Percorrer por autor "Viegas, Carla"
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- Addressing Critical Fungal Pathogens Under a One Health Perspective: Key Insights from the Portuguese Association of Medical MycologyPublication . Sabino, Raquel; Antunes, Francisco; Araujo, Ricardo; Bezerra, A. R.; Brandão, João; Carneiro, Carla; Carvalho, Agostinho; Carvalho, D.; Conceição, I.C.; Cota-Medeiros, Fábio; Cruz, C.; Duarte, Elsa Leclerc; Holum, S.; Matos, O.; Maltez, F.; Mendonça, Alexandre; Moura, G.; Pereira, A.; Fortuna R., Celia; Teixeira, Pedro; de Valdoleiros, Sofia R.; Verissímo, Cristina; Viegas, Carla; Portuguese Association of Medical Mycology (ASPOMM); CarneiroFungal infections have emerged as a significant public health concern, especially with the increasing incidence of severe mycoses caused by pathogens such as Aspergillus fumigatus, Candida auris, Candida albicans, and Cryptococcus neoformans. These fungi, listed as critical priorities by the World Health Organization, pose a heightened risk due to rising antifungal resistance and their severe impact on immunocompromised individuals. This article, coordinated by the Portuguese Association of Medical Mycology, highlights the importance of adopting a One Health perspective to address fungal threats comprehensively. Drawing on interdisciplinary collaboration, the association aims to foster greater awareness, improve diagnostic capabilities, and stimulate research and public health policies in Portugal but also at global level. The paper outlines key strategies for surveillance, prevention, and innovation in fungal diagnostics and therapeutics. Moreover, it emphasizes the urgent need for national coordination and international cooperation in managing fungal infections, advocating for integrative approaches that link human, animal, and environmental health. By presenting aconsolidated overview of current challenges and future priorities, this work seeks to enhance preparedness and response mechanisms in the face of escalating fungal threats.
- Algorithm to assess the presence of Aspergillus fumigatus resistant strains: The case of Norwegian sawmillsPublication . Viegas, Carla; Almeida, Beatriz; Aranha Caetano, Liliana; Afanou, Anani; Straumfors, Anne; Veríssimo, Cristina; Gonçalves, Paulo; Sabino, RaquelAssociation between selection pressure caused by the use of azole fungicides in sawmills and the development of fungal resistance has been described. The aim of this study was to implement an algorithm to assess the presence of Aspergillus section Fumigati resistant strains in sawmills. Eighty-six full-shift inhalable dust samples were collected from eleven industrial sawmills in Norway. Different culture media were used and molecular identification to species level in Aspergillus section Fumigati was done by calmodulin sequencing and TR34/L98H and TR46/Y121F/T289A mutations were screened by real-time PCR assay and confirmed by cyp51A sequencing. Six Fumigati isolates were identified as A. fumigatus sensu stricto and two of these grew on azole-supplemented media and were further analyzed by real-time PCR. One was confirmed to be a TR34/L98H mutant. The obtained results reinforce the need to assess the presence of A. fumigatus sensu stricto resistant isolates at other workplaces with fungicide pressure.
- Algorithm to assess the presence of Aspergillus fumigatus resistant strains: The case of Norwegian sawmillsPublication . Viegas, Carla; Almeida, Beatriz; Aranha Caetano, Liliana; Afanou, Anani; Straumfors, Anne; Veríssimo, Cristina; Gonçalves, Paulo; Sabino, RaquelAssociation between selection pressure caused by the use of azole fungicides in sawmills and the development of fungal resistance has been described. The aim of this study was to implement an algorithm to assess the presence of Aspergillus section Fumigati resistant strains in sawmills. Eighty-six full-shift inhalable dust samples were collected from eleven industrial sawmills in Norway. Different culture media were used and molecular identification to species level in Aspergillus section Fumigati was done by calmodulin sequencing and TR34/L98H and TR46/Y121F/T289A mutations were screened by real-time PCR assay and confirmed by cyp51A sequencing. Six Fumigati isolates were identified as A. fumigatus sensu stricto and two of these grew on azole-supplemented media and were further analyzed by real-time PCR. One was confirmed to be a TR34/L98H mutant. The obtained results reinforce the need to assess the presence of A. fumigatus sensu stricto resistant isolates at other workplaces with fungicide pressure.
- 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
- Analysis of surfaces for characterization of fungal burden - Does it matter?Publication . Viegas, Carla; Faria, Tiago; Meneses, Márcia; Carolino, Elisabete; Viegas, Susana; Gomes, Anita Quintal; Sabino, RaquelMycological contamination of occupational environments can be a result of fungal spores' dispersion in the air and on surfaces. Therefore, it is very important to assess it in both types of the samples. In the present study we assessed fungal contamination in the air and in the surface samples to show relevance of surfaces sampling in complementing the results obtained in the air samples.
- Aspergillus collected in specific indoor settings: their molecular identification and susceptibility patternPublication . Simões, Daniela; Aranha Caetano, Liliana; Veríssimo, Cristina; Viegas, Carla; Sabino, RaquelExposure to Aspergillus conidia is an increased risk factor for the development of respiratory symptoms. The emergence of azole resistance in Aspergillus fumigatus is a major concern for the scientific community. The aim of this study was to perform the molecular identification of Aspergillus species collected from different occupational and non-occupational indoor settings and to study the azole susceptibility profile of the collected Fumigati isolates. The selected Aspergillus isolates were identified as belonging to the sections Fumigati, Nigri Versicolores, Terrei, Clavati and Nidulantes. All the Aspergillus fumigatus were screened for azole resistance using an agar media supplemented with itraconazole, voriconazole and posaconazole. None of the tested isolates showed resistance to those azoles. Knowledge of Aspergillus epidemiology in specific indoor environments allows a better risk characterization regarding Aspergillus burden. This study allowed the analysis of the molecular epidemiology and the determination of the susceptibility pattern of Aspergillus section Fumigati found in the studied indoor settings.
- Aspergillus em ambiente hospitalar: um risco para o desenvolvimento de infeções nosocomiais?Publication . Raquel, Sabino; Veríssimo, Cristina; Viegas, Carla; Brandão, João; Parada, Helena; Martins, Carlos; Furtado, Cristina; Clemons, Karl V.; Stevens, David A.
- Aspergillus section Fumigati – Epidemiological trends - A perspective from a National Reference LaboratoryPublication . Sabino, Raquel; Simões, Helena; Francisco, Mariana; Viegas, Carla; Toscano, Cristina; Batista, JuditeTeresa; Ferreira, Teresa; Veríssimo, CristinaObjectives: Aspergillus fumigatus is the most frequent agent of aspergilosis and reports on infections caused by this species or its siblings are becoming more frequent, together with the increasing number of at risk patients. Nowadays, due to the rising concerns on emerging antifungal resistance, the epidemiological surveillance for clinical and environmental isolates is mandatory. The overall objective of the project is to understand the epidemiology of the Aspergillus isolates (species and antifungal resistance) collected in the Portuguese National Reference Laboratory through our surveillance system on Aspergillus. Methods: During the period 2013-2017, 117 Aspergillus section Fumigati isolates were collected at the National Health Reference Dr. Ricardo Jorge, through the surveillance system on Aspergillus. Isolates were obtained from different patient samples from 15 healthcare institutions of all country, and from different environmental sources (air or surfaces sampling). All isolates were plated for growth as single colonies on malt extract agar with chloramphenicol. These isolates were identified on the basis of macro and microscopic morphology and through the use of molecular tools. Genomic DNA was prepared from each isolate and the sequencing of the Internal Transcribed Spacers (ITS) regions as well as of the gene codifying to calmodulin. Surveillance of azole resistance was performed firstly using Sabouraud dextrose agar supplemented with itraconazole (ICZ), voriconazole (VCZ), and posaconazole (PCZ). When growth was observed, the minimal inhibitory concentration (MIC) was determined by broth microdilution method. In case of doubt, a specific PCR for detection of mutations in the Cyp51A gene of A. fumigatus was performed using the AsperGenius® multiplex real-time PCR assay. Results: From the isolates collected during the study period, 94 were from clinical (human) sources, 2 from animals diagnosed with aspergillosis and 21 from environmental sources Clinical isolates were obtained from 90 patients (53 males, 34 females, 3 not known), with ages ranging from 37 days to 88 years old. Most of these isolates (98%) were from respiratory specimens. The underlying diseases reported are, among others, cystic fibrosis, COPD, HIV, asthma, and neoplasms. In total, 111 A. fumigatus sensu stricto isolates were identified, followed by 3 A. lentulus, 2 A. felis and 1 A. hiratsukae (from hospital environment). In 7 cases, the morphological identification did not matched with the correct species-section. Interestingly, the 5 clinical cryptic species were from the same hospital. Regarding susceptibility, relevant and residual growths were obtained in azole resistance screening media (Table 1). The positive results were then screened by microdilutions and by detection of Cyp51A mutations and resistances were not confirmed. Conclusions: The understanding of local resistance patterns is valuable to assess shifts in the epidemiology of Aspergillus (and therefore, to manage therapeutic approaches). In our collection of Fumigati isolates, 5% of them were cryptic species. Although we did not confirm azole resistance by microdilution or detection of Cyp51A mutations, the MIC values obtained suggest that the median values are higher than what is described in other studies (1.4 to ICZ, 0.4 to PCZ),which may explain the growth in screening media and may suggest a local epidemiology.
- 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.