A phylogenetic study based on sequence analysis of the -tubulin, hydrophobin and calmodulin genes was performed in 19 strains of Aspergillus fumigatus and related species isolated from clinical specimens in Japan. Correlations between detailed morphology and phylogeny were examined.

Although Aspergillus fumigatus is the major agent of invasive aspergillosis, an increasing number of infections are caused by its cryptic species, especially A. lentulus and the A. viridinutans species complex (AVSC). Their identification is clinically relevant because of antifungal drug resistance and refractory infections. Species boundaries in the AVSC are unresolved since most species have uniform morphology and produce interspecific hybrids in vitro. Clinical and environmental strains from six continents (n = 110) were characterized by DNA sequencing of four to six loci. Biological compatibilities were tested within and between major phylogenetic clades, and ascospore morphology was characterised. Species delimitation methods based on the multispecies coalescent model (MSC) supported recognition of ten species including one new species. Four species are confirmed opportunistic pathogens; A. udagawae followed by A. felis and A. pseudoviridinutans are known from opportunistic human infections, while A. felis followed by A. udagawae and A. wyomingensis are agents of feline sino-orbital aspergillosis. Recently described human-pathogenic species A. parafelis and A. pseudofelis are synonymized with A. felis and an epitype is designated for A. udagawae. Intraspecific mating assay showed that only a few of the heterothallic species can readily generate sexual morphs in vitro. Interspecific mating assays revealed that five different species combinations were biologically compatible. Hybrid ascospores had atypical surface ornamentation and significantly different dimensions compared to parental species. This suggests that species limits in the AVSC are maintained by both pre- and post-zygotic barriers and these species display a great potential for rapid adaptation and modulation of virulence. This study highlights that a sufficient number of strains representing genetic diversity within a species is essential for meaningful species boundaries delimitation in cryptic species complexes. MSC-based delimitation methods are robust and suitable tools for evaluation of boundaries between these species.

Cryptic species of , including the species complex, are increasingly reported to be causes of invasive aspergillosis. Their identification is clinically relevant, as these species frequently have intrinsic resistance to common antifungals. We evaluated the susceptibilities of 90 environmental and clinical isolates from the species complex, identified by DNA sequencing of the calmodulin gene, to seven antifungals (voriconazole, posaconazole, itraconazole, amphotericin B, anidulafungin, micafungin, and caspofungin) using the reference European Committee on Antimicrobial Susceptibility Testing (EUCAST) method. The majority of species demonstrated elevated MICs of voriconazole (geometric mean [GM] MIC, 4.46 mg/liter) anditraconazole (GM MIC, 9.85 mg/liter) and had variable susceptibility to amphotericin B (GM MIC, 2.5 mg/liter). Overall, the MICs of posaconazole and the minimum effective concentrations of echinocandins were low. The results obtained by the EUCAST method were compared with the results obtained with Sensititre YeastOne (YO) panels. Overall, there was 67% agreement (95% confidence interval [CI], 62 to 72%) between the results obtained by the EUCAST method and those obtained with YO panels when the results were read at 48 h and 82% agreement (95% CI, 78 to 86%) when the results were read at 72 h. There was a significant difference in agreement between antifungals; agreement was high for amphotericin B, voriconazole, and posaconazole (70 to 86% at 48 h and 88 to 93% at 72 h) but was very low for itraconazole (37% at 48 h and 57% at 72 h). The agreement was also variable between species, with the maximum agreement being observed for isolates (85 and 93% at 48 and 72 h, respectively). Elevated MICs of voriconazole and itraconazole were cross-correlated, but there was no correlation between the other azoles tested.

The incidence of life-threatening fungal infections has steadily increased in immunocompromised hosts such as HIV infected persons and cancer patients. 1) Invasive pulmonary aspergillosis and Pneumocystis carinii pneumonia are a leading cause of deaths in bone marrow transplant recipients and in HIV-infected patients, respectively. Moreover, resistance to the azoles which are the most widely used antifungals today is attracting much attention. Therefore, new antifungal agents are continuously required to overcome the above fungal diseases.

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