Percorrer por autor "Dias, Marta"
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- Aspergillus spp. and azole-resistance characterization on Filtering Respiratory Protective Devices from waste sorting industryPublication . Viegas, Carla; Dias, Marta; Almeida, Beatriz; Gonçalves, Paulo; Veríssimo, Cristina; Sabino, Raquel; Aranha Caetano, LilianaStudies performed on waste management industry have reported Aspergillus as the most frequent genera on waste-sorting, incineration and composting. Filtering Respiratory Protective Devices (FRPD) are disposable after one-day use (workshift) and their use is mandatory in Portuguese waste-sorting industries. During FRPD use, humidity and temperature conditions provide a favorable environment for the growth of retained Aspergillus. The aim of this study was to characterize Aspergillus spp. presence in FRPD interior layer and exhalation valves, as well as to detect possible azole-resistant isolates in this complex indoor environment. Methods The analyzed samples consisted of 120 FRPD (interior layer and exhalation valves). Fungal load was extracted from both matrixes with 10 mL of 0.1% Tween™ 80 saline solution (NaCl 0.9%) for 30 min at 250 rpm, and 150 μL of those extracts were streaked onto malt extract agar (MEA) supplemented with chloramphenicol (0.05%) and dichloran glycerol agar (DG18). After incubation at 27 ºC for 5 to 7 days Aspergillus spp. densities (CFU/m2) were calculated, and Aspergillus sections were identified through macro and microscopic characteristics. The frequency of azole-resistance was determined by inoculation of the extracts onto screening agar plates containing Sabouraud dextrose agar media supplemented with 4 mg/L itraconazole (ITRA), 1 mg/L voriconazole (VORI), and 0.5 mg/L posaconazole (POSA), incubated at 27 °C for 5 days. Results Aspergillus spp. was detected in both interior layers (77 out of 120; 64.17%) and exhalation valves (63 out of 120; 52.5%). Among the Aspergillus genera, section Fumigati presented the highest frequency, both in exhalation valves (76.57% MEA; 87.24% DG18) and in interior layers (75.81% MEA; 51.22% DG18). Fumigati and Nigri were the Aspergillus sections isolated more frequently on MEA. In addition, Flavi, Circumdati and Candidi sections were also frequently isolated on DG18. Restricti and Aspergilli sections were observed occasionally. DG18 allowed the detection of a more diversified set of Aspergillus species than MEA (in both FRPD matrixes). In azole-supplemented media, Aspergillus spp. was the most frequently found genus on exhalation valves (75.0% of the isolates that grew onto ITRA), suggesting that resistant isolates to ITRA at the tested concentration might be present in this occupational environment. Conclusions This study reports contamination of FRPD used by workers at waste industry with Aspergillus and Aspergillus isolates exhibiting reduced susceptibility to azoles. Future trials should be performed to test the protective efficacy of FRPD and to establish deadlines for FRPD replacement. Monitoring of the establishment of azole-resistant strains in this work environment should be continued to reduce the risk of exposure and consequent development of fungal infections.
- 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.
- 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, sampling approach and the culture media should be accurately selected when dealing with Aspergillus spp. exposure assessment.