A new pleosporalean fungus isolated from superficial to deep human clinical specimens.

Human infections by pleosporalean fungi (class Dothideomycetes, phylum Ascomycota) are rarely reported. Because their identification is challenging using morphological characterization, several phylogenetic markers must be sequenced for an accurate identification and taxonomical placement of the isolates. Three isolates of clinical origin were phenotypically characterized, but due to the absence of relevant morphological traits, D1-D2 domains of the 28S nrRNA gene (LSU), the internal transcribed spacer region (ITS) of the nrRNA, and fragments of the RNA polymerase II subunit 2 (rpb2) and translation elongation factor 1-alpha (tef1) genes were sequenced to allow a phylogenetic analysis that would solve their phylogenetic placement. That analysis revealed that these isolates did not match any previously known pleosporalean genera, and they are proposed here as the new fungal genus, Gambiomyces. Unfortunately, the isolates remained sterile, which, consequently, made the morphological description of the reproductive structures impossible. Future studies should try to understand the behaviour of this fungus in nature as well as its characteristics as an opportunistic fungal pathogen. Molecular identification is becoming an essential tool for proper identification of Dothideomycetes of clinical origin. LAY ABSTRACT: We describe a new pleosporalen pathogenic fungus, Gambiomyces profunda, found in superficial to deep samples from a human patient. Because all strains remained sterile, the fungus was finally identified following a phylogenetic analysis by using four different molecular markers.

Neocucurbitaria keratinophila: An emerging opportunistic fungus causing superficial mycosis in Spain.

Although there have been few reports of opportunistic infections (superficial and systemic) caused by coelomycetous fungi, they are becoming more frequent. Neocucurbitaria keratinophila (formerly Pyrenochaeta keratinophila), characterized by producing pycnidial conidiomata and small hyaline conidia, seems to be an emergent opportunistic pathogen in Spain. Since this fungus was first reported from human keratitis, eight strains have been isolates from clinical cases in Spain. This is a retrospective study of these fungal strains, including phenotypic and molecular characterizations, and in vitro antifungal susceptibility assays. These clinical strains were identified by sequencing four phylogenetic markers such as the internal transcribed spacer region (ITS1-5.8S-ITS2) and fragments of the 28S nrRNA (LSU), beta-tubulin (tub2), and RNA polymerase II subunit 2 (rpb2) genes, and by morphological characterization. All the strains tested were susceptible to the majority of antifungals, being isavuconazole the only drug that showed a poor antifungal activity.

Novel Paranannizziopsis species in a Wagler's viper (Tropidolaemus wagleri), tentacled snakes (Erpeton tentaculatum), and a rhinoceros snake (Rhynchophis boulengeri) in a zoological collection.

We report several cases of fungal infections in snakes associated with a new species within the genus Paranannizziopsis. Three juvenile Wagler's vipers (Tropidolaemus wagleri) presented with skin abnormalities or ulcerative dermatitis, and two snakes died. Histologic examination of skin from the living viper revealed hyperplastic, hyperkeratotic, and crusting epidermitis with intralesional fungal elements. The terrestrial Wagler's vipers were housed in a room with fully aquatic tentacled snakes (Erpeton tentaculatum), among which there had been a history of intermittent skin lesions. Approximately 2 months after the biopsy of the viper, a skin sample was collected from one tentacled snake (TS1) with skin abnormalities and revealed a fungal infection with a similar histologic appearance. Fungal isolates were obtained via culture from the Wagler's viper and TS1 and revealed a novel species, Paranannizziopsis tardicrescens, based on phenotypic characterization and molecular analysis. P. tardicrescens was cultured and identified by DNA sequence analysis 8 months later from a dead tentacled snake in an exhibit in an adjacent hallway and 13 months later from a living rhinoceros snake (Rhynchophis boulengeri) with two focal skin lesions. Antifungal susceptibility testing on three of four cultured isolates demonstrated potent in vitro activity for terbinafine and voriconazole.

Secondary Metabolites from the Fungus Dictyosporium sp. and Their MALT1 Inhibitory Activities.

Bioassay-guided separation of an extract from a Dictyosporium sp. isolate led to the identification of six new compounds, 1-6, together with five known compounds, 7-11. The structures of the new compounds were primarily established by extensive 1D and 2D NMR experiments. The absolute configurations of compounds 3-6 were determined by comparison of their experimental electronic circular dichroism (ECD) spectra with DFT quantum mechanical calculated ECD spectra. Compounds 3-5 possess novel structural scaffolds, and biochemical studies revealed that oxepinochromenones 1 and 7 inhibited the activity of MALT1 protease.

Two new species of Gloniopsis (Hysteriales, Ascomycota) from clinical specimens: Morphological and molecular characterisation.

BACKGROUND: The coelomycetes comprise a wide range of fungal species distributed in at least three different classes of the phylum Ascomycota. These are morphologically characterised by producing their conidia inside of fruiting bodies called pycnidia or acervuli, and only a reduced number of species are able to cause human infections. However, their identification in the clinical laboratory is often difficult, due to their few morphological features or because they remain sterile. MATERIALS AND METHODS: In the present study, three isolates of coelomycetes of clinical origin were phenotypically and molecularly studied, by sequencing the D1-D2 fragment of the 28S nuclear ribosomal RNA (nrRNA) (LSU), the internal transcribed spacer region (ITS1-5.8S-ITS2) and a fragment of the translation elongation factor 1-alpha (tef1) genes. RESULTS AND CONCLUSIONS: As result of the molecular analysis, the isolates were identified as belonging to the genus Gloniopsis (order Hysteriales, Dothideomycetes) but without the characteristics of any of the species described so far. Therefore, we propose the new species Gloniopsis percutanea and Gloniopsis pneumoniae. Furthermore, this study revealed that some isolates from clinical specimens identified previously as Rhytidhysteron spp. were misidentified, and considering the few studies in the order Hysteriales and the scarce number of sequences of phylogenetic markers, future revisions of this order should be performed to clarify their taxonomy and obtain a better identification from isolates involved in human mycoses.

DNA sequencing to clarify the taxonomical conundrum of the clinical coelomycetes.

The taxonomy of the fungi that produce human infections and that develop asexual fruiting bodies in culture has become very complex. Recent molecular studies have produced dramatic changes in their classification. Currently, the coelomycetes traditionally included in Sphaeropsidales and Melanconiales are in fact distributed across at least three different classes of the Phylum Ascomycota. Approximately 1000 genera and 7000 species have been grouped in the classes Dothideomycetes, Leotiomycetes and Sordariomycetes and their proper identification can only be made by analysing their DNA sequences and comparing them with those corresponding to type strains available in the adequate databases. To facilitate this task for scientists and clinicians involved in the study of these complex, and every day more numerous taxa, we have updated the knowledge about the taxonomy of the commonest coelomycetes of clinical interest with the aim of improving their identification and antifungal treatment.

Coelomycetous Fungi in the Clinical Setting: Morphological Convergence and Cryptic Diversity.

Human infections by coelomycetous fungi are becoming more frequent and range from superficial to systemic dissemination. Traumatic implantation of contaminated plant material is the most common cause. The typical morphological feature of these fungi is the production of asexual spores (conidia) within fruiting bodies called conidiomata. This study aimed to determine the distribution of the coelomycetes in clinical samples by a phenotypic and molecular study of a large set of isolates received from a U.S. reference mycological institution and by obtaining the in vitro antifungal susceptibility pattern of nine antifungals against a selected group of isolates. A total of 230 isolates were identified by sequencing the D1 and D2 domains of the large subunit (LSU) nuclear ribosomal RNA (nrRNA) gene and by morphological characterization. Eleven orders of the phylum Ascomycota were identified: Pleosporales (the largest group; 66.1%), Botryosphaeriales (19.57%), Glomerellales (4.35%), Diaporthales (3.48%), Xylariales (2.17%), Hysteriales and Valsariales (0.87%), and Capnodiales, Helotiales, Hypocreales and Magnaporthales (0.43% each). The most prevalent species were Neoscytalidium dimidiatum, Paraconiothyrium spp., Phoma herbarum, Didymella heteroderae, and Epicoccum sorghinum The most common anatomical site of isolation was superficial tissue (66.5%), followed by the respiratory tract (17.4%). Most of the isolates tested were susceptible to the majority of antifungals, and only flucytosine showed poor antifungal activity.

New Species Spiromastigoides albida from a Lung Biopsy.

The new species Spiromastigoides albida (Onygenales, Eurotiomycetes, Ascomycota), from a lung biopsy in USA, is proposed and described based on morphological data and the analysis of rRNA, and fragments of actin and ß-tubulin gene sequences. This species is characterized by white colonies and a malbranchea-like asexual morph with profusely branching curved conidiophores forming sporodochia-like structures. Moreover, new combinations for Gymnoascus alatosporus, and for some new species recently described under the generic name Spiromastix, are provided.

A re-evaluation of the genus Myceliophthora (Sordariales, Ascomycota): its segregation into four genera and description of Corynascus fumimontanus sp. nov.

Based on a number of isolates of Myceliophthora (Chaetomiaceae, Sordariales, Ascomycota) recently isolated from soil samples collected in USA, the taxonomy of the genus was re-evaluated through phylogenetic analyses of sequences from the nuc rDNA internal transcribed spacer region and genes for the second largest subunit of RNA polymerase II and translation elongation factor 1α. Members of Myceliophthora were split into four monophyletic clades strongly supported by molecular and phenotypic data. Such clades correspond with Myceliophthora, now restricted only to the type species of the genus Corynascus, which is re-established with five species, the new monotypic genus Crassicarpon and also the new genus Thermothelomyces (comprising four species). Myceliophthora lutea is mesophilic and a permanently asexual morph compared to the members of the other three mentioned genera, which also are able to sexually reproduce morphs with experimentally proven links to their asexual morphs. The asexual morph of M. lutea is characterized by broadly ellipsoidal, smooth-walled conidia with a wide, truncate base. Crassicarpon thermophilum is thermophilic and heterothallic and produces spherical to cuneiform, smooth-walled conidia and cleistothecial ascomata of smooth-walled, angular cells and ascospores with a germ pore at each end. Corynascus spp. are homothallic and mesophilic and produce spherical, mostly ornamented conidia and cleistothecial ascomata with textura epidermoidea composed of ornamented wall cells, and ascospores with one germ pore at each end. Thermothelomyces spp. are thermophilic, heterothallic and characterized by similar ascomata and conidia as Corynascus spp., but its ascospores exhibit only a single germ pore. A dichotomous key to distinguish Myceliophthora from the other mentioned genera are provided, as well as dichotomous keys to identify the species of Corynascus and Thermothelomyces. A new species, namely Corynascus fumimontanus, characterized by verrucose ascomatal wall cells and irregularly shaped ascospores, is described and illustrated.

Changing Epidemiology of Mucoralean Fungi: Chronic Cutaneous Infection Caused by Mucor irregularis.

INTRODUCTION: The fungi pertaining to order Mucorales usually cause an acute form of clinical disease called mucormycosis. A primary chronic presentation in an immunocompetent patient is a rare form of mucormycosis. Mucor irregularis is known for causing chronic cutaneous infections geographically confined to Asia, mainly in China. We describe a case of primary chronic cutaneous mucormycosis caused by M. irregularis from a new geographical niche in India, highlighting changing aspects of its epidemiology. CASE PRESENTATION: The patient was a farmer with a history of skin lesions over the lower limb for the past 6 years. The biopsy taken from the lesions showed pauci-septate hyphae with right-angle branching on KOH wet mount as well as special fungal stains. On fungal culture, greyish-white cottony mycelial growth of Mucormycetes was obtained. The strain was finally identified as M. irregularis on macro- and microscopic features on 2 % MEA and DNA sequencing. The antifungal susceptibility was done using EUCAST broth microdilution method and was found to be susceptible to commonly used antifungal agents. The patient was started on oral itraconazole and saturated solution of potassium iodide (SSKI). While undergoing treatment for 2 months, he was lost to follow-up, however, after a year when he recently visited the hospital; the disease got completely healed with no new crops of skin lesions. CONCLUSION: Mucoralean fungi should also be suspected in cases with chronic presentation, in immunocompetent host, as there is emergence of such fungi in new endemic areas, particularly located in Asia. The role of other antifungal agents apart from amphotericin B for the treatment of chronic mucormycosis needs to be explored.

Primary cutaneous mucormycosis produced by the new species Apophysomyces mexicanus.

A case of fungal necrotizing fasciitis that appeared in an immunocompetent Mexican woman after a car accident is described. The patient did not respond to antifungal treatment and died 4 days later. The fungus was molecularly identified as a new species of Apophysomyces, namely, Apophysomyces mexicanus.

In vitro antifungal susceptibility of clinically relevant species belonging to Aspergillus section Flavi.

The in vitro antifungal susceptibility of 77 isolates belonging to different clinically relevant species of Aspergillus section Flavi, including those of different phylogenetic clades of A. flavus, was tested for nine antifungal agents using a microdilution reference method (CLSI, M38-A2). Terbinafine and the echinocandins demonstrated lower MICs/MECs for all species evaluated, followed by posaconazole. Amphotericin B showed MICs ≥ 2 µg/ml for 38 (49.4%) of the 77 isolates tested.

A new endophytic species of Microthecium (Melanosporales, Sordariomycetes, Pezizomycotina, Ascomycota) from Mallorca (Balearic Islands, Spain).

BACKGROUND: The genus Microthecium contains 31 species worldwide distributed. Most of them are saprobic on soil and plant debris, but a few have been reported as mycoparasites on hypocrealean fungi. By contrast, this genus has never been reported as phytopathogenic, nor endophytic. AIMS: To isolate and identify endophytic fungi from Mediterranean herbaceous plants and trees in order to contribute to the knowledge of the hosts and their geographical location. The present work has been focused on the study of endophytic fungi of hawthorn (Crataegus monogyna). METHODS: The following steps were taken: i, isolation of the fungal strain from living stems of C. monogyna; ii, cultural and micro-morphological study, and iii, sequence comparison of different genetic markers by BLAST search with sequences deposited in GenBank. RESULTS: At the present work we describe a new species of the genus, Microthecium pleomorphosporum, isolated from living stems of C. monogyna in Mallorca (Balearic Islands, Spain). This fungus is characterized by the production of non-ostiolate perithecia and two sorts of ascospores (some smooth-walled, others delicately reticulated) bearing a germ pore at each end which are frequently ornamented by a surrounding donut-like structures, and a phialidic asexual morph and bulbils. The morphologically closest related species is Microthecium tenuissimum, which has bigger ascospores and lacks asexual reproduction. Phylogenetically, M pleomorphosporum is close-related to other species of the genus, although no genetic marker that discriminates this new species from other phylogenetically closer ones could be elucidated as a gold standard. CONCLUSIONS: M. pleomorphosporum, order Melanosporales, is reported here as the first endophytic species of C. monogyna.

Aspergillus novoparasiticus: a new clinical species of the section Flavi.

During a survey on the incidence of Aspergillus in clinical environments, we found some interesting isolates that were morphologically similar to Aspergillus parasiticus, but differed in the color of the colonies and in the pattern of their conidial ornamentation. In the present study, those isolates were characterized using a polyphasic approach. A phylogenetic analysis was carried out, based on partial fragments of the acetamidase (amdS) and O-methyltransferase (omtS) genes and the internal transcribed spacer (ITS) region of rDNA. This information was combined with a detailed morphological and physiological study that included aflatoxin production and assimilation profiles of different carbon and nitrogen sources. The phenotypic and genotypic results support the proposal of a new species, Aspergillus novoparasiticus, phylogenetically placed in a distinct sister clade to that of A. parasiticus. The former has lobate-reticulate conidia and does not produce aspergillic acid on AFPA or organic acids on CREA, while A. parasiticus has echinulate conidia and produces aspergillic and organic acids. In addition, this new species, as well as A. parasiticus, produces aflatoxins B1, B2, G1 and G2.

Two new species of Mucor from clinical samples.

Two new species in the order Mucorales, Mucor velutinosus and Mucor ellipsoideus, isolated from human clinical specimens in the USA, are described and illustrated. The former species is similar to Mucor ramosissimus, from which it can be differentiated by its ability to grow at 37°C and produce verrucose sporangiospores. Mucor ellipsoideus is also able to grow and sporulate at 37°C like M. indicus, the nearest phylogenetic species in this study, however, the former has narrow ellipsoidal sporangiospores in contrast to the subglobose to ellipsoidal sporangiospores of M. indicus. Analysis of the sequences of the ITS and the D1-D2 regions of the rRNA genes confirmed the novelty of these species. The in vitro antifungal susceptibility of the new species showed that amphotericin B was active against all isolates and posaconazole and itraconazole showed low activity.

New Xerophilic Species of Penicillium from Soil.

Soil is one of the main reservoirs of fungi. The aim of this study was to study the richness of ascomycetes in a set of soil samples from Mexico and Spain. Fungi were isolated after 2% w/v phenol treatment of samples. In that way, several strains of the genus Penicillium were recovered. A phylogenetic analysis based on internal transcribed spacer (ITS), beta-tubulin (BenA), calmodulin (CaM), and RNA polymerase II subunit 2 gene (rpb2) sequences showed that four of these strains had not been described before. Penicillium melanosporum produces monoverticillate conidiophores and brownish conidia covered by an ornate brown sheath. Penicillium michoacanense and Penicillium siccitolerans produce sclerotia, and their asexual morph is similar to species in the section Aspergilloides (despite all of them pertaining to section Lanata-Divaricata). P. michoacanense differs from P. siccitolerans in having thick-walled peridial cells (thin-walled in P. siccitolerans). Penicillium sexuale differs from Penicillium cryptum in the section Crypta because it does not produce an asexual morph. Its ascostromata have a peridium composed of thick-walled polygonal cells, and its ascospores are broadly lenticular with two equatorial ridges widely separated by a furrow. All four new species are xerophilic. Despite the genus Penicillium containing more than 480 known species, they are rarely reported as xerophilic.

Three New Derivatives of Zopfinol from Pseudorhypophila Mangenotii gen. et comb. nov.

Triangularia mangenotti was analyzed for the production of secondary metabolites, resulting in the isolation of known zopfinol (1) and its new derivatives zopfinol B-C (2-4), the 10-membered lactones 7-O-acetylmultiplolide A (5) and 8-O-acetylmultiplolide A (6), together with sordarin (7), sordarin B (8), and hypoxysordarin (9). The absolute configuration of 1 was elucidated by the synthesis of MPTA-esters. Compound 1 showed antimicrobial activity against the Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus and the fungus Mucor hiemalis. While 4 was weakly antibacterial, 3 showed stronger antibiotic activity against the Gram-positive bacteria and weak antifungal activity against M. hiemalis and Rhodotorula glutinis. We furthermore observed the cytotoxicity of 1, 3 and 4 against the mammalian cell lines KB3.1 and L929. Moreover, the new genus Pseudorhypophila is introduced herein to accommodate Triangularia mangenotii together with several species of Zopfiella-Z. marina, Z. pilifera, and Z. submersa. These taxa formed a well-supported monophyletic clade in the recently introduced family Navicularisporaceae, located far from the type species of the respective original genera, in a phylogram based on the combined dataset sequences of the internal transcribed spacer region (ITS), the nuclear rDNA large subunit (LSU), and fragments of the ribosomal polymerase II subunit 2 (rpb2) and ß-tubulin (tub2) genes. Zopfiella submersa is synonymized with P. marina due to the phylogenetic and morphological similarity. The isolation of zopfinols 1-4 and sordarins 7-9 confirms the potential of this fungal order as producers of bioactive compounds and suggests these compounds as potential chemotaxonomic markers.

A revision of malbranchea-like fungi from clinical specimens in the United States of America reveals unexpected novelty.

The fungi of the order Onygenales can cause important human infections; however, their taxonomy and worldwide occurrence is still little known. We have studied and identified a representative number of clinical fungi belonging to that order from a reference laboratory in the USA. A total of 22 strains isolated from respiratory tract (40%) and human skin and nails (27.2%) showed a malbranchea-like morphology. Six genera were phenotypically and molecularly identified, i.e. Auxarthron/Malbranchea (68.2%), Arachnomyces (9.1%), Spiromastigoides (9.1%), and Currahmyces (4.5%), and two newly proposed genera (4.5% each). Based on the results of the phylogenetic study, we synonymized Auxarthron with Malbranchea, and erected two new genera: Pseudoarthropsis and Pseudomalbranchea. New species proposed are: Arachnomyces bostrychodes, A. graciliformis, Currahmyces sparsispora, Malbranchea gymnoascoides, M. multiseptata, M. stricta, Pseudoarthropsis crassispora, Pseudomalbranchea gemmata, and Spiromastigoides geomycoides, along with a new combination for Malbranchea gypsea. The echinocandins showed the highest in vitro antifungal activity against the studied isolates, followed by terbinafine and posaconazole; in contrast, amphotericin B, fluconazole, itraconazole and 5-fluorocytosine were less active or lacked in vitro activity against these fungi.

Scedosporium spp. from Clinical Setting in Argentina, with the Proposal of the New Pathogenic Species Scedosporium americanum.

Species of the genus Scedosporium (family Microascaceae, phylum Ascomycota) are responsible for a wide range of opportunistic human infections, and have a low susceptibility to most antifungal drugs. It is well known that the pattern of Scedosporium species distribution varies according to geographic region. To assess the diversity of Scedosporium species in Argentina involved in human infections, we carried out a retrospective study reviewing 49 strains from clinical samples sent for diagnosis to the National Clinical Mycology Reference Laboratory between 1985 and 2019. Then, a phenotypic characterization, a phylogenetic study and and in vitro susceptibility test to antifungals were carried out. An analysis of combined nucleotide sequences dataset of the internal transcribed spacer of the ribosomal DNA (ITS) and of a fragment of the ß-tubulin gene (BT2) demonstrated that 92 % of the strains belonged to the species S. boydii, S. apiospermum and S. angustum, all them pertaining to S. apiospermum species complex. However, two strains (4%) were identified as S. aurantiacum, a species never reported in clinical settings in the Americas'. Surprisingly, one of them displayed a polycytella-like conidiogenesis, up to date only reported for S. apiospermum. In addition, the strain DMic 165285 was phylogenetically located far away from the rest of the species, so is proposed as the novel species Scedosporium americanum. On the other hand, from all seven antifungals tested, voriconazole and posaconazole were the most active drugs against Scedosporium spp.