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Previously, we identified a Chlamydia trachomatis Lymphogranuloma venereum (LGV) recombinant strain possessing a unique non-LGV ompA genotype. Here, culture-independent genome sequencing confirms its circulation in Europe, Middle East and North America, and unveils genetic evidence of emergence of antibiotic resistance. Multi-country and systematic molecular surveillance is needed. This work results in part from the GenomePT project (POCI-01–0145-FEDER-022184), supported by COMPETE 2020— Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e a Tecnologia (FCT). This work was also supported by Fundos FEDER through the Programa Operacional Factores de Competitividade—COMPETE and by Fundos Nacionais through the Fundação para a Ciência e a Tecnologia within the scope of the project UID/BIM/00009/2019 (Centre for Toxicogenomics and Human Health -ToxOmics). info:eu-repo/semantics/publishedVersion

Recent studies have shown that persistent SARS-CoV-2 infections in immunocompromised patients can trigger the accumulation of an unusual high number of mutations with potential relevance at both biological and epidemiological levels. Here, we report a case of an immunocompromised patient (non-Hodgkin lymphoma patient under immunosuppressive therapy) with a persistent SARS-CoV-2 infection (marked by intermittent positivity) over at least 6 months. Viral genome sequencing was performed at days 1, 164, and 171 to evaluate SARS-CoV-2 evolution. Among the 15 single-nucleotide polymorphisms (SNPs) (11 leading to amino acid alterations) and 3 deletions accumulated during this long-term infection, four amino acid changes (V3G, S50L, N87S, and A222V) and two deletions (18-30del and 141-144del) occurred in the virus Spike protein. Although no convalescent plasma therapy was administered, some of the detected mutations have been independently reported in other chronically infected individuals, which supports a scenario of convergent adaptive evolution. This study shows that it is of the utmost relevance to monitor the SARS-CoV-2 evolution in immunocompromised individuals, not only to identify novel potentially adaptive mutations, but also to mitigate the risk of introducing "hyper-evolved" variants in the community. IMPORTANCE Tracking the within-patient evolution of SARS-CoV-2 is key to understanding how this pandemic virus shapes its genome toward immune evasion and survival. In the present study, by monitoring a long-term COVID-19 immunocompromised patient, we observed the concurrent emergence of mutations potentially associated with immune evasion and/or enhanced transmission, mostly targeting the SARS-CoV-2 key host-interacting protein and antigen. These findings show that the frequent oscillation in the immune status in immunocompromised individuals can trigger an accelerated virus evolution, thus consolidating this study model as an accelerated pathway to better understand SARS-CoV-2 adaptive traits and anticipate the emergence of variants of concern. This study was partially cofunded by Fundação para a Ciência e Tecnologia and Agência de Investigação Clínica e Inovação Biomédica (234_596874175) on behalf of the Research 4 COVID-19 call. Some infrastructural resources used in this study come from the GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020-Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Program (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e a Tecnologia (FCT). info:eu-repo/semantics/publishedVersion

We report a multidrug resistant Neisseria gonorrhoeae exhibiting resistance to ceftriaxone and cefixime, isolated in Portugal in 2019. Whole-genome sequencing was performed for typing and identification of genetic determinants of antimicrobial resistance. Due to its antimicrobial susceptibility profile, awareness should be raised for the circulation of this strain. M.P. was supported by the Portuguese Science and Technology Foundation through grant SFRH/BD/109264/2015. info:eu-repo/semantics/publishedVersion

O objetivo deste estudo foi identificar a produção de artigos científicos envolvendo a importância da brinquedoteca para o desenvolvimento infantil da criança hospitalizada, publicados em âmbito nacional entre 2008 até abril de 2018. Utilizou-se os descritores “brinquedo”, “hospital” e “criança” nas seguintes bases de dados: IndexPsi, SciELO, PePSIC, LILACS e Periódicos CAPES . Obteve-se o total de 151 resultados. Após leitura integral dos artigos, a amostra consistiu em 10 artigos científicos. Realizou-se uma análise sistemática dos achados, levantando seis categorias temáticas: 1) continuidade no desenvolvimento infantil; 2) humanização do atendimento; 3) interação social; 4) alívio dos efeitos emocionais decorrentes da internação; 5) o lúdico como fator de aprendizagem; e 6) distração. A brinquedoteca, vista a partir da Psicologia Ambiental, é um importante ambiente restaurador que possibilita a humanização do cuidado e traz benefícios não somente para a criança, mas também para os profissionais da saúde e acompanhantes.

Free PMC article: https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/31950026/ Gastrointestinal infections caused by nontyphoidal Salmonella (NTS) remain one of the main causes of foodborne illness worldwide. Within the multiple existing Salmonella enterica serovars, the serovar Rissen is rarely reported, particularly as a cause of human salmonellosis. Between 2015 and 2017, the Portuguese National Reference Laboratory of Gastrointestinal Infections observed an increase in the number of clinical cases caused by multidrug-resistant (MDR) S. enterica serovar Rissen, particularly from the Azores archipelago. In the present study, we analyzed by whole genome sequencing (WGS) all clinical, animal, food, and environmental isolates received up to 2017 in the Portuguese Reference Laboratories. As such, through a wgMLST-based gene-by-gene analysis, we aimed to identify potential epidemiological clusters linking clinical and samples from multiple sources, while gaining insight into the genetic diversity of S. enterica serovar Rissen. We also investigated the genetic basis driving the observed multidrug resistance. By integrating 60 novel genomes with all publicly available serovar Rissen genomes, we observed a low degree of genetic diversity within this serovar. Nevertheless, the majority of Portuguese isolates showed high degree of genetic relatedness and a potential link to pork production. An in-depth analysis of these isolates revealed the existence of two major clusters from the Azores archipelago composed of MDR isolates, most of which were resistant to at least five antimicrobials. Considering the well-known spread of MDR between gastrointestinal bacteria, the identification of MDR circulating clones should constitute an alert to public health authorities. Finally, this study constitutes the starting point for the implementation of the "One Health" approach for Salmonella surveillance in Portugal. This work was partially funded by the GenomePT project (POCI-01-0145- FEDER-022184), supported by COMPETE 2020–Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), and Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) and by the Fundação para a Ciência e a Tecnologia (FCT) info:eu-repo/semantics/publishedVersion

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.meegid.2018.10.025. The human-restricted bacterium Haemophilus influenzae is responsible for respiratory infections in both children and adults. While colonization begins in the upper airways, it can spread throughout the respiratory tract potentially leading to invasive infections. Although the spread of H. influenzae serotype b (Hib) has been prevented by vaccination, the emergence of infections by other serotypes as well as by non-typeable isolates (NTHi) have been observed, prompting the need for novel prevention strategies. Here, we aimed to study the population structure of H. influenzae and to get some insights into its pan-genome. We studied 305H. influenzae strains, enrolling 217 publicly available genomes, as well as 88 newly sequenced H. influenzae invasive strains isolated in Portugal, spanning a 24-year period. NTHi isolates presented a core-SNP-based genetic diversity about 10-fold higher than the one observed for Hib. The analysis of key factors involved in pathogenesis, such as lipooligosaccharides, hemagglutinating pili and High Molecular Weight-adhesins, suggests that NTHi shape its virulence repertoire, either by acquisition and loss of genes or by SNP-based diversification, likely towards host immune evasion and persistence. Discreet NTHi subpopulations structures are proposed based on core-genome supported with 17 candidate genetic markers identified in the accessory genome. Additionally, this study provides two bioinformatics tools for in silico rapid identification of H. influenzae serotypes and NTHi clades previously proposed, obviating laboratory-based demanding procedures. The present study constitutes an important genomic framework that could lay way for future studies on the genetic determinants underlying invasiveness and disease and population structure of H. influenzae. This work was supported by GenomePT (ref. POCI-01-0145-FEDER022184) from Fundação para a Ciência e Tecnologia, Portugal. info:eu-repo/semantics/publishedVersion

BackgroundDissemination of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in healthcare institutions affects both patients and health-care workers (HCW), as well as the institutional capacity to provide essential health services.MethodsWe conducted an investigation of a cluster of SARS-CoV-2 positive cases detected in a “non-COVID-19” hospital ward during Summer 2020. The magnitude of the nosocomial outbreak was disclosed by massive testing, challenging the retrospective reconstruction of the introduction and transmission events. An in-depth contact tracing investigation was carried out to identify the contacts network during the 15-day period before the screening. In parallel, positive SARS-CoV-2 RNA samples were subjected to virus genome sequencing.ResultsOf the 245 tested individuals, 48 (21 patients and 27 HCWs) tested positive for SARS-CoV-2. HCWs were mostly asymptomatic, but the mortality among the vulnerable patient group reached 57.1% (12/21). Phylogenetic reconstruction revealed that all cases were part of the same transmission chain, thus confirming a single origin behind this nosocomial outbreak. By combining vast epidemiological and genomic data, including analysis of emerging minor variants, we unveiled a scenario of silent SARS-CoV-2 dissemination within the hospital ward, mostly driven by the close contact within the HCWs group and between HCWs and patients. This investigation triggered enhanced prevention and control measures, leading to a more timely detection and containment of novel nosocomial outbreaks.ConclusionsThe present study shows the benefit of combining genomic and epidemiological data for the investigation of complex nosocomial outbreaks, and provides valuable data to minimize the risk of transmission of COVID-19 in healthcare facilities.Short summarySARS-CoV-2 nosocomial outbreaks are of utmost public health concern. Here, we performed an in-depth investigation of a high-fatality rate nosocomial outbreak by combining vast genomic and epidemiological data, providing valuable information to understand cryptic transmission of SARS-CoV-2 within healthcare institutions.

We show that the SARS-CoV-2 B.1.1.7 lineage is highly disseminated in Portugal, with the odds of B.1.1.7 proportion increasing at an estimated 89% (95% confidence interval: 83-95%) per week until week 3 2021. RT-PCR spike gene target late detection (SGTL) can constitute a useful surrogate to track B.1.1.7 spread, besides the spike gene target failure (SGTF) proxy. SGTL/SGTF samples were associated with statistically significant higher viral loads, but not with substantial shift in age distribution compared to non-SGTF/SGTL cases. This study is partially co-funded by Fundação para a Ciência e Tecnologia and Agência de Investigação Clínica e Inovação Biomédica (234_596874175) on behalf of the Research 4 COVID-19 call. Some infrastructural resources used in this study come from GenomePT project (POCI-01-0145- FEDER-022184), supported by COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e a Tecnologia (FCT). info:eu-repo/semantics/publishedVersion

Neisseria gonorrhoeae, the bacterium responsible for the sexually transmitted disease gonorrhoea, has shown an extraordinary ability to develop antimicrobial resistance (AMR) to multiple classes of antimicrobials. With no available vaccine, managingN. gonorrhoeaeinfections demands effective preventive measures, antibiotic treatment and epidemiological surveillance. The latter two are progressively being supported by the generation of whole-genome sequencing (WGS) data on behalf of national and international surveillance programmes. In this context, this study aims to performN. gonorrhoeaeclustering into genogroups based on WGS data, for enhanced prospective laboratory surveillance. Particularly, it aims to identify the major circulating WGS-genogroups in Europe and to establish a relationship between these and AMR. Ultimately, it enriches public databases by contributing with WGS data from Portuguese isolates spanning 15 years of surveillance. A total of 3791 carefully inspectedN. gonorrhoeaegenomes from isolates collected across Europe were analysed using a gene-by-gene approach (i.e. using cgMLST). Analysis of cluster composition and stability allowed the classification of isolates into a two-step hierarchical genogroup level determined by two allelic distance thresholds revealing cluster stability. Genogroup clustering in general agreed with availableN. gonorrhoeaetyping methods [i.e. MLST (multilocus sequence typing), NG-MAST (N. gonorrhoeaemulti-antigen sequence typing) and PubMLST core-genome groups], highlighting the predominant genogroups circulating in Europe, and revealed that the vast majority of the genogroups present a dominant AMR profile. Additionally, a non-static gene-by-gene approach combined with a more discriminatory threshold for potential epidemiological linkage enabled us to match data with previous reports on outbreaks or transmission chains. In conclusion, this genogroup assignment allows a comprehensive analysis ofN. gonorrhoeaegenetic diversity and the identification of the WGS-based genogroups circulating in Europe, while facilitating the assessment (and continuous monitoring) of their frequency, geographical dispersion and potential association with specific AMR signatures. This strategy may benefit public-health actions through the prioritization of genogroups to be controlled, the identification of emerging resistance carriage, and the potential facilitation of data sharing and communication.

Traumatic brain injury (TBI) is the main cause of pediatric trauma death and disability worldwide. Recent studies have sought to identify biomarkers of TBI for the purpose of assessing functional outcomes. The aim of this systematic review was to evaluate the utility of TBI biomarkers in the pediatric population by summarizing recent findings in the medical literature. A total of 303 articles were retrieved from our search. An initial screening to remove duplicate studies yielded 162 articles. After excluding all articles that did not meet the inclusion criteria, 56 studies were gathered. Among the 56 studies, 36 analyzed serum biomarkers; 11, neuroimaging biomarkers; and 9, cerebrospinal fluid (CSF) biomarkers. Most studies assessed biomarkers in the serum, reflecting the feasibility of obtaining blood samples compared to obtaining CSF or performing neuroimaging. S100B was the most studied serum biomarker in TBI, followed by SNE and UCH-L1, whereas in CSF analysis, there was no unanimity. Among the different neuroimaging techniques employed, diffusion tensor imaging (DTI) was the most common, seemingly holding diagnostic power in the pediatric TBI clinical setting. The number of cross-sectional studies was similar to the number of longitudinal studies. Our data suggest that S100B measurement has high sensitivity and great promise in diagnosing pediatric TBI, ideally when associated with head CT examination and clinical decision protocols. Further large-scale longitudinal studies addressing TBI biomarkers in children are required to establish more accurate diagnostic protocols and prognostic tools.

Coronavirus disease, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is mainly transmitted through droplets, but other ways of transmission have been hypothesized. We report a case of vertical transmission of SARS-CoV-2 in a preterm born to an infected mother, confirmed by the presence of the virus in the neonatal blood, nasopharyngeal and oropharyngeal swabs collected in the first half an hour of life. The neonate presented with acute respiratory distress, similar to the findings in severely affected adults. This case highlights the importance of pregnancy, labor and neonatal period surveillance of affected mothers and their newborns. info:eu-repo/semantics/publishedVersion

Multi-walled carbon nanotubes (MWCNT) are engineered nanomaterials widely used in industrial and biomedical applications. Yet, MWCNT inhalation may induce pulmonary adverse effects, and the MWCNT-7 (Mitsui-7) has been classified as possibly carcinogenic to humans. However, its molecular mechanisms of action are poorly understood and there are no biomarkers of exposure for occupational monitoring. Several pulmonary diseases, including lung cancer, have been associated with alterations in microRNA expression that are used as biomarkers of disease progression, and differentially-expressed microRNAs (DE miRNAs) can also allow understanding the molecular effects induced by a toxicant. In this study, we identify DE miRNAs in A549 alveolar epithelial cells following 24 h exposure to MWCNT-7 or crocidolite, as well as their enriched cellular functional pathways. These indicate that both materials change cell survival, differentiation and proliferative properties under the influence of AMPK, FoxO, TGF-β and Hippo pathways, and their metabolic activity and cell-to-cell communication. In addition, MWCNT-7 affects the actin cytoskeleton, ubiquitin mediated proteolysis, and ECM-receptor interactions; crocidolite the PI3K-Akt and mTOR pathways, endocytosis, and central carbon metabolism. Since deregulation of these pathways may be related to carcinogenesis, an interaction network of DE miRNAs and corresponding target cancer-related genes was constructed, highlighting the carcinogenic potential of Mitsui-7. Highlights: Multi-walled carbon nanotubes (MWCNT) are nanomaterials widely used that may induce pulmonary adverse effects upon inhalation; A profile of differentially expressed (DE) miRNAs in A549 alveolar cells was identified following exposure to MWCNT-7 and crocidolite using NGS; Both materials change AMPK, FoxO, TGF-β and Hippo pathways, the cell metabolic activity and cell-to-cell communication; MWCNT-7 affects the actin cytoskeleton, ubiquitin mediated proteolysis, and ECM-receptor interactions; Crocidolite affects the PI3K-Akt and mTOR pathways, endocytosis, and central carbon metabolism; An interaction network of DE miRNAs and corresponding target cancer-related genes highlighted the carcinogenic potential of MWCNT-7, even at low dose and short-term exposure. This work was funded by FCT/MCTES, Project ToxApp4NanoCELFI (PTDC/SAU-PUB/32587/2017) through national funds (PIDDAC) and ToxOmics (UID/BIM/00009/2013). It is also a result of the GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by FCT. info:eu-repo/semantics/publishedVersion

Advances 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. info:eu-repo/semantics/publishedVersion

Research on atypical developing parenting usually investigates only the mother. This happens even if the investigation outcome points to the influence of the parenting style and the child’s relationship with the father on his/her perception of well-being. This investigation aimed to understand the relationships between the behavior of children with Autistic Spectrum Disorder, parenting styles and coparenting of 45 biparental families with children aged, on mean, 59 months, according to defined profiles based on children’s behavioral differences. Data was reviewed by hierarchical clustering. Among the four profiles found, those characterized by behavioral difficulties showed more authoritarian/permissive parenting styles and negative impacts on coparenting; profiles with greater prosociability had a more authoritative parenting style and better coparenting quality. The behavioral assessment of children should drive the formulation of interventions/public policies to promote positive parenting in this population. Resumo Pesquisas sobre parentalidade em desenvolvimento atípico costumam investigar apenas a mãe, mesmo seus resultados apontando para a influência do estilo parental e do relacionamento com o pai na percepção de bem-estar da criança. Esta pesquisa objetivou compreender as relações entre o comportamento de crianças com Transtorno do Espectro Autista, os estilos parentais e o relacionamento coparental de 45 famílias biparentais com crianças com idade média de 59 meses em função dos perfis definidos a partir das diferenças comportamentais infantis. Os dados foram analisados por clusterização hierárquica. Dentre os quatro perfis encontrados, os que se caracterizavam por dificuldades comportamentais apresentaram estilos parentais mais autoritários/permissivos e impactos negativos na coparentalidade; aqueles com maior prossociabilidade evidenciaram um estilo parental mais autoritativo e uma melhor qualidade na coparentalidade. A avaliação comportamental da criança deve pautar a formulação de intervenções/políticas públicas de promoção de parentalidade positiva nessa população.

© 2020 Elsevier Inc. All rights reserved., We investigated biomarkers and genetic modulators of the cerebral vasculopathy (CV) subphenotype in pediatric sickle cell anemia (SCA) patients of sub-Saharan African ancestry. We found that one VCAM1 promoter haplotype (haplotype 7) and VCAM1 single nucleotide variant rs1409419_T were associated with stroke events, stroke risk, as measured by time-averaged mean of maximum velocity in the middle cerebral artery, and with high serum levels of the hemolysis biomarker lactate dehydrogenase. Furthermore, VCAM-1 ligand coding gene ITGA4 variants rs113276800_A and rs3770138_T showed a positive association with stroke events. An additional positive relationship between a genetic variant and stroke risk was observed for ENPP1 rs1044498_A. Conversely, NOS3 variants were negatively associated with silent cerebral infarct events (VNTR 4b_allele and haplotype V) and CV globally (haplotype VII). The -alpha3.7kb–thal deletion did not show association with CV. However, it was associated with higher red blood cell and neutrophil counts, and lower mean corpuscular volume, mean corpuscular hemoglobin and red cell distribution width. Our results underline the importance of genetic modulators of the CV sub-phenotype and their potential as SCA therapeutic targets. We also propose that a biomarker panel comprising biochemical, hematological, imaging and genetic data would be instrumental for CV prediction, and prevention., This work was partially funded by Fundação para a Ciência e a Tecnologia (FCT) grant PIC/IC/83084/2007, ISAMB, and INSA project 2012DGH720. Additionally, it is a result of the GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), FCT.

RESUMO O objetivo desse estudo foi compreender a percepção de pais e mães com filhos diagnosticados com transtorno do espectro autista, de sua relação coparental. Essa investigação caracteriza-se como de caráter exploratória e descritiva com uma abordagem qualitativa. Participaram nove casais, pais (n=9) e mães (n=9) de crianças com TEA com idades entre três a sete anos. Os dados foram coletados em um serviço de atendimento especializado no sul do Brasil. Primeiramente foi aplicado um questionário sociodemográfico e depois foi aplicado com cada membro do casal uma entrevista semiestruturada sobre o relacionamento coparental. Para o tratamento dos dados, utilizou-se o método da análise de conteúdo de Bardin. A análise dessas entrevistas com os casais permitiu a construção de categorias conforme as dimensões do modelo teórico da estrutura interna e contexto ecológico da coparentalidade de Feinberg. O acordo nas práticas parentais se destacou no discurso dos casais, embora as mães destaquem que os companheiros têm uma tendência de apresentar uma disciplina relaxada com a criança com TEA. A divisão do trabalho evidencia um desequilíbrio de tarefas exercidas pelos membros do casal, o que demonstra um sentimento de estresse nas mães sobre os cuidados do filho. Apesar disso, os cônjuges reconhecem o suporte parental e reconhecem os esforços do(a) companheiro(a) nas atividades parentais com poucos momentos de sabotagem do(a) parceiro(a) e exposição da criança aos conflitos do casal. Ao final são apontadas as limitações dessa pesquisa e levantadas algumas sugestões de futuros estudos na área, bem como as implicações dos resultados para a intervenção. RESUMEN El propósito de este estudio fue comprender la percepción de los padres de niños con trastorno del espectro autista (TEA) sobre la relación coparental. Esta investigación se caracteriza como de carácter exploratorio y descriptivo con un abordaje cualitativo. En el caso de los niños con TEA con edades entre 3 y 7 años, participaron 9 parejas, padres (n = 9) y madres (n = 9). Los datos fueron recolectados en un servicio de atención especializada en el sur de Brasil. Primero se aplicó un cuestionario sociodemográfico y luego se aplicó con cada miembro de la pareja una entrevista semiestructurada sobre la relación coparental. Para el tratamiento de los datos, se utilizó el método del análisis de contenido de Bardin. El análisis de estas entrevistas con las parejas permitió la construcción de categorías según las dimensiones del modelo teórico de la Estructura Interna y Contexto Ecológico de la Coparentalidad de Feinberg. El acuerdo en las prácticas parentales se destacó en el discurso de las parejas, aunque las madres destacan que los compañeros tienen una tendencia a presentar una disciplina relajada con el niño con TEA. La división del trabajo evidencia un desequilibrio de tareas ejercidas por los miembros de la pareja, lo que evidencia un sentimiento de estrés en las madres sobre los cuidados del hijo. A pesar de ello, los cónyuges reconocen el apoyo parental y reconocen los esfuerzos del compañero en las actividades parentales con pocos momentos de sabotaje del compañero y exposición del niño a los conflictos de la pareja. Al final se apuntan las limitaciones de esa investigación y se plantean algunas sugerencias de futuros estudios en el área, así como las implicaciones de los resultados para la intervención. ABSTRACT. The purpose of this study was to understand the perception of the parents of children with Autism Spectrum Disorder (ASD) on the coparenting relationship. This research is characterized as exploratory and descriptive with a qualitative approach. Participants included 9 couples, fathers (n = 9) and mothers (n = 9) of children with ASD aged 3 to 7 years. Data were collected in a specialized care service in the south of Brazil. First, a sociodemographic questionnaire was applied and then a semi-structured interview was applied with each member of the couple on the coparenting relationship. For the data treatment, the Bardin content analysis method was used. The analysis of these interviews with the couples allowed the construction of categories according to the dimensions of the theoretical model of the Internal Structure and Ecological Context of Coparentingby Feinberg. Agreement on parenting practices was highlighted in the discourse of couples, although mothers emphasize that partners have a tendency to present a relaxed discipline with the child with ASD. The division of labor evidences an imbalance of tasks performed by the members of the couple, which evidences a feeling of stress in the mothers on the care of their child. Nonetheless, the spouses recognize parental support and recognize the efforts of their partners in parental activities with few moments of sabotage of the partner and exposure of the child to the couple’s conflicts. At the end, the limitations of this research are pointed out and some suggestions for future studies in the area are raised, as well as the implications of the results for the intervention.

Streptococcus agalactiae evasion from the human defense mechanisms has been linked to the production of DNases. These were proposed to contribute to the hypervirulence of S. agalactiae ST17/capsular-type III strains, mostly associated with neonatal meningitis. We performed a comparative genomic analysis between ST17 and ST19 human strains with different cell tropism and distinct DNase production phenotypes. All S. agalactiae ST17 strains, with the exception of 2211-04, were found to display DNase activity, while the opposite scenario was observed for ST19, where 1203-05 was the only DNase(+) strain. The analysis of the genetic variability of the seven genes putatively encoding secreted DNases in S. agalactiae revealed an exclusive amino acid change in the predicted signal peptide of GBS0661 (NucA) of the ST17 DNase(-), and an exclusive amino acid change alteration in GBS0609 of the ST19 DNase(+) strain. Further core-genome analysis identified some specificities (SNVs or indels) differentiating the DNase(-) ST17 2211-04 and the DNase(+) ST19 1203-05 from the remaining strains of each ST. The pan-genomic analysis evidenced an intact phage without homology in S. agalactiae and a transposon homologous to TnGBS2.3 in ST17 DNase(-) 2211-04; the transposon was also found in one ST17 DNase(+) strain, yet with a different site of insertion. A group of nine accessory genes were identified among all ST17 DNase(+) strains, including the Eco47II family restriction endonuclease and the C-5 cytosine-specific DNA methylase. None of these loci was found in any DNase(-) strain, which may suggest that these proteins might contribute to the lack of DNase activity. In summary, we provide novel insights on the genetic diversity between DNase(+) and DNase(-) strains, and identified genetic traits, namely specific mutations affecting predicted DNases (NucA and GBS0609) and differences in the accessory genome, that need further investigation as they may justify distinct DNase-related virulence phenotypes in S. agalactiae. Highlights: Comparative genomic analysis between S. agalactiae ST17 and ST19 human strains; - ST17 strains except one displayed DNase activity which was observed in only one ST19; ST17 DNase(−) revealed exclusive aa change in NucA predicted signal peptide and a TnGBS2.3 homolog; - ST17 DNase(−) revealed an intact phage without homology in S. agalactiae; ST19 DNase(+) revealed an exclusive amino acid change alteration in GBS0609. This work was supported by the GenomePT project (POCI-01-0145- FEDER-022184) from Fundação para a Ciência e a Tecnologia (FCT) info:eu-repo/semantics/publishedVersion

Objective To provide a systematic review investigating the role of inflammatory molecules and neurotrophic factors as biomarkers of neuropsychomotor development in preterm neonates. Data Source: Databases including PubMed, BIREME, and Scopus were systematically searched. Observational studies, as well as transversal, and cohort studies using human subjects published from 1990 to September 2017 were eligible for inclusion. Two authors independently identified eligible studies and analyzed their characteristics, quality, and accuracy in depth. Data synthesis: 11 eligible studies clearly investigated the association between peripheral inflammation and motor and/or cognitive development in preterm infants. However, the selected populations differed in relation to the events associated with prematurity and the risk factors to abnormal motor and/or cognitive development. These studies measured circulating levels of cytokines, chemokines, adhesion molecules, acute phase proteins, and growth factors. The most commonly analyzed proteins were IL-1β, IL-6, TNF, CCL5/RANTES, CXCL8/IL-8, IGFBP-1, and VEGF. In seven of the eligible studies, plasma levels of IL-6 correlated with development delay. Two studies reported correlation between CXCL8/IL-8 plasma levels with cognitive and motor delay. In one study, higher levels of MCP-1/CCL2 were associated with better cognitive and motor outcome. Conclusion: There is preliminary evidence indicating that circulating inflammatory molecules are associated with motor and cognitive development in preterm neonates, even considering different populations.

© Springer-Verlag GmbH Germany, part of Springer Nature 2019, Sickle cell anemia (SCA) is an autosomal recessive monogenic disease with significant clinical variability. Cerebrovascular disease, particularly ischemic stroke, is one of the most severe complications of SCA in children. This study aimed to investigate the influence of genetic variants on the levels of fetal hemoglobin (Hb F) and biochemical parameters related with chronic hemolysis, as well as on ischemic stroke risk, in ninety-one unrelated SCA patients, children of sub-Saharan progenitors. Our results show that a higher Hb F level has an inverse relationship with the occurrence of stroke, since the group of patients who suffered stroke presents a significantly lower mean Hb F level (5.34 ± 4.57% versus 9.36 ± 6.48%; p = 0.024). Furthermore, the co-inheritance of alpha-thalassemia improves the chronic hemolytic pattern, evidenced by a decreased reticulocyte count (8.61 ± 3.58% versus 12.85 ± 4.71%; p, This work was partially funded by Fundação para a Ciência e a Tecnologia (FCT) grant PIC/IC/83084/2007, ISAMB, and INSA project 2012DGH720. Additionally, it is a result of the GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), FCT.

Free PMC article: https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/31130921/ Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli strain C999 was isolated of a Spanish patient with urinary tract infection. Previous genotyping indicated that this strain presented a multidrug-resistance phenotype and carried beta-lactamase genes encoding CTX-M-15, TEM-1, and OXA-1 enzymes. The whole-cell proteome, and the membrane, cytoplasmic, periplasmic and extracellular sub-proteomes of C999 were obtained in this work by two-dimensional gel electrophoresis (2DE) followed by fingerprint sequencing through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). A total of 602 proteins were identified in the different cell fractions, several of which are related to stress response systems, cellular responses, and antibiotic and drug responses, consistent with the multidrug-resistance phenotype. In parallel, whole genome sequencing (WGS) and RNA sequencing (RNA-Seq) was done to identify and quantify the genes present and expressing. The in silico prediction following WGS confirmed our strain as being serotype O25:H4 and sequence type ST131. The presence of proteins related to antibiotic resistance and virulence in an O25:H4-ST131 E. coli clone are serious indicators of the continued threat of antibiotic resistance spread amongst healthcare institutions. On a positive note, a multiomics approach can facilitate surveillance and more detailed characterization of virulent bacterial clones from hospital environments. LP was granted a Ph.D. fellowship by Fundação para a Ciência e a Tecnologia and European Social Fund (SFRH/BD/81307/2011). This work was supported by the Associate Laboratory for Green Chemistry – LAQV which is financed by national funds from FCT/MCTES (UID/QUI/50006/2019). Also, this work is a result of the GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020 – Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e a Tecnologia (FCT). info:eu-repo/semantics/publishedVersion

We studied a commensal colistin-resistant Escherichia coli isolated from a swine cecum sample collected at a slaughter, in Portugal. Antimicrobial susceptibility phenotype of E. coli LV23529 showed resistance to colistin at a minimum inhibitory concentration of 4 mg/L. Whole genome of E. coli LV23529 was sequenced using a MiSeq system and the assembled contigs were analyzed for the presence of antibiotic resistance and plasmid replicon types using bioinformatics tools. We report a novel mcr-1 gene variant (mcr-1.9), carried by an IncX4 plasmid, where one-point mutation at nucleotide T1238C leads to Val413Ala substitution. The mcr-1.9 genetic context was characterized by an IS26 element upstream of the mcr-pap2 element and by the absence of ISApl1. Bioinformatic analysis also revealed genes conferring resistance to β-lactams, sulphamethoxazole, trimethoprim, chloramphenicol and colistin, corresponding to the phenotype noticed. Moreover, we highlight the presence of mcr-1.9 plus blaCTX-M-8, a blaESBL gene rarely detected in Europe in isolates of animal origin; these two genes were located on different plasmids with 33,303 and 89,458 bp, respectively. MCR-1.9-harboring plasmid showed high identity to other X4-type mcr-1-harboring plasmids characterized worldwide, which strongly suggests that the presence of PMCR-encoding genes in food-producing animals, such as MCR-1.9, represent a potential threat to humans, as it is located in mobile genetic elements that have the potential to spread horizontally. VM was supported by Fundação para a Ciência e a Tecnologia (FCT) fellowship (Grant No. SFRH/BPD/77486/2011), financed by the European Social Funds (COMPETE-FEDER), and National Funds of the Portuguese Ministry of Education and Science (POPH-QREN). The authors thank to FCT for the project grant UID/MULTI/00211/2013. info:eu-repo/semantics/publishedVersion

Mitochondrial diseases (MD) are a group of rare inherited disorders, characterized by phenotypic heterogeneity, with hitherto no effective therapeutic options. The aim of this study was to develop a next generation sequencing (NGS) strategy, by using a custom gene panel and whole mitochondrial genome, to identify the disease causing pathogenic variants in 146 patients suspicious of MD. The molecular analysis of this cohort revealed six novel and 15 described pathogenic variants, as well as 26 variants of unknown significance. Our findings are expanding the mutational landscape of these disorders and support the use of a NGS strategy for a higher diagnostic yield. This work was supported by FCT (PTDC/DTP-PIC/2220/2014) and NORTE2020 (NORTE-01-0246-FEDER-000014). Silvia Vilarinho is supported by the National Institute Of Diabetes And Digestive And Kidney Diseases of the National Institutes of Health under Award Number K08DK113109. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. info:eu-repo/semantics/publishedVersion

Introduction: The number of burns in Portugal remains considerably large and therefore constitutes a major public health problem. There is a shortage of studies describing the epidemiological and clinical impact of seasonality on burn centre admissions, particularly in high income countries. Methods: This retrospective analysis included all patients (>18 years old) with acute burns admitted to a specialized Burn Center in Lisbon (Hospital São José) between 20 of September 2010 and 19 of September 2017 (7 years). Clinical data was obtained from electronic medical records. The patients were categorized into four groups, according to each season. Results: Six hundred and sixteen (616) patients were included in this analysis, the majority being male (55.2%). There were no significant differences between the number of patients admitted in each season nor in gender distribution. Older patients were admitted during winter. Hospitalisation time was higher for patients admitted in the winter and surface area injured was higher for those admitted during summer. Fire burns were the most common aetiology in all seasons. Work related burns and suicide attempts were more frequent in the summer. Most patients had more than one anatomical area affected (73.9%) and winter was specifically associated with more frequent isolated lower limb burns. Winter and fire burns were associated with deeper injuries. More patients underwent surgery during winter (80.7%) than in the remaining seasons (average 65%) but the average number of surgeries per season did not change significantly. The overall mortality rate was 6.8% and the need for mechanical ventilation was 28.9%. Concerning both variables we did not find major differences across seasons. Mortality was associated with older patients, deeper and more extensive burns and need for mechanical ventilation. Conclusion: Winter seems to be associated with older patients and with the most severe burns (deeper and more often needing surgery). This study was designed to fill the gap in seasonal burn profile in Portugal and it helps in establishing preventive strategies and better resource allocation. info:eu-repo/semantics/publishedVersion

Background:. Free flap reconstruction in the head and neck region is a complex field in which patient comorbidities, radiation therapy, tumor recurrence, and variability of clinical scenarios make some cases particularly challenging and prone to devastating complications. Despite low free flap failure rates, the impact of flap failure has enormous consequences for the patients. Methods:. Acknowledging and predicting high risk intra- and postoperative situations and having planned strategies on how to deal with them can decrease their rate and improve the patient’s reconstructive journey. Results:. Herein, the authors present 4 examples of significant complications in complex microvascular head and neck cancer reconstruction, encountered for the last 10 years: compression and kinking of the vascular pedicle, lack of planning of external skin coverage in osteoradionecrosis, management of the vessel-depleted neck, and vascular donor site morbidity after fibula harvest. Conclusion:. The authors reflect on the causes and propose preventative strategies in each peri-operative stage.

Hereditary hemochromatosis (HH) is an autosomal recessive disorder characterized by excessive iron absorption resulting in pathologically increased body iron stores. It is typically associated with common HFE gene mutation (p.Cys282Tyr and p.His63Asp). However, in Southern European populations up to one third of HH patients do not carry the risk genotypes. This study aimed to explore the use of next-generation sequencing (NGS) technology to analyse a panel of iron metabolism-related genes (HFE, TFR2, HJV, HAMP, SLC40A1, and FTL) in 87 non-classic HH Portuguese patients. A total of 1241 genetic alterations were detected corresponding to 53 different variants, 13 of which were not described in the available public databases. Among them, five were predicted to be potentially pathogenic: three novel mutations in TFR2 [two missense (p.Leu750Pro and p.Ala777Val) and one intronic splicing mutation (c.967-1G > C)], one missense mutation in HFE (p.Tyr230Cys), and one mutation in the 5′-UTR of HAMP gene (c.-25G > A). The results reported here illustrate the usefulness of NGS for targeted iron metabolism-related gene panels, as a likely cost-effective approach for molecular genetics diagnosis of non-classic HH patients. Simultaneously, it has contributed to the knowledge of the pathophysiology of those rare iron metabolism-related disorders.

A B S T R A C T - Background: We describe the investigation and control of an outbreak of Legionnaires’ disease in Portugal in October, November and December 2014. Methods: Confirmed cases were individuals with pneumonia, laboratory evidence of Legionella pneumophila serogroup 1 and exposure, by residence, occupational or leisure to the affected municipalities. 49 possible sources were reduced to four potential sources, all industries with wet cooling system, following risk assessment. We geo-referenced cases’ residences and the location of cooling towers defining four study areas 10 km buffer centered on each cooling tower system. We compared the number of cases with expected numbers, calculated from the outbreak’s attack rates applied to 2011 census population. Using Stones’ Test, we tested observed to expected ratios for decline in risk, with distance up to 10 km four directions. Isolates of Legionella pneumophila were compared using molecular methods. Results: We identified 403 cases, 377 of which were confirmed, 14 patients died. Patients became ill between 14 October and 2 December. A NE wind and thermal inversion were recorded during the estimated period of exposure. Disease risk was highest in people living south west from all of the industries identified and decreased with distance (p < 0.001). 71 clinical isolates demonstrated an identical SBT profile to an isolate from a cooling tower. Whole genome sequencing identified an unusual L. pneumophila subsp. fraseri serogroup 1 as the outbreak causative strain, and confirmed isolates’ relatedness. Conclusions: Industrial wet cooling systems, bacteria with enhanced survival characteristics and a combination of climatic conditions contributed to the second largest outbreak of Legionnaires’ disease recorded internationally. R E S U M O Contexto: Descrevemos a investigação epidemiológica e medidas de controlo de um surto de doença dos Legionários, ocorrido em Portugal em outubro, novembro e dezembro de 2014. Métodos: A definição de caso englobou doentes com critérios clínicos de pneumonia aguda, com provas imagiológicas compatíveis e confirmação laboratorial para a identificação de Legionella pneumophila (L. pneumophila) serogrupo 1, para além do critério epidemiológico de exposição, quer por motivos de residência, ocupacional ou lazer nas freguesias suspeitas. Quarenta e nove possíveis fontes de infeção foram reduzidas a 4 potenciais fontes, após avaliação de risco, todas as indústrias com sistema de torres de arrefecimento. A georreferenciação por residência dos casos e localização de torres permitiu definir 4 áreas de investigação num perímetro de 10 km centrado em cada uma daquelas 4 torres. Comparouse o número de casos observados com o número de casos esperados, calculados a partir de taxas de ataque do surto aplicadas à população. Usando o teste de Stones, testou-se a razão entre casos observados e casos esperados e declínio do risco em relação à distância de até 10 km em 4 direções. As amostras de L. pneumophila foram comparadas utilizando métodos moleculares. Resultados: Foram identificados 403 casos, dos quais 377 foram confirmados, tendo ocorrido 14 óbitos. Os doentes apresentaram sintomas entre 14 de outubro e 2 de dezembro. Em termos meteorológicos, foram registados ventos NE e inversão térmica durante o período estimado de exposição. O risco de doença foi maior em pessoas que vivem a sudoeste de todas as indústrias identificadas, diminuindo com o aumento da distância (p < 0,001). Amostras de 71 dos casos clínicos demonstraram um perfil SBT idêntico às amostras isoladas a partir de uma torre de arrefecimento. A sequência de genoma de L. pneumophila fraseri serogrupo 1 pouco comum como a estirpe causadora do surto confirmou a relação das amostras isoladas. Conclusões: Torres de arrefecimento industriais, agentes bacterianos com características mais desenvolvidas para elevada sobrevivência e uma rara combinação de condições climáticas, contribuíram para o segundo maior surto de doença dos Legionários registado na literatura. info:eu-repo/semantics/publishedVersion

Free PMC Article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6277554/ In Portugal, the epidemiological stage for the spread of carbapenemase-producing Enterobacteriaceae (CPE) increased from sporadic isolates or single hospital clones (2010-2013), to hospital outbreaks, later. Here we report data from a 6-month study performed under the European Survey on Carbapenemase-Producing Enterobacteriaceae (EuSCAPE). During the study period, 67 isolates (61 Klebsiella pneumoniae and 6 Escherichia coli) non-susceptible to carbapenems were identified in participant hospital laboratories. We detected 37 blaKPC-type (including one new variant: blaKPC-21), 1 blaGES-5, and 1 blaGES-6 plus blaKPC-3, alone or in combination with other bla genes. Bioinformatics analysis of the KPC-21-producing E. coli identified the new variant blaKPC-21 in a 12,748 bp length plasmid. The blaKPC-21 gene was harbored on a non-Tn4401 element, presenting upstream a partial ISKpn6 (ΔISKpn6/ΔtraN) with the related left IR (IRL) and downstream a truncated Tn3 transposon. PFGE and MLST analysis showed an important diversity, as isolates belonged to distinct PFGE and STs profiles. In this study, we highlighted the presence of the high-risk clone E. coli sequence-type (ST) 131 clade C/H30. This worldwide disseminated E. coli lineage was already detected in Portugal among other antibiotic resistance reservoirs. This study highlights the intra- and inter-hospital spread and possible intercontinental circulation of CPE isolates. VM was supported by Fundação para a Ciência e a Tecnologia (FCT) fellowship (Grant SFRH/BPD/ 77486/2011), financed by the European Social Funds (COMPETE-FEDER) and national funds of the Portuguese Ministry of Education and Science (POPH-QREN). The authors thank FCT for Project grant UID/MULTI/00211/2013. info:eu-repo/semantics/publishedVersion

Opuntia ficus-indica (L.) Mill. is a xerophylous plant that originated in tropical and subtropical America. This plant is popularly known in Brazil as &ldquo, palma forrageira&rdquo, (cactus pear) and plays a fundamental role in animal nutrition, mainly in the Northeastern semi-arid region of the country. The plant has several uses since it presents bioactive compounds that confer biological and pharmacological properties. In this context, the cactus pear can also be considered a potential product to combat parasite infections. The objective of this study was to chemically characterize the O. ficus-indica hydroalcoholic extract (OFIEOH) and to determine its efficacy against gastrointestinal parasites using in vitro tests. Initially, the hydroalcoholic extract from cladode peels of O. ficus-indica was produced by maceration for 21 days. For the chemical characterization, colorimetric dosages were performed for carbohydrates, proteins, phenols and condensed tannins. Liquid chromatography coupled to mass spectrometry/electron spray ionization (LC-MS/ESI) was used to characterize the polyphenolic profile of the OFIEOH extract. Fifteen compounds were identified in the OFIEOH extract, such as methyl, glycosylated and aglycone quercetin derivatives and aglycone and glycosylated kaempferol derivatives. Tri-glycosylated methyl quercetin derivatives were the main compounds identified. In vitro egg hatch (EHT) and larval migration tests (LMT) were used in a range of concentrations of OFIEOH from 12.5 to 100 mg/mL for EHT and 12.5 to 200 mg/mL for LMT. In addition, the LMT was used to test ivermectin (IVM) (from 11.4 to 57.1 &micro, M), associated with the inhibitory concentration of 50% (IC50) for OFIEOH. The combination of OFIEOH (12.5 to 200 mg/mL) plus the IC50 of IVM was also tested. The efficacy of OFIEOH alone varied from 19.33 to 90.0% using the EHT. The LMT revealed an efficacy of 5.78 to 77.26% for the extract. Both tests showed a concentration-dependence inhibitory effect. We found a drug-extract antagonistic neutralizing effect when doses of IVM were added to OFIEOH (maximum efficacy of 73.78%), while a positive additive effect was observed when OFIEOH was added to the IC50 of IVM (IC50 of 82.79 for OFIEOH alone against an IC50 of 55.08 of OFIEOH + IVM). The data from this work indicate that OFIEOH alone may be considered as a suitable ecofriendly product to control gastrointestinal parasites of sheep, offering a more holistic approach to improve animal farming and welfare. The drug-extract interaction is also a promising therapeutic alternative, reducing the final dose to the host, with an optimum combination effect.

Whole-genome sequencing (WGS)-based bioinformatics platforms for the rapid prediction of resistance will soon be implemented in the Tuberculosis (TB) laboratory, but their accuracy assessment still needs to be strengthened. Here, we fully-sequenced a total of 54 multidrug-resistant (MDR) and five susceptible TB strains and performed, for the first time, a simultaneous evaluation of the major four free online platforms (TB Profiler, PhyResSE, Mykrobe Predictor and TGS-TB). Overall, the sensitivity of resistance prediction ranged from 84.3% using Mykrobe predictor to 95.2% using TB profiler, while specificity was higher and homogeneous among platforms. TB profiler revealed the best performance robustness (sensitivity, specificity, PPV and NPV above 95%), followed by TGS-TB (all parameters above 90%). We also observed a few discrepancies between phenotype and genotype, where, in some cases, it was possible to pin-point some "candidate" mutations (e.g., in the rpsL promoter region) highlighting the need for their confirmation through mutagenesis assays and potential review of the anti-TB genetic databases. The rampant development of the bioinformatics algorithms and the tremendously reduced time-frame until the clinician may decide for a definitive and most effective treatment will certainly trigger the technological transition where WGS-based bioinformatics platforms could replace phenotypic drug susceptibility testing for TB. This work was supported by Centre for Toxicogenomics and Human Health (ToxOmics, ref. UID/BIM/00009/2013) and GenomePT (ref.POCI-01-0145-FEDER-022184) from Fundação para a Ciência e Tecnologia, Portugal. info:eu-repo/semantics/publishedVersion

Molecular studies on cyanobacteria often involve filtering and freezing of samples leading to loss of cell morphological features. Methanol is often used in preservation of biological materials in association with other fixatives. This study evaluates the application of methanol in the preservation of DNA for molecular studies as well as for the preservation of cell morphology for morphometric analysis in filamentous cyanobacteria. In the present study, both culture and environmental bloom samples were preserved using a cold methanol dehydration series (50, 70, and 100 %) and stored at −20 °C for up to 2 years. The DNA quantity and quality, nucleotide sequence retrieval, and real-time PCR quantification were analyzed over time. Morphometric cell analysis was performed on preserved samples. Results show that the DNA extracted from samples preserved up to 6 months was successfully quantified by real-time PCR. After that period, the DNA quantity decreased with the preservation time. Nevertheless, we were able to detect/amplify the target fragment in samples preserved up to 2 years. The DNA sequence and cell morphology were also maintained during the preservation time. Thus, methanol preservation is an adequate method to preserve molecular information and morphological features after long storage periods. © 2015, Springer Science+Business Media Dordrecht. This research was partially supported by the European Regional Development Fund (ERDF) through the COMPETE - Operational Competitiveness Program - and FCT - Foundation for Science and Technology national funds through projects PPCDT/AMB/67075/2006 and PEst-C/MAR/LA0015/2013, the Post-Doc research grant SFRH/BPD/75922/2011 to E. Valério and Ph.D. research grant SFRH/BD65706/2009 to C. Churro. The authors acknowledge S. Paulino from the Laboratory of Biology and Ecotoxicology of the National Institute of Health Doutor Ricardo Jorge for the assistance in the biovolume measurements.

A new QepA4 variant was detected in an O86:H28 ST156-fimH38 Escherichia coli, showing a multidrug-resistance phenotype. PAβN inhibition ofqepA4-harboring transconjugant resulted in increase of nalidixic acid accumulation. TheqepA4andcatA1genes were clustered in a 26.0-kp contig matching an IncF-type plasmid, and containing a Tn21-type transposon with multiple mobile genetic elements. This QepA variant is worrisome because these determinants might facilitate the selection of higher-level resistance mutants, playing a role in the development of resistance, and/or confer higher-level resistance to fluoroquinolones in association with chromosomal mutations. VM was supported by FCT fellowship (grant SFRH/BPD/77486/2011), financed by the European Social Funds (COMPETE-FEDER) and national funds of the Portuguese Ministry of Education and Science (POPH-QREN). The authors thank Fundação para a Ciência e a Tecnologia (FCT) for project grant PEst-OE/AGR/UI0211/2011-2014, Strategic Project UI211-2011-2014 info:eu-repo/semantics/publishedVersion

Objectives. Development of a simple mutation directed method in order to allow lowering the cost of mutation testing using an easily obtainable biological material. Assessment of the feasibility of such method was tested using a GC-rich amplicon. Design and Methods. A method of denaturing high-performance liquid chromatography (dHPLC) was improved and implemented as a technique for the detection of variants in exon 9 of the IDUA gene. The optimized method was tested in 500 genomic DNA samples obtained from dried blood spots (DBS). Results. With this dHPLC approach it was possible to detect different variants, including the common p.Trp402Ter mutation in the IDUA gene. The high GC content did not interfere with the resolution and reliability of this technique, and discrimination of G-C transversions was also achieved. Conclusion. This PCR-based dHPLC method is proved to be a rapid, a sensitive, and an excellent option for screening numerous samples obtained from DBS. Furthermore, it resulted in the consistent detection of clearly distinguishable profiles of the common p.Trp402Ter IDUA mutation with an advantageous balance of cost and technical requirements.

Morganella morganii is a commensal bacterium and opportunistic pathogen often present in the gut of humans and animals. We report the 4.3 Mbp draft genome sequence of a M. morganii isolated in association with an Escherichia coli from broilers in Portugal that showed macroscopic lesions consistent with colisepticemia. The analysis of the genome matched the multidrug resistance phenotype and enabled the identification of several clinically important and potentially mobile acquired antibiotic resistance genes, including the plasmid-mediated quinolone resistance determinant qnrD1. Mobile genetic elements, prophages, and pathogenicity factors were also detected, improving our understanding toward this human and animal opportunistic pathogen. DJ-D has received research funding from Fundação para a Ciência e a Tecnologia (FCT, grant number SFRH/BD/80001/2011). VM was supported by FCT fellowship (grant SFRH/BPD/77486/2011), financed by the European Social Funds (COMPETE-FEDER) and national funds of the Portuguese Ministry of Education and Science (POPHQREN). We thank the support of FCT grant number PEstOE/AGR/UI0211/2011-2014 and UID/MULTI/00211/2013. info:eu-repo/semantics/publishedVersion

Insights into the genomic adaptive traits of Treponema pallidum, the causative bacterium of syphilis, have long been hampered due to the absence of in vitro culture models and the constraints associated with its propagation in rabbits. Here, we have bypassed the culture bottleneck by means of a targeted strategy never applied to uncultivable bacterial human pathogens to directly capture whole-genome T. pallidum data in the context of human infection. This strategy has unveiled a scenario of discreet T. pallidum interstrain single-nucleotide-polymorphism-based microevolution, contrasting with a rampant within-patient genetic heterogeneity mainly targeting multiple phase-variable loci and a major antigen-coding gene (tprK). TprK demonstrated remarkable variability and redundancy, intra- and interpatient, suggesting ongoing parallel adaptive diversification during human infection. Some bacterial functions (for example, flagella- and chemotaxis-associated) were systematically targeted by both inter- and intrastrain single nucleotide polymorphisms, as well as by ongoing within-patient phase variation events. Finally, patient-derived genomes possess mutations targeting a penicillin-binding protein coding gene (mrcA) that had never been reported, unveiling it as a candidate target to investigate the impact on the susceptibility to penicillin. Our findings decode the major genetic mechanisms by which T. pallidum promotes immune evasion and survival, and demonstrate the exceptional power of characterizing evolving pathogen subpopulations during human infection. This study was partially supported by grant EXPL/BIA-MIC/0309/2013 from the Fundação para a Ciência e a Tecnologia (FCT).

We report here the draft genome sequence of the CTX-M-166-harboring O6:H16 sequence type 48 (ST48)- fimH 34 Escherichia coli strain recovered from a Gallus gallus broiler. Sequence analyses revealed the presence of an IncI1/ST103-IS Ecp1 - bla CTX-M-166 - orf477 plasmid region and of diverse antibiotic resistance and virulence-acquired genes.

The spread of antibiotic resistant bacteria throughout the food chain constitutes a public health concern. To understand the contribution of fresh produce in shaping antibiotic resistance bacteria and integron prevalence in the food chain, 333 antibiotic resistance Gram negative isolates were collected from organic and conventionally produced fruits (pears, apples, and strawberries) and vegetables (lettuces, tomatoes, and carrots). Although low levels of resistance have been detected, the bacterial genera identified in the assessed fresh produce are often described not only as environmental, but mostly as commensals and opportunistic pathogens. The genomic characterization of integron-harboring isolates revealed a high number of mobile genetic elements and clinically relevant antibiotic resistance genes, of which we highlight the presence of as mcr-1, qnrA1, bla GES-11, mphA, and oqxAB. The study of class 1 (n = 8), class 2 (n = 3) and class 3 (n = 1) integrons, harbored by species such as Morganella morganii, Escherichia coli, Klebsiella pneumoniae, led to the identification of different integron promoters (PcW, PcH1, PcS, and PcWTNG-10) and cassette arrays (containing drfA, aadA, cmlA, estX, sat, and bla GES). In fact, the diverse integron backbones were associated with transposable elements (e.g., Tn402, Tn7, ISCR1, Tn2 (*), IS26, IS1326, and IS3) that conferred greater mobility. This is also the first appearance of In1258, In1259, and In3-13, which should be monitored to prevent their establishment as successfully dispersed mobile resistance integrons. These results underscore the growing concern about the dissemination of acquired resistance genes by mobile elements in the food chain. DJ has received research funding from Fundação para a Ciência e a Tecnologia (FCT, grant number SFRH/BD/80001/2011). VM was supported by FCT fellowship (grant SFRH/BPD/77486/2011), financed by the European Social Funds (COMPETEFEDER) and national funds of the Portuguese Ministry of Education and Science (POPH-QREN). We thank the support of FCT grant number PEst-OE/AGR/UI0211/2011-2014 and UID/MULTI/00211/2013. info:eu-repo/semantics/publishedVersion

BACKGROUND: Salmonella enterica is one of the most important causes of gastrointestinal infection in humans, being the great majority of infections related to the consumption of poultry meat and eggs (Foley and Lynne, 2008; EFSA/ECDC, 2015). In animals, infections caused by serotype Enteritidis are rarely responsible for severe disease with animals frequently becoming asymptomatic carriers, except in the case of young chicks and poults, where outbreaks exhibiting clinical disease are often accompanied by high mortality rates (Foley et al., 2008, 2013). Indeed, S. enterica subsp. enterica serovar Enteritidis (S. Enteritidis) has been responsible for severe disease in industrial poultry farming facilities worldwide, posing a potential hazard for public health (Lutful Kabir, 2010). In order to be infectious, Salmonella needs to adapt to different niches and conditions, where virulence and heavy-metal-tolerance factors play an important role, through co-selection events and the formation of pathogenicity islands, respectively (Hensel, 2004; Medardus et al., 2014). Furthermore, antibiotic resistance determinants can also facilitate their survival, with ubiquitous chromosomally encoded efflux mechanisms, playing an important role in both intrinsic, and acquired multidrug resistance. Other resistance mechanisms, such as changes in the membrane permeability, enzymatic modification, and target alterations may increase the levels of bacterial resistance, contributing to the success of the infection (Poole, 2004; Delmar et al., 2014; Li et al., 2015). Both antibiotic susceptibility determination and serotyping constitute very useful tools for the epidemiologic classification of S. enterica isolates. Indeed, in S. enterica, the resistance rates fluctuate according to the serotype and with the antibiotic (Clemente et al., 2015). Classically, serotyping is based on the antigenic reactivity of lipolysaccharide (O antigen) and flagellar proteins (H antigen), followed by a designation using names or formulas (Grimont and Weill, 2007). In this study, we aimed to analyze the genome of a S. Enteritidis isolate responsible for omphalitis in chicks, exploring the molecular features associated with antibiotic resistance and pathogenicity, as well as the ability to spread the respective determinants. DJ has received research funding from Fundação para a Ciência e a Tecnologia (FCT, grant number SFRH/BD/80001/2011). VM was supported by FCT fellowship (grant SFRH/BPD/77486/2011), financed by the European Social Funds (COMPETE-FEDER) and national funds of the Portuguese Ministry of Education and Science (POPH-QREN). We thank the support of FCT grant number PEst-OE/AGR/UI0211/2011-2014 and UID/MULTI/00211/2013. info:eu-repo/semantics/publishedVersion

Background The relationship between Toxoplasma gondii infection and the development of bipolar disorder (BD) has long been investigated, yet to date it is still poorly understood and documented. The aim of this review is to derive a summary estimate of the strength of the association between infection with T. gondii and BD from the available published studies. Methods A systematic review was performed using PubMed, LILACS, PsycINFO, and Embase databases. Studies which included a proportion of seropositive BD patients and controls were further examined in a meta-analysis. Results One hundred eighteen citations were initially retrieved. Thirteen studies were included in our systematic review. Eight out of these thirteen studies were included in our meta-analysis. Statistical analyses showed that T. gondii infection is associated with with BD (OR=1.26). Limitations Small sample size was the major limitation among the studies that carried out serological analyses. In addition, the available studies did not have enough information on disease status/severity or type of bipolar disorder. Also, it was not possible to analyze pregnancy status or perinatal infection. Future studies addressing the aforementioned topics are clearly needed. Conclusions Despite heterogeneous results, patients with BD are more likely to be infected by T. gondii than controls. Early T. gondii infection might predispose the development of BD. T.gondii infection is becoming clinically relevant in psychiatric disorders and future mechanistic studies are required to elucidate the underlying pathophysiological mechanisms.

We present draft genome sequences of 10 Helicobacter pylori clinical strains isolated from children. This will be important for future studies of comparative genomics in order to better understand the virulence determinants underlying peptic ulcer disease. This work was supported by the FCT-PTDC/BIM-MEC/1051/2012 grant from the Fundação para a Ciência e a Tecnologia (FCT) (to M.O.). A.N. and F.F.V. are recipients of postdoctoral fellowships (SFRH/BPD/75295/2010 and SFRH/BPD/95125/2013, respectively) from FCT, and R.R. is a recipient of a fellowship (BRJ-DDI/2012) from the National Institute of Health.

Correspondence to the Editor. Legionnaires’ disease is an often severe form of pneumonia that is typically acquired by susceptible persons (e.g., elderly persons and smokers) through inhalation of aerosols that contain legionella species.1-4 A cluster of cases of this disease occurred in Vila Franca de Xira, Portugal, in 2014.

Burkholderia pseudomallei is a Gram-negative bacillus and the causative agent of melioidosis, a serious infection associated with high mortality rate in humans. It can be naturally found as an environmental saprophyte in soil or stagnant water, and rice paddies that predominate in regions of endemicity such as Northeast Thailand. B. pseudomallei is a Biosafety Level 3 organism due to risks of aerosolization and severe disease and is now included in formal emergency preparedness plans and guidelines issued by various authorities in the United States and Europe. Here, we report the first case of imported melioidosis in Portugal. B. pseudomallei was isolated from the patient's blood as well as from a left gluteal abscess pus. The isolate strain showed the unusual resistance profile to first-line eradication therapy trimethroprim/sulfamethoxazole. Whole genome sequencing revealed its similarity with isolates from Southeast Asia, suggesting the Thai origin of this Portuguese isolate, which is in agreement with a recent patient's travel to Thailand.

Este estudo teve como objetivo identificar os tipos de brinquedos mais usados pelas crianças e verificar as diferenças existentes quanto ao género e à idade de crianças brasileiras e portuguesas. Participaram da pesquisa 259 crianças no Brasil e 172 crianças em Portugal. Todas as crianças frequentavam os quatro primeiros anos do ensino básico e tinham entre 6 e 12 anos de idade. Os dados foram recolhidos por meio de uma escala de brinquedos elaborada especificamente para este estudo. Através da análise dos dados constatou-se que, quanto à caracterização dos brinquedos, muitas semelhanças entre as duas amostras foram encontradas. Os meninos de ambas as amostras apresentaram maior utilização de brinquedos que exigem actividades físicas intensas. As crianças mais jovens da amostra brasileira utilizaram com maior frequência brinquedos que promovem e estimulam o desenvolvimento motor e social. Na amostra portuguesa não foram encontradas diferenças significativas na utilização dos brinquedos entre os diferentes níveis escolares das crianças. This study aims to identify the kinds of toys more used by children and to verify differences about gender and age of Brazilian and Portuguese children. The participants in the research included 259 Brazilian children and 172 Portuguese children. All of these children attended the first four years of elementary school and they are between 6 and 10 years old. Data were collected by a scale of toys designed specifically for this study. By analyzing the data some similarities could be found between these two samples about the characterization of toys. Boys of both samples demonstrated a predilection for the use of toys that require a more intense physical activity. The youngest children of the Brazilian sample showed to be more predisposed to use toys that promote and stimulate the motor and social development. In the Portuguese sample, in turn, significant differences in the use of toys among children from different school levels were not found. info:eu-repo/semantics/publishedVersion

OBJETIVO: comparar as brincadeiras de crianças em idade escolar em amostras de dois países diferentes (Brasil e Portugal). MÉTODO: em cada país 11 crianças participaram do estudo. As crianças freqüentavam o segundo ano do ensino fundamental. As observações foram realizadas dentro do contexto escolar, em uma sala que continha brinquedos. Para registrar os comportamentos das crianças foram utilizadas as técnicas de observação do sujeito focal e amostragem de tempo. RESULTADOS: nas duas amostras foram encontrados resultados similares quanto aos tipos de interação social e escolha de brinquedos e brincadeiras. Quanto à interação social, as crianças preferiam brincar em díades. Os meninos mostraram uma tendência maior do que as meninas para a segregação por sexos em suas atividades. Os tipos de brinquedos mais usados pelas crianças foram os da categoria motora, seguidos pelos cognitivos. Os brinquedos didáticos foram os menos utilizados. A brincadeira proposta pelo objeto foi a atividade mais observada pelos pesquisadores. Contudo, quando as crianças não estavam seguindo a proposta dos objetos, foram encontrados diferentes resultados nas amostras. As crianças brasileiras preferiam os jogos de regras, enquanto que as portuguesas preferiam as brincadeiras de faz-de-conta. Nos dois países os meninos preferiram brincadeiras turbulentas mais do que as meninas. Quando as crianças brasileiras não estavam brincando, elas prefe-riam atividades de conversação, enquanto que as crianças portuguesas atividades de observação. CONCLUSÃO: as similaridades encontradas nas duas amostras sugerem aspectos comuns do comportamento de brincar, enquanto que as diferenças apontam para as particularidades de cada contexto sócio-cultural.

Background: Toxoplasma gondii (T. gondii) infection has been identified as a risk factor for schizophrenia. Objectives: Herein, we sought to evaluate the association between T. gondii infection and clinical symptoms and quality of life in patients with schizophrenia. Methods: We conducted a cross-sectional study with 48 patients with chronic schizophrenia and 40 controls. Peripheral blood was drawn, and IgM and IgG anti-T. gondii antibodies were evaluated by Enzyme-Linked Immunosorbent Assay (ELISA). Depressive, positive and negative symptoms were assessed, respectively, by the Calgary Depression Scale (CDS) and the Positive and Negative Syndrome Scale (PANSS). Cognitive performance was assessed in patients by the Brazilian version of the Schizophrenia Cognition Rating Scale (SCoRS-BR). Quality of life was assessed by the Brazilian version of the Quality of Life in Schizophrenia scale (QLS-BR). Results: The prevalence and titers of T. gondii IgM and IgG antibodies did not differ between patients and controls. The positive serology for T. gondii IgG antibodies was not associated with illness symptoms, cognitive performance, depressive symptoms or quality of life. Discussion: Our findings suggest that toxoplasmosis infection is not associated with severity of symptoms, quality of life, cognitive or depressive symptoms in schizophrenia patients.

8 páginas, 3 figuras, We show the distribution of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genetic clades over time and between countries and outline potential genomic surveillance objectives. We applied three genomic nomenclature systems to all sequence data from the World Health Organization European Region available until 10 July 2020. We highlight the importance of real-time sequencing and data dissemination in a pandemic situation, compare the nomenclatures and lay a foundation for future European genomic surveillance of SARS-CoV-2., We gratefully acknowledge the authors, originating and submitting laboratories of the sequences from GISAID’s EpiCoV Database used in the phylogenetic analysis. We gratefully acknowledge all the staff working with sample collection, sample preparation, sequencing, data analysis and data sharing in all laboratories in the WHO European Region for making this work possible.