Species diversity, antifungal susceptibility and phenotypic and genotypic characterisation of Exophiala spp. infecting patients in different medical centres in Brazil.

The Exophiala genus is responsible for many superficial and invasive infections resulting from black fungi. Identification of Exophiala at the species level is based on morphological observations complemented by molecular tests. The aim of this study was to identify 23 clinical isolates of Exophiala spp. and evaluate the antifungal susceptibility to seven different agents. Molecular identification was based on an analysis of ITS region of rDNA using genomic databases. The micromorphology was evaluated by microculture and scanning electron microscopy. The susceptibility tests were performed using the antifungal agents 5-fluorocytosine (5-FC), amphotericin B (AMB), itraconazole (ITC), voriconazole (VRC), posaconazole (PSC), caspofungin (CFG) and terbinafine (TRB). The ITS analysis identified 100% of the following isolates as: E. dermatitidis (8), E. xenobiotica (6), E. bergeri (4), E. oligosperma (3), E. spinifera (1) and E. mesophila (1). The antifungal susceptibility tests showed that the triazoles compounds were in vitro the most active agents against Exophiala. ITS sequencing enabled the accurate identification of the 23 tested isolates. The triazoles, particularly itraconazole and posaconazole, exhibited MIC values lower than AMB, CAS and 5-FC. Although the guidelines do not indicate AMB for treatment against Exophiala spp., this study showed activity for all of the tested species, except E. mesophila.

Chromoblastomycosis.

Chromoblastomycosis (CBM), also known as chromomycosis, is one of the most prevalent implantation fungal infections, being the most common of the gamut of mycoses caused by melanized or brown-pigmented fungi. CBM is mainly a tropical or subtropical disease that may affect individuals with certain risk factors around the world. The following characteristics are associated with this disease: (i) traumatic inoculation by implantation from an environmental source, leading to an initial cutaneous lesion at the inoculation site; (ii) chronic and progressive cutaneous and subcutaneous tissular involvement associated with fibrotic and granulomatous reactions associated with microabscesses and often with tissue proliferation; (iii) a nonprotective T helper type 2 (Th2) immune response with ineffective humoral involvement; and (iv) the presence of muriform (sclerotic) cells embedded in the affected tissue. CBM lesions are clinically polymorphic and are commonly misdiagnosed as various other infectious and noninfectious diseases. In its more severe clinical forms, CBM may cause an incapacity for labor due to fibrotic sequelae and also due to a series of clinical complications, and if not recognized at an early stage, this disease can be refractory to antifungal therapy.

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.

In vitro susceptibility of environmental isolates of Exophiala dermatitidis to five antifungal drugs.

Several dematiaceous fungi frequently isolated from nature are involved in cases of superficial lesions to lethal cerebral infections. Antifungal susceptibility data on environmental and clinical isolates are still sparse despite the advances in testing methods. The objective of this study was to examine the activities of 5-flucytosine, amphotericin B, itraconazole, voriconazole and terbinafine against environmental isolates of Exophiala strains by minimum inhibition concentration (MIC) determination. The strains were obtained from hydrocarbon-contaminated soil, ant cuticle and fungal pellets from the infrabuccal pocket of attine gynes. Broth microdilution assay using M38-A2 reference methodology for the five antifungal drugs and DNA sequencing for fungal identification were applied. Terbinafine was the most active drug against the tested strains. It was observed that amphotericin B was less effective, notably against Exophiala spinifera, also studied. High MICs of 5-flucytosine against Exophiala dermatitidis occurred. This finding highlights the relevance of studies on the antifungal resistance of these potential opportunistic species. Our results also contribute to a future improvement of the standard methods to access the drug efficacy currently applied to black fungi.

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.