Characterisation of SARS-CoV-2 Lentiviral Pseudotypes and Correlation between Pseudotype-Based Neutralisation Assays and Live Virus-Based Micro Neutralisation Assays

The recent outbreak of a novel Coronavirus (SARS-CoV-2) and its rapid spread across the continents has generated an urgent need for assays to detect the neutralising activity of human sera or human monoclonal antibodies against SARS-CoV-2 spike protein and to evaluate the serological immunity in humans. Since the accessibility of live virus microneutralisation (MN) assays with SARS-CoV-2 is limited and requires enhanced bio-containment, the approach based on "pseudotyping" can be considered a useful complement to other serological assays. After fully characterising lentiviral pseudotypes bearing the SARS-CoV-2 spike protein, we employed them in pseudotype-based neutralisation assays in order to profile the neutralising activity of human serum samples from an Italian sero-epidemiological study. The results obtained with pseudotype-based neutralisation assays mirrored those obtained when the same panel of sera was tested against the wild type virus, showing an evident convergence of the pseudotype-based neutralisation and MN results. The overall results lead to the conclusion that the pseudotype-based neutralisation assay is a valid alternative to using the wild-type strain, and although this system needs to be optimised and standardised, it can not only complement the classical serological methods, but also allows serological assessments to be made when other methods cannot be employed, especially in a human pandemic context

Influenza anti-stalk antibodies: Development of a new method for the evaluation of the immune responses to universal vaccine

Growing interest in universal influenza vaccines and novel administration routes has led to the development of alternative serological assays that are able to detect antibodies against conserved epitopes. We present a competitive ELISA method that is able to accurately determine the ratio of serum immunoglobulin G directed against the different domains of the hemagglutinin, the head and the stalk. Human serum samples were treated with two variants of the hemagglutinin protein from the A/California/7/2009 influenza virus. The signals detected were assigned to different groups of antibodies and presented as a ratio between head and stalk domains. A subset of selected sera was also tested by hemagglutination inhibition, single radial hemolysis, microneutralization, and enzyme-linked lectin assays. Pre-vaccination samples from adults showed a quite high presence of anti-stalk antibodies, and the results were substantially in line with those of the classical serological assays. By contrast, pre-vaccination samples from children did not present anti-stalk antibodies, and the majority of the anti-hemagglutinin antibodies that were detected after vaccination were directed against the head domain. The presented approach, when supported by further assays, can be used to assess the presence of specific anti-stalk antibodies and the potential boost of broadly protective antibodies, especially in the case of novel universal influenza vaccine approaches

Motor and Postural Patterns Concomitant with General Movements Are Associated with Cerebral Palsy at Term and Fidgety Age in Preterm Infants

General movements (GMs) in combination with neurological examination and magnetic resonance imaging at term age can accurately determine the risk of cerebral palsy. The present study aimed to assess whether 11 motor and postural patterns concomitant with GMs were associated with cerebral palsy. Video recordings performed after birth in 79 preterm infants were reviewed retrospectively. Thirty-seven infants developed cerebral palsy at 2 years corrected age and the remaining 42 showed typical development. GMs were assessed from preterm to fidgety age and GM trajectories were defined. The 11 motor and postural patterns were evaluated at each age and longitudinally, alone and in combination with GM trajectories. A logistic regression model was used to assess the association between GMs, concomitant motor and postural patterns, and cerebral palsy. We confirmed that high-risk GM trajectories were associated with cerebral palsy (odds ratio = 44.40, 95% confidence interval = 11.74-167.85). An association between concomitant motor and postural patterns and cerebral palsy was found for some of the patterns at term age and for all of them at fidgety age. Therefore, at term age, concomitant motor and postural patterns can support GMs for the early diagnosis of cerebral palsy

Serratia marcescens in a neonatal intensive care unit: two long-term multiclone outbreaks in a 10-year observational study

We investigated two consecutive Serratia marcescens (S. marcescens) outbreaks which occurred in a neonatal intensive care unit (NICU) of a tertiary level hospital in North Italy in a period of 10 years (January 2003-December 2012). Risk factors associated with S. marcescens acquisition were evaluated by a retrospective case-control study. A total of 21,011 clinical samples was examined: S. marcescens occurred in 127 neonates: 43 developed infection and 3 died. Seven clusters were recorded due to 12 unrelated clones which persisted for years in the ward, although no environmental source was found. The main epidemic clone A sustaining the first cluster in 2003 reappeared in 2010 as an extended spectrum ?-lactamase (ESBL)-producing strain and supporting the second epidemic. Birth weight, gestational age, use of invasive devices and length of stay in the ward were significantly related to S. marcescens acquisition. The opening of a new ward for non-intensive care-requiring neonates, strict adherence to alcoholic hand disinfection, the timely identification and isolation of infected and colonized neonates assisted in containing the epidemics. Genotyping was effective in tracing the evolution and dynamics of the clones demonstrating their long-term persistence in the ward

Comparative analyses of SARS-CoV-2 binding (IgG, IgM, IgA) and neutralizing antibodies from human serum samples

A newly identified coronavirus, named SARS-CoV-2, emerged in December 2019 in Hubei Province, China, and quickly spread throughout the world; so far, it has caused more than 49.7 million cases of disease and 1,2 million deaths. The diagnosis of SARS-CoV-2 infection is currently based on the detection of viral RNA in nasopharyngeal swabs by means of molecular-based assays, such as real-time RT-PCR. Furthermore, serological assays detecting different classes of antibodies constitute an excellent surveillance strategy for gathering information on the humoral immune response to infection and the spread of the virus through the population. In addition, it can contribute to evaluate the immunogenicity of novel future vaccines and medicines for the treatment and prevention of COVID-19 disease.The aim of this study was to determine SARS-CoV-2-specific antibodies in human serum samples by means of different commercial and in-house ELISA kits, in order to evaluate and compare their results first with one another and then with those yielded by functional assays using wild-type virus. It is important to identify the level of SARS-CoV-2-specific IgM, IgG and IgA antibodies in order to predict human population immunity, possible cross-reactivity with other coronaviruses and to identify potentially infectious subjects.In addition, in a small sub-group of samples, a subtyping IgG ELISA has been performed. Our findings showed a notable statistical correlation between the neutralization titers and the IgG, IgM and IgA ELISA responses against the receptor-binding domain of the spike protein. Thus confirming that antibodies against this portion of the virus spike protein are highly neutralizing and that the ELISA Receptor-Binding Domain-based assay can be used as a valid surrogate for the neutralization assay in laboratories that do not have biosecurity level-3 facilities

Clinical features and outcomes of elderly hospitalised patients with chronic obstructive pulmonary disease, heart failure or both

Background and objective: Chronic obstructive pulmonary disease (COPD) and heart failure (HF) mutually increase the risk of being present in the same patient, especially if older. Whether or not this coexistence may be associated with a worse prognosis is debated. Therefore, employing data derived from the REPOSI register, we evaluated the clinical features and outcomes in a population of elderly patients admitted to internal medicine wards and having COPD, HF or COPD + HF. Methods: We measured socio-demographic and anthropometric characteristics, severity and prevalence of comorbidities, clinical and laboratory features during hospitalization, mood disorders, functional independence, drug prescriptions and discharge destination. The primary study outcome was the risk of death. Results: We considered 2,343 elderly hospitalized patients (median age 81 years), of whom 1,154 (49%) had COPD, 813 (35%) HF, and 376 (16%) COPD + HF. Patients with COPD + HF had different characteristics than those with COPD or HF, such as a higher prevalence of previous hospitalizations, comorbidities (especially chronic kidney disease), higher respiratory rate at admission and number of prescribed drugs. Patients with COPD + HF (hazard ratio HR 1.74, 95% confidence intervals CI 1.16-2.61) and patients with dementia (HR 1.75, 95% CI 1.06-2.90) had a higher risk of death at one year. The Kaplan-Meier curves showed a higher mortality risk in the group of patients with COPD + HF for all causes (p = 0.010), respiratory causes (p = 0.006), cardiovascular causes (p = 0.046) and respiratory plus cardiovascular causes (p = 0.009). Conclusion: In this real-life cohort of hospitalized elderly patients, the coexistence of COPD and HF significantly worsened prognosis at one year. This finding may help to better define the care needs of this population

Cell based assays used to quantify neutralizing antibodies against SARS-CoV-2 in human samples

Since the SARS-CoV-2 outbreak at the end of December 2019 in China, it has become extremely important to have well‐established and validated diagnostic and research-use-only assays for this new emerging virus. The microneutralization assay is a fundamentalserological test in virology, immunology, vaccine assessment and epidemiological studies, and represents one of the most used methods to evaluate the immune response induced by SARS-CoV-2 infection or vaccination. In this Phd project different microneutralization methods are presented which can be used to measure anti‐SARS‐CoV‐2 neutralizing antibodies (nAbs) in human serum samples. The aim of the first project of this thesis (Project I) was to compare a microneutralization assay (MN) with a read out based on the cytophatic effect (CPE) and a MN based on a colorimetric read out for the detection of nAbs against SARS-CoV-2 Wild type strain. In the first method the cell monolayers were microscopically inspected for inhibition of CPE at each serum dilution (subjective method), while in the MN based on a colorimetric read out the healthy cell monolayer was stained with neutral red solution, a vital dye. The plates were then read by a spectrophotometer at 540 nm (objective method). A panel of 83 human serum samples were previously tested in enzyme‐linked immunosorbent assay (ELISA) as a pre‐screening. All the samples found to be positive, borderline, and negative in this ELISA were then tested to determine the nAbs titers through the MN CPE and Colorimetric MN. The comparison between log2-trasformed MN titers obtained through these two methods showed comparable values, and the strong agreement in evaluating neutralizing antibodies against SARS-CoV-2 Wild type strain was also confirmed by Correlation (r 2=0,9955), Bland-Altman, and intra-class correlation (ICC) analysis (ICC value of 0.993, which is indicative of an excellent agreement). This suggests the suitability of performing the MN assay using an ‘objective’ colorimetric-based read out method. To better investigate if the classical MN CPE yielded similar results to those obtained with other MN methods, we compared this “classical” MN to a new MN platform: the Virospot MN assay (Project II). This method combines classic virus culture techniques with automated sensitive detection of immunostained virus infected cells. In the Virospot MN, a virus-specific immunostaining was used for plate reading and then the images of all wells were acquired by a CTL ImmunoSpot analyzer. The 80% (MN80) or 90% (MN90) neutralization titers are calculated according to the method described by Zielinska et al. 2005. This titer calculation is based on the serum dilutions above and below the reduction point, 80% or 90% neutralization. The MN CPE and Virospot methods were compared using a panel of 47 human serum samples against SARS-CoV-2 Wild type and Alpha variant. The results of this project showed that the these two different MN assays produce similar titer results against the Wild Type virus, with good correlation values (correlation MN80 r2=0,9091; correlation MN90 r2=0,8900). A lower agreement between the MN CPE and Virospot MN assay was observed when SARS-CoV-2 Alpha variant was used (correlation MN80 r2=0,7226; correlation MN90 r2=0,6673). Overall, these results showed a good agreement between the MN CPE assay and the two different MN methods, Colorimetric MN and Virospot MN assay, in detecting neutralizing antibodies against SARS-CoV-2 in human serum samples. Despite the need for further standardization and/or the differences noticed during the assessment of nAbs against SARS-COV-2 variants, the Colorimetric-based and Virospot MN demonstrate to have advantages over the classical MN CPE, both being completely automated methods, and hence offering a higher throughput, while inspection of each dilution well by means of the optical microscope slows down the process. However, to ensure that these correlation studies can provide meaningful results, further analysis with a bigger number of samples and with other SARS-CoV-2 variants would be an added value. Moreover, to make the data more comparable it would be necessary convert all the results to international standard unit (IU/mL) allowing the accurate calibration of assays to an arbitrary unit, thereby reducing inter-laboratory variation, and creating a common language for reporting data. The SARS-CoV-2 Virospot MN assay offers attractive advantages over the MN assay with a read out based on the cytophatic effect, including the relative insensitivity to variation in amount of infectious virus used in the test, independence from virus replication kinetics and suitability for high throughput analyses. Since many new SARS-CoV-2 variants occurred during the last two years, to make the Virospot more sensitive and robust in detecting neutralizing antibodies against these new variants, the third project (Project III) focused on the optimization study of this MN assay. Several new conditions were adapted to optimize the method and make it more sensitive for the analysis of samples against the Wild Type, Alpha, Beta, and Gamma SARS-CoV-2 variants. Carboxymethyl cellulose (CMC) overlay was introduced to make the spot count more accurate avoiding the viral spread after the first infection. Moreover, different sample matrices (serum and plasma), culture media with and without the CO2 supplementation, and different incubation time points and temperatures were assessed to evaluate and improve the assay performance and robustness. This optimization study has a planned follow-up, which can possibly include samples not only from infected/convalescent individuals but also from vaccinated donors (with two or more doses) or from people with hybrid immunity (such as breakthrough infections). Additionally, further analyses with additional SARS-CoV-2 variants to strengthen these finding will also be part of the next study. This project is worth to be conducted as the Virospot MN assay is likely to have importance for the pre-clinical evaluation and eventual licensing of the SARS-CoV-2 vaccines

Deaf Educators in Brazil: history, training and performance spaces

Esta pesquisa intitulada Educadores Surdos no Brasil: história, formação e espaços de atuação, aborda o percurso de formação de professores e instrutores Surdos ao longo da história e no Brasil e as possibilidades de atuação na contemporaneidade. Assim sendo, o objetivo geral desta pesquisa é conhecer o processo de formação e atuação de profissionais surdos em vários espaços educacionais no Brasil. Desta forma, os objetivos específicos, são: 1) Analisar as funções dos primeiros educadores surdos mencionados na história; 2) Traçar a cronologia dos primeiros professores surdos no Brasil, a partir do século XIX e 3) Apresentar as possibilidades e campos de atuação de educadores surdos na atualidade. Em relação aos procedimentos metodológicos, a pesquisa em tela é de abordagem qualitativa e exploratória. Com o intuito de identificar produções que dialogassem com o tema proposto foi realizado um levantamento bibliográfico utilizando duas bases de dados: o Banco de Teses e Dissertações da Biblioteca Eletrônica Online (Scielo) e da Biblioteca Digital Brasileia de Teses e Dissertações (BDTD), de 2015 a 2019, totalizando cinco anos de produções cientificas publicadas. As buscas foram realizadas a partir de quatro palavras-chave: repetidor surdo, instrutor surdo, formação de professores surdos e professor surdo. Foi realizada a leitura de 586 títulos e resumos de trabalhos publicados nas duas bases de dados consultadas. Esta dissertação está organizada em três capítulos: o Capítulo 1, intitulado A atuação dos repetidores surdos nos Séculos XIX e XX, foi baseado em Rocha (2007, 2009, 2010, 2018) e Sofiato (2011, 2016, 2018), pois tais autoras abordam a atuação dos repetidores surdos, no reinado de D. Pedro II. Além disso, foram abordadas várias gestões do Instituto Nacional de Educação de Surdos em diferentes períodos, bem como, a atuação dos repetidores surdos e regimentos internos. O Capítulo 2, intitulado Formação de Professores de Surdos no Brasil apresenta o contexto da formação de professores no Brasil, envolvendo ações para o público ouvinte e surdo, de acordo com uma cronologia que tem início no século XIX. O Capítulo 3, intitulado A Formação de Professores Surdos no Brasil trata da formação de professores surdos e atuação do professor surdo em diferentes espaços educacionais no Brasil. Como resultado, foram tecidas algumas considerações no que nos convidam a refletir a respeito dos avanços e desafios presentes na formação de profissionais surdos para o campo da educação. Ademais, destaca as funções desempenhadas por tais profissionais atribuições de acordo com o espaço de atuação de/em Libras, como norteadores para o protagonismo de professores surdos, inclusive reforçando a importância da Libras na formação de professores.This research entitled Deaf Educators in Brazil: history, training and performance spaces, addresses the path of training of Deaf teachers and instructors throughout history and in Brazil and the possibilities of performance in contemporary times. Thus, the general objective of this research is to know the process of training and performance of deaf professionals in various educational spaces in Brazil. Thus, the specific objectives are: 1) Analyze the functions of the first deaf educators mentioned in history, 2) Trace the chronology of the first deaf teachers in Brazil, from the nineteenth century and 3) Present the possibilities and fields of action of deaf educators today. Regarding the methodological procedures, the research in question is qualitative and exploratory. In order to identify productions that dialogued with the proposed theme, a bibliographic survey was conducted using two databases: the Electronic Library Online Theses and Dissertations Database (Scielo) and the Brazilian Digital Library of Theses and Dissertations (BDTD), from 2015 to 2019, totaling five years of published scientific productions. The searches were performed from four keywords: deaf repeater, deaf instructor, deaf teacher training and deaf teacher. It was performed the reading of 586 titles and abstracts of published works in the two databases consulted. This dissertation is organized into three chapters: Chapter 1, entitled The performance of deaf repeaters in the 19th and 20th Centuries, was based on Rocha (2007, 2009, 2010, 2018) and Sofiato (2011, 2016, 2018), as these authors address the performance of deaf repeaters, in the reign of D. Pedro II. In addition, several managements of the National Institute for the Education of the Deaf in different periods were addressed, as well as, the performance of deaf repeaters and internal regiments. Chapter 2, entitled Training of Teachers of the Deaf in Brazil presents the context of teacher training in Brazil, involving actions for the hearing and deaf public, according to a chronology that begins in the nineteenth century. Chapter 3, entitled Deaf Teacher Training in Brazil deals with the training of deaf teachers and performance of the deaf teacher in different educational spaces in Brazil. As a result, some considerations were made in what invites us to reflect on the advances and challenges present in the training of deaf professionals for the field of education. Moreover, it highlights the functions performed by such professionals assignments according to the space of performance of / in Libras, as guidelines for the protagonism of deaf teachers, including reinforcing the importance of Libras in teacher training

Diagnóstico del nivel de conocimiento sobre el VIH/SIDA en jóvenes universitarios

Históricamente, las estadísticas revelan que la población mayormente expuesta al VIH/ SIDA son personas con edades entre los 20 a 34 años, es decir, adultos jóvenes en donde el conocimiento y los medios mediante los adquieren se asocian como un factor de riesgo a la enfermedad y que requiere la atención de las autoridades. Objetivo: Diagnosticar el nivel de conocimientos, medios consultados y métodos de prevención empleados para su protección en actividades sexuales. Materiales y métodos: El estudio se centra en la población de jóvenes estudiantes de la Universidad de Panamá matriculados en el año 2016. La muestra estuvo constituida por 310 estudiantes. Para la recolección de los datos se utilizó un instrumento validado por medio de expertos y una prueba piloto. Resultados: Los resultados muestran que la distribución de la muestra de 60% (187) mujeres y 40% (123) hombres. Del total de estudiantes, el 90% eran solteros, un 36.5% (113) hombres solteros y 53.2% (165) mujeres solteras, un 0.6% no respondió. Menos de la mitad de los estudiantes manifestaron tener conocimientos sobre el VIH y más de la mitad ha tenido relaciones sexuales. Destacan las relaciones con la misma pareja como el método de prevención. Los medios de comunicación son la fuente de consulta más popular, sin embargo consideran que en las escuelas se debiera brindar más información sobre el tema. Conclusiones: Los jóvenes requieren más información sobre el VIH y que ésta sea de calidad. Aún persisten conductas de riesgo, teniendo sexo a temprana edad (16 años en promedio), donde apenas el 52% de estos usó el condón en su primera relación sexual como medida de prevención

Changes of intestinal microbiota in early life

There is increasing evidence that the intestinal microbiota plays a pivotal role in the maturation of the immune system and in the prevention of diseases occurring during the neonatal period, childhood and adulthood. A number of nonphysiological conditions during the perinatal period (ie caesarean section, prolonged hospitalization, formula feeding, low gestational age) may negatively affect the normal development of the microbiota, leading to decreased amounts of lactobacilli and bifidobacteria and increased amounts of Clostridia . In addition, perinatal antibiotics can cause intestinal dysbiosis that has been associated with short- and long-term diseases. For example, prolonged early empiric antibiotics increase the risk of necrotizing enterocolitis and late-onset sepsis in preterm neonates, whereas the administration of intrapartum antibiotic prophylaxis has been associated with inflammatory bowel diseases, obesity and atopic conditions, such as eczema and wheezing. Promoting breastfeeding, reducing the length of hospital stay, and reducing unnecessary antibiotic therapies are useful strategies to counterbalance unintended effects of these conditions