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- E se a retirada de um exão pudesse ter um efeito terapêutico? Caminhos de uma abordagem terapêutica molecular para uma doença rara através de exon-skippingPublication . Matos, Liliana; Gonçalves, Mariana; Santos, Juliana Inês; Coutinho, Maria Francisca; Prata, Maria João; Pires, Maria João; Oliveira, Paula; Alves, SandraAtualmente, as abordagens terapêuticas de RNA estão em grande desenvolvimento e algumas destas terapias foram já aprovadas para uso clínico. Uma das estratégias mais conhecidas tem como base a utilização de oligonucleótidos antisense (OA), pequenos oligonucleótidos sintéticos que se ligam aos RNA alvo por meio de emparelhamento de bases. Estas moléculas podem atuar de diferentes formas, sendo a diminuição da expressão génica devido à degradação pela RNase-H dos mRNA alvo e a modulação do processo de splicing para induzir a inclusão ou exclusão de um exão (exon skipping), as mais amplamente aplicadas. Particularmente para as doenças raras, diferentes aplicações de OA estão em desenvolvimento, com vários ensaios clínicos em curso e com 3 destas terapêuticas já em prática clínica. As Doenças Lisossomais de Sobrecarga (DLS) são um grupo de doenças hereditárias do metabolismo raras que podem originar uma patologia grave e progressiva devido a uma disfunção lisossomal específica. Atualmente, não existe cura para as DLS, embora para algumas destas doenças haja estratégias de tratamento. Assim, as abordagens terapêuticas de RNA podem representar uma importante estratégia terapêutica alternativa ou adjuvante. O grupo de investigação em DLS do Departamento de Genética Humana do INSA – Porto, tem contribuído para o desenvolvimento de novas abordagens terapêuticas de RNA tendo sido já obtidos resultados positivos para diferentes DLS. Para a validação pré-clínica de novas terapias, tanto os modelos in vitro como in vivo são necessários e largamente utilizados. Num estudo anterior efetuado in vitro, para a mutação c.3503_3504delTC no gene GNPTAB, que origina a Mucolipidose tipo II α/β, foi testada uma estratégia terapêutica de exon-skipping utilizando diferentes OA em fibroblastos de doentes, tendo resultado na produção de um mRNA in-frame1. Neste estudo, pretendeu-se avaliar o potencial terapêutico desta abordagem tanto in vitro (fibroblastos de murganho C57BL/6) como in vivo (murganhos C57BL/6) e os resultados preliminares obtidos serão apresentados e discutidos. 1. Matos L, Vilela R, Rocha M, et al. Development of an antisense oligonucleotide-mediated exon skipping therapeutic strategy for Mucolipidosis II: validation at RNA level. Hum Gene Ther, 2020, 31(13-14):775-783.
- Cardiovascular risk estimation and management in Familial Hypercholesterolemia patientsPublication . Chora, J.R.; Medeiros, A.M.; Alves, A.C.; Bourbon, M.Objectives and study samples: - Estimate cardiovascular disease (CVD) risk; - What are the lipid-lowering therapy (LLT) strategies; - How many are reaching LDL-C targets; … in Familial Hypercholesterolemia (FH) patients and in the Portuguese general population
- A Cross-Sectional Study of the Street Foods Purchased by Customers in Urban Areas of Central AsiaPublication . Sousa, S.; Lança de Morais, Inês; Albuquerque, Gabriela; Gelormini, Marcello; Casal, Susana; Pinho, Olivia; Motta, Carla; Damasceno, Albertino; Moreira, Pedro; Breda, João; Lunet, Nuno; Padrão, PatriciaThis study aimed to describe street food purchases in cities from Central Asia, considering customers’ characteristics and the nutritional composition of the foods and beverages. Cross-sectional studies were conducted in 2016/2017 in Dushanbe (Tajikistan), Bishkek (Kyrgyzstan), Ashgabat (Turkmenistan) and Almaty (Kazakhstan). Direct observation was used to collect data on the purchases made by street food customers, selected by random and systematic sampling. Nutritional composition was estimated using data from chemical analyses, food composition tables or food labels. A total of 714 customers (56.6% females, 55.5% aged ≥35 years, 23.3% overweight/obese) were observed, who bought 852 foods and beverages, the most frequent being savoury pastries/snacks (23.2%), main dishes (19.0%), sweet pastries/confectionery (17.9%), tea/coffee (11.3%) and soft drinks/juices (9.8%). Fruit was the least purchased food (1.1%). Nearly one-third of customers purchased industrial food items (31.9%). The median energy content of a street food purchase was 529 kcal/serving. Saturated and trans-fat median contents were 4.7 g/serving and 0.36 g/serving (21.4% and 16.5% of maximum daily intake recommendations, respectively). Median sodium and potassium contents were 745 mg/serving (37.3% of maximum recommendation) and 304 mg/serving (8.7% of minimum recommendation), respectively. In general, the purchases observed presented high contents of energy, saturated-fat, trans-fat and sodium, and low levels of potassium. Policies towards the improvement of these urban food environments should be encouraged.
- Results of Portuguese Extra-Analytical Evaluation on SARS-CoV-2 in 2020 schemesPublication . Ventura, Catarina; Cardoso, Ana; Correia, Helena; Guiomar, Raquel; Melo, Aryse; Faria, Ana PaulaThe Portuguese National External Quality Assessment Program (PNAEQ) assembles 3 schemes of extra-analytical issues since 2007 (Pre and Post-Analytical) and 2010 (Laboratory Safety). In 2020, due to the new Coronavirus (SARS-CoV-2), PNAEQ introduced a new SARS-CoV-2 molecular detection scheme with two rounds (May and November). In addition, were included questions in the 3 extra-analytical schemes concerning SARS-CoV-2 and added questions in the 2nd round of molecular detection in collaboration of the World Health Organization. The purpose was checking if laboratories implemented the biological safety recommendations at the entire examination process, including pre- and post-examination procedures. This study aims to evaluate PNAEQ participants in their ability to be aware to the laboratory diagnosis of SARS-CoV-2 in all extra-analytical areas.
- Infográfico - Acidentes Domésticos e de Lazer : Queimadura (crianças e jovens dos 0-18 anos)Publication . Departamento de EpidemiologiaAtendendo a que as Queimaduras não fatais são consideradas uma importante causa de morbilidade a nível mundial, pretende-se destacar esta problemática nas vítimas mais jovens, locais onde ocorrem com maior frequência e principais produtos e, ou, objetos envolvidos. O infográfico apresentado tem como fonte de dados o sistema EVITA – Epidemiologia e Vigilância dos Traumatismos e Acidentes, coordenado pelo Departamento de Epidemiologia do INSA, resultando de informação proveniente das notificações recebidas através da rede de Hospitais do Serviço Nacional de Saúde (SNS) com serviço de urgência que integraram o sistema EVITA durante os anos de 2019 e 2020. Criado em 2000, este instrumento de observação permite obter dados e informação adicional à informação clínica relevante para a monitorização e vigilância dos acidentes que ocorrem em ambiente doméstico, lazer, escolar e desportivo na população portuguesa, que implicaram recurso às urgências de unidades de saúde do SNS. As vítimas são caracterizadas quanto às situações, circunstâncias do local da ocorrência e das lesões sofridas, assim como, quanto aos agentes envolvidos.
- Occupational exposure to metals and PAHs: combining literature-based exposure and in vitro hazard data towards a mixture risk assessment a mixture risk assessmentPublication . Tavares, Ana Maria; Alves, Inês; Moreira, Rodrigo; Louro, Henriqueta; Ladeira, Carina; Viegas, Susana; Loureiro, Susana; Santonen, Tiina; Göen, Thomas; Kortenkamp, Andrew; Luijten, Mirjam; Silva, Maria JoãoThe environment within industrial settings is commonly characterized by the existence of a complex mixture of chemicals from different raw materials and transformation processes. Occupational co-exposure to chromium (Cr), Nickel (Ni), and Polycyclic Aromatic Hydrocarbons (PAHs) may occur in diverse workplaces, such as the aeronautic and waste management, (e.g incineration) sectors. Such co-exposure rais-es concern in terms of occupational health, as these substances are recognized lung carcinogens and mainly act by genotoxic mechanisms, increasing the likelihood of interactive toxic effects. The fact that cur-rent regulatory practices are usually focused on single chemical sub-stances, without integrating the possibility of combined or aggregated exposures and effects, may lead to a risk underestimation.In this work, developed under the scope of HBM4EU Initiative (https://www.hbm4eu.eu), a literature-based mixture risk assess-ment (MRA) exercise for occupational exposure to metals and PAHs was performed. In addition, in vitro toxicity data was obtained for the same mixtures to provide support to its hazard assessment.Human biomonitoring (HBM) data on Cr(VI), Ni and/or PAHs was extracted from occupational studies conducted in the European Un-ion and searched in literature databases. Selected reference values were used to calculate risk quotients (RQ) for each substance based on the retrieved exposure data; the combined risk was given by the sum of the RQ, i.e., the Background Exposure Exceedance Score(BEES).In parallel, we evaluated the combined cyto- and genotoxicity of the same chemicals (assessed by the MTT and micronucleus assays) in the human alveolar A549 cell line.In most of the analysed studies, we observed that BEES levels, estimated from the exposure to metals mixture or to metals and PAHs, exceeded RQ levels considered acceptable for the individual substances. Only two studies, conducted in hazard waste incinerator settings, presented urinary exposure levels for the three substances. They showed a value of BEES of concern (>1) for all exposure sce-narios, even for workers performing activities considered of low ex-posure or no-exposure, such as laboratory and administrative work-ers. In vitro assays supported that A549 cells exposure to these substances resulted in interactive cytotoxic and genotoxic effects that may underlie health effects different from those predicted from single exposures.Our findings show the limitations of applying occupational expo-sure reference values defined on a single substance basis to work-places where exposure to chemical mixtures occur, highlighting the relevance of performing MRA as a more realistic approach to guide suitable risk management measures in occupational settings
- Eumycetoma due to Parathyridaria percutânea in an immunosuppressed patient - an imported case in PortugalPublication . Fraga, Marta; Carvalho, Dinah; Sabino, Raquel; Veríssimo, Cristina; Simões, Helena; Sapinho, Guilherme; Alho, Ana; Espada, Eduardo; Moreno, Raul; Forjaz de Lacerda, J.; Marques Lito, L.; Melo Cristino, J.Objectives: Eumycetoma is a chronic subcutaneous fungal infection characterized by swelling, fistulization and discharge of fungal granules. These infections occur after traumatic inoculation and progress slowly over months or even years into a chronic form. Parathyridaria percutanea is a rare cause of subcutaneous phaeohyphomycoses and there are no published reports of discharging sinus or granuloma formation due to this fungus. We present a rare case of eumycetoma due to Parathyridaria percutanea in a 10-years-old patient from Angola, presenting after allogeneic hematopoietic stem cell transplant (alloHSCT) for severe aplastic anemia. Material and methods: A 10-year-old boy from Angola with severe aplastic anemia was transferred to Portugal and submitted to alloHSCT from a mismatched unrelated donor at a single HLA-locus (9/10). After secondary graft failure due to post-transplant hemophagocytic lymphohistiocytosis, he underwent a second alloHSCT, without engraftment. Twenty-three days after the 3rd alloHSCT from the same donor and seven days under prophylaxis with posaconazole, the patient presented with febrile neutropenia with no clear focus. He complained of painless swelling of the right knee, with slight fluctuation. An ultrasound was performed revealing a heterogeneous hypoechogenic area with imprecise contours, inside of which there was a nodular formation of approximately 10 mm. Two weeks later, a spontaneous drainage of a grossly spherical grain from the right knee occurred. It was characterized as extra-articular, subcutaneous with filamentous aspect. The material was sent to the laboratory where its macroscopic observation allowed the identification of a granule of possible mycotic etiology, consistent with the diagnosis of eumycetoma. This sample was promptly processed for bacteriology (aerobic and anaerobic) and mycology studies. Results: Direct microscopic examination of the grain revealed septate hyphae with irregular hyphal swellings. Cultures for bacteria (aerobic and anaerobic) were sterile. After 1 week, slow growing colonies appeared on Sabouraud dextrose agar (SDA) incubated at 25 °C. Those colonies were flat, spreading with sparse aerial hyphae, become creamy after 5 to 10 days and with orange reverse and then turned dark brown after 4 weeks. Lactophenol cotton blue mount revealed nonsporulating dematiceous hyphae with clamidospores. Phenotypic identification of the organism was uncertain but sequencing of the internal transcribed spacer (ITS) region of ribosomal DNA identified as Parathyridaria percutanea (100% homology, 99% coverage). The isolate showed in vitro resistance to itraconazole, fluconazole and anidulofungin and susceptibility to posaconazole, voriconazole and amphotericin B. As this pathogen is extremely rare, no antifungal guidelines have been proposed so far. The patient received dual coverage with posaconazole and liposomal amphotericin-B but died 2 weeks later of complications of the underlying disease. Conclusions: The presented case maybe the first described case of eumycetoma caused by Parathyridaria percutanea associated with fistulae presentation. Molecular identification of rare fungi is essential, especially when they are poorly sporulated, as in this case. Moreover, rare fungi can present with odd clinical signs, as in this case, emphasizing the relevance of a polyphasic approach to identify the etiological agent.
- A study of quality assessment in clinical parasitology for detection of spiked specimens with Schistosoma mansoniPublication . Aldeia, Cláudia; Correia, Helena; Silva, Susana Pereira; Faria, Ana Paula; Botelho, Mónica; Baptista Fernandes, Teresa; Moutinho, Maria Guilhermina; Santos Costa, Quirina; Santos Gomes, Gabriela; Júlio, CláudiaPNAEQ’s Parasitic Morphology of the National Health Institute Doutor Ricardo Jorge runs three External Quality Assessment (EQA) surveys per year, for microscopy-based detection of parasites. Human-infecting trematode, Schistosoma mansoni, widely occurs in geographical regions across sub-Saharan Africa, South American countries, the Caribbean and the Middle East. In order to mimic patient-like samples, S.mansoni extracted eggs from mice liver, were used to spike-in negative human faeces. Aims: Prepare negative stool specimens spiked with S. mansoni eggs for EQA testing; Evaluate the spiked sample homogeneity and stability; Assess the performance of participant laboratories.
- Isolation of Rasamsonia argillacea species complex in a Cystic Fibrosis Adult Patient - First Case in PortugalPublication . Carvalho, Dinah; Sabino, Raquel; Veríssimo, Cristina; Simões, Helena; Azevedo, Pilar; Marques Lito, Luis; Melo Cristino, JoséObjectives: Cystic fibrosis (CF) is the most common monogenetic autosomal reces sive disease in the human population. An important fungal biota has been described in respiratory secretions of patients suffering from CF being Aspergillus fumigatus and Candida albicans the most common fungi found. We report the isolation, for the first time in Portugal, of the emerging fungal pathogen Rasamsonia argillacea species complex, from a respiratory sample of an adult patient with CF. Material & Methods: A 51-year-old male patient with heterozygous CF due to muatations ΔF508/P205S, is being followed in Cystic Fibrosis Reference Center for about 10 years. In the last years, he has been consistently colonized with Methicilin-susceptible Staphylococcus aureus, Pseudomonas aeruginosa and Aspergillus section Fumigati whereby is under chronic suppression therapy with two inhaled antibiotics. Recently, there has been a progressive clinical respiratory functional deterioration. In a periodic evaluation, a miccrobiology control sputum was requested. Sample was cultured, in parallel, for bacteriol ogy and mycology evaluation. Results: Apart from detection of S. aureus and Raoultella ornithinolytica, after 3-5 days of incubation at 37ºC, the cultures showed several cream-coloured colonies, powdery to velvety. Microscopic examination showed hyaline septate hyphae, Penicillium-like conid iophores with rough wall, ovoid to cylindrical phyalides with a narrow neck and cylin drical unicellular smooth-walled microconidia, arranged in unbranched basipetal chains arising from phialides. The isolate was identified as Rasamsonia argillacea species complex based on its mor phology and confirmed by MALDI-TOF mass spectrometry. As no septate hyphae were seen on direct examination, a new sample was requested to exclude extrinsic contamina tion. The second sample was inoculated as previously, confirming the persistent presence of Rasamsonia argillacea species complex in the sputum of this patient. The identification of this isolate was further confirmed by sequencing the internal transcribed spacer (ITS) region of ribosomal DNA, showing 100% homology with sequences deposited on data bases. Antifungal susceptibility testing showed high minimal inhibitory concentrations (MIC) to almost all tested antifungals (posaconazole, voriconazole, amphotericin B) and low MIC to anidulafungin. The patient had no great exacerbation of his respiratory prob lems and the isolated fungus was interpreted as colonization being the patient under more frequent surveillance. Conclusion: Although colonization of the upper respiratory tract in CF patients by R. argillacea species complex has been described as an emerging situation, the role of these fungi in clinical or functional deterioration of the disease remains controversial. Indeed, data about its real prevalence in the CF population are lacking. However, taking into ac count the ability of this species to predominantly affect the lungs, to induce pneumonia and to disseminate to adjacent organs or even to the central nervous system (CNS) in imunocompromised patients, it is essential to promote its accurate identification that is often misidentified as Penicillium spp. or Paecilomyces species. Antifungal susceptibility testing should be performed for epidemiological purposes and to guide therapy, as Rasamsonia spp. usually presents a marked antifungal resistance profile
- Evaluation of terbinafine resistance of Trichophyton rubrum and T. interdigitale in Portugal and characterization of the associated resistance mechanismsPublication . Henriques, Camila; Simões, Helena; Veríssimo, Cristina; Sabino, RaquelObjectives: Superficial mycoses caused by Trichophyton rubrum are among the most common infections worldwide. In Portugal, it is estimated that 1,510,391 of Portuguese suffer from dermatophytosis, corresponding to an incidence of 14,300 per 1,000,000 inhabitants. Superficial mycoses caused by Trichophyton rubrum are among the most common infections worldwide, being difficult to treat and often associated with recurrences after interruption of the antifungal therapy. Terbinafine is one of the allylamine antifungal agents whose target is squalene epoxidase (SQLE). This agent has been extensively used in the therapy of dermatophytes’ infections. The emergence of resistance to terbinafine in Trichophyton species has been recently described and is associated with point mutations in the SQLE gene. The increasing number of patients with Tinea pedis or Tinea unguium resistant to terbinafine treatment prompted us to screen the terbinafine resistance in Trichophyton isolates obtained by culture at the Mycology Reference Laboratory of the National Institute of Health Doctor Ricardo Jorge. We aimed to determine the frequency of terbinafine resistance and associated mechanisms of resistance. Materials & Methods: Dermatophytes collected during 2017–2020 were identified by culture and mass spectrometry (MALDI-TOF-MS) and/or by sequencing the ITS (Internal Transcribed Spacers) region of the rDNA. All isolates were grown onto agar supplemented with terbinafine (0.06 and 0.125 mg/L) in order to detect potential resistant isolates. Antifungal susceptibility testing was performed following CLSI M38A2 broth microdilution method. The squalene epoxidase gene (SQLE) was sequenced and mutations described as conferring resistance to terbinafine were screened. Results: Among 102 T. rubrum and 17 of T. interdigitale isolates that identified and further tested in what concerns to their antifungal susceptibility, 1 T. rubrum isolate (≈1%) and 3 T. interdigitale isolates (≈18%) showed to be resistant to terbinafine by screening agar. According to the microdilution method, 2.44% of the isolates were less susceptible to terbinafine. Overall, three isolates (1 T. rubrum and 2 T. interdigitale) showed high terbinafine resistance (MICs, 4 to ≥ 8 mg/L) and one isolate (T. interdigitale) displayed moderate terbinafine resistance (1 to 2 mg/L). The sequencing of the SQLE gene allowed the detection of the following single point mutations: in one T. rubrum and in one T. interdigitale, at the position 1189 in the ORF of the SQLE gene (corresponding to a substitution of a phenylalanine at the position 397 by an isoleucine or by a leucine, respectively). In the remaining resistant T. interdigitale isolates, no mutations were found in the gene encoding SQLE. Although the most common substitutions that lead to higher resistance to terbinafine are L393F and F397L, the F397I substitution detected in our study suggests to be associated with high terbinafine resistance. Conclusions: This study allowed us to detect, for the first time in Portugal, Trichophyton isolates resistant to terbinafine. It was also possible to estimate, within the studied isolates, a resistance frequency of 2.4%. Taken together, our results prompt the current knowledge about the necessity of antifungal susceptibility testing to select effective strategies for management of clinical cases of dermatophytosis not only in Portugal but worldwide.