Fungitell assay

Blood cultures were obtained as part of normal clinical practice and processed using a Bactec (BD Diagnostic Systems, Sparks, MD) or BacT/Alert (bioMérieux, Marcy l’Etoile, France) system. After subculturing on Difco Candida bromocresol green (BCG) agar medium, yeast isolates were identified to the species level by matrix-assisted laser desorption ionization-time of flight mass spectrometry (36 (link)). Sera from blood specimens of patients were tested for the presence of β1,3-D-glucan by the Fungitell assay (Associates of Cape Cod Inc., Falmouth, MA; range of the assay from 31.25 to 500 pg/mL), as previously described (37 (link)). The assay was considered positive if the value was ≥80 pg/mL, according to the cutoffs proposed by the manufacturer.

The BDG detection was performed in serum samples using the Fungitell^® assay, which is a commercially available kit (Associates of Cape Cod., East Falmouth, MA, USA). The assay was conducted according to the manufacturer’s protocol. Serum samples were analyzed in duplicate, and the mean value of the duplicates was used for further analysis. The positivity cutoff for the assay was set at ≥80 pg/mL, as per the manufacturer’s instructions. If the BDG results were higher than 523 pg/mL, no further dilutions were carried out.

BG measurements was carried out using the Fungitell^® assay (Associates of Cape Cod, Inc.) as per manufacturer’s instructions[26 ], [27 (link)]. In brief, 5μL of sample was mixed with 20μL pre-treatment buffer (0.125M KOH/6M KCl), in microplate wells, and incubated at 37°C for 10min. Fungitell reagent, reconstituted in 0.1M Tris HCl, pH 7.4, was added to sample and standard curve wells (7.8–500pg/mL, Pachyman). The reactions were read kinetically, at 405nm minus 490nm at 37°C, for 40min. Vmean values (milliabsorbance units/min) were calculated for standards and samples and sample titres interpolated from the standard curve. CV for all assays was <20%. Normal human serum contains low levels of BG, typically 10-40pg/mL[28 (link)]. Levels <60pg/mL are interpreted as negative and between 60-80pg/mL is interpreted as indeterminate. Levels >80pg/mL is interpreted as positive and in at-risk patients is considered a marker of invasive fungal infection[26 ], [29 (link)].

BG testing was performed at MiraVista Diagnostics (Indianapolis, IN) using the Fungitell assay (Associates of Cape Cod Inc., East Falmouth, MA), in accordance with the manufacturer's instructions [21 ]. For the purposes of our study, specimens were tested in duplicate and BG levels were reported numerically (12).