Responses of soil microbiome to copper-based materials (nano and bulk) for agricultural applications: An indoor-mesocosm experiment.

The foreseen increasing application of copper-based nanomaterials (Cu-NMs), replacing or complementing existing Cu-agrochemicals, may negatively impact the soil microbiome. Thus, we studied the effects on soil microbiome function and composition of nano copper oxide (nCuO) or copper hydroxide NMs in a commercial (Kocide®3000) or a lab-synthetized formulation (nCu(OH)2) or bulk copper hydroxide (Cu(OH)2-B), at the commonly recommended Cu dose of 50 mg(Cu)kg-1 soil. Microbial responses were studied over 28 days in a designed indoor mesocosm. On day-28, in comparison to non-treated soil (CT), all Cu-treatments led to a reduction in dehydrogenase (95% to 68%), arylsulfatase (41% to 27%), and urease (40% to 20%) activity. There was a 32% increase in the utilization of carbon substrates in the nCuO-treatment and an increased abundance of viable bacteria in the nCu(OH)2-treatment (75% of heterotrophic and 69% of P-solubilizing bacteria). The relative abundance of Acidobacteria [Kocide®3000, nCuO, and Cu(OH)2-B treatments] and Flavobacteriia [nCu(OH)2-treatment] was negatively affected by Cu exposure. The abundance of Cu-tolerant bacteria increased in soils treated with Kocide®3000 (Clostridia) and nCu(OH)2 (Gemmatimonadetes). All Cu-treated soils exhibited a reduced abundance of denitrification-related genes (0.05% of nosZ gene). The DTPA-extractable pool of ionic Cu(II) varied among treatments: Cu(OH)2-B > Kocide®3000 âˆ¼ nCuO>nCu(OH)2, which may explain changes on the soil microbiome composition, at the genera and OTU levels. Thus, our study revealed that Cu-materials (nano and bulk) influence the soil microbiome with implications on its ecological role. It highlights the importance of assessing the impact of Cu-materials under dynamic and complex exposure scenarios and emphasizes the need for specific regulatory frameworks for NMs.

Effects of the COVID-19 Pandemic on Breastfeeding Initiation and Duration: A Retrospective Cohort Study.

INTRODUCTION AND OBJECTIVES: Several studies support the health benefits of breastfeeding for both the mother and the newborn. However, a significant number of mothers discontinue breastfeeding within the first six months of childbirth, with several factors influencing breastfeeding adherence. The purpose of this study is to assess the impact of the COVID-19 pandemic on the prevention of mother-to-newborn infection transmission, breastfeeding patterns and duration, and the incidence of other infections during the first year of life. METHODS: Data from a sample of 39 mothers who gave birth at the Hospital Pedro Hispano in Porto, Portugual, between March 2020 and November 2021 were collected and a telephone questionnaire was administered. Statistical analysis was conducted using R software, v. 4.2.1 (R Foundation for Statistical Computing, Vienna, Austria). RESULTS AND DISCUSSION: In terms of the impact of the COVID-19 norm 18/2020, which went into effect on March 30th, our research found that the type of feeding during hospitalization was significantly influenced by this norm (X2=10.30, p=0.006). We also confirmed that mothers who received home assistance breastfed for an extra 4.5 months (95% CI: 1-7.5) compared with mothers who did not receive such assistance. Regarding the effect of COVID-19 and breastfeeding on newborn health, our study found that if the total duration of breastfeeding is less than six months, an infection is approximately five times more likely (95% CI = 1.06- 29.56). CONCLUSION: Overall, the findings of this study indicate that the efforts implemented at Hospital Pedro Hispano to limit the effects of the COVID-19 pandemic had some effect on immediate breastfeeding patterns, but not on the total duration of breastfeeding or newborn health. Nonetheless, more continuous assistance at home would have been beneficial.

Differential sensitivity of aquatic life stages of Pelophylax perezi to an acidic metal-contaminated effluent.

Acid mine drainage (AMD) involves complex mixtures of metals and hydrogen ions that can be highly toxic to the biota. Assessing the effects of AMD to aquatic stages of amphibians is key, as this group constitutes the vertebrate class with the highest proportion of species considered as threatened. Thus, the present work aimed at assessing the sensitivity of two aquatic life stages of the green frog Pelophylax perezi to an AMD originated from a cupric-pyrite mine. Embryos (Gosner stages 10-11) and tadpoles (Gosner stages 20-21) of P. perezi were exposed, for 96 h, to six AMD dilutions (1.39% to 7.5%). Endpoints involving responses at different levels of biological organization were monitored: mortality, malformations, hatching rates, body length and growth rate, enzymatic activity related with detoxification metabolism (glutathione S-transferase), and histopathologies (anatomical structures of the digestive, respiratory, and excretory systems). Embryos presented high mortality and malformation rates at AMD levels equal or above 5.36%, as well as premature hatching at 1.95% of AMD or higher. A significant reduction in body length and growth rate occurred in embryos and tadpoles exposed to 1.95% or higher levels of AMD, respectively. At the histological level, several abnormalities were observed for AMD-exposed tadpoles in a variety of tissues. One of the most noticeable histological changes occurred in the intestine that exhibited papillary epithelial hyperplasia along with a yellowish content and was more pronounced in tadpoles exposed to higher AMD levels. FEmbryos were more sensitive to lethal levels of AMD than tadpoles, suggesting embryos as a useful model life stage when performing amphibian risk assessment of mine drainage. Furthermore, AMD was highly toxic for P. perezi aquatic life stages since levels as low as 1.95% induced lethal effects. These results emphasize the importance of implementing efficient remediation methodologies for AMD, given its high toxicity.

Hypermagnesemia in preterm neonates exposed to antenatal magnesium sulfate.

BACKGROUND: One of the possible adverse outcomes of magnesium sulfate (MgSO4) administration for preterm fetal neuroprotection is hypermagnesemia in the newborn. The objectives of this study were to evaluate the association between antenatal MgSO4 administration for neuroprotection and neonatal serum magnesium levels in the first days of life. METHODS: A single-center retrospective case-control study was conducted on preterm neonates born in our institution between January 2017 and December 2019. Prenatal, perinatal, and postnatal parameters were recorded, and demographic information was collected. Comparative analysis between the group of neonates exposed to antenatal MgSO4 and the control group was performed. RESULTS: A total of 98 patients were enrolled in the study, 49 of them were exposed to antenatal MgSO4 for neuroprotection. The serum magnesium levels in the 5 first days of life were higher in the case group, and higher than the normal range of serum magnesium levels (>2.4 mg/dL), with a statistical significance. The number of days needed to normalize serum magnesium levels was higher in the case group compared to the control group (median of 7 days [1-8] versus 2 days [1-5]). CONCLUSIONS: Our findings show that antenatal administration of MgSO4 is associated with high serum magnesium levels in neonates. This group of patients needs close electrolyte monitoring during the first days of life. Parenteral nutrition supplemented with magnesium should only be considered after evaluation of serum magnesium levels.

[Survival and Neurodevelopmental Outcomes of Premature Infants with Severe Peri-Intraventricular Hemorrhage at 24 Months of Age]. / Hemorragia Peri-Intraventricular Grave em Prematuros: Impacto na Mortalidade e no Neurodesenvolvimento aos 24 Meses.

INTRODUCTION: Severe peri-intraventricular haemorrhage has been associated with higher mortality and neurodevelopmental impairment. The impact of peri-intraventricular haemorrhage alone (without white matter injury) remains controversial. The aim of this study was to evaluate the influence of severe peri-intraventricular haemorrhage, associated or not with cystic peri-ventricular leukomalacia, on mortality and neurodevelopment at 24 months. MATERIAL AND METHODS: Retrospective cohort study, that included newborns with severe peri-intraventricular haemorrhage admitted to a maternity hospital with differentiated perinatal support between 2006 and 2015, and two controls with the same gestational age, without peri-intraventricular haemorrhage, who were admitted immediately after the case. Neurodevelopmental assessment, at 24 months, was performed in 99 children, using the Schedule of Growing Skills II scale in 52 and the Ruth Griffiths mental development scale in 47 children. Severe neurodevelopmental deficit was diagnosed in the following conditions: cerebral palsy, delayed psychomotor development, deafness requiring hearing aids and blindness. RESULTS: The study included 41 cases and 82 controls. Out of these, 23 died, 16 (39.0%) in the group of severe peri-intraventricular haemorrhage and seven (8.5%) in the control group (OR 7.6, 95% CI 2.6 - 20.4, p < 0.001). Severe neurodevelopmental deficit was diagnosed in seven (30.4%) in the severe peri-intraventricular haemorrhage group and one (1.3%) in the control group (OR 32; 95% CI 3.7 - 281, p < 0.001). Individualized analysis showed that mortality was higher in peri-intraventricular haemorrhage grade III with associated cystic peri-ventricular leukomalacia (OR 4.4 95% CI 1.3 - 14.2, p = 0.015) and in peri-intraventricular haemorrhage IV (OR 12; 95% CI 3.5 - 41.2, p < 0.001), when compared to controls. Differences were also noticed regarding severe neurodevelopmental deficit when compared with controls (1.3%) in grade III peri-intraventricular haemorrhage with associated cystic peri-ventricular leukomalacia, (75.0%, p < 0.001) and grade IV peri-intraventricular haemorrhage (50.0%, p < 0.001 ). DISCUSSION: This work showed a higher mortality rate and neurodevelopment impairment in preterm newborns with severe peri-ventricular haemorrhage. Analysis by groups stratified according to gestational age and different grades of peri-ventricular haemorrhage displayed the complications associated with peri-ventricular haemorrhage grade IV or grade III, with or without cystic peri-ventricular leukomalacia. CONCLUSION: Preterm newborns with peri-intraventricular haemorrhage grade IV or grade III with cystic peri-ventricular leukomalacia, had a higher risk of mortality and severe neurodevelopmental impairment.

Introdução: A hemorragia peri-intraventricular grave tem sido associada a maior mortalidade e sequelas do neurodesenvolvimento.Mantém-se controverso o impacto da hemorragia peri-intraventricular isolada, sem lesão da substância branca. O objetivo deste trabalho foi avaliar a influência da hemorragia peri-intraventricular grave, associada ou não a leucomalácia peri-ventricular quística, na mortalidade e no neurodesenvolvimento aos 24 meses.Material e Métodos: Estudo de coorte retrospetiva que incluiu os recém-nascidos com hemorragia peri-intraventricular grave, internados numa maternidade de apoio perinatal diferenciado, entre 2006 e 2015, e dois controlos com a mesma idade gestacional, internados logo a seguir ao caso, sem hemorragia peri-intraventricular. A avaliação do neurodesenvolvimento, aos 24 meses, foi realizada em 99 crianças, com recurso à escala The Schedule of Growing Skills Scale II em 52 e à escala de desenvolvimento mental de Ruth Griffiths em 47 crianças. Considerou-se défice grave do neurodesenvolvimento: paralisia cerebral, atraso do desenvolvimento psicomotor, surdez com necessidade de prótese auditiva ou cegueira.Resultados: Foram incluídos 41 recém-nascidos com hemorragia peri-intraventricular grave e 82 controlos. Ocorreram 23 óbitos, 16 (39,0%) nas hemorragias peri-intraventricular graves e sete (8,5%) nos controlos (OR 7,6; IC 95% 2,6 - 20,4; p < 0,001). Verificou-se défice grave do neurodesenvolvimento em sete (30,4%) no grupo de hemorragia peri-intraventricular graves e um (1,3%) no grupo de controlos (OR 32; IC 95% 3,7 - 281; p < 0,001). Na análise individualizada, a mortalidade foi superior quer nas hemorragia peri-intraventricular grau III com leucomalácia peri-ventricular quística associada (OR 4,4 IC 95% 1,3 - 14,2; p = 0,015), quer na hemorragia peri-intraventricular grau IV (OR 12; IC 95% 3,5 - 41,2; p < 0,001), em relação aos controlos. Verificaram-se também diferenças no défice grave do neurodesenvolvimento em relação aos controlos (1,3%) na hemorragia peri-intraventricular grau III com leucomalácia peri-ventricular quística associada (75,0%, p < 0,001) e na hemorragia peri-intraventricular grau IV (50,0%, p < 0,001).Discussão: Este estudo evidenciou maior taxa de mortalidade e de alterações graves do neurodesenvolvimento nos prematuros com hemorragia peri-intraventricular grave. A análise dos grupos estratificados por idade gestacional e a abordagem separada dos vários tipos de hemorragia peri-intraventricular, permitiu evidenciar as complicações associadas à hemorragia peri-intraventriculargrau IV e grau III, associada ou não a leucomalácia peri-ventricular quística.Conclusão: Os recém-nascidos com hemorragia peri-intraventricular de grau IV ou grau III com leucomalácia peri-ventricular quística associaram-se a maior mortalidade e sequelas graves do neurodesenvolvimento.

The impact of silver sulfide nanoparticles and silver ions in soil microbiome.

The use of biosolids as fertilizers in agriculture can lead to the exposure of soil biota to sulfidised silver nanoparticles (Ag2S NPs), generated during the wastewater treatment procedures. Considering the crucial role of microorganisms on soil functions, we aimed to study the effects of 10 mg kg-1 soil of Ag2S NPs or AgNO3 on the soil microbiome, using an indoor mesocosm. After 28 days of exposure, Ag2S NPs induced a significant change in the soil microbiome structure, at class, genera and OTU levels. For instance, a significantly higher abundance of Chitinophagia, known for its lignocellulose-degrading activity, was observed in Ag2S NPs-treated soil toward the control. Nevertheless, stronger effects were observed in AgNO3-treated soil, over time, due to its higher silver dissolution rate in porewater. Additionally, only the AgNO3-treated soil stimulates the abundance of ammonia-oxidizing (AOB; amoA gene) and nitrite-oxidizing (NOB; nxrB gene) bacteria, which are involved in the nitrification process. Distinct variants of amoA and nxrB genes emerged in silver-treated soils, suggesting a potential succession of AOB and NOB with different degree of silver-tolerance. Our study highlights the latter effects of Ag2S NPs on the soil microbiome composition, while AgNO3 exerted a stronger effect in both composition and functional parameters.

Gut and faecal bacterial community of the terrestrial isopod Porcellionides pruinosus: potential use for monitoring exposure scenarios.

This work aimed to characterize the gut and faeces bacterial communities (BC) of Porcellionides pruinosus using high-throughput sequencing. Isopods were collected from the field and kept in laboratory conditions similar to those normally applied in ecotoxicology tests. Faeces and purged guts of isopods (n = 3 × 30) were analysed by pyrosequencing the V3-V4 region of 16 S rRNA encoding gene. Results showed that gut and faecal BCs were dominated by Proteobacteria, particularly by an OTU (Operational Taxonomic Unit) affiliated to genus Coxiella. Diversity and richness values were statistically higher for faecal BC, mainly due to the occurrence of several low-abundance phylotypes. These results may reflect faecal carriage of bacterial groups that cannot settle in the gut. BCs of P. pruinosus comprised: (1) common members of the soil microbiota, (2) bacterial symbionts, (3) bacteria related to host metabolic/ecological features, and (4) bacterial etiological agents. Comparison of BC of this isopod species with the BC from other invertebrates revealed common bacterial groups across taxa. The baseline information provided by this work will assist the design and data interpretation of future ecotoxicological or biomonitoring assays where the analysis of P. pruinosus BC should be included as an additional indicator. CAPSULE: Terrestrial isopods bacterial communities might support ecotoxicological assays and biomonitoring processes as a valuable tool.

Long-term effects of Cu(OH)2 nanopesticide exposure on soil microbial communities.

Copper-based (nano)pesticides in agroecosystems may result in unintended consequences on non-target soil microbial communities, due to their antimicrobial broad spectrum. We studied the impact of a commercial Cu(OH)2-nanopesticide, over 90 days, at single and season agricultural application doses, in the presence and absence of an edaphic organism (the isopod Porcellionides pruinosus), on microbial communities' function, structure and abundance. Results were compared to the effects of Cu(OH)2-ionic. The nanopesticide application resulted in significant changes on both bacterial and fungal communities' structure, particularly at the season application. The exposed bacterial community presented a significantly lower richness, and higher diversity and evenness while the exposed fungal community presented lower diversity and richness. At the functional level, a significant increase on microbial ability of carbon utilization and a significant decrease on the ß-glucosidase activity was observed for communities exposed to the nanopesticide. Regarding Cu forms, less pronounced effects were observed in soils spiked with Cu(OH)2-ionic, which might result from lower Cu concentration in porewater. The presence of P. pruinosus did not induce significant changes in diversity indexes (fungal community) and community-level physiological profiling, suggesting an attenuation of the nanopesticide effect. This study revealed that Cu(OH)2-nanopesticide, at doses applied in agriculture, impact the soil microbial community, possibly affecting its ecological role. On the other hand, invertebrates may attenuate this effect, highlighting the importance of jointly including different interacting communities in the risk assessment of nanopesticides in soils.

Using flow cytometry for bacterioplankton community analysis as a complementary tool to Water Framework Directive to signal putatively impacted sites.

Metal contamination, as well as pesticides, organic matter and nutrient input are main factors leading to freshwater ecosystems degradation. The Water Framework Directive (WFD) was implemented within the European Union with the ultimate goal of promoting a good ecological status in all European waterbodies. However, the broad implementation of the bioassessment behind WFD is costly and time-consuming and the search for complementary methodologies has been given significant attention. In this context, the main goal of this study was to evaluate whether flow cytometry (FCM) and denaturing gradient gel electrophoresis (DGGE) can be used as cellular/molecular tools to efficiently assess riverine bacterioplankton communities and relevantly inform on the ecological quality of these ecosystems. Caima river was chosen as case study using three sampling sites reflecting different levels and types of contamination (point-source organic and metal input). Both bacterioplankton community assessment approaches (DGGE and FCM), as well as macroinvertebrate and periphyton communities were consistent in signaling organic contamination. The putatively metal-loaded site bears some contradictory results depending on the community focused, possibly due to the overall low levels of metals actually found and seasonality. When comparing the two bacterioplankton community analysis tools, DGGE and FCM, the results obtained were essentially coherent, with FCM being simpler, faster and still accurate for screening bacteria communities via quantification of bacteria of high and low DNA content. This highlights the suitability of the FCM approach for prioritization of contaminated sampling sites and reinforces the suitability of using bacterioplankton communities as the focus of rapid tools to complement bioassessment sensu the WFD methodology, e.g. assisting the prioritization of potentially impacted areas.

Using molecular diet analysis to inform invasive species management: A case study of introduced rats consuming endemic New Zealand frogs.

The decline of amphibians has been of international concern for more than two decades, and the global spread of introduced fauna is a major factor in this decline. Conservation management decisions to implement control of introduced fauna are often based on diet studies. One of the most common metrics to report in diet studies is Frequency of Occurrence (FO), but this can be difficult to interpret, as it does not include a temporal perspective. Here, we examine the potential for FO data derived from molecular diet analysis to inform invasive species management, using invasive ship rats (Rattus rattus) and endemic frogs (Leiopelma spp.) in New Zealand as a case study. Only two endemic frog species persist on the mainland. One of these, Leiopelma archeyi, is Critically Endangered (IUCN 2017) and ranked as the world's most evolutionarily distinct and globally endangered amphibian (EDGE, 2018). Ship rat stomach contents were collected by kill-trapping and subjected to three methods of diet analysis (one morphological and two DNA-based). A new primer pair was developed targeting all anuran species that exhibits good coverage, high taxonomic resolution, and reasonable specificity. Incorporating a temporal parameter allowed us to calculate the minimum number of ingestion events per rat per night, providing a more intuitive metric than the more commonly reported FO. We are not aware of other DNA-based diet studies that have incorporated a temporal parameter into FO data. The usefulness of such a metric will depend on the study system, in particular the feeding ecology of the predator. Ship rats are consuming both species of native frogs present on mainland New Zealand, and this study provides the first detections of remains of these species in mammalian stomach contents.

Challenges for assessing vertebrate diversity in turbid Saharan water-bodies using environmental DNA.

The Sahara desert is the largest warm desert in the world and a poorly explored area. Small water-bodies occur across the desert and are crucial habitats for vertebrate biodiversity. Environmental DNA (eDNA) is a powerful tool for species detection and is being increasingly used to conduct biodiversity assessments. However, there are a number of difficulties with sampling eDNA from such turbid water-bodies and it is often not feasible to rely on electrical tools in remote desert environments. We trialled a manually powered filtering method in Mauritania, using pre-filtration to circumvent problems posed by turbid water in remote arid areas. From nine vertebrate species expected in the water-bodies, four were detected visually, two via metabarcoding, and one via both methods. Difficulties filtering turbid water led to severe constraints, limiting the sampling protocol to only one sampling point per study site, which alone may largely explain why many of the expected vertebrate species were not detected. The amplification of human DNA using general vertebrate primers is also likely to have contributed to the low number of taxa identified. Here we highlight a number of challenges that need to be overcome to successfully conduct metabarcoding eDNA studies for vertebrates in desert environments in Africa.

Evaluación de la sensibilización a Der p 1 y Der p 2 en una población pediátrica del Norte de Portugal / Evaluation of sensitization to Der p 1 and Der p 2 in a pediatric population of the North of Portugal

INTRODUCCIÓN: En Portugal los datos del papel de Der p 1 y Der p 2 en niños con alergia al ácaro del polvo son escasos. La inmunoterapia específica (IE) con alérgenos es el único tratamiento que mejora los síntomas, reduce la necesidad de terapia farmacológica y modifica la historia natural de la enfermedad. Con este trabajo, los autores estudian la epidemiología local y buscan aclarar si el análisis molecular de los alérgenos principales es una ventaja para decidir y/o modificar la decisión de iniciar IE en niños con indicación clínica y sensibilizados a Dermatophagoides pteronyssinus. MÉTODOS: Estudio retrospectivo de los niños con asma y/o rinitis. Período de estudio: enero de 2013 a diciembre de 2016. CRITERIOS DE INCLUSIÓN: 1) prick-test positivo a Dermatophagoides pteronyssinus; y 2) enfermedad clínicamente relevante bajo tratamiento. Los valores de Der p 1 y Der p 2 ≥ 0,35kUA/l fueron considerados positivos. La significación estadística se estableció en p < 0,05. RESULTADOS: Se incluyeron en el estudio 279 pacientes. Edad media 9,55 años (min. 4-máx. 17). El asma estuvo presente en 199 niños (71,3%) y rinitis en 245 (87,8%). Der p 1 y Der p 2 fue<0,35kUA/l en 29 (10,4%) pacientes. Der p 1/Der p 2 se correlacionó con el tamaño de la pápula de prick-test, con el valor de los eosinófilos y la IgE total. CONCLUSIONES: Der p 1 y Der p 2 son alérgenos dominantes en nuestra población y puede haber beneficios en la determinación de estos niveles de alérgenos moleculares en pacientes con un prick-test positivo e indicación clínica para IE

INTRODUCTION: In Portugal, data on the role of Der p 1 and Der p 2 in patients with house dust mite (HDM) allergy are scarce. Allergen-specific immunotherapy (sIT) is the only treatment that improves symptoms, reduces the need for pharmacological therapy and modifies the natural history of the disease. With this study, the authors aim to understand the local epidemiology and to clarify if the molecular assay of major allergens is advantageous in deciding and/or modifying the decision to initiate sIT in children with clinical indication which are sensitized to Dermatophagoides pteronyssinus. METHODS: Retrospective study with analysis of patients with asthma and/or rhinitis. Study period: January/2013-December/2016. Inclusion criteria: 1) positive prick-test to Dermatophagoides pteronyssinus; and 2) clinically relevant disease under treatment. Assay Der p 1 and Der p 2 values ≥ 0.35 kUA/L were considered positive. Statistical significance was set at P < .05. RESULTS: The clinical files of 279 patients. Mean ages 9.55 years (min.4-max.17). Asthma was present in 199 children (71.3%) and rhinitis in 245 (87.8%). Der p 1 and Der p 2 was < 0.35kUA/L in 29 (10,4%) patients. The value of Der p 1/Der p 2 correlated with the size of the prick-test papule, the value of the eosinophils and the total IgE. CONCLUSIONS: Der p 1 and Der p 2 are dominant allergens in our population and there may be benefits in determining these molecular allergen levels in patients with a positive prick-test and a clinical indication for sIT prior to a decision of initiating sIT or not

Low-grade intraventricular hemorrhage and neurodevelopment at 24 months of age / Hemorragia intraventricular de baixo grau e neurodesenvolvimento aos 24 meses de idade

AIMS: To evaluate the impact of low-grade intraventricular hemorrhage on neurodevelopmental outcome in preterm infants at 24 months of age. METHODS: We conducted a retrospective case-control study of infants with gestational age less than 34 weeks, admitted to a Neonatal Intensive Care Unit between January/2006 and December/2015. Cases were defined as those with low-grade intraventricular hemorrhage (grades I or II), diagnosed by cranial ultrasonography. For each case, a control with the same gestational age but without intraventricular hemorrhage was selected. Follow-up examinations of neurodevelopment were performed at 24 months of age in cases and controls using the Griffiths Mental Development Scale. Cerebral palsy, neurodevelopmental delay (developmental quotient <2 side deviations below the mean), hearing impairment and/or blindness were considered as severe neurodevelopmental impairment. RESULTS: The study included 172 preterm infants: 86 cases and 86 controls. In the univariate analysis, a difference between the two groups was identified for the following clinical findings: antenatal corticosteroid complete cycle (57% in cases vs. 80% in controls; p=0.001; OR: 0.33, 95%CI 0.17-0.64); male gender (63% cases vs. 41% controls; p=0.004; OR: 2.45, 95%CI 1.3-4.5); outborn (26% cases vs. 9% controls; p=0.005; OR: 3.3 95%CI 1.4-8.0); Clinical Risk Index for Babies higher than 5 (24% in cases vs. 12% in controls; p=0.029; OR: 2.4 95%CI 1.1-5.6); intubation in the delivery room (47% cases vs. 27% controls; p=0.007; OR: 2.38 95%CI 1.3-4.5); and neonatal sepsis (34% in cases vs. 20% in controls; p=0.039; OR: 2.1 95%CI 1.03-4.1). After logistic regression, differences were only maintained for antenatal corticosteroid (p=0.005; OR 0.34, 95%CI 0.16-0.72) and male gender (p=0.002; OR 2.9, 95%CI 1.4-5.8). A severe neurodevelopmental deficit was present in three cases (3.5%) and one control (1.2%). No statistically significant differences in outcome were found between cases and controls. CONCLUSIONS: In this sample, preterm infants with low-grade intraventricular hemorrhage diagnosed by cranial ultrasonography had no difference in early neurodevelopmental outcome when compared with controls.

OBJETIVOS: Avaliar o impacto da hemorragia intraventricular de baixo grau no neurodesenvolvimento de lactentes prematuros aos 24 meses de idade. MÉTODOS: Foi conduzido um estudo de caso-controle retrospectivo em lactentes com idade gestacional inferior a 34 semanas, internados em uma Unidade de Terapia Intensiva Neonatal entre janeiro de 2006 e dezembro de 2015. Os casos foram definidos como aqueles com hemorragia intraventricular de baixo grau (graus I ou II), diagnosticada por ultrassonografia craniana. Para cada caso, foi selecionado um controle com a mesma idade gestacional, mas sem hemorragia intraventricular. A avaliação do neurodesenvolvimento foi realizada aos 24 meses de idade, em casos e controles, com a Escala de Desenvolvimento Mental de Griffiths. Paralisia cerebral, atraso no neurodesenvolvimento (quociente de desenvolvimento <2 desvios padrões abaixo da média para a idade), deficiência auditiva e/ou cegueira foram considerados comprometimento grave do neurodesenvolvimento. RESULTADOS: O estudo incluiu 172 prematuros: 86 casos e 86 controles. Na análise univariada, identificou-se diferença entre os dois grupos para os seguintes achados clínicos: ciclo completo de corticosteroide pré-natal (57% nos casos vs. 80% nos controles; p=0,001; OR: 0,33; IC95% 0,17-0,64); sexo masculino (63% casos vs. 41% controles; p=0,004; OR: 2,45, IC95% 1,3-4,5); nascidos em outro hospital (26% casos vs. 9% controles; p=0,005; OR: 3,3 IC95% 1,4-8,0); Índice de Risco Clínico para Bebês acima de 5 (24% nos casos vs. 12% nos controles; p=0,029; OR: 2,4 IC95% 1,1-5,6); intubação na sala de parto (47% casos vs. 27% controles; p=0,007; OR: 2,38; IC95%: 1,3-4,5); e sepse neonatal (34% nos casos vs. 20% nos controlos; p=0,039; OR: 2,1 95% CI 1,03-4,1). Após a regressão logística, as diferenças foram mantidas apenas para o corticosteróide antenatal (p=0,005; OR 0,34, IC 95% 0,16-0,72) e sexo masculino (p=0,002; OR 2,9, IC95% 1,4-5,8). Um déficit grave de neurodesenvolvimento esteve presente em três casos (3,5%) e um controle (1,2%). Não houve diferenças estatisticamente significativas no desfecho entre casos e controles. CONCLUSÕES: Nesta amostra, os prematuros com hemorragia intraventricular de baixo grau diagnosticados pela ultrassonografia craniana não apresentaram diferença no desenvolvimento neurológico precoce quando comparados aos controles.

Mediterranean Spotted Fever in Children: Study of a Portuguese Endemic Region.

INTRODUCTION: Mediterranean spotted fever is an infectious disease included in the human rickettsiosis group, with its main distribution in the Mediterranean and South European countries. It is an endemic disease in Portugal, with dogs acting as the main domestic reservoir. Children are a particularly vulnerable group due to their close contact with household animals and by frequently playing outdoors. In this study, we aim to describe the local epidemiology and characterize the clinical features and treatment options in a Portuguese endemic region. MATERIALS AND METHODS: We performed a prospective descriptive study of the Mediterranean spotted fever cases admitted to the paediatrics emergency department of a of a group II hospital, between January 1st 2013 and December 31st 2015. All patients were examined by a physician, who was later asked to fill out a detailed questionnaire regarding clinical presentation, diagnostic attitudes and treatment of their patients. Parents were later interviewed and questioned about post-discharge disease evolution. RESULTS: We registered 32 cases (93.9% reported between July and October). After fever, the most frequent reported symptoms were myalgia (37.5%), abdominal pain (25%) and headache (25%). Exanthema was present in 84.4% of cases by the third day of fever:maculonodular (53.1%), papular (37.5%) and macular (9.4%). Eschars were found in 59% of patients, with regional lymphadenopathypresent in 46.9% of cases. Azithromycin (84.4%) and doxycycline (15.5%) were the selected treatments in our population, with no cases of therapeutic failure or side-effects reported. CONCLUSION: The incidence of Mediterranean spotted fever is higher in our population in comparison with the rest of the country. Fever, myalgia, abdominal pain and headache were the most common presenting symptoms, while exanthema was the predominant cutaneous finding. Azithromycin was the preferred treatment and it proved to be successful and safe in all cases.

Introdução: A febre escaro-nodular é uma doença infeciosa aguda incluída no grupo das rickettsioses humanas, que atinge sobretudo os países da bacia do Mediterrâneo e sul da Europa. É uma doença endémica em Portugal com o cão como principal reservatório doméstico. As crianças são um grupo particularmente vulnerável devido ao contato próximo com animais domésticos e a brincarem em campos e jardins. O principal objetivo deste estudo foi perceber e estudar a epidemiologia local, bem como caracterizar a clinica e a resposta terapêutica da nossa população. Material e Métodos: Foi realizado um estudo prospetivo descritivo de todas as crianças com febre escaro-nodular admitidas no serviço de urgência de um hospital grupo II, entre janeiro de 2013 e dezembro de 2015. Todos os doentes foram avaliados por médico, que respondeu a um questionário referente as manifestações clínicas apresentadas e atitudes de diagnóstico e terapêuticas instituídas. Os pais foram posteriormente contatados, por telefone, e questionados sobre a evolução da doença. Resultados: Foram registados 32 casos (93,9% de julho a outubro). Além da febre, as queixas mais frequentes foram mialgias (37,5%), dor abdominal e cefaleias (25%). O exantema estava presente no terceiro dia de febre em 84,4%: maculopapulonodular (53,1%); papular (37,5%) e macular (9,4%). Foi identificada escara em 59% e adenopatia satélite em 46,9%. Azitromicina (84,4%) ou doxiciclina (15,5%) foram os antibióticos de escolha, com sucesso em qualquer dos casos. Não foram relatadas complicações. Conclusão: A incidência de febre escaro-nodular na nossa população é alta, quando comparada com a incidência no restante país. Febre, mialgias e cefaleias foram os principais sintomas e sinais registados enquanto a presença de exantema foi o achado dermatológico predominante. Azitromicina, antibiótico de escolha na maioria dos casos, mostrou ser eficaz.

Encefalite autoimune anti recetor N-metil D-aspartato: relato de dois casos / Auto-immune anti-N-methyl-D-aspartate receptor encephalitis: a two-case report

OBJETIVOS: Relatar dois casos com apresentações diferentes de encefalite antirreceptor N-metil-D-aspartato (NMDA), uma doença autoimune recentemente identificada e caracterizada por alterações da consciência, déficit de memória, convulsões, disfunção autonômica e distúrbios do movimento. DESCRIÇÃO DOS CASOS: Criança de seis anos, sexo feminino, que se apresentou com movimentos distônicos e coreoatetósicos incapacitando a marcha. Adolescente de 17 anos, sexo masculino, com alteração do comportamento, amnésia retrógrada e convulsões. Ambos realizaram eletroencefalograma e ressonância magnética cerebral que não revelaram alterações relevantes. Os anticorpos antirreceptor NMDA foram positivos no líquido cefalorraquidiano em ambos os casos e no sangue no primeiro paciente. Foram administrados metilprednisolona, imunoglobulina e rituximab em diferentes intervalos. Ambos tiveram uma recidiva cerca de seis meses depois, com recuperação ao final de um ano e meio após o diagnóstico. CONCLUSÕES: A encefalite antirreceptor NMDA deve ser considerada quando estamos perante o início súbito de sintomas neuropsiquiátricos. O diagnóstico e terapêutica precoces são fatores prognósticos fundamentais.

AIMS: To report two cases with different presentations of anti-N-methyl-D-aspartate receptor (anti-NMDAR) encephalitis, a newly identified autoimmune disease characterized by consciousness changes, memory deficit, seizures, autonomic dysfunction and movement disorders. CASES DESCRIPTION: A six-year-old female, who presented with dystonic and choreoathetoid movements with refusal to walk. A 17-yearold male, presented with behavioral changes, retrograde amnesia and seizures. Electroencephalogram and brain magnetic resonance imaging did not show any significant findings. Anti-NMDAR antibodies were positive in cerebrospinal fluid in both cases and in serum in the first patient. Methylprednisolone, immunoglobulin and rituximab were given at different intervals. Both had a recurrence about six months later, with recovery at the end of one and a half year of the diagnosis. CONCLUSIONS: Anti-NMDAR encephalitis should be considered in patients with sudden onset of neuropsychiatric symptoms. Early diagnosis and treatment are major prognostic factors.

Cutis marmorata telangiectatica congenita and major lower limb asymmetry.

A 39-week-old male newborn presented at birth with atrophic erythematous and purpuric skin lesions, in a typical right-sided segmental distribution. Lesions were persistent and unaffected by rewarming in the postpartum period. Postnatal echocardiogram showed a predominance of the right cavities and an upper atrial septal defect. Cerebral and abdominal ultrasound were normal along with ophthalmological examination. On follow-up, lower limbs asymmetry was noted. The right lower limb was shorter in length and had a smaller diameter. At 6 months, the right lower limb was 1.5 cm shorter than the left, most likely related to nutritive vessels malformations. The discrepancy was even more pronounced at the age of 9 months. This leg-length asymmetry can lead to severe functional limitations in the future.

[Evaluation of sensitization to Der p 1 and Der p 2 in a pediatric population of the North of Portugal]. / Evaluación de la sensibilización a Der p 1 y Der p 2 en una población pediátrica del Norte de Portugal.

INTRODUCTION: In Portugal, data on the role of Der p 1 and Der p 2 in patients with house dust mite (HDM) allergy are scarce. Allergen-specific immunotherapy (sIT) is the only treatment that improves symptoms, reduces the need for pharmacological therapy and modifies the natural history of the disease. With this study, the authors aim to understand the local epidemiology and to clarify if the molecular assay of major allergens is advantageous in deciding and/or modifying the decision to initiate sIT in children with clinical indication which are sensitized to Dermatophagoides pteronyssinus. METHODS: Retrospective study with analysis of patients with asthma and/or rhinitis. STUDY PERIOD: January/2013-December/2016. INCLUSION CRITERIA: 1) positive prick-test to Dermatophagoides pteronyssinus; and 2) clinically relevant disease under treatment. Assay Der p 1 and Der p 2 values ≥0.35 kUA/L were considered positive. Statistical significance was set at P<.05. RESULTS: The clinical files of 279 patients. Mean ages 9.55 years (min.4-max.17). Asthma was present in 199 children (71.3%) and rhinitis in 245 (87.8%). Der p 1 and Der p 2 was <0.35kUA/L in 29 (10,4%) patients. The value of Der p 1/Der p 2 correlated with the size of the prick-test papule, the value of the eosinophils and the total IgE. CONCLUSIONS: Der p 1 and Der p 2 are dominant allergens in our population and there may be benefits in determining these molecular allergen levels in patients with a positive prick-test and a clinical indication for sIT prior to a decision of initiating sIT or not.

MLPA analysis in a cohort of patients with autism.

BACKGROUND: Autism is a global neurodevelopmental disorder which generally manifests during the first 2 years and continues throughout life, with a range of symptomatic variations. Epidemiological studies show an important role of genetic factors in autism and several susceptible regions and genes have been identified. The aim of our study was to validate a cost-effective set of commercial Multiplex Ligation dependent Probe Amplification (MLPA) and methylation specific multiplex ligation dependent probe amplification (MS-MLPA) test in autistic children refered by the neurodevelopmental center and autism unit of a Paediatric Hospital. RESULTS: In this study 150 unrelated children with autism spectrum disorders were analysed for copy number variation in specific regions of chromosomes 15, 16 and 22, using MLPA. All the patients had been previously studied by conventional karyotype and fluorescence in situ hybridization (FISH) analysis for 15(q11.2q13) and, with these techniques, four alterations were identified. The MLPA technique confirmed these four and identified further six alterations by the combined application of the two different panels. CONCLUSIONS: Our data show that MLPA is a cost effective straightforward and rapid method for detection of imbalances in a clinically characterized population with autism. It contributes to strengthen the relationship between genotype and phenotype of children with autism, showing the clinical difference between deletions and duplications.

Study on the Cytotoxic, Genotoxic and Clastogenic Potential of Attalea phalerata Mart. ex Spreng. Oil Pulp In Vitro and In Vivo Experimental Models.

Attalea phalerata Mart. ex Spreng. (Arecaceae), popularly known as "bacuri", is used in Brazilian folk medicine. Its oil is used orally to relieve pulmonary congestion and joint pain. In topical applications, it is applied as an effective hair tonic and anti-dandruff. The in natura pulp and its nuts are used as food because of its nutritional value. Despite its use in folk medicine, there is a lack of data regarding its in vivo/in vitro cytotoxic/genotoxic and clastogenic effects. Therefore, in this study, we evaluated the cytotoxic, genotoxic and clastogenic effects of Attalea phalerata Mart. ex Spreng. oil (APMO) in vitro and in vivo. For the analysis of cytotoxic potential, the Artemia salina and MTT (3-(4,5-dimethizzol-zyl)-2,5-diphenyltetrazolium bromide) assays were performed. Possible cytotoxic, genotoxic and clastogenic effects of APMO intake were determined by performing the comet and micronucleus assays. Male and female Wistar rats were orally treated with doses of 125, 250, 500 or 1000 mg.kg-1 of the APMO daily for 28 consecutive days (four weeks). The results showed that the APMO did not induce cell death in the experiments of Artemia salina and MTT, indicating that it has no cytotoxicity. The APMO did not cause significant damage to the DNA of the rats in the four doses used when compared to the negative control group (saline + Tween® 80). The APMO did not present any significant increase in micronucleated polychromatic erythrocytes (MNPCEs) for the four tested doses. When compared to the positive control group, all groups (comet and micronucleus tests) were statistically different. These data suggest that the administration of Attalea phalerata Mart oil. ex Spreng does not cause cytotoxicity, genotoxicity and clastogenicity in experimental models in vitro and in vivo following oral administration in this study.

[Magnetic Resonance Imaging Conversion Predictors of Clinically Isolated Syndrome to Multiple Sclerosis]. / Alterações na Ressonância Magnética Preditoras da Conversão da Síndrome Clinicamente Isolada em Esclerose Múltipla.

INTRODUCTION: Clinically isolated syndrome may be the first manifestation of multiple sclerosis, a chronic demyelinating disease of the central nervous system, and it is defined by a single clinical episode suggestive of demyelination. However, patients with this syndrome, even with long term follow up, may not develop new symptoms or demyelinating lesions that fulfils multiple sclerosis diagnostic criteria. We reviewed, in clinically isolated syndrome, what are the best magnetic resonance imaging findings that may predict its conversion to multiple sclerosis. MATERIAL AND METHODS: A search was made in the PubMed database for papers published between January 2010 and June 2015 using the following terms: 'clinically isolated syndrome', 'cis', 'multiple sclerosis', 'magnetic resonance imaging', 'magnetic resonance' and 'mri'. RESULTS: In this review, the following conventional magnetic resonance imaging abnormalities found in literature were included: lesion load, lesion location, Barkhof's criteria and brain atrophy related features. The non conventional magnetic resonance imaging techniques studied were double inversion recovery, magnetization transfer imaging, spectroscopy and diffusion tensor imaging. DISCUSSION: The number and location of demyelinating lesions have a clear role in predicting clinically isolated syndrome conversion to multiple sclerosis. On the other hand, more data are needed to confirm the ability to predict this disease development of non conventional techniques and remaining neuroimaging abnormalities. CONCLUSION: In forthcoming years, in addition to the established predictive value of the above mentioned neuroimaging abnormalities, different clinically isolated syndrome neuroradiological findings may be considered in multiple sclerosis diagnostic criteria and/or change its treatment recommendations.

Introdução: A síndrome clinicamente isolada é uma forma de apresentação da esclerose múltipla, doença desmielinizante crónica do sistema nervoso central, e define-se por um único episódio clínico sugestivo de desmielinização. Porém, doentes com esta síndrome podem não desenvolver novos sintomas ou lesões, mesmo após longos períodos de seguimento, não cumprindo os critérios de diagnóstico da doença. Aqui serão revistas, na síndrome clinicamente isolada, as alterações na ressonância magnética que melhor predizem a conversão em esclerose múltipla. Material e Métodos: Pesquisaram-se na base de dados da PubMed artigos publicados entre janeiro de 2010 e junho de 2015 usando os termos 'clinically isolated syndrome', 'cis', 'multiple sclerosis', 'magnetic resonance imaging', 'magnetic resonance' e 'mri'. Resultados: Carga lesional, localização das lesões, critérios de Barkhof e áreas/volumes de estruturas cerebrais foram as alterações na ressonância magnética convencional encontradas nos artigos incluídos nesta revisão. As técnicas não convencionais estudadas foram a dupla inversão-recuperação, a transferência de magnetização, a espectroscopia e a imagem de tensor de difusão. Discussão: Enquanto o número de lesões e a sua localização têm um papel claro na previsão da conversão da síndrome clinicamente isolada em esclerose múltipla, as técnicas não convencionais e as restantes alterações na ressonância magnética necessitam de mais estudos para confirmar a sua capacidade de prever o desenvolvimento da doença. Conclusão: Para além do valor preditivo das alterações já citadas, no futuro, diferentes achados neurorradiológicos na síndrome clinicamente isolada poderão ser considerados para os critérios de diagnóstico da esclerose múltipla e/ou modificar as recomendações sobre o seu tratamento.