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The diagnosis of invasive fungal diseases (IFD) in critical care patients (CrCP) is difficult. The study investigated the performance of a set of biomarkers for diagnosis of IFD in a mixed specialty critical care unit (CrCU).A prospective observational study in patients receiving critical care for ≥ 7 days was performed. Serum samples were tested for the presence of: (1-3) - β-d-glucan (BDG), galactomannan (GM), and Aspergillus fumigatus DNA. GM antigen detection was also performed on bronchoalveolar lavage (BAL) samples. The patients were classified using published definitions for IFD and a diagnostic algorithm for invasive pulmonary aspergillosis. Performance parameters of the assays were determined.In patients with proven and probable IFD, the sensitivity, specificity, PPV and NPV of a single positive BDG were 63%, 83%, 65% and 83% respectively. Specificity increased to 86% with 2 consecutive positive results. The mean BDG value of patients with proven and probable IFD was significantly higher compared to those with fungal colonization and no IFD (p value < 0.0001).New diagnostic criteria which incorporate these biomarkers, in particular BDG, and host factors unique to critical care patients should enhance diagnosis of IFD and positively impact antifungal stewardship programs.Invasive fungal disease in critical care leads to longer duration of stay and is associated with increased mortality.Diagnostic criteria which incorporate fungal biomarkers and risk factors unique to critical care patients is needed.Fungal biomarkers such as serum 1–3 beta-d-glucan can contribute to antifungal stewardship programs in critical care.