Browsing by Author "Martins, Carlos"
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- Detection of cryptic species of Aspergillus with reduced susceptibility to antifungal agents in hospitalsPublication . Sabino, Raquel; Viegas, Carla; Veríssimo, Cristina; Carolino, Elisabete; Brandão, João; Parada, Helena; Martins, Carlos; Clemons, Karl V.; Stevens, David A.Invasive aspergillosis is a fungal infection caused by Aspergillus spp. affecting mainly the immunocompromised. The mortality rate may reach 85%. Aspergillus identification should be based on molecular methods as there are species morphologically similar but distinct at the molecular level (cryptic species), with variable antifungal susceptibility profiles.
- A new cloning system based on the OprI lipoprotein for the production of recombinant bacterial cell wall-derived immunogenic formulationsPublication . Basto, Afonso P.; Piedade, João; Ramalho, Ruben; Alves, Susana; Soares, Helena; Cornelis, Pierre; Martins, Carlos; Leitão, AlexandreThe conjugation of antigens with ligands of pattern recognition receptors (PRR) is emerging as a promising strategy for the modulation of specific immunity. Here, we describe a new Escherichia coli system for the cloning and expression of heterologous antigens in fusion with the OprI lipoprotein, a TLR ligand from the Pseudomonas aeruginosa outer membrane (OM). Analysis of the OprI expressed by this system reveals a triacylated lipid moiety mainly composed by palmitic acid residues. By offering a tight regulation of expression and allowing for antigen purification by metal affinity chromatography, the new system circumvents the major drawbacks of former versions. In addition, the anchoring of OprI to the OM of the host cell is further explored for the production of novel recombinant bacterial cell wall-derived formulations (OM fragments and OM vesicles) with distinct potential for PRR activation. As an example, the African swine fever virus ORF A104R was cloned and the recombinant antigen was obtained in the three formulations. Overall, our results validate a new system suitable for the production of immunogenic formulations that can be used for the development of experimental vaccines and for studies on the modulation of acquired immunity.
- 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 improving 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 the 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 the 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.