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- Occupational exposure to aflatoxin (AFB1) in poultry productionPublication . Viegas, Susana; Veiga, Luísa; Malta-Vacas, Joana; Sabino, Raquel; Figueiredo, Paula; Almeida, Ana; Viegas, Carla; Carolino, ElisabeteAflatoxin B1 (AFB1) has been recognized to produce cancer in human liver. In addition, epidemiological and laboratory studies demonstrated that the respiratory system was a target for AFB1. Exposure occurs predominantly through the food chain, but inhalation represents an additional route of exposure. The present study aimed to examine AFB1 exposure among poultry workers in Portugal. Blood samples were collected from a total of 31 poultry workers from six poultry farms. In addition, a control group (n = 30) was included comprised of workers who undertook administrative tasks. Measurement of AFB1 in serum was performed by enzyme-linked immunosorbent assay (ELISA). For examining fungi contamination, air samples were collected through an impaction method. Air sampling was obtained in pavilion interior and outside the premises, since this was the place regarded as the reference location. Using molecular methods, toxicogenic strains (aflatoxin-producing) were investigated within the group of species belonging to Aspergillus flavus complex. Eighteen poultry workers (59%) had detectable levels of AFB1 with values ranging from <1 ng/ml to4.23 ng/ml and with a mean value of 2 ± 0.98ng/ml. AFB1 was not detected in the serum sampled from any of the controls. Aspergillus flavus was the fungal species third most frequently found in the indoor air samples analyzed (7.2%) and was the most frequently isolated species in air samples containing only Aspergillus genus (74.5%). The presence of aflatoxigenic strains was only confirmed in outdoor air samples from one of the units, indicating the presence of a source inside the building in at least one case. Data indicate that AFB1 inhalation represents an additional risk in this occupational setting that needs to be recognized, assessed, and prevented.
- Prevalence of fungi in indoor air with reference to gymnasiums with swimming poolsPublication . Viegas, Carla; Alves, Célia; Carolino, Elisabete; Rosado, Laura; Santos, Carlos SilvaFungal contamination of air in 10 gymnasiums with swimming pools was monitored. Fifty air samples of 200 L each were collected, using a Millipore air tester, from the area surrounding the pool, in training studios, in showers and changing rooms for both sexes, and also, outside premises, since these are the places regarded as reference. Simultaneously, environmental parameters – temperature and humidity – were also monitored. Some 25 different species of fungi were identified. The six most commonly isolated genera were the following: Cladosporium sp. (36.6%), Penicillium sp. (19.0%), Aspergillus sp. (10.2%), Mucor sp. (7%), Phoma sp. and Chrysonilia sp. (3.3%). For yeasts, three different genera were identified, namely, Rhodotorula sp. (70%), Trichosporon mucoides and Cryptococcus uniguttulattus (10%).
- Sampling protocol to assess Aspergillus section Fumigati in woodworking environmentsPublication . Dias, Marta; Gomes, Bianca; Pena, Pedro; Cervantes, Renata; Rodriguez, Margarida; Riesenberger, Bruna; Marques, Liliana; Ribeiro, Edna; Viegas, CarlaPortugal’s wood industry employs over 80,000 people and comprises about 16,600 enterprises in 2021. Sawmill workers are exposed to several occupational hazards, including microorganisms. Health problems have been documented by workers exposed to wood dust infected with fungi. A fungal priority pathogens list (WHO FPPL) was recently released by the WHO. It prioritizes fungal pathogens based on their perceived public health importance and unmet research and development needs. Some of the species on this list are frequently found in sawmills. Azole fungicides are commonly used by sawmills to protect wood. Aspergillus section Fumigati azole antifungal resistance has previously been observed in this environment. This study aimed to establish a sampling protocol to effectively isolate Aspergillus section Fumigati in woodworking environments.
- Trends on Aspergillus epidemiology: perspectives from a national reference laboratory surveillance programPublication . Sabino, Raquel; Gonçalves, Paulo; Martins-Melo, Aryse; Simões, Daniela; Oliveira, Mariana; Francisco, Mariana; Viegas, Carla; Carvalho, Dinah; Martins, Carlos; Ferreira, Teresa; Toscano, Cristina; Simões, Helena; Veríssimo, CristinaIdentification of Aspergillus to species level is important since sibling species may display variable susceptibilities to multiple antifungal drugs and also because correct identification contributes to improving the knowledge of epidemiological studies. Two retrospective laboratory studies were conducted on Aspergillus surveillance at the Portuguese National Mycology Reference Laboratory. The first, covering the period 2017–2018, aimed to study the molecular epidemiology of 256 Aspergillus isolates obtained from patients with respiratory, subcutaneous, or systemic infections and from environmental samples. The second, using our entire collection of clinical and environmental A. fumigatus isolates (N = 337), collected between 2012 and 2019, aimed to determine the frequency of azole-resistant A. fumigatus isolates. Aspergillus fumigatus sensu stricto was the most frequent species in both clinical and environmental samples. Overall, and considering all Aspergillus sections identified, a high frequency of cryptic species was detected, based on beta-tubulin or calmodulin sequencing (37% in clinical and 51% in environmental isolates). Regarding all Fumigati isolates recovered from 2012–2019, the frequency of cryptic species was 5.3% (18/337), with the identification of A. felis (complex), A. lentulus, A. udagawae, A. hiratsukae, and A. oerlinghauensis. To determine the frequency of azole resistance of A. fumigatus, isolates were screened for azole resistance using azole-agars, and 53 possible resistant isolates were tested by the CLSI microdilution reference method. Nine A. fumigatus sensu stricto and six Fumigati cryptic isolates showed high minimal inhibitory concentrations to itraconazole, voriconazole, and/or posaconazole. Real-time PCR to detect cyp51A mutations and sequencing of the cyp51A gene and its promoter were performed. The overall frequency of resistance to azoles in A. fumigatus sensu stricto was 3.0%. With this retrospective analysis, we were able to detect one azole-resistant G54R mutant A. fumigatus environmental isolate, collected in 2015. The TR34/L98H mutation, linked to the environmental transmission route of azole resistance, was the most frequently detected mutation (N = 4; 1.4%). Our findings underline the demand for correct identification and susceptibility testing of Aspergillus isolates.
- 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.
- Possíveis implicações da contaminação fúngica num aviárioPublication . Viegas, Carla; Viegas, Susana; Veríssimo, C.; Rosado, Laura; Santos, Carlos SilvaIntrodução – Apesar de em Portugal se verificar o aumento da indústria da produção de aves para consumo humano, apenas alguns estudos incidem sobre a qualidade do ar interior e as implicações da sua degradação. Objectivos – Descrever a contaminação fúngica num aviário, analisar possíveis associações com a temperatura ambiente e a humidade relativa e o possível impacto na saúde dos consumidores e trabalhadores desta unidade. Métodos – Foi desenvolvido um estudo descritivo para avaliar a contaminação fúngica num aviário. Colheram‑se 5 amostras de ar de 100 litros através do método de compactação e 4 amostras de superfícies, utilizando a técnica da zaragatoa e um quadrado de 10 cm de lado de metal. Simultaneamente, os parâmetros ambientais – temperatura ambiente e humidade relativa – também foram medidos. Resultados – Foram identificadas vinte espécies de fungos no ar, sendo os seguintes os quatro géneros mais comummente isolados: Cladosporium (40,5%), Alternaria (10,8%), Chrysosporium e Aspergillus (6,8%). Nas superfícies, 21 espécies de fungos foram identificadas, sendo os 4 géneros mais identificados Penicillium (51,8%), Cladosporium (25,4%), Alternaria (6,1%) e Aspergillus (4,2%). Importa referir o facto de Aspergillus flavus, também isolado no ar, ser reconhecido como produtor de micotoxinas (aflatoxina) e Aspergillus fumigatus, uma das espécies isoladas no ar e superfícies, ser capaz de causar aspergilose grave ou fatal. Não se verificou relação significativa (p> 0,05) entre a contaminação fúngica e as variáveis ambientais. Conclusão – Caracterizou‑se a distribuição fúngica no ar e superfícies do aviário e analisou‑se a possível influência das variáveis ambientais. Foi reconhecido um potencial problema de Saúde Pública devido à contaminação fúngica e à possível produção de micotoxinas com a eventual contaminação dos produtos alimentares. A contaminação fúngica, particularmente causada pelo Aspergillus fumigatus, e a possível presença de micotoxinas no ar, devem ser encaradas também como fatores de risco neste contexto ocupacional. ABSTRACT - Background – Although there is an increasingly industry that produce whole chickens for domestic consumption in Portugal, only few investigations have reported on the indoor air of these plants and the consequences of their degradation. Objectives – Describe one poultry environmental fungal contamination analyse possible associations between temperature and relative humidity and its possible impact on the health of consumers and of the poultry workers. Methods – A descriptive study was developed to monitor one poultry fungal contamination. Five air samples of 100 litres through impaction method were collected and 4 swab samples from surfaces were also collected using a 10 cm square of metal. Simultaneously, environmental parameters – temperature and relative humidity – were also measured. Results – Twenty species of fungi in air were identified, being the 4 most commonly isolated the following genera: Cladosporium (40.5%), Alternaria (10.8%), Chrysosporium and Aspergillus (6.8%). In surfaces, 21 species of fungi were identified, being the 4 genera more identified Penicillium (51.8%), Cladosporium (25.4%), Alternaria (6.1%) and Aspergillus (4.2%). In addition, Aspergillus flavus also isolated in the poultry air is a well‑known producer of potent mycotoxins (aflatoxin), and Aspergillus fumigatus, one of the species isolated in air and surfaces, is capable of causing severe or fatal aspergillosis. There was no significant relationship (p>0,05) between fungal contamination and environmental variables. Conclusions – Was characterized fungal distribution in poultry air and surfaces and analyzed the association of environmental variables. It was recognized the Public Health problem because of fungal contamination and also due to probable mycotoxins production with the possible contamination of food products. Fungal contamination, particularly due to the presence of Aspergillus fumigatus and also the possible presence of mycotoxins in the air, should be seen as risk factor in this occupational setting.
- Culture media and sampling collection method for Aspergillus spp. assessment: tackling the gap between recommendations and the scientific evidencePublication . Viegas, Carla; Dias, Marta; Carolino, Elisabete; Sabino, RaquelCulturing is still the most widely used method for determining fungal growth. Thus, is important to identify the most suitable culture media to assess Aspergillus spp. The aim of this study was to analyze data obtained from previous studies, aiming at identifying the most suitable culture media (malt extract agar (MEA) or dichloran-glycerol agar (DG18) to assess Aspergillus spp. isolation and growth. This study was conducted by using environmental samples (n = 1153). Most of the active sampling methods (air samples) were impacted directly onto both culture media. As for passive sampling methods, fungi were extracted from environmental matrices inoculated onto both media. Overall, total Aspergillus counts were higher in MEA (n = 617, 53.5%) than in DG18 (n = 536, 46.5%). Regarding Aspergillus sections, significant associations were detected with the media (χ2 (7) = 241.118, p < 0.001), the sampling approach (p < 0.001, 95% CI = (0.3 × 10−4), and the indoor environment (p < 0.001, 95% CI = (0.3 × 10−4)). As such, the sampling approach and the culture media should be accurately selected when dealing with Aspergillus spp. exposure assessment.
- Sampling protocol to assess occupational exposure to bioburden in clinical facilitiesPublication . Viegas, Carla; Almeida, B.; Monteiro, Ana; Paciência, I.; Rufo, J.; Carolino, Elisabete; Viegas, SusanaBioburden (comprising fungi and bacteria) is associated with a wide range of adverse health effects and besides patients, clinical staff is also at risk to be exposed. Most of the studies worldwide focusing on bioburden for Indoor Air Quality (IAQ) studies or for occupational exposure assessments purposes rely mostly on air sampling (active methods). In Portugal, occupational health concern has been increasing, and efforts have been made to standardize exposure assessment procedures in occupational environments. However, these are currently not adapted to health care facilities. Therefore, a national and international network - EXPOSE Project - of academic institutions, in direct collaboration with public health institutions, developed a project aiming to propose a sampling protocol to access occupational exposure to bioburden in clinical facilities.
- Tea contamination by mycotoxins and azole-resistant mycobiota: the need of a One Health approach to tackle exposuresPublication . Viegas, Carla; Simões, Ana Beatriz; Faria, Mariana; Gomes, Bianca; Cervantes, Renata; Dias, Marta; Carolino, Elisabete; Twaruzek, Magdalena; Kosicki, Robert; Viegas, Susana; Caetano, Liliana AranhaDespite tea beneficial health effects, there is a substantial risk of tea contamination by harmful pathogens and mycotoxins. A total of 40 tea samples (17 green (raw) tea; 13 black (fermented) tea; 10 herbal infusions or white tea) were purchased from different markets located in the Lisbon district in 2020. All products were directly available to consumers either in bulk (13) and or in individual packages (27). Bacterial analysis was performed by inoculating 150 μL of samples extracts in tryptic soy agar (TSA) supplemented with 0.2 % nystatin medium for mesophilic bacteria, and in Violet Red bile agar (VRBA) medium for coliforms (Gram-negative bacteria). Fungal research was performed by spreading 150 μL of samples in malt extract agar (MEA) supplemented with 0.05 % chloramphenicol and in dichloran-glycerol agar (DG18) media. The molecular detection of the Aspergillus sections Fumigati, Nidulantes, Circumdati, and Flavi was carried out by Real-Time PCR (qPCR). Detection of mycotoxins was performed using a high-performance liquid chromatograph (HPLC) with a mass spectrometry detector. Azole resistance screening was achieved following the EUCAST guidelines. The highest counts of total bacteria (TSA) were obtained in green raw tea (81.6 %), while coliform counts (VRBA) were found in samples from black raw tea (96.2 %). The highest fungal counts were obtained in green raw tea (87.7 % MEA; 69.6 % DG18). Aspergillus sp. was the most prevalent genus in all samples on MEA (54.3 %) and on DG18 (56.2 %). In the raw tea 23 of the samples (57.5 %) presented contamination by one to five mycotoxins in the same sample. One Aspergillus section Fumigati isolate from a green tea beverage recovered from itraconazole-Sabouraud dextrose agar (SDA) medium, presented itraconazole and posaconazole E-test MICs above MIC90 values. Our findings open further discussion regarding the One-Health approach and the necessary investment in researching biological hazards and azole resistance associated with the production and consumption of tea (in particular green tea).
- Budget-friendly protocol for TR34/L98H and TR46/Y121F/T289A mutation detection in Aspergillus section Fumigati isolatesPublication . Dias, Marta; Rodriguez, Margarida; Vasques, C.; Riesenberger, Bruna; Marques, Liliana; Gomes, Bianca; Pena, Pedro; Cervantes, Renata; Viegas, Susana; Viegas, CarlaAspergillus section Fumigati is one of the most common sections, in the environment. It has been found in different occupational environments, such as sawmills and waste sorting. Its cryptic species show intrinsic resistance to several antifungals. Resistance in A. fumigatus is emerging due to selective pressure caused by the prolonged use of azoles. It is often associated with mutations in the Cyp51A gene. The fungal priority pathogens list (WHO), includes A. fumigatus with critical priority. Further analysis to identify potential resistance mechanisms and mutations is needed. Objective: This evaluation aims to offer a protocol for mutation detection in Aspergillus section Fumigati isolates. It will contribute to the development of guidance that can support future occupational exposure assessments.