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.

Multicenter Evaluation of the Vitek MS v3.0 System for the Identification of Filamentous Fungi.

Invasive fungal infections are an important cause of morbidity and mortality affecting primarily immunocompromised patients. While fungal identification to the species level is critical to providing appropriate therapy, it can be slow and laborious and often relies on subjective morphological criteria. The use of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry has the potential to speed up and improve the accuracy of identification. In this multicenter study, we evaluated the accuracy of the Vitek MS v3.0 system in identifying 1,601 clinical mold isolates compared to identification by DNA sequence analysis and supported by morphological and phenotypic testing. Among the 1,519 isolates representing organisms in the v3.0 database, 91% (n = 1,387) were correctly identified to the species level. An additional 27 isolates (2%) were correctly identified to the genus level. Fifteen isolates were incorrectly identified, due to either a single incorrect identification (n = 13) or multiple identifications from different genera (n = 2). In those cases, when a single identification was provided that was not correct, the misidentification was within the same genus. The Vitek MS v3.0 was unable to identify 91 (6%) isolates, despite repeat testing. These isolates were distributed among all the genera. When considering all isolates tested, even those that were not represented in the database, the Vitek MS v3.0 provided a single correct identification 98% of the time. These findings demonstrate that the Vitek MS v3.0 system is highly accurate for the identification of common molds encountered in the clinical mycology laboratory.

Species of Aspergillus section Aspergillus from clinical samples in the United States.

The diversity of Aspergillus species in clinical samples is continuously increasing. Species under the former name Eurotium, currently accommodated in section Aspergillus of the genus Aspergillus, are xerophilic fungi widely found in the human environment and able to grow on substrates with low water activity. However, their prevalence in the clinical setting is poorly known. We have studied the presence of these species in a set of clinical samples from the United States using a multilocus sequence analysis based on the internal transcribed spacer (ITS) region of the rRNA, and fragments of the genes ß-tubulin (BenA), calmodulin (CaM), and polymerase II second largest subunit (RPB2). A total of 25 isolates were studied and identified as follows: A. montevidensis (44%), A. chevalieri (36%), A. pseudoglaucus (8%), and A. costiformis (4%). A new species Aspergillus microperforatus is also proposed, which represented 8% of the isolates studied and is characterized by uniseriate conidial heads, subglobose to pyriform vesicles, rough conidia, globose to subglobose cleistothecia, and lenticular and smooth ascospores. The in vitro antifungal activity of eight clinically available antifungals was also determined against these isolates, with the echinocandins and posaconazole having the most potent activity.

Multi-locus sequence typing provides epidemiological insights for diseased sharks infected with fungi belonging to the Fusarium solani species complex.

Fusarium spp. are saprobic moulds that are responsible for severe opportunistic infections in humans and animals. However, we need epidemiological tools to reliably trace the circulation of such fungal strains within medical or veterinary facilities, to recognize environmental contaminations that might lead to infection and to improve our understanding of factors responsible for the onset of outbreaks. In this study, we used molecular genotyping to investigate clustered cases of Fusarium solani species complex (FSSC) infection that occurred in eight Sphyrnidae sharks under managed care at a public aquarium. Genetic relationships between fungal strains were determined by multi-locus sequence typing (MLST) analysis based on DNA sequencing at five loci, followed by comparison with sequences of 50 epidemiologically unrelated FSSC strains. Our genotyping approach revealed that F. keratoplasticum and F. solani haplotype 9x were most commonly isolated. In one case, the infection proved to be with another Hypocrealian rare opportunistic pathogen Metarhizium robertsii. Twice, sharks proved to be infected with FSSC strains with the same MLST sequence type, supporting the hypothesis the hypothesis that common environmental populations of fungi existed for these sharks and would suggest the longtime persistence of the two clonal strains within the environment, perhaps in holding pools and life support systems of the aquarium. This study highlights how molecular tools like MLST can be used to investigate outbreaks of microbiological disease. This work reinforces the need for regular controls of water quality to reduce microbiological contamination due to waterborne microorganisms.

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.

Four new species of Talaromyces from clinical sources.

The genus Talaromyces constitutes an important group of molds with species that are mainly found in soil, indoor environments and food products. Traditionally, it has been considered, together with Eupenicillium, the teleomorphic state of Penicillium. However, the taxonomy of these fungi has changed considerably, and Talaromyces currently includes sexually and asexually reproducing species. In a previous study of the occurrence of penicillium-like fungi from clinical samples in the USA, we used the combined phylogeny of the internal transcribed spacer (ITS) region of the rDNA and ß-tubulin (BenA) gene to identify 31 isolates of Talaromyces, 85 of Penicillium and two of Rasamsonia. However, seven isolates of Talaromyces were assigned to the corresponding sections but not to any particular species. In this study, we have resolved the taxonomy of these isolates through a multilocus sequence analysis of the ITS, fragments of the BenA, calmodulin (CaM), and RNA polymerase II second largest subunit (RPB2) genes, and a detailed phenotypic study. As a result, four new species are described and illustrated, ie Talaromyces alveolaris, T. georgiensis, T. minnesotensis and T. rapidus.

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.

Fungal Keratitis Secondary to Trametes betulina: A Case Report and Review of Literature.

PURPOSE: We report the first case of human infection and keratitis secondary to Trametes betulina, a rare filamentous fungus. METHODS: Clinical examination including external and slit-lamp examination and corneal scrapings with microbiologic evaluation were performed on a patient with chronic allergic conjunctivitis, entropion and a long-standing corneal ulcer resistant to treatment. RESULTS: The culture from the corneal scraping revealed a basidiomycetous fungus which was submitted for identification. DNA extraction with sequencing and analysis of the ITS and D1/D2 regions were performed on the isolate and demonstrated 100% similarity to Lenzites betulina/Trametes betulina. Susceptibility testing demonstrated potent in vitro activity of voriconazole (MIC < 0.03 µg/ml). The patient was treated with voriconazole, and the corneal ulcer and infiltrate resolved. The infection resulted in corneal thinning and a dense central corneal scar. Penetrating keratoplasty was performed 5 months after diagnosis and treatment and revealed stromal scarring without fungal elements. CONCLUSION: This is the first reported case of keratitis caused by Trametes betulina. This organism should be considered in the differential diagnosis for rare filamentous fungal keratitis and its treatment with voriconazole also noted.

[A case of coccidioidomycosis in Turkey imported from the United States of America]. / Türkiye'de Amerika Birlesik Devletleri kaynakli bir koksidiyoidomikoz olgusu.

Coccidioidomycosis caused by Coccidioides immitis or Coccidioides posadasii is a rare infectious disease except in endemic regions. In this report the third documented imported case of coccidioidomycosis in Turkey was presented. A thirty-year-old male patient was admitted to our hospital with fever and purulent drainage from his chest tube. He had worked in Arizona, USA, until 4 months before this presentation. While in Arizona, he experienced cough and hemoptysis and was diagnosed as pulmonary coccidioidomycosis. He was treated with itraconazole for two months and he had no symptoms for 3 years. He then returned to Turkey and 2 months after his return to Turkey, he was admitted to another hospital in Istanbul with dyspnea and diagnosed as hydro-pneumothorax, and pleural fluid obtained from the inserted chest tube was found to be purulent. One gram of BID amoxicillin-clavulanate was given. Physical examination on admission revealed a purulent drainage on the right side chest tube, a temperature of 38.5°C and decreased breath sounds on the right lung. Piperacillin-tazobactam 3 x 4.5 g intravenous and fluconazole 400 mg intravenous once daily were started. Human immunodeficiency virus test was negative. Gram-negative diplococci and rods, gram-positive cocci and septate hyphae were seen in the Gram stain of his pleural fluid. Pleural fluid culture revealed Moraxella catarrhalis after 24 hours incubation and a mold after 72 hours of incubation. Anti-coccidioidal antibodies were found positive in a titer of 1/2. Hydro-pneumothorax, atelectasis and a 3 mm nodules in the right lung were seen in his thorax CT. The patient's pleural fluid and the culture plates were sent to the Public Health Institute of Turkey, Mycology Reference Laboratory (PHIT-MRL), with a clinical suspicion of coccidioidomycosis. The specimen and plates were submitted to the PHIT-MRL Bio Safety Level-3 laboratory for mycological evaluation. The microscopic examination of 15% KOH preparations of pleural fluid specimens revealed septate hyphae which appear to be in the early stages of forming arthroconidia. The pleural fluid culture grew buff-white coloured colonies with aerial hyphae, which were suspected of being a Coccidioides spp. The strain was identified as C.immitis/posadasii by direct microscopy and culture, and subsequently confirmed by the FDA-approved DNA probe. DNA sequence analysis of the ITS and D1/D2 rDNA regions confirmed the isolate to be C.posadasii species [ITS 100% match to GenBank Accession No. AB232901 (630/630 base pair match), and D1/D2 100% match to GenBank Accession No. AB232884 (617/617 base pair match)]. ITS1 and ITS2 barcode analysis also confirmed the species to be C.posadasii, which is the species endemic in Arizona. Susceptibility testing was performed according to Clinical and Laboratory Standards Institute M38-A2 guidelines in the Fungus Testing Laboratory of the University of Texas Health Science Center at San Antonio and minimal inhibitory concentration values were; 0.125 µg/ml for amphotericin B, posaconazole and voriconazole, 0.5 µg/ml for itraconazole and 8 µg/ml for fluconazole. He had decortication of the pleura and was discharged from hospital after six weeks treatment with intravenous fluconazole which was continued orally for one year. Anti-coccidioidal antibodies were negative after two months of treatment. The patient is currently asymptomatic. The presented case is the third case reported from Turkey and provides additional contribution to the existing literature with regard to the appearance of arthroconidium, which is the unusual hyphal form, instead of the expected spherules in the infected tissue.

Veterinary Fusarioses within the United States.

Multilocus DNA sequence data were used to assess the genetic diversity and evolutionary relationships of 67 Fusarium strains from veterinary sources, most of which were from the United States. Molecular phylogenetic analyses revealed that the strains comprised 23 phylogenetically distinct species, all but two of which were previously known to infect humans, distributed among eight species complexes. The majority of the veterinary isolates (47/67 = 70.1%) were nested within the Fusarium solani species complex (FSSC), and these included 8 phylospecies and 33 unique 3-locus sequence types (STs). Three of the FSSC species (Fusarium falciforme, Fusarium keratoplasticum, and Fusarium sp. FSSC 12) accounted for four-fifths of the veterinary strains (38/47) and STs (27/33) within this clade. Most of the F. falciforme strains (12/15) were recovered from equine keratitis infections; however, strains of F. keratoplasticum and Fusarium sp. FSSC 12 were mostly (25/27) isolated from marine vertebrates and invertebrates. Our sampling suggests that the Fusarium incarnatum-equiseti species complex (FIESC), with eight mycoses-associated species, may represent the second most important clade of veterinary relevance within Fusarium Six of the multilocus STs within the FSSC (3+4-eee, 1-b, 12-a, 12-b, 12-f, and 12-h) and one each within the FIESC (1-a) and the Fusarium oxysporum species complex (ST-33) were widespread geographically, including three STs with transoceanic disjunctions. In conclusion, fusaria associated with veterinary mycoses are phylogenetically diverse and typically can only be identified to the species level using DNA sequence data from portions of one or more informative genes.

Identification and Antifungal Susceptibility of Penicillium-Like Fungi from Clinical Samples in the United States.

Penicillium species are some of the most common fungi observed worldwide and have an important economic impact as well as being occasional agents of human and animal mycoses. A total of 118 isolates thought to belong to the genus Penicillium based on morphological features were obtained from the Fungus Testing Laboratory at the University of Texas Health Science Center in San Antonio (United States). The isolates were studied phenotypically using standard growth conditions. Molecular identification was made using two genetic markers, the internal transcribed spacer (ITS) and a fragment of the ß-tubulin gene. In order to assess phylogenetic relationships, maximum likelihood and Bayesian inference assessments were used. Antifungal susceptibility testing was performed according to CLSI document M38-A2 for nine antifungal drugs. The isolates were identified within three genera, i.e., Penicillium, Talaromyces, and Rasamsonia The most frequent species in our study were Penicillium rubens, P. citrinum, and Talaromyces amestolkiae The potent in vitro activity of amphotericin B (AMB) and terbinafine (TRB) and of the echinocandins against Penicillium and Talaromyces species might offer a good therapeutic alternative for the treatment of infections caused by these fungi.

First Detection of TR34 L98H and TR46 Y121F T289A Cyp51 Mutations in Aspergillus fumigatus Isolates in the United States.

Azole resistance in Aspergillus fumigatus is an increasing problem. The TR34 L98H and TR46 Y121F T289A mutations that can occur in patients without previous azole exposure have been reported in Europe, Asia, the Middle East, Africa, and Australia. Here, we report the detection of both the TR34 L98H and TR46 Y121F T289A mutations in confirmed A. fumigatus isolates collected in institutions in the United States. These mutations, other mutations known to cause azole resistance, and azole MICs are reported here.

Ustilago echinata: Infection in a Mixed Martial Artist Following an Open Fracture.

Ustilago, a common fungal parasite of grains, is infrequently isolated as a pathogen in humans. We describe a case of Ustilago echinata infection following an open distal tibia fracture, review the current literature of this genus as a cause of invasive fungal infection in humans, and discuss management issues.

Necrotizing cutaneous mucormycosis after a tornado in Joplin, Missouri, in 2011.

BACKGROUND: Mucormycosis is a fungal infection caused by environmentally acquired molds. We investigated a cluster of cases of cutaneous mucormycosis among persons injured during the May 22, 2011, tornado in Joplin, Missouri. METHODS: We defined a case as a soft-tissue infection in a person injured during the tornado, with evidence of a mucormycete on culture or immunohistochemical testing plus DNA sequencing. We conducted a case-control study by reviewing medical records and conducting interviews with case patients and hospitalized controls. DNA sequencing and whole-genome sequencing were performed on clinical specimens to identify species and assess strain-level differences, respectively. RESULTS: A total of 13 case patients were identified, 5 of whom (38%) died. The patients had a median of 5 wounds (range, 1 to 7); 11 patients (85%) had at least one fracture, 9 (69%) had blunt trauma, and 5 (38%) had penetrating trauma. All case patients had been located in the zone that sustained the most severe damage during the tornado. On multivariate analysis, infection was associated with penetrating trauma (adjusted odds ratio for case patients vs. controls, 8.8; 95% confidence interval [CI], 1.1 to 69.2) and an increased number of wounds (adjusted odds ratio, 2.0 for each additional wound; 95% CI, 1.2 to 3.2). Sequencing of the D1-D2 region of the 28S ribosomal DNA yielded Apophysomyces trapeziformis in all 13 case patients. Whole-genome sequencing showed that the apophysomyces isolates were four separate strains. CONCLUSIONS: We report a cluster of cases of cutaneous mucormycosis among Joplin tornado survivors that were associated with substantial morbidity and mortality. Increased awareness of fungi as a cause of necrotizing soft-tissue infections after a natural disaster is warranted.

Acrophialophora, a poorly known fungus with clinical significance.

Acrophialophora fusispora is an emerging opportunistic fungus capable of causing human infections. The taxonomy of the genus is not yet resolved and, in order to facilitate identification of clinical specimens, we have studied a set of clinical and environmental Acrophialophora isolates by morphological and molecular analyses. This set included the available type strains of Acrophialophora species and similar fungi, some of which were considered by various authors to be synonyms of A. fusispora. Sequence analysis of the large subunit (LSU) and internal transcribed spacer (ITS) regions of the nuclear ribosomal DNA and a fragment of the ß-tubulin (Tub) gene revealed that Acrophialophora belongs in the family Chaetomiaceae and comprises three different species, i.e., A. fusispora, Acrophialophora levis, and Acrophialophora seudatica; the latter was previously included in the genus Ampullifera. The most prevalent species among clinical isolates was A. levis (72.7%), followed by A. fusispora (27.3%), both of which were isolated mostly from respiratory specimens (72.7%), as well as subcutaneous and corneal tissue samples. In general, of the eight antifungal drugs tested, voriconazole had the greatest in vitro activity, while all other agents showed poor in vitro activity against these fungi.

Cladosporium Species Recovered from Clinical Samples in the United States.

Cladosporium species are ubiquitous, saprobic, dematiaceous fungi, only infrequently associated with human and animal opportunistic infections. We have studied a large set of Cladosporium isolates recovered from clinical samples in the United States to ascertain the predominant species there in light of recent taxonomic changes in this genus and to determine whether some could possibly be rare potential pathogens. A total of 92 isolates were identified using phenotypic and molecular methods, which included sequence analysis of the internal transcribed spacer (ITS) region and a fragment of the large subunit (LSU) of the nuclear ribosomal DNA (rDNA), as well as fragments of the translation elongation factor 1 alpha (EF-1α) and actin (Act) genes. The most frequent species was Cladosporium halotolerans (14.8%), followed by C. tenuissimum (10.2%), C. subuliforme (5.7%), and C. pseudocladosporioides (4.5%). However, 39.8% of the isolates did not correspond to any known species and were deemed to comprise at least 17 new lineages for Cladosporium. The most frequent anatomic site of isolation was the respiratory tract (54.5%), followed by superficial (28.4%) and deep tissues and fluids (14.7%). Species of the two recently described cladosporiumlike genera Toxicocladosporium and Penidiella are reported for the first time from clinical samples. In vitro susceptibility testing of 92 isolates against nine antifungal drugs showed a variety of results but high activity overall for the azoles, echinocandins, and terbinafine.

Morphological and Molecular Characterization of Exophiala polymorpha sp. nov. Isolated from Sporotrichoid Lymphocutaneous Lesions in a Patient with Myasthenia Gravis.

Exophiala species are capable of causing cutaneous and subcutaneous infections in immunocompromised patients. An Exophiala isolate was cultured from a biopsy specimen of a lesion on the forearm of a patient with myasthenia gravis. The patient also had lesions on the palm and distal aspects of the hand, which were successfully treated with a long-term course of itraconazole. A detailed morphological and molecular characterization of the isolate was undertaken. Phylogenetic analysis of the internal transcribed spacer region and portions of the ß-tubulin and translation elongation factor 1-alpha genes indicated that the isolate was a novel species closely related to but genetically distinct from species within the Exophiala spinifera clade; the name Exophiala polymorpha sp. nov. is proposed. Morphologically, E. polymorpha most closely resembles E. xenobiotica but it differs in possessing phialides bearing prominent, wide collarettes, and it does not produce chlamydospores.

Pulmonary Fungal Infection Caused by Neoscytalidium dimidiatum.

Neoscytalidium dimidiatum is a mold known to cause onychomycosis and dermatomycosis; however, it is an extremely rare cause of systemic infection. We report a case of pulmonary infection with Neoscytalidium dimidiatum in an immunocompromised patient and discuss in vitro susceptibility data from this case and previous literature.

Combat-Related Pythium aphanidermatum Invasive Wound Infection: Case Report and Discussion of Utility of Molecular Diagnostics.

We describe a 22-year-old soldier with 19% total body surface area burns, polytrauma, and sequence- and culture-confirmed Pythium aphanidermatum wound infection. Antemortem histopathology suggested disseminated Pythium infection, including brain involvement; however, postmortem PCR revealed Cunninghamella elegans, Lichtheimia corymbifera, and Saksenaea vasiformis coinfection. The utility of molecular diagnostics in invasive fungal infections is discussed.

First Human Case of Fungal Keratitis Caused by a Putatively Novel Species of Lophotrichus.

We report an aggressive fungal keratitis caused by a putatively novel species of Lophotrichus in a patient with traumatic injury to the cornea from a dog paw. The organism was isolated from the patient's necrotic cornea, which perforated despite coverage with hourly fortified broad-spectrum topical antibiotic therapy. This report represents the first case of human infection caused by this species.