Molecular identification of melanised non-sporulating moulds: a useful tool for studying the epidemiology of phaeohyphomycosis.

Subcutaneous infections caused by melanised fungi have been increasingly reported among transplant patients, and these infections have the potential for blood and visceral dissemination. Some moulds, such as Mycelia sterilia, cannot grow and sporulate on different media, making their identification impossible by conventional methods. The fast and accurate identification of melanised fungi at the species level is important because species may have tropism to different organs and different susceptibilities to antifungal agents. Molecular tools have been reported to be helpful for the species identification of non-sporulating moulds. Our goal was to identify the species of M. sterilia isolates obtained from clinical samples of transplant patients using sequences of ITS and the D1/D2 regions of rDNA. Clinical samples were obtained from eight kidney transplant recipients who developed subcutaneous fungal infections. The diagnosis was confirmed by histopathology and conventional culture. Histopathology showed septated, melanised hyphae, and the cultures identified non-sporulating fungi. Therefore, the DNA from the M. sterilia isolates was subjected to PCR amplification and sequencing of the ITS and D1/D2 regions. Genus/species identification was obtained by comparison with gene banks. We obtained the following identifications: Alternaria sp. (2), Cochliobolus lunatus/Curvularia lunata (2), Cochliobolus hawaiiensis/Bipolaris hawaiiensis (1), Ochroconis sp. (1), Medicocopsis romeroi/Pyrenochaeta romeroi (1) and Nigrograna mackinnonii/Pyrenochaeta mackinnonii (1).