2. Materials and Methods

2.1. Population of Patients
Forty-five intubated and mechanically ventilated patients hospitalized in a “COVID-19 ICU” of Rennes teaching hospital were screened for this study and benefited from a systematic monitoring to detect Aspergillus.
The hospital’s ethics committee (N 20-56 obtained the 30 April 2020) approved the study. The presence of SARS-CoV-2 in respiratory specimens (nasal and pharyngeal swabs or sputum) was detected by real time reverse transcription-polymerase chain reaction (RT-PCR) methods.
The following data were recorded: age, patient’s preexisting condition (current smoking, diabetes, hypertension, cardiovascular disease, pulmonary disease, and kidney disease), body mass index, ICU length of stay, duration of mechanical ventilation, ventilator-free days at day 28, need for prone position ventilation, and death in the ICU. Initial clinical laboratory workup included a complete blood count and serum biochemical tests. Chest CT scans were performed during the ICU hospitalization. The Simplified Acute Physiology Score (SAPS II) and the Sepsis-Related Organ Failure Assessment (SOFA) score at admission in ICU, at day 7 and 14 days after admission were used to assess severity [7,8].

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.

2.3. Statistical Analysis
Continuous variables were expressed as median (interquartile range, IQR) and compared using the nonparametric Mann–Whitney U or Kruskal–Wallis test. Dunn’s correction tests were performed if multiple comparisons were requested. Qualitative data were compared using Chi-square test. Tests were two-sided with significance set at α less than 0.05.
Concordance between categorical results from diagnostic tests was performed using the percent agreement coefficient and Cohen’s kappa coefficient (κ). When comparing quantitative data, an ANOVA test was performed. All data were analyzed with GraphPad Prism 8.4 (GraphPad Software, La Jolla, CA, USA).