The genus Anthopsis and its phylogenetic position in Chaetothyriales.

The genus Anthopsis was introduced for a black fungus with peculiar, inverted phialides and triangular conidia. The genus accommodates, in addition to the type species Anthopsis deltoidea, which once was reported as a cause of human phaeohyphomycosis, two further taxa: A. catenata and A. microspora. Current taxonomy is mainly based on microscopic structures of phialides. To assess the phylogenetic position of the genus, sequences of the internal transcribed spacer region and partial LSU rDNA were obtained for Anthopsis spp. and compared with sequences from public databases. Phylogenetic analyses based on both loci were used to assess the evolutionary relationships of Anthopsis spp. at the family and ordinal levels. Anthopsis s.str. was found to cluster in Chaetothyriales, while A. catenata proved to be of helotialean affinity. Thermotolerance and morphology of each species were recorded.

Barcoding markers for Pneumocystis species in wildlife.

Lung specimens (n = 216) from six wildlife species were examined for occurrence of Pneumocystis species in pulmonary tissues. Among small mammals the shrew Sorex antinorii (80 %) were most frequently colonized. In contrast, foxes and badgers did not yield positive amplification. Host-specificity was noted, at least at the level of the host genus. Phylogenetic trees based on partial mtLSU and mtSSU showed high diversity of species corresponding to animal host diversity. Nuclear rDNA ITS data confirmed unambiguous separation of species. In conclusion, ITS is an excellent marker to distinguish species of the genus Pneumocystis.

Molecular identification of Histoplasma capsulatum using rolling circle amplification.

Histoplasmosis is a systemic fungal disease that occurs worldwide, causing symptomatic infection mostly in immunocompromised hosts. Etiological agent is the dimorphic fungus, Histoplasma capsulatum, which occurs in soil contaminated with bird or bat droppings. Major limitation in recognition of H. capsulatum infections is the low awareness, since other diseases may have similar symptomatology. The molecular methods have gained importance because of unambiguous diagnostic ability and efficiency. The aim of this study was to develop and evaluate a padlock probe in view of rolling circle amplification (RCA) detection method which targets ITS (Internal Transcribed Spacer) rDNA of H. capsulatum enabling rapid and specific detection of the fungus in clinical samples. Two padlock probes were designed and one of these (HcPL2) allowed specific amplification of H. capsulatum DNA while no cross-reactivity was observed with fungi used as negative controls. This method proved to be effective for H. capsulatum specific identification and demonstrated to be faster than the traditional method of microbiological identification.

Differentiation of clinically relevant Mucorales Rhizopus microsporus and R. arrhizus by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).

This study addresses the usefulness of matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS for reliable identification of the two most frequently occurring clinical species of Rhizopus, namely Rhizopus arrhizus with its two varieties, arrhizus and delemar, and Rhizopus microsporus. The test-set comprised 38 isolates of clinical and environmental origin previously identified by internal transcribed spacer (ITS) sequencing of rDNA. Multi-locus sequence data targeting three gene markers (ITS, ACT, TEF ) showed two monophylic clades for Rhizopus arrhizus and Rhizopus microsporus (bootstrap values of 99 %). Cluster analysis confirmed the presence of two distinct clades within Rhizopus arrhizus representing its varieties arrhizus and delemar. The MALDI Biotyper 3.0 Microflex LT platform (Bruker Daltonics) was used to confirm the distinction between Rhizopus arrhizus and Rhizopus microsporus and the presence of two varieties within the species Rhizopus arrhizus. An in-house database of 30 reference main spectra (MSPs) was initially tested for correctness using commercially available databases of Bruker Daltonics. By challenging the database with the same strains of which an in-house database was created, automatic identification runs confirmed that MALDI-TOF MS is able to recognize the strains at the variety level. Based on principal component analysis, two MSP dendrograms were created and showed concordance with the multi-locus tree; thus, MALDI-TOF MS is a useful tool for diagnostics of mucoralean species.

Bradymyces gen. nov. (Chaetothyriales, Trichomeriaceae), a new ascomycete genus accommodating poorly differentiated melanized fungi.

Three slow growing, melanized and morphologically poorly differentiated fungal strains were isolated from a hyperaemic focus near the enlarged spleen of a farmed rainbow trout (Oncorhynchus mykiss) and from a rock collected at 3,200 m a. s. l. (Alps, Italy). Two phylogenetic analyses of the combined nuc18S and nuc28S rDNA and ITS rDNA and ß-tubulin sequences showed that these isolates belong to the Trichomeriaceae, a family of the ascomycete order Chaetothyriales containing black yeasts that cause infections in humans and animals. The strains form a well-supported monophyletic clade. The new genus Bradymyces, with two new species, Bradymyces oncorhynchi and Bradymyces alpinus, is proposed based on phylogenetic, ecophysiological and morphological data. It is characterized by the presence of moniliform hyphae, blastic proliferation, endoconidia, multicellular and muriform bodies, and bodies with dark fragmented incrustations on the surface. Bradymyces most closely resembles members of Knufia. The ex-type isolate of B. oncorhynchi CCF 4369(T) ( = CBS 133066(T) = CCFEE 6134(T)) represents the first case of a Trichomeriaceae member isolated from cold-blooded water vertebrates. B. alpinus [ex-type strain CCFEE 5493(T) ( = CBS 138368(T) = CCF 4803(T))] is represented by two isolates from a single locality in the Alps and in contrast to B. oncorhynchi shows overall slower growth parameters and does not grow at 25 °C.

Efficacy of a selective isolation procedure for members of the Pseudallescheria boydii complex.

Members of the P. boydii species complex (Microascaceae) are frequently involved in human opportunistic disease. Studies indicate that the prevalent habitat of P. boydii sensu lato is in agriculturally exploited or otherwise human-impacted soils. Quantitative analysis of fungal indicators in the environment can be exploited for monitoring of general environmental changes, as well as for understanding local population changes and its epidemiological consequences. In this study we present the development and testing of a semi-selective isolation procedure for P. boydii and related species. Three general media, DG18, rose bengal agar and five variations of modified Leonian's agar with and without benomyl were tested. Germination percentages of P. boydii, S. prolificans, Petriella spp. and Aspergillus fumigatus (control) were evaluated. Tests were carried out on the success of P. boydii isolation from inoculum mixed with A. fumigatus. Subsequently the procedure was applied to water, sediment and soil samples. On the newly introduced semi-selective medium (SceSel+), the germination of P. boydii was superior or similar to that seen on the other media tested. P. boydii was isolated from mixed cultures only on SceSel+ but not on SceSel without benomyl. Isolation from environmental sources with SceSel+ was successful, and human impacted soil was confirmed as the predominant habitat of P. boydii.