Title: 
Virulence evaluation of Aspergillus fumigatus Clinical Isolates in Galleria mellonella model.

Objectives:
Aspergillus spp. are responsible for causing opportunistic diseases in immunocompromised patients, including Pulmonary Invasive Aspergillosis (PIA), a disease with a high mortality rate characterized by invasion of the lung tissue, inflammation and possible dissemination to other organs. In this study, we analyzed virulence of Aspergillus fumigatus clinical isolates from São Paulo Hospital (HSP, UNIFESP) evaluating the percentage of survival and health index post-infection in Galleria mellonella model.

Methods:
The virulence of 12 strains (2 standard strains and 10 HSP clinical isolates) was evaluated using the experimental model of infection in Galleria mellonella, generating a survival and morbidity curve. For infection, 10 μL of a conidia inoculum
adjusted to 108 CFU/mL was inoculated in the left hemocele of the larvae (2.0 - 2.5 cm in length). The larvae (n = 16) were incubated at 37 ° C and observed daily up to 7 days for survivor count and morbidity assessment by health index post-infection. Post-infection, Galleria mellonella larvae were monitored for the following attributes: activity, cocoon
formation, melanization, and survival.

Results:
A significant statistical difference between the survival of larvae infected with A. fumigatus and the control group (PBS) (p<0.0001). Seven strains (ATCC 16913, 1220, 1238, 1304, 1341, 1343, 1359, and 1392) showed a high virulent profile with 100 % of mortality after 2 or 3 days’ post-infection. The clinical isolates 1213, 1228 and 1232 presented a mild virulent profile. Larvae infected with 1213 succumbed on the sixth day, while the ones infected with 1228 or 1232 presented percentage of survival of 6.25% and 17.65%, respectively (Figure 1A). The survival curve results may be correlated with the morbidity data, wherein A. fumigatus strains with high virulent profile also promoted higher morbidity. The same was observed for 1213, 1228 and 1232 strains (Figure 1B).

Conclusion:
In conclusion, this study demonstrates the differences in the virulence of clinical isolates of Aspergillus fumigatus determined by the analysis of the percentage of survival and health index post-infection. We could divide those strains into two profiles according to their virulence, a high-virulence profile (ATCC 16913, 1220, 1238, 1304, 1341, 1343, 1359 and 1392) and a mild-virulence profile (1213, 1228 and 1232).