Exophiala amphawaensis sp. nov., a novel black yeast isolated from the gut of amphipods in Thailand.

The genus Exophiala is polymorphic, able to transition between yeast, hyphal and pseudohyphal forms. Species of the genus Exophiala are ubiquitous fungi that are distributed in various environments around the world. During a survey of fungal diversity in the gut of amphipods (Floresorchestia amphawaensis and undescribed Dogielinotid amphipods) from the Amphawa estuary, Samut Songkhram province, Thailand, five black yeast strains (DMKU-MG01, DMKU-MG07, DMKU-MG08, DMKU-HG10 and DMKU-FG04) were identified as representing a novel taxon on the basis of a combination of morphological and molecular phylogenetic features. The five strains did not produce filamentous hyphae or pseudohyphae. Only budding yeast cells were observed. On the basis of the phenotypic characteristics and the results of molecular analyses of the D1/D2 region of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) region, the five strains were identified as representing a novel species via applied nucleotide pairwise analysis. They differed from the most closely related species Exophiala alcalophiala by 3.54 % nucleotide substitutions (20 nucleotide substitutions in 572 bp) in the D1/D2 domains of the LSU rRNA gene. Moreover, the sequences of the ITS region of the five strains differed from those of the most closely related species E. alcalophiala, by 7.44-9.62 % nucleotide substitutions, and Exophiala halophiala, by 7.2-7.53 % nucleotide substitutions. The results of phylogenetic analyses based on the concatenated sequences of the ITS regions and the D1/D2 domains of the LSU rRNA gene confirmed that the five black yeast strains represented a single novel species of the genus Exophiala. In this study, Exophiala amphawaensis sp. nov. is proposed to accommodate these strains. The holotype is TBRC 15626T and the isotype is PYCC9020. The MycoBank accession number of the novel species is MB 851477.

Doubly Misdiagnosed: Exophiala Masquerading as Squamous Cell Carcinoma and Chromoblastomycosis.

ABSTRACT: Deep cutaneous fungal infections (DCFI) can arise in the setting of skin trauma and immunosuppression. DCFI may be secondary to chromoblastomycosis, which is typically characterized by pseudoepitheliomatous hyperplasia histologically and can be mistaken for squamous cell carcinoma. In addition, "copper penny" spore-like pigmented yeast forms on Grocott's methenamine silver stain can suggest chromoblastomycosis, but this finding is not specific. By contrast, phaeohyphomycosis characteristically exhibits circumscribed pseudocyst or abscess on histopathology, and both yeast and hyphae can be seen. Our case reports a DCFI with pseudoepitheliomatous hyperplasia and "copper penny" yeast forms, ultimately diagnosed as phaeohyphomycosis after isolating Exophiala spinifera on fungal culture.

Retrospective study of phaeohyphomycosis in aquarium-housed fish, with first descriptions of Exophiala lecanii-corni and Neodevriesia cladophorae in fish.

A broadening fish host range is affected by novel and known pigmented fungal pathogens. A review of 2,250 piscine submissions received by the Aquatic Pathology Service, University of Georgia, revealed 47 phaeohyphomycosis cases (2.1%), representing 34 bony and cartilaginous fish species. The majority involved bony fish (45/47, 95.7%) and were predominantly marine (41/47, 87.2%), with only a few freshwater species (4/47, 8.5%). Cartilaginous fish cases included two zebra sharks (Stegostoma fasciatum) (2/47, 4.3%). Northern seahorses (Hippocampus erectus) had the highest incidence overall (7/47, 14.9%). Culture and sequencing of the internal-transcribed spacer region of the rDNA (ITS), large ribosomal subunit gene D1/D2 domains (LSU) and the DNA polymerase II gene (RPB2) were performed for fungal identification when fresh tissue was obtainable. Exophiala, Ochroconis and Neodevriesia spp. were identified, with Exophiala as the most common fungal genus (8/11, 72.7%). Exophiala lecanii-corni and Neodevriesia cladophorae were described for the first time from fish. Microscopically, lesions were characterized by necrosis, granulomatous inflammation and angioinvasion most frequently affecting the skin/fin, skeletal muscle and kidneys. In this study of diverse aquarium-housed fish species, phaeohyphomycosis cases occurred sporadically and in rare outbreaks with variable pathologic presentations, tissue distributions and severities.

Impact of airway Exophiala spp. on children with cystic fibrosis.

INTRODUCTION: Isolation of Exophiala species from sputum samples has become increasingly reported in Cystic Fibrosis (CF). However, the clinical significance of Exophiala spp. with regards to the paediatric CF population is unknown. METHODS: A case control study was undertaken to compare CF children with and without chronic Exophiala spp. in their sputum samples. Demographic and clinical data were collected retrospectively for each case from the date of Exophiala isolation and for 12 months preceding isolation. Each case was compared to three age and year-matched controls. To determine the effect of Exophiala on clinical course, patients were then followed for 12 months post isolation. RESULTS: In total, 27 of 244 eligible paediatric CF patients (11%) isolated Exophiala spp. on more than one occasion. There were no significant differences in the key clinical parameters: spirometry, mean number of intravenous (IV) antibiotic days and body mass index (BMI), between cases and controls (p = 0.91, p = 0.56 and p = 0.63 respectively). A higher proportion of cases isolated Candida spp. (67% vs 21%, p < 0.0001) and Aspergillus fumigatus (37% vs 26%, p = 0.37). There was no clinically significant difference in spirometry, mean number of IV antibiotic days and BMI in cases pre and post Exophiala spp. isolation. Posaconazole was the only drug used that successfully eradicated Exophiala. CONCLUSION: Despite the frequent isolation of Exophiala spp. in this cohort, in most patients it is not associated with significant clinical deterioration. It does however seem to be associated with isolation of other fungi.

Attempted Isolation of Cryptococcus Species and Incidental Isolation of Exophiala dermatitidis from Human Oral Cavities.

Recent molecular studies suggest that Cryptococcus may inhabit the normal human mouth. We attempted to isolate Cryptococcus from 21 adult non-acutely ill patients and 40 volunteer medical and non-medical staff in Southeastern Wisconsin, USA. An upper lip sulcus culture and an oral rinse specimen were inoculated separately onto Staib (birdseed) agar containing chloramphenicol and incubated in gas impermeable zip lock bags at 35 °C. No cryptococci were grown from any of the 122 samples from the 61 subjects. Both specimens from a woman with no risk factors for fungal disease yielded a black yeast at 4 days on Staib agar. This isolate was shown to be Exophiala dermatitidis by colony and microscopic morphology, analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and sequencing through the internal transcribed spacer ribosomal RNA gene. This appears to be a novel isolation of E. dermatitidis from the oral cavity of a generally healthy human.

Orbital abscess caused by Exophiala dermatitidis following posterior subtenon injection of triamcinolone acetonide: a case report and a review of literature related to Exophiala eye infections.

BACKGROUND: Subtenon injection of triamcinolone acetonide (STTA) has been widely adopted in the clinical setting of ophthalmology and its infectious complications are rare. However, orbital abscess following STTA has been reported in seven cases. Furthermore, although eye infections due to Exophiala species are uncommon, there have been 19 cases to date. E. jeanselmei, E. phaeomuriformis, E. werneckii, and E. dermatitidis have been reported to cause human eye infections; however, to the best of our knowledge, orbital abscess caused by E. dermatitidis has not yet been reported. We describe the first documented case of fungal orbital abscess caused by E. dermatitidis following STTA. We also review the related literature of orbital abscess following STTA, as well as eye infections caused by the four Exophiala species. CASE PRESENTATION: The patient was a 69-year-old Japanese woman with diabetic mellitus. She had a macular oedema in her right eye, which occurred secondary to branch retinal vein occlusion. An orbital abscess caused by E. dermatitidis occurred 4 months after the second STTA for the macular oedema, which was successfully treated by a surgical debridement and systemic administration of voriconazole. CONCLUSIONS: Our findings in the patient and from our literature survey caution ophthalmologists to the fact that STTA can cause fungal orbital infections, especially in diabetic patients. Furthermore, surgical treatment is one of the most important risk factors.

In vitro activity of olorofim (F901318) against fungi of the genus, Scedosporium and Rasamsonia as well as against Lomentospora prolificans, Exophiala dermatitidis and azole-resistant Aspergillus fumigatus.

In recent decades, invasive infections caused by fungal pathogens have been reported with increasing frequency. Concurrently, the rates of detected resistance mechanisms against commonly used antifungal agents in fungi are increasing. The need for novel antifungal drugs is thus imminent. In this study, the novel drug olorofim (F901318) was tested for its antifungal activity against the human fungal pathogens Lomentospora prolificans (n = 20), Scedosporium aurantiacum (n = 2), Scedosporium apiospermum (n = 6), Rasamsonia argillacea species complex (n = 23), Exophiala dermatitidis (n = 10) and azole-resistant Aspergillus fumigatus (ARAF) (n = 25) in an in vitro broth microdilution assay according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations. Whilst olorofim was ascertained to be effective against R. argillacea species complex [minimum inhibitory concentrations (MICs) of ≤0.008 mg/L], Scedosporium spp. (MICs of 0.032-0.5 mg/L), L. prolificans (MICs of 0.032-0.5 mg/L) and ARAF (MICs of ≤0.008-0.032 mg/L), the drug had an MIC of >4 mg/L against E. dermatitidis. These data demonstrate the antifungal activity of olorofim against a broad range of filamentous fungal pathogens.

First Report of Environmental Isolation of Exophiala spp. in Malaysia.

The opportunistic pathogen Exophiala dermatitidis has been frequently isolated from tropical regions of the world. However, there is no report of environmental isolation of Exophiala spp. from Malaysia. The information regarding the ecology of this microbe is important for a better understanding of the opportunism. This study aims to conduct a survey of natural distribution of Exophiala spp. in Malaysia. Forty-seven strains of Exophiala-like was isolated by using selective media. These isolates from the fields were molecularly identified based on the ITS region. The biochemical activity of these microbes was tested by conducting various tests, i.e. DNase test, proteinase activity, and urea hydrolysis. Overall, 22 strains of E. dermatitidis were successfully obtained and identified from burnt tree bark, oil dripped soil sample, hot spring biofilm, railway track stones, tar road contaminated with petrol hydrocarbon, drain and deep mud of Sungai Pinang besides the new discovery from pigeon droppings. A single strain of E. heteromorpha was identified from tar road contaminated with petrol hydrocarbon. Genotypes of the isolated E. dermatitidis were identified by the neighbor-joining tree and grouped into Genotype A, A2 and B. The existence of new Genotype A4 was confirmed by a similar cladogram position in both neighbor-joining and maximum likelihood tree. The survival of E. dermatitidis in the hydrocarbon contaminated environment was studied by supplying engine oil and observing the growth pattern. The results of this study suggest that the opportunistic Exophiala spp. was isolated from nutrient limited and harsh conditions in the natural environment.

Clinical relevance of Scedosporium spp. and Exophiala dermatitidis in patients with cystic fibrosis: A nationwide study.

An increased prevalence of various filamentous fungi in sputum samples of patients with cystic fibrosis (CF) has been reported. The clinical significance, however, is mostly unclear. The aim of this study was to investigate the clinical relevance of Scedosporium spp. and Exophiala dermatitidis from sputum samples of patients with CF in the Netherlands. In this cross-sectional study, all CF patients of the Dutch national CF registry who were treated at five of the seven recognized CF centers during a 3-year period were included. We linked clinical data of the national CF registry with the national Dutch filamentous fungal database. We investigated the association between clinical characteristics and a positive sputum sample for Scedosporium spp. and E. dermatitidis, using logistic regression. Positive cultures for fungi were obtained from 3787 sputum samples from 699 of the 1312 patients with CF. Scedosporium spp. was associated with severe genotype, CF-related diabetes, several microorganisms, and inhaled antibiotics. E. dermatitidis was associated with older age, female sex, and Aspergillus spp. CF patients with and without Scedosporium spp. or E. dermatitidis seemed comparable in body mass index and lung function. This study suggests that Scedosporium spp. and E. dermatitidis are probably no major pathogens in CF patients in the Netherlands. Greater understanding of epidemiologic trends, risk factors, and pathogenicity of filamentous fungi in the respiratory tracts of patients with CF is needed.

Identification of opportunistic and nonopportunistic Exophiala species using high resolution melting analysis.

Exophiala is a genus comprising several species of opportunistic black yeasts. Exophiala species identification by morphological, physiological, and biochemical characteristics is challenging because of the low degree of phenotypic differences between species and its polyphyletic nature. We aimed to develop a high-resolution melting (HRM) assay based on the internal transcribed spacer (ITS) region to differentiate between pairs of clinical and environmental Exophiala species. HRM primers were designed based on the conserved ITS region of five Exophiala species (E. dermatitidis, E. phaeomuriformis, E. heteromorpha, E. xenobiotica, and E. crusticola). Environmental and clinical Exophiala isolates representing these five species (n = 109) were analyzed. The HRM assay was optimized using clinical and environmental reference isolates (n = 22), and then the results were compared with those obtained with nonreference isolates of Exophiala (n = 87) using two designed primer sets. The designed HRM assay was based on the normalized melting peak approach and two primer sets, and successfully distinguished between the five Exophiala species. The HRM1 primer set provided sufficient resolution, with a melting temperature (Tm) difference of approximately 2.5°C among the analyzed species and of approximately 1°C between E. dermatitidis and E. phaeomuriformis. HRM typing results were in agreement with those of ITS-sequence typing (100% sensitivity and specificity). The developed HRM assay can be used to ascertain the identity of Exophiala species, which may differ in clinical significance, with high accuracy. Its application to identify species directly in clinical samples and/or environmental niches may be possible in the future.

Isolated leptomeningeal carcinomatosis and possible fungal meningitis as late sequelae of oesophageal adenocarcinoma.

We describe a case of a 67-year-old man with known chronic obstructive pulmonary disease, type 2 diabetes mellitus, hypertension, osteoarthritis, previous history of excess alcohol intake, and oesophagectomy 3 years earlier for T3N0 adenocarcinoma, referred by his general practitioner with confusion, weight loss and several recent falls. CT of the chest, abdomen and pelvis revealed a right middle-lobe pulmonary embolism, while CT of the head revealed a communicating hydrocephalus. Lumbar puncture was performed, and empirical treatment for tuberculous and fungal meningitis was commenced. Unfortunately, he suffered a rapid neurological deterioration with markedly elevated cerebrospinal fluid (CSF) pressures, leading to an external ventricular drain. Cytological analysis of a CSF sample revealed a cellular infiltrate consistent with leptomeningeal carcinomatosis (adenocarcinoma), with the previous oesophageal malignancy the likely primary. He passed away 17 days after hospital admission. Prolonged culture of CSF later produced evidence of two distinct phaeomycotic moulds (Cladosporium sp and Exophiala sp), suggesting that fungal meningitis may also have contributed to the clinical picture.

A Comparison of Isolation Methods for Black Fungi Degrading Aromatic Toxins.

The prevalence of black fungi in the order Chaetothyriales has often been underestimated due to the difficulty of their isolation. In this study, three methods which are often used to isolate black fungi are compared. Enrichment on aromatic hydrocarbon appears effective in inhibiting growth of cosmopolitan microbial species and allows appearance of black fungi. We miniaturized the method for high-throughput purposes. The new procedure saves time, consumes less space and can process multiple samples simultaneously.

A fatal case of Exophiala dermatitidis disseminated infection in an allogenic hematopoietic stem cell transplant recipient during micafungin therapy.

Exophiala dermatitidis is a dematiaceous fungus that is increasingly becoming the cause of fungal infection in immunocompromised patients. However, the risk factors and optimal treatment modality for E. dermatitidis infection are unknown to date. Herein, we present a fatal case of E. dermatitidis infection in an adult patient that developed after allogeneic hematopoietic stem cell transplantation for chronic active Epstein-Barr virus infection. The dematiaceous fungus caused a breakthrough fungemia despite prophylactic administration of micafungin. Although the patient was intensively treated with liposomal-amphotericin B and voriconazole, serum level of beta-D-glucan continuously increased, and the patient eventually died because of cerebral hemorrhage. An autopsy found multiple involvements of the fungal infection at the bilateral lungs, thoracic cavities, diaphragm, and thyroid. To the best of our knowledge, this is the first reported case of E. dermatitidis infection involving these tissues as determined via autopsy. This case highlights the importance of attention for Exophiala infection in immunocompromised individuals in those given antifungal therapy with echinocandins.

Invasive Fungal Infection Caused by Exophiala dermatitidis in a Patient After Lung Transplantation: Case Report and Literature Review.

This report describes a case of invasive Exophiala dermatitidis infection after double lung transplantation in a 76-year-old man. After thoracotomy, the patient's wound showed dehiscence with purulent secretion. The black yeast was isolated from cultures taken from the wound, and species identification was confirmed by sequence analysis of the internal transcribed spacer (ITS-S2) region. The results of the susceptibility testing showed voriconazole as the most active drug. Despite adaptation of the antifungal therapy the clinical condition worsened, and the patient died. In addition, we evaluated the fungicidal activity of antiseptics towards E. dermatitidis and aimed to provide a brief literature review of previously reported infections caused by this rare fungus. To the best of our knowledge, this is the first report of a rapidly progressing invasive fungal infection with E. dermatitidis originating from a colonized wound after lung transplantation.