Analyzing and Differentiating Immune Dysregulation in COVID-19 and Influenza Patients Predisposing to Mold Infections

Patients suffering from respiratory viral infections are susceptible to developing secondary fungal infections. During the SARS-CoV-2 pandemic, COVID-19-associated aspergillosis (CAPA) has been diagnosed in up to 39% of COVID-19 patients admitted to the ICU, resulting in a significant concern for the healthcare system. Therapeutic approaches for CAPA have been mainly influenced by the treatment strategies for influenza-associated aspergillosis (IAPA), although there may be distinct features caused by the underlying viral infection. To enhance patient therapy, it is crucial to evaluate the infection-specific or shared immune alterations that contribute to fungal susceptibility.

Utilizing our previously published whole blood (WB) stimulation assays, we examine the immune response of influenza, COVID-19, or non-infected control patients to the human pathogenic mold Aspergillus fumigatus. Additionally, we identify underlying mechanistic impairments in COVID-19 and influenza patients by stimulating WB with various TLR agonists.

For innate immune cell stimulation, Hirudin anticoagulated WB is incubated with stimuli for 6 hours under constant rotation. T-cell immune responses are evaluated using Lithium-Heparinized whole WB incubated for 24 hours. Plasma from stimulated WB is collected for multiplex cytokine analysis (e.g., IFN-γ, TNF-α, IL-17A, G-CSF), and cells are lysed for subsequent RNA isolation. Gene expression analysis is performed using the NanoString nCounter Host Response Panel with 785 genes. Furthermore, we are performing flow cytometry to phenotype immune cells and to evaluate Aspergillus stimulated T cells, monocytes, and granulocytes. Additionally, we quantify the direct fungicidal activity of PMNs by measuring hyphal length using live-cell imaging.

This enables us to identify generic impairments in host immunity against A. fumigatus among patients suffering from viral infections. This analysis include discerning infection-specific or shared motifs and improved understanding of signaling pathways involved in the mold susceptibility of patients with an underlying SARS-CoV-2 or Influenza infection.