In a large Phase III trial conducted in 10 Latin American countries, the safety and efficacy of the live attenuated monovalent rotavirus vaccine RIX4414 was evaluated in 15,183 healthy infants followed up during the first two years of life. Belém was the only site in Brazil included in this multicentre trial. The study in Belém included a subset of 653 infants who were followed up until 24 months of age for protection against severe rotavirus gastroenteritis. These subjects were randomly assigned in a 1:1 ratio to receive two doses of vaccine (n = 328) or two doses of placebo (n = 325) at approximately two and four months of age. Of the 653 enrolled infants, 23 dropped out during the study period. For the combined two-year period, the efficacy of RIX4414 was 72.3% [95% confidence interval (CI) 37.5-89.1%] against severe rotavirus-related gastroenteritis, reaching a protection rate of 81.8% (95% CI 36.4-96.6%) against circulating wild-type G9 rotavirus strains. It is concluded that two doses of RIX4414 are highly efficacious against severe rotavirus gastroenteritis in Belém during the first two years of life and provide high protection against the worldwide emergence and spread of G9P[8] strains.

This study describes the genetic relationships of the first human astrovirus type-8 (HAstV-8) detected in Belém-Brazil, during a public hospital-based study. This strain was compared with other HAstV-8 strains identified elsewhere which have sequences available at GeneBank. The regions ORF1a (primers Mon348/Mon340) and ORF2 (primers Mon269/Mon270) were analyzed by nucleotide sequencing and a high similarity rate was observed among the Belém strain and other HAstV-8 strains. In ORF1a, homology values of 93-100% were detected, and in ORF2 96-99%. Considering the sequence variation (7%) observed in ORF2 region, it was suggested that HAstV-8 strains could be divided in three different lineages.

Human astroviruses (HAstV) have been increasingly identified as important etiological agents of acute gastroenteritis in children up to five years old. The aim of this study was to determine the prevalence and genotype diversity of HAstV in children with symptomatic and asymptomatic infections in São Luís, Maranhão, Brazil. From June 1997 to July 1999 a total of 183 fecal samples 84 from symptomatic and 99 from asymptomatic children were tested by enzyme immunoassay for HAstV. Prevalence rates were found to be 11 and 3% for symptomatic and asymptomatic children, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) was carried out in 46 specimens (26 symptomatic and 20 asymptomatic) including the 12 samples that were positive by enzyme immunoassay (EIA). The overall positivity yielded by both methods was 8% (15/184); of these, 11% (9/84) for symptomatic and 5% (5/99) for those without symptoms or signs. Sequence analysis of amplicons revealed that HAstV-1 genotype was the most prevalent, accounting for 60% of isolates. Genotypes 2, 3, 4, and 5 were also detected, as one single isolate (10%) for each type. Variations in the sequences were observed when Brazilian isolates were compared to prototype strains identified in the United Kingdom. No seasonal pattern of occurrence was observed during these two years of study, and peak detection rate was observed in children aged between 3 and 6 months in the symptomatic group, and between 18 and 24 months in the controls.

De junho de 1997 a junho de 1999, pesquisou-se a infecção por rotavírus entre crianças até 2 anos de idade internadas com quadro diarréico agudo em São Luís, nordeste do Brasil. Coletaram-se 128 espécimes fecais oriundos de pacientes diarréicos. Paralelamente, obtiveram-se 122 amostras de um contingente caracterizado como controle, comparável ao anterior no tocante às idades e distribuição temporal. As freqüências de positividade para rotavírus alcançaram 32,0% (41/128) e 9,8% (12/122), respectivamente (p < 0,001). Procedeu-se à determinação dos sorotipos e eletroferotipos dos rotavírus em 42 (79,2%) das 53 amostras reativas para rotavírus. Identificaram-se eletroferotipos "longo" e "curto" em freqüências similares - 38,1% e 40,5%, respectivamente. De um modo geral, caracterizou-se o sorotipo G em 35 (83,3%) das amostras positivas, a maioria, revelando especificidade para o tipo G1. Considerando o conjunto dos eletroferotipos e sorotipos, rotavírus classificados como G1 exibiram padrões eletroforéticos "longo" e "curto" nas freqüências de 30,9% e 19%, respectivamente. Todos os rotavírus do tipo G2 apresentaram eletroferotipo de configuração "curta". No tocante ao perfil temporal, observou-se que as gastroenterites por rotavírus naquela região ocorrem ao longo de todo o ano, denotando-se tendência quanto à mais expressiva concentração no segundo semestre de vida das crianças, se comparado ao primeiro; em síntese, 45,2% e 26,1% (p = 0,13), respectivamente. As infecções por rotavírus configuraram picos quanto à distribuição durante o segundo semestre de vida, com freqüências de 30,1% e 13,5%, respectivamente. Aqueles do tipo G2 circularam durante todo o período de estudo, enquanto o sorotipo G1 (n = 27) emergiu a partir de junho de 1998. Aliás, detectaram-se 20 (74,1%) das amostras virais com essa última especificidade ao longo de 1998. Os dados acima sustentam a importância dos rotavírus na etiologia das gastroenterites graves no nordeste brasileiro e consubstanciam o conceito de que uma futura vacina contra esses enteropatógenos necessariamente deve conferir proteção frente aos múltiplos sorotipos circulantes.

During June 1997-June 1999 rotavirus infection was screened in infants aged up to 2 years and hospitalised with acute diarrhoea in São Luís, Northeastern Brazil. Altogether, 128 stool samples were collected from diarrhoeic patients and additional 122 faecal specimens from age- and- temporal matched inpatients without diarrhoea were obtained; rotavirus positivity rates for these groups were 32.0% (41/128) and 9.8% (12/122), respectively (p < 0.001). Both electropherotyping and serotyping could be performed in 42 (79.2%) of the 53 rotavirus-positive stool samples. Long and short electropherotypes were detected at similar rates - 38.1% and 40.5% of specimens, respectively. Overall, a G serotype could be assigned for 35 (83.3%) of specimens, the majority of them (66.7%) bearing G1-serotype specificity. Taking both electropherotypes and serotypes together, G1 rotavirus strains displaying long and short RNA patterns accounted for 30.9% and 19.0% of tested specimens, respectively; all G2 strains had short electropherotype. Rotavirus gastroenteritis was detected year-round and, in 1998, the incidence rates tended to be higher during the second semester than in the first semester: 45.2% and 26.1% (p = 0.13), respectively. Rotavirus infections peaked at the second semester of life with frequencies of 30.1% and 13.5% for diarrhoeic children and controls, respectively. While the six rotavirus strains bearing G2-type specificity were circulating throughout the whole study period, G1 serotypes (n = 27) emerged as from June 1998 onwards, 20 (74.1%) of which clustering in 1998. These data underscore the importance of rotaviruses in the aetiology of severe infantile gastroenteritis in Northeastern Brazil and sustain the concept that a future vaccine should confer protection against more than one serotype.

A vacina tetravalente contra rotavírus de origem símio-humana, geneticamente rearranjada (RRV-TV), foi licenciada para uso rotineiro nos Estados Unidos da América do Norte; entretanto, tal imunizante foi removido do mercado, uma vez que a intussuscepção emergiu como possível evento adverso vacinal. A eficácia da RRV-TV - em sua formulação menos concentrada (10 x 10(4) pfu/dose) - foi avaliada no tocante ao estado nutricional das crianças que integraram estudo caracterizado como de fase III, levado a efeito em Belém, região norte do Brasil. Observou-se proteção (moderada) apenas entre os indivíduos com escore "Z" (peso-por-idade, WAZ) superior a -1, com eficácias de 38% (p = 0,04) e 40% (p = 0,04) para todos os episódios de diarréia por rotavírus e os caracterizados como "puros" (sem outro enteropatógeno identificado no espécime clínico), respectivamente. A par disso, denotou-se tendência a mais elevados níveis protetores (43%, p = 0.05) entre crianças que apresentavam escore "Z" (estatura-por-idade, HAZ) > -1. Considerando-se conjuntamente os escores "Z" [WAZ, HAZ e peso-por-estatura (WHZ)] menores ou iguais a -1, não se registrou proteção significativa (p > 0,05), uma vez comparadas as crianças que receberam placebo àquelas vacinadas. Também não resultaram expressivas as diferenças, se as infecções "puras" e "mistas" por rotavírus são comparadas relativamente às variáveis antropométricas HAZ, WAZ e WHZ. Não obstante o reduzido número de infantes desnutridos no presente estudo, os resultados disponíveis oferecem evidências de que o estado nutricional pode interferir na eficácia das vacinas contra rotavírus nos países em desenvolvimento.

The rhesus-human reassortant, tetravalent rotavirus vaccine (RRV-TV) was licensed for routine use in the United States of America but it was recently withdrawn from the market because of its possible association with intussusception as an adverse event. The protective efficacy of 3 doses of RRV-TV, in its lower-titer (4 x 10(4) pfu/dose) formulation, was evaluated according to the nutritional status of infants who participated in a phase III trial in Belém, Northern Brazil. A moderate protection conferred by RRV-TV was related to weight-for-age Z-scores (WAZ) greater than -1 only, with rates of 38% (p = 0.04) and 40% (p = 0.04) for all- and- pure rotavirus diarrhoeal cases, respectively. In addition, there was a trend for greater efficacy (43%, p = 0.05) among infants reaching an height-for-age Z-score (HAZ) of > -1. Taking WAZ, HAZ and weight-for-height Z-score (WHZ) indices <= -1 together, there was no significant protection (p > 0.05) if both placebo and vaccine groups are compared. There was no significant difference if rates of mixed and pure rotavirus diarrhoeal cases are compared in relation to HAZ, WAZ and weight-for-height Z-score (WHZ) indices. Although a low number of malnourished infants could be identified in the present study, our data show some evidence that malnutrition may interfere with the efficacy of rotavirus vaccines in developing countries.

Faecal samples were obtained from 190 children, aged 0 to 5 years, admitted to a public hospital in Belém, Pará, Brazil. These patients were placed in a pediatric ward with 40 beds distributed in six rooms. Case were classified into three groups: (a) nosocomial: children who developed gastroenteritis 72 hr or later after admission; (b) community-acquired: patients admitted either with diarrhoea or who had diarrhoea within 72 hr following admission; (c) non-diarrhoeic: those children who had no diarrhoea three days before and three days after collection of formed faecal sample. Specimens were routinely processed for the presence of rotaviruses, bacteria and parasites. Rotaviruses were detected through enzyme-linked immunosorbent assay (ELISA) and subsequently serotyped/electrophoretyped. Rotaviruses were the most prevalent enteropathogens among nosocomial cases, accounting for 39 % (9/23) of diarrhoeal episodes; on the other hand, rotaviruses ocurred in 8.3 % (11/133) and 9 % (3/34) of community-acquired and non-diarrhoeic categories, respectively. Mixed infections involving rotavirus and Giardia intestinalis and rotavirus plus G. intestinalis and Entamoeba histolytica were detected in frequencies of 8.6 and 4.3 %, respectively, in the nosocomial group. The absence of bacterial pathogens in this category, and the unusual low prevalence of these agents in the other two groups may reflect the early and routine administration of antibiotics following admission to this hospital. Rotavirus serotype 2 prevailed over the other types, accounting for 77.8 % of isolates from nosocomial diarrhoeal episodes. In addition, at least five different genomic profiles could be observed, of which one displayed an unusual five-segment first RNA cluster. Dehydration was recorded in all cases of hospital-acquired, rotavirus-associated diarrhoea, whereas in only 57 % of nosocomial cases of other aetiology. It was also noted that nosocomial, rotavirus-associated diarrhoeal episodes occur earlier (7 days), following admission, if compared with those hospital-acquired cases of other aetiology (14 days).

Rotavírus atípicos foram detectados nas fezes de duas crianças diarreícas residentes em Belém, Brasil. Partículas de rotavírus foram visualizadas por microscopia eletrônica nos espécimes fecais de ambos os pacientes, tendo a eletroforese do ácido ribonucleico (ARN) exibido padrões compatíveis com rotavírus do grupo C. Testes imunoenzimáticos (ELISA) foram negativos quanto à presença de antígenos do grupo A. As duas crianças apresentaram três infecções sucessivas por esse agente, sendo que, em ambos os casos, os rotavírus atípicos foram excretados por ocasião da terceira infecção, produzindo sintomas brandos e de pouca duração.

Atypical rotaviruses were detected in faeces from two diarrhoeic children living in Belém, Pará, Brazil. Rotavirus particles were detected by electron microscopy and the RNA electrophoresis showed patterns which were compatible with group C rotaviruses. Tests for the presence of group A antigen by enzyme-linked-immunosorbent assay (ELISA) were negative. The two children had three successive rotavirus infection and in both cases the atypical strains were excreted at the time of the third infection, causing a mild and short-lasting disease.

Amostras de soro, em número de 948, foram coletadas de 83 crianças, ao longo de seus três primeiros anos de vida, e testadas quanto àpresença de anticorpos grupo-especificos para rotavirus por um teste de bloqueio, utilizando-se o ensaio imunoenzimático (ELISA). Observou-se que anticorpos transferidos passivamente pela mãe persistiram por cerca de dois e meio meses; subseqüentemente, baixos níveis de anticorpos para rotavirus começaram a aparecer aos sete meses de idade, atingindo um pico por volta dos onze. A partir de um ano apositividade aumentou gradualmente, alcançando os mais altos valores aos 34 meses de vida. Respostas imunes individuais foram avaliadas em 61 crianças acompanhadas desde o seu nascimento até os três anos de idade, obtendo-se os seguintes resultados: 38 (62,3%) delas desenvolveram imunidade de longa duração, após a primo-infecção; 11 (18,0%) indivíduos apresentaram anticorpos específicos que se mantiveram por um intervalo de tempo relativamente curto; 07 (11,5%) não exibiram qualquer resposta imune ao agente em questão, ao longo dos seus três primeiros anos de vida; e 05 (8,2%) mostraram-se positivos quanto à presença de anticorpos para rotavirus, desde o nascimento até os três anos de idade.

Nine hundred and forty-eight serum samples from 83 children living in Belem, Brazil, collected'within their first three years of life, were testedfor the presence of group- specific rotavirus-antibody by an enzyme-linked immunosorbent assay (ELISA) blocking-test. Passively transferred maternal antibody lasted about two and half months; subsequentely, low levels of rotavirus antibody started to appear at seven months, reaching a peak at eleven months of age. From one year onwards positivity gradually increased, reaching highest values at 34 months of life. Individual responses were examined in sera from 61 children who were followed up since birth to three years of age: 38 (62,3%) ofthem developed a long-term immunity following first infection; eleven (18.0%) children developed a short-term immunity after first infection by rotavirus; seven (11.5%) had no antibody response within their first three years of life; and 5 (8.2%) showed positive antibody response from birth to three years old.

Amostras de rotavírus provenientes de crianças habitantes na periferia de Belém, Pará, foram analisadas por eletroforese em gel de policrilamida (PAGE). As bandas correspondentes aos 11 segmentos de ARN foram detectadas em 46 (76,7%) das 60 amostras de rotavírus. Das amostras classificadas 5 (109%) foram relativas ao subgrupo I, 41 (89,1%) ao subgrupo II e, em 23,3%) não foi possível classificação face a ausência das bandas 10 e 11. As amostras de rotavírus sub-grupo II e codificadas como "1N2L" foram as mais freqüentes, ocorrendo em 30 (65,2%) das 46 classificadas.

Electrophoresis of rotavirus RNA in polyacrylamide gel was casrried out by using faecal samples collected from children living in the peripheral area of Belém, Brazil. Typical rotavirus profiles (eleven bands) were noted in 46(76.7%) out of 60 ELISA rotavirus positive specimens. According to the eletrophoretic patterns, 5(10.9%) were classified as subgroup I, while 41(89.1%) of them as subgroup II. Lacking of bands 10 and 11 in 14(23.3%) samples did not allow us to classify their electropherotypes. The profile coded as "1N2L" was the predominant one, ccouting for 65.2% of the classified strains.

Infecções sucessivas causadas por rotavírus foram detectadas em quatro crianças habitantes de Belém, acompanhadas desde seu nascimento até os três anos de idade. Em uma delas (Reg. 23.983) três infecções sintomáticas foram observadas, duas por sorotipo não especificado e outra pelo 2: a primeira aos 4 meses, a segunda aos 20, e a terceira aos 27. Outro indivíduo (Reg. 24.384) apresentou dois episódios diarréicos, com intervalo de oito meses, o primeiro por sorotipo 1 e o segundo por sorotipo não identificado. Infecções também aparentes foram observadas em duas ocasiões envolvendo a mesma criança (Reg. 24.004), a primeira por sorotipo 1 e, doze meses após. uma segunda pelo 2. Em uma outra criança (Reg. 24.097) duas infecções, ambas inaparentes e por sorotipo nao especincado, foram detectadas: a primeira aos 24 meses de vida e a segunda, aos 28.

Repeated infections involving different rotavirus serotypes were detected in four children living in Belém, who were followed up since birth to three years of age. In one child (Reg. 23.983) three successive symptomatic infections (one of them associated with serotype 2) were noted: the first, at four months of age, the second at 20 months and the third at 27 months. Another child had two subsequent infections, the first one by rotavirus serotype 1, and the second by a not identified rotavirus serotype. In this case two episodes of rotavirus-related diarrhoea were recorded, occurring eight months apart. Apparent infections were detected on two occasions involving the same child (Reg. 24.004), the first being associated with serotype 1, and the second with serotype 2. The fourth child (Reg. 24.097) had two successive infections by not determined rotavirus serotypes, without clinical manifestations; the first occurred at 24 months and the second at 28 months.