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- Portuguese cork industry: filling the knowledge gap regarding occupational exposure to fungi and related health effectsPublication . Viegas, Carla; Dias, Marta; Pacífico, Cátia; Faria, Tiago; Clérigo, Anália Matos; Dias, Hermínia Brites; Caetano, Liliana Aranha; Carolino, Elisabete; Gomes, Anita Q.; Viegas, SusanaIntroduction: The presence of the Penicillium section Aspergilloides (formerly known as Penicillium glabrum) in the cork industry involves the risk of respiratory diseases such as suberosis. Methods: This study aimed to corroborate the predominant fungi presents in this occupational environment by performing a mycological analysis of 360 workers’ nasal exudates collected by nasal swabs. Additionally, evaluation of respiratory disorders among the cork workers was also performed by spirometry. Results: Penicillium section Aspergilloides was detected by qPCR in 37 out of the 360 nasal swabs collected from workers’ samples. From those, 25 remained negative for Penicillium sp. when using culture-based methods. A significant association was found between ventilatory defects and years of work in the cork industry, with those people working for 10 or more years in this industry having an approximately two-fold increased risk of having ventilatory defects compared to those working less time in this setting. Among the workers who detected the presence of Penicillium section Aspergilloides, those with symptoms presented slightly higher average values of CFU. Discussion: Overall, the results obtained in this study show that working in the cork industry may have adverse effects on worker’s respiratory health. Nevertheless, more studies are needed (e.g., using serological assays) to clarify the impact of each risk factor (fungi and dust) on disease etiology.
- Commercial tea samples: a natural health ally or an Aspergillus infusionPublication . Gomes, Bianca; Dias, Marta; Cervantes, Renata; Caetano, Liliana Aranha; Viegas, CarlaIntroduction and Purpose - Tea consumption has been increasing in the last few years. The lack of specific legislation concerning microbial contamination of teas and infusions might represent a risk for consumers’ health. This study characterizes fungal contamination and azole resistance on green and black tea and other infusions commercially available in Portugal.
- Organic dust exposure in veterinary clinics: a case study of a small-animal practice in PortugalPublication . Viegas, Carla; Monteiro, Ana; Ribeiro, Edna; Caetano, Liliana Aranha; Carolino, Elisabete; Assunção, Ricardo; Viegas, SusanaLiterature about occupational health in small-animal veterinary practices is scarce, but most of it has recognised a number of risks to be considered, including organic dust exposure. The aim of this pilot study was to assess organic dust, bacterial, and fungal contamination in the indoor environment of a typical Portuguese veterinary clinic but also to screen for azoleresistant fungi. To complement these findings we also analysed workers’ nasal exudates for resistant bacteriota. Particles measurements included mass concentrations (PMC) of five particle sizes (PM0.5, PM1, PM2.5, PM5, PM10) and their counts (PNC). Indoor air samples were obtained from six locations as well as before and during cat dental cleaning and cultured on four media for bacterial and fungal assessment. An outdoor sample was also collected for reference Surface samples were taken from the same indoor locations using swabs and we also use electrostatic dust cloths as passive methods. PM10 showed the highest concentrations across the locations. Indoor air fungal loads ranged from 88 to 504 CFU m−3. The azole-resistant Aspergillus section Nigri was identified in one sample. Indoor air bacterial loads ranged from 84 to 328 CFU m-3. Nasopharyngeal findings in the 14 veterinary clinic workers showed a remarkably low prevalence of Staphylococcus aureus (7.1 %). Our results point to contamination with organic dusts above the WHO limits and to the need for better ventilation. Future studies should combine the same sampling protocol (active and passive methods) with molecular tools to obtain more accurate risk characterisation. In terms of prevention, animals should be caged in rooms separate from where procedures take place, and worker protection should be observed at all times.
- Occupational exposure to fungi and mycotoxins: overview of different studies developed in PortugalPublication . Viegas, Carla; Cervantes, Renata; Dias, Marta; Gomes, Bianca; Pena, Pedro; Carolino, Elisabete; Twarużek, Magdalena; Kosicki, Robert; Soszczyńska, Ewelina; Caetano, Liliana Aranha; Viegas, SusanaAs in all European countries, Portuguese employers are obliged by legal requirements to assess and prevent exposure to chemical, physical and biological risks (Directive 89/391/EEC). However, the health risks of biological origin are frequently less recognized and, thus, underreported. This study intends to present an overview of the assessments of fungal contamination and mycotoxins performed in three different occupational environments (Firefighters headquarters - FFH; Waste collection trucks – WCT and; Cemeteries - CM). Active sampling (impaction and impingement methods) and several passive sampling methods (swabs, settled dust, settled dust filters, electrostatic dust cloths – EDC, cleaning materials,…) were employed. Azole resistance screening, the molecular detection of Aspergillus sections, as well as mycotoxin analysis were also conducted. Regarding FFH it was raised concern regarding the following findings: a) microbial contamination didn´t comply with Portuguese IAQ legal requirements in most of the FFH (fungal load 45.45%); b) fungal exposure through inhalation underlining a possible risk factor for respiratory diseases; c) prevalence of Aspergillus sp. in cleaning materials that might represent a potential source of cross-contamination; d) widespread of Aspergillus section Fumigati in all the FFH analysed; e) trend of multidrug resistance found among fungi with a focus on Fumigati isolates; and f) detection of several mycotoxins (fumonisin B2, nivalenol, mycophenolic acid, and sterigmatocystin). Concerning WCT Aspergillus sp. (4.18%) was one of the most prevalent species. Aspergillus section Fumigati was detected in 5 samples. Mycotoxins were detected in filters (N=1) and in settled dust samples (N=16). The mycotoxin detected in the filter was fumonisin B1 and the most detected in settled dust was mycophenolic acid. Sterigmatocystin, deoxynivalenol, and zearalenon were detected in only one sample each. In what concerns CM, Aspergillus sp. presented the highest counts in DG18 (18.38%) and it wasn't observed in azole-supplemented SDA media. Mycophenolic acid was detected in one settled dust sample. Overall, our study reveals that a comprehensive sampling approach using active and passive sampling methods and combined analytic methods (i.e., culture-based and molecular) is an important asset in microbial exposure assessments. Furthermore, in all three occupational environments mycotoxins were detected unveiling this occupational health threat.
- Fungal and mycotoxins contamination in non-alcoholic beverages: the case of tea from Portuguese market and coffee beans from BrazilPublication . Viegas, Carla; Gomes, Bianca; Cervantes, Renata; Dias, Marta; Pena, Pedro; Oliveira, Filipe; Andrade, Ednilton Tavares de; Caetano, Liliana Aranha; Twaruzek, Magdalena; Kosicki, Robert; Viegas, SusanaAgriculture constitutes one of the most sensitive sectors that could be affected by climate change. Among the xenobiotics contaminating agricultural crops, fungi and mycotoxins are the most challenging since their presence represents an economic burden due to crop loss and serious health effects related to animals and humans with severe repercussions. This study intends to characterize the fungal and mycotoxin contamination in tea available in the Portuguese market and in coffee from two Brazilian industries.
- Commercial green tea from Portugal: comprehensive microbiologic analysesPublication . Viegas, Carla; Sá, Flávio; Mateus, Margarida; Santos, Patrícia; Almeida, Beatriz; Aranha Caetano, Liliana; Gomes, Anita Q.; Viegas, SusanaIn 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 teabags 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 significant 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 of how tea is packed can influence the fungal diversity and this variable should be further investigated.
- Portuguese groceries: a potential indoor environment to be used as public health sentinel for fungal and mycotoxins contaminationPublication . Viegas, Carla; Cervantes, Renata; Gomes, Bianca; Moreira, Silvia; Dias, Marta; Pena, Pedro; Carolino, Elisabete; Twaruzek, Magdalena; Kosicki, Robert; Caetano, Liliana Aranha; Viegas, SusanaThis study aimed to characterize microbial contamination in Portuguese grocery stores. The research was carried out in 15 grocery stores in Cascais, Portugal. Electrostatic dust cloths (EDC) and surface swabs were used on 3 sampling sites: checkout, fruits/vegetables, and warehouse/dispenser. Fungal contamination was characterized by culture-based methods, through the inoculation onto two different culture media: malt extract agar (MEA) supplemented with chloramphenicol (0.05 %) and dichloran-glycerol agar (DG18). Screening of azole resistance was performed by inoculation of EDC extracts' in azole-supplemented Sabouraud dextrose agar (SDA) media, according to EUCAST guidelines. A total of 39 samples were screened for mycotoxin contamination. The highest fungal contamination was obtained from fruits/vegetables in swabs (76% MEA; 71% DG18). The most prevalent genera in swabs was Cladosporium sp. (54% MEA). Regarding EDC, besides Penicillium sp. (63%) the most prevalent were Aspergillus sections Circumdati (25%) and Aspergilli (35%). Penicillium sp. was highly prevalent in the checkout (61% SDA; 87% voriconazole), and warehouse/dispenser sectors (73% voriconazole; 52% posaconazole). Circumdati and Fumigati were the most prevalent Aspergillus sections in fruits/vegetables (73% and 64% in voriconazole, respectively). Four mycotoxins (fumonisin B1, B2, B3) were detected simultaneously in most of the 36 samples. Identifying the most critical workplaces in groceries concerning fungal and mycotoxin contamination is of utmost importance to human health (workers and consumers) when using a One Health approach.
- Exposure to azole-resistant Aspergillus fumigatus in Portuguese sawmillsPublication . Dias, Marta; Gomes, Bianca; Pena, Pedro; Cervantes, Renata; Caetano, Liliana Aranha; Carolino, Elisabete; Viegas, CarlaSawmill workers have an increased risk of adverse respiratory health effects due to wood dust, microorganisms, and metabolite exposure. Knowledge about exposure-response relationships is scarce, with information lacking regarding this transformation sector in Portugal. Among microbial components, fungi and fungal spores and fragments stand as allergens and irritants, due to their high prevalence, under the right circumstances, on wood products (planks, chips) that have been preserved. Aspergillus species can infect the lungs, causing pneumonia-like symptoms that can progress into more severe sickness and are reported as the most common found in sawmills. Moreover, Aspergillus section Fumigati has recently entered the WHO ranking list of most critical fungal pathogens, based on the impact of the fungal disease on public health. While antifungal resistance has been reported in sawmills, there is no data, to date, on occupational exposure to azole-resistant Aspergillus fumigatus (ARAF) in Portuguese sawmills. This study aims to determine the prevalence of azole-resistant Aspergillus fumigatus, followed by the characterization of the distribution of azole-resistance mutations in Aspergillus fumigatus from Portuguese sawmills. The correlation of the exposures with work tasks handled wood products, and seasonal variation will also be made to suggest the most suitable procedures to ensure sawmill workers’ safety.
- Portuguese groceries: a comprehensive occupational exposure assessment to fungal contaminationPublication . Viegas, Carla; Cervantes, Renata; Gomes, Bianca; Moreira, Sílvia; Dias, Marta; Pena, Pedro; Carolino, Elisabete; Twaruzek, Magdalena; Kosicki, Robert; Soszczyńska, Ewelina; Caetano, Liliana Aranha; Viegas, SusanaTo our knowledge, there has been no research on the occupational exposure of grocery employees. This study aimed to characterize the microbial contamination in 15 grocery stores in Cascais, Portugal, by passive sampling. The molecular detection of Aspergillus sections, as well as mycotoxin analysis, screening of azole resistance, and cytotoxicity measurement was also conducted to better estimate the health risks of exposure and to identify possible relations between the risk factors. Electrostatic dust cloths (EDC) and surface swabs were used on 3 sampling sites: checkout, fruits/vegetable, and warehouse/dispenser. Fungal contamination was characterized by culture-based methods, through the inoculation onto two different culture media: malt extract agar (MEA) supplemented with chloramphenicol (0.05 %) and dichloran-glycerol agar (DG18). Screening of azole resistance was performed by inoculation of EDC extracts' in azole-supplemented Sabouraud dextrose agar (SDA) media, according to EUCAST guidelines. A total of 39 samples were screened for mycotoxins contamination. EDC’s cytotoxicity (IC50) was assessed in lung epithelial (A549) and liver carcinoma (HepG2) human cells, in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The highest fungal contamination was obtained from fruits/vegetables in swabs (76 % MEA; 71% DG18). Cladosporium sp. (54% MEA) was the most prevalent genera in swabs. Regarding EDC, besides Penicillium sp. (63%) the most prevalent were Aspergillus sections Circumdati (25%) and Aspergilli (35%). Penicillium sp. was highly prevalent in the checkout (61% SDA; 87% voriconazole), and warehouse/dispenser sectors (73% voriconazole; 52% posaconazole). Circumdati and Fumigati were the most prevalent Aspergillus sections in fruits/vegetables (73% and 64% in voriconazole, respectively). Four mycotoxins (fumonisin B1, B2, B3) were detected simultaneously in most 36 samples. Two out of 39 EDC samples exhibited an IC50 of 10 mm2/ml: one (M7F/L) in A549 cells, and another (M8A) in HepG2 cells. Identifying the variables that influence the fungal and mycotoxins contamination of groceries environment is of utmost importance to prevent human exposure (workers and consumers) to these risk factors when using a One Health approach.
- Assessment of the microbial contamination of mechanical protection gloves used on waste sorting industry: a contribution for the risk characterizationPublication . Viegas, Carla; Twarużek, Magdalena; Dias, Marta; Almeida, Beatriz; Carolino, Elisabete; Kosicki, Robert; Soszczyńska, Ewelina; Grajewski, Jan; Aranha Caetano, Liliana; Viegas, SusanaIn Portugal, mechanical protection gloves (MPG) are of mandatory use and during their use sweat is released and, consequently, the humidity of the material increases leading to conditions favorable to the growth of microorganisms. However, no studies have been conducted in MPG to assess the bioburden. This study intended to determine the bioburden present in MPG and their biological effects and to discuss the possibility to use MPG as a passive method to assess occupational exposure to microbial contamination. Fungal burden was characterized through molecular tools for fungal toxigenic species, and antifungal resistance and mycotoxin profiles were determined. Cell viability was determined in swine kidney (SK) monolayer and hepatocellular carcinoma (Hep G2) cell lines. All MPG samples presented Gram-negative bacteria. The fungal contamination ranged from 0 CFU.m−2 in both MEA and DG18, to 5.09 × 106 and 2.75 × 106, and the most common fungi found was Aspergillus spp. (50.46%). Azole resistant Aspergillus sections were found in azole supplemented media. Aspergillus sections (Circumdati, Flavi, Fumigati and Versicolores) were detected by molecular tools in 66 out of 67 samples. The most-reported mycotoxin was mycophenolic acid (89.6%). HepG2 cells appear to be more sensitive to MPG contamination, with high cytotoxicity (IC50 < 0.05 mm2/ml) observed for 18 out of 57 gloves. MPG can be used in passive sampling to assess occupational exposure to bioburden in waste sorting industries and contribute to risk characterization. Some contaminants of MPG had cytotoxic potential and affected the biology of hepatic cells more than renal cells.
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