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- A familial partial AZFb-c microdeletion associated with diferente fértile phenotypesPublication . Pereira, I.; Aguiar, A.; Correia, S.; Pinto, M.G.; Calhaz Jorge, C.; Gonçalves, J.After the Klinefelter syndrome, Y chromosome microdeletions are the second most frequent genetic cause of spermatogenic failure resulting in male infertility. Y chromosome microdeletions, encompassing one or more of the three AZF regions, are associated with diverse testicular histology, ranging from Sertoli-cell-only syndrome (AZFa del), maturation arrest (AZFb del) to hypospermatogenesis (AZFc del). The molecular screening of these regions is routinely performed in the work-up of infertile patients with azoospermia or severe oligozoospermia as each one has different prognostic values, both in terms of clinical decision-making and appropriate genetic counselling as well as for understanding the etiology of spermatogenesis impairment. Different partial AZFc deletions were already described, although it is still controversial if these are truly a genetic risk factor for spermatogenesis impairment or a deletional variant without phenotypic consequences. Here we present the molecular results obtained after AZF analysis of two infertile brothers (both diagnosed with oligoteratoastenozoospermia), and of their fertile father. Several multiplex-PCR assays were performed with distinct sets of STS markers, specific for the three AZF regions. The molecular analysis revealed that all three men presented the same partial AZFb/c microdeletion, namely the absence of the sY1197, sY1291 and sY1192 STSs. This microdeletion probably results from the recombination of amplicons b1/b3, reducing the gene copy number of PRY, BPY, DAZ, and RBMY. The b1/b3 deletion is rare and its influence on spermatogenesis is still not clear since it can be found in men with severe oligozoospermia or with normal sperm counts. Our result suggests that b1/b3 del is most likely a risk factor predisposing to spermatogenic failure, but is not sufficient alone. The different (in)fertile phenotypes associated with it, a fertile father opposed to his two infertile sons, can be possibly influenced by genetic background, environmental and epigenetic factors, contributing to different phenotypic expressions of individual/specific genomes.
- Efeitos adversos de contaminantes estuarinos em células humanas: avaliação da genotoxicidade e desregulação endócrinaPublication . Vicente, Ana Margarida Moreira; Dias, Deodália Maria Antunes; Silva, Maria João[POR]O Estuário do Sado é um ecossistema de elevado valor ecológico e económico, devido às várias atividades de pesca que suporta. Contudo, esses valores estão ameaçados pela contaminação dos sedimentos e da água do estuário por compostos com um reconhecido potencial mutagénico e carcinogénico. Este trabalho pretendeu contribuir para a avaliação do risco para a saúde humana decorrente da contaminação do Estuário do Sado, através da caracterização do potencial citotóxico e genotóxico de extratos fracionados obtidos a partir de amostras de sedimentos. Para além disso, pretendeu-se implementar um ensaio de avaliação das propriedades de desregulação endócrina das mesmas amostras. Os sedimentos recolhidos em três locais de pesca do Estuário do Sado (A, C e P) foram fracionados com solventes de diferentes polaridades: n-hexano (fração 2), diclorometano (fração 3) e metanol (fração 4). Tentou implementar-se o ensaio E-screen em células MCF-7, utilizando-se o 17-β-estradiol e o Bisfenol A, como controlo positivo. A caracterização da citotoxicidade e da genotoxicidade foi realizada em células HepG2, recorrendo-se, respetivamente, ao ensaio do vermelho neutro e ao ensaio cometa. As células MCF-7 foram expostas durante um período de 5 dias a várias concentrações dos controlos positivos, de modo, a se medir a proliferação celular. Não se conseguiu no entanto obter nos diferentes ensaios realizados uma reprodutibilidade do método. No ensaio do vermelho neutro foi observada uma diminuição da viabilidade celular principalmente nas frações A4, P3 e P4 o que sugere a existência de contaminantes nestas frações com capacidade de induzir citotoxicidade. No que diz respeito aos efeitos ao nível do ADN, observou-se uma elevação significativa do nível de lesões no ADN apenas nas frações 2 e 4 da amostra P. As diferenças registadas nos efeitos citotóxicos e genotóxicos entre as frações e as várias amostras sugerem que existem diferentes contaminantes nestas amostras, e além disso que diferentes contaminantes são extraídos com cada um dos compostos usados na preparação das diferentes frações. Estes resultados refletem as diferentes pressões exercidas ao longo do Estuário do Sado, e salientam a importância de estabelecer associações entre a identificação e quantificação dos contaminantes presentes nas amostras com os seus efeitos biológicos de modo a tentar tirar ilações sobre um potencial impacto na saúde humana.
- Occurrence of extended-spectrum beta-lactamases in Salmonella enterica strains isolated from broilers and food of animal origin in PortugalPublication . Clemente, Lurdes; Correia, Ivone; Themudo, Patrícia; Albuquerque, Teresa; Manageiro, Vera; Jones-Dias, Daniela; Ferreira, Eugénia; Caniça, ManuelaSalmonella enterica is a zoonotic bacteria transmitted through the food chain and isolates harbouring extended-spectrum beta-lactamases (ESBLs) have emerged worldwide during the last decade, with the CTX-M group being particularly important. The aim of the present study was to determine the antimicrobial susceptibility of S. enterica strains isolated from broilers and food of animal origin and to characterize ESBLs producers. On the scope of the national antimicrobial resistance surveillance programme on Salmonella, a total of 283 strains isolated from broilers (n=100) and food of animal origin (n=183), were received at the National Laboratory of Veterinary Research in 2011. The minimum inhibitory concentration (MIC) of 11 antimicrobials (nalidixic acid, ciprofloxacin, ampicillin, cefotaxime, chloramphenicol, florfenicol, streptomycin, gentamicin, tetracycline, sulphamethoxazole and trimethoprim) for all isolates was determined by agar dilution method. Susceptibility towards cefoxitin was determined through disk diffusion method. Breakpoints were interpreted accordingly to EUCAST epidemiological cut-off values. ‘Non-wild type’ (‘NWT’) isolates for cefotaxime (MIC>0.5mg/L) and cefoxitin (<19mm) were screened for the presence of ESBL- (blaTEM, blaOXA, blaSHV, blaCTX) and PMA_-encoding genes, using PCR method. Sequencing was applied to fully identify beta-lactamases. Among broilers, we identified 62% of ‘NWT’ isolates for ciprofloxacin, 57% for nalidixic acid and 28% for sulphamethoxazole, whereas in isolates from food of animal origin, 71%, 63% and 56% were ‘NWT’ isolates for tetracycline, sulphamethoxazole and ampicillin, respectively. Among all, 5/283 (1.8%) strains presented ‘NWT’ MICs for cefotaxime and were multidrug resistant: 2 Salmonella Havana isolated from broilers and 3 Salmonella S. 4,[5],12:i:- isolated from food of animal origin (swine); these isolates had one blaCTX-M-type gene, and 2 from food of animal origin presented 1 blaTEM-type gene and 1 blaSHV-type gene, respectively; they were ‘wild type’ for cefoxitin and no PMAB-encoding gene was detected. To our knowledge, this is the first time in Portugal that ESBL-encoding genes, particularly from blaCTXM family, were detected in isolates of Salmonella Havana, a very common serotype isolated from our broiler population. It should also be emphasised that third generation cephalosporins are not allowed in the national poultry production, contrary to the large animal production, which may explain the detection of ESBL-encoding genes in our strains from swine origin. Horizontal gene transfer may be responsible for the coresistance of strains to non-beta-lactam antibiotics. This study shows that national animal health monitoring systems play an important role and should be improved in an international level.
- Estudo de Comunidades Fúngicas em Arquivos: Implicações na Conservação e na SaúdePublication . Pinheiro, A.C.; Viegas, C.; Jurado, V.; Sabino, R.; Veríssimo, C.; Rosado, L.; Macedo, M.F.A influência da contaminação fúngica para a saúde ambiental e para a conservação do património é o tema premente e actual que suscitou a hipótese de estudo aqui apresentada. Os fungos, dada a sua extrema capacidade de adaptação, podem colonizar diversos materiais – orgânicos ou não – e a sua acção pode ser mecânica, por intermédio das suas hifas ou química, através dos seus metabolitos.Em termos de conservação do património, os estudos sobre fungos têm suscitado grande interesse dada a sua elevada capacidade de biodeterioração. Tendo inicialmente assentado em técnicas tradicionais de cultur), os estudos mais recentes já incluem técnicas modernas de biologia molecular. O estudo apresentado utiliza ambas as técnicas: a tradicional, recorrendo a meios de cultura e a mais recente, usando o DNA e a amplificação genómica para conseguir identificar os fungos até à espécie. Para conseguir realizar este intuito foi desenvolvida a aplicação da recente técnica de cromatografia líquida desnaturante de alta resolução (DHPLC) à análise de amostras complexas de fungos filamentosos e leveduriformes.A investigação executada permitiu identificar focos de contaminação fúngica que ultrapassam os limites aconselháveis em termos de qualidade do ar interior. As espécies Aspergillus niger, Aspergillus fumigatus ou Stachybotrys atra, todos eles encontrados em ambiente arquivístico são presenças relevantes em termos de qualidade do ar interior pois são indicadoras de más condições ambientais.A optimização da aplicação de cromatografia desnaturante à mistura de leveduras e fungos filamentos permite identificar os componentes da mistura e pode ser usada no contexto clínico e no contexto ambiental, em amostras de papel ou noutro tipo de amostras.
- Detection of mosquito-only flaviviruses in EuropePublication . Calzolari, M.; Zé-Zé, Líbia; Ruzek, D.; Vazquez, A.; Jeffries, C.; Defilippo, F.; Osório, Hugo Costa; Kilian, P.; Ruíz, S.; Fooks, A.R.; Maioli, G.; Amaro, Fátima; Tlusty, M.; Figuerola, J.; Medlock, J.M.; Bonilauri, P.; Alves, M.J.; Sebesta, O,; Tenorio, A.; Vaux, A.G.; Bellini, R.; Gelbic, I.; Sánchez-Seco, M.P.; Johnson, N.; Dottori, M.The genus Flavivirus, family Flaviviridae, includes a number of important arthropod-transmitted human pathogens such as dengue viruses, West Nile virus, Japanese encephalitis virus and yellow fever virus. In addition, the genus includes flaviviruses without a known vertebrate reservoir, which have been detected only in insects, particularly in mosquitoes, such as cell fusing agent virus, Kamiti River virus, Culex flavivirus, Aedes flavivirus, Quang Binh virus, Nakiwogo virus and Calbertado virus. Reports of the detection of these viruses with no recognized pathogenic role in humans are increasing in mosquitoes collected around the world, particularly in those sampled in entomological surveys targeting pathogenic flaviviruses. The presence of six potential flaviviruses, detected from independent European arbovirus surveys undertaken in the Czech Republic, Italy,Portugal, Spain and the UK between 2007 and 2010, is reported in this work. Whilst the Aedes flaviviruses, detected in Italy from Aedes albopictus mosquitoes, had already been isolated in Japan, the remaining five viruses have not been reported previously: one was detected in Italy, Portugal and Spain from Aedes mosquitoes (particularly from Aedes caspius), one in Portugal and Spain from Culex theileri mosquitoes, one in the Czech Republic and Italy from Aedes vexans, one in the Czech Republic from Aedes vexans and the last in the UK from Aedes cinereus. Phylogenetic analysis confirmed the close relationship of these putative viruses to other insect-only flaviviruses.
- Air contaminants in animal production – poultry casePublication . Viegas, C.; Viegas, S.; Monteiro, A.; Carolino, E.; Sabino, R.; Verissimo, C.A descriptive study was developed in order to assess air contamination caused by fungi and particles in seven poultry units. Twenty seven air samples of 25 litters were collected through impaction method. Air sampling and particle concentration measurement were performed in the pavilions’ interior and also outside premises, since this was the place regarded as reference. Simultaneously, temperature and relative humidity were also registered. Regarding fungal load in the air from the seven poultry farms, the highest value obtained was 24040 CFU/m3 and the lowest was 320 CFU/m3. Twenty eight species/genera of fungi were identified, being Scopulariopsis brevicaulis (39.0%) the most commonly isolated species and Rhizopus sp. (30.0%) the most commonly isolated genus. From the Aspergillus genus, Aspergillus flavus (74.5%) was the most frequently detected species. There was a significant correlation (r=0.487; p=0.014) between temperature and the level of fungal contamination (CFU/m3). Considering contamination caused by particles, in this study, particles with larger dimensions (PM5.0 and PM10) have higher concentrations. There was also a significant correlation between relative humidity and concentration of smaller particles namely, PM0.5 (r=0.438; p=0.025) and PM1.0 (r=0.537; p=0.005). Characterizing typical exposure levels to these contaminants in this specific occupational setting is required to allow a more detailed risk assessment analysis and to set exposure limits to protect workers’ health.
- Positive impact of the Portuguese smoking law on respiratory health of restaurant workersPublication . Madureira, Joana; Mendes, Ana; Almeida, Sophia; Teixeira, João PauloThe impact of smoke-free law on the respiratory and sensory symptoms among restaurant workers was evaluated. Fifty-two workers in 10 Portuguese restaurants were interviewed before and 2 years after implementation of the smoke-free law. A significant reduction in self-reported workplace environmental tobacco smoke (ETS) exposure was observed after the enforcement of the law, as well as a marked reduction in adverse respiratory and sensory symptoms such as dry, itching, irritated, or watery eyes, nasal problems, and sore or dry throat or cough, between pre- and post-ban. This study demonstrates that the smoking ban was effective in diminishing the exposure symptoms among workers and consequently in improving their respiratory health. These observations may have implications for policymakers and legislators in other countries currently considering the nature and extent of their smoke-free workplace legislation.
- Phenotypic and molecular characterization of CMY-46 and CMY-50, two novel plasmid-mediated AmpC β-lactamase carried by Escherichia coliPublication . Manageiro, Vera; Louro, Deolinda; Ferreira, Eugénia; Caniça, ManuelaObjectives: The identification of isolates containing AmpC β-lactamases is epidemiologically and clinically relevant. With this study we performed the phenotypic and molecular characterization of two new CMY-2-types, designated CMY-46 and CMY-50, encountered among a total of 1664 clinical non-duplicate isolates of various Enterobacteriaceae species. Methods: E. coli INSRA1169 and INSRA3413 were isolated from the urine of patients with 77 years and 7 months old, hospitalized in the ward and in pediatrics, respectively. The blaCMY genes were cloned in the plasmid pBK-CMV and transformed into electrocompetent E. coli DH5α ∆ampC by electroporation. Antimicrobial susceptibility (MIC) was determined by a microdilution method. E. coli INSRA6015, a CMY-2-producer, was used for phenotype comparison. PCR-mapping of the genetic environment of new blaCMY genes was performed using primers for known antibiotic and mercury resistance genes. Results: Antimicrobial susceptibly tests showed that all isolates and respective transformants were nonsusceptible to amoxicillin, amoxicillin plus clavulanic acid, cephalothin, cefoxitin, ceftazidime and cefotaxime. INSRA1169 and INSRA6015 were also nonsusceptible to ciprofloxacin and to trimethoprim. Regarding gentamycin, only INSRA1169 was resistant. Its noteworthy that the transformants EcDH5a(pBK-CMY-2) and EcDH5a(pBK-CMY-46) exhibited higher values for extended-spectrum cephalosporins than the respective isolates. All strains were susceptible to cefepime and imipenem, showing synergy between cloxacilin and cefoxitin and/or ceftazidime. No phenotypic alterations were found comparing the new CMY-type with the parental CMY-2. The genetic characterization of CMY-46 and CMY-50-encoding genes revealed a Citrobacter freundii chromosome-type structure, encompassing a blc-sugE-blaCMY-2-type-ampR platform in both isolates. In addition, a sul1-type class 1 integron and a truncated mercury resistance operon were encountered. Conclusion: Although the CMY-type enzymes studied conferred resistance to extended-spectrum cephalosporins, the susceptibility to cefepime lead us to assume that those enzymes are not extended-spectrum cephalosporinases. Otherwise, the presence of three genetic resistance-encoding regions is of great concern, namely the truncated mercury resistance operon, which may help to promote antibiotic resistance through indirect selection.
- Prevalence of overweight and obesity among Portuguese children (6-8 years old) using three definition criteria: COSI PORTUGAL, 2008Publication . Rito, Ana; Wijnhoven, Trudy M.A.; Rutter, Harry; Carvalho, Maria Ana; Paixão, Eleonora; Ramos, Carlos; Cláudio, Débora; Espanca, Rosa; Sancho, Teresa; Cerqueira, Zélia; Carvalho, Rita; Faria, Carmo; Feliciano, Elsa; Breda, JoãoIntroduction: Previous studies places Portugal among the five countries with the highest prevalence of childhood obesity in Europe. This paper describes the prevalence of thinness, overweight and obesity in Portuguese children of 6-8 years of age, based on the first data collection from COSI Portugal, which took place during the 2007/2008 school year. Methods: Semi-longitudinal design with repeated cross-sectional national representative samples. Specific prevalence of overweight (including obesity) and obesity was determined using three different diagnostic criteria. Across the seven geographic regions 3765 children were enrolled from 181 schools with 50.3% of boys. Results: Using the IOTF reference the prevalences of thinness, overweight and obesity were 4.8%, 28.1%, and 8.9% respectively; using the CDC reference they were 2.1%, 32.2%, and 14.6%; and according to the WHO reference they were 1.0%, 37.9%, and 15.3%. Univariate analysis showed a higher risk of obesity in older children, in boys and in the Azores region. The islands of Madeira and the Azores were the regions with the highest prevalence of overweight at 39.4% and 46.6% respectively, and Algarve was the one with the lowest (21.4%). Conclusion:. These findings demonstrate the need for urgent action in Portugal, and provide policy makers with comprehensive and detailed information to assist with this.