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In conventional DNA fingerprinting, hypervariable and repetitive sequences (minisatellite or microsatellite DNA) are detected with hybridization probes. As demonstrated here, these probes can be used as single primers in the polymerase chain reaction (PCR) to generate individual fingerprints. Several conventional DNA fingerprinting probes were used to prime the PCR, yielding distinctive, hypervariable multifragment profiles for different strains of Cryptococcus neoformans. PCR fingerprinting with the oligonucleotide primers (GTG)5, (GACA)4, and the phage M13 core sequence (GAGGGTGGXGGXTCT), but not with (CA)8 or (CT)8, generated DNA polymorphisms with all 42 strains of C. neoformans investigated. PCR fingerprints produced by priming with (GTG)5, (GACA)4, or the M13 core sequence differentiated the two varieties of C. neoformans, C. neoformans var. neoformans (serotypes A and D) and C. neoformans var. gattii (serotypes B and C). Furthermore, strains of serotypes A, D, and B or C could be distinguished from each other by specific PCR fingerprint patterns. These primers, which also successfully amplified hypervariable DNA segments from other species, provide a convenient method of identification at the species or individual level. Amplification of polymorphic DNA patterns by PCR with these primers offers several advantages over classical DNA fingerprinting techniques, appears to be more reliable than other PCR-based methods for detecting polymorphic DNA, such as analysis of random-amplified polymorphic DNA, and should be applicable to many other organisms.

Several Scedosporium species are clinically important emerging pathogens. Scedosporium prolificans is reported to be the most virulent of the species, while the recently described species Scedosporium aurantiacum, which accounts for a substantial proportion of Australian clinical isolates is capable of causing a range of serious infections. In addition, environmental surveys have revealed a high prevalence of S. aurantiacum in the urban Sydney region. This study was conducted to assess the virulence of selected S. aurantiacum strains recovered from patients who are colonized or have invasive disease, as well as those from environmental sources, in comparison with S. prolificans. PCR fingerprinting with the primer M13 revealed high genetic variation among the S. aurantiacum strains. We evaluated the virulence of eight S. aurantiacum and two S. prolificans strains in a murine model using an infectious dose of 2 × 10⁵ conidia. S. aurantiacum was noted to be as virulent as S. prolificans, causing death in 60-100% of mice (P0.05). There were significant strain-specific virulence differences (P0.005), indicating a possible link between genotype and virulence in S. aurantiacum.

PCR fingerprinting and multilocus sequence typing were applied to determine the major molecular types of the Cryptococcus neoformans/Cryptococcus gattii species complex in the Republic of Korea. Of the 78 strains isolated from patients diagnosed with cryptococcosis between 1990 and 2008, 96% were C. neoformans serotype A, mating type MATalpha and molecular type VNI. The remaining 4% were C. gattii, serotype B, mating type MATalpha and either molecular type VGIIb or VGIII. Of the 62 strains with known HIV status, only 14 (22.6%) were isolated from HIV-positive patients and belonged to molecular type VNI. Remarkably, 93% of the C. neoformans isolates had identical PCR fingerprint profiles with the VNIc genotype that has been identified recently as the major genotype among C. neoformans strains in China. Most strains (81.8%) of the VNIc genotype were associated with non-HIV patients compared with strains of the non-VNIc genotype (20%) (P=0.009). Unlike the Chinese strains, a majority (60%) of the non-HIV patients infected with strains of the VNIc genotype in the Republic of Korea had serious underlying conditions, with cancer and liver disease being the most common. This study affirms VNIc to be the most prevalent genotype of C. neoformans isolated from non-HIV patients with cryptococcosis.

Genomic DNA was prepared from a total of 16 different isolates of the rape seed pathogen Leptosphaeria maculans , the teleomorph of Phoma lingam . These DNA preparations were digested with the restriction endonucleases Bam H I, Eco R I, Hpa II, Mbo I, Msp I and Sau3 A I, separated on agarose gels and analysed by hybridization with the radioactively labelled synthetic oligonucleotides (GTG) 5 , (GACA) 4 or (CT) 8 or with labelled DNA of phage M13. These probes reveal a high degree of DNA polymorphism within the species. Although all investigated isolates can clearly and unequivocally be distinguished from each other, aggressive and nonaggressive strains appear as consistent groups with high degrees of similarity. The fingerprinting technique is thus an appropriate tool for molecular diagnosis of this important plant pathogen. In general, oligonucleotide fingerprinting may be regarded as a convenient technique to identify individual fungal strains with a reliability similar to that of mammalian systems.

We have analyzed nine different species of the filamentous fungus Trichoderma and three strains of T. reesei for the presence of hypervariable loci in their genomes by hybridization with simple repeat oligonucleotides [(CT)8, (GTG)5, and (GACA)4]. On the basis of the DNA-fingerprints obtained, the Trichoderma aggregate is re-classified into five groups: I (T. reesei, T. todica), II (T. polysporum, T. longibrachiatum, T. koningii, and T. pseudokoningii), III (T. virgatum), IV (T. saturnisporum) and V (T. harzianum). These results contradict the claim that T. reesei is a subspecies of T. longibrachiatum. Furthermore, hybridization with (CA)8 allowed a subdivision of group II, wherein T. pseudokoningii formed a subgroup, IIb, which is highly homologous with, but distinct from subgroup IIa. The results show that RFLP analysis may be used to re-classify the Trichoderma aggregate.

Respiratory samples from cystic fibrosis outpatients were cultured on Sabouraud's dextrose agar (SABD) containing antibiotics, Mycosel, and Scedosporium -selective medium (SceSel+). Thirty-two (14.7%) of 218 specimens from 11/69 (15.9%) patients yielded a Scedosporium sp., most frequently Scedosporium aurantiacum (17/218). Scedosporium was recovered on SceSel+, Mycosel, and SABD from 90.6%, 50.0%, and 46.9% of the specimens tested, respectively.

Suspected nosocomial Aspergillus fumigatus infections in an Australian hematology unit were investigated by molecular typing of clinical and environmental isolates using polymerase chain reaction fingerprinting, CSP typing, and multilocus microsatellite typing. Only multilocus microsatellite typing revealed that all isolates were genetically distinct. The selection of an appropriate typing method is essential for effective outbreak investigations.

A model of pulmonary cryptococcosis in immunocompetent rats was developed to better understand the virulence of Cryptococcus gattii. Six isolates were studied, representing four molecular genotypes (VGI-MATα, VGIIa-MATα, VGIIa-MAT a, VGIIb-MATα), obtained from Australia, Vancouver (Canada) and Colombia. These originated from human patients, a cat and the environment and were administered intratracheally (i.t.) or transthoracically into Fischer 344 or Wistar-Furth rats in doses varying from 10(4) to 10(7) colony-forming units (CFU) in 0.1 ml of saline. With the exception of animals given the VGIIa-MAT a isolate, rats consistently became ill or died of progressive cryptococcal pneumonia following i.t. doses exceeding 10(7) CFU. Affected lungs increased in weight up to tenfold and contained numerous circumscribed, gelatinous lesions. These became larger and more extensive, progressing from limited hilar and/or tracheal lesions, to virtually confluent gelatinous masses. Disease was localized to the lungs for at least 3-4 weeks, with dissemination to the brain occurring in some animals after day 29. The dose-response relationship was steep for two VGI isolates studied (human WM179, environmental WM276); doses up to 10(6) CFU i.t. did not produce lesions, while 10(7) or more yeast cells produced progressive pneumonia. Intratracheal inoculation of rats with C. gattii provides an excellent model of human pulmonary cryptococcosis in healthy hosts, mimicking natural infections. Disease produced by C. gattii in rats is distinct from that caused by C. neoformans in that infections are progressive and ultimately fatal.

The trehalose pathway is essential for stress tolerance and virulence in fungi. We investigated the importance of this pathway for virulence of the pathogenic yeast Cryptococcus gattii using the highly virulent Vancouver Island, Canada, outbreak strain R265. Three genes putatively involved in trehalose biosynthesis, TPS1 (trehalose-6-phosphate [T6P] synthase) and TPS2 (T6P phosphatase), and degradation, NTH1 (neutral trehalose), were deleted in this strain, creating the R265 tps1 Δ, R265 tps2 Δ, and R265 nth1 Δ mutants. As in Cryptococcus neoformans , cellular trehalose was reduced in the R265 tps1 Δ and R265 tps2 Δ mutants, which could not grow and died, respectively, at 37°C on yeast extract-peptone-dextrose agar, suggesting that T6P accumulation in R265 tps2 Δ is directly toxic. Characterizations of the cryptococcal hexokinases and trehalose mutants support their linkage to the control of glycolysis in this species. However, unlike C. neoformans , the C. gattii R265 tps1 Δ mutant demonstrated, in addition, defects in melanin and capsule production, supporting an influence of T6P on these virulence pathways. Attenuated virulence of the R265 tps1 Δ mutant was not due solely to its 37°C growth defect, as shown in worm studies and confirmed by suppressor mutants. Furthermore, an intact trehalose pathway controls protein secretion, mating, and cell wall integrity in C. gattii . Thus, the trehalose synthesis pathway plays a central role in the virulence composites of C. gattii through multiple mechanisms. Deletion of NTH1 had no effect on virulence, but inactivation of the synthesis genes, TPS1 and TPS2 , has profound effects on survival of C. gattii in the invertebrate and mammalian hosts. These results highlight the central importance of this pathway in the virulence composites of both pathogenic cryptococcal species.

Australia-wide population-based surveillance for scedosporiosis identified 180 cases, with 118 (65.6%) cases of colonization and 62 (34.4%) cases of infection. Predisposing factors for isolation of Scedosporium spp. included chronic lung disease in 37.8% and malignancy in 21.7% of cases. Predictors of invasive disease (n = 62) included haematological stem cell transplantation (n = 7), leukaemia (n = 16) and diabetes mellitus (n = 8). Of 183 phenotypically-speciated isolates, 75 (41%) were Scedosporium prolificans (risk factors: haematologic cancer (n = 17), neutropaenia (n = 14)) and 108 (59%) had Scedosporium apiospermum/Pseudallescheria boydii phenotype [risk factor: diabetes (n = 15)]. Scedosporium prolificans (p 0.01) and leukaemia (p 0.03) independently predicted death. Epidemiological and antifungal susceptibility profiles of Scedosporium aurantiacum (prevalence ≥15.8%) and S. apiospermum were similar. No patient with S. aurantiacum infection (n = 6) died. This is the first description of clinical features associated with S. aurantiacum.

Pneumocystis jirovecii is an important opportunistic pathogen in immunocompromised patients. Molecular typing is employed to study this pathogen, as no culture system exists. No Australian P. jirovecii strains have been previously studied. Direct sequencing, targeting the internal transcribed spacer (ITS) regions of the nuclear rRNA operon, the mitochondrial large-subunit rRNA (mt LSU rRNA), and the dihydropteroate synthase (DHPS) gene, was performed on 68 Australian samples, collected between 2001 and 2007. Seven novel Australian ITS haplotypes (a composite of the ITS1 and ITS2 regions) were identified (SYD1m, SYD1g, Isyd2, Esyd3, Osyd4, Ag, and Hc). A dendrogram of published ITS haplotypes revealed that of the seven novel haplotypes, three (SYD1m, SYD1g, and Osyd4) are closely related to the haplotype Eg. Applying statistical parsimony, an Australian haplotype network was constructed which identified Eg as the ancestral haplotype, with two unresolved loops encountered. This suggests that the ITS lacks the resolution required for evolutionary analysis. Only two mt LSU rRNA genotypes were detected, with genotype 1 predominating. Mutant DHPS genotypes were present in 13% (8/60) of the samples. The novel haplotype Isyd2 was associated with less severe disease than the other Australian haplotypes. In contrast, patients with mutant DHPS genotypes were more likely to have severe disease, require invasive ventilation, and have a poor outcome than patients with wild-type DHPS genotypes. In conclusion, genetic clinical correlates continue to be found for Pneumocystis pneumonia; however, they remain controversial and warrant further study.

The identification of fungal species and determination of their significance in the clinical laboratory are complex practices that help establish or exclude a fungal cause of disease. In the past, the clinical mycologist utilized a limited array of phenotypic measurements for categorizing isolates

This communication describes the consensus multi-locus typing scheme established by the Cryptococcal Working Group I (Genotyping of Cryptococcus neoformans and C. gattii) of the International Society for Human and Animal Mycology (ISHAM) using seven unlinked genetic loci for global strain genotyping. These genetic loci include the housekeeping genes CAP59,GPD1, LAC1, PLB1, SOD1, URA5 and the IGS1 region. Allele and sequence type information are accessible at http://www.mlst.net/ .

We have analyzed 11 strains and clones, representing five species (Penicillium janthinellum, P. citrioviridae, P. chrysogenum, Aspergillus niger, Trichoderma harzianum) and three genera of filamentous fungi, for the presence of hypervariable loci in their genomes by hybridization with simple repeat oligonucleotides and the DNA of phage M13. The oligonucleotide probes (CT)8, (GTG)5 and (GACA)4, as well as M13 DNA, are informative probes for fingerprinting in all genera and species tested. The probe (GATA)4 produced informative fingerprints only with the genomic DNA of A. niger. There was no similarity between the fingerprints originating from fungi of different genera and also little similarity between the fingerprints of different species belonging to the same genus. Fingerprints of strains of the same species differed only slightly from each other. Fingerprints of clones originating from one strain were identical. The results indicate that DNA fingerprinting is a powerful method to differentiate species and strains of filamentous fungi.

Scedosporium species are increasingly encountered opportunistic fungal pathogens not only in immunocompromised patients but are also significant primary pathogens in immunocompetent individuals. The environmental reservoir of these fungi is uncertain and the epidemiology and mode of transmission are not well-defined. Conventional phenotypic methods are of limited use for epidemiological purposes since they are insensitive and inadequately discriminatory. Molecular techniques not only enable accurate phylogenetic delineation of species but also provide the means for rapid, reliable genotyping of strains for epidemiological and population genetic studies. This review discusses the methods that have been applied for genotyping of these increasingly important pathogens.

In order to study the infectious agents causing human disseminated cryptococcosis in the state of Para, North Brazil, 56 isolates of Cryptococcusspp. (54 isolated from cerebral spinal fluid and two from blood cultures) from 43 cases diagnosed between 2003-2007 were analysed. The species were determined through morphological and physiological tests and genotypes were determined by URA5-RFLP and PCR-fingerprinting (wild-type phage M13). The following species and genotypes were identified: Cryptococcus neoformans VNI (28/56, 50%), Cryptococcus gattii VGII (25/56, 44.64%) and C. gattii VGI (3/56, 5.26%). The genotype VNI occurred in 12 out of 14 HIV-positive adults, whereas the genotype VGII occurred in 11 out of 21 HIV-negative adults (p < 0.02, OR = 6.6 IC95% 0.98-56.0). All patients less than 12 years old were HIV negative and six cases were caused by the VGII genotype, one by the VGI and one by VNI. Therefore, endemic primary mycosis in HIV-negative individuals, including an unexpectedly high number of children, caused by the VGII genotype deserves further study and suggests the need for surveillance on cryptococcal infection in the state of Para, Eastern Amazon.

The basidiomycetous yeast Cryptococcus gattii, is a primary pathogen which causes disease in apparently healthy humans and a wide range of animals. Recently, an outbreak of cryptococcosis caused by a previously uncommon genotype of C. gattii, VGII, emerged on Vancouver Island, British Columbia, Canada. Two pathogenic sub-types of VGII (designated VGIIa and VGIIb) were identified among these isolates. All of the isolates proved to be mating type alpha and had exceptionally high sporulation capacity. The common subtype, VGIIa, was more virulent than VGIIb in mice, suggesting a linkage between subtype and fertility/virulence. To test this hypothesis, we compared the fertility of 91 isolates from the Vancouver Island outbreak with that of 72 VGII isolates selected globally. Of all isolates, 69.94% were found to be fertile and exhibited clamp connections and basidiospores. The Vancouver isolates showed a high fertility rate of 84.2% as compared to only 29% of the 21 Australian isolates investigated. Mating type alpha strains were more fertile (72.79%) than mating type a (43.75%) (p0.022). Amongst the two subtypes of VGII a much higher proportion of VGIIa (91.7%) than VGIIb (33.3%) was fertile (p0.001). These results suggest that there is a clear correlation between the VGII subtypes of C. gattii and their mating/fertility. Further in vitro and in vivo investigations of more strains and congenic pairs are warranted.

Fifteen randomly selected microsatellites (simple sequence repeats; SSRs), from the H99 Cryptococcus neoformans var. grubii (serotype A) genome, were sequenced, characterized and applied to sequence 87 clinical and environmental C. neoformans var. grubii isolates from 12 different countries based on Multilocus Microsatellite Typing (MLMT). Among the 15 SSR loci, three (designated CNG1, CNG2 and CNG3) were polymorphic, while the remaining 12 SSR loci showed no variations. The specific PCR primers of the polymorphic microsatellites, i.e., CNG1, CNG2 and CNG3, amplified those loci only from strains of C. neoformans (C. neoformans var. grubii, C. neoformans var. neoformans and the AD hybrid) but not from Cryptococcus gattii, suggesting a species-specific association. The three polymorphic microsatellites are useful markers for strain genotyping, population genetic analysis, epidemiological studies, and may be helpful for the diagnosis of cryptococcosis due to C. neoformans.

The Cryptococcus species complex contains two closely related basidiomycetous yeasts: Cryptococcus neoformans and C. gattii, which cause cryptococcosis in humans and other animals. The species and varieties are characterized, by different clinical, epidemiological, biochemical and molecular features. The currently used identification methods are either time-consuming or not anymore commercially available. However, a rapid, sensitive and robust assay for the detection of these pathogens is vital for early diagnosis and appropriate treatment decisions. To overcome those limitations, four padlock probes targeting species-specific single nucleotide polymorphisms at the internal transcribed spacers (ITSs) of the RNA gene locus were developed and applied during isothermal hyperbranched rolling circle amplification (HRCA). The probes were tested against 99 samples, including 94 clinical cryptococcal cultures, three closely related Cryptococcus species, and two clinical specimens. The use of the padlock probes and the combination of probe signal amplification by HRCA provided a quick and sensitive assay for the accurate identification of C. neoformans var. grubii, C. neoformans var. neoformans and C. gattii. HRCA was also useful to detect hybrids, when they were heterozygous at the ITS locus. The HRCA results were in agreement with previous genotyping data based on PCR fingerprinting, amplified fragment length polymorphism and ITS sequencing.

Molecular typing showed genetic diversity, dismissed 2 suspected outbreaks of scedosporiosis, and identified multiple strains of the newly described species S. aurantiacum., One hundred clinical isolates from a prospective nationwide study of scedosporiosis in Australia (2003–2005) and 46 additional isolates were genotyped by internal transcribed spacer–restriction fragment length polymorphism (ITS-RFLP) analysis, ITS sequencing, and M13 PCR fingerprinting. ITS-RFLP and PCR fingerprinting identified 3 distinct genetic groups. The first group corresponded to Scedosporium prolificans (n = 83), and the other 2 comprised isolates previously identified as S. apiospermum: one of these corresponded to S. apiospermum (n = 33) and the other to the newly described species S. aurantiacum (n = 30). Intraspecies variation was highest for S. apiospermum (58%), followed by S. prolificans (45%) and S. aurantiacum (28%) as determined by PCR fingerprinting. ITS sequence variation of 2.2% was observed among S. apiospermum isolates. No correlation was found between genotype of strains and their geographic origin, body site from which they were cultured, or colonization versus invasive disease. Twelve S. prolificans isolates from 2 suspected case clusters were examined by amplified fragment length polymorphism analysis. No specific clusters were confirmed.

Yeasts of the artificial genus Candida include plant endophytes, insect symbionts, and opportunistic human pathogens. Phylogenies based on rRNA gene and actin sequences confirmed that the genus is not monophyletic, and the relationships among Candida species and allied teleomorph genera are not clearly resolved. Protein-coding genes have been useful to resolve taxonomic positions among a broad range of fungi. Over 70 taxa of the genus Candida and its allied sexually reproducing genera were therefore selected, and their phylogenetic relationships were investigated using nuclear sequences of the largest subunit and second largest subunit of RNA polymerase II gene, actin, the second subunit of the mitochondrial cytochrome oxidase gene, and D1/D2 LSU rRNA gene. The DNA sequences were analysed by maximum parsimony and Bayesian inference, resulting in the recognition of six major phylogenetic groups (A-F). Group A contains six facultative pathogenic Candida species, which seem to have derived from nonpathogenic species, while Group B contains species of Clavispora, Metschnikowia, and Pichia guilliermondii. Species of Debaryomyces form an independent group C that is related to groups A and B. Pichia fermentans and other environmental species are concentrated in Group D. Group E, containing Pichia anomala, may be a sibling to group F, which is represented by the Saccharomyces species complex.

Within the Cryptococcus neoformans species complex, two species and five serotypes are recognized: C. neoformans (var. grubii, serotype A; var. neoformans, serotype D and a hybrid, serotype AD) and C. gattii (serotypes B and C). Mating types a and alpha are designated by a single locus, with the mating type alpha being most prevalent in serotype A and D strains.To evaluate the ability of Colombian isolates of the C. neoformans species complex to mate in vitro with tester strains of the opposite mating type.Fifty three clinical isolates were included in this study, 33 C. neoformans var. grubii serotype A, 4 C. neoformans var. neoformans serotype D, all mating type alpha, and 16 C. gattii, 13 serotype B (mating type a) and 3 serotype C (mating type alpha), were mixed on V8 juice agar, using a modified method, with the appropriate tester strains to determine the mating types in vitro.Mating studies revealed that 9 of 33 (27.3%) serotype A isolates and 6 of 13 (46.2%) serotype B isolates were able to mate. Clamp connections and basidia with basidiospores were observed microscopically, indicating that the mating process had occurred. All mating competent serotype A strains were mating type alpha and the serotype B mating competent strains were mating type a.This is the first report of the determination of the mating ability of Colombian Cryptococcus neoformans isolates to mate in vitro with appropriate tester strains, which is of great importance to study the propagation of the fungus around the globe.

The epidemiology of invasive fungal disease (IFD) due to filamentous fungi other than Aspergillus may be changing. We analysed clinical, microbiological and outcome data in Australian patients to determine the predisposing factors and identify determinants of mortality. Proven and probable non-Aspergillus mould infections (defined according to modified European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria) from 2004 to 2012 were evaluated in a multicentre study. Variables associated with infection and mortality were determined. Of 162 episodes of non-Aspergillus IFD, 145 (89.5%) were proven infections and 17 (10.5%) were probable infections. The pathogens included 29 fungal species/species complexes; mucormycetes (45.7%) and Scedosporium species (33.3%) were most common. The commonest comorbidities were haematological malignancies (HMs) (46.3%) diabetes mellitus (23.5%), and chronic pulmonary disease (16%); antecedent trauma was present in 21% of cases. Twenty-five (15.4%) patients had no immunocompromised status or comorbidity, and were more likely to have acquired infection following major trauma (p

A 17-year-old desexed male Birman cat presented with a fleshy mass protruding from the left ear canal. A culture from the mass revealed a heavy growth of Cryptococcus gattii (molecular type VGII, serotype B). The lesion resolved with antifungal therapy over 8 weeks. Itraconazole was continued indefinitely due to persistent high serum cryptococcal antigen titres. The cat was euthanased 12 months later due to the acute development of hindlimb ataxia and collapse which may or may not have been attributable to cryptococcosis. This cat had first presented when 4 years of age with a 3-week history of inappetance, sneezing and serous nasal discharge. Culture of swabs from both nostrils were positive for C. gattii (VGII). Fluconazole therapy produced steady improvement over a 6 month period, at which time therapy was discontinued. The cat presented 9 months later for sneezing, again with a positive culture of C. gattii from the nasal cavity. Antifungal therapy was continued for 8 months, after which time cultures were negative and symptoms resolved. Three episodes of cryptococcosis in a cat over a 13-year period were thus documented. Importantly, the two C. gattii isolates, obtained 13 years apart, were identical using DNA fingerprinting and random amplification of polymorphic DNA (RAPD) analysis.

Cryptococcus gattii is a pathogenic yeast that together with Cryptococcus neoformans causes cryptococcosis in humans and animals. High numbers of viable C. gattii propagules can be obtained from certain species of Australian Eucalyptus camaldulensis trees, and an epidemiological link between Eucalyptus colonization and human exposure has been proposed. However, the highest prevalence of C. gattii cryptococcosis occurs in Papua New Guinea and in regions of Australia where the eucalypt species implicated to date are not endemic. This study investigated the population structure of three geographically distinct clinical and veterinary populations of C. gattii from Australia and Papua New Guinea. All populations that consisted of a genotype found frequently in Australia (VGI) were strongly clonal and were highly differentiated from one another. Two populations of the less common VGII genotype from Sydney and the Northern Territory had population structures inferring recombination. In addition, there was some evidence of reduced genetic differentiation between these geographically remote regions. In a companion study presented in this issue, VGII isolates were overwhelmingly more fertile than those of the VGI genotype, giving biological support to the indirect assessment of sexual exchange. It appears that the VGI genotype propagates clonally on eucalypts in Australia and on an unknown substrate in Papua New Guinea, with infection initiated by an unidentified infectious propagule. VGII isolates are completing their life cycles and may be dispersed via sexually produced basidiospores, which are also likely to initiate respiratory infection.

The use of simple sequence repeats or microsatellites as genetic markers has become very popular because of their abundance and length variation between different individuals. SSRs are tandem repeat units of 1 to 6 base pairs that are found abundantly in many prokaryotic and eukaryotic genomes. This is the first study examining and comparing SSRs in completely sequenced fungal genomes. We analyzed and compared the occurrences, relative abundance, relative density, most common, and longest SSRs in nine taxonomically different fungal species: Aspergillus nidulans, Cryptococcus neoformans, Encephalitozoon cuniculi, Fusarium graminearum, Magnaporthe grisea, Neurospora crassa, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Ustilago maydis. Our analysis revealed that, in all of the genomes studied, the occurrence, abundance, and relative density of SSRs varied and was not influenced by the genome sizes. No correlation between relative abundance and the genome sizes was observed, but it was shown that N. crassa, the largest genome analyzed had the highest relative abundance of SSRs. In most genomes, mononucleotide, dinucleotide, and trinucleotide repeats were more abundant than the longer repeated SSRs. Generally, in each organism, the occurrence, relative abundance, and relative density of SSRs decreased as the repeat unit increased. Furthermore, each organism had its own common and longest SSRs. Our analysis showed that the relative abundance of SSRs in fungi is low compared with the human genome and that longer SSRs in fungi are rare. In addition to providing new information concerning the abundance of SSRs for each of these fungi, the results provide a general source of molecular markers that could be useful for a variety of applications such as population genetics and strain identification of fungal organisms.

A high biodiversity of Cryptococcus neoformans isolates is known to exist in some Brazilian urban areas, raising the possibility that patients may encounter multiple inoculum sources in their daily life. C. neoformans isolates from two groups of AIDS patients with cryptococcosis from Rio de Janeiro were studied by polymerase chain reaction (PCR) fingerprinting and randomly amplified polymorphic DNA (RAPD) analysis. The first group contained 60 serial isolates obtained from 19 patients over periods ranging from 18 to 461 days; the intent was to determine whether the original strain persisted or whether reinfection with a new strain occurred. The second group was made up of 22 isolates from 11 patients, and consisted of a pair of isolates collected from blood and cerebrospinal fluid from each patient either before or shortly after treatment was initiated. The aim was to determine if the patient was infected by different strains simultaneously. All isolates were subtyped by PCR fingerprinting, using minisatellite (M13), and microsatellite [(GACA)4 and (GTG)5] specific primers, and RAPD analysis employing the combined primers 5SOR and CN1. The majority of isolates were C. neoformans var. grubii, specifically, molecular types VNI or VNII, but numerous distinguishable subtypes were found. Only three isolates were C. n. var. gattii (molecular types VGI or VGII). Except in two cases, all isolates obtained from the same patient showed identical PCR profiles independent of time of isolation or body site. Almost all patients, however, carried unique genotypes not found in any other patient. Our results confirm that persistent cryptococcal infection is caused by relapse rather than reinfection, but they also show that in exceptional cases, patients may be infected with more than one C. neoformans strain.

Cryptococcus neoformans var. gattii has regularly been the cause of serious human disease. However, the environmental sources of these infections often remain unclear. During an environmental sampling study, two different strains of C. neoformans var. gattii were isolated from fresh insect frass (order Lepidoptera; family Oecophoridae) in a shallow cavity in the bark of a living Eucalyptus tereticornis tree, one molecular type VGI and the other VGII. This is the first published report of the isolation of two different molecular types of C. neoformans var. gattii from a single source, and the third of isolation of molecular type VGII from an environmental source. The potential association with insect frass is consistent with categorising C. neoformans var. gattii within the Tremellales, containing mycoparasitic fungi.

Highly similar gene sequences of the 5' region of the large subunit (LSU) are commonly interpreted to predict the organism's identity. However, it was recognised that closely related taxa do not always show sufficiently diverged D1/D2 LSU sequences to differentiate between them. The effectiveness of species separation using D1/D2 LSU sequences, small subunit (SSU) sequences and actin gene sequences was determined by pair-wise comparisons. The LSU data showed coinciding similarities among and within species. The actin data resolved all investigated species. Examples strengthened the value of almost complete SSU sequences for species separation. The larger number of differences in the highly conserved actin gene, compared to the overall more variable LSU gene, is due to the tolerance of protein coding genes to synonymous nucleotide changes. In contrast, the pairing in secondary structures of the rRNA, ensuring the functionality of the molecule, relies on longer and uninterrupted sequence sections. In conclusion, D1/D2 LSU sequences are not specific enough to identify closely related taxa. The actin gene is a better marker in these cases. However, because of the availability of a large database of fungal D1/D2 LSU sequences, this gene region is currently still the preferred target for sequence-based identification. (C) 2003 Elsevier B.V. All rights reserved.

In a continuation of studies started by de Ruiter et al. (1993), all known species of the Mortierella isabellina-group (Micromucor/Umbelopsis clade of O'Donnell et al. 2001) and a few other Mucorales and species of Mortierella were investigated by RFLP (including ITS1, 5.8S, ITS2 and the 5' end of the large subunit rDNA gene) and ITS1 sequence analyses. This monophyletic group is unrelated to Mortierella and is only distantly related to the core group of the Mucoraceae. M. longicollis falls outside the Umbelopsis clade. Molecular data resolved two subclades within the M. isabellina-group; however, they are not correlated with any differences in sporangial wall and shape, spore pigmentation and shape, or sporangiophore branching. Therefore we subsume all taxa in one genus, Umbelopsis. The new family Umbelopsidaceae and the new combinations U. isabellina, U. ramanniana, and U. autotrophica are proposed.

In Brazil, 4.5% of the AIDS-related opportunistic infections are caused by Cryptococcus neoformans. This pathogen is a ubiquitous environmental basidiomycetous encapsulated yeast, commonly found in soil and avian excreta. The present study investigates further the population structure of clinical and environmental C. neoformans isolates from south Brazil. One hundred five clinical and 19 environmental (pigeon excreta and Eucalyptus spp.) isolates from the Brazilian state Rio Grande do Sul were characterized based on morphological, biochemical, molecular and serological data. The majority of the clinical and environmental isolates analyzed belonged to C. neoformans var. grubii serotype A (89.5 and 52.6%, respectively), were mating type alpha (98.1 and 94.7%, respectively) and were phospholipase-positive (94.3 and 73.7%, respectively). PCR-fingerprinting with the microsatellite-specific primer M13 and the minisatellite-specific primer (GACA)(4) grouped the majority of the isolates into the molecular type VNI (89.5 of the clinical and 52.6% of the environmental isolates). Our results add considerable new information to the few available data on ecology, molecular biology and epidemiology of C. neoformans in the southern region of Brazil.

Cryptococcus neoformans is a pathogenic yeast that is currently divided into three varieties, five serotypes, and eight molecular types. The following report describes the use of PCR-restriction fragment length polymorphism (RFLP) analysis of the phospholipase B gene ( PLB1 ) as a simple tool to differentiate between C. neoformans subgroups. A PLB1 fragment, 1,970 bp, was amplified and digested with either Ava I or Hin dIII. Both sets of profiles grouped the isolates into their respective varieties, but only the Ava I profiles allowed for the identification of the eight molecular types via the corresponding RFLP profiles A1 to A8. Digestion of the same fragments with Hin dIII resulted in RFLP profiles H1 to H5, which distinguished only between serotype A, AD, D, and B/C. Neither enzyme distinguished serotype B from serotype C. The serotype AD profile was a composite of the serotype A and D profiles. Further investigation showed that the serotype AD isolates used in this study are heterozygous, with one allele of PLB1 originating from a serotype A parent and the other from a serotype D parent.

The Westmead Medical Mycology Collection is completing20 years of existence. During this time there have been10,073 strains deposited representing 437 species, whicharecurrentlymaintainedinthecollection.Establishedorig-inally under the curation of Professor Wieland Meyer at theMolecular Mycology Research Laboratory, in the Centre forInfectiousDiseasesandMicrobiologyattheSydneyMedicalSchool – Westmead Hospital, The University of Sydney, itrecently moved to the new Westmead Millennium InstituteforMedicalResearchinWestmead,Australia.Itsprimaryaimis to preserve Australian human and animal pathogenicfungal biodiversity while providing reference and clinicalstrains for the mycology community. The stored strains areidentified phenotypically, biochemically and molecularly.Theyarestoredeitherlyophilised,inglycerolat 808Corasliving culture at 148C. The majority of the stored strains arethe result of specific clinical, molecular epidemiologicaland basic science projects. As such, the pathogenic yeastsCryptococcus neoformans and C. gattii account for 54%of the specimens deposited. To further characterisethe maintained strains specific MultiLocus SequenceTyping schemes have been developed for C. neoformans,C. gattii, Scedosporium apiospermum, S. aurantiacum,S. boydii and Pneumocystis jirovecii, which are publicallyaccessible at http://mlst.mycologylab.org. The collectionalso formed the basis for the development of the qualitycontrolled ISHAM-ITS sequence database for human andanimal pathogenic fungi accessible at http://its.mycologylab.org.

Molecular Mycology ResearchLaboratory, Centre for InfectiousDiseases and Microbiology, SydneyMedicalSchool–WestmeadHospitalMarie Bashir Institute for InfectiousDiseases and BiosecurityThe University of SydneyWestmead Millennium Institute176 Hawkesbury Road, WestmeadSydney, NSW 2145, AustraliaTel: +61 2 8627 3430Fax: +61 2 9891 5317Email: wieland.meyer@sydney.edu.au

Wieland Meyer, Laszlo Irinyi and Tania Sorrell Molecular Mycology Research Laboratory, Centre for Infectious Diseases and Microbiology, Sydney Medical School–Westmead Hospital, MarieBashir Institute for InfectiousDiseases andBiosecurity, University of Sydney, Sydney,WestmeadMillenniumInstitute,Westmead,NSW,Australia Email: wieland.meyer@sydney.edu.au Email: laszlo.irinyi@sydney.edu.au Email: tania.sorrell@sydney.edu.au

Invasive fungal diseases (IFDs) caused by the filamentous fungal pathogens of the genera Aspergillus, fusarium, zygomycetes, scedosporium and the yeast candida are the most important causes of fungus related deaths in immunosuppressed hematology patients1. Clinical efficacy of A. fumigatus specific T cells has been demonstrated in haploidentical hematopoietic stem cell transplant (HSCT) recipients2. We recently published a method to culture A. fumigatus specific T cells from normal allogeneic donors using procedures compliant with the code of Good Manufacturing Practice3. The safety and efficacy of cells generated using our method is currently being tested in a Phase 1 study in HSCT recipients. Here, we investigated the possibility of manufacturing a single T cell product with activity against a broad target range of fungal pathogens for clinical adoptive immunotherapy. We made water-soluble lysates from germinated spores of fungal pathogens, A. flavus, A. terreus, C. albicans, C. krusei, F. oxysporum, F. solani, R. oryzae and S. prolificans and used these to pulse monocyte derived dendritic cells (MoDC) from PBMC of 4 normal donors. Pulsed MoDC were washed, irradiated and used to stimulate autologous PBMC on Days 0 and 7. Cells were expanded with the addition of IL-2, IL-7 and IL-15 from Days 7 to 21. Cell numbers, phenotype and fungus-specificity were assessed on Day 21. Cross-reactivity of cultured T cells was tested against lysates of other fungi to identify a combination of antigens likely to induce broad anti-fungal T cell reactivity. Products were generated from PBMC using either no selection or TNFα capture on Day 7 of culture to select antigen-specific cells. Three products were generated from G-CSF mobilized peripheral blood stem cell products (PBSC) without selection. Antifungal activity was mapped to specific HLA molecules using blocking antibodies to HLA-DR, -DP and –DQ. In cultures from normal donor PBMC, expansion occurred with lysates from all fungi (range 2.3-109.6 fold) generating 85-97% T cells of which >80% were CD4+ T cells. The percentage of fungus-specific (TNFα+) CD4+ cells were A flavus 6.8±5.5%, A terreus 13.2±12.8%, C albicans 10.5±8.5%, C krusei 6.4±6.8%, F oxysporum 6.4±3.8%, F solani 5.2±7.8%, R oryzae 7.6±6.4 and S prolificans 4.4±3.8% (n=4). T cells also produced IFNγ and IL-2. T cells from cultures generated with Aspergillus, Fusarium and Scedosporium cross-reacted with one another and with lysates from most other fungi. We selected a combination of A. terreus, C. krusei and R. oryzae to generate multifungus T cell products from PBMC and PBSC. All but one multifungus culture generated >89% T cells. The majority of T cells had terminally differentiated effector and effector memory phenotypes. Regulatory T cells were Blocking HLA-DR, but not -DP or -DQ on antigen presenting cells abrogated fungus-specific cytokine production by cultured T cells (n=7). Antifungal activity was maintained when MoDC from partially HLA-DRB1 matched allogeneic donors were used to present fungal antigens to cultured T cells (n=7). In contrast, no antifungal activity was observed when MoDC from completely HLA-DRB1 mismatched donors were used to stimulate fungal T cells (n=4). We demonstrate that similarly to A. fumigatus specific T cells, T cells specific for other clinically relevant fungi can be expanded in vitro. We have developed a method to manufacture a T cell product with activity against multiple clinically relevant fungi, using blood or stem cells of healthy donors as starting material. The use of TNFα capture did not increase the number or purity of fungus-specific cells in cultures. The clinical utility of infusions of multifungus responsive T cell products in prophylaxis and treatment of invasive fungal disease needs to be tested clinically. References 1Neofytos D, Horn D et al, Clinical Infectious Diseases 2009 2Perruccio K, Tosti A et al, Blood 2005 3Gaundar S, Clancy L et al, Cytotherapy 2012 Disclosures No relevant conflicts of interest to declare.

Surveillance for fungal diseases is essential to improve our understanding of their epidemiology and to enable research and prevention efforts to be prioritized. In order to conduct better surveillance for fungal diseases, it is important to develop more accurate and timely diagnostic tests, to follow rigorous epidemiological methods and to have adequate support from public health agencies and the pharmaceutical industry. Investigations of nosocomial and community outbreaks of fungal infection have also resulted in a better understanding of the sources and routes of transmission of these diseases, and of the risk factors for infection. This has led to more effective prevention and control strategies. In addition, outbreak investigations have offered excellent opportunities to develop new molecular sub-typing methods, and to evaluate and validate older ones. For example, results obtained from a global epidemiological study of the genomic structure of Cryptococcus neoformans have led to a better understanding of the epidemiology of cryptococcosis. Similarly, a study of variations in the genotype of Trichophyton rubrum has found that patients may become infected with multiple strains, which has important implications for study design when looking at the epidemiology of dermatophyte infections.

Amplified fragment length polymorphism (AFLP) genotyping of isolates of the pathogenic fungus Cryptococcus neoformans suggested a considerable genetic divergence between the varieties C. neoformans var. neoformans and C. neoformans var. grubii on the one hand versus C. neoformans var. gattii on the other. This divergence is supported by additional phenotypic, biochemical, clinical and molecular differences. Therefore, the authors propose the existence of two species, C. neoformans (Sanfelice) Vuillemin and C. bacillisporus Kwon-Chung, which differ in geographical distribution, serotypes and ecological origin. Within each species three AFLP genotypes occur, which differ in geographical distribution and serotypes. Differences in ecological origin (AIDS patients, non-AIDS patients, animals or the environment) were found to be statistically not significant. In C. neoformans as well as in C. bacillisporus one of the genotypes represented a hybrid. The occurrence of hybridization has consequences for the reproductive biology of the species, as new genotypes with altered virulence or susceptibility to antifungal drugs may arise through the exchange of genetic material.

1 Proteomics Technology Centre of Expertise, University of Technology Sydney, ultimo, NSW, Australia 2 School of Molecular and Microbial Biosciences, University of Sydney, Sydney, NSW, Australia 3 Faculty of Veterinary Science, University of Sydney, Sydney, NSW, Australia 4 Molecular Mycology Research Laboratory, Centre for Infectious Diseases and Micro, Westmead Hospital, Sydney, NSW, Australia 5 Institute for the Biotechnology of Infectious Diseases, University of Technology Sydney, Sydney, NSW, Australia

validity of current antimicrobial prophylactic regimensused, if any. This article outlines the clusters in Australia,hypothesises why this may have occurred and presents therecent consensus proposal for outbreak containment andprophylaxis against PCP. Details of the outbreaks in otherregions are not reviewed.In organ transplant recipients, PCP typically presents with acutebreathlessness, dry cough and progressive respiratory failure withnormal lung auscultation; severe infection is common

Sixty-one clinical and forty-nine environmental isolates of Cryptococcus neoformans var. gattii from Australia and the United States were analyzed by random amplification of polymorphic DNA (RAPD), using 12- to 22-mer primers in pairs, and/or PCR fingerprinting with a single primer derived from the microsatellite core sequence of the wild-type phage M13 (5' GAGGGTGGCGGTTCT 3'). Three major genetic profiles were identified by both typing techniques. A single RAPD profile (VGI) predominated among clinical isolates (44 of 48, 92%) and isolates from host eucalypts (45 of 45, 100%) from Australia. Of the 94 Australian isolates, 4 (3 clinical and 1 environmental) were assigned to profile VGII; 2 of these were recovered from patients and one was recovered from plant debris from Western Australia. Only one Australian clinical isolate was assigned to profile VGIII. A different distribution of RAPD profiles (four VGIII, two VGII, and one VGI) was found among four clinical and three environmental isolates from the United States. RAPD profiles of 8 of the 101 isolates studied revealed minor genetic variants, 4 of profile VGI and 4 of profile VGII. Genetic concordance between the majority of clinical and environmental isolates in Australia is consistent with the hypothesis that human disease is acquired from exposure to host eucalypts. Profiles of clinical isolates were independent of body site of infection, and profiles of all isolates were stable over time. Analysis by PCR fingerprinting confirmed the RAPD results. A second RAPD profile (VGII) was associated with infection in southwest Western Australia, where the two host eucalypts do not occur naturally. This raises the possibility of an alternative and as yet unidentified natural habitat of C. neoformans var. gattii. Our results indicate that RAPD analysis is a sensitive and useful method for investigating environmental sources of human infection with this biotype.

The increased incidence of nosocomial infections especially in immunocompromised patients has stimulated interest in developing more definite procedures for epidemiological studies of the etiological agents, such as pathogenic strains of different bacterial and fungal species. To determine the origins of infection, the routes of acquisition and transmission as well as the persistence of pathologic strains, precise and reproducible diagnostic methods are required. Recently, Williams et al (1990) and Welsh and McClelland (1990) described a PCR-based method for assessing DNA polymorphisms by amplifying genomic DNA with single primers of arbitrary nucleotide sequence. The use of the PCR with primers differing in length and nucleotide composition detected polymorphisms in the absence of specific sequence information in DNA from bacteria (Jayarao et al 1992), fungi (Crowhurst et al 1991), plants (Wilde et al 1992), animals (Welsh et al 1991) and humans (Williams et al 1990). Polymorphisms generated by this method have been termed arbitrarily primed-polymerase chain reaction (AP-PCR) fingerprinting (Welsh and McClelland 1990), random amplification of polymorphic DNA (RAPD) markers (Williams et al 1990) and DNA amplification fingerprinting (Jayarao et al 1992). The term PCR fingerprinting is used here because single primers, which were originally applied as hybridization probes in conventional DNA fingerprinting to detect minisatellite and microsatellite DNA such as the core sequence of phage M 13 (5’GAGGGTGGCGGTTCT), and the simple repeat sequences (GACA)4 and (GATA)4 (Meyer et al.1991; Ali et al 1986) were used to amplify DNA sequences in the genome of different bacterial and yeast species. Besides, in our experiments primers were tested which were already used in other labs for AP-PCR (universal sequence of M 13; 5’TTATGAAACGACGGCCAGT; Welsh et al 1991) or RAPD (10-mer oligonucleotide; 5’TCACGATGCA; Williams et al).

DNA polymorphisms generated by the polymerase chain reaction (PCR) were used to differentiate clinical isolates of Candida. This PCR method employed single primers that were originally designed as hybridization probes for DNA fingerprinting experiments to probe minisatellite and microsatellite DNA sequences. To evaluate this procedure, 35 isolates from 20 patients in several intensive care units and 12 isolates obtained from the oral cavities of healthy dental patients were fingerprinted. The PCR-fingerprint patterns of isolates of Candida albicans from the immunocompromised patients revealed fewer differences than isolates from the dental service. Multiple isolates from different body sites of the same patients revealed that patients may harbour isolates of Candida with the same or different PCR-fingerprints. Since this method is generally simpler and faster than established methods of biotyping medically important yeasts, PCR-fingerprinting may prove useful for the surveying of large numbers of pathogens for epidemiological studies.

DNA-fingerprinting has been successfully used to detect hypervariable, repetitive DNA sequences (minisatellites and microsatellites) in fungi. Combined with methods used to identify random amplified polymorphic DNA (RAPD), conventional DNA-fingerprinting hybridization probes can also be used as single primers to detect DNA polymorphisms among fungal species and strains. The oligonucleotides (CA)8, (CT)8, (CAC)5, (GTG)5, (GACA)4 and (GATA)4, as well as the phage M13 and its core sequence, have been used as specific probes in hybridization experiments and as primers for PCR analysis. Both methods have enabled the differentiation of all the fungal species and strains that were examined, including species of Penicillium, Trichoderma, Leptosphaeria, Saccharomyces, Candida and Cryptococcus. These methods have been used 1) to clarify the taxonomic relationships among relevant species of the Trichoderma aggregate, 2) to discriminate between aggressive and non-aggressive isolates of the rape seed phytopathogen, Leptosphaeria maculans, and 3) to identify strains of the pathogenic yeasts, Cryptococcus neoformans and Candida albicans. PCR-fingerprinting allowed serotypes of C. neoformans to be distinguished. The application of DNA- and PCR-fingerprinting to fungal DNA should aid in clarification of their taxonomy and improved diagnosis of mycotic disease.

Scedosporium species are emerging fungal pathogens in Australia and elsewhere. The reason for this increase in infections is unclear. Since it is assumed that the infections are caused by inhalation of fungal spores, we sampled the urban and rural environment of the greater Sydney region for the presence of the human pathogenic species. Our findings indicate that there may be species-specific associations with areas of high human activity, hinting of a possible link between the environment and the emergence of infections.