Rapid Detection and Quantification of Aspergillus fumigatus in Environmental Air Samples Using Solid-Phase Cytometry

Abstract

Aspergillus fumigatus is an ubiquitous fungus capable of causing severe infections such as aspergilloma, allergic bronchopulmonary aspergillosis, and invasive aspergillosis, especially in immunocompromised patients. Monitoring the number of Aspergillus fumigatus spores in the air is crucial for infection control. In the present study, a novel approach for the quantification of Aspergillus fumigatus, based on solid-phase cytometry (SPC) and immunofluorescent labeling, was developed. The sensitivity and specificity of the assay were confirmed by testing pure cultures. Paecilomyces variotii and Rhizopus stolonifer were codetected but could be excluded on the basis of morphology of the microcolonies. The SPC method has considerable advantages compared to the culture-based method, including its low detection limit (4 cells/m3), its speed (results are obtained within 24 h), and the straightforward microscopic identification of Aspergillus fumigatus. Additionally, comparison of results obtained with both methods demonstrated that they are equally accurate for the quantification of Aspergillus fumigatus in environmental air samples.

Bioaerosols from a Food Waste Composting Plant Affect Human Airway Epithelial Cell Remodeling Genes

“…Vanhee et al . [39] determined the number of A . fumigatus using solid-phase cytometry, and reported a low 5 ± 3 to high 57 ± 38 number of conidia in environmental air samples.…”

Genetic and virulence variation in an environmental population of the opportunistic pathogen Aspergillus fumigatus

“…5). (Spreadbury et al, 1990), restriction enzyme analysis (Denning et al, 1990), amplified fragment length polymorphism (Vos et al, 1995) and sequence-specific DNA primer analysis (Mondon et al, 1997), and more recently microsatellite length polymorphism (Balajee et al, 2008;Bart-Delabesse et al, 1998;de Valk et al, 2007), MLST (Bain et al, 2007) and variable number of short tandem repeat typing (Vanhee et al, 2009 Guinea et al, 2011), as well as to study genotypic diversity of strains in avian disease (Arné et al, 2011;Lair-Fulleringer et al, 2003;Olias et al, 2011). The recent discovery of a functional sexual cycle in A. fumigatus probably accounts for the genetic diversity observed in the population (O'Gorman et al, 2009), and much of this diversity appears to be concentrated in discrete genomic islands within the DNA that encode for proteins involved in heterokaryon incompatibility and cell-wall-associated proteins that may be involved in virulence (Fedorova et al, 2008).…”

Rapid quantification of itraconazole-resistant Aspergillus fumigatus in air

“…In order to rapidly quantify A. fumigatus cells in air samples we developed a method based on SPC and immunofluorescence labelling as an alternative to culturebased methods (Vanhee et al 2009). The protocol included impacting air on a water soluble polyvinyl alcohol film (PVA), dissolution of the polymer, filtration of the sample, microcolony formation at 47°C, immunofluorescence labelling, scanning of the filter membrane with a solid-phase cytometer and microscopic validation.…”

“…In a first part of the present study, this method was adapted to allow the quantification of airborne, itraconazole resistant A. fumigatus. To that end, itraconazole was added to the growth medium during the microcolony formation step and the remainder of the procedure was performed as described previously (Vanhee et al, 2009). In order to determine the optimal concentration of itraconazole that should be added to the medium, the MIC obtained with the CLSI M38-A2 microdilution test for 30 A. fumigatus reference strains was compared with the lowest concentration leading to the absence of microcolony formation.…”