Introdução: Uma das importantes causas de morbidade e mortalidade relacionadas aos cuidados na assistência continua sendo as infecções, conhecidas como Infecções Relacionadas à Assistência à Saúde (IRAS). Dentre elas, inclui-se a gastroenterite que é uma infecção causada por bactérias, vírus ou protozoários. Uma gastroenterite que tem dispertado atenção como IRAS, são as noroviroses causadas pelo Norovirus Humano (HuNoV). Esta apresenta alta infectividade a partir do baixo inóculo, alto potencial patogênico acometendo indivíduos sadios e persistência no ambiente. No contexto de precauções padrão, deve-se admitir a presença de indivíduos infectados por HuNoV nos Serviços de Saúde (SS), sintomáticos ou não. O HuNoV é eliminado para o ambiente a partir dos disseminadores por meio do vômito e fezes que na maioria das vezes contamina o piso. Nesse sentido, as superfícies hospitalares contaminadas por estas matérias orgânicas, têm merecido atenção dos controladores de infecção no que diz respeito à transferência deste para o ambiente. A padronização do cuidado seguro do local onde houve a contaminação pelo material fecal ou vômito torna-se de fundamental importância para evitar contaminação cruzada, que pode facilmente resultar em surtos. Embora o Centers for Disease Control and Prevention (2003) recomende apenas a limpeza para piso, justificado por ser uma superfície onde as mãos dos profissionais não tocam durante os procedimentos assistenciais, não existe um consenso quando se trata de vômito e fezes como dejetos derramadas no piso, surgindo a polêmica da necessidade de também usar desinfetantes em função do HuNoV. Objetivo: Evidenciar a necessidade do uso de desinfetante no piso após a limpeza para prevenir a transferência de HuNoV para o ambiente a partir de vômito e fezes de pacientes presumivelmente contaminados. Método: Trata-se de uma Revisão Sistemática (RS) cujo procedimento metodológico, seguiu as recomendações do Manual da Colaboração Cochrane sendo relatada de acordo com o Preferred Reporting Items for Systematic Reviews and Meta-Analysis - PRISMA. A busca das evidências, foi realizada nas bases de dados Cochrane Library, BVS, Medline, Web of Science e Cinahal, acessando artigos publicados e indexados sem restrição de idiomas e de período, até fevereiro do ano de 2015. Foram utilizados descritores oficiais e adaptados de diferentes bases de dados e sua combinação foi realizada por meio do operador booleano AND e OR. Resultados: Após a leitura dos títulos, resumos e textos na íntegra por duas avaliadoras, selecionaram-se três estudos. Os dados foram sintetizados e apresentados de forma descritiva por meio de quadros e tabelas. Ressalta-se que para estudos laboratoriais, não existem checklist específicos para avaliar a qualidade das evidências, por isso, foi utilizado o sistema Grading of Recommendations, Assessment, Development and Evaluation - GRADE. A análise da adequação dos delineamentos do estudo para o objeto da presente RS, assim como presença de vieses, foram independentemente julgados por três especialistas no método e no assunto, seguida de uma discussão grupal em busca de consenso. Conclusão: Essa revisão confirmou por meio dos estudos encontrados a necessidade da utilização do desinfetante posteriormente a limpeza, para eliminar o HuNoV. No entanto, considerando a possibilidade da sobrevivência do HuNoV decorrente das práticas atuais não seguras da desinfecção do piso, essa RS não encontrou dados suficiente se HuNoV residuais terão a capacidade de aerolizar contaminando superfícies tocadas ou o ar, havendo risco destes serem deglutidos o que terá que ser respondida em pesquisas futuras. / Introduction: Infections remain one of the major causes of morbidity and mortality related to care, referred as Infections Related to Health Care. Among them, gastroenteritis is a common infection which may be caused by bacteria, viruses or protozoa. A gastroenteritis that has been highlighted as related to health care are noroviruses caused by Human Norovirus (HuNoV). It has high infectivity from low inoculum, high pathogenic potential affecting healthy individuals and environmental patency. Considering the context of standard precautions, it must be admitted the presence of patients infected by HuNoV in Health Services, symptomatic or not. HuNoV is eliminated to the environment from the scatterers through vomit and feces that, most of the time, contaminate the floor. So hospital surfaces contaminated by these organic materials have received attention of infection controllers related to its transfer to the environment. The stardization of safe care of the place where there was contamination by feces or vomit becomes crucial to avoid cross-contamination, which can easily lead to outbreaks. While Centers for Disease Control and Prevention (2003) recommend cleaning of floor only, as it is a surface where individuals dont touch their hands during assistance proceedings, there is no consensus related to vomit and feces wastes into the environment because of HuNoV, resulting in the controversy of also use desinfectants based on the NuNov. Purpose: Highlighting the need of using disinfectant on the floor after cleaning to prevent transferring HuNoV from vomit and feces of infected patients to the environment. Method: It is a systematic review whose methodological procedure followed the Cochrane Collaboration Handbook recommendations reported according to PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analysis). The search of evidence was carried out through databases Cochrane Library, BVS, PubMed, Web of Science and Cinahal considering articles published and indexed with no restriction to languages and publication date until February 2015. Adapted descriptors were used for data bases and its combination was performed using the Boolean operators AND and OR. Results: Once titles, abstracts and full texts were read by two reviewers, three studies have been selected. Data were synthesized and descriptively presented through charts and tables. Importantly, when it comes to laboratory investigations, there are no specific checklists to assess the quality of evidence, so the GRADE system was used. The analysis of the adequacy of the study designs for the object of the present systematic review, as well as the presence of bias, were independently judged by three experts in the method and subject, followed by a group looking for consensus. Conclusion: This review has confirmed the need of using disinfectant after cleaning to eliminate HuNoV. However, considering the HuNoV survival due to unsafe current practices of floor disinfection, this systematic review havent found enough data to prove if wasted HuNoV have the ability to aerosolize contaminating touched surfaces or air, with a risk of these being swallowed. This question will be answered by future researches.

The Centers for Disease Control and Prevention estimated 23,000,000 cases of viral gastroenteritis caused by Norovirus in 2000, 40% of which were transmitted by food including: a variety of fresh produce, cake, deli meats, fruit salad, cheeses and ice. (CDC, 2003). An estimated 83,391 cases of Hepatitis A virus was reported in 2000, of which 5% was attributed to foodborne transmission (CDC, 2003). These figures underscore an urgent need for a method that can isolate virus from a variety of food matrices. The aim of this study was to develop an overall assessment of the inhibitory effects of a variety of food matrices on Real Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). Additionally, to compare a sequence specific hybridization probe amplification format to a non sequence specific SYBR Green format using the Roche LightCycler. The secondary aim was to evaluate the effectiveness of a food virus concentration and isolation protocol under development at the Florida Department of Health Bureau of Laboratories, Tampa. Three food specimens consisting of prepackaged smoked ham, fresh cilantro, and Thompson's green grapes were seeded with three dilutions of poliovirus 3 (Sabin strain). A viral concentration procedure under development at the Florida Department of Health Bureau of Laboratories, Tampa was used to isolate the virus. Real Time RT-PCR was carried out on the Roche LightCycler in SYBR Green and Hybridization probe formats. Spiking the virus-negative samples of each matrix with a dilution series of poliovirus 3 created post flocculation spikes. This post-flocculation dilution series amplification allowed a standard curve to be created unique to each food matrix. The flocculation and concentrations specimens were then amplified and the standard curves from the post-flocculation seed were used to calculate the loss associated with the concentration procedure. This study reports significant differences (p<0.05) in recovery detected between the various matrices, and Real Time RT-PCR formats. The concentration protocol under development at the Florida Department of Health Bureau of Laboratories, Tampa, demonstrates a 12-78% recovery of seeded virus in a simulated “real world” virus contamination event among the various matrices.

Les Norovirus (NoV), appartenant à la famille des Caliciviridae, sont une cause majeure dépidémies et de cas sporadiques de gastroentérites hautement contagieuses chez lhomme. Leur transmission emprunte la voie fécale-orale et ils sont à l'origine dune part importante des toxi-infections humaines d'origine alimentaire, en particulier dues à la consommation de mollusques bivalves. Ils possèdent un génome constitué dARN monocaténaire de polarité positive et sont classeés par analyse de proximité génétique en cinq génogroupes, contenant chacun plusieurs génotypes. Un problème majeur réside dans lincapacité à multiplier facilement les NoV en culture de cellules. La RT-PCR est devenue la méthode de choix pour leur détection dans les échantillons de matières fécales, les denrées alimentaires et les prélèvements effectués dans lenvironnement. Il est important de disposer de techniques à la fois sensibles et permettant également la détection dun large panel de NoV. La quantification de la charge virale est possible par lutilisation des techniques de RT-PCR en temps réel et est primordiale pour non seulement déterminer le niveau de contamination dun prélèvement, mais également pour étudier et caractériser la pathogénie de linfection à NoV. Des NoV ont été détectés dans diverses espèces animales, dont lespèce bovine. Ces découvertes ont soulevé d'importantes questions sur une éventuelle transmission zoonotique et l'existence d'un réservoir animal pour les NoV. La caractérisation moléculaire des deux prototypes de NoV bovins, nommément le virus Newbury2 et le virus Jena, a révélé qu'ils étaient génétiquement proches et associés aux NoV humains. Parmi les hypothèses évoquées, les animaux pourraient être soit des porteurs passifs de NoV, soit infectés de manière active par ces virus, responsables dès lors d'une zoonose. Caractériser les NoV circulant chez lhomme et les espèces animales est intéressant dans le but détudier leurs voies de transmission et léventuel passage inter-espèce de ces virus. Un mécanisme important d'évolution des NoV est la recombinaison, dun grand intérêt dans létude des NoV, générant des modifications du génome viral aboutissant à la création dun génome « chimère » à partir de deux génomes parentaux différents. Elle crée ainsi de la variation génétique et par là lémergence de nouveaux virus. En effet, il est bien documenté que la recombinaison se produit souvent parmi les NoV et contribue à la diversité génétique de ces virus ainsi quà lapparition de nouvelles épidémies. La prévalence des souches de NoV recombinants peut être sous-estimée par le fait que la caractérisation des NoV est habituellement basée sur le séquençage partiel du gène de lARN polymérase-ARN dépendante uniquement, alors quidéalement il faudrait séquencer différentes parties du génome, principalement lARN polymérase-ARN dépendante et la protéine de capside, pour identifier de tels virus. Il est important de déterminer précisément l'implication exacte de la recombinaison sur lévolution des NoV afin de comprendre les mécanismes dévolution des souches et l'avantage sélectif conféré pour certaines dentre elles. Etudier ce mécanisme permettra de mieux comprendre lavantage sélectif observé pour certains NoV et aidera à élucider les voies de transmission des NoV. Létude de ces deux points (transmission zoonotique et virus recombinants) est primordiale. En effet, le franchissement de la barrière despèce affecterait à la fois l'épidémiologie et l'évolution de ces virus, et compliqueraient également la capacité au développement dun vaccin ou dun traitement. Dans d'autres espèces animales, comme les chevaux ou les oiseaux, aucun NoV na été détecté à ce jour mais ces dernières années, des NoV ont été décrits dans de nombreuses espèces animales (chien, lion, mouton). Cela laisse donc présager dune gamme despèce cible encore plus étendue. Ce travail sinscrit dans le cadre de létude des voies de transmission des NoV avec comme objectif, après la mise au point de méthodes rapides et sensibles de détection et de quantification, dapporter un éclaircissement aux questions importantes relatives à lévolution des NoV et à leur catégorie dhôte. Dans un premier temps, la RT-PCR conventionnelle a été utilisée afin de détecter les NoV dans les espèces humaine et bovine. Ensuite, une RT-PCR utilisant la technologie SYBR Green a été développée et utilise un contrôle interne constitué dARN ajouté au même tube. Ce test est capable de détecter des NoV humains et bovins appartenant aux génogroupes I, II et III et permet de faire la distinction entre les NoV et le contrôle interne par lanalyse des courbes de dissociation. Une dilution 10 fois des échantillons sest révélée la méthode de choix pour lever les inhibitions. Afin de pouvoir confirmer directement le résultat et de permettre la quantification des NoV, une RT-PCR en temps réel utilisant la technologie TaqMan a été développée. Elle utilise un contrôle interne dARN et un standard dARN. De façon très intéressante, cette méthode peut détecter les NoV humains appartenant au génogroupes I, II et bovins du génogroupe III. Les inhibitions furent efficacement levées par une dilution 10 fois de léchantillon ou lajout de sérum albumine bovine au mix de RT-PCR. Ces deux RT-PCR en temps réel ont montré une sensibilité supérieure comparée à la RT-PCR conventionnelle. Avec pour objectif de comprendre les voies de transmission des NoV, la situation en Belgique a été investiguée et des NoV humains et bovins ont été détectés et analysés par séquençage partiel. Des NoV appartenant à différents génogroupes ont été détectés : GI et GII chez lhomme et GIII chez les bovins. Par analyse de la proximité génétique, les NoV bovins se sont révélés de génotype GIII.2 et les NoV humains de différents génotypes, mais majoritairement de génotype GII.4. Ces analyses ont permis également lidentification dune co-infection et de deux recombinants naturels, ces derniers étant proches de génotypes différents en fonction de la région du génome analysée (polymérase ou capside). L'identification de zones privilégiées pour la recombinaison dans la région située à la jonction de l'ORF (open reading frame) 1 et de lORF2 confirme l'importance de cette région dans ce phénomène. Afin détudier lévolution des NoV bovins, un NoV bovin détecté en Belgique fut séquencé complètement (Bo/B309/2003/BE) et comparé à la souche originale Newbury2 (isolée dans les années 80). Dune part, cette études a permis daboutir à la mise au point et la validation doutils permettant la détection et létude les NoV humains et animaux, tant pour leur pathogénie, que leur évolution ou leurs voies de transmission. Dautre part, basée sur le panel déchantillons recueillis durant cette étude, lanalyse phylogénétique des NoV détectés va dans le sens des études réalisées dans dautres pays tendant à montrer que les NoV bovins constituent un groupe de virus distincts, différents des NoV humains. Cela suggère que les NoV bovins ne représentent pas un risque pour la santé humaine. Néanmoins, la possibilité dune infection zoonotique ou dun réservoir animal ne peut pas être exclue vu la proximité de séquence entre les NoV humains et animaux et aussi la relation étroite et proche entre les populations humaines et les élevages danimaux. La détection dune co-infection et de deux recombinants naturels démontre les possibilités dévolution des NoV et limportance dune analyse complète de leur génome pour leur caractérisation. Ce travail a été pionnier dans létude des NoV au laboratoire et a ouvert la voie à dautres sujets de recherche sur les NoV et à de nouvelles thèses de doctorat, notamment sur létude de linteraction NoV-hôte (NoV dans lespèce bovine) et létude de la recombinaison des NoV in vitro (NoV murins).

Numerous toxins and pathogens gain entry into mammalian cells using cell surface glycans. The Iyer group at Georgia State University is working on the development of glycoconjugates for the accurate detection of infectious agents. In this thesis, I have focused on the development of glycans to detect influenza virus and norovirus. In the first section, I have focused on influenza viruses. A panel of synthetic glycans was synthesized as receptor mimics for the specific capture of influenza viruses. The synthetic glycans were printed onto commercial glass slides using a free amine at the end of a spacer to generate a small focused microarray. This glycan printed microarray was evaluated for its ability to capture three strains of influenza viruses. The analytical limit of detection is ~10 pfu/ml, (plaque forming units/milliliter) which is clinical relevant as 102 viral particles are typically required to cause infection. We also tested the drug susceptibility of current antivirals, Zanamivir and Ostelamivir using the microarray and determined the feasibility of this system to determine antiviral resistance for different strains. In addition to optical detection, I developed an electrochemical assay to rapidly detect influenza viruses. Here, we utilized an unique property of influenza viral surface enzyme, Neuraminidase (NA), which cleaves terminal N-Acetyl Neuraminic acid (sialic acid) from cell surfaces and proteins. We designed an electrochemical assay that uses glucose bearing sialic acid substrates. Glucose is released when exposed to viral NA or intact viruses. The released glucose can be detected using repurposed glucose meters. Thus, personal glucose meters that were designed to assist diabetics and prediabetics monitor blood glucose can potentially be used to detect pathogens. Using this approach, we have detected 19 unique strains of influenza viruses. We also demonstrated drug susceptibility using this assay. The limit of detection of this assay is 102 pfu/sample, which is clinically relevant. The results were validated plaque assays and polymerase chain reaction (PCR). In the second part of this thesis, I focused on norovirus detection. I developed a focused glycan microarray that comprised of a library of histo blood group antigens (HBGAs). The HBGAs were attached to a carrier protein and printed onto activated glass slides. A panel of norovirus virus like particles (VLPs) and strains that included different genogroups was exposed to the microarray. We found that different VLPs and strains give rise to unique binding patterns. When the binding pattern of VLPs for a particular strain were compared to the corresponding intact virus, the binding patterns didn't match well, presumably because the virus does not recognize the same antibody as the VLPs. Unfortunately, antibodies for the virus cannot be generated because the virus cannot be grown in a laboratory setting. Indeed, all norovirus samples are obtained from human challenge studies. I also used surface plasmon resonance (SPR) studies in an effort to determine the binding affinities. Divalent biotinylated H type glycans were synthesized and their binding affinities with different VLPs and viral strains were determined. Initial studies suggest that the binding affinities are strain specific. These results demonstrate that glycans can be used to capture and isolate norovirus, although more research is required to develop glycan based norovirus detection kits.

Made available in DSpace on 2015-07-08T12:28:18Z (GMT). No. of bitstreams: 2 38.pdf: 779592 bytes, checksum: bb7860a5216579fd5bbccc4e3c3d8a6d (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2012 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / As hortaliças como a alface (Lactuca sativa) têm sido associadas a diversos surtos de doenças de origem alimentar. Dentre os patógenos envolvidos, os norovírus (NoV) são reconhecidos como os principais agentes etiológicos de gastrenterite, sendo as estirpes do genogrupo II (GII) mais prevalentes. Dessa forma, muito esforço tem sido realizado no desenvolvimento de métodos para detecção desses vírus em hortaliças. Os estudos têm focado na padronização dos métodos e otimização das etapas de extração, concentração e detecção do ácido nucléico viral. O uso de outros vírus como controle interno também tem sido estudado, para identificação de falhas durante a análise e evitar resultados falso-negativos. O presente estudo teve como objetivo investigar a contaminação de NoV GII pelo método de concentração por filtração em membrana negativa seguida de semi-nested PCR e PCR em tempo real e avaliar a qualidade microbiológica (pesquisa de Salmonella e enumeração de coliformes) de 90 amostras de alface (30 in natura, 30 minimamente processadas e 30 de serviços de alimentação) no Estado do Rio de Janeiro.O bacteriófago PP7 foi utilizado como controle interno e uma otimização do método comparando a solução salina fosfatada tamponada (PBS) e o tampão glicina (TG) foi realizada. Nenhuma amostra apresentou contaminação por NoV GII e Salmonella. As amostras in natura apresentaram qualidade microbiológica satisfatória de acordo com a legislação. Uma amostra minimamente processada (3,3%) e seis de serviços de alimentação (20%) apresentaram condições higiênico-sanitárias insatisfatórias devido ao número de coliformes termotolerantes acima do permitido, indicando que os procedimentos de higienização realizados nos serviços de alimentação não foram eficazes para eliminação dos micro-organismos ou que a contaminação pode ocorrer, por parte dos manipuladores. / Green leafy vegetables, as lettuce (Lactuca sativa) have been linked to diverse foodborne outbreaks worldwide. Among several pathogens involved, norovirus (NoV) are recognized as one of the most important etiological agent associated with gastroenteritis, being genotype II (GII) the most prevalent. In this manner, efforts have been made to develop methods to detect these viruses in green leafy vegetables. Researches have focused on standardizing methods and optimize stages of extraction, concentration and detection of viral nucleic acid. The use of others viruses as internal control has also been studied, to identifying possible errors during analysis and avoid false-negatives results. This study aimed to verify the contamination by NoV GII through concentration methodology by negative-membrane filtration followed by detection by semi-nested PCR and quantification by Real Time PCR and microbiological quality evaluation (Salmonella research and enumeration of total and thermo tolerant coliform) of 90 samples of lettuce (30 in natura, 30 minimally processed and 30 from food services) in the state of Rio de Janeiro. Bacteriophage PP7 was utilized as internal control and a method optimization comparing phosphate buffered saline (PBS) and Glycine buffer (GB) was carried out. No sample showed contamination by NoV GII and Salmonella. Samples of in natura lettuce exhibited satisfactory microbiology quality according Brazilian resolution. One minimally processed sample (3.3%) and six (20%) from food service showed unsatisfactory hygienic-sanitary conditions because of the number of thermotolerant coliforms above of allowed, indicating that hygienic proceedings in restaurants were not effective for eliminating those microorganisms or that the contamination may occur by employers. PP7 bacteriophage was detected in 40, 86.7 and 76.7% of in natura, minimally processed and food service, respectively. Using GB increased PP7 bacteriophage recovery (p = 0.029), but did not demonstrate significant difference in NoV GII recovery (p = 0.57), and increased sensitivity of semi nested PCR technique for detecting NoV GII in samples artificially contaminated. This last one exhibited lower sensitivity than Real Time PCR for NoV GII detection. Results point out that GB is an elution buffer more efficient and that PP7 bacteriophage is applicable as an internal control of this method.

京都大学 / 新制・論文博士 / 博士(社会健康医学) / 乙第13455号 / 論社医博第17号 / 新制||社医||11(附属図書館) / 京都大学大学院医学研究科社会健康医学系専攻 / (主査)教授 長尾 美紀, 教授 近藤 尚己, 教授 中川 一路 / 学位規則第4条第2項該当 / Doctor of Public Health / Kyoto University / DFAM

Norovirus Gastroenteritis Leading to Partial Small Bowel Obstruction David Berry DO, Arthur Cecchini DO, Koushik Sanku MD, Bhavesh Gajjar MD Berrydw@etsu.edu, Cecchini@etsu.edu, Sankuk@etsu.edu, Gajjarb@etsu.edu Department of Internal Medicine, Quillen College of Medicine, East Tennessee State University BACKGROUND Acute gastroenteritis (AGE) is a common problem in both inpatient and outpatient settings. Most cases are viral in origin, with norovirus being the most cited. Typical symptoms include low-grade fever, chills, nausea, vomiting, and abdominal discomfort. The physical examination is usually unremarkable, but abdominal tenderness or signs of volume depletion may be present in severe disease. Most patients have spontaneous remission within a few days and do not require hospitalization or diagnostic evaluation. Laboratory evaluation is often helpful in severe disease, immunocompromised patients, or when bloody or mucoid diarrhea is present. Polymerase chain reaction (PCR) gastrointestinal multiplex testing is often the preferred evaluation as it has a high sensitivity, specificity, and turnaround time when compared to traditional stool studies of enzyme-immunoassay studies. Treatment is often supportive, but specific bacterial and parasitic pathogens should prompt treatment with antimicrobial therapy. CASE PRESENTATION This case presents a 47-year-old male with no known previous medical history or history of intraabdominal surgeries. He presented with four days of progressive nausea, vomiting, diarrhea, and abdominal discomfort. The physical examination revealed a distended and tender abdomen. The metabolic panel did not show any electrolyte derangements. Computed tomography with intravenous contrast revealed partial small bowel obstruction versus less likely ileus. Gastrointestinal pathogen PCR returned positive for norovirus. The patient was given intravenous fluid, nausea control, and pain control, his diet was advanced, and his symptoms subsequently resolved. We believe this case to be unusual, as most cases of viral gastroenteritis are uncomplicated, and this patient presented with radiographic evidence of ileus versus partial small bowel obstruction. PCR testing revealed positivity for norovirus. He had no previous abdominal surgeries or family history of early intestinal malignancies, and the symptoms spontaneously resolved with several days of conservative management, making another etiology much less likely. CONCLUSION AGE is a common diagnosis seen in the primary care clinic, and most patients have an uneventful recovery. However, suspicion of partial obstruction or intestinal ileus should arise when severe abdominal pain and prolonged vomiting are present.

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