ABSTRACT Rotavirus is the main global cause of severe childhood diarrhoea among children. In 2006, Rotarix® (G1P[8]) was introduced into Brazil’s National Immunization Program. The vaccine coverage rate was 84.4% in 2009. Evidences of increasing G2P[4] after 2006 opened up the discussion about the vaccine effectiveness to non-G1 strains. The aim of this study was to identify the circulating rotavirus genotypes in two Brazilian regions during 2009. A total of 223 positive samples by immunochromatography and latex agglutination assay from the Northeast (Bahia/Pernambuco States) and Southeast (São Paulo/Rio de Janeiro States) regions were included in the study. The samples were submitted to genotyping by nested-PCR according to VP7(G) and VP4(P) and 175 samples (78.5%) were able to be characterized. Considering the characterization of VP7, the G-types detected were G1, G2, and G4 in the Northeast, and G2, G3, G5, and G9 in the Southeast. Considering the characterization of VP4, the P-types detected were P[4], P[8], and P[6]/P[9] in the Northeast and the Southeast. The most frequent mixed types found were G2P[4]/G2P[NT](81.4%), G2P[6](5.2%), G1P[6](5.2%) in the Northeast, and G2P[4]/G2P[NT](78.8%), G2P[6](8.2%), G9P[8](4.7%) in the Southeast. Among immunized individuals whose age ranged from 0-4 years, the G2P[4]/G2P[NT] genotype was identified in 91,0% of cases, and among non-immunized individuals of the same age, the G2P[4]/G2P[NT] genotype was identified in 85.7% of the cases. In accordance with the high level of vaccine coverage, the data suggest that the circulation of G2P[4] in these regions had a considerable increase after the introduction of Rotarix®.

RESUMO Em relação à saúde pública no Brasil, um novo cenário emergiu com o estabelecimento dos programas universais de vacinação contra o rotavírus (RV). Os resultados de cinco anos (2007-2012) de vigilância dos genótipos G e P de cepas de RV detectadas em indivíduos com gastroenterite aguda no Brasil são descritos no presente estudo. Um total de 6196 amostras fecais foi investigado utilizando ELISA e RT-PCR. RVs foram detectados em 19,1% (1181/6196). O pico de incidência de RV se deslocou de junho-agosto para setembro. RV foi detectado com menor frequência entre crianças ≤ 5 anos (19,5%) quando comparado às crianças mais velhas e adolescentes (6-18 anos) (40,6%). A distribuição genotípica mostrou um perfil diferente a cada ano: a cepa G2P[4] foi prevalente durante 2007-2010, G9P[8] em 2011 e G12P[8] em 2012. Infecções mistas (G1+G2P[4], G2+G3P[4]+P[8], G2+G12P[8]), combinações não usuais (G1P[4], G2P[6]) e cepas atípicas (G3P[3]) também foram identificadas em todo o período do estudo. A vacinação em massa pode alterar o padrão sazonal do RV. A tendência do RV em infectar crianças mais velhas e adolescentes após a implementação da vacina tem sido relatada em todo o mundo. A emergência de G2P[4] segue provavelmente a tendência mundial e, aparentemente, não está relacionada à vacinação. G12 também parece estar emergindo na população brasileira. As rápidas mudanças nos padrões de genótipos dos RVs observados durante o período desse estudo ilustram a existência de uma população dinâmica de cepas selvagens co-circulando no Brasil.

SUMMARY Regarding public health in Brazil, a new scenario emerged with the establishment of universal rotavirus (RV) vaccination programs. Herein, the data from the five years of surveillance (2007-2012) of G- and P-type RV strains isolated from individuals with acute gastroenteritis in Brazil are reported. A total of 6,196 fecal specimens were investigated by ELISA and RT-PCR. RVs were detected in 19.1% (1,181/6,196). The peak of RV incidence moved from June-August to September. RV was detected less frequently (19.5%) among children ≤ 5 years than in older children and adolescents (6-18 years) (40.6%). Genotype distribution showed a different profile for each year: G2P[4] strains were most prevalent during 2007-2010, G9P[8] in 2011, and G12P[8] in 2012. Mixed infections (G1+G2P[4], G2+G3P[4]+P[8], G2+G12P[8]), unusual combinations (G1P[4], G2P[6]), and rare strains (G3P[3]) were also identified throughout the study period. Widespread vaccination may alter the RV seasonal pattern. The finding of RV disease affecting older children and adolescents after vaccine implementation has been reported worldwide. G2P[4] emergence most likely follows a global trend seemingly unrelated to vaccination, and G12, apparently, is emerging in the Brazilian population. The rapidly changing RV genotype patterns detected during this study illustrate a dynamic population of co-circulating wildtype RVs in Brazil.

Objectives:The aim of this study was to monitor rotavirus (RV) infections in adults >18 years with acute gastroenteritis during 2004–2011 national Brazilian RV surveillance. In addition, to characterize the RV group A (RVA) strains in order to gain insight into the supposed vaccine selective pressure imposed to Brazilian children population.Methods:A total of 2102 convenient fecal specimens were investigated by ELISA, PAGE, and RT-PCR.Results:RV was detected in 203 (9.6%) of 2102 specimens, and showed a marked peak of detection in September. RVA infection was detected in 9.4% (197/2102) and RV group C (RVC) in 0.3% (6/2102). The most frequent genotypes detected in 2004 and 2005 were G9P[8] (38.5%; 5/13) and G1P[8] (54.5%; 6/11), respectively. The dominant genotype identified from 2006 to 2011 was G2P[4] (64.4%; 116/180). Detection rate varied during the 8-year period of the study from 0.7% to 12.9%.Conclusion:The high detection rate of G2P[4] in adults provides further evidence that its dominance reflects the seasonality of RVA strains instead of the supposed selective advantage created by vaccination program. It also can be suggested that adult infections may serve as a reservoir to maintain RVA strains in childhood gastroenteritis. Considering the detection rate, the evident reduction of RVA frequency observed in children after vaccine introduction was not present in adults.

IntroductionThis study aimed to monitor the seasonality of rotavirus infection, and gain insight into the variability of Brazilian strains.MethodsA total of 28 stool samples were analyzed from 698 revised cases of gastroenteritis during a norovirus outbreak in the summer of 2010 in Guarujá, Brazil. Diagnosis was performed using enzyme-linked immunosorbent assay (ELISA), reverse transcription polymerase chain reaction (RT-PCR), and sequencing.ResultsRotavirus infection was detected in 17.9% (5/28) of samples; 4 samples were G2P[4] genotype, and one G2P[4]+P[6] genotype. G2 and P[4] sequences showed a genetic relationship to strains from India and Russia, respectively.ConclusionsThe seasonal pattern of rotavirus may be a consequence of human activity apart from climate factors.

RESUMO Objetivo: Descrever um surto de norovírus ocorrido em uma instituição de longa permanência no Brasil, de 8 a 29 de julho 2005. Métodos: Nas primeiras 48 a 72 horas após o início dos sintomas entre moradores da instituição de longa permanência e funcionários, as principais estratégias de controle da infecção foram: educação da equipe, reforço na higienização das mãos, implementação de precauções de contato até 48 a 72 horas após o término de sintomas, limpeza “terminal” dos quartos dos moradores e afastamento dos funcionários sintomáticos até 48 a 72 horas após o término dos sintomas. As características clínicas e epidemiológicas das infecções por norovírus foram descritas baseadas nos dados dos prontuários. Resultados: A incidência foi 41,3% entre moradores e 16,25% entre funcionários. O principal sintoma foi diarreia, acometendo 100% dos casos; 44% das amostras de fezes foram positivas pela análise RIDASCREEN® Norovirus, com a identificação do norovírus genogrupo II. Setenta por cento dos moradores eram do sexo feminino, com idade de 51 a 98 anos. Os moradores tinham, em média, duas comorbidades, sendo 87,3% com doenças cardiovasculares ou pulmonares e 47,6% com demência. Não houve recidiva do surto ou óbitos. Conclusões: As medidas precoces de prevenção associada à vigilância são estratégias que mantém as instituições de longa permanência livres de infecções por norovírus e protegem aqueles indivíduos mais vulneráveis.

ABSTRACT Objective: To describe a norovirus outbreak in a Brazilian long-term care facility from July 8 to 29, 2005. Methods: In the first 48 to 72 hours after onset of symptoms in inpatients and employees, the main infection control strategies were staff education, emphasis on hand washing, implementing contact precautions up to 48-72 hours after resolution of symptoms, complete cleaning of the rooms and exclusion of symptomatic employees from work until 48-72 hours after resolution of their symptoms. Epidemiological and clinical characteristics of the norovirus infections were described based on chart review. Results: The incidence among inpatients and employees was 41.3% and 16.25%, respectively. The main symptom was diarrhea, affecting 100% of inpatients and employees. Forty-four percent of specimens were positive by RIDASCREEN® Norovirus analyses, and identified as norovirus genogroup GII. Seventy percent of inpatients were women and their age range was 51-98 years. Inpatients had in average two comorbid conditions – 87.3% with cardiovascular or chronic pulmonary condition and 47.6% with dementia. There was not relapse or death. Conclusions: The early infection-control measures associated to surveillance are required to keep long-term care facilities free of noroviruses and to protect those who are most vulnerable.

Norovírus (NoV), anteriormente chamado Norwalk-like vírus, constituem um importante grupo de patógeno humano associado a surtos de gastroenterites não bacterianas. Levantamentos epidemiológicos de surtos demonstram que as mais importantes vias de transmissão são contatos pessoa-pessoa, água e alimentos contaminados, afetando adultos e crianças. Os NoV são classificados em genogrupos, sendo os genogrupos GI, GII e GIV encontrados em humanos. A grande diversidade genética dos NoV e a falta de informação sobre esses vírus no Brasil determinaram o tema deste estudo que teve como objetivo a identificação e caracterização molecular de NoV em amostras fecais de pacientes com gastroenterites, provenientes de surtos de diarréia no estado de São Paulo. Neste estudo, 204 amostras de fezes coletadas durante surto de diarréia foram analisadas por RT-PCR, e 12 foram seqüenciadas para confirmar o genogrupo. O método one step PCR foi empregado para a amplificação da região B da ORF1 com o pool de primers MON 431, 432, 433 e 434. Do total, 32 (15,7%) amostras foram positivas para norovírus genogrupo GII. A comparação das seqüências dos produtos de PCR às seqüências do GenBank demonstrou 88,8% a 98,8% de identidade, sugerindo a presença de norovírus genogrupo GII em surtos de gastroenterites no Estado de São Paulo.

Norovirus (NoV), previously called Norwalk-like virus, represents an important group of human pathogens associated with outbreaks of nonbacterial gastroenteritis. Epidemiological surveys of outbreaks have shown that the most important routes of transmission are person-to-person contacts and contaminated food and water, with the virus affecting adults and children. NoV is classified into genogroups, being genogroups GI, GII and GIV found in humans. In view of the high genetic diversity of NoV and the lack of information about this virus in Brazil, the aim of the present study was the molecular characterization of NoV isolated from diarrheic stool samples of patients from São Paulo State. In this study, 204 stool specimens collected during diarrhea outbreaks were analyzed by RT-PCR, and 12 were sequenced for genogroup confirmation. One-step PCR was performed in order to amplify the B region of ORF 1 using the MON 431, 432, 433 and 434 primer pool. From total, 32 (15.7%) stool specimens were positive for NoV genogroup GII. Comparison of the sequences of the PCR products to GenBank sequences showed 88.8% to 98.8% identity, suggesting the presence of genogroup GII in gastroenteritis outbreaks in São Paulo State.

Exantema viral é considerado problema comum em regiões tropicais, afetando principalmente crianças. Diversos exantemas cutâneos estão associados a infecções por Enterovirus. Amostras biológicas provenientes de uma criança apresentando exantema generalizado foram enviadas ao Laboratório de Vírus Entéricos do Instituto Adolfo Lutz para a realização do diagnóstico laboratorial. Amostra viral isolada em RD (human rhabdomyosarcoma cells) foi submetida à reação em cadeia pela polimerase apresentando um produto de 437 pares de base, característico de gênero Enterovirus. O sorotipo echovirus 6 (E-6) foi identificado por ensaio de imunofluorescência indireta. Em adição, as amostras pareadas de soro apresentaram soroconversão para E-6. Até o momento, não há relatos do envolvimento de E-6 associado a doenças exantemáticas no Brasil, enfatizando a importância da vigilância epidemiológica para essas doenças e suas complicações.

Viral exanthems are a common problem in tropical regions, particularly affecting children. Various skin rashes have been reported in acute infections caused by Enterovirus. Biological samples from a child who presented generalized rashes were sent to the Enteric Virus Laboratory of the Adolfo Lutz Institute for laboratory diagnosis to be performed. A viral sample isolated from RD (human rhabdomyosarcoma cells) was subjected to the polymerase chain reaction and showed a 437-base pair product that was characteristic of the Enterovirus genus. Echovirus 6 (E-6) serotype was identified using the indirect immunofluorescence test. In addition, paired serum samples presented seroconversion to E-6. So far, there have not been any reports of E-6 involvement in exanthematic diseases in Brazil. Thus, the importance of epidemiological surveillance for these diseases and their complications is emphasized.

Entre fevereiro e maio de 2004, em São Joaquim da Barra, Estado de São Paulo, foi notificado ao Centro de Vigilância Epidemiológica (CVE) um surto de meningite asséptica (MA) envolvendo 40 indivíduos. Foram enviadas ao Instituto Adolfo Lutz amostras de líquido cefalorraquidiano (LCR) de 23 pacientes com MA para tentativa de isolamento viral. Estas amostras foram inoculadas em 3 linhagens celulares: RD, HEp2 e Vero. Culturas celulares que apresentaram efeito citopático (ECP) foram submetidas a ensaio de Imunofluorescência Indireta (IFI), reação de Neutralização (Nt) e RT-PCR (Transcrição Reversa Reação em Cadeia da Polimerase). Em 52,2% (12/23) das amostras foi observado ECP. Todos os vírus isolados foram identificados como gênero HEV por IFI e RT-PCR e o sorotipo echovirus 6 (E-6) por IFI e Nt. Nossos resultados confirmam a participação e importância dos echovirus como agente etiológico responsável pelo surto ocorrido e a diversidade de sorotipos circulantes no Estado de São Paulo.

An aseptic meningitis outbreak occurred during a period from February to May 2004 in São Joaquim da Barra, a town in the northern region of São Paulo State. A total of 40 cases were reported to the Epidemiological Surveillance Center of São Paulo State. Cerebrospinal fluid samples obtained from 23 patients were sent to the Adolfo Lutz Institute for isolation of the virus. These samples were inoculated into RD, HEp2 and Vero cell lineages and those presenting a cytopathogenic effect were selected for analysis by indirect immunofluorescence assay (IFA), neutralization testing (Nt) and reverse transcriptase-polymerase chain reaction (RT-PCR). Cytopathogenic effects were observed in 52.2% (12/23) of these samples. All isolated viruses were identified as human enterovirus by IFA and RT-PCR and echovirus 6 was typed by IFA and Nt. Our results confirmed the participation and importance of echovirus as the etiological agent responsible for this outbreak and the serotype diversity of human enteroviruses circulating in São Paulo State.

O objetivo deste estudo foi identificar e caracterizar quanto aos aspectos imunológicos e moleculares os Enterovirus humanos associados a casos de meningite asséptica. Amostras clínicas (líquido cefaloraquidiano, fezes e soro) originárias de 21 casos suspeitos de meningite viral, provenientes do estado de São Paulo no período de 1998 a 1999 foram analisadas. Os vírus isolados foram identificados como pertencentes ao gênero Enterovirus e mais especificamente ao sorotipo echovírus 30 (EV-30), utilizando-se técnicas imunológicas como Imunofluorescência Indireta e Neutralização e técnica molecular da transcrição reversa - reação em cadeia da polimerase (RT-PCR) direcionada para as regiões 5' não traduzida (5'NTR) e Proteína Viral 1(VP1) do genoma viral. Os resultados obtidos contribuíram com o sistema de vigilância epidemiológica do estado de São Paulo na geração de informações sobre a circulação de EV-30 associado à meningite asséptica nessa região do mundo.

The objective of this study was to identify and characterize the immunological and molecular aspects of Human Enterovirus (HEV) associated with aseptic meningitis cases in São Paulo State, Brazil. Clinical samples (cerebrospinal fluid CSF, stool and sera) were analyzed from 21 viral meningitis suspected cases collected in the period from 1998 to 1999. The isolated virus were identified as belonging to Enterovirus genus and specifically to echovirus serotype 30 (EV-30), using immunological techniques as Indirect Immunofluorescense Assay (IFA) and Neutralization (Nt) and by molecular technique of Reverse Transcription Polymerase Chain Reaction (RT-PCR) directed to the 5' NTR and VP1 regions of viral genome. The results obtained contributed with to the surveillance system of São Paulo State and they are important due to a shortage of epidemiological data about the circulation of EV-30 associated to aseptic meningitis in this region of the world.