title: Molecular identification of zygomycetes from culture and experimentally infected tissues.
abstract: Mucormycosis is an emerging infection associated with a high mortality rate. Identification of the causative agents remains difficult and time-consuming by standard mycological procedures. In this study, internal transcribed spacer (ITS) sequencing was validated as a reliable technique for identification of Zygomycetes to the species level. Furthermore, species identification directly from infected tissues was evaluated in experimentally infected mice. Fifty-four Zygomycetes strains belonging to 16 species, including the most common pathogenic species of Rhizopus spp., Absidia spp., Mucor spp., and Rhizomucor spp., were used to assess intra- and interspecies variability. Ribosomal DNA including the complete ITS1-5.8S-ITS2 region was amplified with fungal universal primers, sequenced, and compared. Overall, for a given species, sequence similarities between isolates were >98%. In contrast, ITS sequences were very different between species, allowing an accurate identification of Zygomycetes to the species level in most cases. Six species (Rhizopus oryzae, Rhizopus microsporus, Rhizomucor pusillus, Mucor circinelloides, and Mucor indicus) were also used to induce disseminated mucormycosis in mice and to demonstrate that DNA extraction, amplification of fungal DNA, sequencing, and molecular identification were possible directly from frozen tissues.
authors.affiliation: Centre National de Référence Mycologie et Antifongiques, Unité de Mycologie Moléculaire, CNRS FRE2849, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France.
authors.forename: Patrick
authors.initials: P
authors.lastname: Schwarz
authors.forename: Stéphane
authors.initials: S
authors.lastname: Bretagne
authors.forename: Jean-Charles
authors.initials: JC
authors.lastname: Gantier
authors.forename: Dea
authors.initials: D
authors.lastname: Garcia-Hermoso
authors.forename: Olivier
authors.initials: O
authors.lastname: Lortholary
authors.forename: Françoise
authors.initials: F
authors.lastname: Dromer
authors.forename: Eric
authors.initials: E
authors.lastname: Dannaoui
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003470:Culture Media; D004271:DNA, Fungal / Q000032:analysis; D021903:DNA, Ribosomal Spacer / Q000032:analysis; D005658:Fungi / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D051379:Mice; D008969:Molecular Sequence Data; D009090:Mucorales / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D009091:Mucormycosis / Q000382:microbiology*; D016533:Mycological Typing Techniques; D012340:RNA, Ribosomal, 5.8S; D017422:Sequence Analysis, DNA; D013045:Species Specificity
chemical_list: D003470:Culture Media; D004271:DNA, Fungal; D021903:DNA, Ribosomal Spacer; D012340:RNA, Ribosomal, 5.8S