Intraspecific Diversity and Taxonomy of Emmonsia crescens.

Emmonsia crescens is known as an environmental pathogen causing adiaspiromycosis in small rodents. As the generic name Emmonsia is no longer available for this species, its taxonomic position is re-evaluated. The intraspecific variation of Emmonsia crescens was analyzed using molecular, morphological, and physiological data, and the relationship between frequency of adiaspiromycosis and body temperature of host animals was explored. A North American and a pan-global lineage could be discerned, each with subclusters at low genetic distance. European strains produced the classical type of very large adiaspores, while in the North American lineage adiaspores relatively small, resembling the broad-based budding cells of Blastomyces. Members of the closely related genus Emergomyces may exhibit large, broad-based in addition to small, narrow-based budding cells. We conclude that the morphology of the pathogenic phase in these fungi differs gradationally between species and even populations, and is therefore less suitable as a diagnostic criterion for generic delimitation. Two Emmonsia species are reclassified in Emergomyces.

Molecular Diagnostics of Arthroconidial Yeasts, Frequent Pulmonary Opportunists.

Magnusiomyces capitatus and Saprochaete clavata are members of the clade of arthroconidial yeasts that represent emerging opportunistic pulmonary pathogens in immunocompromised patients. Given that standard ribosomal DNA (rDNA) identification often provides confusing results, in this study, we analyzed 34 isolates with the goal of finding new genetic markers for classification using multilocus sequencing and amplified fragment length polymorphism (AFLP). The interspecific similarity obtained using rDNA markers (the internal transcribed spacer [ITS] and large subunit regions) was in the range of 96 to 99%, whereas that obtained using protein-coding loci (Rbp2, Act, and Tef1α) was lower at 89.4 to 95.2%. Ultimately, Rbp2 was selected as the best marker for species distinction. On the basis of cloned ITS data, some strains proved to be misidentified in comparison with the identities obtained with phenotypic characters, protein sequences, and AFLP profiles, indicating that different copies of the ribosomal operon were present in a single species. Antifungal susceptibility testing revealed that voriconazole had the lowest MIC against M. capitatus, while amphotericin B had the lowest MIC against S. clavata Both species exhibited in vitro resistance to fluconazole and micafungin.

Identification of chromoblastomycosis agents by PCR based reverse line blot (PCR-RLB) hybridization assay.

Chromoblastomycosis is one of the most prevalent implantation fungal infections caused by melanized fungi, affecting individuals with certain risk factors with high morbidity due to its recalcitrant nature. It is difficult to identify the etiological agents and thus a suitable reproductive molecular identification method applicable in developing countries has been investigated. We report the identification of four different fungal causative agents of chromoblastomycosis by reverse line blotting hybridization (RLB) based on biotin-labeled PCR products and amine labeled probes to hybridize. Sixty five reference strains, including type strains, i.e. Fonsecaea pedrosoi, F. monophora, F. nubica, and Phialophora verrucosa, obtained from the CBS-KNAW were included in this study. Internal transcribed spacer 1 (ITS1) regions of relevant species were aligned and adjusted using BIONUMERICS v. 4.61 in order to design four specific probes to identify informative nucleotide polymorphisms. The final identification of these species by RLB assay was concordant with ITS sequencing and showed 100% specificity with no cross hybridization, able to identify all tested strains. The time and cost were less compare to other routine identification methods such as sequencing. This assay allows sensitive and specific simultaneous detection and identification of a different fungal species.

Direct detection of Exophiala and Scedosporium species in sputa of patients with cystic fibrosis.

Detection of species of Exophiala and Scedosporium in the respiratory tracts of cystic fibrosis (CF) patients remains controversial because of highly variable results. The results of our study suggested a significantly higher prevalence and more complex colonization than previously estimated. Approximately 17% (27/162) of clinical sputum samples were found to be positive for Exophiala dermatitidis and 30% (49/162) were positive for Scedosporium apiospermum / S. boydii species complex determined by reverse line blot (RLB) hybridization. In contrast, only 14.2% (23/162) and 1.2% (2/162) of clinical sputa were positive for E. dermatitidis and S. apiospermum / S. boydii species complex when tested by culture, respectively. Molecular detection methods, such as loop-mediated isothermal amplification (LAMP) or reverse line blot (RLB) hybridization, have the potential to become powerful alternatives to selective culture, providing a more realistic understanding on the prevalence of E. dermatitidis and S. apiospermum / S. boydii species complex in the respiratory tract of CF patients.

Nomenclatural notes on Nadsoniella and the human opportunist black yeast genus Exophiala.

The opportunistic black yeast are particularly known through the genus Exophiala, characterised by annellidic budding cells. However, this phenotype is polyphyletic within the order Chaetothyriales. Seventeen generic names are available in the family Herpotrichiellaceae, one of which is Exophiala. Future taxonomy will be based on molecular phylogeny; each multi-species clade may qualify for one of these names. This paper focuses on the genus Nadsoniella, which is the oldest valid name in the Herpotrichiellaceae. Despite its exophiala-like phenotype, the type species of Nadsoniella clusters in the jeanselmei-clade, competing with the sympodial genus Rhinocladiella. In contrast, Exophiala competes with morphologically pronounced genera Thysanorea and Veronaea. Replacing the current phenotypic system for phylogenetic nomenclature requires highly stable phylogenies, which currently are not available.

Combination of Amphotericin B and Flucytosine against Neurotropic Species of Melanized Fungi Causing Primary Cerebral Phaeohyphomycosis.

Primary central nervous system phaeohyphomycosis is a fatal fungal infection due mainly to the neurotropic melanized fungiCladophialophora bantiana,Rhinocladiella mackenziei, andExophiala dermatitidis.Despite the combination of surgery with antifungal treatment, the prognosis continues to be poor, with mortality rates ranging from 50 to 70%. Therefore, a search for a more-appropriate therapeutic approach is urgently needed. Ourin vitrostudies showed that with the combination of amphotericin B and flucytosine against these species, the median fractional inhibitory concentration (FIC) indices for strains ranged from 0.25 to 0.38, indicating synergy. By use of Bliss independence analysis, a significant degree of synergy was confirmed for all strains, with the sum ΔE ranging from 90.2 to 698.61%. No antagonism was observed. These results indicate that amphotericin B, in combination with flucytosine, may have a role in the treatment of primary cerebral infections caused by melanized fungi belonging to the orderChaetothyriales Furtherin vivostudies and clinical investigations to elucidate and confirm these observations are warranted.

DNA barcoding of clinically relevant Cunninghamella species.

Mucormycosis caused, in part, by representatives of the genus Cunninghamella is a severe infection with high mortality in patients with impaired immunity. Several species have been described in the literature as etiologic agents. A DNA barcoding study using ITS rDNA and tef-1α provided concordance of molecular data with conventional characters. The currently accepted Cunninghamella species were well supported in phylogenetic trees of both markers except for C. septata with ITS that clustered in the C. echinulata clade. Sequence variability was distinctly higher for the ITS than for tef-1α. Intraspecific ITS variability of some of the species exceeded that between some closely related species, but the marker remained applicable for species identification. The most variable species for both markers was C. echinulata. Cunninghamella bertholletiae is the main pathogenic species; infections by C. blakesleeana, C. echinulata, and C. elegans are highly exceptional.

Three New Species of the Genus Ochroconis.

Ochroconis bacilliformis, O. phaeophora and O. robusta, three novel species of the melanized genus Ochroconis (Sympoventuriaceae, Venturiales), are described, illustrated and distinguished phenotypically and molecularly from previously described species in the genus Ochroconis. Their potential significance for infection of cold-blooded vertebrates is discussed.

Susceptibility and diversity in the therapy-refractory genus scedosporium.

Scedosporium species show decreased susceptibility to the majority of systemic antifungal drugs. Acquired resistance is likely to disseminate differentially with the mode of exchange of genetic material between lineages. Inter- and intraspecific diversities of Scedosporium species were analyzed for three partitions (rDNA internal transcribed spacer gene [ITS], partial ß-tubulin gene, and amplified fragment length polymorphism profiles), with the aim to establish distribution of resistance between species, populations, and strains. Heterogeneity of and recombination between lineages were determined, and distances between clusters were calculated using a centroid approach. Clinical, geographic, and antifungal data were plotted on diversity networks. Scedosporium minutisporum, Scedosporium desertorum, and Scedosporium aurantiacum were distinguished unambiguously in all partitions and had differential antifungal susceptibility profiles (ASP). Pseudallescheria fusoidea and Pseudallescheria ellipsoidea were indistinguishable from Scedosporium boydii. Pseudallescheria angusta took an intermediate position between Scedosporium apiospermum and S. boydii. Scedosporium boydii and S. apiospermum had identical ASP. Differences in (multi)resistance were linked to individual strains. S. apiospermum and S. boydii showed limited interbreeding and were recognized as valid, sympatric species. The S. apiospermum/S. boydii group, comprising the main clinically relevant Scedosporium species, consists of separate lineages and is interpreted as a complex undergoing sympatric evolution with incomplete lineage sorting. In routine diagnostics, the lineages in S. apiospermum/S. boydii are indicated with the umbrella descriptor "S. apiospermum complex"; individual species can be identified with rDNA ITS with 96.3% confidence. Voriconazole is recommended as the first-line treatment; resistance against this compound is rare.

Identification and typing of isolates of Cyphellophora and relatives by use of amplified fragment length polymorphism and rolling circle amplification.

The species diversity and identification of black fungi belonging to Cyphellophora and Phialophora, which colonize and infect human skin and nails, were studied using amplified fragment length polymorphism (AFLP). A total of 76 Cyphellophora and Phialophora isolates were evaluated, and their delimitation was compared to earlier studies using multilocus sequencing. The results of the AFLP analysis and sequencing were in complete agreement with each other. Seven species-specific padlock probes for the most prevalent species were designed on the basis of the ribosomal DNA internal transcribed spacer region, and identification of the respective species could easily be achieved with the aid of rolling circle amplification.

Taxonomy and epidemiology of Mucor irregularis, agent of chronic cutaneous mucormycosis.

Mucormycosis usually presents as a progressive infection with significant angio-invasion. Mucormycosis due to Mucor irregularis (formerly Rhizomucor variabilis var. variabilis), however, is exceptional in causing chronic cutaneous infection in immunocompetent humans, ultimately leading to severe morbidity if left untreated. More than 90 % of the cases known to date were reported from Asia, mainly from China. The nearest neighbour of M. irregularis is the saprobic species M. hiemalis. The aim of this study was to evaluate the taxonomic position, epidemiology, and intra- and inter-species diversity of M. irregularis based on 21 strains (clinical n = 17) by multilocus analysis using ITS, LSU, RPB1 and RPB2 genes, compared to results of cluster analysis with amplified fragment length polymorphism (AFLP) data. By combining MLST and AFLP analyses, M. irregularis was found to be monophyletic with high bootstrap support, and consisted of five subgroups, which were not concordant in all partitions. It was thus confirmed that M. irregularis is a single species at 96.1-100 % ITS similarity and low recombination rates between populations. Some geographic structuring was noted with some localised populations, which may be explained by limited air-dispersal. The natural habitat of the species is likely to be in soil and decomposing plant material.

Pleurostomophora ochracea, a novel agent of human eumycetoma with yellow grains.

The first yellow-grain fungal mycetoma, in a 60-year-old man from Central Sudan, is reported. Morphological and phylogenetic analysis of the ribosomal small subunit (SSU), large subunit (LSU), internal transcribed spacer (ITS), ß-tubulin (BT2), actin (ACT1), and elongation factor (TEF1) genes revealed that the isolate deviated from any known agent of mycetoma; it clustered in the genus Pleurostoma (anamorph genus, Pleurostomophora) in the order Calosphaeriales. The novel species, here named Pleurostomophora ochracea, is characterized by phenotypic features. The species proved to be highly susceptible to itraconazole, ketoconazole, posaconazole, and voriconazole, but not to fluconazole. The fungus was inhibited by caspofungin at 8 µg/ml, while no inhibition was found with 5-flucytosine (MIC > 64 µg/ml). Compared to other members of the genus Pleurostomophora, P. ochracea is slow growing, with a relatively high optimum growth temperature (36 to 37°C). This is the first case of a yellow-grain fungal mycetoma; yellow grains are otherwise of bacterial nature. Our case emphasizes that identification of mycetoma agents by the color of the grain only is not sufficient and may lead to inappropriate therapy.

Molecular epidemiology of Fonsecaea species.

To assess population diversities among 81 strains of fungi in the genus Fonsecaea that had been identified down to species level, we applied amplified fragment-length polymorphism (AFLP) technology and sequenced the internal transcribed spacer regions and the partial cell division cycle, beta-tubulin, and actin genes. Many species of the genus Fonsecaea cause human chromoblastomycosis. Strains originated from a global sampling of clinical and environmental sources in the Western Hemisphere, Asia, Africa, and Europe. According to AFLP fingerprinting, Fonsecaea isolates clustered in 5 groups corresponding with F. pedrosoi, F. monophora, and F. nubica: the latter 2 species each comprised 2 groups, and F. pedrosoi appeared to be of monophyletic origin. F. pedrosoi was found nearly exclusively in Central and South America. F. monophora and F. nubica were distributed worldwide, but both showed substantial geographic structuring. Clinical cases outside areas where Fonsecaea is endemic were probably distributed by human migration.

Identification of Pseudallescheria and Scedosporium species by three molecular methods.

The major clinically relevant species in Scedosporium (teleomorph Pseudallescheria) are Pseudallescheria boydii, Scedosporium aurantiacum, Scedosporium apiospermum, and Scedosporium prolificans, while Pseudallescheria minutispora, Petriellopsis desertorum, and Scedosporium dehoogii are exceptional agents of disease. Three molecular methods targeting the partial ß-tubulin gene were developed and evaluated to identify six closely related species of the S. apiospermum complex using quantitative real-time PCR (qPCR), PCR-based reverse line blot (PCR-RLB), and loop-mediated isothermal amplification (LAMP). qPCR was not specific enough for the identification of all species but had the highest sensitivity. The PCR-RLB assay was efficient for the identification of five species. LAMP distinguished all six species unambiguously. The analytical sensitivities of qPCR, PCR-RLB, and LAMP combined with MagNAPure, CTAB (cetyltrimethylammonium bromide), and FTA filter (Whatman) extraction were 50, 5 × 10(3), and 5 × 10(2) cells/µl, respectively. When LAMP was combined with a simplified DNA extraction method using an FTA filter, identification to the species level was achieved within 2 h, including DNA extraction. The FTA-LAMP assay is therefore recommended as a cost-effective, simple, and rapid method for the identification of Scedosporium species.

Diversity of opportunistic black fungi on babassu coconut shells, a rich source of esters and hydrocarbons.

The present study assessed the diversity of black yeast-like fungi present on babassu coconut shells, a substrate rich in lipids and several volatile organic compounds (VOCs) including aromatic hydrocarbons. Using different isolation methods, one-hundred-six isolates were obtained and were identified by ITS sequencing as members of the genera Exophiala, Cladophialophora, Veronaea, and Rhinocladiella. Two novel species were discovered. Eight strains were selected for assessing their ability to grow on toluene and phenyl acetate as the sole carbon and energy source. All strains tested were able to assimilate phenyl acetate, while two out of eight were able to use toluene. VOCs profiling in babassu samples was also investigated by GC-ToF MS, revealing that a complex mixture of VOCs was emitted, which included alkylbenzenes such as toluene. Assimilation of alkylbenzenes by the black yeasts might therefore be the result of evolutionary adaptation to symbiotic interactions with higher plants. The potential relationship between lipid/aromatic hydrocarbon metabolism and pathogenicity is also discussed.

The 'species complex' issue in clinically relevant fungi: A case study in Scedosporium apiospermum.

The genus Scedosporium currently comprises six species, Scedosporium apiospermum, Scedosporium boydii, Pseudallescheria angusta, Scedosporium minutisporum, Scedosporium dehoogii, and Scedosporium aurantiacum, most of which can be distinguished with the primary fungal DNA barcode, the ITS1/2 region of the rDNA gene cluster. In the present study, four additional genetic loci were explored from a phylogenetic point of view enabling a barcoding approach based on K2P pairwise distances to resolve the taxa Scedosporium. We included partial γ-actin (ACT), ß-tubulin (BT2), elongation factor 1α (TEF1), and the small ribosomal protein 60S L10 (L1) (RP60S). Phylogenetic inference of each marker individually showed that four out of six species within Scedosporium can be distinguished unambiguously, while strains of S. apiospermum, S. boydii, and P. angusta showed occasional recombination, and accordingly, no genealogical concordance between markers was obtainable. We defined S. apiospermum, S. boydii, and P. angusta as the 'S. apiospermum species complex' since observed differences were not consistent between lineages, and no clinical differences are known between entities within the complex. While BT2 revealed the best performance among the genetic loci tested at the lineage level, barcoding of the ITS region is sufficient for distinction of all entities in Scedosporium at the species or 'complex' level.

Global Spread of Human Chromoblastomycosis Is Driven by Recombinant Cladophialophora carrionii and Predominantly Clonal Fonsecaea Species.

Global distribution patterns of Cladophialophora carrionii, agent of human chromoblastomycosis in arid climates of Africa, Asia, Australia, Central-and South-America, were compared with similar data of the vicarious Fonsecaea spp., agents of the disease in tropical rain forests. Population diversities among 73 C. carrionii strains and 60 strains of three Fonsecaea species were analyzed for rDNA ITS, partial ß-tubulin, and amplified fragment-length polymorphism (AFLP) fingerprints. Populations differed significantly between continents. Lowest haplotype diversity was found in South American populations, while African strains were the most diverse. Gene flow was noted between the African population and all other continents. The general pattern of Fonsecaea agents of chromoblastomycosis differed significantly from that of C. carrionii and revealed deeper divergence among three differentiated species with smaller numbers of haplotypes, indicating a longer evolutionary history.

Elucidation of distribution patterns and possible infection routes of the neurotropic black yeast Exophiala dermatitidis using AFLP.

Distribution of populations of the opportunistic black yeast Exophiala dermatitidis was studied using AFLP. This fungus has been hypothesized to have a natural habitat in association with frugivorous birds and bats in the tropical rain forest, and to emerge in the human-dominated environment, where it occasionally causes human pulmonary or fatal disseminated and neurotropic disease. The hypothesis of its natural niche was investigated by comparing a set of 178 strains from natural and human-dominated environments in Thailand with a worldwide selection of 107 strains from the reference collection of the CBS Fungal Biodiversity Centre, comprising 75.7% clinical isolates. Many isolates had unique AFLP patterns and were too remote for confident comparison. Eight populations containing multiple isolates could be distinguished, enabling determination of geographic distributions of these populations. Some of the populations were confined to Thailand, while others occurred worldwide. The local populations from Thailand contained strains from natural and urban environments, suggesting an environmental jump of the fungus. Strains from human brain belonged to widely dispersed populations. In some cases cerebral isolates were identical to isolates from the human intestinal tract. The possibility of cerebral infection through intestinal translocation was thus not excluded.

Rapid screening for genotypes as possible markers of virulence in the neurotropic black yeast Exophiala dermatitidis using PCR-RFLP.

A simple method for fungal genotype screening was developed for the black yeast Exophiala dermatitidis based on RFLP of ribosomal ITS regions currently used as potential virulence markers. In a study set of 502 strains of the species, two main genotypes were recognized. Only 0.97% of lanes were difficult to interpret as they did not clearly present one of the expected genotypes. Twenty strains were deviating and proved to be E. spinifera after sequencing. Eight common, related species (based on SSU data) with clinical significance yielded different patterns with TaqI digestion, and thus the method is also usable for routine diagnostics.

Cladophialophora saturnica sp. nov., a new opportunistic species of Chaetothyriales revealed using molecular data.

While many members of the black yeasts genus Cladophialophora have been reported to cause diseases in humans, understanding of their natural niche is frequently lacking. Some species can be recovered from the natural environment by means of selective isolation techniques. The present study focuses on a Cladophialophora strain that caused an interdigital tinea nigra-like lesion in a HIV-positive Brazilian child. The fungal infection was successfully treated with oxiconazole. Similar strains had been recovered from the environment in Brazil, Uruguay and the Netherlands. The strains were characterized by sequencing the Internal Transcribed Spacer (ITS) regions and the small subunit (SSU) of the nuclear ribosomal RNA gene, as well as the elongation factor 1-alpha (EF1alpha) gene. Since no match with any known species was found, it is described as the new species, Cladophialophora saturnica.