Molecular identification of black-grain mycetoma agents

Abstract

Black-grain mycetomas are subcutaneous devastating chronic infections due to several dematiaceous fungi. They are diagnosed mostly in tropical countries. Identification of these fungi with standard mycological procedures is difficult because of their poor or delayed sporulation. The aim of this study was thus to assess the accuracy of molecular identification of these fungi. A total of 54 strains, mostly of clinical origin, were used, including 15 Madurella mycetomatis, 6 Madurella grisea, 12 Leptosphaeria senegalensis, 4 Leptosphaeria tompkinsii, 6 Pyrenochaeta spp., 4 Curvularia lunata, and 7 Exophiala jeanselmei strains. The internal transcribed spacer 1 (ITS1)-5.8S-ITS2 DNA region was amplified by using universal fungal primers and sequenced. Both intra- and interspecies sequence similarities were assessed. Madurella mycetomatis appeared to be a homogeneous species. More intraspecies variations were found for C. lunata and E. jeanselmei, leading, in some instances, to changes in the initial identification. L. senegalensis and L. tompkinsii showed intraspecies similarities of >99%, but similarity between the two species was <88%. Intergenera and interspecies variations were important, with sequence homologies of <81% between genera. In contrast, Pyrenochaeta romeroi and M. grisea appeared to be heterogeneous, with intraspecies similarities of 40 to 100% and 53 to 100%, respectively, which suggest either erroneous identification or the need for taxonomic revision. Epidemiological and therapeutic studies could benefit from a precise identification of the fungi responsible for black-grain mycetoma based not only on phenotypical characteristics but also on ITS sequencing.

FIG. 1.

Schematic representation of the rRNA gene region and positions of primers used in the study. SSU, small subunit; LSU, large subunit.

FIG. 2.

Distance tree of ITS2 rRNA gene sequences of 54 strains belonging to species known as black-grain mycetoma agents. The tree was constructed using the neighbor-joining method with the Phylip software package and visualized with TreeView. Pseudallescheria boydii (GenBank accession number AY228119) was selected as the outgroup. CNRMA, National Reference Center for Mycoses and Antifungals, Pasteur Institute, Paris, France; IP, Pasteur Institute Collection of Fungi, Pasteur Institute, Paris, France; CBS, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands.

FIG. 3.

RFLP analysis of three strains of Leptosphaeria senegalensis (lanes 1 and 2, IP 1155.77; lanes 3 and 4, IP 614.60; lanes 5 and 6, IP 612.60) and three strains of L. tompkinsii (lanes 7 and 8, IP 1151.76; lanes 9 and 10, IP 559.60; lanes 11 and 12, IP 1156.77). After PCR amplification using primers ITS4 and ITS5 (lanes 1, 3, 5, 7, 9, and 11), restriction profiles were generated with the endonuclease SmaI (lanes 2, 4, 6, 8, 10, and 12). M, 100-bp DNA ladder.