Association between selection pressure caused by the use of azole fungicides in sawmills and the development of fungal resistance has been described. The aim of this study was to implement an algorithm to assess the presence of Aspergillus section Fumigati resistant strains in sawmills.Eighty-six full-shift inhalable dust samples were collected from eleven industrial sawmills in Norway. Different culture media were used and molecular identification to species level in Aspergillus section Fumigati was done by calmodulin sequencing and TR 34 /L98H and TR 46 /Y121F/ T289A mutations were screened by real-time PCR assay and confirmed by cyp51A sequencing. Six Fumigati isolates were identified as A. fumigatus sensu stricto and two of these grew on azolesupplemented media and were further analyzed by real-time PCR. One was confirmed to be a TR 34 /L98H mutant.The obtained results reinforce the need to assess the presence of A. fumigatus sensu stricto resistant isolates at other workplaces with fungicide pressure.

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dSubmicronic particles released from fungal cultures have been suggested to be additional sources of personal exposure in moldcontaminated buildings. In vitro generation of these particles has been studied with particle counters, eventually supplemented by autofluorescence, that recognize fragments by size and discriminate biotic from abiotic particles. However, the fungal origin of submicronic particles remains unclear. In this study, submicronic fungal particles derived from Aspergillus fumigatus, A. versicolor, and Penicillium chrysogenum cultures grown on agar and gypsum board were aerosolized and enumerated using field emission scanning electron microscopy (FESEM). A novel bioaerosol generator and a fungal spores source strength tester were compared at 12 and 20 liters min ؊1 airflow. The overall median numbers of aerosolized submicronic particles were 2 ؋ 10 5 cm ؊2 , 2.6 ؋ 10 3 cm ؊2 , and 0.9 ؋ 10 3 cm ؊2 for A. fumigatus, A. versicolor, and P. chrysogenum, respectively. A. fumigatus released significantly (P < 0.001) more particles than A. versicolor and P. chrysogenum. The ratios of submicronic fragments to larger particles, regardless of media type, were 1:3, 5:1, and 1:2 for A. fumigatus, A. versicolor, and P. chrysogenum, respectively. Spore fragments identified by the presence of rodlets amounted to 13%, 2%, and 0% of the submicronic particles released from A. fumigatus, A. versicolor, and P. chrysogenum, respectively. Submicronic particles with and without rodlets were also aerosolized from cultures grown on cellophane-covered media, indirectly confirming their fungal origin. Both hyphae and conidia could fragment into submicronic particles and aerosolize in vitro. These findings further highlight the potential contribution of fungal fragments to personal fungal exposure.

Employees at grain elevators and compound feed mills are exposed to large amounts of grain dust during work, frequently leading to airway symptoms and asthma. Although the exposure to grain dust, microorganisms, β-1→3-glucans and endotoxins has been extensively studied, the focus on the mycotoxin content of grain dust has previously been limited to one or few mycotoxins. Our objective was therefore to screen settled grain dust from grain elevators and compound feed mills for fungal metabolites by LC/MS-MS and explore differences between work places, seasons and climatic zones. Seventy fungal metabolites and two bacterial metabolites were detected. Trichothecenes, depsipeptides, ergot alkaloids, and other metabolites from Fusarium, Claviceps, Alternaria, Penicillium, Aspergillus, and other fungi were represented. The prevalence of individual metabolites was highly variable, and the concentration of each metabolite varied considerably between samples. The prevalence and concentration of most metabolites were higher in grain elevators compared to compound feed mills. Differences between seasons and climatic zones were inconclusive. All samples contained multiple mycotoxins, indicating a highly complex pattern of possible inhalational exposure. A mean exposure of 20 ng/m3 of fungal metabolites was estimated, whereas a worst case scenario estimated as much as 10 ?g/m3. Although many of these compounds may be linked to toxicological and immunological effects through experimental or epidemiological studies, it still remains to be determined whether the detected concentrations implicate adverse health outcomes when inhaled.

Mold particles from Aspergillus fumigatus, Penicillium chrysogenum, Aspergillus versicolor, and Stachybotrys chartarum have been linked to respiratory-related diseases. We characterized X-ray-inactivated spores and hyphae fragments from these species by number of particles, morphology, and mycotoxin, β-glucan and protease content/activity. The pro-inflammatory properties of mold particles were examined in human bronchial epithelial cells (BEAS-2B) and THP-1 monocytes and phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1. Spores from P. chrysogenum and S. chartarum contained some hyphae fragments, whereas the other preparations contained either spores or hyphae. Each mold species produced mainly one gelatin-degrading protease that was either of the metallo-or serine type, while one remains unclassified.Mycotoxin levels were generally low. Detectable levels of β-glucans were found mainly in hyphae particle preparations. PMA-differentiated THP-1 macrophages were by far the most sensitive model with effects in the order of 10 ng/cm 2 . Hyphae preparations of A. fumigatus and P. chrysogenum were more potent than respective spore preparations, whereas the opposite seems to be true for A. versicolor and S. chartarum.Hyphae fragments of A. fumigatus, P. chrysogenum, and A. versicolor enhanced the release of metalloprotease (proMMP-9) most markedly. In conclusion, species, growth stage, and characteristics are all important factors for pro-inflammatory potential. K E Y W O R D Sβ-glucans, cytokines, mold particles, morphology, mycotoxins, proteases | BACKGROUNDThere is sufficient evidence from epidemiological studies of associations between indoor dampness/mold and adverse health effects including respiratory symptoms, respiratory infections, and exacerbation of asthma. 1,2 Several kinds of indoor air pollution agents may contribute. Mold has been suggested to be particularly important, as it may not only cause infection and toxic effects, but also trigger allergic and non-allergic inflammatory reactions that may be linked to various respiratory-related diseases. [3][4][5] Quantitative guidelines (thresholds) for acceptable levels of indoor contamination of microorganisms/mold have not been suggested. 1 However, for the work environment where exposure levels can be much higher, a proposal has been made. 6 Species that commonly occur in moist indoor environments into tangled mass of networks known as mycelia. Spores from many species, but not all, are easily aerosolized. Spores from some species, for example, S. chartarum are produced in slimy aggregates which are dispersed by water and may become airborne after secondary dispersion. 6 Furthermore, experimental studies have demonstrated that not only spores but also hyphae fragments can be liberated from fungal cultures. [7][8][9] Recently, an immune-microscopic method for their detection has been described. 10 In addition to direct microscopic quantification, 9 components like ergosterol, 11 polysaccharides such as β(1→3)-glucans 12 and enzymes such as p...

Sawmill workers are exposed to wood dust (a well-known carcinogen), microorganisms, endotoxins, resin acids (diterpenes), and vapours containing terpenes, which may cause skin irritation, allergy, and respiratory symptoms including asthma. The health effects of most of these exposures are poorly understood as most studies measure only wood dust. The present study assessed these exposures in the Norwegian sawmill industry, which processes predominantly spruce and pine. Personal exposures of wood dust, resin acids, endotoxin, fungal spores and fragments, mono-, and sesquiterpenes were measured in 10 departments in 11 saw and planer mills. The geometric mean (GM) and geometric standard deviation (GSD) thoracic exposures were: 0.09 mg m−3 dust (GSD 2.6), 3.0 endotoxin units (EU) m−3 (GSD 4.9), 0.4 × 105 fungal spores m−3 (GSD 4.2), 2 × 105 fungal fragments m−3 (GSD 3.2), and 1560 ng m−3 of resin acids (GSD 5.5). The GM (GSD) inhalable exposures were: 0.72 mg m−3 dust (2.6), 17 EU m−3 (4.3), 0.4 × 105 fungal spores m−3 (3.8), and 7508 ng m−3 (4.4) of resin acids. The overall correlation between the thoracic and inhalable exposure was strong for resin acid (rp = 0.84), but moderate for all other components (rp = 0.34–0.64). The GM (GSD) exposure to monoterpenes and sesquiterpenes were 1105 µg m−3 (7.8) and 40 µg m−3 (3.9), respectively. Although mean exposures were relatively low, the variance was large, with exposures regularly exceeding the recommended occupational exposure limits. The exposures to spores and endotoxins were relatively high in the dry timber departments, but exposures to microbial components and mono-and sesquiterpenes were generally highest in areas where green (undried) timber was handled. Dust and resin acid exposure were highest in the dry areas of the sawmills. Low to moderate correlation between components (rp ranging from 0.02 to 0.65) suggests that investigations of exposure–response associations for these components (both individually and combined) are feasible in future epidemiological studies.

Assessment of exposure to fungi has commonly been limited to fungal spore measurements that have shown associations between fungi and development or exacerbation of different airway diseases. Because large numbers of submicronic fragments can be aerosolized from fungal cultures under laboratory conditions, it has been suggested that fungal exposure is more complex and higher than that commonly revealed by spore measurements. However, the assessment of fungal fragments in complex environmental matrix remain limited due to methodological challenges. With a recently developed immunolabeling method for field emission scanning electron microscope (FESEM), we could assess the complex composition of fungal aerosols present in personal thoracic samples collected from two Norwegian sawmills. We found that large fungal fragments (length >1 µm) dominated the fungal aerosols indicating that the traditional monitoring approach of spores severely underestimate fungal exposure. The composition of fungal aerosols comprised in average 9% submicronic fragments, 62% large fragments, and 29% spores. The average concentrations of large and submicronic fragments (0.2–1 µm) were 3 × 105 and 0.6 × 105 particles m−3, respectively, and correlated weakly with spores (1.4 × 105 particles m−3). The levels of fragments were 2.6 times higher than the average spore concentration that was close to the proposed hazardous level of 105 spores per m3. The season influenced significantly the fungal aerosol concentrations but not the composition. Furthermore, the ratio of spores in the heterogeneous fungal aerosol composition was significantly higher in saw departments as compared to sorting of green timber departments where the fungal fragments were most prevalent. Being the dominating particles of fungal aerosols in sawmills, fungal fragments should be included in exposure-response studies to elucidate their importance for health impairments. Likewise, the use of fungal aerosol composition in such studies should be considered.

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