Multilocus Sequence Typing Reveals Extensive Genetic Diversity of the Emerging Fungal Pathogen Scedosporium aurantiacum.

Scedosporium spp. are the second most prevalent filamentous fungi after Aspergillus spp. recovered from cystic fibrosis (CF) patients in various regions of the world. Although invasive infection is uncommon prior to lung transplantation, fungal colonization may be a risk factor for invasive disease with attendant high mortality post-transplantation. Abundant in the environment, Scedosporium aurantiacum has emerged as an important fungal pathogen in a range of clinical settings. To investigate the population genetic structure of S. aurantiacum, a MultiLocus Sequence Typing (MLST) scheme was developed, screening 24 genetic loci for polymorphisms on a tester strain set. The six most polymorphic loci were selected to form the S. aurantiacum MLST scheme: actin (ACT), calmodulin (CAL), elongation factor-1α (EF1α), RNA polymerase subunit II (RPB2), manganese superoxide dismutase (SOD2), and ß-tubulin (TUB). Among 188 global clinical, veterinary, and environmental strains, 5 to 18 variable sites per locus were revealed, resulting in 8 to 23 alleles per locus. MLST analysis observed a markedly high genetic diversity, reflected by 159 unique sequence types. Network analysis revealed a separation between Australian and non-Australian strains. Phylogenetic analysis showed two major clusters, indicating correlation with geographic origin. Linkage disequilibrium analysis revealed evidence of recombination. There was no clustering according to the source of the strains: clinical, veterinary, or environmental. The high diversity, especially amongst the Australian strains, suggests that S. aurantiacum may have originated within the Australian continent and was subsequently dispersed to other regions, as shown by the close phylogenetic relationships between some of the Australian sequence types and those found in other parts of the world. The MLST data are accessible at http://mlst.mycologylab.org. This is a joined publication of the ISHAM/ECMM working groups on "Scedosporium/Pseudallescheria Infections" and "Fungal Respiratory Infections in Cystic Fibrosis".

Molecular Epidemiology Reveals Low Genetic Diversity among Cryptococcus neoformans Isolates from People Living with HIV in Lima, Peru, during the Pre-HAART Era.

Cryptococcosis, a mycosis presenting mostly as meningoencephalitis, affecting predominantly human immunodeficiency virus (HIV)-infected people, is mainly caused by Cryptococcus neoformans. The genetic variation of 48 C. neoformans isolates, recovered from 20 HIV-positive people in Lima, Peru, during the pre-highly active antiretroviral therapy (HAART) era, was studied retrospectively. The mating type of the isolates was determined by PCR, and the serotype by agglutination and CAP59-restriction fragment length polymorphism (RFLP). Genetic diversity was assessed by URA5-RFLP, PCR-fingerprinting, amplified fragment length polymorphism (AFLP), and multilocus sequence typing (MLST). All isolates were mating type alpha, with 39 molecular type VNI, seven VNII, corresponding to C. neoformans var. grubii serotype A, and two VNIII AD hybrids. Overall, the cryptococcal population from HIV-positive people in Lima shows a low degree of genetic diversity. In most patients with persistent cryptococcal infection, the same genotype was recovered during the follow-up. In four patients with relapse and one with therapy failure, different genotypes were found in isolates from the re-infection and from the isolate recovered at the end of the treatment. In one patient, two genotypes were found in the first cryptococcosis episode. This study contributes data from Peru to the ongoing worldwide population genetic analysis of Cryptococcus.

Cryptococcus gattii in North American Pacific Northwest: whole-population genome analysis provides insights into species evolution and dispersal.

The emergence of distinct populations of Cryptococcus gattii in the temperate North American Pacific Northwest (PNW) was surprising, as this species was previously thought to be confined to tropical and semitropical regions. Beyond a new habitat niche, the dominant emergent population displayed increased virulence and caused primary pulmonary disease, as opposed to the predominantly neurologic disease seen previously elsewhere. Whole-genome sequencing was performed on 118 C. gattii isolates, including the PNW subtypes and the global diversity of molecular type VGII, to better ascertain the natural source and genomic adaptations leading to the emergence of infection in the PNW. Overall, the VGII population was highly diverse, demonstrating large numbers of mutational and recombinational events; however, the three dominant subtypes from the PNW were of low diversity and were completely clonal. Although strains of VGII were found on at least five continents, all genetic subpopulations were represented or were most closely related to strains from South America. The phylogenetic data are consistent with multiple dispersal events from South America to North America and elsewhere. Numerous gene content differences were identified between the emergent clones and other VGII lineages, including genes potentially related to habitat adaptation, virulence, and pathology. Evidence was also found for possible gene introgression from Cryptococcus neoformans var. grubii that is rarely seen in global C. gattii but that was present in all PNW populations. These findings provide greater understanding of C. gattii evolution in North America and support extensive evolution in, and dispersal from, South America. Importance: Cryptococcus gattii emerged in the temperate North American Pacific Northwest (PNW) in the late 1990s. Beyond a new environmental niche, these emergent populations displayed increased virulence and resulted in a different pattern of clinical disease. In particular, severe pulmonary infections predominated in contrast to presentation with neurologic disease as seen previously elsewhere. We employed population-level whole-genome sequencing and analysis to explore the genetic relationships and gene content of the PNW C. gattii populations. We provide evidence that the PNW strains originated from South America and identified numerous genes potentially related to habitat adaptation, virulence expression, and clinical presentation. Characterization of these genetic features may lead to improved diagnostics and therapies for such fungal infections. The data indicate that there were multiple recent introductions of C. gattii into the PNW. Public health vigilance is warranted for emergence in regions where C. gattii is not thought to be endemic.

Clonality and α-a recombination in the Australian Cryptococcus gattii VGII population--an emerging outbreak in Australia.

BACKGROUND: Cryptococcus gattii is a basidiomycetous yeast that causes life-threatening disease in humans and animals. Within C. gattii, four molecular types are recognized (VGI to VGIV). The Australian VGII population has been in the spotlight since 2005, when it was suggested as the possible origin for the ongoing outbreak at Vancouver Island (British Columbia, Canada), with same-sex mating being suggested as the driving force behind the emergence of this outbreak, and is nowadays hypothesized as a widespread phenomenon in C. gattii. However, an in-depth characterization of the Australian VGII population is still lacking. The present work aimed to define the genetic variability within the Australian VGII population and determine processes shaping its population structure. METHODOLOGY/PRINCIPAL FINDINGS: A total of 54 clinical, veterinary and environmental VGII isolates from different parts of the Australian continent were studied. To place the Australian population in a global context, 17 isolates from North America, Europe, Asia and South America were included. Genetic variability was assessed using the newly adopted international consensus multi-locus sequence typing (MLST) scheme, including seven genetic loci: CAP59, GPD1, LAC1, PLB1, SOD1, URA5 and IGS1. Despite the overall clonality observed, the presence of MATa VGII isolates in Australia was demonstrated for the first time in association with recombination in MATα-MATa populations. Our results also support the hypothesis of a "smouldering" outbreak throughout the Australian continent, involving a limited number of VGII genotypes, which is possibly caused by a founder effect followed by a clonal expansion. CONCLUSIONS/SIGNIFICANCE: The detection of sexual recombination in MATα-MATa population in Australia is in accordance with the natural life cycle of C. gattii involving opposite mating types and presents an alternative to the same-sex mating strategy suggested elsewhere. The potential for an Australian wide outbreak highlights the crucial issue to develop active surveillance procedures.

Development and validation of a multiplex PCR for detection of Scedosporium spp. in respiratory tract specimens from patients with cystic fibrosis.

The emergence of Scedosporium infections in diverse groups of individuals, which are often treatment refractory, warrants timely and accurate laboratory diagnosis. Species- or group-specific primers based on internal transcribed spacer (ITS) sequence polymorphisms were designed for Scedosporium aurantiacum, Scedosporium dehoogii, Scedosporium prolificans, Pseudallescheria boydii species complex (former clade 5)/Pseudallescheria apiosperma (formerly classified as S. apiospermum sensu lato) and Pseudallescheria minutispora. Primers for S. aurantiacum, S. prolificans, and P. boydii species complex/P. apiosperma were incorporated into a multiplex PCR assay for the detection and identification of the three major clinically important Scedosporium species and validated using sputum specimens collected from patients seen at a major Australian cystic fibrosis clinic. The multiplex PCR assay showed 100% specificity in identifying the three major clinically relevant Scedosporium species from pure culture. When evaluated using DNA extracts from sputa, sensitivity and specificity of the multiplex PCR assay were 62.1% and 97.2%, respectively. This highly species-specific multiplex PCR assay offers a rapid and simple method of detection of the most clinically important Scedosporium species in respiratory tract specimens.

Scedosporium aurantiacum is as virulent as S. prolificans, and shows strain-specific virulence differences, in a mouse model.

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 (P > 0.05). There were significant strain-specific virulence differences (P < 0.005), indicating a possible link between genotype and virulence in S. aurantiacum.

Abundance of Pseudallescheria/Scedosporium species in the Australian urban environment suggests a possible source for scedosporiosis including the colonization of airways in cystic fibrosis.

Members of the Pseudallescheria/Scedosporium species complex are emerging opportunistic fungal pathogens which have the capacity to colonize patients with damaged airways, including those with cystic fibrosis (CF). Assuming human infection is acquired via inhalation of fungal spores from the environment, we performed a qualitative environmental survey encompassing 25 urban, semirural and rural sites in the greater Sydney region to determine the prevalence of Pseudallescheria/Scedosporium species. Soil sampling revealed an abundance of Pseudallescheria/Scedosporium, particularly in locations associated with high human activity. No variation was noted during repeated sampling at different times of the year. Strains of Scedosporium aurantiacum were most frequently isolated (54.6%), followed by Scedosporium prolificans (43%), P. boydii (2.1%) and S. dehoogii (0.3%). The findings coincide with the relatively high prevalence of Scedosporium infections in Australia and their presence as colonizers in CF patients. They emphasize the importance of environmental studies to assess the clinical risk of infection.

Heterothallism in Scedosporium apiospermum and description of its teleomorph Pseudallescheria apiosperma sp. nov.

Scedosporium apiospermum has traditionally been thought of as the anamorph of Pseudallescheria boydii (Microascaceae, Ascomycota), but recent molecular studies has demonstrated that they are different species. Since a teleomorph was not observed among isolates recently identified as S. apiospermum, we investigated whether this species could be heterothallic. In this study, 15 isolates of S. apiospermum were paired in all possible combinations, including self-pairings. Several combinations produced fertile ascomata typical of the genus Pseudallescheria, while all isolates were self-sterile. The isolates were grouped into two different mating types. Crosses among F1 progeny ascospores demonstrated a bi-allelic heterothallic mating system. The new species Pseudallescheria apiosperma, teleomorph of S. apiospermum, is proposed and described.

Genetic diversity of the Cryptococcus species complex suggests that Cryptococcus gattii deserves to have varieties.

The Cryptococcus species complex contains two sibling taxa, Cryptococcus neoformans and Cryptococcus gattii. Both species are basidiomycetous yeasts and major pathogens of humans and other mammals. Genotyping methods have identified major haploid molecular types of C. neoformans (VNI, VNII, VNB and VNIV) and of C. gattii (VGI, VGII, VGIII and VGIV). To investigate the phylogenetic relationships among these haploid genotypes, we selected 73 strains from 2000 globally collected isolates investigated in our previous typing studies, representing each of these genotypes and carried out multigene sequence analyses using four genetically unlinked nuclear loci, ACT1, IDE, PLB1 and URA5. The separate or combined sequence analyses of all four loci revealed seven clades with significant support for each molecular type. However, three strains of each species revealed some incongruence between the original molecular type and the sequence-based type obtained here. The topology of the individual gene trees was identical for each clade of C. neoformans but incongruent for the clades of C. gattii indicating recent recombination events within C. gattii. There was strong evidence of recombination in the global VGII population. Both parsimony and likelihood analyses supported three major clades of C. neoformans (VNI/VNB, VNII and VNIV) and four major clades of C. gattii (VGI, VGII, VGIII and VGIV). The sequence variation between VGI, VGIII and VGIV was similar to that between VNI/VNB and VNII. MATa was for the first time identified for VGIV. The VNIV and VGII clades are basal to the C. neoformans or the C. gattii clade, respectively. Divergence times among the seven haploid monophyletic lineages in the Cryptococcus species complex were estimated by applying the hypothesis of the molecular clock. The genetic variation found among all of these haploid monophyletic lineages indicates that they warrant varietal status.

Consensus multi-locus sequence typing scheme for Cryptococcus neoformans and Cryptococcus gattii.

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/ .

Clinical significance and phylogenetic relationship of novel Australian Pneumocystis jirovecii genotypes.

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.

Genotyping of Scedosporium species: a review of molecular approaches.

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.

Different virulence of the species of the Pseudallescheria boydii complex.

Pseudallescheria boydii sensu lato is a complex of species involved in severe human infections. We have evaluated, using a murine model, the virulence of 2 strains of each of the most representative species of the complex, i.e., P. boydii sensu stricto, P. minutispora, Scedosporium apiospermum, S. aurantiacum and S. dehoogii. We used two different inocula, i.e., 5 x 10(4) conidia/ml (for immunosuppressed animals) and 1 x 10(6) conidia/ml (for immunocompetent animals), which were administered intravenously. Scedosporium aurantiacum and S. dehoogii were the most virulent species, causing the death of 80% and 70% of the immunocompetent mice, respectively. The remaining species only killed 0-20% of the animals.

Molecular and phenotypic data supporting distinct species statuses for Scedosporium apiospermum and Pseudallescheria boydii and the proposed new species Scedosporium dehoogii.

Based on the morphological, physiologic, and molecular (beta-tubulin gene) study of 141 isolates of the Pseudallescheria boydii species complex (including several synonyms) and relatives, the new species Scedosporium dehoogii is proposed. Scedosporium apiospermum and P. boydii are considered two different species and the new name Scedosporium boydii is proposed for the anamorph of the latter species. A summary of the key morphological and physiological features for distinguishing the species of Pseudallescheria/Scedosporium is provided.

Reclassification of Graphium tectonae as Parascedosporium tectonae gen. nov., comb. nov., Pseudallescheria africana as Petriellopsis africana gen. nov., comb. nov. and Pseudallescheria fimeti as Lophotrichus fimeti comb. nov.

During a biodiversity survey of Argentinian soil fungi, we recovered a rare Scedosporium-like fungus which was proven to be genetically and morphologically different from known species of Scedosporium (anamorph of Pseudallescheria) and relatives and is proposed here as representing a new genus. This genus is mainly characterized by producing sympodial conidia from denticulate conidiogenous cells. This isolate was morphologically identical to Graphium tectonae and thus the new combination Parascedosporium tectonae gen. nov., comb. nov. is proposed. Sequence analysis of four regions of three genes, i.e. beta-tubulin (two loci), calmodulin and the internal transcribed spacer region of the 5.8S rRNA, confirmed our proposal. Both the phylogenetic analysis and morphological studies excluded Pseudallescheria africana and Pseudallescheria fimeti from the genus Pseudallescheria. The former is proposed as a member of the new genus Petriellopsis, and the latter has been accommodated in Lophotrichus. The type strains of Parascedosporium tectonae gen. nov., comb. nov., Petriellopsis africana gen. nov., comb. nov. and Lophotrichus fimeti comb. nov. are respectively CBS 127.84(T), CBS 311.72(T) and CBS 129.78(T).

Antifungal susceptibilities of the species of the Pseudallescheria boydii complex.

Eighty-four isolates belonging to eight species that constitute the Pseudallescheria boydii complex were tested against 11 antifungal agents by using the microdilution method. There were significant differences among the species, with Scedosporium aurantiacum being the most resistant. In general, voriconazole was the most active drug, followed by posaconazole.

Efficacy of voriconazole in a guinea pig model of invasive trichosporonosis.

We have evaluated the efficacy of voriconazole (VRC) in a systemic infection by Trichosporon asahii in immunosuppressed guinea pigs. VRC was more effective than amphotericin B in prolonging survival and reducing tissue burden. The best results were obtained with VRC at 10 mg/kg of body weight/day.

Molecular phylogeny of the Pseudallescheria boydii species complex: proposal of two new species.

Pseudallescheria boydii (anamorph Scedosporium apiospermum) is the species responsible for human scedosporiosis, a fungal infection with a high mortality rate and which is difficult to treat. Recently, it has been demonstrated that high genetic variation exists within this species. We have performed a morphological and molecular study involving numerous strains of clinical or environmental origins and from different countries. The analysis of partial sequences of the beta-tubulin (two loci) and calmodulin genes and the internal transcribed spacer region of the rRNA gene has demonstrated that P. boydii is a species complex. The combined analysis of the sequences of the four loci of 60 strains has showed the presence of 44 haplotypes in the in group. Three species morphologically related to P. boydii sensu stricto, i.e., Pseudallescheria angusta, Pseudallescheria ellipsoidea, and Pseudallescheria fusoidea, which had previously been considered synonyms, could be differentiated genetically from P. boydii in our study. It is relevant that two of the three strains now included in P. ellipsoidea have caused invasive infections. The species Pseudallescheria minutispora and Scedosporium aurantiacum are clearly phylogenetically separated from the other species studied and are here proposed as new. Morphological features support this proposal. All the strains included in S. aurantiacum species have a clinical origin, while those included in P. minutispora are environmental. Further studies are needed to demonstrate whether all the species included in the P. boydii complex have different clinical spectra and antifungal susceptibility.

Efficacy of micafungin in combination with other drugs in a murine model of disseminated trichosporonosis.

Using a murine model of disseminated infection caused by Trichosporon asahii, we have evaluated the efficacies of amphotericin B (AMB; 1 mg/kg of body weight/day), fluconazole (FLC; 20 mg/kg/twice a day), and micafungin (MFG; 5 mg/kg/twice a day). We tested these drugs alone and in combination (MFG with AMB and MFG with FLC). MFG with AMB showed a synergistic effect and demonstrated a higher degree of efficacy in prolonging survival and reducing the kidney fungal burden than either agent alone. The combination MFG with FLC was able to reduce significantly the kidney fungal burden in comparison to that achieved with either drug administered alone.