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- 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.
- Antifungal-resistant mucorales in different indoor environmentsPublication . Aranha Caetano, Liliana; Faria, Tiago; Springer, Jan; Loeffler, Juergen; Viegas, CarlaThis paper sought to address the prevalence of Mucorales in different indoor environments in Portugal. Environmental samples (183 in total) were collected at dwellings (n = 79) and workplaces (bakeries, swine farms, taxis, waste-sorting plants) (n = 93) by passive sampling using electrostatic dust collector (EDC), air-conditioning filters, litter, and/or raw materials. Samples were inoculated onto non-selective MEA and DG18 media and were screened for antifungal drug-resistance in azole-supplemented agar Sabouraud media. A probe-based Mucorales-specific real-time PCR assay (Muc18S) was used to detect Mucorales in complement to conventional culture-based methods. Mucorales order was found as more prevalent in air-conditioning filters from waste-sorting fork lifters (35.7%). Amongst Mucorales isolates able to grow in azole-supplemented media, 16 isolates of Mucor sp., Rhizopus sp. or Rhizomucor sp. were not susceptible to 1 mg/L voriconazole, and four isolates of Mucor sp. or Rhizopus sp. were not susceptible to 4 mg/L itraconazole. In conclusion, a combination of the culture-based and molecular methods proved to be reliable for Mucorales order identification in complex environmental samples.
- Assessment of azole resistance in clinical settings by passive samplingPublication . Caetano, Liliana Aranha; Almeida, Beatriz; Viegas, CarlaIntroduction - Clinical settings (hospitals, primary health care centers – PHCC), with high occupation rates, present microbiologic agents in their environment. Exposure to mycobiota in indoor environments is related to several adverse human health effects, such as respiratory symptoms. Azole resistance in fungal species and consequent failure of antifungal therapy is a major concern for public health. Portuguese legislation recommends active air sampling for bioburden assessment indoors. Passive sampling can be used in complement to determine bioburden levels from longer periods. Aim of the study - To assess the fungal burden and prevalence of azole resistance in clinical settings in Portugal using passive sampling methods.
- Characterization of occupational exposure to fungal burden in Portuguese bakeriesPublication . Viegas, Carla; Faria, Tiago; Aranha Caetano, Liliana; Carolino, Elisabete; Quintal-Gomes, Anita; Twarużek, Magdalena; Kosicki, Robert; Viegas, SusanaSeveral studies reported adverse respiratory health effects in workers exposed to ambient contaminants in bakeries. The aim of this study was to examine worker exposure to fungi and mycotoxins in Portuguese bakeries in order to develop new policies in occupational health. Environmental samples such as air, surfaces, settled dust and electrostatic dust collector (EDC) were collected in 13 bakeries for fungal and mycotoxins assessment. Air samples obtained by impaction were performed applying malt extract agar (MEA) supplemented with chloramphenicol (0.05%) and dichloran glycerol (DG18) agar-based media. Air samples collected through impinger method were determined as well for fungal detection by molecular tools of Aspergillus sections and mycotoxins. The highest median value for fungal load was 1053 CFU·m-3 and 65.3% (32 out of 49) of the sampling sites displayed higher fungal load than limits imposed by the World Health Organization. Aspergillus genera was found in air, surface swabs and EDC. Molecular tools were effective in measuring Aspergillus section Fumigati in 22.4% on air, 27.8% on surface swabs and in 7.4% in EDC and Aspergillus section Versicolores in one air sample. All settled dust samples showed contamination with six to eight mycotoxins in each sample. The mycotoxins detected were deoxynivalenol-3-glucoside, deoxynivalenol, zearalenone, 15-acetyldeoxynivalenol, monoacetoxyscirpenol, diacetoxyscirpenol, fumonisin B1, fumonisin B2, griseofulvin, HT2, ochratoxin A, ochratoxin B and mycophenolic acid. Industrial hygienists and exposure assessors should rely on different sampling methods (active and passive) and different assays (culture based and molecular methods) to obtain an accurate risk characterization regarding fungal burden (fungi and mycotoxins). Additionally, the awareness for the raw material as a potential mycotoxins indoor contamination source is important.
- Nasal swab as a tool to access occupational exposure to fungi in a cork industryPublication . Viegas, Carla; Pacífico, Cátia; Faria, Tiago; Aranha Caetano, Liliana; Gomes, Anita Quintal; Viegas, SusanaApproximately 49% of all cork produced worldwide in 2016 was from Portugal, where there are 650 companies working in this production sector. Additionally, two thirds of the world exportation comes from Portugal, with Portuguese cork industry employing about ten thousands workers (MTSS 2009). The presence of the Penicillium section Aspergilloides (formerly known as Penicillium glabrum) in this industry involves the risk of respiratory diseases such as suberosis, a type of hypersensitivity pneumonitis that is one of the most prevalent diseases among cork workers. Besides Penicillium section Aspergilloides, Aspergillus section Fumigati was also reported in cork industries. This is one of the most ubiquitous saprophytic fungi and is also considered one of the potentially pathogenic species with the highest clinical relevance. The use of the nasal swab procedure is of particular importance since it allows to determine the fungal presence in the nose cavity, being an easy and painless collection method. The aim of this study was to determine the exposure to the dominant mycobiota of this occupational environment through the mycological analysis of nasal exudates from cork industry workers. Nasal mucus samples from 360 workers from 3 companies (plant A - 41workers; plant B – 165 workers; and plant C – 154 workers) and also from a control group (38 individuals) with administrative tasks were performed. Duplicate samples were taken with sterilized cotton swabs from one nostril of each worker. The swabs were rotated against the internal anterior walls of the nostril and then placed in the provided transport tube. One of the swab samples was then plated onto malt extract agar (MEA) supplemented with chloramphenicol (0.05 %). All the collected samples were incubated at 27 °C for 5 to 7 days. The other swab sample was used for DNA extraction following molecular identification of Penicillium section Aspergilloides and Aspergillus section Fumigati by Real-Time PCR (qPCR). Among the 360 workers subjected to the nasal swab assay only 50 (13.9%) did not present fungal contamination. Around 36.6% of the workers' nasal swabs presented Penicillium genus contamination, 9.9% with Aspergillus sp. and 29.1% with more than one fungal genera. Among the 38 subjects from the control group, 16 (42.1%) did not present fungal contamination, 44.7% present Penicillium sp. and 18.4% Cladosporium sp. One subject presented Mucor sp. and other Geotrichum sp. contamination. DNA from Penicillium section Aspergilloides was successfully amplified by qPCR in 37 cork workers. From those, it was only possible to identify in 12 samples the genus Penicillium by culture based-methods. Aspergillus section Fumigati was also co-amplified with Penicillium section Aspergilloides in one worker, while in another one was detected singularly. As expected, in the 38 controls analyzed none were positive for Penicillium section Aspergilloides nor Aspergillus section Fumigati. The fungal species identified and detected in the collected nose swabs presented the same trend described for this very specific occupational environment. This approach allowed us to estimate the risk associated with the performance of the tasks since high dust contamination is expected to promote the exposure to fungi playing a role as carriers to the worker’s nose. As observed in previous environmental assessments, culture-based methods coupled with molecular tools allowed to obtain a wider spectrum of the workers' nasal mycobiota.