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- Creatine deficiency syndromes: biochemical and molecular aspectsPublication . Valongo, Carla; Almeida, Lígia; Ramos, Altina; Salomons, Gajja; Jacobs, Cornelis; Vilarinho, LauraIntroduction: Creatine deficiency syndromes (CDS) represent a group of inborn errors of creatine biosynthesis: L-arginine-glycine amidinotransferase - AGAT and guanidinoacetate methyltransferase - GAMT deficiencies and transport (creatine transporter - SLC6A8 deficiency). Patients with CDS may present with mental retardation (MR), expressive speech and language delay, and epilepsy. Patients with GAMT deficiency or SLC6A8 deficiency may also exhibit autistic-like behavior. The common denominator of these disorders is the depletion of the brain creatine pool, as demonstrated by in vivo 1H-MRS. Patients and Methods: The authors studied 6,600 urine samples from Portuguese autistic children and young adults for defects in creatine metabolism. We started with the determination of guanidinoacetate and creatine in urine by GC-MS-SIM. Based on these findings, enzyme assays or DNA mutation analysis may be performed. Molecular genetic analysis for GAMT deficiency and creatine transporter deficiency is also available in our laboratory. Results: A marked excretion of guanidinoacetate in urine compatible with GAMT deficiency was observed in seven cases. Furthermore, other 15 patients showed high urinary levels of creatine/creatinine ratio what suggests a defect of SLC6A8. All GAMT deficient patients show the same mutation (c.59G>C) which suggests a founder effect in our population. Molecular genetic analysis of the SLC6A8 deficiency patients revealed a large spectrum of mutations. Discussion: So far, 22 patients with CDS were identified in our laboratory (1:300). We believe these defects are still under diagnosed, so the possibility should be considered in all children affected by unexplained MR, seizures, and speech delay. SLC6A8 defect should also be considered in males with MR and negative fragile-X testing. GAMT deficiency is treatable with oral creatine monohydrate and ornithine supplementation with arginine dietary restriction.
- Características socioeconómicas e obesidade infantil no âmbito do projeto comunitário MUN-SI 2008-2011Publication . Doroana, Érica; Silva, Ana Lúcia; Carvalho, Maria Ana; Rito, Ana Isabel
- Letalidade intra-hospitalar ajustada por ano, grupo etário e sexo; significado, implicações e robustez da análisePublication . Nunes, BaltazarLetalidade intra-hospitalar ajustada por ano, grupo etário e sexo; significado, implicações e robustez da análise
- Programa Nacional de Diagnóstico Precoce em Portugal- Casuística de 2011Publication . Lopes, Lurdes; Sousa, Carmen; Fonseca, Helena; Carvalho, Ivone; Marcão, Ana; Rocha, Hugo; Vilarinho, LauraO Programa Nacional de Diagnóstico Precoce (PNDP) visa identificar doenças nas primeiras semanas de vida do bebé de forma a possibilitar uma intervenção precoce e a impedir a ocorrência de atraso mental, doença grave irreversível ou morte da criança. O PNDP iniciou-se em 1979 com o rastreio da fenilcetonúria tendo-se implementado em 1981 o rastreio do hipotiroidismo congénito. As novas tecnologias de espectrometria de massa, permitindo a análise simultânea de vários parâmetros numa só amostra de sangue, possibilitaram, a partir de 2004, o aumento das doenças rastreadas até às atuais 25 (24 doenças hereditárias do metabolismo e o hipotiroidismo congénito). O rastreio é feito sobre o sangue colhido por picada no pé do bebé, entre o 3º e o 6º dia de vida, para uma ficha com papel de filtro adequado, sendo a totalidade das análises efetuadas na Unidade de Rastreio Neonatal Metabolismo e Genética do INSA. A taxa de cobertura do PNDP é 100% dos recém - nascidos, sendo o tempo médio de início de tratamento de 9,87 dias. Em Portugal, durante o ano de 2011, foram rastreados 97.116 recém-nascidos tendo-se identificado 44 casos de Hipotiroidismo Congénito (1/2.207) e 31 casos de doenças hereditárias do metabolismo (1/3.133). Os programas de rastreio neonatal são sistemas dinâmicos que devem ser continuamente avaliados e atualizados. Nesta conformidade, há duas doenças cuja integração no Programa Nacional de Diagnóstico Precoce, deve ser considerada e avaliada, atendendo à realidade atual em termos demográficos e de tratamento efetivo - a fibrose quística e a anemia falciforme (drepanocitose). Serão assim integradas no projeto de desenvolvimento futuro do rastreio neonatal em Portugal.
- Autosomal Recessive Cerebellar Ataxia and Low Mitocondrial Complex III in a Portuguese FamilyPublication . Nogueira, Célia; Nesti, Claudia; Meschini, Maria Chiara; Carrozzo, Rosalba; Barros, Jose; Sá, Maria José; Azevedo, Luisa; Vilarinho, Laura; Santorelli, FilippoIntroduction: Defects of mitochondrial complex III (CIII) are a relatively rare cause of mitochondrial dysfunction. The complex catalyzes the electron transfer from reduced coenzyme Q to cytochrome c and is composed of 11 subunits, one of which (MT-CYB) is mtDNA encoded. Mutations in MT-CYB and in assembly factor BCS1L account for the vast majority of cases with low CIII, and are associated with a wide range of neurological disorders. The gene coding for human tetratricopeptide 19 (TTC19) produces a poorly characterized protein thought to be involved in the correct assembly of CIII. Recently, mutations in TTC19 have been described in three unrelated Italian kindred in association with a severe neurodegenerative disease. Objectives: We studied a consanguineous Portuguese family where a severe neurometabolic disorder occurred in four siblings (three men and one woman) in association with a slowly progressive disorder characterized by dystonia of hands and feet, ataxic gait, severe olivo-ponto-cerebellar atrophy documented at brain MRI, and relentless psychiatric manifestations. Variability in age at onset and disease course was observed. Methods: The enzymatic activity of CIII was determined in muscle using a reported spectrophotometric method. Sequence analysis of genomic DNA was performed to identify disease-causing mutations in TTC19. Immunodetection analysis in muscle homogenate and skin fibroblasts allowed the detection of the amount TTC19 protein using a commercially available anti-TTC19 antibody. Results: In this family, we identified a novel homozygous TTC19 mutation predicting frameshift and early protein truncation. The mutation was heterozygous in parents and healthy siblings, and it was absent in ethnically-matched controls. The protein was undetectable in tissues by Western blot analyses. Conclusion: This is the fourth kindred presenting mutations in TTC19. The clinical phenotype of such condition is severe, embraces neurological and psychiatric symptoms, and represents a further example of autosomal recessive ataxia of metabolic origin.
- Characterization of Clostridium difficile 027 strains from an outbreak in a Portuguese hospitalPublication . Antunes, Wilson; Serrano, Mónica; Santos, Andrea; Rodrigues, João; Pereira, Fátima; Oleastro, Mónica; Henriques, Adriano O.C. difficile infection (CDI) is the cause of an intestinal disease mediated by two potent cytotoxins, TcdA and TcdB. Symptoms of CDI can range from asymptomatic colonization or mild diarrhea, to life-threatening inflammatory lesions such as pseudomembraneous colitis, toxic megacolon or bowel perforation. In part because of the recent emergence of so-called hypervirulent strains, especially (but not exclusively) those belonging to ribotype 027, C. difficile is now considered a main nosocomial enteric pathogen. Hypervirulent epidemic strains have been associated with more severe disease conditions, with higher relapse rates and increased mortality. Health care-associated CDI develops in hospitalized patients undergoing antibiotic treatment because C. difficile can colonize the gut if the normal intestinal microbiota is disturbed. However, C. difficile is also emerging as an important pathogen in the community, as well as in animal husbandry. The organism is an obligate anaerobe, and has the ability to form spores. Spores are extremely resilient and can accumulate and remain viable in the environment or in the host for long periods of time. Spores that remain latent in the gut are responsible for the recurrence of C. difficile-associated disease (CDAD) when antibiotic therapy is stopped. At least some of the hypervirulent epidemic strains show a greater sporulation capacity in vitro, as well as robust toxin production. The first detection of C. difficile 027 hypervirulent epidemic strains implicated in a hospital outbreak in Portugal dates from January 2012, involving 12 patients, with a crude mortality rate of 50%. Here we report on the genetic characterization of those strains as well as the antibiotic resistance profile, toxin production, and rate and efficiency of spore formation. In parallel, C. difficile 027 non-outbreak strains isolated from other Portuguese health care facilities are also investigated.
- Cascade Screening in Familial Hypercholesterolemia importance in early detectionPublication . Leitão, F.; Medeiros, A.M.; Berguete, S.; Alves, A.C.; Bourbon, M.Familial hypercholesterolemia (FH) is a genetic condition mainly caused by mutations in LDLR but missense mutations in APOB and PCSK9 can cause similar phenotypes. FH is characterized by increased levels of LDL cholesterol, leading to premature cardiovascular diseases (CVD). Cascade screening (CS) allows the rapid identification of new FH cases within a family. The main goal of this work is to perform CS of FH families and to understand the importance of this approach to identify prematurely individuals that are at risk to develop CVD.
- Active Packaging Produced by Extrusion with Shrimp Waste: Migration of Astaxanthin into Food SimulantsPublication . Sanches-Silva, A.; Ribeiro, T.; Albuquerque, T.G.; Paseiro, P.; Sendón, R.; Bernaldo de Quirós, A.; López-Cervantes, J.; Sánchez-Machado, D.; Soto Valdez, H.; Angulo, I.; Pardo Aurrekoetxea, G.; Costa, H.S.Introduction: Astaxanthin (3,3’-dihydroxy-β-β´-carotene-4-4´-dione), a potent antioxidant, is one of the major carotenoids in crustaceans. In the frame of the project ‘Preparation of active packaging with antioxidant and antimicrobial activity based on astaxanthin and chitosan’, a methodology for the incorporation of compounds obtained from shrimp waste in plastic matrices was developed to produce an active packaging with antioxidant properties. The aim of the present work was to develop and optimize a method to determine astaxanthin by ultra-high pressure liquid chromatography in fermented shrimp waste. Moreover, the method was also applied to determine the migration of astaxanthin from plastic films containing different amounts of shrimp waste to food simulants. Material and Methods: The method was optimized to determine astaxanthin by ultra-high pressure liquid chromatography (UHPLC) with diode array detection (DAD). The chromatographic separation was achieved using a vanguard pre-column (UPLCÒ BEH, 1.7 µm particle size) and a column (UPLCÒ BEH, 2.1 x 50 mm, 1.7 µm particle size) at 20 °C. The mobile phase was a gradient of A (dichloromethane/methanol with ammonium acetate/acetonitrile 5:20:75 (v/v)) and B (ultrapure water) with a flow rate of 0.5 mL/min. The optimized UPLC method allowed an excellent resolution of astaxanthin. The method was also evaluated in what concerns to validation parameters such as linearity, precision, limit of detection, limit of quantification and recovery. Low density polyethylene plastic films produced by extrusion with different amounts of the lipid fraction of shrimp waste were prepared and tested regarding migration into fatty food stimulants (isooctane and ethanol 95%, v/v). Results and conclusion: The proposed method to determine astaxanthin in shrimp waste is simple and has a low detection level (0.054 μg/mL). The concentration of astaxanthin found in the lipid fraction of fermented shrimp waste was 453.8 μg/g. The films produced by extrusion with the lipid fraction of the fermented shrimp waste did not originate astaxanthin migration into the tested fatty food simulants. Further studies could be made in order to evaluate the capacity of these films in protecting packed food from oxidation.
- Polyclonal KPC-3-producing Enterobacteriaceae in PortugalPublication . Manageiro, Vera; Ferreira, Eugénia; Louro, Deolinda; Caniça, Manuela; Antimicrobial Resistance Surveillance Program in Portugal (ARSIP)Water has been recognized as a reservoir for antibiotic resistance genes (ARG), where the presence of mobile genetic elements, including plasmids, favors their dissemination. It is noteworthy that nonpathogenic environmental organisms, where plasmids encoding multiple ARG are prevalent, can provide resistance to most classes of antimicrobials including beta-lactams, aminoglycosides, chloramphenicol, trimethoprim, streptomycin, fosfomycin, quinolones, among others. The main goal of this study was to evaluate the presence of ARGs, related with -lactam and quinolone resistance, in Gram-negative bacteria isolates from surface and raw and treated waste water environments. Water samples were collected from different environments within an urban water cycle in the region of Northern Portugal, which included treated and raw wastewater, water to the consumers and water surface. Screening of antimicrobial susceptibility of 56 Gram-negative isolates (20 Escherichia coli, 8 Citrobacter spp., 7 Klebsiella spp., 6 Kluyvera spp., 4 Sphingomonas panni, 2 Enterobacter spp., 1 Acinetobacter johnsonii, 3 Aeromonas veronii, 1 Hafnia alvei, 1 Pantoea agglomerans, 1 Roultella ornithinolytica, 1 Serratia sp., 1 Stenotrophomonas maltophilia), identified by 16S rRNA gene sequencing analysis using universal primers, was performed by disk diffusion method. Interpretative reading of susceptibilities allowed to direct the search for antibiotic resistant genes. PCR and sequencing were used to screen and identify beta-lactamase- and plasmidmediated quinolone resistance (PMQRs)-encoding genes. All isolates were also screened for the presence of class 1 integrons. PCR-based replicon typing (PBRT) was used to type the resistance plasmids of the blaGES-5- producing isolate among the major incompatibility (Inc) groups, specifically FIA, FIB, FIC, HI1, HI2, I1-I , L/M, N, P, W, T, A/C, K, B/O, X, Y, F, and FIIA. Multilocus sequence typing (MLST) of the GES-5 K. pneumoniae-producing isolate was performed according to the Institute Pasteur scheme (http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html). Overall, 16/56 isolates were multidrug-resistant (MDR), i.e. presenting a reduced susceptibility to 3 or more structurally unrelated antibiotics, suggesting a great diversity of resistance mechanisms. Noteworthy, 10 isolates (4 S. panni, 1 A. johnsonii, 3 A. veronii, 1 K. pneumoniae, and 1 S. maltophilia) showed nonsusceptibility to carbapenems, which constitutes one of the last resorts on the antimicrobial therapy. Their phenotypic and molecular characterization revealed the expression of several enzymes: the naturally occurring carbapenemase in one S. maltophilia, ImiS in three A. veronii, both MBLs, and OXA-type carbapenemase in one A. johnsonii, responsible for their intrinsic resistance; the class A GES-5-producing K. pneumoniae isolate belonged to a novel MLST sequence type, the ST961 (18-22-18-90-142-13-179). PBRT of the plasmid-carrying blaGES-5 gene showed that it did not belong to any of the Inc groups tested. No carbapenemases were found in the 4 S. panni isolates. The beta-lactam resistance, carbapenem susceptibility, found in 33 isolates was justified by the presence of various Class A (12 blaTEM-1 with distinct promoters, 6 blaSHV) and different Class C -lactamase-encoding genes (blaCMY, blaACC, blaACT), some here firstly described: blaCMY-65 (JF780936), blaCMY-89 (HE819403), blaCMY-90 (HE819404), blaACT-13 (HE819402) and blaACC-5 (HE819401). Class 1 integrons were detected among 6 of TEM- 1-producing isolates. Together, the beta-lactamases identified explain the level of beta-lactam resistance. Besides quinolone resistance detected, none PMQR were identified, suggesting chromosomal alterations in the quinolone resistance-determining region. This study identified ARGs related not only to commonly used antibiotics, but also to carbapenems, providing, at our knowledge, the first description of a GES-5-producing Enterobacteriaceae recovered in an environmental setting. The study highlights the need of surveillance of these antibiotic resistance mechanisms in environmental backgrounds, since it represents a liable reservoir of potential pathogenic resistant bacteria. Worryingly, recent studies demonstrated that while the WWTP reduced the bacterial load, the treatment is inefficient to remove antibiotic resistant bacteria.
- Migration of chitosan films prepared by solvent evaporation and extrusionPublication . Sanches-Silva, A.; Ribeiro, T.; Albuquerque, T.G.; Paseiro, P.; Sendón, R.; Bernaldo de Quirós, A.; López-Cervantes, J.; Sánchez-Machado, D.; Soto Valdez, H.; Angulo, I.; Pardo Aurrekoetxea, G.; Costa, H.S.Introduction: Chitosan has multiple applications and as inhibitor of microbial growth, there is interest in developing new methodologies to its incorporation into plastics, which would avoid microbial growth in foods. In the frame of the project “Preparation of active packaging with antioxidant and antimicrobial activity based on astaxanthin and chitosan” (PAPAAABAC), chitosan was extracted from shrimp by-products and used to prepare plastic films by solvent evaporation (casting) and extrusion. Material and Methods: Chitosan films were prepared by extrusion of polyamide and by casting. By casting, films were prepared at different concentrations (1%, 2%, and 3%) by dissolving chitosan in acetic acid aqueous solution 1% (w/v), with and without plasticizer (1% glycerol). By extrusion, chitosan pellets were made of polyamide 6 at 2%, 5%, 6%, 8%, and 10% with two different particle sizes (180 and 300 µm). Then, they were rolled in an extruder using a specific screw for polyamide. In order to determine chitosan in food simulants (ultrapure water and ethanol 95% (v/v)), it was degraded into the glucosamine units by hydrolysis and quantified by ultra high pressure liquid chromatography coupled with diode array detection after its derivatization with 9-fluorenylmethyl chloroformate. Results and conclusion: From the plastic films prepared by casting and by extrusion, only those prepared by casting without plasticizer presented chitosan migration that increases with the amount of chitosan added. These films could not be used to pack aqueous foodstuffs. However, the addition of a plasticizer (glycerol) has avoided the migration of chitosan. Therefore, the use of casting films with chitosan shall include a plasticizer in the formulation. Films prepared by extrusion presented no migration into both simulants indicating suitability to pack both aqueous and fatty foodstuffs.