Platelia aspergillus ag kit

Diagnostic procedures for the diagnosis of IFD routinely used galactomannan enzyme immunoassay in serum, bronchoalveolar lavage (BAL), and cerebrospinal fluid (CSF) besides histology, direct examination and culture on clinical specimens according to standard method [34 (link)]. Briefly, identification of fungi was done using classical phenotypically methods until implementation of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS, Microflex, Bruker Daltonics): yeasts were identified using MALDI Biotyper V1.3 Software (Bruker Daltonics) since April 2013 and filamentous fungi were identified at the species level using an extended reference spectra library [35 (link)] since October 2014. Mucormycosis diagnosis was carried out using PCR on serum in the Mycology Laboratory of Besancon University Hospital [36 (link)]. Serum GM tests were performed twice a week until neutropenia recovery, yielding a total of 2297 collected sera. Galactomannan detection was performed using the Platelia™ Aspergillus Ag kit (Bio-Rad Laboratories, Marnes-la-Coquette, France) according to the manufacturer's instructions. Serum, BAL and CSF were treated under a class 2 biological safety cabinet. Positive GM tests were controlled on the same sample and always verified on another sample. GM assay in BAL were performed in duplicate.

Eight- to ten-week-old female BALB/c mice (Charles River, Senneville, Quebec, Canada) were neutrophil depleted by intraperitoneal injection of 200 μg of anti-Ly6G antibody (clone 1A8, BioXcell) every 48 h, beginning 1 day prior to infection. Mice were then infected intratracheally with 1 × 10⁷A. fumigatus conidia in 50 μl of PBS plus 0.01% (vol/vol) Tween 80 (PBS-T) or with PBS-T alone for uninfected controls. Doxycycline (Sigma) was administered by intraperitoneal injection at doses ranging from 10 to 25 mg/kg by oral gavage (100 mg/kg Doxycycline every 12 h) or by supplementation of drinking water (500 μg/ml Doxycycline and 5% sucrose) and chow (625 mg/kg Doxycycline; Envigo-Teklad) as indicated. Doxycycline-free control mice were given equal volumes of PBS. Serum Doxycycline levels were determined by ultra-high-performance liquid chromatography coupled to mass-spectrometry (uHPLC-MS/MS) analyses at the Drug Discovery Platform of the Research Institute of the McGill University Health Centre (Montreal, Canada). For fungal burden studies, mice were euthanized 36 h postinfection, and their lungs were harvested and homogenized in PBS. The fungal burden was determined by quantification of pulmonary galactomannan (GM) content using a Platelia Aspergillus Ag kit (Bio-Rad) as previously described (55 (link)).