Browsing by Author "Francisco, Mariana"
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- 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.
- New approach to study the real exposure to fungi in cork industry: nasal swabs mycobiota investigation coupled with screening on fungal resistance to azolesPublication . Viegas, Carla; Clérigo, Anália; Pacífico, Cátia; Faria, Tiago; Sabino, Raquel; Francisco, Mariana; Verissimo, Cristina; Viegas, SusanaThe presence of fungi belonging to the Penicillium glabrum complex has been associated with the development of respiratory diseases such as suberosis, one of the most prevalent diseases among workers from cork industries, besides occupational asthma. Azoles are used as pesticides but also the first line therapy in the treatment of Aspergillus infections; azole-resistance as been described as to have also an environmental source and is considered an emerging public health problem. The aim of this work was to characterize fungal distribution and to evaluate the presence of azole-resistant Aspergillus isolates in nose swab samples from the cork industry workers.
- Trends on Aspergillus Epidemiology-Perspectives from a National Reference Laboratory Surveillance ProgramPublication . Sabino, Raquel; Gonçalves, Paulo; Martins Melo, Aryse; Simões, Daniela; Oliveira, Mariana; Francisco, Mariana; Viegas, Carla; Carvalho, Dinah; Martins, Carlos; Ferreira, Teresa; Toscano, Cristina; Simões, Helena; Veríssimo, CristinaIdentification of Aspergillus to species level is important since sibling species may display variable susceptibilities to multiple antifungal drugs and also because correct identification contributes to improve the knowledge of epidemiological studies. Two retrospective laboratory studies were conducted on Aspergillus surveillance at the Portuguese National Mycology Reference Laboratory. The first, covering the period 2017-2018, aimed to study the molecular epidemiology of 256 Aspergillus isolates obtained from patients with respiratory, subcutaneous, or systemic infections and from environmental samples. The second, using our entire collection of clinical and environmental A. fumigatus isolates (N = 337), collected between 2012 and 2019, aimed to determine the frequency of azole-resistant A. fumigatus isolates. Aspergillus fumigatus sensu stricto was the most frequent species in both clinical and environmental samples. Overall, and considering all Aspergillus sections identified, a high frequency of cryptic species was detected, based on beta-tubulin or calmodulin sequencing (37% in clinical and 51% in environmental isolates). Regarding all Fumigati isolates recovered from 2012-2019, the frequency of cryptic species was 5.3% (18/337), with the identification of A. felis (complex), A. lentulus, A. udagawae, A. hiratsukae, and A. oerlinghauensis. To determine the frequency of azole resistance of A. fumigatus, isolates were screened for azole resistance using azole-agars, and 53 possible resistant isolates were tested by the CLSI microdilution reference method. Nine A. fumigatus sensu stricto and six Fumigati cryptic isolates showed high minimal inhibitory concentrations to itraconazole, voriconazole, and/or posaconazole. Real-time PCR to detect cyp51A mutations and sequencing of cyp51A gene and its promoter were performed. The overall frequency of resistance to azoles in A. fumigatus sensu stricto was 3.0%. With this retrospective analysis, we were able to detect one azole-resistant G54R mutant A. fumigatus environmental isolate, collected in 2015. The TR34/L98H mutation, linked to environmental transmission route of azole resistance, was the most frequently detected mutation (N = 4; 1.4%). Our findings underline the demand for correct identification and susceptibility testing of Aspergillus isolates.