Chromoblastomycosis by Exophiala jeanselmei associated with squamous cell carcinoma.

Chromoblastomycosis is a subcutaneous, chronic, granulomatous mycosis that occurs more frequently in tropical and subtropical countries. Herein, we describe a case of a 90-year-old female patient with diagnosis of chromoblastomycosis by Exophiala jeanselmei with a 22-year evolution who developed a squamous cell carcinoma. In the meantime, She underwent two misdiagnoses and an unnecessary operation. This case is also the fifth case of E. jeanselmei caused CBM in history.

Protocol for Genome-Scale Reconstruction and Melanogenesis Analysis of Exophiala dermatitidis.

Exophiala dermatitidis is a polyextremotolerant fungus with a small genome, thus suitable as a model system for melanogenesis and carotenogensis. A genome-scale model, iEde2091, is reconstructed to increase metabolic understanding and used in a shadow price analysis of pigments, as detailed here. Important to this reconstruction is OptFill, a recently developed alternative gap-filling method useful in the holistic and conservative reconstruction of genome-scale models of metabolism, particularly for understudied organisms like E. dermatitidis where gaps in metabolic knowledge are abundant. For complete details on the use and execution of this protocol, please refer to Schroeder and Saha (2020) and Schroeder et al. (2020).

A screening of the MMV Pathogen Box® reveals new potential antifungal drugs against the etiologic agents of chromoblastomycosis.

Chromoblastomycosis (CBM) is a chronic subcutaneous mycosis caused by traumatic implantation of many species of black fungi. Due to the refractoriness of some cases and common recurrence of CBM, a more effective and less time-consuming treatment is mandatory. The aim of this study was to identify compounds with in vitro antifungal activity in the Pathogen Box® compound collection against different CBM agents. Synergism of these compounds with drugs currently used to treat CBM was also assessed. An initial screening of the drugs present in this collection at 1 µM was performed with a Fonsecaea pedrosoi clinical strain according to the EUCAST protocol. The compounds with activity against this fungus were also tested against other seven etiologic agents of CBM (Cladophialophora carrionii, Phialophora verrucosa, Exophiala jeanselmei, Exophiala dermatitidis, Fonsecaea monophora, Fonsecaea nubica, and Rhinocladiella similis) at concentrations ranging from 0.039 to 10 µM. The analysis of potential synergism of these compounds with itraconazole and terbinafine was performed by the checkerboard method. Eight compounds inhibited more than 60% of the F. pedrosoi growth: difenoconazole, bitertanol, iodoquinol, azoxystrobin, MMV688179, MMV021013, trifloxystrobin, and auranofin. Iodoquinol produced the lowest MIC values (1.25-2.5 µM) and MMV688179 showed MICs that were higher than all compounds tested (5 - >10 µM). When auranofin and itraconazole were tested in combination, a synergistic interaction (FICI = 0.37) was observed against the C. carrionii isolate. Toxicity analysis revealed that MMV021013 showed high selectivity indices (SI ≥ 10) against the fungi tested. In summary, auranofin, iodoquinol, and MMV021013 were identified as promising compounds to be tested in CBM models of infection.

The Neurotropic Black Yeast Exophiala dermatitidis Induces Neurocytotoxicity in Neuroblastoma Cells and Progressive Cell Death.

The neurotropic and extremophilic black yeast Exophiala dermatitidis (Herpotrichellaceae) inhabits diverse indoor environments, in particular bathrooms, steam baths, and dishwashers. Here, we show that the selected strain, EXF-10123, is polymorphic, can grow at 37 °C, is able to assimilate aromatic hydrocarbons (toluene, mineral oil, n-hexadecane), and shows abundant growth with selected neurotransmitters (acetylcholine, gamma-aminobutyric acid, glycine, glutamate, and dopamine) as sole carbon sources. We have for the first time demonstrated the effect of E. dermatitidis on neuroblastoma cell model SH-SY5Y. Aqueous and organic extracts of E. dermatitidis biomass reduced SH-SY5Y viability by 51% and 37%, respectively. Melanized extracellular vesicles (EVs) prepared from this strain reduced viability of the SH-SY5Y to 21%, while non-melanized EVs were considerably less neurotoxic (79% viability). We also demonstrated direct interactions of E. dermatitidis with SH-SY5Y by scanning electron and confocal fluorescence microscopy. The observed invasion and penetration of neuroblastoma cells by E. dermatitidis hyphae presumably causes the degradation of most neuroblastoma cells in only three days. This may represent a so far unknown indirect or direct cause for the development of some neurodegenerative diseases such as Alzheimer's.

Exophiala dermatitidis: Key issues of an opportunistic fungal pathogen.

The black yeast Exophiala dermatitidis is an opportunistic pathogen, causing phaeohyphomycosis in immunosuppressed patients, chromoblastomycosis and fatal infections of the central nervous system in otherwise healthy Asian patients. In addition, it is also regularly isolated from respiratory samples from cystic fibrosis patients, with rates varying between 1% and 19%.Melanin, as part of the cell wall of black yeasts, is one major factor known contributing to the pathogenicity of E. dermatitidis and increased resistance against host defense and anti-infective therapeutics. Further virulence factors, e.g. the capability to adhere to surfaces and to form biofilm were reported. A better understanding of the pathogenicity of E. dermatitidis is essential for the development of novel preventive and therapeutic strategies. In this review, the current knowledge of E. dermatitidis prevalence, clinical importance, diagnosis, microbiological characteristics, virulence attributes, susceptibility, and resistances as well as therapeutically strategies are discussed.

Mating type (MAT) locus and possible sexuality of the opportunistic pathogen Exophiala dermatitidis.

Sexual reproduction among the black yeasts is generally limited to environmental saprobic species and is rarely observed among opportunists in humans. To date, a complete sexual cycle has not been observed in Exophiala dermatitidis. In this study, we aimed to gain insight into the reproductive mode of E. dermatitidis by characterizing its mating type (MAT) locus, conducting MAT screening of environmental and clinical isolates, examining the expression of the MAT genes and analyzing the virulence of the isolates of different mating types. Similar to other members of the Pezizomycotina, the E. dermatitidis genome harbors a high mobility group (HMG) domain gene (MAT1-2-1) in the vicinity of the SLA2 and APN2 genes. The MAT loci of 74 E. dermatitidis isolates (11 clinical and 63 environmental) were screened by PCR, and the surrounding region was amplified using long-range PCR. Sequencing of the ∼ 12-kb PCR product of a MAT1-1 isolate revealed an α-box gene (MAT1-1-1). The MAT1-1 idiomorph was 3544-bp long and harbored the MAT1-1-1 and MAT1-1-4 genes. The MAT1-2 idiomorph was longer, 3771-bp, and harbored only the MAT1-2-1 gene. This structure suggests a heterothallic reproduction mode. The distribution of MAT among 74 isolates was ∼ 1:1 with a MAT1-1:MAT1-2 ratio of 35:39. RT-PCR analysis indicated that the MAT genes are transcribed. No significant difference was detected in the virulence of isolates representing different mating types using a Galleria mellonella model (P > 0.05). Collectively, E. dermatitidis is the first opportunistic black yeast in which both MAT idiomorphs have been characterized. The occurrence of isolates bearing both idiomorphs, their approximately equal distribution, and the expression of the MAT genes suggest that E. dermatitidis might reproduce sexually.

Exophiala dermatitidis isolates from various sources: using alternative invertebrate host organisms (Caenorhabditis elegans and Galleria mellonella) to determine virulence.

Exophiala dermatitidis causes chromoblastomycosis, phaeohyphomycosis and fatal infections of the central nervous system of patients with Asian background. It is also found in respiratory secretions from cystic fibrosis (CF) patients. In this study a variety of E. dermatitidis strains (isolates from Asia, environmental and CF) were characterized in their pathogenicity by survival analyzes using two different invertebrate host organisms, Caenorhabditis elegans and Galleria mellonella. Furthermore, the morphological development of hyphal formation was analyzed. E. dermatitidis exhibited pathogenicity in C. elegans. The virulence varied in a strain-dependent manner, but the nematodes were a limited model to study hyphal formation. Analysis of a melanin-deficient mutant (Mel-3) indicates that melanin plays a role during virulence processes in C. elegans. The strains isolated from Asian patients exhibited significantly higher virulence in G. mellonella compared to strains from other sources. Histological analyzes also revealed a higher potential of invasive hyphal growth in strains isolated from Asian patients. Interestingly, no significant difference was found in virulence between the Mel-3 mutant and their wild type counterpart during infection in G. mellonella. In conclusion, invasive hyphal formation of E. dermatitidis was associated with increased virulence. This work is the basis for future studies concerning E. dermatitidis virulence.

Disseminated Exophiala dermatitidis causing septic arthritis and osteomyelitis.

BACKGROUND: Exophiala dermatitidis is a melanized fungus isolated from many environmental sources. Infections caused by Exophiala species are typically seen in immunocompromised hosts and manifest most commonly as cutaneous or subcutaneous disease. Systemic infections are exceedingly rare and associated with significant morbidity and mortality CASE PRESENTATION: A 28-year-old female originally from India presented with fevers, chills, weight loss and increasing back pain. She had a recent diffuse maculopapular rash that resulted in skin biopsy and a tentative diagnosis of sarcoidosis, leading to administration of azathioprine and prednisone. An MRI of her spine revealed a large paraspinal abscess requiring surgical intervention and hardware placement. Cultures from the paraspinal abscess grew a colony of dark pigmented mold. Microscopy of the culture revealed a melanized fungus, identified as Exophiala dermatitidis. Voriconazole was initially utilized, but due to relapse of infection involving the right iliac crest and left proximal humerus, she received a prolonged course of amphotericin B and posaconazole in combination and required 7 separate surgical interventions. Prolonged disease stability following discontinuation of therapy was achieved. CONCLUSIONS: Described is the first identified case of disseminated Exophiala dermatitidis causing osteomyelitis and septic arthritis in a patient on immunosuppressive therapy. A positive outcome was achieved through aggressive surgical intervention and prolonged treatment with broad-spectrum antifungal agents.

Swim bladder mycosis in pretty tetra (Hemigrammus pulcher) caused by Exophiala pisciphila and Phaeophleospora hymenocallidicola, and experimental verification of pathogenicity.

Spontaneous invasive and chronic disseminated mycosis affected Hemigrammus pulcher kept in a public aquarium, and infection was manifested by inappetence, exophthalmia, erratic swimming, eroded scales, anaemia of the gills and abdominal distension. Internally, there was a grossly swollen swim bladder with a thickened wall filled with a dark mass. The body cavities contained a clear, light amber fluid and a swollen intestine which was full of a watery fluid containing small gas bubbles. Histopathology revealed a granulomatous inflammatory response with fungal hyphae in the lumen and wall of the swim bladder, hepatopancreas, spleen and kidneys with signs of nephrohydrosis. Exophiala pisciphila and Phaeophleospora hymenocallidicola were isolated from the swim bladder, abdominal cavity and gastrointestinal tract. The exogenous source of infection was probably the ample wooden decoration and plants inside the aquarium. Koch's postulates were fulfilled by re-isolation of both fungal species from fish artificially infected under laboratory conditions. As P. hymenocallidicola is less capable of defence against phagocytosis, E. pisciphila probably played a major role. Severe clinical manifestations with 100% mortality developed in two fish species infected by E. pisciphila. A significant increase in the plasma levels of amino acids was observed as a result of the activation of proteolysis.

Overview of selected virulence attributes in Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans, Trichophyton rubrum, and Exophiala dermatitidis.

The incidence of fungal diseases has been increasing since 1980, and is associated with excessive morbidity and mortality, particularly among immunosuppressed patients. Of the known 625 pathogenic fungal species, infections caused by the genera Aspergillus, Candida, Cryptococcus, and Trichophyton are responsible for more than 300 million estimated episodes of acute or chronic infections worldwide. In addition, a rather neglected group of opportunistic fungi known as black yeasts and their filamentous relatives cause a wide variety of recalcitrant infections in both immunocompetent and immunosuppressed hosts. This article provides an overview of selected virulence factors that are known to suppress host immunity and enhance the infectivity of these fungi.

Is Marine Dispersion of the Lethargic Crab Disease Possible? Assessing the Tolerance of Exophiala cancerae to a Broad Combination of Salinities, Temperatures, and Exposure Times.

Since 1997, an emergent fungal disease named lethargic crab disease (LCD) has decimated stocks of the edible mangrove land crab Ucides cordatus (Linnaeus, 1763) (Brachyura: Ocypodidae) along the Brazilian coast, threatening the mangrove ecosystem and causing socioeconomic impacts. Evidence from a variety of sources suggests that the black yeast Exophiala cancerae (Herpotrichiellaceae, Chaetothyriales) has been responsible for such epizootic events. Based on the spatiotemporal patterns of the LCD outbreaks, the well-established surface ocean currents, and the range of ecological traits of Exophiala spp., a marine dispersal hypothesis may be proposed. Using in vitro experiments, we tested the survival and growth of E. cancerae CBS 120420 in a broad combination of salinities, temperatures, and exposure times. While variation in salinity did not significantly affect the growth of colony-forming units (CFUs) (P > 0.05), long exposure times visibly influenced an increase in CFUs growth (P < 0.05). However, higher temperature (30 °C) caused a reduction of about 1.2-fold in CFUs growth (P < 0.05). This result suggests that sea surface temperatures either above or below the optimum growth range of E. cancerae could play a key role in the apparent north-south limits in the geographical distribution of LCD outbreaks. In light of our results, we conclude that a fundamental step toward the understanding of LCD epidemiological dynamics should comprise a systematic screening of E. cancerae in estuarine and coastal waters.

Virulence markers of opportunistic black yeast in Exophiala.

The black yeast genus Exophiala is known to cause a wide variety of diseases in severely ill individuals but can also affect immunocompetent individuals. Virulence markers and other physiological parameters were tested in eight clinical and 218 environmental strains, with a specific focus on human-dominated habitats for the latter. Urease and catalase were consistently present in all samples; four strains expressed proteinase and three strains expressed DNase, whereas none of the strains showed phospholipase, haemolysis, or co-haemolysis activities. Biofilm formation was identified in 30 (13.8%) of the environmental isolates, particularly in strains from dishwashers, and was noted in only two (25%) of the clinical strains. These results indicate that virulence factors are inconsistently present in the investigated Exophiala species, suggesting opportunism rather than pathogenicity.

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.

Infrequent Production of Xanthomegnin by Fungal Strains Recovered from Patients with Ocular Mycoses.

Mycotoxins are putative virulence factors of fungi that play an important role in the pathogenesis of fungal infections. Mycotoxin production has been used as a diagnostic marker for the early diagnosis of fungal diseases. Using high-performance liquid chromatography, we investigated whether the fungal strains recovered from eye tissue samples obtained from patients with ocular mycoses produced the mycotoxin xanthomegnin. We tested 62 well-characterized strains of fungi, including Aspergillus spp. (n = 14), Exophiala spp. (n = 9), Fusarium spp. (n = 15), and several molds (n = 24). All isolates were identified to the species level using PCR and DNA sequencing of rRNA genes. We detected xanthomegnin activity (0.02 µg/ml) in one of the three Aspergillus flavus strains. However, we were unable to detect xanthomegnin in any of the other 61 fungal strains. Our result suggests that xanthomegnin production was infrequent in fungal strains recovered from patients with ocular mycoses.

Exophiala angulospora Causes Systemic Mycosis in Atlantic Halibut: a Case Report.

Filamentous black yeasts from the genus Exophiala are ubiquitous, opportunistic pathogens causing both superficial and systemic mycoses in warm- and cold-blooded animals. Infections by black yeasts have been reported relatively frequently in a variety of captive and farmed freshwater and marine fishes. In November 2012, moribund and recently dead, farm-raised Atlantic Halibut Hippoglossus hippoglossus were necropsied to determine the cause of death. Histopathology revealed that three of seven fish were affected by a combination of an ascending trans-ductual granulomatous mycotic nephritis, necrotizing histiocytic encephalitis, and in one fish the addition of a fibrogranulomatous submucosal branchitis. Microbial cultures of kidney using selective mycotic media revealed pure growth of a black-pigmenting septated agent. Application of molecular and phenotypic taxonomy methodologies determined that all three isolates were genetically consistent with Exophiala angulospora. This is the first report of E. angulospora as the causal agent of systemic mycosis in Atlantic Halibut.

Cerebral phaeohyphomycosis by Exophiala dermatitidis.

Cerebral phaeohyphomycosis is a rare and frequently fatal disease. We report a case of cerebral phaeohyphomycosis caused by Exophiala dermatitidis in a young immuno competent male presenting to a tertiary care hospital in Jaipur.

[Phaeomycotic cyst caused by Exophiala xenobiotica in a patient with rheumatoid arthritis and lung cancer].

In black fungal infections, Exophiala species are frequently encountered as causative agents of human mycosis, particularly in immunocompromised patients. Among them, Exophiala jenselmei was previously reported as the most common etiological agent. Advances in molecular taxonomy proved this taxon to be heterogeneous, and led to newly introduced or redefined species. Exophiala xenobiotica is one of the novel species differentiated from E. jenselmei on the basis of molecular phylogeny.Here, we report a case of pheomycotic cyst caused by E. xenobiotica, which was well controlled via drainage and local thermotherapy. A 70-year-old man developed a cystic nodular lesion on the dorsum of his right thumb over the previous 3 months. He had been treated with prednisolone and methotrexate for 4 years for rheumatoid arthritis. The patient also had lung cancer with vertebral bone metastasis. Direct microscopic examination of the greenish pus aspirated from the cyst revealed mycelial elements. Culture of the pus on blood and Sabouraud dextrose agar yielded numerous black colonies multiple times. Histopathological examination of a biopsy specimen showed subcutaneous abscess formation surrounded by granulomatous tissues. Faintly pigmented pseudohyphae were seen within the abscess. The presence of melanin in the fungal cells was determined by Fontana-Masson staining. Initial microscopic examination of the isolate revealed annellidic conidiogenous cells, suggestive of E. jenselmei. This strain was further identified as E. xenobiotica by sequence analysis of the internal transcribed spacer (ITS) region of ribosomal RNA, showing a 100% sequence homology with the strain type.Pheomycotic cysts should be considered on identifying a slowly developing chronic subcutaneous abscess in immunocompromised patients. Sequencing is recommended for accurate species identification of causative pathogens.

Diversidad y polimorfismo en el género Exophiala: manejo de las especies comunes en el laboratorio de baja complejidad / Diversity and polimorphism in the genus Exophiala: handling of common species in the low complexity laboratory

Exophiala es un género fúngico poco común, conocido como integrante de las levaduras negras y sus respectivas formas filamentosas que pertenece al orden Chaetothyriales. Estos hongos son causantes de cromoblastomicosis o diferentes tipos de faeohifomicosis en pacientes sanos o con compromiso inmune (cutáneas, subcutáneas, diseminadas y quistes de faeohifomicosis). Sus miembros son a menudo difíciles de clasificar a nivel de especie solo por métodos fenéticos, debido a su polimorfismo en cultivo y la gran diversidad molecular que exhiben, la cual se confirma en la revisión de la literatura en estos últimos 15 años. El objetivo de este estudio, se basa en la descripción y recopilación de las especies más comunes del género, presente en ambientes diversos y específicos, con especial atención a los principales aportes morfo fisiológicos útiles en su diagnóstico en un laboratorio de baja complejidad, sin olvidar que debido a su alto polimorfismo y la aparente similitud entre especie, el micólogo debe confirmar su hallazgo mediante el envío de su cepa a un laboratorio de referencia internacional para evitar el subdiagnóstico.

Exophiala is an uncommon fungic genus, known as integrant of black yeast and its filamentous relatives that belong to the Chaetothyriales order. This fungi produce cromoblastomicosis or different kinds of phaeohyfomycosis in healthy patients or immunocompromissed patients (cutaneous, subcutaneous, scattered and phaeohyfomycosis’s cysts). Its members are often hard to classify to species by only using phenetics methods, because they present polimorfisms in culture and shows great molecular diversity, wich is confirmed in the review of literature in the last 15 years. The aim of this study is the description and recopilation of the most common species of the genus, that is present in a variety or specific enviroments, with special enphasis onto the principal morfophysiologic contributions that are usefull to diagnose in a low complexity laboratory, given its high polymorfism and similar look between species, the mycologist must confirm its strain by sending it to an international reference laboratory in order to avoid underdiagnosis.

[Cutaneous and subcutaneous phaeohyphomycosis caused by Exophiala jeanselmei after renal transplantation: a case report].

OBJECTIVE: We report a case of cutaneous and subcutaneous phaeohyphomycosis caused by Exophiala jeanselmei after renal transplantation in Guangdong. A 66-year-old man who had a renal transplantation 6 years ago was admitted in October 2011 for the presence of 16 nodules (0.5-1.5 cm) found on his right middle finger, wrist and forearm for 5 months. Microscopic examination of the purulent exudate showed segmented and branched brown mycelium, and tissue biopsy and PAS staining showed fungal hyphae. The isolate was processed for morphological identification and molecular sequence analysis. A black colony was found after culture of the isolate on SDA at 26 degrees Celsius;, and small culture identified the isolate as Exophiala jeanselmei. ITS sequence analysis of the isolate showed a 100% homology with Exophiala jeanselmei. E-test strip was used in drug sensitivity test, and the isolate was sensitive to amphotericin B, voriconazole, itraconazole and fluconazole, but resistant to 5-flucytosine and caspofungin. Good response was obtained with surgical intervention, local injection and systemic antifungal treatment.

Black yeast-like fungi associated with Lethargic Crab Disease (LCD) in the mangrove-land crab, Ucides cordatus (Ocypodidae).

Lethargic Crab Disease (LCD) caused extensive epizootic mortality of the mangrove land crab Ucides cordatus (Brachyura: Ocypodidae) along the Brazilian coast, mainly in the Northeastern region. The disease was named after the symptoms of slow movement of infected crabs. Causative agents were suspected to be two black yeast-like fungi of the family Herpotrichiellaceae (ascomycete order Chaetothyriales), judged by infected tissue biopsies from moribund U. cordatus. The aim of the present study is to prove that two species are involved in the disease: the recently described black yeast Exophiala cancerae, but also a less virulent, hitherto undescribed fonsecaea-like species, introduced here as the novel species Fonsecaea brasiliensis. Strains were identified by ITS rDNA sequencing, and species borderlines were established by multilocus sequencing and AFLP analysis. Fonsecaea brasiliensis proved to be closely related to the pathogenic species Cladophialophora devriesii which originally was isolated from a systemic infection in a human patient. The virulence of F. brasiliensis is lower than that of E. cancerae, as established by artificial inoculation of mangrove crabs.