The ribosomal Internal Transcribed Spacer (ITS) regions of the two recognized species of Coccidioides were studied using a reference set of strains that had been previously identified with species defining microsatellite polymorphisms. Unambiguous identification of the two species proved to be possible by amplifying and sequencing the ITS region. PCR-reactions are sensitive to amplification conditions requiring their careful optimization. Stable amplification and sequencing was achieved with primers ITS3 and 4, enabling species diagnosis. Alternatively, Restriction Fragment Length Polymorphism (RFLP) of the entire ITS region using an annealing temperature of 52 degrees C with the restriction enzymes BsrI and XcmI can also distinguish the species. Three strains typifying the species, Glenospora meteuropaea, G. metamericana and Geotrichum louisianoideum, were analyzed and found to be conspecific with C. posadasii. Although these species have nomenclatural priority over C. posadasii, the latter will be proposed for conservation as it has been included in the US select agent list. In addition, Coccidioides immitis is neotypified in this report. Results of antifungal susceptibility testing did not reveal differences between the two species.

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The present survey in Austria, Germany and Switzerland continued the survey of cryptococcosis set up by the European Confederation of Medical Mycology (ECMM) in 1997. From 2000 to 2003 77 cases have been reported. An HIV infection is still the most important risk factor (68%). Young HIV+ women from ASIA contributed to the increase of cryptococcosis in females. A total of 129 clinical isolates of both surveys were genotyped by PCR fingerprinting to study the prevalence of different genotypes. The prevalence of Cryptococcus neoformans var. grubii (serotype A) with the genotypes VNA1 and VNA2 was higher in Germany and Austria (74.5%) than in Switzerland (52%), while in Switzerland the Cr. neoformans hybrids AD (26%) and Cr. neoformans var. neoformans (serotype D) (22%) were more prevalent compared with Germany and Austria (8 and 17.5% respectively). Cryptococcus gattii isolates were studied by FT-IR spectroscopy. DNA in the ITS region was sequenced to get further information about Cr. neoformans serotype AD strains and about the geographical origin of the Cr. gattii isolates. The ITS sequence of the serotype AD isolates of the genotypes VNAD1, VNAD2 and VNAD4 is usually identical to serotype A or serotype D respectively. In the three isolates of the genotype VNAD3 a genotype-specific sequence pattern was detected. Two autochthonous infections due to Cr. gattii could indicate that the genotype VGIV with the ITS type 'Asia 2' might be endemic in Europe.

Scedosporium spp. are the second most prevalent filamentous fungi after Aspergillus spp. recovered from cystic fibrosis (CF) patients in various regions of the world. Although invasive infection is uncommon prior to lung transplantation, fungal colonization may be a risk factor for invasive disease with attendant high mortality post-transplantation. Abundant in the environment, Scedosporium aurantiacum has emerged as an important fungal pathogen in a range of clinical settings. To investigate the population genetic structure of S. aurantiacum, a MultiLocus Sequence Typing (MLST) scheme was developed, screening 24 genetic loci for polymorphisms on a tester strain set. The six most polymorphic loci were selected to form the S. aurantiacum MLST scheme: actin (ACT), calmodulin (CAL), elongation factor-1α (EF1α), RNA polymerase subunit II (RPB2), manganese superoxide dismutase (SOD2), and β-tubulin (TUB). Among 188 global clinical, veterinary, and environmental strains, 5 to 18 variable sites per locus were revealed, resulting in 8 to 23 alleles per locus. MLST analysis observed a markedly high genetic diversity, reflected by 159 unique sequence types. Network analysis revealed a separation between Australian and non-Australian strains. Phylogenetic analysis showed two major clusters, indicating correlation with geographic origin. Linkage disequilibrium analysis revealed evidence of recombination. There was no clustering according to the source of the strains: clinical, veterinary, or environmental. The high diversity, especially amongst the Australian strains, suggests that S. aurantiacum may have originated within the Australian continent and was subsequently dispersed to other regions, as shown by the close phylogenetic relationships between some of the Australian sequence types and those found in other parts of the world. The MLST data are accessible at http://mlst.mycologylab.org. This is a joined publication of the ISHAM/ECMM working groups on “Scedosporium/Pseudallescheria Infections” and “Fungal Respiratory Infections in Cystic Fibrosis”.

Among the genus Cunninghamella, so far C. bertholletiae is known to be the only clinically relevant species. Correct identification of C. bertholletiae is not possible with classical methods. PCR and sequencing of the internal transcribed spacer (ITS) region was used to identify seven of nine clinical isolates as C. bertholletiae and two as C. echinulata. Also an isolate of the surrounding area of one patient infected with C. echinulata could be identified as C. echinulata. High homology in the ITS region was found within the isolates of C. bertholletiae. Within the species C. echinulata and C. elegans a differentiation on subspecies level was achieved by an analysis of restriction fragment length polymorphism of the ITS amplicons after incubation with TaqI and HinfI. Similar results were obtained by PCR fingerprinting of the complete DNA with the single microsatellite DNA primers (GTG)5 and (GAC)5. For the first time C. echinulata could be identified as agent of zygomycosis in humans.