2.2. Aspergillus Detection
Respiratory samples, either bronchial or endotracheal aspirates or bronchoalveolar lavages, were systematic twice weekly and Aspergillus detection was performed using culture and real-time quantitative PCR, but GM was not performed to avoid any risk of lab contamination.
Briefly, respiratory samples were first digested using v/v digestEUR (Eurobio) for 30 min under shaking. Mycological culture were performed after centrifugation of fluidified samples by inoculation of 100–200 µL of pellet on Sabouraud-Chloramphenicol dextrose Agar plates, and incubated for 7 days at 30 °C and 37 °C. Mold identification at genus or species complex level was performed microscopically, and confirmed at species level using MALDI-ToF mass spectrometry (MALDI Biotyper, Bruker France, Marne-la-Vallée, France), after fungal material extraction [9]. Spectrum profiles were then submitted to the Mass Spectrometry Identification (MSI) online database for definitive identification (https://msi.happy-dev.fr/ [10]).
For molecular detection, 200 µL of plain fluidified respiratory sample underwent immediate SARS-CoV-2 inactivation by heating at 56 °C overnight in ATL Lysis buffer (Qiagen, Saint-Quentin Fallavier, France), before DNA extraction using the EZ1 DSP virus kit (Qiagen) on a EZ1 Advanced XL device (Qiagen). Molecular detection of A. fumigatus was done using a 28S rDNA Aspergillus-targeted PCR, as previously published [11,12].
In case of Aspergillus positive culture and/or positive PCR in respiratory samples, additional tests were performed on serum, i.e., detection of GM (Platelia GM Aspergillus, Biorad, Marnes-la-Coquette, France), Aspergillus PCR and detection of anti-Aspergillus antibody by ELISA (Platelia IgG Aspergillus, Biorad) and in-house immunoelectrophoresis. Briefly, Aspergillus PCR was performed on 1 mL of serum extracted using MagNA Pure 24 Total NA Isolation kit (Roche diagnostics, Meylan, France) on a MagNA Pure 24 device (Roche diagnostics), according to manufacturer recommendations. DNA was eluted in a volume of 50 µL.