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No. of bitstreams: 2 Dissertação - Nathânia Dábilla Alves Silva - 2016.pdf: 1926312 bytes, checksum: 08f15d1c8a44d1ed6118f432a6917f2d (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-04-13 / The norovirus (NoVs) are important viral causative agents of acute gastroenteritis (AGE), affecting individuals of all ages in distinct parts of the word; however, the highest morbi-mortality rates occur mainly in children under five years of age and the elderly. The aim of this study was to to screening NoV by Polymerase Chain Reaction Post Reverse Transcription (RT-qPCR) and real-time (RT-qPCR) in fecal and nasal swab of children up to six years of age, with and without AGE symptoms. Samples were obtained at the Materno Infantil Hospital, from May/2014 to May/2015. Secretor status of children was also determined by enzyme immunoassay and genotyping (FUT2 gene) from the sediment of nasal swab epithelial cells. A global positivity index of 17% (37/219) for NoV in feces, and from these positive children, 48.6% (18/37) had AGE symptoms. Mean viral load in fecal samples was 2.59 x 1010 CG/g from symptomatic and 1.37 x 109 CG/g in asymptomatic. A higher positivity rate (70%) was observed GII NoV, compared to GI NoV (30%) and a higher positivity in children up to 24 month old (67.5%), although not statistically significant. As for the secretor status of children positive for NoV in fecal samples, 94.6% positive secretory status. The NoV were detected in practically every month of the study, and no particular pattern of circulation in relation to dry or rainy seasons was observed. Most children positive for NoV (70%) had the record they have received at least the first dose of the vaccine against Rotavirus, being the highest viral load detected among vaccinated children. The NoV RNA was detected in 8.7% of nasal swab samples of the children and of these, 58% had AGE symptoms. The mean viral load in swab samples from symptomatic children was 2.10 x 108 and in the asymptomatic children was 2.41 x 107 CG/mL. A high NoV genotype variability was found in the study (GI.2, GI.3, GI.5, GII.3, GII.4 and GII.6), with a predominance of GII.4 (28.6%), with this being the first report of NoV GI.5 in Brazil. The data obtained in this study reveal a high frequency, viral load, and genetic variability of NoVs among children attended in a hospital of Goiânia, Goiás. The results are important for a better understanding of NoV epidemiology in nosocomial environment, and may constitute useful information on the advent of the development of an effective vaccine. The viral load in nasal swab samples is a novel data that may contribute for the elucidation of a possible alternative rout of NoV transmission. / Os norovírus (NoVs) são importantes agentes virais causadores de gastroenterite aguda (GEA), atingindo indivíduos de todas as idades em diversas partes do mundo, porém os maiores índices de morbimortalidade ocorrem principalmente nas crianças menores de cinco anos e idosos. O objetivo do presente estudo foi realizar a pesquisa de NoV, através da Reação em Cadeia pela Polimerase Pós-Transcrição Reversa (RT-PCR) e por tempo real (RT-qPCR) em amostras fecais e swab nasal de crianças com até seis anos de idade, com e sem sintomas de GEA. As amostras foram coletadas no Hospital Materno Infantil, entre maio/2014 e maio/2015. Procedeu-se ainda à determinação do status secretor das crianças, por Ensaio Imunoenzimático e genotipagem (gene FUT2), em sedimento de células epiteliais do swab nasal. Foi observado um índice de positividade global de 17% (37/219) para NoV nas fezes, sendo que destas crianças positivas, 48,6% (18/37) apresentavam sintomas de GEA. A carga viral média nas amostras de fezes de crianças com sintomas foi 2,59 x 1010 CG/g e 1,37 x 109 CG/g nas assintomáticas. Observou-se maior positividade para NoV GII (70%) quando comparado ao GI (30%) e maior positividade nas crianças de até 24 meses de idade (67,5%), entretanto este dado não foi estatisticamente significativo. Quanto ao status secretor das crianças positivas para NoV nas fezes, 94,6% status secretor positivo. Os NoV foram detectados em praticamente todos meses do estudo, não sendo observado padrão de circulação definido em relação às estações seca e chuvosa. A maioria das crianças positivas para NoV (70%) tinham o registro de terem recebido pelo menos a primeira dose da vacina contra Rotavírus, sendo a carga viral mais elevada detectada entre crianças vacinadas. O RNA de NoV foi ainda detectado em 8,7% (19/219) das amostras de swab nasal das crianças e destas, 58% apresentavam sintomas de GEA. A carga viral média nas amostras de swab das crianças sintomáticas foi 2,10 x 108 CG/mL e nas assintomáticas 2,41 x 107 CG/mL. Foi observada elevada variabilidade de genótipos de NoV no estudo (GI.2, GI.3, GI.5, GII.3, GII.4 e GII.6), com maior predominância de GII.4 (28,6%), sendo este o primeiro relato de NoV GI.5 no Brasil. Os dados obtidos neste estudo revelam elevada frequência, carga viral e variabilidade genética de NoVs entre crianças atendidas em um hospital de Goiânia, Goiás. Os resultados são importantes para o melhor entendimento da epidemiologia dos NoVs em ambiente nosocomial, e poderão ser uteis como informação no advento do desenvolvimento de uma vacina eficaz. A determinação da carga viral de NoV em xii amostras de swab nasal é um dado novo, podendo este contribuir para a elucidação de uma possível rota alternativa de transmissão dos NoV.

MSc (Microbiology) / Department of Microbiology

Abstract Background: Gastroenteritis is a common infection and the leading cause of morbidity worldwide and is mostly caused by viruses. Outbreaks appear in both developed and developing countries and result in large economic costs. Rapid detection is important for appropriate treatment, control and to prevent the spread of infection.  Objective: Evaluation and performance comparison between the BioFire®FilmArray® Torch System gastrointestinal panel and the Molecular BD MAXTMenteric viral panel to indicate a multiplex method for viral gastroenteritis diagnostic in a routine laboratory setting.  Material and methods: In this study, 58 different samples were used which consisted of selected stool specimens from patients who were tested and treated for gastroenteritis infection at Uppsala Academic Hospital and Norrlands University Hospital in Umeå during 2018-2021, samples from Quality control for molecular diagnostics viral gastroenteritis EQA pilot study during 2018-2019 and cultivated strains of different adenovirus species from 2018. All samples were analyzed with both systems for comparison of detected pathogens.  Results: Sensitivity and specificity values were 95% and 100% respectively for the BioFire®FilmArray®Torch System and 100% and 93.3% for the BD MAXTMSystem.   Conclusions: Bothsystems are rapid and adequate diagnostic tools. The BioFire®FilmArray®Torch System with greater coverage has the ability of detecting more pathogens and is more promising particularly in the occasional infection circumstance. The BD MAXTMSystem demonstrated almost the same results and seems to be a better option in times of an outbreak when the numbers of patients are significantly higher.

No description available.

No description available.

PhD (Microbiology) / Department of Microbiology / Globally, one in ten child deaths before the age of 5 years is due to diarrheal disease, causing almost 800,000 mortalities worldwide, which mostly occur in Sub-Saharan Africa and South Asia. Eighty-eight percent (88%) of diarrheal deaths worldwide are attributable to unsafe water, inadequate sanitation and poor hygiene. Unsanitary environments and poor hygiene practices allow diarrhea causing pathogens including viruses, bacteria and parasites to spread more easily. Norovirus (NoV) are now considered the most common cause of outbreaks of nonbacterial gastroenteritis. However, the factors which control the genetic diversity, the sources of sporadic NoV infections, the transmission and persistence of infection are poorly understood. Limited data are available for NoVs strains in South Africa, especially in rural and peri-urban areas. Despite the excessive burden of diarrhea disease in developing countries, NoVs outbreaks have been to date mostly reported in developed countries. Given that the contribution of the various pathogens to diarrhea may differ substantially between regions depending on local meteorological, geographic, and socio-economic conditions, there is a need to investigate intensively the role of viral agents associated with diarrhea in different settings in Africa continent. How would poor living conditions in rural setting impact the prevalence and genetic characteristics of Norovirus strains circulating Limpopo province is the research question of this study. ix To determine the prevalence and genetic diversity of NoVs strains circulating in the rural communities in the Limpopo Province, South Africa and investigate the genetic relationship between NoVs strains, a cross-sectional study was performed on human stools collected from rural communities. We used qualitative variables of poor living environmental conditions including type of water used at the household of child’s parent or guardian, use of toilet seat, presence of livestock at the household and parent employment status to assess possible environmental risk factors of NoV infection within the study area. Prior to this prospective study, we conducted a systematic review of the PubMed and EMBASE databases for published articles of Human NoVs in Africa between 1990 and 2013 in order to assess the contribution of Human NoVs to diarrhoeal diseases in Africa. This review provides a picture of Human NoVs studies in Africa and reveals that unreported sporadic gastroenteritis cases of Human NoVs are common in Africa. Most are community-associated infections reported from urban settings. Possible environmental transmission routes have been documented. Combined environmental and clinical studies are required for targeted actions to control transmission of Human NoVs in Africa. Between July 2014 and April 2015, outpatient children under 5 years of age from rural communities of Vhembe district, South Africa, were enrolled for the study. A total of 303 stool specimens were collected from those with diarrhea (n=253) and without (n=50) diarrhea. NoVs were identified using real-time one-step RT-PCR. Nucleotide sequencing methods were performed to genotype the strains. Phylogenetic analyses x were performed to compare identified NoVs genotypes to the worldwide circulating strains. One hundred and four (41.1%) NoVs were detected. NoV detection rates in symptomatic and asymptomatic children (OR = 1.24; 95% CI 0.66 – 2.33) were not significantly different. Comparison of the median CT values for NoV in symptomatic and asymptomatic children revealed significant statistical difference of estimated GII viral load from both groups, with a much higher viral burden in symptomatic children to our knowledge this is the first study reporting on the differences in estimated viral load of GII and GI NoV positive cases and controls. The study findings may have implications for the diagnosis of NoV disease and future vaccine development, which may only need to consider GII as the genogroup associated with diarrhea in the South African population. Sequence analyses demonstrated multiple NoV genotypes identified in rural communities of Vhembe district. The most prevalent NoV genotypes were GII.4 Sydney 2012 variants (n=7) among the capsid genotypes, GII.Pe (n=9) among the polymerase genotypes and GII.Pe/GII.4 Sydney 2012 (n=8) putative recombinants among the RdRp/Capsid genotypes. Two unassigned GII.4 variants and an unusual RdRp genotype GII.P15 were found. With note, the rare GII.P15 identified in this study, has a common ancestor with GII.P15 strain from Japan previously reported as GII / untypeable recombinant strain implicated in a gastroenteritis outbreak. To our knowledge this is the first report of this unusual genotype in the African continent. Though not proven predictive of diarrhea disease in this study, the high detection rate of NoV reflects the substantial exposure of children from rural communities to enteric xi pathogens possibly. However in this study no risk factor has been found between NoV positive and qualitative environmental variables of poor living conditions in rural setting. The results also suggest that the difference between asymptomatic and symptomatic children with NoV may be at the level of the viral load of NoV genogroups involved. The findings highlighted NoV genetic diversity and revealed continuous pandemic spread and predominance of GII.Pe/GII.4 Sydney 2012, indicative of increased NoV activity. An unusual RdRp genotype GII.P15 and two unassigned GII.4 variants were also identified from rural settings of the Vhembe district/South Africa. NoV surveillance / NRF

Aptamerer är nukleinsyra-baserade molekyler som binder specifikt till en målstruktur. Aptamerer har flera fördelar över antikroppar, så som snabb och billig framtagning och produktion. Multimera aptamer-baserade strukturer har visats ge bättre resultat än monomera aptamerer, men framtagningen av sådana strukturer är tidskrävande och icke-skalbar. Denna studie utforskar generering och selektion av multimera aptamer-baserade strukturer genom slumpmässig ligering och in vitro-evolution, med virusliknande partiklar av norovirus GII.2 och GII.4 som målstruktur. Optimering av ligeringsförhållanden visade att en större andel aptamerer, flerarmade fragment, och linjära fragment resulterade i störst diversitet i den ursprungliga strukturblandingen. Selektionsexperiment uppvisade kraftig positiv selektion för strukturer innehållande aptameren Buf-2, vilket indikerar att den har hög affinitet för båda genotyper. Aptameren SMV21 uppvisade också positiv selektion för båda genotyper. Studien finner också positiv selektion av multimera aptamer-baserade strukturer, vilket bekräftar att de binder starkare till sin målstruktur. Potentiella sekvenser med hög affinitet togs fram genom att generera konsensus-sekvenser från sekvenseringsdatan av de selekterade strukturerna. SPR användes för att mäta affiniteten av de selekterade strukturerna till norovirus, men på grund av ospecifik binding kunde inga slutsatser dras. / Aptamers are nucleic acid-based targeted binders that hold advantages over antibodies, such as cheap and fast development and production. Multimeric aptamer-based structures have shown improved performance compared to monomeric aptamers, but the development of such structures is time-consuming and unscalable. This study explores the generation and selection of multimeric aptamer-based structures through random ligation and in vitro evolution, targeting norovirus GII.2 and GII.4. Optimization of ligation conditions was performed, revealing that a bigger proportion of aptamers, multi-armed fragments, and linear fragments ensures a diverse initial structure pool. Selection experiments demonstrated a strong positive selection for structures containing the Buf-2 aptamer, indicating its high affinity for both norovirus genotypes. The SMV21 aptamer also showed positive selection for both genotypes. The study further found that multimeric aptamer-based structures experience positive selection, confirming their stronger binding to the desired target. Potential strong-binding sequences were obtained by generating consensus sequences from sequencing data of the selected strong binders. SPR was employed to determine the affinity of the selected binders to the norovirus, but the results were inconclusive due to unspecific binding.

Les norovirus sont l'une des causes principales de gastroentérite. Depuis 2002, des variants de norovirus GII.4 successifs ont circulé dans la population par cycle de 2-3 ans, ce qui suscite des interrogations quant au rôle de leurs ligands, les antigènes tissulaires de groupes sanguins (HBGA), dans leur évolution. Nous avons analysé l'interaction entre des variants de GII.4 représentatifs et des HBGA, et déterminé le rôle d'acides aminés (aa) clés. Par mutagénèse dirigée, nous avons montré qu'une configuration stricte des aa directement impliqués dans l'accroche est indispensable. La suppression de la thréonine 395, caractéristique des variants après 2002, confère la capacité de se lier à Lex et Si-Lex, démontrant que les aa en dehors du site de liaison peuvent modifier les propriétés d'attachement. L'analyse de l'accroche de VLP de 6 variants isolés de 1987 à 2007 à des échantillons de salive phénotypés et des HBGA synthétiques montre que tous les variants sont capables de s'attacher à la salive des sécréteurs indépendamment du phénotype ABO et aux oligosaccharides propres au phénotype sécréteur. Deux variants récents ont pu également s'accrocher aux sucres présents dans la salive des nonsécréteurs Le(+). Nos données suggèrent que la capacité de se lier à Lex et Si-Lex serait une conséquence de la variation génétique des aa situés à proximité du site de liaison. L'analyse des propriétés d'attachement par résonance plasmonique de surface a montré que seuls les variants après 2002 présentent une affinité forte pour les antigènes A et B, suggérant que l'accélération évolutive des GII.4 pourrait être liée à une affinité accrue des variants pour les HBGA après 2002.

Norovirus (NoV) is a significant cause of gastroenteritis globally, and the consumption of oysters is frequently linked to outbreaks. Depuration is the principle means employed to reduce levels of potentially harmful agents or toxins in shellfish. The aim of this thesis is to construct mathematical models which can describe the depuration dynamics of water-borne pathogens, and specifically examine the dynamics of NoV during depuration for a population shellfish. Legislation is currently under consideration within the EU by the Directorate-General for Health and Consumers (DG SANCO) to limit the maximum level of NoV that consumers are exposed to via this route. Therefore it is important to the utility of the thesis that any models constructed should incorporate control measures which could be used to implement minimum NoV levels. Doing so allowed calculation of minimum depuration times that would be required to adhere to the control measures incorporated into the models. In addition to modelling the impact on pathogens during the depuration, we wished to gain some insight into how the variability, and not just the mean levels, of water-borne pathogens can be as important with respect to the length of depuration required to minimise any food safety risks to the consumer. This proved difficult in the absence of any data sets that can be used to calculate variability measures, as little data is currently available to inform these values for NoV. However, our modelling techniques were able to calculate an upper limit on the variability of water-borne pathogens that can be well approximated by lognormal distributions. Finally we construct a model which provided linkage between the depuration process and the accretion of pathogens by shellfish while still within farming waters. This model proposed that the pulses of untreated waste waters released by sewage treatment works due to high levels of rainfall would be transmitted into shellfish whilst filter-feeding.

Os norovírus (NV) são uma importante causa de hospitalização infantil. Crianças internadas por gastroenterite por NV (GENV) são consideradas portadoras de diarreia grave. O objetivo desse estudo, realizado na cidade do Rio de Janeiro, Brasil, é descrever as características clínicas e a frequência da diarreia por NV em crianças hospitalizadas, comparando as taxas de detecção de NV em crianças vacinadas e não vacinadas contra rotavírus (Rotarix). Foram coletadas 659 amostras de fezes de igual número de crianças e encaminhadas para análise pela reação em cadeia pela polimerase, precedida de transcrição reversa no período de janeiro de 2004 a dezembro de 2009. O percentual de amostras positivas para os NV foi de 27,3% nesse período. Das 180 amostras positivas para NV, 55% tiveram origem na comunidade (aqCo) e 45% foram de aquisição nosocomial (aqNo). O percentual de GENV nos dois anos anteriores (2004 e 2005) à introdução da vacina Rotarix foi de 28,3%, sendo 11,3% o percentual de amostras aqCo. Nos dois anos posteriores (2008 e 2009), a GENV significou 24,4%, e as amostras aqCo foram 14,9% (p<0,05). Em 647 crianças, 494 não receberam a vacina Rotarix, enquanto 151 crianças receberam, pelo menos, uma dose. O percentual de GENV foi de 23,8% e 39,7%, respectivamente (p<0,05). Apesar do comportamento sazonal dos casos de GENV aqCo, esse fato não teve significância estatística. Das 180 crianças, 61,6% tinham peso &#8804; p10 do NCHS, 82,2% tinham idade &#8804; 5anos. As crianças com idade &#8804; 2 anos foram mais acometidas nos casos de aqCo do que àquelas de aqNo (p<0,05). Foram observados em 82 crianças: vômitos (73,2%), febre (54,9%), tosse (20,7%), coriza (2,2%), sangue nas fezes (8,5%), erupção cutânea (4,9%) e broncoespasmo (7,3%). Houve significância estatística com relação à frequência maior de febre, coriza, tosse e broncoespasmo nas crianças com GENV de aqCo do que naquelas de aqNo (p<0,05). De 69 crianças, 73,9% apresentaram desidratação e, dessas, 76,5% necessitaram de hidratação venosa. Esses dados tiveram significância estatística, representada por maiores percentuais nas crianças com GENV de aqCo do que naquelas de aqNo (p<0,05). Esse estudo demonstra que os NV foram um importante agente etiológico nos casos de gastroenterites em crianças hospitalizadas e responsável por altas taxas de infecções nosocomiais. Estatisticamente, não foi comprovada uma tendência de aumento dos casos de GENV no período do estudo, como também do aumento da frequência de GENV nos anos posteriores em relação aos anos anteriores à introdução da vacina Rotarix no Brasil em 2006. No entanto, houve significância estatística quando foi avaliado o percentual de GENV em crianças hospitalizadas vacinadas e não vacinadas contra RV. Um aumento dos casos de GENV em crianças poderá vir a acontecer nos próximos anos, quando é esperado que um número maior de crianças será vacinado contra RV. Tosse, coriza e broncoespasmo são sintomas que devem ser mais detalhadamente investigados. Estratégias de prevenção contra a disseminação dos NV são condutas importantes em unidades de internação. Uma vacina eficaz contra norovírus pode ser um benefício significativo para reduzir o percentual de crianças hospitalizadas por diarreia. / Noroviruses (NV) are a major cause of infant hospitalization. Children hospitalized for gastroenteritis NV (NVGE) are considered to have severe diarrhea. The purpose of this study, conducted in the city of Rio de Janeiro, Brazil, is to describe the clinical characteristics and frequency of norovirus diarrhea in hospitalized children, comparing the rates of detection of NV in vaccinated and unvaccinated children against rotavirus (RV). We collected 659 fecal samples from equal numbers of children and sent for analysis by polymerase chain reaction, reverse transcription from January 2004 to December 2009. The percentage of samples positive for NV was 27.3% in this period. Of the 180 samples positive for NV, 55% meant source community (Coaq) and 45% of nosocomial acquisition (Noaq). The percentage of NVGE the previous two years (2004 and 2005) the introduction of the vaccine against RV was 28.3% and 11.3% represented the percentage of samples Coaq. In the two subsequent years (2008 and 2009), NVGE meant to 24.4%, and the samples were Coaq 14.9% (p <0.05). In 647 children, 494 received no vaccine against RV while 151 children received at least one dose. The percentage of NVGE was 23.8% and 39.7%, respectively (p <0.05). Despite the seasonal behavior of NVGE aqCo cases, this did not reach statistical significance. Of the 180 children, 61.6% had weight &#8804; p10 NCHS, 82.2% were aged &#8804; 5 years old. Children aged &#8804; 2 years were most affected in cases of Coaq than those of Noaq (p <0.05). Were observed in 82 children: vomiting (73.2%), fever (54.9%), cough (20.7%), coryza (2.2%), blood in stools (8.5%), rash (4.9%) and bronchospasm (7.3%). There was statistical significance with respect to the higher frequency of fever, coryza, coughing and wheezing in children with NVGE of Coaq than those of Noaq (p <0.05). Of 69 children, 73.9% had dehydration and of these, 76.5% required intravenous hydration. These data were statistically significant, represented by the highest percentage of children with NVGE of Coaq than those of Noaq (p <0.05). This study demonstrates that the NV were an important etiologic agent in cases of gastroenteritis in hospitalized children and responsible for high rates of nosocomial infections. Statistically, , it was not shown a tendency to increase in cases of NVGE during the study period, as well as the increased frequency NVGE in later years relative to years prior to the introduction of RV vaccine in Brazil. However, statistical significance was assessed as the percentage of NVGE in hospitalized children vaccinated and unvaccinated against RV. An increase in cases of children in NVGE could happen in the next few years, when it is expected that a greater number of children will be vaccinated against RV. Cough, coryza and wheezing are symptoms that should be further investigated. Strategies for preventing the spread of NV are important conduits in inpatient units. An effective vaccine against norovirus can be a significant benefit to reduce the percentage of children hospitalized for diarrhea.