New possibilities for chromoblastomycosis and phaeohyphomycosis treatment: identification of two compounds from the MMV Pathogen Box® that present synergism with itraconazole

BACKGROUND Black fungi of the Herpotrichiellaceae family are agents of chromoblastomycosis and phaeohyphomycosis. There are few therapeutic options for these infections and it is common to associate antifungal drugs in their treatment. OBJECTIVES To investigate the Medicines for Malaria Venture (MMV) Pathogen Box® for possible compounds presenting synergism with antifungal drugs used to treat black fungal infections. METHODS An initial screening of the Pathogen Box® compounds was performed in combination with itraconazole or terbinafine at sub-inhibitory concentrations against Fonsecaea pedrosoi. Hits were further tested against eight Herpotrichiellaceae using the checkerboard method. FINDINGS No synergism was observed with terbinafine. MMV687273 (SQ109) and MMV688415 showed synergism with itraconazole against F. pedrosoi. Synergism of these compounds was confirmed with some black fungi by the checkerboard method. SQ109 and itraconazole presented synergism for Exophiala dermatitidis, F. pedrosoi, F. monophora and F. nubica, with fungicidal activity for F. pedrosoi and F. monophora. MMV688415 presented synergism with itraconazole only for F. pedrosoi, with fungicidal activity. The synergic compounds had high selectivity index values when combined with itraconazole. MAIN CONCLUSIONS These compounds in combination, particularly SQ109, are promising candidates to treat Fonsecaea spp. and E. dermatitidis infections, which account for most cases of chromoblastomycosis and phaeohyphomycosis.

Curso clínico de micosis producidas por Exophiala xenobiotica en paciente trasplantado renal en el Hospital Universitario de Burgos / Clinical course of micosis produced by exophiala xenobiotica in renal transplanted patient at the University Hospital of Burgos

Resumen Las micosis por Exophiala xenobiotica comprenden un amplio espectro clínico en pacientes inmunosuprimidos, desde infecciones localizadas, hasta diseminadas. Son incluidas como etiología de las feohifomicosis, actualmente consideradas como infecciones fúngicas emergentes en pacientes trasplantados de órgano sólido. Presentamos 2 casos de micosis por Eexophiala xenobiotica en paciente trasplantado renal, una micosis cutánea localizada y una infección sistémica con afectación del sistema nervioso central.

Abstract Mycosis by exophiala xenobiotica comprise a broad clinical spectrum in immunosuppressed patients, from localized to disseminated infections. They are a recognized etiology of phaeohyphomycosis, currently considered as emerging fungal infections in transplanted solid organ recipients. We present 2 cases of mycosis by exophiala xenobiotica in kidney transplant recipients, a localized cutaneous mycosis and a systemic infection with central nervous system involvement.

Effects of photodynamic therapy alone or in combination with antifungal agents on the apoptosis of planktonic and biofilm cells of Exophiala dermatitidis / 中华皮肤科杂志

Objective To evaluate the in vitro effects of photodynamic therapy alone or in combination with antifungal agents on the apoptosis of planktonic and biofilm cells of Exophiala dermatitidis (E.dermatitidis).Methods The planktonic suspensions of E.dermatitidis were prepared,and the biofilms of E.dermatitidis were prepared via a modified 96-well plate-based methods.Planktonic and biofilm cells of E.dermatitidis were separately divided into several groups:antifungal agent groups treated with antifungal agents alone,photodynamic therapy group receiving photodynamic therapy alone,combination groups receiving photodynamic therapy followed by the treatment with antifungal agents,and blank control group receiving no treatment.These antifungal agents included amphotericin B,posaconazole,voriconazole and itraconazole.The concentrations of these antifungal agents were all 1 mg/L,and the treatment with antifungal agents lasted 2 hours.Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to detect the apoptosis of planktonic and biofilm cells of E.dermatitidis in all the groups.Results The antifungal agents and photodynamic therapy both affected the apoptosis of planktonic (both P ＜ 0.001) and biofilm cells (beth P ＜ 0.05) of E.dermatitidis.The apoptosis rates of E.dermatitidis planktonic cells in the control group,amphotericin B group,posaconazole group,voriconazole group and itraconazole group were 11.67％ ± 0.21％,13.30％ ± 1.78％,14.30％ ± 3.61％,14.51％ ± 1.91％and 36.17％ ± 4.00％ respectively.The apoptosis rate of E.dermatitidis planktonic cells was significantly higher in the itraconazole group than in the control group (P ＜ 0.05),but no significant differences were observed between the other 3 antifungal agent groups and control group (all P ＞ 0.05).The photodynamic therapy group also showed a significantly higher apoptosis rate of E.dermatitidis planktonic cells (41.37％ ±7.80％) compared with the control group (P ＜ 0.05).After the treatment with photodynamic therapy combined with amphotericin B,posaconazole,voriconazole or itraconazole,the apoptosis rates of E.dermatitidis planktonic cells were 29.23％ ± 6.71％,37.23％ ± 10.86％,43.57％ ± 6.42％ and 69.87％ ± 3.53％ respectively.Moreover,the photodynamic therapy + voriconazole group and photodynamic therapy + itraconazole group both showed significantly higher apoptosis rates compared with the voriconazole group and itraconazole group respectively (both P ＜ 0.05).The apoptosis rate of E.dermatitidis biofilm cells was significantly higher in the photodynamic therapy group than in the control group (32.00％ ± 0.43％ vs.25.30％ ± 1.31％,P ＜ 0.05),as well as in the photodynamic therapy + amphotericin B than in the amphotericin B group (P ＜ 0.05).Conclusion Photodynamic therapy combined with antifungal agents can markedly promote the apoptosis of planktonic and biofilm cells of E.dermatitidis.

In vitro antifungal activity of four antifungal agents alone or in combination against Exophiala dermatitidis biofilms / 中华皮肤科杂志

Objective To evaluate the in vitro antifungal activity of 4 antifungal agents alone or in combination against Exophiala dermatitidis (E.dermatitidis) biofilms.Methods E.dermatitidis biofilms were prepared by using a modified 96-well plate-based method.The in vitro antifungal activity of amphotericin B,voriconazole,itraconazole and caspofungin alone or in combination against E.dermatitidis biofilms were investigated via the broth microdilution checkerboard technique.Results The sessile minimum inhibitory concentration ranges resulting in 50％ (SMICS0) and 80％ inhibition (SMIC80) of E.dermatitidis biofilms were all ＞ 32 mg/L for itraconazole,voriconazole and caspofungin,and the SMIC50 and SMIC80 ranges of amphotericin B were 1-2 mg/L and 4-8 mg/L respectively.The combination of amphotericin B with voriconazole showed synergistic inhibitory effects against E.dermatitidis biofilms,while the combination of amphotericin B with itraconazole or caspofungin,as well as the combination of voriconazole with caspofungin,revealed no synergistic effects.No antagonistic effect was observed in any of the combinations.Conclusion Amphotericin B appears more active against E.dermatitidis biofilms,and the combination with voriconazole can enhance the anti-biofilm effects against E.dermatitidis biofilms.

In vitro antifungal activity of tacrolimus alone or in combination with itraconazole or terbinafine against Exophiala dermatitidis / 中华皮肤科杂志

Objective To evaluate in vitro antifungal activity of tacrolimus combined with itraconazole or terbinafine against Exophiala dermatitidis (E.dermatitidis).Methods The minimum inhibitory concentrations (MICs) of itraconazole and terbinafine against 12 strains of E.dermatitidis were determined using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution susceptibility method (M38-A2 Document).A broth microdilution checkerboard method was used to evaluate the in vitro antifungal activity of tacrolimus combined with itraconazole or terbinafine against E.dermatitidis.Results The MIC ranges of terbinafine and itraconazole against E.dermatitidis were 0.060-0.125 mg/L and 0.5-1 mg/L,respectively.The combination of tacrolimus with terbinafine showed synergistic inhibitory effects against 5 strains of E.dermatitidis,while the combination of tacrolimus with itraconazole revealed synergistic effects against 10 strains of E.dermatitidis.No antagonism was observed in either of the two combinations.Conclusion In vitro combination of tacrolimus with itraconazole or terbinafine can enhance the antifungal activity of itraconazole or terbinafine against E.dermatitidis.

A Case of Subcutaneous Phaeohyphomycosis Caused by Exophiala oligosperma Showing Multiple Cysts / 대한피부과학회지

Exophiala species are dark pigmented fungi associated with phaeohyphomycosis that exhibit yeast-like or hyphal forms of the fungus in superficial subcutaneous locations or systemic disease. The incidence of subcutaneous tissue infection of Exophiala species has recently increased, particularly in immunocompromised hosts. In Korea, E. jeanselmei, E. dermatitidis, and E. salmonis were isolated from patients with phaeohyphomycosis. However, there have been no case reports of phaeohyphomycosis caused by E. oligosperma. Here we report the first case of phaeohyphomycosis caused by E. oligosperma in Korea. An 82-year-old female with competent immune function presented with multiple subcutaneous masses on the dorsum of the left hand and wrist. A histopathological examination of the skin specimen revealed granulomatous inflammation with fungal hyphae and yeast. Molecular identification was performed using internal transcribed spacer sequences analysis. The sequence showed 100% identity with the E. oligosperma strains. She was treated with oral itraconazole and showed improvement.

Cutaneous phaeohyphomycosis caused by Exophiala dermatitidis: A case report and literature review.

We report a case of cutaneous phaeohyphomycosis caused by Exophiala dermatitidis. An adult male presented with a 1 month history of erythematous swelling and ulcer on the right forearm. E. dermatitidis was identifi ed from the lesion through microscopic examination, in vitro culture, cutaneous biopsy and molecular analysis. He was treated with oral itraconazole (400 mg/day) and showed improvement.

Phaeohyphomycosis in renal transplantation: report of two cases

Abstract Phaeohyphomycosis is an infection caused by a filamentous fungus that contains pigment melanin in its cell wall. We report two cases caused by Exophiala sp. emphasizing the clinical variability of the disease, as well as diagnostic and therapeutic difficulties of this opportunistic infection in immunosuppressed patients (kidney transplant).

A Case of Phaeohyphomycosis from Exophiala Species Mimicking Facial Cutaneous Tumor / 대한의진균학회지

Phaeohyphomycosis is an infection of humans and other animals caused by a number of phaeoid fungi. It is characterized by the development of dark-colored hyphae and other types of fungal elements in invaded tissues. Phaeohyphomycosis caused by Exophiala species is an unusual infection, but it has been reported with increasing frequency as laboratory methods for diagnosis have improved. A 68-year-old woman presented with yellowish round crusted plaque which is similar to actinic keratosis or keratoacanthoma on her right cheek. Histopathologic examination showed mononuclear cell infiltrate and dark-brown hyphae. The nucleotide sequence of internal transcribed spacer-2 was identical to that of Exophiala species. We report a case of phaeohyphomycosis mimicking skin cancer.

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.

Pulmonary phaeohyphomycosis due to Exophiala jeanselmei：a case report and review of literature / 中国感染与化疗杂志

Objective To better understanding the clinical presentations of phaeohyphomycosis,and improve the diagnosis and management of the disease.Methods We reported a case of pulmonary phaeohyphomycosis caused by Exophiala jeanselmei at the First Affiliated Hospital of Guangzhou Medical University in 2008,and reviewed the relevant literature.The clinical,radio-logical and etiological features were summarized based on this case and the other 23 phaeohyphomycosis patients reported in China from January 1995 to August 2013.Results 24 Chinese cases of phaeohyphomycosis have been reported to date,including 15 males and 9 females.The age of these patients ranged from 4 to 76 (mean 40.0±21 .8)years old.Seventeen patients were otherwise healthy.The other 7 patients had complications.Clinical presentations of phaeohyphomycosis vary widely,including cutaneous and subcutaneous infection in 18 cases,pulmonary and central nervous system involvement in two cases each,para-nasal sinus and palpebral conjunctiva infection one case each.The diagnosis of 18 cases were confirmed both microbiologically and histologically.One case was confirmed histologically alone.Five cases were identified microbiologically alone.The samples for culture were collected from skin abscess (1/5 ),pulmonary tissue (2/5 ),and cervical spinal fluid (2/5 ),respectively. Twenty-two strains of causative organisms were identified,7 of which were Exophiala jeanselmei .Twenty-three patients received treatment.They were cured by antifungal agents alone (18)or in conjunction with surgical resection (4 ),or assisted with XD-635AB-based photodynamic laser therapy (1).Specifically,10 pa-tients were cured by itraconazole alone.Conclusions In China, most patients of phaeohyphomycosis have concurrent conditions or have previously received immunosuppressive agents and cor-ticosteroids.Cutaneous and subcutaneous infection were most common,located mainly on limbs,face,chest and abdominal skin.The most frequently isolated pathogen is Exophiala jeanselmei ,followed by Phialophora verrucosa and Exophiala spinifera .Itraconazole therapy would be very effective.Susceptibility testing is very useful in case of refractory infection.

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.

Phaeohyphomycosis: a clinical-epidemiological and diagnostic study of eighteen cases in Rio Grande do Sul, Brazil

The goal of this study was to review 18 cases of phaeohyphomycosis in Rio Grande do Sul. The records of all of the patients with a diagnosis of phaeohyphomycosis between 1995-2010 were reviewed. Twelve of the 18 patients (66.6%) were male. The average age of the patients was 50 years old (range: 16-74 years). Eleven patients (61%) presented with subcutaneous lesions. Seven patients (38.8%) had received a solid organ transplant. In all of the cases, the presence of melanin in the fungal cells was determined by Fontana-Masson staining of tissue sections and documented. Among the 18 patients, a total of 11 different fungal species were isolated. The causative organisms included Exophiala jeanselmei, Alternaria, Curvularia, Cladophialophora and Colletotrichum gloeosporioides. To our knowledge, this review reports the first case of subcutaneous phaeohyphomycosis caused by C. gloeosporioides in a lung transplant patient. The number of reported cases of phaeohyphomycosis has increased in the last decade. In a number of cases, this increased incidence may be primarily attributed to iatrogenic immunodeficiency.

Molecular Phylogenetics of Exophiala Species Isolated from Korea

BACKGROUND: Recently, identification of fungi have been supplemented by molecular tools, such as ribosomal internal transcribed spacer (ITS) sequence analysis. According to these tools, morphological Exophiala species was newly introduced or redefined. OBJECTIVE: This study was designed to investigate the phylogenetics based on ribosomal ITS sequence analysis from clinical Exophiala species isolated in Korea. METHODS: The strains of Exophiala species were 4 clinical isolates of phaeohyphomycosis agents kept in the department of dermatology, Dongguk University Medical Center(DUMC), Gyeongju, Korea. The DNAs of total 5 strains of Exophiala species were extracted by bead-beating method. Polymerase chain reaction of ITS region using the primer pairs ITS1-ITS4, was done and phylogenetic tree contributed from sequences of ITS region from 5 Korean isolates including E. dermatitidis CBS 109154 and comparative related strains deposited in GenBank. RESULTS: The strains of Exophiala species were 3 strains of E. dermatitidis, 1 strain of E. jeanselmei and 1 strain of Exophiala new species. Among the 3 subtypes (type A, B, C) of E. jeanselmei, E. jeanselmei DUMC 9901 belonged to type B. Of the 2 main types of E. dermatitidis (type A, B) and 3 subtypes of E. dermatitidis type A (A0, A1 and A2), two strains (E. dermatitidis CBS 709.95, E. dermatitidis CBS 109154) belonged to A0 subtypes, 1 strain (E. dermatitidis DUMC 9902) A1 subtype, respectively. CONCLUSION: Phylogenetic analysis of ITS region sequence provided useful information not only for new species identification but for the subtyping and origin of Exophiala species.

Subcutaneous Phaeohyphomycosis Caused by Exophiala salmonis

We report a case of subcutaneous infection in a 55-yr-old Korean diabetic patient who presented with a cystic mass of the ankle. Black fungal colonies were observed after culturing on blood and Sabouraud dextrose agar. On microscopic observation, septated ellipsoidal or cylindrical conidia accumulating on an annellide were visualized after staining with lactophenol cotton blue. The organism was identified as Exophiala salmonis by sequencing of the ribosomal DNA internal transcribed spacer region. Phaeohyphomycosis is a heterogeneous group of mycotic infections caused by dematiaceous fungi and is commonly associated with immunocompromised patients. The most common clinical manifestations of subcutaneous lesions are abscesses or cystic masses. To the best of our knowledge, this is the first reported case in Korea of subcutaneous phaeohyphomycosis caused by E. salmonis that was confirmed by molecular analysis and identification of morphological characteristics. This case suggests that E. salmonis infections are no longer restricted to fish.

Infecções causadas por fungos demácios e suas correlações anátomo-clinicas / Infections caused by dematiaceous fungi and their anatomoclinical correlations

O gênero Exophiala é composto por fungos melanizados dimórficos, responsáveis por um espectro de doenças, incluindo feohifomicoses, micetomas, cromoblastomicoses e fungemia. A espécie E. jeanselmei é a predominante nestas infecções, seguida de E. dermatitidis. Este trabalho tem como objetivo relatar quatro casos e discutir aspectos clínicos, histológicos, micológicos e epidemiológicos para o seu diagnóstico.

The Exophiala genus comprises dimorphic melanized fungi responsible for a spectrum of diseases including phaeohyphomycosis, mycetoma, chromoblastomycosis and fungemia. The E. jeanselmei species is predominant in such infections, followed by E. dermatitidis. This paper aims at reporting four cases and at discussing clinical, histologycal, mycologycal and epidemiologycal aspects for its diagnosis.

Phaeohyphomycosis caused by Exophiala spinifera in a patient with systemic lupus erythematosus / 中华皮肤科杂志

A 27-year-old woman who suffered from a 2-year history of systemic lupus erythematosus (SLE) presented with a 6-month history of nodules and ulcer on the right lower extremity. Direct microscopic examination of the pus showed branched and septate hyphae and spores in a chain-like arrangement.Histopathological examination revealed yellowish brown hyphae and spores. Dark green velvety colony grew on Sabouraud dextrose agar (SDA). Slide culture showed branched, septate hyphae and spine-like annellated conidiophores. The isolate was identified as Exophiala spinifera by DNA sequence analysis. The strain was unable to liquefy gelatin, could grow at 25 ℃ to 39 ℃, and was sensitive to itraconazole, amphotericin B and terbinafine. Animal test revealed that the infection induced by Exophiala spinifera in immunocompromised mice was more severe than that in normal controls. Based on the clinical features, histopathological, fungal culture and DNA sequencing results, the patient was diagnosed with systemic lupus erythematosus accompanied by subcutaneous phaeohyphomycosis caused by Exophiala spinifera.

Feoifomicose subcutânea / Subcutaneous phaeohyphomycosis

São apresentadas imagens ilustrativas de um caso de feoifomicose subcutânea causada pela Exophiala jeanselmei num paciente transplantado renal. Breves comentários sobre a doença encontram-se no texto. Ressalta-se a necessidade de essa micose entrar no diagnóstico diferencial de outras dermatoses, inclusive as não infecciosas.

This report shows images of a case of subcutaneous phaeohyphomycosis caused by Exophiala jeanselmei in a patient who has undergone renal transplantation. The paper comments briefly on the disease and emphasizes the need to take this mycosis into account in the differential diagnosis of other dermatoses, including non-infectious dermatoses.

Subcutaneous phaeohyphomycosis due to Exophiala spinifera in an immunocompromised host.

A case of phaeohyphomycosis presenting as multiple subcutaneous abscesses in a young lady with deteriorating liver function was reported here. The lesion started as a solitary abscess in the neck, mimicking tuberculous cold abscess and rapidly involved the face, chest, arms, and legs within six months with ulceration and discharge of thick brownish foul smelling pus. Potassium hydroxide mount of pus from various sites revealed septate dematiaceous hyphae and pseudohyphae. Culture yielded pure growth of Exophiala spinifera. Tissue debridement was done along with initiation of antifungal therapy with ketoconazole. As liver function deteriorated, antifungal therapy was withdrawn after seven days. Patient expired three weeks after admission due to hepatic failure.