Molecular characterization and antifungal susceptibility of Aspergillus spp. isolated from critically ill COVID-19 patients: implications for suspected COVID-19-associated pulmonary aspergillosis (CAPA) in a Brazilian tertiary referral hospital.

COVID-19-associated pulmonary aspergillosis (CAPA) is a severe complication in critically ill patients, but species identification and antifungal resistance data from middle-income countries remain limited. We analyzed 17 Aspergillus isolates from respiratory specimens of 15 critically ill patients with COVID-19 in São Paulo State, Brazil, during 2020-2021. Patients were retrospectively classified according to ECMM/ISHAM criteria as probable CAPA (n = 5), possible CAPA (n = 7), or non-CAPA (n = 3). Complementary reassessment using the 2024 FUNDICU definitions classified patients as probable invasive pulmonary aspergillosis (IPA; n = 3), probable tracheobronchial aspergillosis (TBA; n = 1), or as not meeting these definitions (n = 9); two were ineligible due to classical EORTC/MSGERC host factors (n = 2). Species identification was performed by benA sequencing and MALDI-TOF MS. Susceptibility to amphotericin B (AMB), itraconazole (ITZ), voriconazole (VCZ), and posaconazole (PCZ) was determined according to CLSI M38-A2, and cyp51A and its promoter were analyzed in A. fumigatus. The predominant species were A. fumigatus (n = 7) and A. flavus (n = 5), followed by A. tamarii (n = 2), A. parasiticus, A. terreus, and A. welwitschiae (n = 1 each). All A. fumigatus isolates exhibited non-wild-type phenotypes (NWT) against PCZ, with some also showing reduced susceptibility to ITZ and AMB. Silent cyp51A mutations occurred in three A. fumigatus isolates, while several section Flavi isolates displayed multiazole NWT. These findings highlight diagnostic limitations and emerging azole resistance, supporting susceptibility testing and surveillance.