Cytotoxicity of Aspergillus Section Fumigati Isolates Recovered from Protection Devices Used on Waste Sorting Industry.

Safe working conditions must be guaranteed during waste sorting, which is crucial to maximizing recycling and reuse, in order to minimize workers' exposure to chemical and biological hazards. This study determines the contribution of Aspergillus section Fumigati to the overall cytotoxicity of filtering respiratory protection devices (FRPD) and mechanic protection gloves (MPG) collected in 2019 from different workstations in one waste sorting industry in Portugal. The cytotoxicity of 133 Aspergillus section Fumigati isolates was determined as IC50 in human A549 epithelial lung cells and swine kidney cells, using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Aspergillus section Fumigati cytotoxicity results were compared with previous total cytotoxicity data from FRPD and MPG samples. A significant correlation was detected between the total cytotoxicity of samples and cytotoxicity of Aspergillus section Fumigati isolates in A549 cells (rS = -0.339, p = 0.030). The cytotoxicity of Aspergillus section Fumigati isolates explained 10.7% of the total cytotoxicity of the sample. On the basis of the comparison of cytotoxicity levels, it was possible to determine the contribution of Aspergillus section Fumigati isolates for the total cytotoxicity of protection devices used in the waste sorting industry. The results support in vitro toxicology as a relevant approach in risk assessments regarding cytotoxicity in passive sampling, and thus, useful in determining the contribution of relevant microbial contaminants to overall cytotoxicity. This approach can provide valuable answers in dose/response studies, and support innovations in risk characterization and their translation into occupational policies.

Are In Vitro Cytotoxicity Assessments of Environmental Samples Useful for Characterizing the Risk of Exposure to Multiple Contaminants at the Workplace? A Systematic Review.

In some occupational environments risk characterization is challenging or impossible to achieve due to the presence of multiple pollutants and contaminants. Thus, in vitro testing using the most relevant cell lines will provide information concerning health effects due to the co-exposure to multiple stressors. The aim of this review article is to identify studies where the cytotoxicity assessment was performed in environmental samples, as well as to describe the main outputs and challenges regarding risk characterization and management. This study is based on a study of the available information/data on cytotoxicity assessment performed on environmental samples following the PRISMA methodology. Different cell lines were used depending on the environment assessed and exposure routes implicated. The A549 alveolar epithelial cell line was applied in four studies for occupational exposure in the waste sorting industry and for outdoor environments; lymphocytes were used in two studies for occupational and outdoor environments; swine kidney cells were used in three studies performed in the waste industry and hepatocellular/Hep G2 in one study in the waste industry. Cytotoxicity assessments in environmental samples should have a more prominent role due to their contribution for identifying and better understanding the associations between co-exposure to environmental contaminants and adverse human health effects as a prioritization for risk management.

Algorithm to assess the presence of Aspergillus fumigatus resistant strains: The case of Norwegian sawmills.

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 TR34/L98H and TR46/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 azole-supplemented media and were further analyzed by real-time PCR. One was confirmed to be a TR34/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.

Aspergillus Section Fumigati in Firefighter Headquarters.

BACKGROUND: Aspergillus section Fumigati is one of the Aspergillus sections more frequently related to respiratory symptoms and by other health outcomes. This study aimed to characterize Aspergillus section Fumigati distribution in eleven firefighter headquarters (FFHs) to obtain an accurate occupational exposure assessment. METHODS: A sampling approach protocol was performed using active (impaction method) and passive sampling methods (floor surfaces swabs, electrostatic dust collectors (EDCs), and settled dust). All samples were analysed by culture-based methods and passive sampling was used for molecular detection of Aspergillus section Fumigati. Results: Of all the matrices, the highest counts of Aspergillus sp. were obtained on settled dust filters (3.37% malt extract agar-MEA, 19.09% dichloran glycerol-DG18) followed by cleaning cloths (1.67% MEA; 7.07% DG18). Among the Aspergillus genus, the Fumigati section was predominant in Millipore and EDC samples in MEA (79.77% and 28.57%, respectively), and in swabs and settled dust filters in DG18 (44.76% and 30%, respectively). The Fumigati section was detected more frequently in DG18 (33.01%) compared to MEA (0.33%). The Fumigati section was observed in azole supplemented media (itraconazole and voriconazole) in several passive sampling methods employed and detected by qPCR in almost all passive samples, with EDCs being the matrix with the highest prevalence (n = 61; 67.8%). CONCLUSION: This study confirms that Aspergillus sp. is widespread and the Fumigati section is present in all FFHs. The presence of fungi potentially resistant to azoles in the FFHs was also observed. Further studies are needed to identify the best corrective and preventive measures to avoid this section contamination in this specific occupational environment.

Cytotoxicity of filtering respiratory protective devices from the waste sorting industry: A comparative study between interior layer and exhalation valve.

Filtering respiratory protection devices (FRPD) are mandatory for workers to wear in the Portuguese waste-sorting industry. Previous results regarding microbial contamination found on FRPD interior layer raised the question of whether microbial contamination from the exhalation valve would also have cytotoxicity effects. Since the FRPD exhalation valves are very close to workers' nose and mouth, they represent a source of exposure to bioburden by inhalation. This study aimed to evaluate the cytotoxicity of the microbial contamination present in the FRPD exhalation valves. For this purpose, the cytotoxicity effects were determined through the MTT assay in two different cell lines (human A549 epithelial lung cells, and swine kidney cells) and compared with previous results obtained with FRPD interior layers. The contamination present in the FRPD exhalation valves presented some cytotoxicity on epithelial lung cells, suggesting the inhalation route as a potential route of exposure through the use of FRPD in the waste-sorting industry. Half-maximal (50%) inhibitory concentration (IC50) values were lower for FRPD interior layer than exhalation valves in lung cells, with overall cytotoxicity lower in exhalation valves when compared to interior layer (z = -4.455, p = 0.000). Higher bacterial counts in TSA were correlated with lower IC50 values, thus, higher cytotoxicity effect in lung cells. No statistically significant differences were detected among different workplaces.

Aspergillus collected in specific indoor settings: their molecular identification and susceptibility pattern.

Exposure to Aspergillus conidia is an increased risk factor for the development of respiratory symptoms. The emergence of azole resistance in Aspergillus fumigatus is a major concern for the scientific community. The aim of this study was to perform the molecular identification of Aspergillus species collected from different occupational and non-occupational indoor settings and to study the azole susceptibility profile of the collected Fumigati isolates. The selected Aspergillus isolates were identified as belonging to the sections Fumigati, Nigri Versicolores, Terrei, Clavati and Nidulantes. All the Aspergillus fumigatus were screened for azole resistance using an agar media supplemented with itraconazole, voriconazole and posaconazole. None of the tested isolates showed resistance to those azoles. Knowledge of Aspergillus epidemiology in specific indoor environments allows a better risk characterization regarding Aspergillus burden. This study allowed the analysis of the molecular epidemiology and the determination of the susceptibility pattern of Aspergillus section Fumigati found in the studied indoor settings.

Settled dust assessment in clinical environment: useful for the evaluation of a wider bioburden spectrum.

The collection and analysis of settled dust samples from indoor environments has become one of several environmental sampling methods used to assess bioburden indoors. The aim of the study was to characterize the bioburden in vacuumed settled dust from 10 Primary Health Care Centers by culture based and molecular methods. Results for bacterial load ranged from 1 to 12 CFU.g-1 of dust and Gram-negative bacteria ranged between 1 to 344 CFU.g-1 of dust. Fungal load ranged from 0 CFU.g-1 of dust to uncountable. Aspergillus section Fumigati was detected in 4 sampling sites where culture base-methods could not identify this section. Mucorales (Rhizopus sp.) was observed on 1 mg/L voriconazole. Three out of 10 settled dust samples were contaminated by mycotoxins. Settled dust sampling coupled with air sampling in a routine way might provide useful information about bioburden exposure.

Commercial green tea from Portugal: Comprehensive microbiologic analyses.

In recent times green tea (GT) consumption has increased, due to the numerous studies that indicate a wide variety of health benefits following its regular consumption. The aim of this study was to assess the bioburden (bacteria and fungi) of bulk and bags of GT marketed in Lisbon and to obtain a more refined fungal burden characterization, including azole resistance profile. The bacteriota in tea bags before boiling ranged from lower than the detection limit to 1770 CFU.g-1, whereas in brew samples ranged from lower than the detection limit to 54.55 CFU.mL-1. In bulk samples before boiling ranged from lower than the detection limit to 2636 CFU.g-1, while after boiling ranged from lower than the detection limit to 72.73 CFU.mL-1. Fungal contamination on tea bags before boiling ranged from lower than the detection limit to 66.67 CFU.g-1 and after boiling, all samples presented results lower than the detection limit. Concerning bulk samples before boiling ranged from lower than the detection limit to 96.97 CFU.g-1, whereas after boiling ranged from lower the detection limit to 30.3 CFU.mL-1. Before boiling, the most common fungal species in the bagged tea (90.91 CFU.g-1; 45.45%) and bulk samples (66.67 CFU.g-1; 91.67%) was Aspergillus section Nigri. Fungal diversity was higher on bulk samples than in tea bags. Aspergillus section Nigri and Rhizopus sp. growth was observed mostly on itraconazole-supplemented Sabouraud dextrose agar media, which require further investigation. Aspergillus sections Fumigati and Nidulantes were detected by using real time PCR, but not in the GT samples in which they were identified through culture-based methods. A significantly reduction of bacterial contamination after boiling was observed, however fungal contamination with toxigenic potential was observed before and after boiling. Future research work needs to characterize in detail the mycotoxins contamination to allow a risk-benefit assessment to estimate the human health benefits and risks following tea consumption and to support policy-actions, if and when needed. The results also suggest that the conditions how tea is packed can influence the fungal diversity and this variable should be further investigated.

Are workers from waste sorting industry really protected by wearing Filtering Respiratory Protective Devices? The gap between the myth and reality.

One of the solutions for decreasing the workers' exposure to bioburden is the use of Filtering Respiratory Protective Devices (FRPD). As such it is important to determine whether these devices are fulfilling their protective role. This is the basis of the current study, aimed at characterizing bioburden retained by 120 FRPD (both in interior layers and in exhalation valves) through culture based-methods and molecular tools and also via analysis of antifungal resistance and mycotoxins profile. Our results show that Gram - Bacteria are present at a higher prevalence than total bacteria in both matrixes. Regarding fungal identification, Chrysonilia sitophila presented the highest prevalence on interior layers (55.1% on malt extract agar (MEA) supplemented with chloramphenicol (0.05%); 59.6% on dichloran-glycerol agar (DG18)), whereas on exhalation valves Aspergillus sp. presented the highest prevalence on MEA (6.8%) and C. sitophila on DG18 (36.3%). Among Aspergillus genera, section Fumigati was the one with the highest prevalence in both matrices. Aspergillus sp. was the most prevalent on exhalation valves (75.0% ITRA) in the screening of azole resistance. Fumigati section was the most abundant Aspergillus sp. detected on the interior layers (33.33%, 40 samples out of 120) and on the exhalation valves (1.66%, 2 samples out of 120). The interior layers and exhalation valves from workers with more waste contact showed an increased exposure to bioburden. This study showed that FRPD can have high levels of bioburden, toxigenic fungal strains and Aspergillus sections with reduced susceptibility to the tested azoles and can be used as a passive sampling method since it mimics the results obtained by active methods in previous studies. The gathered information will be useful to prioritize multiple interventions on workers' education or even on FRPD replacement frequency.