INTRODUÇÃO: A doença diarreica aguda é a segunda causa de morte em crianças abaixo de 5 anos de idade. No Brasil, entre 2003 e 2009, a diarreia aguda foi responsável por cerca de 100.000 internações por ano, e por 4% das mortes em crianças abaixo de 5 anos de idade. O rotavírus é a principal etiologia de diarreia aguda grave no mundo todo, sendo responsável por 40% das internações por diarreia aguda, e 29% de todas as mortes por diarreia aguda. A vacina monovalente (RV1) contra o rotavírus foi introduzida no Programa Nacional de Imunizações em 2006. OBJETIVOS: Verificar o impacto da vacina monovalente contra rotavírus nas consultas de pronto-socorro e internações por doença diarreica aguda em crianças menores de 5 anos de idade, verificar a positividade do exame \"pesquisa de rotavírus nas fezes\", e verificar a presença ao ausência de imunidade de rebanho. METODOLOGIA: Foi realizado um estudo ecológico retrospectivo no Hospital Universitário da Universidade de São Paulo. O período foi dividido em pré-vacina (2003 a 2005) e pós-vacina (2007 a 2009). Foram incluídas todas as crianças abaixo de 5 anos que passaram em consulta no pronto-socorro e verificado o diagnóstico do atendimento e internação através de registro eletrônico. Foram obtidas as taxas de consultas no pronto-socorro e internações por doença diarreica aguda, foram selecionadas as crianças não vacinadas para cálculo da imunidade de rebanho, e verificado se houve coleta do exame pesquisa de rotavírus nas fezes. A redução nas taxas foi obtida através da fórmula: redução (%) = (1 - odds ratio) x 100. RESULTADOS: No período pré-vacina a taxa de consultas por diarreia aguda foi de 85,8 consultas por 1.000 consultas gerais, no período pós vacina a taxa de consultas por diarreia aguda foi 80,9 por 1.000, e a redução foi 6% (IC 95%, 4% a 9%, p < 0,001), chegando a 40% (IC 95%, 36% a 44%, p<0,001) nos meses de maio e junho. A taxa de internação por diarreia aguda era 40,8 internações por 1.000 e caiu para 24,9 por 1.000, redução de 40% (IC 95%, 22% a 54%, p < 0,001), chegando a 82% (IC 95%, 62% a 92%, p < 0,001) nos meses de maio e junho. Nas crianças não vacinadas não houve redução na taxa de consultas de pronto-socorro (IC 95%, -4% a 5%, p=0,903), e não se pode afirmar se houve redução ou aumento das internações por diarreia aguda (IC 95%, -212% a 35%, p=0,381). Houve queda da positividade do exame pesquisa de rotavírus em 2009 (redução de 70%, IC 95%, 26% a 88%, p=0,007). CONCLUSÕES: Após a introdução da vacina contra rotavírus (RV1) houve uma redução de 6% nas consultas por diarreia aguda no pronto-socorro, de 40% nas internações por diarreia aguda e de 70% na positividade do exame pesquisa de rotavírus nas fezes. Não foi detectada imunidade de rebanho / INTRODUCTION: Acute diarrheal disease is the second cause of death in children under five years. In Brazil, from 2003 to 2009, acute diarrhea was responsible for nearly 100,000 deaths per year and 4% of the deaths in children under five years. Rotavirus is the leading cause of severe acute diarrhea worldwide, accounting for 40% of hospital admissions and 29% of all the acute diarrhea-related deaths. In 2006, the rotavirus monovalent vaccine (RV1) was added to the Brazilian National Immunization Program. OBJECTIVES: To analyze the impact of the RV1 on Emergency Department (ED) visits and hospital admissions for acute diarrhea, the rates of positivity of the stool rotavirus tests and the presence or absence of herd immunity. METHODS: A retrospective ecologic study at the University Hospital, University of Sao Paulo. The study analyzed two periods: the pre-vaccine (2003-2005) and the post-vaccine (2007-2009) periods. We screened the main diagnosis of all ED attendances and hospital admissions of children under five years in an electronic registry system database and calculated the rates of ED visits, hospital admissions and positivity of the stool rotavirus test in patients with acute diarrhea. Herd immunization was evaluated in non-vaccinated children. The reduction rate was analyzed according to the formula: reduction (%) = (1- odds ratio) x 100. RESULTS: The rates of ED visits for acute diarrhea was 85.8 and and 80.9 per 1,000 total ED visits in the pre and post vaccination periods, respectively, resulting in 6% reduction (95% CI, 4% to 9%, p< 0.001) in the overall period and reaching 40% reduction on May and June. The rates of hospital admissions for acute diarrhea was 40.8 per 1,000 in the pre-vaccine period and dropped to 24.9 per 1,000 hospitalizations, resulting in 40% reduction (95% CI, 22% to 54%, p < 0.001) in the overall period and 82% (95% CI, 62% to 92%, p < 0.001) on May and June. Among non-vaccinated children, no reduction on ED visits was observed (95% CI, -4% to 5%, p=0.903), while an increase or reduction in the hospitalization rates could not be determined (95% CI, -212% to 35%, p=0.381). There was a decrease in the positivity of the stool rotavirus tests in 2009 (70% reduction, 95% CI, 26% to 88%, p=0.007). CONCLUSIONS: The introduction of the RV1 vaccine resulted in 6% reduction in the ED visits and 40% reduction in hospital admissions for acute diarrhea, and 70% reduction in the rates of positive stool rotavirus tests. No herd immunity was observed

Submitted by Maria Creuza Silva (mariakreuza@yahoo.com.br) on 2014-10-03T17:40:16Z No. of bitstreams: 1 Tese Maria Yury Ichihara. 2014.pdf: 2248119 bytes, checksum: 821a02dd8be59ca78df05111c9fdd559 (MD5) / Approved for entry into archive by Maria Creuza Silva (mariakreuza@yahoo.com.br) on 2014-10-07T13:52:13Z (GMT) No. of bitstreams: 1 Tese Maria Yury Ichihara. 2014.pdf: 2248119 bytes, checksum: 821a02dd8be59ca78df05111c9fdd559 (MD5) / Made available in DSpace on 2014-10-07T13:52:13Z (GMT). No. of bitstreams: 1 Tese Maria Yury Ichihara. 2014.pdf: 2248119 bytes, checksum: 821a02dd8be59ca78df05111c9fdd559 (MD5) / A diarréia é uma das causas mais freqüentes de atendimentos ambulatoriais e de hospitalização em menores de 5 anos. Bactérias e o rotavírus são os principais agentes etiológicos envolvidos nas diarréias graves, sendo o rotavírus responsável por 22% a 38% das admissões hospitalares. Para abordar o tema sobre a internação de crianças brasileiras menores de 2 anos devido a diarréia foram realizados três estudos casos-controles com base hospitalar. Inicialmente, foi estimada a associação dos fatores de risco e a internação por diarréia aguda (exceto àquela causada por rotavírus) de acordo com as rotas de transmissão dos agentes etiológicos, as várias fontes de infecção e as condições de vida das populações. Foi demonstrado que os principais fatores de risco associados à internação por diarréia foram a falta de esgotamento sanitário e de água de boa qualidade e ter uma ou mais internações prévias devido à diarréia. Em relação à diarréia aguda causada por rotavírus, a OMS recomenda o uso de duas vacinas licenciadas no mundo (Rotarix® e RotaTeq®). A vacina oral monovalente contra rotavirus (G1P[8], Rotarix®) foi introduzida no Programa Nacional de Imunização do Brasil em 2006. A eficácia e efetividade da vacina variam entre países com renda alta e baixa, embora exista forte evidência de proteção cruzada para os genótipos G1-G4 e G9. Avaliamos a efetividade global e genótipo-específica da vacina oral monovalente na prevenção de internação de crianças brasileiras com diarréia causada por rotavirus. Além disso, estimamos a efetividade da vacina global e genótipo-específica por tempo de vacinação após a segunda dose da vacina (até dois anos) e EV para as Regiões brasileiras. Elevadas efetividades geral e genótipo-específica da vacina foram observadas, mesmo num contexto de grande diversidade genotípica e com predominância do genótipo G2P[4]. A duração da proteção global e genótipo-específica da vacina permaneceu até dois anos e foi maior para G1P[8] do que para G2P[4]. Por outro lado, consideramos plausível que a EV poderia variar em diferentes populações e em diferentes períodos de tempo, mediante a grande diversidade genotípica, a ocorrência de genótipos incomuns, de combinações mistas de G e P e de emergência de novas cepas advindas de combinações inter-espécies (homem e animal). Analisamos a EV estratificada por Regiões brasileiras e ficou demonstrado que a EV para a Região Norte foi similar à EV global. Porém a EV para as outras Regiões foi menor, talvez devido ao pequeno número de casos. Baseado nos resultados dos estudos nós recomendamos: 1) implementar ações voltadas para o domínio público (ambiente, saneamento, higiene na comunidade e acesso a serviços de saúde) para reduzir a morbidade por diarréia; 2) a continuidade do uso da vacina oral monovalente no Programa Nacional de Imunização; e 3) o monitoramento de genótipos para detecção precoce de cepas novas e incomuns. Além disso, novos estudos precisam ser conduzidos para avaliar variações da efetividade da vacina entre as Regiões, as sub-regiões e as áreas mais vulneráveis do Brasil. Será importante realizar estudos de custo-efetividade para subsidiar a política nacional de imunização. / Diarrhea has been a frequent reason of visits to the health services and hospitalization among children under five. Bacteria and rotavirus are the main agents involved in severe diarrhea, in which rotavirus is responsible from 22% to 38% of children hospital admissions. To address the issue of hospitalization of Brazilian children under 2 years due to diarrhea, we conducted three hospital based case-control study. Initially, we aimed to estimate the association of risk factors and acute diarrhea hospitalization (except those caused by rotavirus) according to the routes of transmission of etiologic agents, the various sources of infection and the living conditions of populations. It was demonstrated that the main risk factors were lack of sewage and water of good quality, and already having one or more hospitalizations due to diarrhea. In relation to the rotavirus acute diarrhea, the World Health Organization has been recommended the use of two licensed vaccines worldwide (Rotarix ® and RotaTeq ®). The oral monovalent rotavirus vaccine (G1[P8] strain, Rotarix®) was introduced in Brazilian National Immunization Program in 2006. The vaccine efficacy and effectiveness vary between high and low income countries, although there is strong evidence of cross-protection for G1-G4 and G9 genotypes. We evaluated overall and genotype-specific oral monovalent rotavirus VE in preventing RV-A diarrhea hospital admission of Brazilian children. Also, we estimated overall and genotype-specific VE by time since second dose vaccination (up to two years) and VE according to Brazilian Regions. High overall and genotype-specific VE were observed, even though there was a great diversity of rotavirus genotypes circulating in Brazil and a predominance of G2P[4] genotype. The overall and genotype-specific VE lasted for two years after second dose vaccination and it was higher for G1P[8] than G2P[4]. Besides, we considered that it was plausible that RV-A VE could vary in different populations (Regions) and in different periods of time, since there was a great genotype diversity, an occurrence of unusual genotypes, mixed combinations of G and P and emergence of new strains from combinations of inter-species (human and animal). We analyzed the VE for Brazilian Regions and we demonstrated that the VE for Northern Region was similar to the overall VE. However, the VE for other Regions was lower than VE for Northern Region, maybe because of the small number of the cases. Based on the findings of the studies we recommend: 1) to implement actions of the public domain (environment, sanitation, hygiene in the community and access to health services) to reduce the diarrhea morbidity; 2) the continued use of oral monovalent rotavirus vaccine in the National Immunization Program; and 3) the monitoring for early detection of unusual and novel rotavirus genotypes. In addition, new studies should be conducted to evaluate the variations of rotavirus VE in different Regions, sub-Regions and vulnerable areas in Brazil. It might be useful to conduct cost-effectiveness studies to inform national immunization policy.

Um total de 8.961 amostras fecais coletadas de pacientes com diarréia aguda, no período de 1996 a 2006, no Estado de São Paulo foi testado para rotavírus por EIE. Destas, 20,0% foram positivas e posteriormente realizadas a caracterização dos rotavírus em genótipos G e P por nested RT-PCR. O genótipo G1 de rotavírus foi o mais freqüente, detectado em 35,2% das amostras, seguidos dos tipos G9, G2, G3, G4, infecção mista e G12. A associação mais freqüente foi a P[8]G1 e P[8]G9. Foi realizada a sequencia nucleotídica do gene 9 (VP7) de 38 rotavirus genótipo G1. Duas cepas foram analisadas dos anos de 1997, 1998, 2001 e 2002, três cepas dos anos 1996, 1999 e 2003, quatro cepas em 2000, sete cepas em 2004 e 2005, e cinco em 2006. Os 38 rotavírus G1 foram classificados em duas linhagens distintas, linhagem G1-I e linhagem G1-II. A linhagem G1-I foi detectada durante seis anos, 1996-1997, 2001-2002 e 2004-2006, e a linhagem G1-II foi detectada durante os anos de 1998-2001, e 2003-2005. Análises preliminares mostraram que Rotarix ® foi eficiente contra estas linhagens G1. / Rotavirus (RV) infections are recognized as a major cause of severe gastroenteritis in children worldwide. In March 2006, a monovalente P[8]G1, human RV vaccine (Rotarix® GlaxoSmithKline Biologicals) was introduced in Brazil into the routine childhood immunization schedule. Therefore, the study of genetic diversity among rotavirus strains before and after the introduction of this vaccine may be important for the development of vaccination strategies. A total of 8,961 fecal samples collected from patients with acute diarrhea, during the 11-year period surveillance in São Paulo State (1996 to 2006) were tested for rotavirus by ELISA. One thousand seven hundred eighty- four (1,784, 20.0%) were positive, and the characterization of the G and P genotypes was performed on 1,300 rotavirus samples by nested RT-PCR. The G1 type was the most prevalent rotavirus strain (35.2%). The second most prevalente was the G9 type (31.2%), followed by G2 (4.0%), G3 (3.5%), G4 (2.2%), mix infection (1.8%) and G12 (0.5%). The more frequent association was P[8]G1 and P[8]G9. We performed a sequence analysis of 38 P[8]G1 rotavirus strains, selected from a total of 341 P[8]G1.Two strains from 1997, 1998, 2001, and 2002 were analyzed; three strains from 1996, 1999, and 2003; four strains from 2000; seven strains from 2004, and 2005; and five strains from 2006. All 38 rotavirus G1 sequence in this study were found to be classified into two distinct lineages, lineage I with 44.7% (17/38) and lineage II with 55.3% (21/38). The G1I lineages were detected during six rotavirus seasons 1996-1997, 2001- 2002, and 2004-2006 whereas and lineage G1- II was detected during 1998-2001, and 2003-2005. Preliminary analyses 4 demonstrated that Rotarix® has been efficacious against these G1 lineages.

Os rotavírus são um dos principais agentes causadores de doenças entéricas em várias espécies animais, com ocorrência generalizada na suinocultura do Brasil. O seu diagnóstico é um componente essencial para estudos epidemiológicos e delimitação de medidas profiláticas visando o controle da doença. Apesar da relevância, não existem testes, tais como PCR em tempo real desenvolvido para detectar a diversidade genética de rotavírus suíno do grupo A (RVA). Este trabalho descreve o desenvolvimento de uma PCR em tempo real com SYBR® Green, para a detecção de rotavírus suíno e os seus resultados comparados com a PCR convencional e ELISA. Foram desenhados primers visando o segmento codificador da proteína NSP5 (137pb) e também foram utilizados primers visando o mRNA do gene mitocondrial bovino NADH5 (191pb) para o controle interno exógeno. Amostras de fezes de suínos de até 60 dias de idade de suínos do estado de São Paulo foram usadas para compor um painel de teste. Foram utilizadas como amostras de referencia o isolado 32/00 (controle positivo de rotavírus suíno do grupo A), concentrado de células MDBK (controle positivo do controle interno exógeno) e água tratada com DEPC (controle negativo). A extração do RNA total foi realizado com Trizol a partir de suspensões fecais contendo MDBK e o cDNA foi sintetizado utilizando primers aleatórios e M-MLV. Para as reações de PCR em tempo real utilizou-se o reagente MaximaTM SYBR Green qPCR Master Mix (Fermentas Life Science). Durante a padronização da PCR convencional, a temperatura de 54°C foi definida como a Tm ótima para a reação. O desempenho do ensaio foi validado em sete amostras positivas inicialmente testadas pelos métodos ELISA e PAGE. O isolado viral RV8209 foi utilizado para determinar o limiar de detecção da PCR em tempo real através de diluições seriadas sendo defino como ponto de corte Ct=33,5 (10-12,28TCID50%). O segmento codificador da NSP5 foi clonado no vetor pTZ57R/T submetido a restrição enzimática e usado como alvo para gerar uma curva padrão, onde obteve-se uma eficiência de 93,39%, slope de -3,49 e R2 de 0,993. A detecção do controle interno exógeno mostrou 82,9% de positividade para PCR convencional e 76,31% para a PCR em tempo real, com correlação significativa (0,718). O ensaio de ELISA para RVA apresentou 10,5% (8/78) de positividade, enquanto que as taxas de detecção da PCR em tempo real e PCR convencional foram de 50% (29/58) e 30,1% (24/63), respectivamente. Foi encontrada uma correlação moderada (0,546) entre PCR convencional e PCR em tempo real; baixa (0,056) entre a PCR convencional e ELISA; ausente (0,0) entre a PCR em tempo real e ELISA. Os resultados obtidos sugerem que a detecção por PCR em tempo real para a detecção do rotavírus suíno do grupo A em amostras fecais possa ser utilizada como diagnostico rápido e eficiente aumentando o repertório dos testes já estabelecidos, de modo a proporcionar uma maior sensibilidade para o diagnóstico clínico e epidemiológico. / Rotavirus is one of the main causative agents of enteric diseases in several animal species with widespread occurrence in Brazilian pig farm. Diagnosis is an essential component for epidemiological studies and delineation of prophylactic measures aiming disease control. Despite the relevance, there are no assays such as real-time PCR developed to detect the genetic diversity of porcine rotavirus from group A (RVA). This work describes the development of SYBR Green real-time PCR assay for the detection of porcine rotavirus and the results were compared with conventional PCR and ELISA. Primers were designed targeting the coding segment of the protein NSP5 (137pb) and also primers targeting the bovine mitochondrial gene mRNA NAD5 (191pb) were used for the exogenous internal control. Fecal samples from pigs up to 60 days of age from São Paulo state were used to compose a panel test. The reference sample was the isolate 32/00 (positive control for porcine rotavirus group A), concentrated MDBK cells (Exogenous Internal Positive Control) and DEPC-treated water (negative control). Total RNA extraction from supernatants of fecal samples containing MDBK cells were carried out with TRIzol reagent and cDNA was synthesized using random primers and M-MLV Reverse Transcriptase. For real-time PCR reactions were used MaximaTM SYBR Green qPCR Master Mix (Fermentas Life Science). For conventional PCR optimization, 54oC was defined as the optimum reaction temperature (Tm). The performance of the assay was validated on seven samples initially tested positive by ELISA and PAGE methods. The limit of detection of the developed real-time-PCR assay was determined using serial dilutions of the isolated RV8209 with Ct=33,5 (10-12,28TCID50%). The NSP5 gene segment was cloned into vector pTZ57R/T submitted to enzymatic restriction and used as template to generate a standard curve which Efficiency of 93.39%, slope of -3.49 and R2 &#9633; 0.993. The Exogenous internal control showed 82.9% positivity for conventional PCR and 76.31% for real-time PCR with substantial correlation (0.718). The ELISA assay detected porcine RVA in 10,5% (8/78) of fecal samples, whereas the detection rates of both SYBR Green real-time PCR and conventional PCR assays were 50% (29 of 58) and 30.1% (24 of 63), respectively. A moderate correlation (0.546) was found between conventional PCR and real-time PCR; low (0.056) between the conventional PCR and ELISA; absent (0.0) between the real-time PCR and ELISA. Our findings suggest that detection of Group A porcine rotavirus in fecal samples by use of the real-time PCR assay may be fast and efficient increasing the repertoire of tests established to improve sensitivity for epidemiology and clinical diagnosis.

No description available.

No description available.

Rotavirus infection causes gastroenteritis, specifically severe gastroenteritis, affecting children younger than five globally, regardless of hygiene and water quality. Current licensed, live, attenuated vaccines do not contain the G9 genotype, which is a prevalent rotavirus strain circulating in sub-Saharan Africa, a region that carries a high rotavirus disease burden. Rotavirus-like particles (RV-VLPs) is an attractive non-live vaccine candidate, which has shown promising results in animal studies. Previously, dsRNA was extracted from a stool sample containing a South African human G9P[6] neonatal strain, and amplified cDNA using a sequence-independent procedure. The consensus sequence was obtained for the genome segments using 454® pyrosequencing. The insect-cell-codon-optimized genome segments 2 (VP2), 4 (VP4), 6 (VP6) and 9 (VP7) were cloned into a modified pFASTBACquad vector (pFBq). Several combinations of the genome segments were cloned to produce double-layered particles (DLP; pFBqVP2VP6) or triple-layered particles (TLP; pFBqVP2VP6VP7). In the current study, a ΔTLP (pFBqdVP2-VP8*VP6VP7) construct was generated. The first 92 amino acids of VP2 are not necessary for the conformation of recombinant RV-VLPs. The ORF of VP8*, which contains immune important epitopes, was fused to the 5’ end of the dVP2 coding region resulting in a dVP2-VP8* fused protein which was expressed in the presence of VP6 and VP7 to produce ΔTLPs. The Bac-to-Bac® Baculovirus Expression System and Spodoptera frugiperda (Sf) 9 insect cells were used for expression. All the proteins were successfully expressed. VP2, VP6, VP4 and the dVP2-VP8* fused protein were visible on Coomassie stained SDS-PAGE. Expression of VP7 could only be confirmed with western blot analysis. Particle formation, as assessed by transmission electron microscopy (TEM), was observed for DLPs. No TLPs of dVP2-8*/6/7 or VP2/6/7 were visualized due to the lower expression level of VP7 and the lack of calcium supplements during the assembly process. In conclusion, it was possible to produce RV-DLPs derived from the consensus sequence determined for a G9P[6] rotavirus directly from stool without prior propagation in cell culture or virus isolation. This strain contains both the G9 and P[6] genotypes that are currently prevalent in sub-Saharan Africa. / Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2013.

Rotavirus infection causes gastroenteritis, specifically severe gastroenteritis, affecting children younger than five globally, regardless of hygiene and water quality. Current licensed, live, attenuated vaccines do not contain the G9 genotype, which is a prevalent rotavirus strain circulating in sub-Saharan Africa, a region that carries a high rotavirus disease burden. Rotavirus-like particles (RV-VLPs) is an attractive non-live vaccine candidate, which has shown promising results in animal studies. Previously, dsRNA was extracted from a stool sample containing a South African human G9P[6] neonatal strain, and amplified cDNA using a sequence-independent procedure. The consensus sequence was obtained for the genome segments using 454® pyrosequencing. The insect-cell-codon-optimized genome segments 2 (VP2), 4 (VP4), 6 (VP6) and 9 (VP7) were cloned into a modified pFASTBACquad vector (pFBq). Several combinations of the genome segments were cloned to produce double-layered particles (DLP; pFBqVP2VP6) or triple-layered particles (TLP; pFBqVP2VP6VP7). In the current study, a ΔTLP (pFBqdVP2-VP8*VP6VP7) construct was generated. The first 92 amino acids of VP2 are not necessary for the conformation of recombinant RV-VLPs. The ORF of VP8*, which contains immune important epitopes, was fused to the 5’ end of the dVP2 coding region resulting in a dVP2-VP8* fused protein which was expressed in the presence of VP6 and VP7 to produce ΔTLPs. The Bac-to-Bac® Baculovirus Expression System and Spodoptera frugiperda (Sf) 9 insect cells were used for expression. All the proteins were successfully expressed. VP2, VP6, VP4 and the dVP2-VP8* fused protein were visible on Coomassie stained SDS-PAGE. Expression of VP7 could only be confirmed with western blot analysis. Particle formation, as assessed by transmission electron microscopy (TEM), was observed for DLPs. No TLPs of dVP2-8*/6/7 or VP2/6/7 were visualized due to the lower expression level of VP7 and the lack of calcium supplements during the assembly process. In conclusion, it was possible to produce RV-DLPs derived from the consensus sequence determined for a G9P[6] rotavirus directly from stool without prior propagation in cell culture or virus isolation. This strain contains both the G9 and P[6] genotypes that are currently prevalent in sub-Saharan Africa. / Thesis (MSc (Biochemistry))--North-West University, Potchefstroom Campus, 2013.

<p>Greywater is domestic household wastewater without inputfrom the toilet, i.e. wastewater from sinks, the shower,washing machine and dishwasher in a home. Source separation ofgreywater can be a strategy to enhance recirculation of plantnutrients and/or improve water use. The risk for transmissionof disease when reusing greywater is largely dependent on thecross-contamination by faeces. High levels of faecalindicators, mainly thermotolerant coliform bacteria, have beenreported in greywater, indicating substantial faecal pollution.However, growth of indicator bacteria within the system leadsto an overestimation of thefaecal input and thus the hygienerisk. The faecal input of the greywater in Vibyåsen,Sollentuna, North of Stockholm, was estimated to be 0.04 ±0.02 g faeces person-1 day-1 from the quantification of thefaecal sterol coprostanol, compared to 65 g, 5.2 g and 0.22 gp-1 d-1 using E. coli, enterococci and cholesterolrespectively.</p><p>Prevalence of pathogens in the population and the faecalload based on coprostanol concentrations were used to form thebasis of a screening-level quantitative microbial riskassessment (QMRA) that was undertaken for rotavirus, Salmonellatyphimurium, Campylobacter jejuni, Giardia intestinalis andCryptosporidium parvum, looking at the treatment required to bebelow an acceptable level of risk (10-3) for reuse or dischargeof the greywater. The different exposure scenarios simulated–groundwater recharge, direct contact, irrigation andrecreational water–showed that a reduction of 0.7–3.7 log was needed for rotavirus, with the measured level offaecal load in Vibyåsen. The other pathogen of concern wasCampylobacter, where a 2.2 log reduction was needed forgroundwater recharge. The infectious dose of Salmonella is highand the excretion numbers of Giardia cysts and Cryptosporidiumoocysts low, resulting in no treatment requirements for theseorganisms under these circumstances. Pathogen input fromcontaminated food via the kitchen sink had a minor effect onthe microbiological quality of the greywater. Studies on virusoccurrence in greywater as well as validation of the faecalload of greywater at another site would give valuable input forfuture QMRAs.</p><p>Greywater treatment efficiency studies, especially on virusremoval, are scarce and more investigations are warranted.Active sludge may not be a suitable technique for greywater dueto the low carbon content in this flow. Chemical precipitationhas the advantage of removing phosphorus as well as virusesefficiently and it is suggested as one possible method fortreating greywater. Otherwise the most common practice forgreywater treatment in Sweden is soil infiltration. However, itis suggested that the recommendations for wastewaterinfiltration also be observed for greywater, despite the lowfaecal load, due to the simulated results on virus reductionneeded.</p><p><b>Key words:</b>greywater, greywater reuse, greywatertreatment, microbial risk assessment, groundwater recharge,irrigation, recreational water, faecal contamination, indicatorbacteria, index organisms, faecal sterols, bacteriophages,enteric pathogens, rotavirus, Salmonella, Campylobacter,Giardia, Cryptosporidium, Legionella</p>

O objetivo deste estudo foi avaliar a qualidade virológica da água de reúso produzida em uma das estações de tratamento de esgoto da cidade de São Paulo. Para tanto, foram coletadas concomitantemente 177 amostras de esgoto tratado (100L) e bruto (15L) e os vírus concentrados utilizando método Viradel-ultracentrifugação. Em seguida as amostras foram tratadas com Vertrel XF e os ácidos nucléicos extraídos para a detecção de adenovírus (HAdV), rotavírus (RV-A), norovírus e vírus da hepatite A (VHA). A detecção por PCR e/ou RT-PCR evidenciou RV-A (G1-G5), VHA e HAdV incluindo os da espécie F tanto no esgoto bruto quanto no tratado, no entanto norovírus não foram detectados em ambos os efluentes. A infectividade de RV-A e HAdV foi avaliada por cultivo celular e os rotavírus RV-A foram também quantificados por reação de imunoperoxidase direta. PCR em tempo real foi padronizada para quantificação de vírus não cultiváveis ou de difícil cultivo como os VHA. Com base nos resultados obtidos foi verificada a ocorrência e a distribuição anual de cada vírus nas águas de reúso. / The aim of the study was to evaluate the virological quality from one Sewage Treatment Plant in the state of São Paulo. From January/2005 to November/2006, 177 samples (15L) of raw sewage were collected at the entrance and another 177 (100L) at the end of treatment, twice a week. Viruses were concentrated by filtration through positively charged microporous filters, followed by ultracentrifugation. Virus concentrates were treated by using Vertrel-XF and the viral genomes extracted for detection of adenovirus (HAdV), rotavirus (RV-A), norovirus and hepatitis A virus (HAV). PCR and RT-PCR revealed RV-A (G1-G5), HAV and HAdV, including the enteric ones (species F) in sewage and treated wastewater samples. Norovirus was not detected in any samples. The infectivity of HAdV and RV-A was assayed by inoculating onto suitable cell line. Immunoperoxidase assay was used to calculate the rotavirus FFU/L in the samples. Real time-PCR was standardized for enumeration of non-cultivable virus. The occurrence and annual distribution of each virus in reuse water were analyzed.