An alternative host model of a mixed fungal infection by azole susceptible and resistant Aspergillus spp strains

Aspergillus fumigatus is the most common mold involved in human infections. However, the number of non-fumigatus species able to cause disease is continuously increasing. Among them, Aspergillus lentulus is reported in hematological and cystic fibrosis patients and in those treated with corticosteroids. A. lentulus differs from A. fumigatus in some clinically relevant aspects such as virulence and antifungal susceptibility, showing high MICs to most antifungals. Previous studies proved that A. lentulus was pathogenic in immunocompromised mice, although the course of the infection was delayed compared to A. fumigatus. These differences could explain why A. lentulus is mostly found in mixed infections with A. fumigatus challenging the diagnosis and treatment. We used the alternative model host Galleria mellonella to compare virulence, host interaction, fungal burden and antifungal response when larvae were infected with A. fumigatus or A. lentulus alone, and with a mixture of both species. A. lentulus was pathogenic in G. mellonella but infected larvae did not respond to therapeutic doses of voriconazole. We were able to simultaneously detect A. fumigatus and A. lentulus by a multiplex Nested Real Time PCR (MN-PCR). Comparative analysis of larvae histological sections showed melanization of both species but presented a different pattern of immune response by haemocytes. Analysis of fungal burden and histology showed that A. lentulus survived in the G. mellonella despite the antifungal treatment in single and mixed infections. We conclude that the simultaneous presence of antifungal susceptible and resistant Aspergillus species would likely complicate the management of these infections.