Browsing by Author "Caetano, Liliana Aranha"
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- Analysis of Aspergillus spp. burden by culture and molecular based methods in different occupational environments: what needs to be done?Publication . Quintal Gomes, Anita; Faria, Tiago; Caetano, Liliana Aranha; Sabino, Raquel; Viegas, CarlaFungal burden have traditionally being detected by conventional culture analysis, which despite its limitations, is widely used by the scientific community. Alternatively, quantitative real-time PCR (qPCR), based on the amplification of genomic regions specific to certain fungal species, has been associated with increased sensivity, allowing the detection of dormant forms of fungi, such as spores. We present several studies where both methods were used to detect the presence of toxigenic fungi, namely Aspergillus, particularly from the Fumigati, Flavi and Circumdati sections
- Azole-Resistant Aspergillus fumigatus Harboring the TR34/L98H Mutation: First Report in Portugal in Environmental SamplesPublication . Gonçalves, Paulo; Melo, Aryse; Dias, Marta; Almeida, Beatriz; Caetano, Liliana Aranha; Veríssimo, Cristina; Viegas, Carla; Sabino, RaquelIntroduction: The frequency in detection of azole-resistant Aspergillus fumigatus isolates has increased since 2010. In Portugal, the section Fumigati is one of the most frequent, and resistant strains to have been found in clinical and environmental contexts. Although several cryptic species within the Fumigati section show intrinsic resistance to azoles, one factor driving (acquired) resistance is selective pressure deriving from the extensive use of azoles. This is particularly problematic in occupational environments where high fungal loads are expected, and where there is an increased risk of human exposure and infection, with impact on treatment success and disease outcome. The mechanisms of resistance are diverse, but mainly associated with mutations in the cyp51A gene. Despite TR34/L98H being the most frequent mutation described, it has only been detected in clinical specimens in Portugal. Methods: We analyzed 99 A. fumigatus isolates from indoor environments (healthcare facilities, spas, one dairy and one waste sorting unit) collected from January 2018 to February 2019 in different regions of Portugal. Isolates were screened for resistance to itraconazole, voriconazole and posaconazole by culture, and resistance was confirmed by broth microdilution. Sequencing of the cyp51A gene and its promoter was performed to detect mutations associated with resistance. Results: Overall, 8.1% of isolates were able to grow in the presence of at least one azole, and 3% (isolated from the air in a dairy and from filtering respiratory protective devices in a waste sorting industry) were pan-azole-resistant, bearing the TR34/L98H mutation. Conclusion: For the first time in Portugal, we report environmental isolates bearing the TR34/L98H mutation, isolated from occupational environments. Environmental surveillance of the emergence of azole-resistant A. fumigatus sensu stricto strains is needed, to ensure proper and timely implementation of control policies that may have a positive impact on public and occupational health.
- Exposure assessment in one central hospital: A multi-approach protocol to achieve an accurate risk characterizationPublication . Viegas, Carla; Almeida, Beatriz; Monteiro, Ana; Paciência, Inês; Rufo, João; Aguiar, Lívia; Lage, Bruna; Diogo Gonçalves, Lídia Maria; Caetano, Liliana Aranha; Carolino, Elisabete; Gomes, Anita Quintal; Twarużek, Magdalena; Kosicki, Robert; Grajewski, Jan; Teixeira, João Paulo; Viegas, Susana; Pereira, CristianaThe bioburden in a Hospital building originates not only from patients, visitors and staff, but is also disseminated by several indoor hospital characteristics and outdoor environmental sources. This study intends to assess the exposure to bioburden in one central Hospital with a multi-approach protocol using active and passive sampling methods. The microbial contamination was also characterized through molecular tools for toxigenic species, antifungal resistance and mycotoxins and endotoxins profile. Two cytotoxicity assays (MTT and resazurin) were conducted with two cell lines (Calu-3 and THP-1), and in vitro pro-inflammatory potential was assessed in THP-1 cell line. Out of the 15 sampling locations 33.3% did not comply with Portuguese legislation regarding bacterial contamination, whereas concerning fungal contamination 60% presented I/O > 1. Toxigenic fungal species were observed in 27% of the sampled rooms (4 out of 15) and qPCR analysis successfully amplified DNA from the Aspergillus sections Flavi and Fumigati, although mycotoxins were not detected. Growth of distinct fungal species was observed on Sabouraud dextrose agar with triazole drugs, such as Aspergillus section Versicolores on 1 mg/L VORI. The highest concentrations of endotoxins were found in settled dust samples and ranged from 5.72 to 23.0 EU.mg-1. While a considerable cytotoxic effect (cell viability < 30%) was observed in one HVAC filter sample with Calu-3 cell line, it was not observed with THP-1 cell line. In air samples a medium cytotoxic effect (61-68% cell viability) was observed in 3 out of 15 samples. The cytokine responses produced a more potent average cell response (46.8 ± 12.3 ρg/mL IL-1β; 90.8 ± 58.5 ρg/mL TNF-α) on passive samples than air samples (25.5 ± 5.2 ρg/mL IL-1β and of 19.4 ± 5.2 ρg/mL TNF-α). A multi-approach regarding parameters to assess, sampling and analysis methods should be followed to characterize the biorburden in the Hospital indoor environment. This study supports the importance of considering exposure to complex mixtures in indoor environments.
- Fungal burden exposure assessment in podiatry clinics from IrelandPublication . Viegas, Carla; Coggins, Ann Marie; Faria, Tiago; Caetano, Liliana Aranha; Quintal Gomes, Anita; Sabino, Raquel; Veríssimo, Cristina; Roberts, Nigel; Watterson, David; MacGilchrist, Claire; Fleming, Gerard T.AFungi are amongst the bioaerosols of most importance, as indicated by the growing interest in this field of research. The aim was to characterize the exposure to fungal burden in podiatry clinics using culture-based and molecular methods. Methods: Airborne fungi were collected using an impaction air sampler and surface samples were also performed. Fourteen air samples were collected for direct detection of fungal DNA from filamentous fungi and dermatophytes. Overall, 63.6 % of the evening samples and 46 % of the morning samples surpassed the threshold values (150 CFU/ m3). Molecular detection, by real time PCR, of the target fungal species/ strains (Aspergillus and Stachybotrys species) was negative for all samples collected. Trichophyton rubrum was detected by PCR analysis in one DNA sample collected on day six. Results suggest the use of both culture-based and molecular methodologies are desirable for a complete evaluation of fungal burden in this particular health care setting.
- Molecular characterization and antifungal susceptibility of Aspergillus collected in indoor settingsPublication . Simões, Daniela; Viegas, Carla; Caetano, Liliana Aranha; Veríssimo, Cristina; Sabino, RaquelIntroduction: The exposure to Aspergillus conidia is an increased risk factor for the development of respiratory symptoms, such as asthma, in occupational and non-occupational settings. Aspergillus identification should be based on molecular methods as there are species with similar morphology but distinct at the molecular level (cryptic species), with variable antifungal susceptibility profiles. The aim of this study was to perform the molecular identification of Aspergillus species collected from different occupational and non-occupational settings. Due to the emergence of azole-resistance in A. fumigatus, the susceptibility of the collected Fumigati isolates was determined. Methods: Sixty Aspergillus spp. isolates were studied. The isolates were collected from 28 samples of non-occupational settings (houses) and 32 from occupational settings (veterinary clinic, faculty environment, waste industry, dairy farms, bakery and taxis). All the isolates were plated onto malt extract agar with chloramphenicol and incubated at 27ºC, during 5 days. These isolates were identified on the basis of macro and micromorphology and also by sequencing the calmodulin gene. All the Aspergillus of the Fumigati section were screened (for azole resistance detection) using media supplemented with itraconazole, voriconazole and posaconazole. Results: From the 60 isolates, 41 (48%) were identified as belonging to the Fumigati section, 15 (25%) to the Nigri section and 1 (2%) to the Versicolores, Terrei, Clavati and Nidulantes sections. In the majority of the settings, Aspergillus fumigatus sensu stricto was the most frequently isolated species (55%). However, in the waste industry the majority of the isolates (92%) belong to the Nigri section, 82% of them being Aspergillus tubingensis. None of the tested isolates belonging to Fumigati section presented resistance to the tested azoles. Discussion: Environments contaminated with Aspergillus may be the cause or enhance respiratory problems in workers of specific settings. The most frequent species found in the studied environments was A. fumigatus sensu stricto, described as the major etiological fungal agent of respiratory infections/allergies. In the waste industry setting, however, the most frequent species found was A. tubingensis, described as less susceptible to azoles. The knowledge of the Aspergillus epidemiology in specific settings will allow a better risk assessment analysis of these environments.
- 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.