ENTEROPATHOGENS DETECTED IN A DAYCARE CENTER, SOUTHEASTERN BRAZIL: BACTERIA, VIRUS, AND PARASITE RESEARCH

Introduction: The objective of this study was to determine the prevalence and etiological profile of enteropathogens in children from a daycare center. Methods: From October 2010 to February 2011 stool samples from 100 children enrolled in a government daycare center in the municipality of São José do Rio Preto, in the state of São Paulo, were collected and analyzed. Results: A total of 246 bacteria were isolated in 99% of the fecal samples; 129 were in the diarrheal group and 117 in the non-diarrheal group. Seventy-three strains of Escherichia coli were isolated, 19 of Enterobacter, one of Alcaligenes and one of Proteus. There were 14 cases of mixed colonization with Enterobacter and E. coli. Norovirus and Astrovirus were detected in children with clinical signs suggestive of diarrhea. These viruses were detected exclusively among children residing in urban areas. All fecal samples were negative for the presence of the rotavirus species A and C. The presence of Giardia lamblia, Entamoeba coli, Endolimax nana and hookworm was observed. A significant association was found between food consumption outside home and daycare center and the presence of intestinal parasites. Conclusions: For children of this daycare center, intestinal infection due to pathogens does not seem to have contributed to the occurrence of diarrhea or other intestinal symptoms. The observed differences may be due to the wide diversity of geographical, social and economic characteristics and the climate of Brazil, all of which have been reported as critical factors in the modulation of the frequency of different enteropathogens.

Genomic Signatures and Antiviral Drug Susceptibility Profile of A(H1N1)pdm09

Background: Genetic changes in influenza surface and internal genes can alter viral fitness and virulence. Mutation trend analysis and antiviral drug susceptibility profiling of A(H1N1)pdm09 viruses is essential for risk assessment of emergent strains and disease management. Objective: To profile genomic signatures and antiviral drug resistance of A(H1N1)pdm09 viruses and to discuss the potential role of mutated residues in human host adaptation and virulence. Study design: A(H1N1)pdm09 viruses circulating in Portugal during pandemic and post-pandemic periods and 2009/2010 season. Viruses were isolated in MDCK-SIAT1 cell culture and subjected to mutation analysis of surface and internal proteins, and to antiviral drug susceptibility profiling. Results: The A(H1N1)pdm09 strains circulating during the epidemic period in Portugal were resistant to amantadine. The majority of the strains were found to be susceptible to oseltamivir and zanamivir, with five outliers to neuraminidase inhibitors (NAIs) identified. Specific mutation patterns were detected within the functional domains of internal proteins PB2, PB1, PA, NP, NS1, M1 and NS2/NEP, which were common to all isolates and also some cluster-specific. Discussion: Modification of viral genome transcription, replication and apoptosis kinetics, changes in antigenicity and antiviral drug susceptibility are known determinants of virulence. We report several point mutations with putative roles in viral fitness and virulence, and discuss their potential to result in more virulent phenotypes. Monitoring of specific mutations and genetic patterns in influenza viral genes is essential for risk assessing emergent strains, disease epidemiology and public health implications.

SAINT LOUIS ENCEPHALITIS VIRUS IN MATO GROSSO, CENTRAL-WESTERN BRAZIL

The dengue virus (DENV), which is frequently involved in large epidemics, and the yellow fever virus (YFV), which is responsible for sporadic sylvatic outbreaks, are considered the most important flaviviruses circulating in Brazil. Because of that, laboratorial diagnosis of acute undifferentiated febrile illness during epidemic periods is frequently directed towards these viruses, which may eventually hinder the detection of other circulating flaviviruses, including the Saint Louis encephalitis virus (SLEV), which is widely dispersed across the Americas. The aim of this study was to conduct a molecular investigation of 11 flaviviruses using 604 serum samples obtained from patients during a large dengue fever outbreak in the state of Mato Grosso (MT) between 2011 and 2012. Simultaneously, 3,433 female Culex spp. collected with Nasci aspirators in the city of Cuiabá, MT, in 2013, and allocated to 409 pools containing 1-10 mosquitoes, were also tested by multiplex semi-nested reverse transcription PCR for the same flaviviruses. SLEV was detected in three patients co-infected with DENV-4 from the cities of Cuiabá and Várzea Grande. One of them was a triple co-infection with DENV-1. None of them mentioned recent travel or access to sylvatic/rural regions, indicating that transmission might have occurred within the metropolitan area. Regarding mosquito samples, one pool containing one Culex quinquefasciatus female was positive for SLEV, with a minimum infection rate (MIR) of 0.29 per 1000 specimens of this species. Phylogenetic analysis indicates both human and mosquito SLEV cluster, with isolates from genotype V-A obtained from animals in the Amazon region, in the state of Pará. This is the first report of SLEV molecular identification in MT.

Evolution of the Human Immunodeficiency Virus Type 2 Envelope in the First Years of Infection is Associated with the Dynamics of the Neutralizing Antibody Response

Background: Differently from HIV-1, HIV-2 disease progression usually takes decades without antiretroviral therapy and the majority of HIV-2 infected individuals survive as elite controllers with normal CD4+ T cell counts and low or undetectable plasma viral load. Neutralizing antibodies (Nabs) are thought to play a central role in HIV-2 evolution and pathogenesis. However, the dynamic of the Nab response and resulting HIV-2 escape during acute infection and their impact in HIV-2 evolution and disease progression remain largely unknown. Our objective was to characterize the Nab response and the molecular and phenotypic evolution of HIV-2 in association with Nab escape in the first years of infection in two children infected at birth. Results: CD4+ T cells decreased from about 50% to below 30% in both children in the first five years of infection and the infecting R5 viruses were replaced by X4 viruses within the same period. With antiretroviral therapy, viral load in child 1 decreased to undetectable levels and CD4+ T cells recovered to normal levels, which have been sustained at least until the age of 12. In contrast, viral load increased in child 2 and she progressed to AIDS and death at age 9. Beginning in the first year of life, child 1 raised high titers of antibodies that neutralized primary R5 isolates more effectively than X4 isolates, both autologous and heterologous. Child 2 raised a weak X4-specific Nab response that decreased sharply as disease progressed. Rate of evolution, nucleotide and amino acid diversity, and positive selection, were significantly higher in the envelope of child 1 compared to child 2. Rates of R5-to-X4 tropism switch, of V1 and V3 sequence diversification, and of convergence of V3 to a β-hairpin structure were related with rate of escape from the neutralizing antibodies. Conclusion: Our data suggests that the molecular and phenotypic evolution of the human immunodeficiency virus type 2 envelope are related with the dynamics of the neutralizing antibody response providing further support for a model in which Nabs play an important role in HIV-2 pathogenesis.

Improving Influenza Surveillance in Portuguese Preschool Children by Parents' Report

Influenza surveillance is usually based on nationally organized sentinel networks of physicians and on hospital reports. This study aimed to test a different report system, based on parents' phone contact to the research team and in home collection of samples by a dedicated team. The identification of influenza and other respiratory viruses in children who attended a Hospital Emergency Department was also recorded. Real-time PCR and reverse transcription PCR were performed for influenza A and B, parainfluenza 1-4, adenovirus, human metapneumovirus, respiratory syncytial virus A and B, rhinovirus, enterovirus, group 1 coronaviruses, group 2 coronaviruses, and human bocavirus. One hundred children were included, 64 from the day care centers and 36 from the Hospital. Overall, 79 samples were positive for at least one respiratory virus. Influenza A (H3) was the virus most frequently detected: 25 cases, 20 of these in children under 5 years of age (ten from day care centers and ten who went to the hospital) which was higher than those reported by the National Influenza Surveillance Programme for this age. CONCLUSION: The results obtained in this study suggest that a surveillance system based on parents' reports could complement the implanted system of the National Influenza Surveillance Programme.

Pandemia influenza A/H1N1 2009 em Portugal : estratégias de intervenção e saúde pública

RESUMO - Introdução: A necessidade de gestão da ameaça de uma pandemia obriga à gestão da incerteza absoluta. O desconhecimento científico quanto a uma série de factores tais como, as características dos vírus causadores de infecções, a efectividade das medidas de prevenção e de tratamento, contribuiu para a dificuldade de actuação a vários níveis. Face à evolução da situação epidemiológica mundial no campo da gripe, Portugal reviu e adaptou o seu plano de contingência para a gripe tendo sido homologado, em Janeiro de 2006, um novo plano – Plano de Contingência Nacional do Sector da Saúde para a Pandemia. Objectivos: O presente estudo apresenta como principais objectivos (1) a avaliação das estratégias de intervenção e respectiva implementação das medidas de saúde pública não farmacológicas, durante a pandemia da gripe de 2009; (2) a identificação dos pontos críticos na gestão das medidas de saúde pública não farmacológicas, para preparação e resposta a futuras pandemias da gripe e, por último, (3) a definição de um modelo de acompanhamento, de monitorização e de avaliação das medidas de saúde pública não farmacológicas, que permita identificar os pontos importantes a ter em conta na implementação de boas práticas de saúde pública, nos diferentes níveis de prestação de cuidados de saúde, tendo como limitação o estado da arte dos sistemas de informação e avaliação, nacionais e internacionais, existentes. Material e Métodos: Para a avaliação das estratégias de intervenção e respectiva implementação das medidas de saúde pública não farmacológicas bem como, para a identificação dos pontos críticos na gestão das respectivas medidas foram consideradas duas perspectivas: a avaliação dos resultados através do Relatório da Pandemia da Gripe e a avaliação dos resultados através das avaliações da OMS e da Euro OMS. É apresentado um modelo de acompanhamento, de monitorização e de avaliação das medidas de saúde pública não farmacológicas, a implementar na classe médica que esteve na “linha da frente”, durante a pandemia H1N1 2009 em Portugal, nos cuidados primários, hospitalares e intensivos. Resultados: Deverá existir uma representação dos profissionais de saúde para suporte das decisões de forma a assegurar uma representação alargada não só na preparação mas também, na implementação e na aplicação do Plano de Contingência. O processo de planemanto deverá ter acesso público com possibilidade de participação activa do cidadão na sua implementação. O Plano deve ter a capacidade para se adaptar à situação epidemiológica e, no terreno os exercícios de simulações durante o processo de planeamento são importantes. As orientações técnicas devem ser dinâmicas, práticas, específicas e úteis e, dever-se investir numa cultura de confiança (“right people communicating to right people”). As alterações ao plano devem partir da coordenação, comando e controlo que, nos vários níveis de decisão, devem definir claramente quem toma as decisões. Conclusões: As estratégias para as intervenções ao nível da saúde pública, continuam, no mundo hodierno a desempenhar um papel fundamental e crucial na contenção das pandemias. O modelo apresentado neste estudo procura uma abordagem objectiva para destacar não só, os elementos essenciais de actividades bem-sucedidas, mas também as áreas em que a experiência pandémica sugere que o futuro planeamento deve dar maior ênfase.

Autism Spectrum Disorder Secondary to Enterovirus Encephalitis

Millions of children are infected by enteroviruses each year, usually exhibiting only mild symptoms. Nevertheless, these viruses are also associated with severe and life-threatening infections, such as meningitis and encephalitis. We describe a 32-month-old patient with enteroviral encephalitis confirmed by polymerase chain reaction in cerebrospinal fluid, with unfavorable clinical course with marked developmental regression, autistic features, persistent stereotypes and aphasia. She experienced slow clinical improvement, with mild residual neurologic and developmental deficits at follow-up. Viral central nervous system infections in early childhood have been associated with autism spectrum disorders but the underlying mechanisms are still poorly understood. This case report is significant in presenting a case of developmental regression with autistic features and loss of language improving on follow-up. To our knowledge, this is the first published report of enterovirus encephalitis leading to an autism spectrum disorder.

The Intensive Care Global Study on Severe Acute Respiratory Infection (IC-GLOSSARI): a Multicenter, Multinational, 14-Day Inception Cohort Study

PURPOSE: In this prospective, multicenter, 14-day inception cohort study, we investigated the epidemiology, patterns of infections, and outcome in patients admitted to the intensive care unit (ICU) as a result of severe acute respiratory infections (SARIs). METHODS: All patients admitted to one of 206 participating ICUs during two study weeks, one in November 2013 and the other in January 2014, were screened. SARI was defined as possible, probable, or microbiologically confirmed respiratory tract infection with recent onset dyspnea and/or fever. The primary outcome parameter was in-hospital mortality within 60 days of admission to the ICU. RESULTS: Among the 5550 patients admitted during the study periods, 663 (11.9 %) had SARI. On admission to the ICU, Gram-positive and Gram-negative bacteria were found in 29.6 and 26.2 % of SARI patients but rarely atypical bacteria (1.0 %); viruses were present in 7.7 % of patients. Organ failure occurred in 74.7 % of patients in the ICU, mostly respiratory (53.8 %), cardiovascular (44.5 %), and renal (44.6 %). ICU and in-hospital mortality rates in patients with SARI were 20.2 and 27.2 %, respectively. In multivariable analysis, older age, greater severity scores at ICU admission, and hematologic malignancy or liver disease were independently associated with an increased risk of in-hospital death, whereas influenza vaccination prior to ICU admission and adequate antibiotic administration on ICU admission were associated with a lower risk. CONCLUSIONS: Admission to the ICU for SARI is common and associated with high morbidity and mortality rates. We identified several risk factors for in-hospital death that may be useful for risk stratification in these patients.

Influenza A(H1N1)pdm09 Resistance and Cross-Decreased Susceptibility to Oseltamivir and Zanamivir Antiviral Drugs

Neuraminidase inhibitors (NAIs) oseltamivir and zanamivir are currently the only effective antiviral drugs available worldwide for the management of influenza. The potential development of resistance is continually threatening their use, rationalizing and highlighting the need for a close and sustained evaluation of virus susceptibility. This study aimed to analyze and characterize the phenotypic and genotypic NAIs susceptibility profiles of A(H1N1)pdm09 viruses circulating in Portugal from 2009 to 2010/2011. A total of 144 cases of A(H1N1)pdm09 virus infection from community and hospitalized patients were studied, including three suspected cases of clinical resistance to oseltamivir. Oseltamivir resistance was confirmed for two of the suspected cases. Neuraminidase (NA) H275Y resistant marker was found in viruses from both cases but for one it was only present in 26.2% of virus population, raising questions about the minimal percentage of resistant virus that should be considered relevant. Cross-decreased susceptibility to oseltamivir and zanamivir (2-4 IC50 fold-change) was detected on viruses from two potentially linked community patients from 2009. Both viruses harbored the NA I223V mutation. NA Y155H mutation was found in 18 statistical non-outlier viruses from 2009, having no impact on virus susceptibility. The mutations at NA N369K and V241I may have contributed to the significantly higher baseline IC50 value obtained to oseltamivir for 2010/2011 viruses, compared to viruses from the pandemic period. These results may contribute to a better understanding of the relationship between phenotype and genotype, which is currently challenging, and to the global assessment of A(H1N1)pdm09 virus susceptibility profile and baseline level to NAIs.

Etiologia das infeções respiratórias virais em criançass em idade pré-escolar

RESUMO: Os vírus respiratórios continuam a ocupar um papel relevante na morbilidade e mortalidade infantil, tendo na última década sido alargado o espectro de vírus potencialmente causadores das infeções respiratórias. O diagnóstico destas infeções pode ser efetuado por várias metodologias, sendo as técnicas de biologia molecular consideradas as mais sensíveis para este fim. No âmbito do Projeto Ambiente e Saúde em Creches e Infantários (ENVIRH) foi efetuada uma comparação da prevalência dos principais vírus respiratórios em crianças em idade pré-escolar, com critérios de infeção respiratória, recorrendo a técnicas de biologia molecular, em duas populações: crianças que se encontravam na escola/domicilio e crianças que recorreram a uma urgência hospitalar. O estudo decorreu em dois períodos, de Fevereiro a Maio de 2011 e de Outubro de 2011 a Abril de 2012. Foram efetuadas duas colheitas de zaragatoas, uma nasal e outra orofaríngea. A metodologia utilizada para a identificação viral nas amostras foi a PCR e RT-PCR multiplex em tempo real. Os vírus pesquisados foram: Influenza A e B, Parainfluenza 1-4, Metapneumovirus humano, Vírus Sincicial respiratório (VSR), Rinovírus, Enterovírus, Coronavírus e Bocavirus. Foram realizadas 100 colheitas em crianças com idades compreendidas entre os 5 meses e os 5 anos. Foram obtidas 64 amostras dos infantários/domicílios, das quais 47 foram positivas. Da urgência Hospitalar obtiveram-se 36 amostras, em que 32 foram positivas. O vírus da gripe A (H3) foi o mais frequentemente detetado nas duas populações, mas apenas durante o surto de 2012. O VSR e os adenovírus foram mais frequentes nas crianças que recorreram ao hospital, ao contrário dos enterovirus e dos coronavírus, que não foram detetados nesta população. Os bocavirus nunca foram detetados isoladamente. Este estudo reforça a importância de se utilizarem técnicas de biologia molecular para o diagnóstico etiológico das infeções respiratórias, devido à elevada sensibilidade das mesmas, o que se reflete na elevada percentagem de amostras positivas. O facto de se utilizarem técnicas “multiplex”, que permitem a pesquisa simultânea de vários vírus, facilita a deteção de um maior espectro destes agentes. A elevada prevalência de Influenza A H3N2 deveu-se ao facto de grande parte do estudo ter coincidido com um período de surto por este vírus. O sistema de alerta montado durante o projeto ENVIRH pareceu promissor para uma eventual utilização futura em períodos de atividade gripal.--------------ABSTRACT: In the last decade, as respiratory viruses keep representing a relevant factor in child morbidity and mortality, the spectrum of viruses that may potentially cause respiratory infections has been widened. Within the several methodologies that may be applied in the diagnosis of these types of infections, the ones that use molecular biology are considered to be the most sensitive. The Environment and Health in Daycares and Nurseries Project (ENVIRH) arranged for a study, by means of molecular biology techniques, on the main respiratory viruses' influence in pre-school aged children with respiratory infection symptoms. This study compared children in two different populations: children at school or at home and children that were taken to a hospital emergency service. The study was conducted in two different time periods, one from February to May 2011 and the other from October 2011 to April 2012. During this time, two swab collections were held, one nasal and one oropharyngeal. PCR and RT-PCR multiplex in real time techniques were used for viral identification of the samples, searching for the viruses Influenza A and B, Parainfluenza 1-4, human Metapneumovirus, Respiratory Sincytial Virus (RSV), Rhinovirus, Enterovirus, Coronavirus and Bocavirus. One hundred (100) collections were held in children between the ages of 5 months and 5 years, sixty-four (64) at home/school and thirty-six (36) at the hospital's emergency service. From a total of seventy-nine (79) positive samples, forty-seven (47) were obtained at home/school and thirty-two (32) at the hospital. The virus detected the most in both populations was the Influenza A (H3), but only during the outbreak of 2012. Unlike the enteroviruses and coronaviruses, that were not detected within this population, the RSV and the adenoviruses were most common within the children at the hospital. Bocaviruses were never detected isolated from other viruses. The high percentage of positive samples reinforces the significance of using molecular biology techniques for the etiological diagnosis of respiratory infections. The use of multiplex techniques, that make the simultaneous search for multiple viruses possible, enhances the detection of a larger spectrum of such agents. Most of the study coincided with an outbreak of the Influenza A H3N2 virus, thus explaining the high number of its cases identified. The alert system set up during the ENVIRH project looked promising enough for eventual periods of flu activity in the future.

Detection of HTLV-IIa in blood donors in an urban area of the Amazon Region of Brazil (Belém, PA)

The human lymphotropic viruses type I (HTLV-I) and type II (HTLV-II) are members of a group of mammalian retroviruses with similar biological properties, and blood transfusion is an important route of transmission. HTLV-I is endemic in a number of different geographical areas and is associated with several clinical disorders. HTLV-II is endemic in several Indian groups of the Americas and intravenous drug abusers in North and South America, Europe and Southeast Asia. During the year of 1995, all blood donors tested positive to HTLV-I/II in the State Blood Bank (HEMOPA), were directed to a physician and to the Virus Laboratory at the Universidade Federal do Pará for counselling and laboratory diagnosis confirmation. Thirty-five sera were tested by an enzyme immune assay, and a Western blot that discriminates HTLV-I and HTLV-II infection. Two HTLV-II positive samples were submitted to PCR analysis of pX and env genomic region, and confirmed to be of subtype IIa. This is the first detection in Belém of the presence of HTLV-IIa infection among blood donors. This result emphasizes that HTLV-II is also present in urban areas of the Amazon region of Brazil and highlights the need to include screening tests that are capable to detect antibodies for both types of HTLV.

Purification of complex biopharmaceuticals with new processes, advanced analytics and computer-aided process design tools

Dissertação para obtenção do Grau de Mestre em Biotecnologia

A longitudinal study on enteropathogenic infections of livestock in Trinidad

A longitudinal study was conducted on selected livestock farms to determine the prevalence of enteropathogens in diarrhoeic and non-diarrhoeic animals. The enteropathogens assayed from faecal samples and rectal swabs were bacteria (Escherichia coli, Campylobacter spp. Salmonella spp. and Yersinia enterocolitica), parasites (coccidia, gastrointestinal nematodes and Cryptosporidium spp.) and viruses (group A rotavirus and parvovirus). The prevalence of the enteropathogens in various animal species was related to age and month of the year. Generally, younger animals presented a higher prevalence of infection by enteropathogens than older animals while most infections occurred between the months of January and April.

Integrated functional genomics for improved manufacture of recombinant enveloped virus

Dissertation presented to obtain the Ph.D degree in Engineering and Technology Sciences-Biotechnology

Fulminant hepatic failure in children and adolescents in Northern Brazil

The histological findings of fulminant hepatic failure were correlated to the demographic, clinical, biochemical and virological features in children and adolescents, native to the Amazonas State in Northern Brazil. 96.2% had evidence of infection by primary hepatotrophic viruses. Histological analysis revealed three distinct patterns of fulminant hepatic failure.