Galactoxylomannans of Cryptococcus neoformans.

Galactoxylomannans (GalXMs) from single isolates of Cryptococcus neoformans serotypes A, B, and D were isolated from culture supernatants and then purified by affinity, ion-exchange, and gel-filtration chromatography. GalXMs are a group of closely related complex polysaccharides. GalXMs from serotypes A (9759 A) and C (3183 C) and an acapsular mutant of serotype D (Cap67 D) have similar galactose, xylose, and mannose molar ratios, but each has some unique structural features. GalXM9759 A and GalXM 3183 C were associated with a starchlike glucan that was removed during purification. Only a trace of glucose was detected in the Cap67 D GalXM. Gas-liquid chromatography-mass spectroscopy of per-O-methylated polysaccharides and 13C nuclear magnetic resonance spectroscopy showed that GalXM is a complex branched polysaccharide. The main chain consists of mannose or galactose or alternating mannose and galactose residues. Xylose is present only as nonreducing termini. Galactofuranose occurs only in 3183 C and Cap67 D, and it is always present as nonreducing termini.     

Intracellular Fate of Cryptococcus neoformans

Human peripheral leukocytes were found to engulf and kill cells of Cryptococcus neoformans. Fewer encapsulated than nonencapsulated cells met this fate, since cryptococcal capsular polysaccharide inhibited phagocytosis. During 10 to 12 hr of incubation of nonencapsulated cells in human serum, sufficient polysaccharide was produced to inhibit phagocytosis by 50%. The polysaccharide inhibitor was found in the sera of four patients with cryptococcosis, but not on the surfaces of their leukocytes. Additional experiments indicated that serum is not essential for effective phagocytosis. However, normal human serum contains anticryptococcal activity which is not inhibited by capsular material. Preliminary findings indicate that the phagocytic index of a patient with cryptococcosis may be correlated with the severity of his disease.     

Origin of Cryptococcus neoformans var. neoformans Diploid Strains

The basidiomycetous yeast Cryptococcus neoformans is an important human fungal pathogen. Two varieties, C. neoformans var. neoformans and C. neoformans var. gattii, have been identified. Both are heterothallic with two mating types, MATa and MATalpha. Some rare isolates are self-fertile and are considered occasional diploid or aneuploid strains. In the present study, 133 isolates, mostly from Italian patients, were investigated to detect the presence of diploid strains in the Igiene Università Milano culture collection. All of the diploid isolates were further investigated by different methods to elucidate their origins. Forty-nine diploid strains were identified by flow cytometry. PCR fingerprinting using the (GACA)(4) primer showed that the diploid state was associated with two specific genotypes identified as VN3 and VN4. Determination of mating type on V8 juice medium confirmed that the majority of the strains were sterile. PCR and dot blotting using the two pheromone genes (MFa and MFalpha) as probes identified 36 of the 49 diploid isolates as MATa/alpha. The results of pheromone gene sequencing showed that two allelic MFalpha genes exist and are distinct for serotypes A and D. In contrast, the MFa gene sequence was conserved in both serotype alleles. Amplification of serotype-specific STE20 alleles demonstrated that the diploid strains contained one mating locus inherited from a serotype A parent and one inherited from a serotype D parent. The present results suggest that diploid isolates may be common among the C. neoformans population and that in Italy and other European countries serotype A and D populations are not genetically isolated but are able to recombine by sexual reproduction.     

Catecholamines and virulence of Cryptococcus neoformans.

Cryptococcus neoformans was unable to utilize catecholamines (epinephrine, norepinephrine, or dopamine) as sole carbon or nitrogen sources. Therefore, catecholamines are not essential growth factors for this fungus and the brain is not a preferred nutritional niche for its growth with regard to catecholamines. To establish whether the brain is a survival niche for C. neoformans and to explain the role of phenoloxidase as a virulence factor, a wild-type strain that had phenoloxidase activity and mutants which lacked it were exposed to an epinephrine oxidative system, and the survival of both strains was tested. The oxidative system contained epinephrine as an electron donor, Fe3+ as the catalytic transition metal ion, and hydrogen peroxide as an electron acceptor. The wild-type strain was found to be resistant to this oxidative system, whereas under the same conditions the mutant strain was susceptible and its survival decreased at a rate of 4 logs per h. Damage to high-molecular-weight DNA seems to be a causative factor of cell death after exposure of the mutants to the oxidative system. These results suggest that C. neoformans may survive in the brain because of its ability to utilize catecholamines for melanogenesis and thus neutralize the harmful effects of catecholamines which are manifested in the presence of hydrogen peroxide and transition metal ions. The role of phenoloxidase in resistance to the epinephrine oxidative system is also discussed.     

Development of a Singleplex PCR Assay for Rapid Identification and Differentiation of Cryptococcus neoformans var. grubii,Cryptococcus neoformans var. neoformans,Cryptococcus gattii,and Hybrids

A singleplex PCR assay using a single primer pair targeting the putative sugar transporter gene was developed here to distinguish Cryptococcus neoformans var. grubii, Cryptococcus neoformans var. neoformans, and Cryptococcus gattii according to the distinct size of the amplicon. The interspecies and intravarietal hybrids were also characterized on the basis of distinct combined profiles of amplicons. This PCR assay is a rapid, simple, and reliable approach suitable for laboratory diagnoses and large-scale epidemiologic studies.     

Receptor-mediated recognition of Cryptococcus neoformans.

Cryptococcus neoformans, a facultative intracellular pathogen of macrophages, is unique among medically important fungi in its possession of a polysaccharide capsule. Capsule represents the organism's major virulence factor. In the absence of opsonins, binding of encapsulated C. neoformans to macrophages is minimal. Following incubation in serum, C. neoformans potently activates complement, resulting in surface deposition of the third component of complement. Macrophages bind and phagocytose opsonized C. neoformans via three major complement receptors (CR) for C3 fragments, designated CD35 (CR1), CD11b/CD18 (CR3), and CD11c/CD18 (CR4). Antibody in normal human serum generally lacks opsonic activity, although vaccination can elicit anticapsular antibodies that are opsonic. The major component of cryptococcal capsule, glucuronoxylomannan (GXM), is shed from the fungus and circulates in the blood and cerebrospinal fluid of patients with cryptococcosis. Cellular receptors defined for GXM include CD14, toll-like receptor-2, toll-like receptor-4, and CD18. GXM binding to macrophage receptors triggers activation of nuclear factor-kB, but not mitogen-activated protein kinases. This results in no proinflammatory gene expression or release. C. neoformans also secretes mannoproteins, which are recognized by mannose receptors as well as by mannose-binding lectin, perhaps in conjunction with CD14. Strategies directed at modulating how intact C. neoformans and its released components are recognized by phagocytes could lead to novel approaches to treating cryptococcosis     

Molecular identification of Cryptococcus neoformans serotypes

Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections primarily in immunocompromised hosts. Based on the genetic characteristics and serologic properties of capsular polysaccharides, three varieties and five serotypes have been defined: C. neoformans var. neoformans (serotype D), C. neoformans var. grubii (serotype A), hybrid serotype AD, and C. neoformans var. gattii (serotypes B and C). Epidemiologic features, such as geographic distribution and ecologic niche, and clinical characteristics have been shown to be associated with serotypes. At the present time, serotyping is based on agglutination tests with either commercial or "homemade" antisera or on immunofluorescence assays using a monoclonal antibody directed against the capsule polysaccharide. In this paper, we describe two molecular methods (PCR-restriction enzyme analysis and length polymorphism analysis) for C. neoformans serotype identification. Both are based on the sequence characteristics of a fragment of the CAP59 gene required for capsule biosynthesis. Testing of 72 C. neoformans strains including representatives of the five serotypes demonstrated the reliability of these methods.     

Kinetics of lymphocyte transformation in mice immunized with viable avirulent forms of Cryptococcus neoformans.

A murine model was developed to study the cell-mediated immune response of mice immunized with one of two live, avirulent forms of Cryptococcus neoformans: a nonencapsulated mutant and a thinly encapsulated pseudohyphal variant. A lymphocyte transformation assay was used to evaluate the cellular response of control and sensitized spleen cells after in vitro incubation with three merthiolate-killed whole-cell antigens of C. neoformans. An antigen-to-spleen cell ratio of 10:1 and 5 days of incubation of antigen-spleen cell mixtures were established as optimal conditions for maximum lymphocyte transformation. Maximum responses occurred from 2 to 3 weeks after the last of eight weekly intraperitoneal inoculations of C. neoformans. This assay provided an accurate, reproducible method of studying cell-mediated immunity to C. neoformans, and applications to the study of cryptococcal pathogenesis are proposed.     

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.     

Laccase and melanization in clinically important Cryptococcus species other than Cryptococcus neoformans

The laccase enzyme and melanin synthesis have been implicated as contributors to virulence in Cryptococcus neoformans. Since isolations of Cryptococcus species other than C. neoformans from clinical specimens have been increasing, we examined the laccase activities of C. albidus, C. laurentii, C. curvatus, and C. humicola. Incubation of cells with epinephrine produced adrenochrome color in C. albidus, C. laurentii, and C. curvatus but not in C. humicola. Activity was always less than in C. neoformans. Laccase was detected in the soluble fractions of disrupted C. albidus, C. laurentii, and C. curvatus cells. Activity staining of partially purified enzyme after nondenaturing polyacrylamide gel electrophoresis revealed that laccases from C. albidus, C. laurentii, and C. curvatus migrated more slowly than that from C. neoformans. One strain of C. curvatus exhibited two melanin bands. Thus, several clinically emerging Cryptococcus species express laccase and can synthesize melanin.     

Gene disruption in Cryptococcus neoformans and Cryptococcus gattii by in vitro transposition

Cryptococcus neoformans and Cryptococcus gattii are basidiomycetous fungi that infect immunocompromised and immunocompetent people. We developed an insertional mutagenesis strategy for these species based on in vitro transposition and we tested the method by disrupting the URA5 gene in a strain of C. neoformans and the CAP10 gene in three strains of C. gattii. We targeted plasmid DNA containing the URA5 gene or plasmid DNA containing the CAP10 gene from genomic libraries from the shotgun sequencing project for the C. gatti strain WM276. In the latter case, the availability of the end sequences of the clones from the assembled genomic sequence allows rapid selection of target genes for disruption. Modified transposons containing the nourseothricin (NAT) or neomycin (Neo) resistance cassettes were randomly inserted into the target DNA by in vitro transposition. The disrupted genes were used for biolistic transformation and homologous integration was subsequently confirmed by PCR and Southern blot analysis. These results demonstrate that the emerging genomic resources, combined with in vitro transposition into plasmid DNAs from shotgun sequencing libraries or cloned PCR products, will facilitate high-throughput genetic analysis in Cryptococcus species.     

Rapid presumptive identification of Cryptococcus neoformans.

Carbohydrate-containing extracts were prepared from mature yeast colonies grown on Sabouraud dextrose agar by mixing a 0.001-ml loopful of yeast cells for 30 s in phenolized saline and removing the cells by centrifugation. Extracts were prepared from 54 Cryptococcus neoformans isolates, 29 isolates of other Cryptococcus species, 16 isolates of Candida species, 2 Rhodotorula, 2 Torulopsis, and 1 Saccharomyces species. Initially the carbohydrate content of each extract was estimated (Molisch method) and adjusted to 1, 5, and 10 microgram/ml. Twofold dilutions of each extract were tested for reactivity with the cryptococcal latex agglutination reagent of Bloomfield et al. (N. Bloomfield, M.A. Gordon, and D.F. Elmendorf, Jr., Proc. Soc. Exp. Biol. Med. 114:64-67, 1963). All 54 C. neoformans extracts gave strong agglutinations (3+ to 4+) in dilutions of 1:4 or greater. None of the other yeasts produced any agglutination, except for 1 of 15 C. laurentii isolates, which showed a 1+ reaction that disappeared at a dilution of 1:4 and above. Subsequent testing established that a single extract made from 0.001 ml of yeast cells in 6 ml of phenolized saline contained less than 5 microgram of carbohydrate per ml, was suitable for a single rapid screening dilution, and eliminated any cross-reaction from the C. laurentii isolates. In our hands this method has provided a reliable differentiation of C. neoformans from other unknown yeast colonies in less than 20 min exclusive of a Molisch determination.     

Binding of cryptococcal polysaccharide to Cryptococcus neoformans

Radioiodinated cryptococcal polysaccharide was used to study binding of the soluble polysaccharide to encapsulated and non-encapsulated cryptoccoci. Binding of polysaccharide to non-encapsulated cryptococci occurred rapidly over a 30-min period and was largely complete after 2 h. Bound, labeled polysaccharide was slowly eluted from Cryptococcus neoformans after the addition of unlabeled polysaccharide, indicating reversibility of binding. Non-encapsulated cryptococci bound polysaccharide in two ways. Specific binding to the yeast was saturable by ca. 82 ng of polysaccharide per 10(6) yeast cells. Nonspecific binding also occurred which was not saturable under the conditions used in our experiments. Phagocytosis of the non-encapsulated yeast strain was inhibited when the specific binding was ca. 50% saturated. Binding of polysaccharide to an encapsulated strain showed nonspecific, nonsaturable binding, but little specific binding occurred. Presumably the specific binding sites were saturated in the encapsulated strain. Polysaccharides obtained from a hypocapsular mutant (A61) and a normally encapsulated strain competed effectively with labeled serotype D polysaccharide for binding sites on non-encapsulated cryptococci and had identical phagocytosis-inhibiting properties. Similarly, polysaccharides from all four cryptococcal serotypes competed effectively with labeled serotype D polysaccharide for binding sites on the non-encapsulated strain, and all four polysaccharides inhibited phagocytosis of non-encapsulated Cryptococcus neoformans. Unmodified, de-O-acetylated, carboxyl-reduced, periodate-oxidized and reduced (polyalcohol), and Smith-degraded polysaccharides competed with labeled polysaccharide for binding sites on the cell. The unmodified, de-O-acetylated and carboxyl-reduced polysaccharides inhibited phagocytosis of non-encapsulated cells, but the polyalcohol and Smith product were unable to inhibit phagocytosis.     

Extracellular proteinase activity of Cryptococcus neoformans.

Extracellular proteinase activity was studied for eight strains of Cryptococcus neoformans var. neoformans and two strains of Cryptococcus neoformans var. gattii. Proteinase activity was measured by protein agar clearance, azoalbumin hydrolysis, gelatin liquefaction, and protein substrate polyacrylamide gel electrophoresis. All strains of C. neoformans produced extracellular proteolytic activity. Maximal extracellular proteinase activity in supernatants of C. neoformans cultures was associated with late logarithmic- and stationary-phase cultures. C. neoformans was able to utilize murine immunoglobulin G1, bovine immunoglobulin G, and human complement factor 5 for growth in media containing these proteins as the sole sources of carbon and nitrogen, suggesting a capacity to degrade immunologically important proteins. Protein substrate polyacrylamide gel electrophoresis revealed several bands with proteolytic activity at apparent molecular masses of 200, 100, and 50 kDa. The results confirm the existence of extracellular proteinase activity for C. neoformans.     

Combined Fontana-Masson-mucin staining of Cryptococcus neoformans.

Cryptococci react positively with various histochemical stains, including the Fontana-Masson (FM), which stains the cell wall, and mucin stains, such as alcian blue and mucicarmine, which stain the capsule. Combinations of the FM stain with both the alcian blue and mucicarmine stains were performed on paraffin-embedded tissue specimens that were obtained from 15 patients who had culture-proved cryptococcosis. Combined FM-mucicarmine and FM-alcian blue stains were compared with other individual fungal stains. The FM stain, followed by either the mucicarmine or alcian blue stain, distinctively demonstrated both the cell wall and capsule of most organisms. More organisms were recognized in the combined stains than with either stain done individually. No interference between the stains was noted. Combining the FM stain with either of these two mucin stains appears to be helpful for identifying cryptococci.     

Construction of stable episomes in Cryptococcus neoformans

We report the generation of stable plasmids constructed by inserting specific DNA sequences into previously known unstable vectors. These sequences were obtained from a DNA library recovered from a previously reported stable minichromosome created by electroporative transformation in Cryptococcus neoformans (Varma and Kwon-Chung 1994). A 6-kb insert from this minichromosome significantly enhanced both the frequencies at which URA5 transformants were obtained as well as the stability of their uracil prototrophy on non-selective media. A 1.5-kb sequence of this insert contained telomeric sequence repeats which when introduced into plasmids resulted in significant increases in transformation frequency. A 1081-bp sequence (STAB), present in the remainder of the insert, had an ARS-like function enhancing the episomal maintenance of plasmids in the transformants regardless of the gene (ADE2/URA5) used as a selection marker. Received: 4 November 1997 / 11 May 1998     

Spores as Infectious Propagules of Cryptococcus neoformans

Cryptococcus neoformans and Cryptococcus gattii are closely related pathogenic fungi that cause pneumonia and meningitis in both immunocompromised and immunocompetent hosts and are a significant global infectious disease risk. Both species are found in the environment and are acquired via inhalation, leading to an initial pulmonary infection. The infectious propagule is unknown but is hypothesized to be small desiccated yeast cells or spores produced by sexual reproduction (opposite- or same-sex mating). Here we characterize the morphology, germination properties, and virulence of spores. A comparative morphological analysis of hyphae and spores produced by opposite-sex mating, same-sex mating, and self-fertile diploid strains was conducted by scanning electron microscopy, yielding insight into hyphal/basidial morphology and spore size, structure, and surface properties. Spores isolated by microdissection were found to readily germinate even on water agarose medium. Thus, nutritional signals do not appear to be required to stimulate spore germination, and as-yet-unknown environmental factors may normally constrain germination in nature. As few as 500 CFU of a spore-enriched infectious inoculum (∼95% spores) of serotype A C. neoformans var. grubii were fully virulent (100% lethal infection) in both a murine inhalation virulence model and the invertebrate model host Galleria mellonella. In contrast to a previous report on C. neoformans var. neoformans, spores of C. neoformans var. grubii were not more infectious than yeast cells. Molecular analysis of isolates recovered from tissues of infected mice (lung, spleen, and brain) provides evidence for infection and dissemination by recombinant spore products. These studies provide a detailed morphological and physiological analysis of the spore and document that spores can serve as infectious propagules.Humans are exposed to infectious agents via inhalation and cutaneous exposure and from the microbiota. Transmission can involve direct human-human transmission, intermediate animal or insect vectors, or exposure to environmental sources. Animals are exposed to pathogenic fungi via direct/fomite transmission of dermatophytes that infect skin and/or nails, animal-animal transmission by the inhalation of the obligate pathogen Pneumocystis, and bloodstream penetration by Candida species from the gastrointestinal microbiota (46).Many pathogenic fungi (dimorphic fungal pathogens, molds such as Aspergillus fumigatus, and the basidiomycete Cryptococcus) are environmental, and exposure occurs via the inhalation of conidia and/or spores, hyphal fragments, or yeast cells. Particles of >5 μm are subject to efficient mucociliary airway clearance, and smaller infectious propagules more readily deposit deep in the lungs and alveoli (18). As such, spores and conidia represent known or suspected infectious propagules for many pathogenic fungi. Moreover, as spores are often stress resistant, abundant, and readily aerially dispersed, animals may encounter spores more often than other infectious forms. Studies of Schizophyllum commune revealed abundant spores present in air above the ocean at distances of up to a mile from shore (24).Cryptococcus neoformans is a common, opportunistic human fungal pathogen that causes meningoencephalitis in immunocompromised individuals and, if untreated, is uniformly fatal (6, 40). Cryptococcosis is caused by three varieties or sibling species that diverged 10 to 40 million years ago and that exhibit different environmental distributions and virulence properties (32, 43). C. neoformans var. grubii (serotype A) is the major cause (95%) of infections worldwide and >99% of infections in AIDS patients (6). C. neoformans var. neoformans (serotype D) strains account for <5% of infections worldwide, but serotype AD hybrids cause up to 20% of clinical infections in Europe (2, 9, 31). The serotype A and D lineages are globally distributed, associated with pigeon guano, and typically infect immunocompromised hosts.The closely related species Cryptococcus gattii (serotypes B and C) is more geographically restricted to tropical and/or subtropical regions, associated with trees, and commonly infects immunocompetent hosts and less frequently AIDS patients and other immunocompromised hosts (6, 29). A C. gattii outbreak has been occurring on Vancouver Island since 1999 and recently expanded to the Canadian mainland and the United States (5, 12, 25, 38, 52).The infectious propagules for many human fungal pathogens are thought to be spores. Humans are exposed to Cryptococcus by inhalation, leading to an initial pulmonary infection that can be asymptomatic or limited or can disseminate. For many individuals, the initial pulmonary infection is cleared; in others, the organism establishes a dormant latent granulomatous form in the hilar lymph nodes (10, 17). Individuals can die harboring granulomas without overt disease (1, 19, 48). In response to immunosuppression, either primary infection or recrudescence of latent infection results in dissemination via the bloodstream. The organism can infect many organs and tissues but exhibits a predilection to infect the central nervous system (45). The suspected infectious propagules for Cryptococcus are spores or small, desiccated, less encapsulated yeast cells that are an ideal size for alveolar deposition. C. neoformans var. neoformans serotype D spores have been reported to be up to 100 times more infectious than yeast cells in an immunocompromised murine inhalation model (50). Studies to purify spores and analyze their pathogenicity in animal models using different infection methods were previously reported, documenting that spores can serve as infectious propagules under certain conditions, including when directly inoculated intracerebrally, intravenously, or by inhalation (7, 50, 59).Cryptococcus neoformans and C. gattii have defined sexual cycles that produce abundant basidiospores, which could represent infectious propagules (13, 23, 27, 28, 39, 42). However, if spores are the infectious propagule, and spores are produced by mating between a and α cells, how and where would this occur in the largely α unisexual population? A potential resolution was the discovery that α isolates can produce hyphae, basidia, and basidiospores via monokaryotic fruiting (11, 54), a modified sexual cycle that requires only one of the two mating types (4, 12, 21, 33-36, 49). This might be a route by which infectious propagules are produced in nature; however, serotype A C. neoformans var. grubii and C. gattii strains do not reproducibly undergo monokaryotic fruiting under laboratory conditions. Population genetic studies provided evidence that they do so in nature (4, 21, 36, 49), but this remains to be established under defined laboratory conditions.As most previous studies of spore infectivity focused on serotype D, we focused our investigations on serotype A spores and virulence properties. Our analysis examined spore size, shape, and surface properties and conditions supporting spore germination. Importantly, we demonstrate that infectious inocula highly enriched for spores serve as efficient infectious propagules in both a murine inhalation model and an invertebrate host. Analyses of isolates recovered from infected animals provide evidence that recombinant spore progeny are infectious. Spores were not more virulent than yeast cells in either virulence assay. Taken together, our studies provide electron microscopic views of the spores and demonstrate that both spores and yeast cells can serve as infectious propagules.     

Environmental prevalence of Cryptococcus neoformans and Cryptococcus gattii in India: an update

An overview of work done to-date in India on environmental prevalence, population structure, seasonal variations and antifungal susceptibility of Cryptococcus neoformans and Cryptococcus gattii is presented. The primary ecologic niche of both pathogens is decayed wood in trunk hollows of a wide spectrum of host trees, representing 18 species. Overall, C. neoformans showed a higher environmental prevalence than that of C. gattii which was not found in the avian habitats. Apart from their arboreal habitat, both species were demonstrated in soil and air in close vicinity of their tree hosts. In addition, C. neoformans showed a strong association with desiccated avian excreta. An overwhelming number of C. neoformans strains belonged to genotype AFLP1/VNI, var. grubii (serotype A), whereas C. gattii strains were genotype AFLP4/VGI, serotype B. All of the environmental strains of C. neoformans and C. gattii were mating type α (MATα). Contrary to the Australian experience, Eucalyptus trees were among the epidemiologically least important and, therefore, the hypothesis of global spread of C. gattii through Australian export of infected Eucalyptus seeds is rebutted. Reference is made to long-term colonization of an abandoned, old timber beam of sal wood (Shorea robusta) by a melanin positive (Mel(+)) variant of Cryptococcus laurentii that was pathogenic to laboratory mice.     

Determination of Cryptococcus neoformans var. neoformans mating type by multiplex PCR

Mating type plays an important role in the epidemiology and virulence of Cryptococcus neoformans. The present study designed a multiplex PCR method to distinguish the six mating type patterns (Aa, Da, Aalpha, Dalpha, Aa/Dalpha, and Aalpha/Da) of C. neoformans var. neoformans. PCR amplification identified one fragment for Aa (860 bp), Dalpha (413 bp) and Da (645 bp) strains, two fragments for Aalpha (320 and 400 bp) and Aa/Dalpha (860 and 413 bp) strains, and three fragments (645, 400, 320 bp) for an Aalpha/Da strain. The method appears to be a valid, simple and relatively inexpensive tool for epidemiological and virulence studies.