. Strains were grown on modified Sabouraud dextrose agar containing 2% glucose, 1% polypeptone, 0.5% yeast extract, and 1.5% agar for 2 days at 27°C. Several strains that did not grow at 27°C were cultured at 20°C. The cells were harvested in physiological saline solution (PSS), heated at 100°C for 1 h, washed with PSS, and resuspended in 0.5% formalinized saline solution adjusted to McFarland no. 10 turbidity.

Slide agglutination test. Eight factor sera were prepared according to the method described in our previous paper (6). Our previous study used both heated and formalinized cells for immunization and antigenic analyses, but we found no differences between the two types of cells; we therefore used only heat-killed cells in this study. Two milliliters of anti-C. neoformans serum and 1 ml of heat-killed packed cells were mixed. The suspension was then incubated at 37°C for 2 h and then at 4°C overnight. After centrifugation, the supernatant was tested for antibody by the slide agglutination test. The combinations of serotypes of antisera and cells for adsorption are listed elsewhere (6). The agglutination titers of the factor sera used were 1:8 to 16 against serotype A, 1:16 to 32 against serotype A, 1:16 to 32 against serotype A, 1:4 to 8 against serotype B, 1:4 against serotype B, 1:8 against serotype C, 1:8 against serotype A, and 1:8 against serotype D for factors 1, 2, 3, 4, 5, 6, 7, and 8, respectively. Five factor sera in a commercially available kit, Crypto Check (Iatron Laboratories, Inc., Tokyo, Japan) (8), were also used for comparative study. To determine the antigenic formulas of Cryptococcus species, equal volumes of factor serum and heat-killed cell suspension were mixed on a glass slide and rotated for 5 min, and then the results of agglutination were observed. The formation of aggregates within 5 min was considered positive. Smaller clumps were recorded as weakly positive. PSS was used for a negative control. Very small aggregates observed in comparison with the negative controls were recorded as very weakly positive.

	RESULTS

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Antigenic formulas of 34 Cryptococcus species. As shown in Table 1, the agglutination patterns of the 34 Cryptococcus species other than C. neoformans were classified into eight groups. Eighteen of the species in groups 1 to 7 cross-reacted with C. neoformans serotypes. The patterns of groups 1, 2, 3, 5, and 6 were the same as the patterns of C. neoformans serotypes A, D, A-D, B, and C, respectively. The species belonging to group 4, which reacted with factor sera 1, 2, and 3, were closely related serologically to C. neoformans serotype A or D; however, they were not identical to serotype A or D because they lacked factor 7 or 8. C. dimennae, which reacted only with factor serum 1, was placed in group 7. The 15 strains in group 8 were serologically different from the species in groups 1 to 7.

Serological intraspecies diversity. (i) C. curvatus. As shown in Table 3, all nine strains of C. curvatus used possess the major antigens 1, 2, and 3, although small differences in their reactivities to factor sera 7 and 8 were seen. Strains CBS570, CBS2829, and CBS5324 reacted to both factor serum 7 and factor serum 8; strains CBS2744, CBS2755, and CBS5162 reacted to factor serum 8; and strains CBS2176, CBS2754, and CBS5163 did not react to either factor serum 7 or factor serum 8. Our previous study (19) considered these strains of C. curvatus identical species. The C. curvatus strains were homogeneous by both serological and genetic data.

(ii) C. humicolus. The strains of C. humicolus were serologically heterogeneous (Table 4). Molecular biological studies have also demonstrated the heterogeneity of C. humicolus (19). Strains CBS571, CBS2042, and CBS4283 possessed major antigens 1, 2, and 3 and have the same ITS sequence. Although CBS1896 and CBS1897 reacted to factor sera 1, 2, and 3, they also reacted to factor serum I prepared from anti-Trichosporon cutaneum serum (7). The ITS sequences of these two strains were identical. Interestingly, CBS2043 did not react with any of the eight sera but did react to Trichosporon factor I serum. The serological pattern of CBS2043 was identical to that of the type strain of T. cutaneum. These patterns positioned the lineage with T. cutaneum with high bootstrap values on the tree. As for the other strains, the serological pattern of JCM1531 was the same as the pattern of the type strain of C. humicolus. However, since it has a unique sequence, it should be reclassified as another species. JCM1530 lacked antigen 1, and the reactivities of JCM1460 and JCM5123 were very weak. These results show that strains identified as C. humicolus actually belong to several species.

(iii) C. laurentii. Ten strains of C. laurentii were used (Table 5). Eight strains, including the type strain, did not react to any of the factor sera. The type strain did not belong to the Filobasidium or Filobasidiella lineages but was a member of the Bulleromyces lineage (20). Only 2 of 10 strains reacted with factor sera 1 and 2. As with C. humicolus, the C. laurentii strains showed intraspecies variation. These 10 strains were grouped into two major phylogenetic groups (18): one group contained CBS139, CBS2174, CBS8648, CBS318, CBS8645, and CBS942, and the other group contained CBS973, CBS2409, CBS2993, and CBS6578. Although strains CBS942 and CBS8648, which reacted to antisera 1 and 2, belong to the same group, they belong to a different cluster. Sugita et al. suggested that the 10 strains should be further divided among at least seven species (18).

	DISCUSSION

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This report describes the antigenic formulas of the 34 Cryptococcus species accepted in the fourth edition of The Yeasts: A Taxonomic Study (2). The strains used were divided into eight serological groups based on agglutination patterns using eight factor sera derived from serotypes of C. neoformans. This experiment added one new group to those used in our previous studies (6), which corresponded to serotype B, containing C. amylolentus. Consequently, all five serotypes of C. neoformans are found in species of Cryptococcus. The grouping was considered to depend on the chemical structure of the cell surface polysaccharides. Many strains belonging to groups 1, 2, 3, 4, 5, and 6 would possess the same epitopes as those of the C. neoformans serotypes. The surface antigens of strains in groups 7 and 8 could be structurally different, although they are also members of the genus Cryptococcus.

Furthermore, we demonstrated relationships between the antigenic patterns and the molecular phylogenetic data. Previously, we reported that T. cutaneum and related species are serologically classified into four groups: I, II, III, and I-III. Serotypes I, II, and III react with specific sera, named factors I, II, and III, respectively. Serotype I-III reacts with both factors I and III. This grouping correlated well with a molecular phylogenetic tree based on the LSU rDNA sequences (13, 16, 17). We compared our results using Cryptococcus species with recently reported phylogenetic relationships (4, 20). Takashima and Nakase determined the 18S rDNA sequences of the type strains of 33 Cryptococcus species and basidiomycetous yeasts and showed that phylogenetically 23 species were included in five lineages: Bulleromyces, Filobasidiella, Filobasidium, C. humicolus-Trichosporon, and C. luteolus. Fifteen of the 23 species were included in the Filobasidium lineage. These were further divided into three main branches: the C. albidus-C. vishniacii, Filobasidium-related species, and C. terreus branches. Fonseca et al. named the three corresponding groups the albidus, magnus, and aerius clades, respectively, after sequencing the LSU rDNA (D1-D2 region). Interestingly, as shown in Table 2, our serological data correlate well with their classification. The chemical structures of the cell surface antigenic polysaccharides responsible for antigens 1, 2, and 3 of the species in the albidus clade might be very similar.

Since Candida curvata and Candida humicola were closely related to Cryptococcus based on biochemical and serological characteristics, these species were reclassified into the genus Cryptococcus. However, the strains identified as belonging to C. humicolus could be heterogeneous (18). This paper demonstrated heterogeneity of serological characteristics. The phylogenetic position of C. humicolus was located in the C. humicolus-Trichosporon lineage (20). We found that several isolates identified as C. humicolus reacted with factor serum for Trichosporon serotype I. Serologically, the taxonomic position of C. humicolus is near Trichosporon.

C. luteolus and C. hungaricus, which both belong to the C. luteolus lineage, did not react with any of the eight factor sera. Cystofilobasidiales (3) contains four Cryptococcus species: C. huempii, C. aquaticus, C. feraegula, and C. macerans. The first two are on the same branch and did not react with any factor sera. The antigenic patterns of C. feraegula and C. macerans, which belong to another branch in Cystofilobasidiales, were 1, 2, 3, and 7w and 1, 4, and 6, respectively. In a significant number of the species, the serological characteristics were correlated with the molecular phylogenetic data, although several species in isolated phylogenetic positions also shared antigens. This might be a result of differences in the observed sequences. The results of serological studies depend on the genes for enzymes synthesizing cell surface antigens.

Several cases of cryptococcosis due to non-neoformans Cryptococcus species have been reported. C. albidus and C. laurentii have been isolated from lung, cerebrospinal fluid, and blood samples. Recently, these species were isolated from patients with human immunodeficiency virus infection (9-12). C. humicolus and C. curvatus have also been isolated from AIDS patients (1, 14). The number of non-neoformans Cryptococcus infections could increase as opportunistic infections become more common. Rapid, accurate procedures for identifying varieties and serotypes of C. neoformans have been established (6, 8). Taxonomic research and taxonomy-based identification of non-neoformans isolates are required. Sugita et al. (18, 19) and Fonseca et al. (4) have reported the intraspecies diversity of C. humicolus, C. laurentii, and C. albidus using ITS and rDNA sequences. Serological data could provide additional information for taxonomic studies of Cryptococcus and Trichosporon species.

This study used eight factor sera derived from C. neoformans serotypes. Although there are many difficulties in preparing antisera to Cryptococcus species, a future study should prepare antisera using Cryptococcus species antigens to better understand the serological characteristics of Cryptococcus.

In summary, we determined the antigenic patterns of 34 species in the genus Cryptococcus using type strains and eight factor sera prepared from adsorption experiments with C. neoformans serotypes. Comparison of the serological characteristics with the reported molecular phylogenetic tree showed that the serological and phylogenetic data were well correlated in the Filobasidium lineage. Furthermore, serological heterogeneity was observed in the species C. humicolus and C. laurentii, as well as in their phylogenetic relationships.