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- Contributo dos genes NOS e ECA para a suscetibilidade de elevar a glicemia em hipertensosPublication . Aguiar, Laura; Semente, Ildegário; Ferreira, Joana; Matos, Andreia; Mascarenhas, Mário Rui; Menezes Falcão, Luíz; Faustino, Paula; Bicho, Manuel; Inácio, ÂngelaIntrodução: A Hipertensão Arterial (HTA) é um fator de risco cardiovascular muito prevalente em Portugal. Esta patologia é multifatorial, envolvendo fatores genéticos e ambientais. A resistência à insulina e a hipertensão são componentes do síndroma metabólico e frequentemente coexistem. Níveis aumentados de glicemia associados a hipertensão aumentam significativamente o risco de doença cardiovascular. Objetivo: Este estudo teve como objetivo investigar a potencial implicação de polimorfismos genéticos nos genes da sintase do óxido nítrico endotelial (eNOS) e da enzima conversora da angiotensina (ECA) no desenvolvimento da HTA e hiperglicemia, na população portuguesa. Material e métodos: Foi realizado um estudo de caso-controlo para uma amostra de 377 indivíduos portugueses, dos quais 243 hipertensos e 134 normotensos. As análises polimórficas do número variável de repetições em tandem (VNTR) no intrão 4 (repetição em tandem de 27 pb) do gene eNOS e do polimorfismo ECA I/D (inserção/ deleção) foram realizadas por reação em cadeia da polimerase (PCR). Todas as análises estatísticas foram realizadas recorrendo ao software SPSS, versão 24.0, sendo o nível de significância estatística estabelecido para p <0,05. Resultados: Encontrou-se uma associação entre o alelo 4a do gene eNOS com a hipertensão (p =0,001), verificando-se ainda que na hipertensão, a presença do alelo 4a está associada a valores superiores de HbA1c (p=0,031). Em relação ao gene ECA, não se encontram diferenças estatisticamente significativas entre doentes e controlos, contudo verificou-se que a presença do alelo D em hipertensos está associada a níveis mais elevados de glicemia (p=0,017). Conclusão: Os nossos resultados mostram uma associação entre os genes eNOS e ECA com fenótipos clínicos associados a hiperglicemia, na população portuguesa. Indivíduos com níveis elevados de glicemia têm maior risco de desenvolver hipertensão. A identificação de polimorfismos genéticos que possam influenciar o desenvolvimento e gravidade da HTA, pode permitir um diagnóstico mais precoce e específico, que pode proporcionar melhores estratégias terapêuticas e de prevenção, para esta doença tão prevalente em Portugal.
- Avaliação da exposição das mulheres portuguesas em idade fértil ao metilmercúrio: um estudo preliminar de biomonitorização humanaPublication . Santiago, Susana; Assunção, Ricardo Manuel Abreu de; Carvalho, Cristina Maria LeitãoO metilmercúrio (MeHg) é um composto tóxico para os seres humanos sendo que o consumo de peixe é habitualmente considerado a principal fonte de exposição humana. As espécies de peixe predadoras, como o atum ou o espadarte, assumem particular importância neste contexto, devido maioritariamente aos processos de bioacumulação e biomagnificação. Portugal apresenta o maior consumo de produtos da pesca e da aquicultura na União Europeia (UE), acima da média da UE e do mundo. O principal alvo da toxicidade associada ao MeHg é o sistema nervoso central e o período pré-natal representa um momento de maior vulnerabilidade relativamente aos potenciais efeitos no neurodesenvolvimento do feto. A avaliação da exposição da população bem como do risco associado deverá ser feita de forma sistemática e adequada por forma a estabelecer medidas, quando necessárias, que previnam os potenciais efeitos tóxicos decorrentes desta exposição. A biomonitorização humana (BMH) permite avaliar, de forma direta, a exposição humana a compostos químicos e desta forma caracterizar o risco associado. A concentração total de mercúrio no sangue é geralmente considerada um biomarcador adequado para estimar a exposição interna a curto prazo ao MeHg, em indivíduos com consumo regular de pescado. O presente estudo teve como objetivo avaliar a exposição ao MeHg de mulheres portuguesas em idade fértil através da BMH, atendendo a que este grupo populacional apresenta elevada suscetibilidade a este composto, considerando os potenciais efeitos tóxicos na descendência. Para este estudo, 300 mulheres portuguesas em idade fértil (25 a 44 anos) foram selecionadas aleatoriamente entre as participantes de um estudo epidemiológico transversal realizado em Portugal (INSEF, http://www.insef.pt/). Foi determinada a concentração de mercúrio total nas amostras de sangue total das participantes por espectrofotometria de absorção atómica com decomposição térmica e amalgamação (TDA/AAS), após validação do método de ensaio. Duas amostras apresentaram concentração de mercúrio total abaixo do limite de quantificação (0,5 μg/L) e, nas restantes (n = 298), os níveis de mercúrio variaram de 0,6 a 35,0 μg/L. Cerca de 52% das amostras apresentaram valores abaixo de 5 μg/L, um valor de BMH abaixo do qual não são esperados efeitos adversos na saúde. No entanto, 48% das amostras revelaram níveis acima de 5 μg/L e, portanto, apresentaram risco de efeitos adversos para a saúde. Os valores obtidos de concentração de mercúrio no sangue foram superiores nas mulheres com mais idade, maior nível de escolaridade e residentes na Região Autónoma da Madeira, com diferenças estatisticamente significativas relativamente aos restantes grupos (p<0,05).Este estudo reforça a necessidade de desenvolver e implementar em Portugal estratégias de comunicação de risco focadas na seleção de espécies de peixes com menor concentração de MeHg, a fim de evitar a exposição humana a esse composto, principalmente em populações particularmente suscetíveis.
- Dietary exposure to aflatoxins in the Portuguese population – the use of biomonitoring data to assess the associated burdenPublication . Martins, Carla; Vidal, Arnau; De Boevre, M.; De Saeger, Sarah; Nunes, Carla; Torres, Duarte; Goios, Ana; Lopes, Carla; Alvito, Paula; Assunção, RicardoBackground: Human biomonitoring (HBM) is an important tool to assess the human exposure to chemicals, contributing to describe trends of exposure over time and to identify population groups that could be under risk. Aflatoxins (AFTs) are genotoxic and carcinogenic compounds causing hepatocellular carcinoma (HCC), the third leading cause of cancer deaths worldwide. In Portugal, scarce data are available regarding exposure to AFTs and none previous study used HBM data to characterize comprehensively the associated burden. Methods: 24h-urine samples (n=94) were analyzed by liquid chromatography–mass spectrometry (LC-MS/MS) for the determination of AFTs (B1, B2, G1, G2, M1). Regarding left-censored data (< LOD) a substitution approach was considered (< LOD = 0). Deterministic and probabilistic models were developed to estimate the health impact of the exposure to aflatoxins, estimating the DALYs attributed to AFTs exposure for the Portuguese population (10291k). Results: AFTs were detected in 13% (AFB1), 16% (AFB2), 1% (AFG1), 2% (AFG2) and 19% (AFM1) of the 24h-urine samples. The mean probable daily intake estimated was 16.7 and 13.4 ng/kg body weight/day, calculated mean DALYs/100k were 1.7 (0-10.7) and 1.68 (0.04-6.23) considering the deterministic and probabilistic models, respectively. Conclusion: The present study generated, for the first time and within a HBM study, reliable and crucial data to characterize the burden associated to Portuguese population exposure to aflatoxins. The obtained results constitute an important contribution to support risk managers in the establishment of preventive policy measures that contribute to ensure the public health protection.
- Activity of Diphenyl diselenide against Aspergillus isolatesPublication . Melo, Aryse Martins; Poester, Vanice Rodrigues; Munhoz, Lívia; Trápaga, Mariana; Roca, Beatriz; Klafke, Gabriel Baracy; Sabino, Raquel; Stevens, David A.; Xavier, Melissa OrzechowskiOrganoselenium compounds have been showing promising antimicrobial activity against bacteria and some fungal species. Among these compounds, diphenyl diselenide (PhSe)2 is a simple and chemically stable molecule with proven low toxicity to animal hosts. Although the mechanisms of action of this molecule are not totally clear, it has been reported that it has a prooxidative activity for microorganisms, due to glutathione depletion. Given the emergence of azole resistance Aspergillus sp. isolates is a global and rising concern, research towards new molecules with antifungal potential are necessary. Thus, the aim of this study was to evaluate the in vitro susceptibility of Aspergillus spp. clinical isolates to (PhSe)2.
- Remote Audit for Quality Control Evaluation in Four Clinical Laboratories focus in hematologic parameters - Portugal and Brazil, 2018Publication . Carletto, Aline; Miranda, Armandina; Faria, Ana Paula; Marques, CristinaThe external audit is an evaluation feature of the clinical laboratories Quality Management System (QMS), which aims to determine if the quality requirements, according to an implemented normative reference, comply with good laboratory practices. Analytical Quality Control (AQC) in clinical laboratory is an analytical method error detection system that includes Internal Quality Control (IQC) and External Quality Control (EQC), allowing the transmission of precise and accurate results. Count Blood Cells (CBC) is one of the most frequently requested laboratory tests, and is the basis of numerous medical interventions, and therefore the transmission of reliable and appropriate analytical results is very important. The results presented is part of the thesis for conclusion the clinical analysis master's. The assessment simulates a remote quality audit according to ISO NP EN ISO 15189:2014. The main objective is to validate online questionnaire and spreadsheets, used to collect data of QMS and to evaluate IQC and EQC results in the area of hematology, as quality indicators from four clinical laboratories.
- Molecular identification of Aspergillus isolates from Magellanic penguinsPublication . Melo, Aryse Martins; da Silva Filho, Rodolfo Pinho; Poester, Vanice Rodrigues; von Groll, Andrea; Stevens, David A.; Sabino, Raquel; Xavier, Melissa OrzechowskiAspergillosis is an important disease in marine birds and has a mortality rate of 50% in Magellanic penguins (Spheniscus magellanicus) in captivity. Molecular biology allows the precise identification of Aspergillus to species level, which is important since cryptic species may show differences in their virulence attributes and in their antifungal susceptibility. This work aimed to perform molecular identification and the itraconazole susceptibility profile of Aspergillus clinical isolates collected from Magellanic penguins with proven aspergillosis.
- 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.
- TAD-GConTool and CNV-ConTool to assist prediction of phenotypic outcome of chromosomal rearrangementsPublication . Fino, Joana; David, DezsoWith the advance of genome sequencing technologies, it is currently possible to identify a large number of chromosomal or genomic structural variants in a single individual. Therefore, the validation and manual assessment of structural variants clinical significance becomes unpractical and time consuming when performed with previous methodologies. In order to assist the validation process, we developed two clinically inspired bioinformatics tools - TADGConTool and CNV-ConTool. They were developed in python with a Common Gateway Interface that allows easy and user-friendly access through any standards compliant web browser (available at: http://dgrctools.insa.min- saude.pt/). TAD-GConTool collects genomic information of breakpoint regions, using topological associated domains (TADs) as reference. It then accesses public databases to retrieve elements found inside TADs, and the associated clinical phenotypes, highlighting those causing dominant disorders. CNV-ConTool searches for overlaps between patient-specific breakpoints and CNVs, and those reported in several public databases. These tools were already successfully applied to about 40 cases studied under the project “Next-gen cytogenetics enters clinical care and annotates the human genome” (HMSPICT/0016/2013) and are now being made available to the broader scientific community. These tools allowed a faster and more informed evaluation of the genomic structural variants, helping select potential pathogenic variants, either by identifying phenotype- associated genes, or by overlapping deletions and duplications with already described benign or pathogenic CNVs. As genome sequencing is becoming more and more a routine method for identification of chromosomal and genomic structural variants, such clinically oriented bioinformatics tools are crucial and represent the first level of analysis toward personalized genomic medicine. This research was supported by national funds through FCT - Fundação para a Ciência e a Tecnologia, Research Grant HMSP-ICT/0016/2013.
- Towards a rapid sequencing-based molecular surveillance and mosaicism investigation of Toxoplasma gondiiPublication . Vilares, Anabela; Borges, Vítor; Sampaio, Daniel; Ferreira, Idalina; Martins, Susana; Vieira, Luis; Gargaté, Maria João; Gomes, João PauloAdvances in molecular epidemiology of Toxoplasma gondii are hampered by technical and cost-associated hurdles underlying the acquisition of genomic data from parasites. In order to implement an enhanced genotyping approach for molecular surveillance of T. gondii, we applied a multi-locus amplicon-based sequencing strategy to samples associated with human infection. This approach, targeting genome-dispersed polymorphic loci potentially involved in adaptation and virulence, genetically discriminated almost all 68 studied strains and revealed a scenario of marked genomic mosaicism. Two-thirds (n = 43) of all strains were classified as recombinant, although recombination seemed to be linked to the classical archetypal lineage. While 92% of the Sag2 archetype I strains revealed genetic mosaicism, only 45% of Sag2 archetype II strains were identified as recombinant. Contrarily to the virulence-associated archetype I, most type II strains (regardless of their recombination background) were non-virulent in mouse. Besides Sag2, some of the newly studied loci (namely the type I/I-like alleles of Sag1, B17, PK1, and Sag3 and type III/III-like alleles of TgM-A) constitute promising candidates to rapidly infer T. gondii mouse virulence. Our successful attempt to capture microsatellite length variation launches good perspectives for the straightforward transition from the laborious intensive historical method to more informative next-generation sequencing (NGS)/bioinformatics-based methodologies. Overall, while T. gondii whole-genome sequencing will be hardly feasible in most laboratories, this study shows that a discrete loci panel has the potential to improve the molecular epidemiology of T. gondii towards a better monitoring of circulating genotypes with clinical importance.
- Molecular detection of Aspergillus in samples collected from patients with suspicion of respiratory fungal infectionPublication . Oliveira, Mariana; Simões, Helena; Veríssimo, Cristina; Sabino, RaquelThe aim of this study was to determine the potential of the real-time multiplex PCR to detect Aspergillus spp. DNA in respiratory samples from a cohort of patients with suspicion of respiratory fungal infection. This is of particular importance in patients who are HIV positive and frequently misdiagnosed with pulmonary tuberculosis, when they suffer from aspergillosis. In this study, we have tested patients with a higher risk to develop chronic pulmonary aspergillosis (CPA): HIV+ patients as well as patients with active tuberculosis or with a previous Mycobacterium infection.