Caracterización molecular de norovirus, sapovirus y astrovirus en niños con gastroenteritis aguda en Belém (Pará, Brasil)

Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.A importância dos norovírus (NoVs), sapovírus (SaVs) e astrovírus humanos (HAstVs) como causa de surtos de gastrenterites já está bem definida. Entretanto, poucos estudos têm descrito casos esporádicos de gastrenterite aguda causados por esses agentes. O objetivo deste estudo foi determinar o papel destes vírus na etiologia das gastrenterites agudas em crianças atendidas durante uma vigilância intensiva realizada em hospitais e ambulatórios de Belém, Brasil, de março a setembro de 2003. Um total de 305 espécimes fecais de pacientes com gastrenterite grave foram coletados e testados por reação em cadeia da polimerase precedida de transcrição reversa (RT-PCR), utilizando iniciadores específicos Mon 269 e Mon 270 para os HAstVs; p289 e p290 para os calicivírus humanos (HuCVs); e Mon 431/433 e Mon 432/434 para os NoVs. Sequenciamento dos amplicons de HAstV, HuCVs e NoVs, obtidos por RT-PCR, foi realizado usando os mesmos iniciadores. Das 305 amostras testadas, 96 (31,5%) apresentaram resultados positivos, sendo que 51 diagnosticadas como HuCVs, 40 como HAstVs e cinco infecções mistas. Das 56 (18,4%) amostras de HuCVs sequenciadas, 30 foram NoVs (9,8%) pertencentes aos genogrupos GI-4 e GII-4, e 15 (4,9%) SaVs dos grupos GI-1, GI-2 e GII-1. HAstVs foram detectados em 45 (14,7%) das amostras, incluindo os genótipos 1, 8 e 2. Esta pesquisa ressalta a importância destas viroses como causa de gastrenterite aguda e demonstra a circulação de diferentes genótipos durante o período de estudo. Estes resultados reforçam a necessidade de se estabelecer uma vigilância intensiva das gastrenterite causadas por estes vírus, de forma a poder avaliar o impacto da doença e monitorar os genótipos circulantes.The importance of norovirus (NoVs), sapovirus (SaVs) and human astrovirus (HAstVs) as causes of gastroenteritis outbreaks is already well-defined, but a few studies have described sporadic cases of acute gastroenteritis caused by these viral entities. The aim of this study was to determine the role of these viruses in the etiology of acute gastroenteritis in children enrolled to participate in hospital – and emergency department – based intensive surveillance carried out in Belém, Brazil, from March to September 2003. A total of 305 stool specimens from patients with severe gastroenteritis were collected and screened by reverse transcription followed by polymerase chain reaction (RT-PCR), using the specific primers Mon 269 and Mon 270 for HAstVs, p289 and p290 for human calicivirus (HuCVs), and Mon 431/433 and Mon 432/434 for NoVs. Sequencing of RT-PCR HAstV, HuCV and NoV amplicons was carried out using the same primers. Of the 305 samples tested, 96 (31.5%) were positive, with 51 diagnosed as HuCVs, 40 as HAstVs and five as mixed infections. Of the 56 (18.4%) HuCVs sequenced, 30 were NoVs (9.8%) of genogroups GI-4 and GII-4, and 15 (4.9%) were SaVs of types GI-1, GI-2 and GII-1. HAstVs, including genotypes 1, 8 and 2, were detected in 45 (14.7%) samples. This study has highlighted the importance of these viruses as causes of acute gastroenteritis and established the circulation of different genotypes during the study period. These results reinforce the need for establishing an intensive surveillance for gastroenteritis caused by these viruses to assess the burden of disease and to monitor the circulation of genotypes

Detection of a novel recombinant strain of norovirus in na African-descendant community from the Amazon region of Brazil in 2008

This study was funded by the Foudation for Research Support of the State of Pará (Fundação de Amparo à Pesquisa do Estado do Pará - Secretaria de Estado de Desenvolvimento, Ciência e Tecnologia) grant code MS/CNPQ/SECTAM – 001/2006, agreement 032/2007, and by Evandro Chagas Institute, Secretary of Health Surveillance (IEC/ SVS), Ministry of Health, BrazilMinistério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Noroviruses, a major cause of acute gastroenteritis outbreaks worldwide, are constantly evolving. Thisability is reflected in the speed and efficiency with which these viruses spread and remain in the human population.The present study reports the detection of a novel recombination event among norovirus genotypes in Brazil in2008. A strain detected in a stool sample from a child with norovirus-associated gastroenteritis, residing in an Africandescendant semi-closed community of Paraº State, wascharacterized as a novel intergenotype recombinant, GII.7/ GII.20, as determined by partial sequencing and SimPlot analysi

Norovirus infection in children admitted to hospital for acute gastroenteritis in Belém, Pará, Northern Brazil

Coordination of Improvement of Higher Education Personnel (CAPES) Evandro Chagas Institute, SVS, MSFederal University of Pará State. Tropical Medicine Center. Postgraduate Program in Tropical Diseases. Belém, PA, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Federal University of Pará State. Institute of Health Sciences. Belém, PA, Brazil.University of the Pará State. Postgraduate Program in Parasite Biology in the Amazon, Center for Biological and Health Sciences. Belém, PA, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Federal University of Pará State. Tropical Medicine Center. Belém, PA, Brazil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Noroviruses are the leading cause of epidemic, non-bacterial outbreaks of acute gastroenteritis, and are also a major cause of sporadic acute gastroenteritis in infants. The aim of the present study was to identify norovirus infections in children not infected by rotavirus admitted to hospital for acute gastroenteritis in Belém. A total of 348 fecal specimens were obtained from children with diarrhea aged less than 5 years, all of whom had tested negative for rotavirus, between May 2008 and April 2010. Fecal samples were screened for norovirus antigen using enzyme-immunoassay (EIA). Specimens were subjected to reverse-transcription polymerase chain reaction (RT-PCR) using the primers Mon432/434-Mon431/433 for detection of the GI and GII norovirus strains, respectively. Based on both methods, the overall norovirus positivity rate was 36.5% (127/348). Of the 169 samples collected in the first year, 44.4% (n = 75) tested positive for norovirus using both methods, 35.5% (n = 60) by EIA and 40.8% (n = 69) by RT-PCR. Using RT-PCR as a reference standard, a sensitivity of 78.3%, specificity of 94%, and agreement of 87.6% were recorded. Genome sequencing was obtained for 22 (31.9%) of the 69 positive samples, of which 90.9% (20/22) were genotype GII.4d and 9.1% (2/22) were genotype GII.b. Norovirus infection was most frequent in children under 2 years of age (41.5%-115/277). The peak incidence (62.1%) of norovirus-related acute gastroenteritis in these patients (not infected by rotavirus) was observed in February 2010. These findings emphasize the importance of norovirus as a cause of severe acute gastroenteritis among children in Belém, Pará, Northern Brazil

Surto de norovírus em um navio cruzeiro ao longo da costa brasileira, março de 2011

Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Programa de Pós-Graduação em Virologia. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Universidade do Estado do Pará. Centro de Ciências Biológicas e da Saúde. Programa de Pós-Graduação em Biologia Parasitária na Amazônia. Belém, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Vigilância em Saúde do Estado do Pará. Belém, PA, Brasil.Centro de Informações Estratégicas em Vigilância em Saúde. Belém, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Ministério da Saúde. Secretaria de Vigilância em Saúde. Instituto Evandro Chagas. Ananindeua, PA, Brasil.Outbreaks of gastrointestinal disease may occur on board of cruise ships due to the consumption of contaminated water or food, with a fast person-to-person transmission. Epidemiologic investigations carried out by Centers for Disease Control and Prevention in USA, have confirmed that more than 95% of gastroenteritis outbreaks in cruise ships are caused by norovirus (NoV). In March 2011 an outbreak of acute gastroenteritis (AGE) occurred on a cruise ship with 1,224 passengers and 554 crew members that sailed from Rio de Janeiro, Rio de Janeiro State (Southeast, Brazil) to Manaus, Amazonas State (North) and stopovers in Recife, Pernambuco State, and Fortaleza, Ceará State (Northeast) and Belém, Pará State (North). Epidemiological data were obtained and seven rectal swab samples were collected and tested for NoV detection and characterization by molecular techniques. A total of 53 persons (42 passengers and 11 crew members) developed AGE, 75.5% of whom were older than 60 years old. The symptoms duration was less than 48 h, most of the patients presenting vomiting (79.2%) and diarrhea (73.6%). Most of the cases varied from mild to moderate and only one patient needed parenteral rehydration. Cases of AGE were recorded in eight of 12 vessel floors, especially in the recreational areas. The seven rectal samples collected were all NoV-positive by RT-PCR and all NoV strains were genogrouped as GII by semi-nested PCR. The quantitative real-time PCR produced a 57.1% NoV positivity rate. The partial nucleotide sequencing classified five (71.4%) of these samples as GII.P4. Our findings highlight the need for continuous viral enteropathogens surveillance including cruise ships considering the increase of this kind of touristic option in Brazil.Os surtos da doença gastrointestinal podem ocorrer em navios cruzeiros devido ao consumo de água e comida contaminadas, com rápida transmissão de uma pessoa a outra. Investigações epidemiológicas realizadas pelos Centros de Controle e Prevenção de Doenças, nos EUA, confirmaram que mais de 95% dos surtos de gastroenterite em navios são causados por norovírus (NoV). Em março de 2011, um surto de gastroenterite aguda (GEA) ocorreu em um cruzeiro com 1.224 passageiros e 554 tripulantes que partiu do Rio de Janeiro, Estado do Rio de Janeiro (Sudeste do Brasil) para Manaus, Estado do Amazonas (Norte) com paradas em Recife, Estado do Pernambuco, e Fortaleza, Estado do Ceará (Nordeste) e Belém, Estado do Pará (Norte). Dados epidemiológicos foram obtidos e sete amostras por swab retal foram coletadas e testadas para a detecção e caracterização do NoV por meio de técnicas moleculares. Um total de 53 pessoas (42 passageiros e 11 tripulantes) desenvolveram AGE, dos quais 75,5% eram maiores de 60 anos de idade. Os sintomas duraram menos de 48 h, a maioria dos pacientes apresentou vômito (79,2%) e diarreia (73,6%). A maior parte dos casos obteve variação entre leve e moderado e apenas um paciente necessitou de reidratação parental. Casos de AGE foram registrados em oito dos 12 andares do navio, principalmente nas áreas de recreação. As sete amostras de swab retal coletadas revelaram resultado positivo para NoV por meio de RT-PCR e todas as cepas foram genogrupadas como GII pelo semi-nested PCR. O PCR quantitativo em tempo real produziu uma taxa de positividade de 57,1% para NoV. O sequenciamento parcial de nucleotídeos classificou cinco (71,4%) das sete amostras como GII.P4. Os resultados destacam a necessidade de vigilância contínua dos enteropatógenos virais, incluindo navios cruzeiros, considerando o aumento deste tipo de opção turística no Brasil

Norovirus diversity in diarrheic children from an African-descendant settlement in Belém, Northern Brazil.

Norovirus (NoV), sapovirus (SaV) and human astrovirus (HAstV) are viral pathogens that are associated with outbreaks and sporadic cases of gastroenteritis. However, little is known about the occurrence of these pathogens in relatively isolated communities, such as the remnants of African-descendant villages ("Quilombola"). The objective of this study was the frequency determination of these viruses in children under 10 years, with and without gastroenteritis, from a "Quilombola" Community, Northern Brazil. A total of 159 stool samples were obtained from April/2008 to July/2010 and tested by an enzyme immunoassay (EIA) and reverse transcription-polymerase chain reaction (RT-PCR) to detect NoV, SaV and HAstV, and further molecular characterization was performed. These viruses were detected only in the diarrheic group. NoV was the most frequent viral agent detected (19.7%-16/81), followed by SaV (2.5%-2/81) and HAstV (1.2%-1/81). Of the 16 NoV-positive samples, 14 were sequenced with primers targeting the B region of the polymerase (ORF1) and the D region of the capsid (ORF2). The results showed a broad genetic diversity of NoV, with 12 strains being classified as GII-4 (5-41.7%), GII-6 (3-25%), GII-7 (2-16.7%), GII-17 (1-8.3%) and GI-2 (1-8.3%), as based on the polymerase region; 12 samples were classified, based on the capsid region, as GII-4 (6-50%, being 3-2006b variant and 3-2010 variant), GII-6 (3-25%), GII-17 (2-16.7%) and GII-20 (1-8.3%). One NoV-strain showed dual genotype specificity, based on the polymerase and capsid region (GII-7/GII-20). This study provides, for the first time, epidemiological and molecular information on the circulation of NoV, SaV and HAstV in African-descendant communities in Northern Brazil and identifies NoV genotypes that were different from those detected previously in studies conducted in the urban area of Belém. It remains to be determined why a broader NoV diversity was observed in such a semi-isolated community

Dendograms constructed using the partial region of the norovirus RNA polymerase sequence (ORF1-B) (A) and capsid region (ORF2-D) (B) amplified from 14 strains from diarrheic children of Abacatal “Quilombola” Community, metropolitan region of Belém, Pará State, Brazil.

<p>April/2008 to July/2010. Prototype strains are presented together with strains from other locations. The number above each branch corresponds to the bootstrap value (2,000 replicates). The scale bar is proportional to the genetic distance. Study samples were marked (♦): PA-local/Qui-code in the study+F_x-Number of the stool/country/month+year of collection.</p

Map showing the location of the Abacatal “Quilombola” Community, in the metropolitan region of Belém city, Pará State, Amazon region, Brazil.

<p>Map showing the location of the Abacatal “Quilombola” Community, in the metropolitan region of Belém city, Pará State, Amazon region, Brazil.</p

Monthly distribution of the positive cases of norovirus, sapovirus and astrovirus (including their genotypes) detected in stool specimens from diarrheic children of the Abacatal “Quilombola” Community, Pará State, Brazil.

<p>April/2008 to July/2010.</p