Rotavirus analyses by SYBR Green real-time PCR and microbiological contamination in bivalves cultivated in coastal water of Amazonian Brazil.

The contamination of mussels and oysters by viruses and bacteria is often associated with water contamination and gastroenteritis in humans. The present study evaluated viral and bacterial contamination in 380 samples, from nine mollusk-producing regions in coastal water north of the Brazilian Amazon. Rotavirus contamination was studied for groups A to H, using a two-step SYBR Green RT-qPCR (quantitative reverse transcription polymerase chain reaction), and bacterial families Enterobacteriaceae, Vibrionaceae, and Aeromonadaceae by classical and molecular methods. From the 19 pools analyzed, 26.3% (5/19) were positive for group A Rotavirus, I2 genotype for VP6 region, without amplifications for groups B-H. Bacteriological analysis identified Escherichia coli isolates in 89.5% (17/19) with identification of atypical enteropathogenic E. coli aEPEC in 10.5% (2/19), Salmonella (Groups C1 and G) (10.5%, 2/19), Vibrio alginolyticus (57.9%, 11/19) V. parahaemolyticus (63.2%, 12/19), V. fluvialis (42.1%, 8/19), V. vulnificus (10.5%, 2/19), V. cholerae non-O1, non O139(10.5%, 2/19) and Aeromonas salmonicida (52.6%, 10/19). All the samples investigated presented some level of contamination by enterobacteria, rotavirus, or both, and these results may reflect the level of contamination in the Northern Amazon Region, due to the natural maintenance of some of these agents or by the proximity with human populations and their sewer.

G1P[8] Rotavirus in children with severe diarrhea in the post-vaccine introduction era in Brazil: Evidence of reassortments and structural modifications of the antigenic VP7 and VP4 regions.

Worldwide rotaviruses A (RVA) are responsible for approximately 215,000 deaths annually among children aged <5 years. RVA G1P[8] remains associated with >50% of gastroenteritis cases in this age group. The aim of this study was to assess the genetic variability of G1P[8] strains detected in children with severe diarrhea in Belém, Pará, Brazil, during the post-rotavirus vaccine introduction era. Phylogenetic analysis clustered the VP4 and VP7 genes of 40 samples selected between 2009 and 2011 into lineages found to be different from the Rotarix® vaccine strain. A detailed investigation of their complete genotype constellations identified 2 reassortant viruses (5%), resulting from reassortments between the genogroups Wa-like and DS-1-like (G1-P[8]-I1-R2-C1-M1-A1-N1-T2-E1-H1) and Wa-like and AU-1-like (G1-P[8]-I1-R3-C1-M1-A1-N1-T1-E1-H1) genotype constellations. A comparison of the amino acid residues presents in the antigenic epitopes of VP7 and VP4, showed differences in the electrostatic charges distribution, between wild type Brazilian strains and the Rotarix® and RotaTeq® vaccine strains. These findings reflect the structural analyses of the antigenic regions of VP7 and VP4 of the RVA G1P[8] in children with gastroenteritis in Northern Brazil raising the hypothesis that structural modifications at these sites over time may account for the emergence of new strains that could possibly pose a challenge to current vaccines.

Naturally occurring serotype 2/subgroup II rotavirus reassortants in northern Brazil.

Nine serotype 2 human rotavirus strains were isolated in a community-based longitudinal study in Northern Brazil. Five of these strains had a 'long' RNA electrophoretic pattern and all five strains were determined to belong to subgroup II by ELISA assay, in contrast to properties common to serotype 2 human rotaviruses previously characterized. Hybridization studies of one of these unusual strains with 32P-labelled mRNAs derived from the prototype human strains Wa (serotype 1, subgroup II) and S2 (serotype 2, subgroup I) suggested that it was generated by a reassortment event in nature, in which a subgroup II, 'long' electropherotype rotavirus exchanged its serotype-specific gene and gene number 10 for the equivalent genes from a serotype 2, 'short' electropherotype virus.

Concomitant rotavirus serotypes 1 and 4 infections in a diarrhoeic child from Belém, Brazil.

Concomitant serotypes 1 and 4 infections were detected in a 15-month old female child with community-acquired diarrhoea which lasted 7 days and coursed with moderate dehydration. The evidence for dual rotavirus infection was offered by the following findings: a) enzyme-linked immunosorbent assay (ELISA) positive reactions to both 1 and 4 serotypes; and b) extra-migrating bands at electrophoresis of RNA in polyacrylamide gel (PAGE). These results suggest that children living under poor sanitation conditions are heavily exposed to rotavirus infections; in addition, the co-circulation of different serotypes in the same setting sustains the current concept that a rotavirus vaccine should be multivalent, in order to protect children against the four epidemiologically important rotavirus G serotypes.

Pattern of acquisition of rotavirus antibody in children followed up from birth to the age of three years.

Nine hundred and forty-eight serum samples from 83 children living in Belem, Brazil, collected within their first three years of life, were tested for 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; subsequently, 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%) of them 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.

[Prospective study of rotavirus infections in Belém, Pará, Brazil: clinical and epidemiological features]. / Estudo prospectivo das infecções por rotavírus em Belém, Pará, Brasil: uma abordagem clínico-epidemiológica.

A prospective study of acute diarrhoeal diseases was carried out from April 1990 to September 1992 with the purpose of assessing the immunogenicity, safety and efficacy of a Rhesus-human reassortant rotavirus ("RRV-TV") vaccine, involving 540 children living in Belém, Pará, Brazil. As half of the children received placebo, this trial provided the opportunity of broadening the knowledge on both clinical and epidemiological aspects of rotavirus infection in the Amazon region. There were 2,789 diarrhoeal episodes during the above mentioned period, of which 86 (3.1%) associated with rotavirus; serotype 1 was the more prevalent, accounting for 67.9% of serotyped strains. Rates of 5.9 and 0.2 episodes of diarrhoea per child/year were noted for all cases and the rotavirus-related ones, respectively. This agent was the only pathogen found in 70.9% of the 86 rotavirus-related episodes of acute diarrhoea, whereas the most frequent associations involved Giardia intestinalis and enteropathogenic Escherichia coli, accounting for 7.0% and 11.6% of mixed infections,respectively. The monthly rates of rotavirus-related episodes of diarrhoea ranged from 0.8% to 9.6%, reaching the highest peaks during the dry months of the year. Means of clinical severity scores of 9.4 and 5.3 were recorded for the rotavirus-related episodes of diarrhoea and those of other aetiology, respectively.

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 Brazil's 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.

Atypical rotavirus among diarrhoeic children living in Belém, Brazil.

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 infections and in both cases the atypical strains were excreted at the time of the third infection, causing a mild and short-lasting disease.

[Temporal distribution of electrophoretic profiles of nucleic acid from rotavirus in the feces of children in Belém, Pará, Brazil]. / Distribuição temporal de perfis eletroforéticos de ácido nucleico de rotavírus em fezes de crianças em Belém, Pará.

Electrophoresis of rotavirus RNA in polyacrylamide gel was carried 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 electrophoretic 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, accounting for 65.2% of the classified strains.

Nosocomial transmission of an avian-like rotavirus strain among children in Belém, Brazil.

An atypical group-A rotavirus strain, with an electrophoretype displaying 5 segments in the first dsRNA size class, was detected among 3 hospitalized children less than 2 years old. Detection occurred initially 24 h after admission in a non-diarrhoeic child hospitalized because of acute respiratory infection. The second detection involved a child who occupied a different room within the same ward and who developed nosocomial diarrhoea 48 h later. A third case, also of hospital-acquired diarrhoea, was recorded in a child who occupied a bed in the same room as the second case and developed gastroenteritis 24 h following the second case's detection. In addition to the unusual, avian-like genomic profile, the strain was classified as serotype 2, based on a human VP7-specific enzyme-linked immunosorbent assay (ELISA). The question of whether these events reflect either a genomic rearrangement of a human rotavirus strain or a possible interspecies transmission will be further investigated through hybridization assays.

Detection and characterization of rotavirus G and P types from children participating in a rotavirus vaccine trial in Belém, Brazil.

This study sought the characterization of rotaviruses in a trial with a tetravalent rhesus-human rotavirus vaccine in Belém, Brazil in children who received three doses of vaccine or placebo in the 1st, 3rd and 5th months of life. Rotavirus electropherotypes, subgroups, G serotypes, G, [P] and [P], G genotypes were determined in 93.3%, 95.9%, 93.3%, 73.3%, 95.5% and 92.2% of isolates, respectively. Serotypes G1, G2 and G4 were detected in 58.9%, 30% and 4.4% of the cases, respectively. Rotavirus genotype G5 was detected for the first time in Northern region in 4.4% of the infections. Rotavirus genotypes P[8], P[4], P[6] and P[8 + 6] were detected in 54.5%, 26.7%, 12.2%, and 2.2% of the cases, respectively. The predominant genotypes were P[8], G1 and P[4], G2 with 53% and 26.6% of the infections, respectively. Unusual strains accounted for 20.5% including P[4], G1, P[6], G1, P[6], G4, P[6], G5, P[8], G2, P[8], G5. Mixed infections involving P[8 + 6], G2 and P[8 + 6], G1 were also noted. The neonatal P[6] strains associated with diarrhea were detected among children aged 9-24 months. To our knowledge, this study represents the first in Brazil to analyse, on molecular basis, rotavirus genotypes from children participating in a rotavirus vaccine trial. These results are of potential importance regarding future rotavirus vaccination strategies in Brazil.

Tetravalent rhesus-human rotavirus vaccine (RRV-TV) in Belém, Brazil: efficacy against prevailing P and G genotypes.

To determine the efficacy of a reassortant rhesus-human tetravalent rotavirus vaccine (RRV-TV) (4 x 10(4) pfu/dose) against P and G rotavirus genotypes, 90 positive samples were tested using reverse transcription-polymerase chain reaction. The efficacy of the RRV-TV vaccine against P[8] and G1 individually or in binary combination P[8], G1 was 72 per cent (p < 0.005) 61 per cent (p < 0.013), and 70 per cent (p < 0.009), respectively, only for the first year of follow-up. In the second year, as well as after 2 years of follow-up, no efficacy was observed to these genotypes. These data indicate that further studies with rotavirus vaccines should focus on the molecular characterization of rotaviruses genotypes, in order to see whether or not cross-protection among different G and P genotypes may occur as a result of common bearing of VP4 specificities.

Epidemiology of rotavirus subgroups and serotypes in Belem, Brazil: a three-year study.

Rotavirus subgroups and serotypes were determined in 61 rotavirus-positive faecal samples obtained from children living in Belém, Brazil, followed up from birth to 3 years of age. Fifty-five (90%) of the specimens were subgrouped and the serotypes of 30 (49%) of them were determined. Subgroup II was detected in 49 (89%) of the 55 subgrouped strains. Serotype 1 was present in 15 (50%) of the 30 serotyped samples; serotypes 2, 3 and 4 were found in 30%, 3.3% and 16.7% respectively, of these specimens. Absence of Vp7, the major outer capsid glycoprotein, did not allow serotyping in 21 (34.4%) of the 61 rotavirus-positive specimens, and an unidentifiable new serotype was found in faeces of one child. In addition, 4 samples were classified as subgroup II serotype 2 (which is very unusual). Twelve (80%) of the 15 serotype 1 (subgroup II) specimens were collected from children (5 of them asymptomatic) during their first year of life. All 9 serotype 2 (subgroups I, II, or not determined) samples were detected during the second and third years of life, 7 (77.8%) of them were related to apparent infections. The 5 serotype 4 (subgroup II) samples were obtained throughout the study period, and were associated with both symptomatic (3 cases) and asymptomatic infections. Thirteen children had more than 1 rotavirus infection. Three had 3 successive infections. In 3 cases, the initial infection (either symptomatic or asymptomatic) caused by serotype 1, was followed by a subsequent diarrhoeic episode associated with serotype 2.

Rotavirus G and P types in children from Belém, northern Brazil, as determined by RT-PCR: occurrence of mixed P type infections.

Fifty-four group A rotavirus-positive stool samples, obtained from children aged less than three years during a longitudinal (December 1982 to March 1986) study in Belém, Brazil, were re-examined. The samples were tested by reverse-transcription and polymerase chain reaction to determine their G-type and P-type specificity. Only 17 (32%) of these rotavirus strains could be successfully G- and P-genotyped. While 10 (59%) of the 17 strains showed single G- and P-type specificity, the remaining belonged to single G- and mixed P-genotypes. Rotavirus strains P[8], G1 and P[4], G1 predominated, accounting for 29% and 18% of the typed strains respectively. Mixed P-type infections caused by rotaviruses classified as P[8] + P[4], G1 were identified in 23% of cases. All but 3 of the 54 rotavirus strains displayed long genomic profiles, as demonstrated by the analysis of RNA by polyacrylamide gel electrophoresis. Most (70%) rotavirus strains with single G- and P-type specificity were detected during the first year of life, whereas 5 (71%) of the seven mixed P-type infections occurred throughout the second or third year of age. Reinfections were noted in two children, both of them being infected with P[8] + P[4], G1 rotavirus strains when aged 20 months. The high proportion of untypeable rotavirus strains suggests that unusual types may be circulating in Belém. In addition, the occurrence of mixed P-type infections in our region indicates the potential for reassortment between different rotavirus genogroups. Monitoring of these rotavirus strains may have important implication in the context of future strategies of rotavirus vaccination in Brazil.

Longitudinal study of rotavirus infections among children from Belém, Brazil.

From December 1982 to March 1986 a group of 80 children between 0 and 3 years old who lived in the peripheral area of Belém, Brazil, were followed up for episodes of diarrhoea. A total of 441 diarrhoeal episodes were recorded and 36 (8.2%) were associated with rotavirus. This agent was the only pathogen in 50% of rotavirus-related episodes of acute diarrhoea, and strains were characterized by analysis of RNA in polyacrylamide gels. Forty-one belonged to subgroup II (long pattern) and five to subgroup I. Reinfections by rotavirus were noted in 12 children involving either the same or different subgroups. Ten distinct electrophoretypes were detected in the study period and the predominant one had the '1N2L' profile. The cumulative age-specific attack rate for diarrhoea reached 2.8 by the end of the first year of life; a frequency of 2.3 episodes of diarrhoea per child per year was observed throughout the complete investigation. In comparing the age-specific attack rates for diarrhoea between breast-fed and bottle-fed children, a peak at 6 months of age was noted in the former, and at 1 month in the latter. A comparison by Fischer's exact test (P = 0.21) provided no evidence for protection against clinical rotavirus disease by maternal milk. By the same test, however (P = 0.021), we found significant evidence that early rotavirus infections were more likely to be asymptomatic and that infections after 4 months were more likely to be symptomatic. The clinical picture in children with rotavirus-related diarrhoea was more severe than in those suffering from acute diarrhoea due to another agent.

Prevalence of antibodies to Norwalk virus among Amerindians in isolated Amazonian communities.

The seroepidemiology of Norwalk virus infections was examined among Amerindians belonging to eight relatively isolated communities in the Amazon region by means of a new enzyme immunoassay using recombinant Norwalk virus antigen. The seroprevalence of antibodies to Norwalk virus ranged from 39% in the Maiogong to 100% in the Kubenkrankrein. The distribution of antibody levels varied greatly among groups; five of the eight communities had an antibody prevalence greater than 90% with many high values (> 100 units), while three had both a low seroprevalence and a preponderance of low values (< 100 units). While few children less than 5 years of age were sampled, no significant differences in antibody prevalence were noted among age groups, and the prevalence of antibody among children 5-10 years of age approached that of the older age groups. The low prevalence of titers of antibodies to Norwalk virus in several tribes living in these isolated Indian communities suggests that Norwalk virus may have been only recently introduced.

Rotavirus serotypes and electrophoretypes among children attending three paediatric hospitals in Belem, Brazil.

From March to September 1988 stool specimens of 101 hospitalized diarrhoeic infants and children, aged 1-24 months were examined by enzyme-linked immunosorbent assay (ELISA) for the presence of rotavirus antigen. This agent was found in 40 (40 per cent) of the 101 episodes of acute diarrhoea, and strains were both characterized by analysis of RNA in polyacrilamide gel and serotyped by ELISA using serotype-specific monoclonal antibodies. The highest frequency of rotavirus positivity was 80 per cent in the 16-18 month age group. All 11 (28 per cent) serotyped strains belonged to serotype 3 whereas absence of Vp7, the major outer capsid glycoprotein, did not allow serotyping in 29 (73 per cent) of the 40 rotavirus-positive specimens. Four distinct electrophoretypes were detected and the predominant one had the IbIIbIIIgIV a profile, accounting for 62 per cent of the classified strains. Unclear patterns did not allow us to determine the electrophoretype of six rotavirus strains. The clinical picture in study children was in general of moderate/severe nature.

Rotaviruses as a cause of nosocomial, infantile diarrhoea in northern Brazil: pilot study.

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. Cases 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 occurred 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).