Clinical spectrum of Exophiala infections and a novel Exophiala species, Exophiala hongkongensis

Abstract

Background: <i>Exophiala</i> species are saprophytic fungi which have been isolated from environments rich in hydrocarbons or from hot, humid, and oligotrophic environments. These fungi are considered as dematiaceous moulds; and due to their phenotypic characteristics at the beginning of colony formation, they are also often referred to as ‘black yeasts’, a misnomer which sometimes may mislead the choice of antifungal agents. When the cultures mature, brown hyphae are formed bearing conidiogenous cells referred to as annellides, a typical characteristic of this fungal genus. Although <i>Exophiala</i> species are environmental fungi, they should not be disregarded as contaminants when they are isolated from clinical specimens. These fungi are causative agents of skin and subcutaneous tissue infections and of systemic infections, such as prosthetic valve endocarditis, dialysis-associated peritonitis, and disseminated infections, especially in immunocompromised patients. Unfortunately, <i>Exophiala</i> species can often only be identified to the genus level by phenotypic characterisation. Objectives: The aims of this study were to study the clinical spectrum of <i>Exophiala</i> infections in Queen Mary Hospital, Hong Kong by a polyphasic approach, and to characterise a potentially novel <i>Exophiala</i> species, <i>Exophiala hongkongensis</i> (ex-type strain HKU32<font size=-1>^T</font>). Methods: All <i>Exophiala</i> strains characterised in this study were isolated from patients during a 15-year period (1998-2012) and were retrieved from the collection in the clinical microbiology laboratory at Queen Mary Hospital, Hong Kong. The strains were characterised phenotypically by microscopic examination of fungal structure using the agar block smear preparation method and phylogenetically using the internal transcribed spacer (ITS) region and Rpb1 gene. In addition, a unique strain, HKU32<font size=-1>^T</font>, was further characterised phenotypically by scanning electron microscopy, enzyme activity test using the API-ZYM system, and growth tests on different temperatures and culture media. HKU32<font size=-1>^T</font> was also further phylogenetically characterised using β-tubulin and β-actin genes. All the phylogenetic analyses were performed by the maximum likelihood method using MEGA 5.0.5. Results: Microscopic examination of the young cultures of all the 12 strains showed subspherical, budding, yeast-like cells. Sequencing of the ITS region and partial <i>Rpb1</i> gene showed 11 of the 12 strains were known <i>Exophiala</i> species, including <i>E. oligosperma</i> [n = 3], <i>E. jeanselmei</i> [n = 2], <i>E. lecanii-corni</i> [n = 2], <i>E. bergeri</i> [n = 1], <i>E. cancerae</i> [n = 1], <i>E. dermatitidis</i> [n = 1], and <i>E. xenobiotica</i> [n = 1]). As for HKU32<font size=-1>^T</font>, it displayed unique morphological features and was positive for eight enzymes in the API-ZYM test. Optimal growth was observed at 30°C on potato dextrose agar or at 24°C on cornmeal agar. HKU32<font size=-1>^T</font> also occupied unique phylogenetic positions in all the phylogenetic analyses, with <i>Exophiala nishimurae</i> being the most closely related species. Clinical spectrum of <i>Exophiala</i> infections in Hong Kong included chronic skin infection, colonisation of gastrointestinal tract, continuous ambulatory peritoneal dialysis (CAPD) peritonitis, onychomycosis, pneumonia, tinea pedis, and wrist or finger nodule. Conclusion: <i>Exophiala</i> species could cause a wide range of infections and the most frequent species isolated from patients in Hong Kong was <i>Exophiala oligosperma</i>. <i>Exophiala hongkongensis</i> sp. nov. is proposed to describe the unique strain HKU32<font size=-1>^T</font>.