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Journal of Clinical Microbiology, February 2006, p. 340-349, Vol. 44, No. 2
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.2.340-349.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Molecular Identification of Zygomycetes from Culture and Experimentally Infected Tissues

Patrick Schwarz,1 Stéphane Bretagne,1,2 Jean-Charles Gantier,1 Dea Garcia-Hermoso,1 Olivier Lortholary,1,3 Françoise Dromer,1 and Eric Dannaoui1,4*

Unité de Mycologie Moléculaire, Centre National de Référence Mycologie et Antifongiques, CNRS FRE2849, Institut Pasteur, Paris,1 Laboratoire de Parasitologie—Mycologie, AP-HP, Hôpital Henri Mondor, Créteil,2 Université Paris Descartes, Faculté de Médecine, AP-HP, Hôpital Necker-Enfants-Malades, Service des Maladies Infectieuses et Tropicales, Paris,3 Université Paris Descartes, Faculté de Médecine, AP-HP, Hôpital Européen Georges Pompidou, Unité de Parasitologie—Mycologie, Paris, France4

Received 18 July 2005/ Returned for modification 5 October 2005/ Accepted 8 November 2005

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.

* Corresponding author. Mailing address: 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. Phone: 33 1 40 61 32 50. Fax: 33 1 45 68 84 20. E-mail: dannaoui{at}pasteur.fr.

Journal of Clinical Microbiology, February 2006, p. 340-349, Vol. 44, No. 2
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.2.340-349.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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