Alexandre Madi Fialho

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).

A novel avian virus with trisegmented double-stranded RNA and further observations on previously described similar viruses with bisegmented genome.

The occurrence in chickens of small viruses with bisegmented double-stranded RNA (dsRNA) genome is confirmed and a new virus with similar properties but with three genome segments is described. Both differ from birnaviruses (Intervirology 25, 141-143, 1986) in having indistinct surface structure, smaller diameters (35 nm), and higher buoyant density (1.4 g/ml) in CsCl but are similar in these respects to viruses previously described in several mammals (Lancet 2, 103-104, 1988; J. Gen. Virol. 69, 2749-2754, 1988; Res. Vet. Sci, in press) under the tentative name of picobirnaviruses (PBV). Genome segment length estimations gave values of 2.6 and 1.9 kbp for the avian PBV and 2.9, 2.4 and 0.9 kbp for the trisegmented viruses. The source and pathogenic potential of these viruses remain to be established.

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.

Noroviruses associated with outbreaks of acute gastroenteritis in the State of Rio Grande do Sul, Brazil, 2004-2011.

BACKGROUND Acute gastroenteritis norovirus (NoV) in a country of continental dimensions like Brazil has resulted in under-reporting of the number of outbreaks, as well as the genotypes associated. OBJECTIVES To demonstrate the role of NoV in outbreaks occurring in the State of Rio Grande do Sul, Southern Brazil, we determined its prevalence, as well as the genotypes associated, and evaluated clinical and epidemiological aspects. STUDY DESIGN NoV investigation was carried out in rotavirus group A negative stool samples from 2265 patients from 741 outbreaks that occurred in the State of Rio Grande do Sul, Brazil, during a period of eight years (2004-2011). NoV detection and nucleotide sequencing for genotype characterization was carried by using sets of primers targeting a conservative Rd-Rp polymerase genome region and the viral capsid gene, respectively. RESULTS NoVs were detected in 817 stool samples (36.1%) and associated with 327 outbreaks (44.1%). NoV GII.2, GII.3, GII.4, GII.6, GII.12, GII.13, GII.14, GII.15, GII.17, GII.21; and GI.1 and GI.3 were characterized. GII.4 was the most frequently detected (72.3%), with five variants identified (Asia_2003, Hunter_2004, Yerseke_2006a, Den_Haag_2006b, New Orleans_2009). This study describes the first detection of GI.1 and GII.13 and GII.15 in Brazil and demonstrates NoV winter-spring seasonality in this region of the country. CONCLUSIONS NoVs were responsible for almost 50% of outbreaks, with about 70% of them resulting from genotype GII.4 and its variants. The seasonality observed could help health authorities to establish a system of active surveillance in order to reduce NoV impact especially in congregate settings.

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.

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.

Detection and Molecular Characterization of Human Group C Rotavirus in Brazil

Objective: The aim of the present study was to characterize 24 isolates of group C rotavirus (RV-C) by analysis of DNA sequences of the VP4, VP6, VP7 and NSP4 genes of several Brazilian states. Methods: All RV-C strains were confirmed by polyacrylamide gel electrophoresis and were characterized by sequence and phylogenetic analysis of the genes encoding NSP4, VP6, VP4, and VP7 proteins. Results: Analysis of the NSP4 gene from Brazilian strains revealed that the isolates are more closely related to each other than to those of other strains previously published. The VP6 gene showed high homology to Indian strains. The sequences of VP4 and VP7 genes showed two lineages circulating in Brazil in the same region and at the same time. Conclusion: These results confirm the transmission of RV-C in Brazil. RV-C infection appears to occur occasionally despite the low detection rate of the virus.

Factors associated with rotavirus diarrhoea in children living in a socially diverse urban centre in Brazil

A case-control study, aimed at identifying factors associated with rotavirus diarrhoea cases presenting to health facilities, was conducted in children from low-income and middle-low-income families in Brazil. Cases were 390 children with diarrhoea and rotavirus in stools; controls were 1674 children without diarrhoea presenting to the same facilities. Data were collected by questionnaire and observations during home visits. Explanatory variables were grouped according to a conceptual model of causation. The ORs by non-conditional logistic regression and population-attributable fractions were calculated. Socioeconomic factors contributed a third of cases, followed by contact with diarrhoea cases and by not being breast fed. In cases aged <1 year, not being breast fed was the main determinant, followed by socioeconomic factors, and crowding and contact outside the home; in older children, socioeconomic factors followed by contact inside and outside the home were the main determinants. Environmental and sanitation variables were not associated with diarrhoea in the final model, and socioeconomic factors were only partly mediated by proximal variables. Transmission of rotavirus appears to be mostly by person-to-person contact, and shows marked social differentials not explained by the biological factors studied. The rotavirus vaccine is unlikely to protect against the full range of circulating genotypes of rotavirus, and understanding rotavirus epidemiology remains essential to the development of control policies.