Platelia aspergillus assay

The viability of infected A549 cells in the presence of voriconazole was evaluated by measuring the cytosolic enzyme lactate dehydrogenase (LDH) released into the culture medium after 24 h of incubation using an LDH cytotoxicity assay kit (Beyotime Institute of Biotechnology, Haimen, Jiangsu, China). The growth of A. fumigatus was quantified by collecting culture supernatants and measuring the levels of galactomannan using the Platelia^® Aspergillus assay (Bio-Rad, Marnes la Coquette, France) according to the manufacturer’s instructions.

GM was measured using the Platelia Aspergillus assay (Bio-Rad Laboratories, Hercules, CA). Absorbance values were used to calculate the ng/mL GM, based on validated standards, so that GM could be analyzed on a continuous basis rather than as a dichotomous variable. This was done to explore results with a broad dynamic range and to maximize sensitivity to changes over time. To define the lower level of quantification (LLOQ), a patient serum sample with low GM value, near the limit of detection, was assayed in duplicate four times and had an optical density index (ODI) value > 0.1; the mean ODI was 0.194, which yielded a GM of 0.426 ng/mL using standard curve. Subtracting 2 standard deviations from this value gave 0.355 ng/mL as the LLOQ. The serum GM ODI (GMI) was determined as part of the post-hoc analysis, with a single value of ≥0.5 considered positive. BDG was measured using the Fungitell assay (Associates of Cape Cod, Cape Cod, MA) within a range from non-detectable (<31 pg/mL) to >500 pg/mL; for values >500 pg/mL, samples were diluted in reagent-grade water and re-tested to obtain accurate results [23 (link)]. A cutoff of 80 pg/mL was used to define positivity [24 (link)].