Rosane Maria Santos de Assis

Laboratory-based rotavirus surveillance during the introduction of a vaccination program, Brazil, 2005-2009

Background: Brazil introduced universal antirotavirus vaccination in March 2006. This article reports the results of rotavirus A (RV-A) surveillance from January 2005 to December 2009. Methods: A total of 6109 fecal samples were collected in 18 Brazilian states. RV-A was detected by enzyme immunoassay and/or polyacrylamide gel electrophoresis, and genotyped through seminested reverse transcription-polymerase chain reaction. Results: RV-A was detected in 20.3% (n = 1242) of the samples. Among children less than 2 years old, regardless the antirotavirus vaccination status, the rates of RV-A detection were 33.8% in 2005, 23.7% in 2006, 16.8% in 2007, 22.9% in 2008, and 18.3% in 2009 (P < 0.001; χ2 test for linear trend). Among RV-A-positive samples, genotype G1P[8] or G1P[not typed(NT)] was detected in 14% in 2005, 12.3% in 2006, 9.5% in 2007, 0.7% in 2008, and 20.4% in 2009; G2P[4]/G2P[NT] was characterized in 9% in 2005, 49% in 2006, 66% in 2007, 85% in 2008, and 37.5% in 2009; G3P[8]/G3P[NT] was observed in 8.7% in 2005, 3.5% in 2006, and 5.7% in 2009; G9P[8]/G9P[NT] was observed in 52% in 2005, 22% in 2006, 12.3% in 2007, 3.2% in 2008, and 3.4% in 2009. Conclusions: Our data demonstrate the reemergence of RV-A genotype G2P[4] in Brazil from 2005 to 2008, and that the rate of G2P[4] detection decreased in 2009, probably reflecting natural oscillations of RV-A genotypes.

Rotavirus Genotype Distribution after Vaccine Introduction, Rio de Janeiro, Brazil

Brazil introduced rotavirus vaccination in March 2006. We studied 133 rotavirus-positive fecal samples collected from February 2005 through December 2007. Genotype G2P[4] was found in 1.4% of samples in 2005, in 44% in 2006, and in 96% in 2007. Rotavirus detection rate decreased from 38% in 2005 to 24% in 2007 (p = 0.012).

Detection and molecular characterization of group A rotavirus from hospitalized children in Rio de Janeiro, Brazil, 2004

Rotavirus is a major cause of infantile acute diarrhea, causing about 440,000 deaths per year, mainly in developing countries. The World Health Organization has been recommending the assessment of rotavirus burden and strain characterization as part of the strategies of immunization programs against this pathogen. In this context, a prospective study was made on a sample of 134 children with acute diarrhea and severe dehydration admitted to venous fluid therapy in two state hospitals in Rio de Janeiro, Brazil, from February to September 2004. Rotavirus where detected by polyacrylamide gel electrophoresis (PAGE) and by an enzyme-linked immunoassay to rotavirus and adenovirus (EIARA) in 48% of the children. Positive samples for group A rotavirus (n = 65) were analyzed by reverse transcription/heminested multiplex polymerase chain reaction to determine the frequency of G and [P] genotypes and, from these, 64 samples could be typed. The most frequent G genotype was G1 (58%) followed by G9 (40%). One mixed infection (G1/G9) was detected. The only [P] genotype identified was [8]. In order to estimate the rotavirus infection frequency in children who acquired diarrhea as hospital infection in those hospitals, we studied 24 patients, detecting the pathogen in 41% of them. This data suggest that genotype G9 is an important genotype in Rio de Janeiro, with implications to the future strategies of vaccination against rotavirus, reinforcing the need of continuous monitoring of circulating strains of the pathogen, in a surveillance context.

Rotavirus Strain Diversity in Rio de Janeiro, Brazil: Characterization of VP4 and VP7 Genotypes in Hospitalized Children

Rotavirus strains from 91 patients treated at a childrens hospital from 1996 to 1998 in Rio de Janeiro, Brazil, were characterized by electropherotyping, reverse transcription-PCR amplification for P and G genotypes, and Southern hybridization. Results obtained showed that following predominant [P],G type combination: P[4], G2 (21 per cent), P[8], G1 (17 per cent), P[8], G3 (13 per cent), which are prevalent throughout the world. However, an unexpected number of cases were associated with uncommon genotypes: P[8], G2 (13 per cent), P[8], G5 (11 per cent), P[8], G9 (7 per cent), P[8], G10 (4 per cent), P[6], G4 (3 per cent), P[6], G3 (1 per cent), P[4], G9 (1 per cent), and P[6], G9 (1 per cent). Mixed infections with more than one type were identified in only two cases and 16 per cent of the samples were not G and/or P typeable. A subset of G types was confirmed by Southern hybridization and chemiluminescent detection. Rotavirus seasonal distribution was observed between April and July. The contribution of the results obtained in the present investigation corroborates the required epidemiological surveillance for rotavirus infection in Brazil.

Rotavirus epidemiology before and after vaccine introduction

OBJECTIVES To evaluate the prevalence and circulation of rotavirus genotypes before and after the introduction of oral vaccine against human rotavirus, and to check for a possible change in the age of occurence of the infection by RV-A. METHODS This was a cross-sectional study conducted between 2002-2011, in the city of Juiz de Fora, state of Minas Gerais, Brazil. A total of 1,144 diarrheal stool specimens were obtained from nonhospitalized children aged between 0 and 5 years, and analyzed by polyacrylamide gel electrophoresis and reverse-transcription polymerase chain reaction for genotype characterization. Data on prevalence and age distribution of rotavirus cases were analyzed through the chi-squared test (p < 0.05), using SPSS, release 13.0. RESULTS Rotavirus infection was detected in 9.35% (107/1,144) samples, with prevalence rates ranging from 11.12% (90/809) in the pre-vaccine to 5.07% (17/335) in the post-vaccine period (p = 0.001). Among the samples tested, the most frequently detected genotypes were G1P[6] (6/33 = 18.2%) in the period between 2002 and 2005 and G2P[4] in 2006 (11/33 = 33.3%) and in the period between 2007 and 2011 (5/33 = 15.2%). There was also a significant reduction in the number of cases of rotavirus disease in children aged between 0 and 36 months after the vaccine introduction. CONCLUSIONS The study evidenced a significant decrease in the prevalence of rotavirus, mainly in children aged between 0 and 36 months in the 2007-2011 period, as well as a reduction in G1 genotype circulation.

Outbreak of acute gastroenteritis in young children with death due to rotavirus genotype G9 in Rio Branco, Brazilian Amazon region, 2005

BACKGROUND An epidemic of acute gastroenteritis occurred in Rio Branco City, Acre State, in Brazils Amazon region in 2005. An investigation was conducted to confirm the etiology and identify possible risk factors for death. METHODS Rio Branco municipality surveillance data for the period May to October 2005 were reviewed. In a case-control study, children who died following acute gastroenteritis were compared to age-matched controls with acute gastroenteritis who survived. Rotavirus A (RV-A) was investigated in 799 stool samples and genotyped by reverse transcription polymerase chain reaction (RT-PCR). RESULTS The cumulative incidence of diarrhea in children aged <5 years was 21%. A fatal outcome was significantly associated with uncovered household water storage containers. RV-A was identified in 88% of samples and G9 was the prevalent genotype (71%). CONCLUSIONS Oral rehydration solution and boiling or chlorinating drinking water likely limited mortality. This epidemic was caused by RV-A genotype G9. After the outbreak, a rotavirus vaccine was introduced into the official childhood immunization schedule in Brazil.

An outbreak of gastroenteritis associated with astrovirus serotype 1 in a day care center, in Rio de Janeiro, Brazil

Between June 4th and June 20th 1996 rotavirus, adenovirus, and astrovirus (HAstrV) were investigated in fecal samples from 27 children under three years old with acute diarrhea, attending the Bertha Lutz day care center, in Rio de Janeiro. All fecal samples were analyzed by polyacrylamide gel electrophoresis (PAGE), reverse transcriptase polymerase chain reaction (RT-PCR), enzyme immunoassays (EIA), and electron microscopy (EM). Nine of them (33%) showed positive results for HAstrV by at least one of the employed methodologies. Eight were positive by RT-PCR and EIA, and six by EM. All positive samples were inoculated onto HT-29 (human colon adenocarcinoma) cultured cells for HAstrV isolation and seven were positive after three passages. The sequencing analysis of eight RT-PCR products (449 bp) from gene that codifies VP2 protein, showed a total nucleotide identity among them and 98% with HAstrV-1 (strain Oxford type 1). This is the first report of a gastroenteritis outbreak associated with HAstrv-1 in a day care center in Rio de Janeiro and it reinforces the importance of this virus in association with infantile acute gastroenteritis.

Prevalence and genomic characterization of G2P(4) group A rotavirus strains during monovalent vaccine introduction in Brazil

This study aims to: estimate the prevalence of G2P[4] rotaviruses in Brazil between 2001-2011 from patients with acute gastroenteritis; perform phylogenetic analyses of G2P[4] Brazilian strains (from vaccinated and non-vaccinated children) based on VP7 and VP8(∗) encoding genes and analyze the antigenic regions of these proteins comparing with RV1; and assess the full genetic background of eleven selected Brazilian strains. The G2P[4] detection rate among RVA positive samples was 0/157 in 2001, 3/226 (1.3%) in 2002, 0/514 in 2003, 0/651 in 2004, 31/344 (9%)/2005, 112/227 (49%)/2006, 139/211 (66%)/2007, 240/284 (85%)/2008, 66/176 (37.5%)/2009, 367/422 (87%)/2010 and 75/149 (50%)/2011. For the VP7 and VP8(∗) encoding genes, 52 sequences were analyzed and shared up to 99% nucleotide identity with other contemporary G2P[4] strains detected worldwide, grouping into different clusters. Most differences inside antigenic epitopes of VP7 and VP8(∗) have been maintained in the G2P[4] Brazilian strains along the years, and all were present before RV1 introduction. Eleven G2P[4] strains (4-vaccinated/7-non-vaccinated) were completely characterized and possessed the typical DS-1-like genotype constellation (G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2) sharing up to 99% of nucleotide identity with contemporary worldwide strains. Reassortments between Brazilian G2P[4] human strains were observed. In conclusion, the data obtained in the current study suggests that implementation of RV1 vaccination might not influence the genetic diversity observed in G2P[4] analyzed strains. Several factors might have contributed to the increased prevalence of this genotype in Brazil since 2005: the introduction of RV1 into the Brazilian National Immunization Program has resulted in a decrease in the relative prevalence of predominant Wa-like RVA strains facilitating the increase of the heterotypic (DS-1-like) RVA strain G2P[4] in the Brazilian population; the genetic diversity found in different geographical regions throughout the years before, and after the introduction of RV1; the long period of low or no circulation of this genotype in Brazil previous to RV1 introduction could have created favorable conditions for the accumulation of immunological susceptible individuals.

Phylogenetic analysis of human P[8]G9 rotavirus strains circulating in Brazil reveals the presence of a novel genetic variant

BACKGROUND Group A rotavirus (RV-A) genotype P[8]G9 has emerged as one of the leading causes of gastroenteritis in children worldwide and currently is recognized as one of the five most common genotypes detected in humans. High intragenotype diversity in G9 RV-A has been observed, and nowadays, based on the genetic variability of the VP7 gene, six different phylogenetic lineages and eleven sublineages were described. OBJECTIVES To study the degree of genetic variation and evolution of Brazilian P[8]G9 RV-A strains. STUDY DESIGN Phylogenetic analysis of 19 P[8]G9 RV-A strains isolated from 2004 to 2007 in five different Brazilian states was conducted using the NSP1, NSP3, NSP5, VP4 and VP7 genes. For the VP4 and VP7 genes, 3D protein structure predictions were generated to analyze the spatial distribution of amino acid substitutions observed in Brazilian strains. RESULTS Based on the phylogenetic analyses, all Brazilian strains clustered within lineage G9-III and P[8]-3 for VP7 and VP4, respectively, and were classified as genotype A1, T1 and H1 for the NSP1, NSP3 and NSP5 genes, respectively. Interestingly, all the strains isolated in Acre State (Northern Brazil) formed a closely related cluster clearly separated from the other Brazilian and prototype strains with regard to the five genes studied. Unique amino acid substitutions were observed in Acre strains in comparison with the prototype and Brazilian strains. CONCLUSION Inclusion of Acre strains in the phylogenetic analysis revealed the presence of a novel genetic variant and demonstrated a diversification of P[8]G9 rotaviruses in Brazil.

G1P[8] species A rotavirus over 27 years--pre- and post-vaccination eras--in Brazil: full genomic constellation analysis and no evidence for selection pressure by Rotarix® vaccine.

Epidemiological data on species A rotavirus (RVA) infections have demonstrated the genetic diversity of strains circulating worldwide. Many G and P genotype combinations have been described over the years, varying regionally and temporally, especially in developing countries. However, the most common G and P genotype combinations identified in RVA human strains worldwide are G1P[8], G2P[4], G3P[8], G4P[8] and G9P[8]. RVA genotype G1P[8] strains are responsible for more than 50% of child infections worldwide and component of the two vaccines (Rotarix® [RV1] and RotaTeq® [RV5]) licensed globally. For a better understanding of the evolutionary mechanisms of this genotype in Brazil, phylogenetic analyses based on the 11 RVA genome segments (genomic constellation) from 90 G1P[8] RVA strains collected in two eras - (i) pre-vaccination with RV1 (1996-February 2006); (ii) post-vaccination (March 2006-2013) - in different Brazilian states were performed. The results showed the Wa-like genomic constellation of the Brazilian G1P[8] strains with a I1-R1-C1-M1-A1-N1-T1-E1-H1 specificity, except for two strains (rj14055-07 and ba19030-10) that belong to a I1-R1-C1-M1-A1-N1-T3-E1-H1 genomic constellation, evidencing the occurrence of reassortment (Wa-like×AU-1-like) of the NSP3 gene. Reassortment events were also demonstrated between Brazilian G1P[8] strains and the RV1 vaccine strain in some genes in vaccinated and unvaccinated children. VP7 and VP8* antigenic site analysis showed that the amino acid substitutions observed in samples collected after the introduction of RV1 in Brazil were already detected in samples collected in the 1980s and 1990s, suggesting that mass Brazilian RV1 vaccination had no impact on the diversity observed inside antigenic sites for these two proteins.