Browsing by Author "Aranha Caetano, Liliana"
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- 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.
- 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 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.
- Detection of dermatophytes in the environment of a podiatry clinicPublication . Sabino, Raquel; Aranha Caetano, Liliana; Veríssimo, Cristina; Coggins, Ann Marie; Fleming, Gerard T.; Roberts, Nigel; MacGilchrist, Claire; Viegas, CarlaObjectives: Podiatry is a healthcare profession that specializes in the management of disorders of the lower limb and foot. Podiatric treatments have the potential to generate substantial concentrations of organic dusts. Occupational exposure to bioaerosols in podiatry clinics has been studied, but it was never accessed in a deeply manner for fungi. The detection of dermatophytes in podiatric clinics is a matter of concern since the environmental presence of these fungi can contribute to spread the infection to podiatry workers and to other patients consulted in the podiatry clinics. The aim of the present study was to characterize the dermatophyte burden during podiatric activities by the use of cultural methods but also molecular methodologies for fungal DNA detection directly from the collected samples. Methods: During the period of 4 weeks, environmental samples from a podiatric clinic were collected for both conventional and molecular methodologies. For culture, 44 air samples and 39 swabs from surfaces were inoculated in Mycosel agar. Fourteen air samples from the same sampling sites were collected for direct detection of fungal DNA. Air samples ranging from 88 to 300L were collected using a calibrated impinger device (Midget ImpingeR WITH Universal Sample Pump, SKC (PA, USA), at 2.2L/min airflow rate. Five milliliters of the collection liquid was used for DNA extraction using the ZR Fungal/Bacterial DNA MiniPrep Kit Detection of dermatophytes species (in general) and Trichophyton rubrum (in particular) were both achieved by using the Dermatophyte PCR kit, (SSI Diagnostica, Herredsvejen Hillerød, Denmark). PCR amplifications were performed using 2 μL and 5 μL of the extracted DNA. Results: In the first week, 1 out of 17 samples had a positive result for Trichophyton rubrum (detected in an air sample), whereas in the remaining weeks, no dermatophytes were identified in the remaining 129 samples (weeks 2, 3 and 4). The molecular detection of dermatophytes was performed in the 14 air samples using two different DNA volumes: 2 and 5 µl. Using 2 µl of the extracted DNA, 1 out 14 samples gave a positive result for T. rubrum and none of the PCR reactions was inhibited. Using a 5 µl volume of DNA, 5 samples were positive for T. rubrum but the PCR reaction was inhibited in 6 of them (Table 1). Globally, 5 out of the 14 (36%) samples analyzed showed positive results for the detection of T. rubrum DNA. Conclusions: Giving the ratio of positive/inhibited PCR reactions, we cannot rule out the hypothesis of more positive samples. Nevertheless, the obtained results emphasize the importance of the application of new methodologies for an air quality assessment approach and reinforce the complementarity of both cultural and conventional methodologies. To our knowledge, this study presents for the first time, the application of the Dermatophyte PCR kit for dermatophyte DNA detection directly from environmental samples (air). The promising results indicate the need of optimization of this procedure specifically in this type of samples in order to use this methodology in a routine basis, for occupational and indoor air quality exposure assessments.