Detection of a novel species A, DS-1-like, G4P[6] rotavirus strain from a Brazilian child with gastroenteritis.

Rotaviruses belonging to species A (RVA) remain among the most common causes of severe gastroenteritis in children aged <5 years, leading to substantial morbidity and mortality worldwide. Genome reassortment events between two human strains or human and animal strains represent one of the mechanisms which appear to generate the broad genetic variability of circulating. According to a nucleotide, sequence-based classification system, RVA strains are currently classified into three genotype constellations including Wa-like (genogroup I), DS-1-like (genogroup II), and AU-like (genogroup III). The present study reports the detection of an unusual RVA G4P[6] strain (coded as strain HSE005), which might have originated from a natural reassortment event between human and animal RVA strains. Molecular characterization of this isolate showed that it belonged to genogroup II, genotype G4P[6]. In addition, two genes (VP3 and NSP4) of this strain denoted evidence of reassortment events involving strains of distinct zoonotic evolutionary origins. Therefore, we propose that a new G4P[6] strain was identified, highlighting a possible first zoonotic transmission including a reassortment event that involved the VP3 gene.

Emergence of G12P[6] rotavirus strains among hospitalised children with acute gastroenteritis in Belém, Northern Brazil, following introduction of a rotavirus vaccine.

Species A rotavirus still remains a major cause of acute gastroenteritis in infants and young children. Globally, six genotypes (G1P[8], G2P[4], G3P[8], G4P[8], G9P[8] and G12P[8]) account for >90% of circulating strains; however, genotype G12 in combination with P[6] or P[9] has been detected at increasing rates. We sought to broaden our knowledge about the rotavirus strains circulating during the early post-vaccine-introduction period. Stool samples were obtained from children hospitalised for acute gastroenteritis in Belém, Northern Brazil, from May 2008 to May 2011 and examined by reverse transcription polymerase chain reaction and nucleotide sequencing. A total of 122 out of the original 1076 rotavirus strains were judged to be non-typeable in the first analysis and were therefore re-examined. G2P[4] was the most prevalent genotype (58.0%), followed by G1P[8] (16.9%), and G12P[6] (7.5%). G12P[6] strains were identified at similar rates during the first (2.5%) and second (3.9%) years, and the rate jumped to 15.6% in the third year. Analysis of VP7 sequences of the G12P[6] strains showed that they belonged to lineage III. In addition, co-circulating G12P[6] strains displaying long and short RNA patterns were found to belong to the Wa-like and DS-1-like constellation, respectively. Additional unusual circulating strains G12P[9] and G3P[9] were also identified. This hospital-based study showed a high prevalence of G12P[6] strains in the third year of surveillance. Our results highlight the need for continuous longitudinal monitoring of circulating rotavirus strains after introduction of rotavirus vaccines in Brazil and elsewhere.

Rotavirus antigenemia as a common event among children hospitalised for severe, acute gastroenteritis in Belém, northern Brazil.

BACKGROUND: Rotavirus antigenemia and RNAemia (the presence of rotavirus RNA in serum) have been commonly identified among paediatric patients with acute gastroenteritis. In this study we examined the association between rotavirus antigenemia and clinical features, and sought to determine the genotypes of rotaviruses detected in paired stool and serum samples. METHODS: Paired stool and serum samples were obtained from children hospitalised for acute gastroenteritis in Belém, Brazil, between June 2012 and June 2015. The 20-point Vesikari scoring system was used to assess the disease severity upon a retrospective medical record review. Stool and serum samples were primarily screened for the presence of rotavirus antigen using a commercial ELISA assay. The rotavirus isolates from stool and serum samples were genotyped by using the classical reverse-transcriptase polymerase chain reaction (RT-PCR) and/or through nucleotide sequencing of VP4 and VP7 genes. Viral load was estimated using real-time RT-PCR. RESULTS: In total rotavirus antigen was detected in 109 (24.2%) stool samples from 451 children, whereas antigenemia occurred in 38.5% (42/109) of these patients. We demonstrated that patients positive for rotavirus RNA in paired stool and serum samples were more likely to have a higher frequency of vomiting episodes in a 24-h period (p = 0.0035). Our findings also suggested that children not vaccinated against rotavirus are more likely to develop antigenemia, as compared to those given at least one vaccine dose (p = 0.0151). G12P [8] and G2P [4] genotypes were predominant throughout the study period, accounting for 52.3% (57/109) and 27.5% (30/109) of the typed isolates, respectively. Ten stool-serum pairs could be typed for VP4 and VP7 genes. Seven of these pairs showed concordant results with G2P [4] genotype being detected in stool and serum samples, whereas discrepancies between genotypes (G2P [4]/G2P[NT] and G12P [8]/G2P[NT]) were seen in three pairs. CONCLUSIONS: Rotavirus antigenemia and RNAemia occur in a significant number of children hospitalised for acute gastroenteritis in Belém, Brazil, and may contribute to a greater disease severity, particularly translated into a greater number of vomiting episodes. This study documented a high concordance of genotypes detected in a subgroup of paired stool and serum samples.

Analysis of a genotype G3P[9] rotavirus a strain that shows evidence of multiple reassortment events between animal and human rotaviruses.

The species A rotaviruses (RVA) are important gastroenteric pathogens that infect humans and animals. RVA genotype G3P[9] has been described in human-animal reassortment events, and the complexity of its hosts motivates the genetic investigation of this strain. Therefore, the aim of this study is to analyse a G3P[9] sample that was detected in a child with acute gastroenteritis. The 1A3739 sample featured the constellation G3P[9]-I18-R3-C3-Mx-A19-N3-T3-E3-H6. The sequence for VP3 gene was not obtained. The phylogeny showed a closer relationship among genes VP7, VP1, NSP3, NSP4, and NSP5 with genes of animal origin, such as chiropter, alpaca, equine, and simian. In addition, the genes VP6 and NSP1 belong to the new genotypes I18 and A19, respectively. The emergence of strains such as these can interfere with the effectiveness of the RVA vaccine, and continuous monitoring is therefore important. Additional studies are needed to determine the evolutionary source and to identify a possible reservoir of RVA in nature.

Efficacy, safety and effectiveness of licensed rotavirus vaccines: a systematic review and meta-analysis for Latin America and the Caribbean.

BACKGROUND: RotaTeq™ (RV5; Merck & Co. Inc., USA) and Rotarix™ (RV1, GlaxoSmithKline, Belgium) vaccines, developed to prevent rotavirus diarrhea in children under five years old, were both introduced into national immunization programs in 2006. As many countries in Latin America and the Caribbean have included either RV5 or RV1 in their routine childhood vaccination programs, we conducted a systematic review and meta-analysis to analyze efficacy, safety and effectiveness data from the region. METHODS: We conducted a systematic search in PubMed, EMBASE, Scielo, Lilacs and the Cochrane Central Register, for controlled efficacy, safety and effectiveness studies published between January 2000 until December 2011, on RV5 and RV1 across Latin America (where both vaccines are available since 2006). The primary outcome measures were: rotavirus-related gastroenteritis of any severity; rotavirus emergency department visits and hospitalization; and severe adverse events. RESULTS: The results of the meta-analysis for efficacy show that RV1 reduced the risk of any-severity rotavirus-related gastroenteritis by 65% (relative risk (RR) 0.35, 95% confidence interval (CI) 0.25; 0.50), and of severe gastroenteritis by 82% (RR 0.18, 95%CI 0.12; 0.26) versus placebo. In trials, both vaccines significantly reduced the risk of hospitalization and emergency visits by 85% (RR 0.15, 95%CI 0.09; 0.25) for RV1 and by 90% (RR 0.099, 95%CI 0.012; 0.77) for RV5. Vaccination with RV5 or RV1 did not increase the risk of death, intussusception, or other severe adverse events which were previously associated with the first licensed rotavirus vaccine. Real-world effectiveness studies showed that both vaccines reduced rotavirus hospitalization in the region by around 45-50% for RV5 (for 1 to 3 doses, respectively), and, by around 50-80% for RV1 (for 1 to 2 doses, respectively). For RV1, effectiveness against hospitalization was highest (around 80-96%) for children vaccinated before 12 months of age, compared with 5-60% effectiveness in older children. Both vaccines were most effective in preventing more severe gastroenteritis (70% for RV5 and 80-90% for RV1) and severe gastroenteritis (50% for RV5 and 70-80% for RV1). CONCLUSION: This systematic literature review confirms rotavirus vaccination has been proven effective and well tolerated in protecting children in Latin America and the Caribbean.

Phylogenetic analysis of human group C rotavirus in hospitalized children with gastroenteritis in Belém, Brazil.

Group C rotavirus (RVC) is potentially an important pathogen associated with acute gastroenteritis (AG), especially in outbreaks. This study aims to detect and molecularly characterize RVC in hospitalized children with AG in Belém, Brazil. From May 2008 to April 2011, 279 stools were subjected to reverse-transcription polymerase chain reaction targeting VP7, VP6, VP4, and NSP4 genes. RVC positivity rate was 2.1% (6/279) and phylogenetic analysis of positive samples yields genotype G4-P[2]-I2-E2. No evidence of zoonotic transmission and VP7 gene demonstrated close relationship with Asian strains. RVC surveillance is worth to expand information on evolutionary and epidemiological features of this virus.

High prevalence of G12P[8] rotavirus strains in Rio Branco, Acre, Western Amazon, in the post-rotavirus vaccine introduction period.

The present study aimed to provide a molecular characterization of circulating rotavirus (RVA) strains in Rio Branco, Acre, in the post-rotavirus vaccination period, particularly with regard to the emerging, increasingly prevalent G12P[8] genotype. A total of 488 fecal specimens from diarrheic and non-diarrheic children were obtained between January and December 2012. RVA detection was initially performed using enzyme-linked immunosorbent assay (ELISA) method, followed by reverse-transcription polymerase chain reaction (RT-PCR) using specific primers. RVA was detected in 18.3% (44/241) of the children with acute diarrhea and in 1.2% (3/247) of the non-diarrheic children (P < 0.001), with overall RVA-positivity of 9.6% (47/488). The most common genotype was G2P[4] with 43.2% (19/44) of the diarrheic cases, followed by G12P[8] (27.3%, 12/44), G3P[6] (18.2%, 8/44), G3P[8] (4.5%, 2/44), and G12P[6] (2.3%, 1/44). G12 samples belonged to lineage III and were from children aged 4-52 months. All of these children had acute diarrhea associated with fever (83.3%, 10/12) and vomiting (66.7%, 8/12). Most of the cases occurred in August (58.3%, 7/12), 75% (9/12) of which having received the full vaccination scheme with Rotarix™. For the first time G12 was reported at relative high prevalence in Brazil. Our findings warrant further monitoring studies on the molecular characterization of circulating RVA strains after rotavirus vaccine introduction in Brazil and elsewhere, since the occurrence of either unusual our emerging genotypes may pose a challenge to vaccination strategies.

Clinical Severity and Rotavirus Vaccination among Children Hospitalized for Acute Gastroenteritis in Belém, Northern Brazil.

In March 2006, Brazil introduced the monovalent rotavirus (RV) vaccine (Rotarix™) into the public sector. This study assessed the severity of rotavirus gastroenteritis (RVGE) according to the vaccination status among hospitalized children. We identified 1023 RVGE episodes among not vaccinated (n = 252), partially vaccinated (n = 156) and fully vaccinated (n = 615) children. Very severe gastroenteritis (scored ≥ 15) was reported in 16.7, 17.9 and 13.5% of not vaccinated, partially vaccinated and fully vaccinated children, respectively. There was a trend for a shorter duration of RV diarrhoea among vaccinated children than in not vaccinated children (p = 0.07). A protective effect of vaccination was noted when mean duration of symptoms and hospital stay are analysed, comparing unvaccinated, partially vaccinated and fully vaccinated children (p < 0.05). We showed a vaccination dose effect trend, with fully vaccinated children having less-severe RVGE than not vaccinated and partially vaccinated children.

Rotavirus strain surveillance for three years following the introduction of rotavirus vaccine into Belém, Brazil.

The monovalent human rotavirus (RV) vaccine, RIX4414 (Rotarix™, GlaxoSmithKline Biologicals) was introduced into Brazil's Expanded Program on Immunization in March 2006. One year after vaccine introduction, the G2P[4] strain was found to be predominant, with an apparent extinction of many non-G2 strains. This study investigated the diversity of circulating strains in the three years following RIX4414 introduction. Between May 2008 and May 2011, stool samples were collected from children aged ≥12 weeks who were hospitalized for severe lab confirmed RV-gastroenteritis (≥3 liquid or semi-liquid motions over a 24-h period for <14 days, requiring ≥1 overnight hospital stay and intravenous rehydration therapy) in Belém, Brazil. RV-gastroenteritis was detected by ELISA and the G- and P-types were determined by RT-PCR assays. During the first year of surveillance nucleotide sequencing was used for typing those samples not previously typed by RT-PCR. A total of 1,726 of 10,030 severe gastroentertis hospitalizations (17.2%) were due to severe RVGE. G2P[4] was detected in 57.2% of circulating strains over the whole study period, however it predominated during the first 20 months from May 2008 to January 2009. G1P[8] increased in the last part of the study period from May 2010 to May 2011 and represented 36.6% (112/306) of the circulating strains. G2P[4] was the predominant RV strain circulating during the first 20 months of the study, followed by G1P[8]. These findings probably reflect a natural fluctuation in RV strains over time, rather than a vaccine-induced selective pressure.

Diversity of rotavirus strains circulating in Northern Brazil after introduction of a rotavirus vaccine: high prevalence of G3P[6] genotype.

Rotavirus A (RVA) is the most common cause of severe acute gastroenteritis in infants and young children worldwide, causing 453,000 deaths annually. In Brazil, the most frequent genotype identified was G1 during almost three decades in the pre-vaccination period; however, after anti-rotavirus vaccine introduction, there was a predominance of G2 genotype. The aim of this study was to determine the G and P genotypes of rotaviruses isolated from children under 5 years of age with acute gastroenteritis in the Northern region of Brazil, and discuss the emergence of G3P[6] genotype. A total of 783 stool specimens were obtained between January 2011 and March 2012. RVA antigen was detected in 33% (272/783) of samples using a commercial enzyme-linked immunosorbent assay and type-specificity was determined by reverse-transcription polymerase chain reaction. The most common binary combination was G2P[4], representing 41% of cases, followed by G3P[6] (15%), G1P[8] (8%), G3P[8] (4%), G9P[8] (3%), and G12P[6] (2%). G3P[6] strains were analyzed further and phylogenetic analysis of VP7 gene showed that G3 strains clustered into lineage I and showed a high degree of amino acid identity with vaccine strain RV3 (95.1-95.6%). For VP4 sequences, G3P[6] clustered into lineage Ia. It was demonstrated by the first time the emergence of unusual genotype G3P[6] in the Amazon region of Brazil. This genotype shares neither VP7 nor VP4 specificity with the used vaccine and may represent a challenge to vaccination strategies. A continuous monitoring of circulating strains is therefore needed during the post-vaccine era in Brazil.

A multi-country study of intussusception in children under 2 years of age in Latin America: analysis of prospective surveillance data.

BACKGROUND: Intussusception (IS) is a form of acute intestinal obstruction that occurs mainly in infants and is usually of unknown cause. An association between IS and the first licensed rotavirus vaccine, a reassortant-tetravalent, rhesus-based rotavirus vaccine (RRV-TV), led to the withdrawal of the vaccine. New rotavirus vaccines have now been developed and extensively studied for their potential association with IS. This study aimed to describe the epidemiology and to estimate the incidence of IS in Latin American infants prior to new vaccine introduction. METHODS: Children under 2 years of age representing potential IS cases were enrolled in 16 centers in 11 Latin American countries from January 2003 to May 2005. IS cases were classified as definite, probable, possible or suspected as stated on the Brighton Collaboration Working Group guidelines. RESULTS: From 517 potential cases identified, 476 (92%) cases were classified as definite, 21 probable, 10 possible and 10 suspected for intussusception. Among the 476 definite IS cases, the median age at presentation was 6.4 months with 89% of cases aged <1 year. The male to female ratio was 1.5:1. The incidence of definite IS per 100,000 subject-years ranged from 1.9 in Brazil to 62.4 in Argentina for children <2 years of age, and from 3.8 in Brazil to 105.3 in Argentina for children aged <1 year. Median hospital stay was 4 days with a high prevalence of surgery as the primary treatment (65%). Most cases (88%) made a complete recovery, but 13 (3%) died. No clear seasonal pattern of IS cases emerged. CONCLUSIONS: This study describes the epidemiology and estimates the incidence of IS in Latin American infants prior to the introduction of new rotavirus vaccines. The incidence of IS was found to vary between different countries, as observed in previous studies. TRIAL REGISTRATION: Clinical study identifier 999910/204 (SERO-EPI-IS-204).

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

Noroviruses, a major cause of acute gastroenteritis outbreaks worldwide, are constantly evolving. This ability 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 in 2008. A strain detected in a stool sample from a child with norovirus-associated gastroenteritis, residing in an African-descendant semi-closed community of Pará State, was characterized as a novel intergenotype recombinant, GII.7/GII.20, as determined by partial sequencing and SimPlot analysis.

Burden and typing of rotavirus group A in Latin America and the Caribbean: systematic review and meta-analysis.

The efficacy of licensed rotavirus vaccines has only been shown against certain rotavirus group A (RV-A) types. It is critical to understand the burden of rotavirus gastroenteritis (RVGE) and its prevalent types to assess the potential impact of these vaccines in Latin America and the Caribbean (LA&C). We performed a systematic review and meta-analyses of all the available evidence reported from 1990 to 2009 on the burden of rotavirus disease and strains circulating in LA&C. Eligible studies--185 country-level reports, 174 951 faecal samples--were selected from MEDLINE, Cochrane Library, EMBASE, LILACS, regional Ministries of Health, PAHO, regional proceedings, doctoral theses, reference lists of included studies and consulting experts. Arc-sine transformations and DerSimonian-Laird random-effects model were used for meta-analyses. The proportion of gastroenteritis cases due to rotavirus was 24.3% (95%CI 22.3-26.4) and the incidence of RVGE was 170 per 1000 children-years (95%CI 130-210). We estimated a global annual mortality for 22 countries of 88.2 (95%CI 79.3-97.1) deaths per 100 000 under 5 years (47 000 deaths).The most common G type detected was G1 (34.2%), followed by G9 (14.6%), and G2 (14.4%). The most common P types detected were P[8] (56.2%), P[4] (22.1%) and P[1] 5.4%, and the most prevalent P-G type associations were P[8]G1 17.9%, P[4]G2 9.1% and P[8]G9 8.8%. In the last 10 years, G9 circulation increased remarkably and G5 almost disappeared. More recently, G12 appeared and P[4]G2 re-emerged. To our knowledge, this is the first meta-analysis of rotavirus infection and burden of disease in LA&C.

Oral live attenuated human rotavirus vaccine (Rotarix™) offers sustained high protection against severe G9P[8] rotavirus gastroenteritis during the first two years of life in Brazilian children.

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.

The burden of norovirus disease in children: a multi-country study in Chile, Brazil, Thailand and the Philippines.

BACKGROUND: Noroviruses (NoVs) cause acute gastroenteritis (AGE) worldwide, affecting children in particular. We aimed to estimate the burden of disease due to NoV among children aged <6 years in Brazil, Chile, Philippines and Thailand. METHODS: This was a prospective, hospital-based, observational study. Children were recruited over one year between 2014 and 2017. Four cohorts were analysed: community-acquired AGE outpatients and inpatients, nosocomial AGE inpatients, and asymptomatic outpatients. We collected demographic and clinical data, and a stool sample that was tested for NoV. Positive samples were tested for Rotavirus (RV) and NoV-genotyped. Disease severity was assessed by the Vesikari and modified Vesikari scores. Prevalence and incidence of NoV-AGE were estimated by cohort and country. RESULTS: 1637 participants yielded valid laboratory results. The proportion of NoV-positive cases was 23.8% (95% CI 20.8-27.2) in the outpatient cohort, 17.9% (15.0-21.3) in the hospital cohort, 21.4% (12.7-33.8) in the nosocomial cohort and 9.6% (6.9-13.2) in the asymptomatic cohort. Genotype GII.4 was predominant (58%). Less than 4% samples had RV coinfection. In general, NoV-positive subjects had more severe presentations than NoV-negative subjects. CONCLUSIONS: NoV caused AGE with substantial burden throughout the studied settings, with higher relative frequency in Brazil where RV vaccination coverage is high.

Efficacy and safety of an oral live attenuated human rotavirus vaccine against rotavirus gastroenteritis during the first 2 years of life in Latin American infants: a randomised, double-blind, placebo-controlled phase III study.

BACKGROUND: Peak incidence of rotavirus gastroenteritis is seen in infants between 6 and 24 months of age. We therefore aimed to assess the 2-year efficacy and safety of an oral live attenuated human rotavirus vaccine for prevention of severe gastroenteritis in infants. METHODS: 15 183 healthy infants aged 6-13 weeks from ten Latin American countries randomly assigned in a 1 to 1 ratio to receive two oral doses of RIX4414 or placebo at about 2 and 4 months of age in a double-blind, placebo-controlled phase III study were followed up until about 2 years of age. Primary endpoint was vaccine efficacy from 2 weeks after dose two until 1 year of age. Treatment allocation was concealed from investigators and parents of participating infants. Efficacy follow-up for gastroenteritis episodes was undertaken from 2 weeks after dose two until about 2 years of age. Analysis was according to protocol. This study is registered with ClinicalTrials.gov, number NCT00140673 (eTrack444563-023). FINDINGS: 897 infants were excluded from the according-to-protocol analysis. Fewer cases (p<0.0001) of severe rotavirus gastroenteritis were recorded for the combined 2-year period in the RIX4414 group (32 [0.4%] of 7205; 95% CI 0.3-0.6) than in the placebo group (161 [2.3%] of 7081; 1.9-2.6), resulting in a vaccine efficacy of 80.5% (71.3-87.1) to 82.1% (64.6-91.9) against wild-type G1, 77.5% (64.7-86.2) against pooled non-G1 strains, and 80.5% (67.9-88.8) against pooled non-G1 P[8] strains. Vaccine efficacy for hospital admission for rotavirus gastroenteritis was 83.0% (73.1-89.7) and for admission for diarrhoea of any cause was 39.3% (29.1-48.1). No cases of intussusception were reported during the second year of follow-up. INTERPRETATION: Two doses of RIX4414 were effective against severe rotavirus gastroenteritis during the first 2 years of life in a Latin American setting. Inclusion of RIX4414 in routine paediatric immunisations should reduce the burden of rotavirus gastroenteritis worldwide.

Safety and efficacy of an attenuated vaccine against severe rotavirus gastroenteritis.

BACKGROUND: The safety and efficacy of an attenuated G1P[8] human rotavirus (HRV) vaccine were tested in a randomized, double-blind, phase 3 trial. METHODS: We studied 63,225 healthy infants from 11 Latin American countries and Finland who received two oral doses of either the HRV vaccine (31,673 infants) or placebo (31,552 infants) at approximately two months and four months of age. Severe gastroenteritis episodes were identified by active surveillance. The severity of disease was graded with the use of the 20-point Vesikari scale. Vaccine efficacy was evaluated in a subgroup of 20,169 infants (10,159 vaccinees and 10,010 placebo recipients). RESULTS: The efficacy of the vaccine against severe rotavirus gastroenteritis and against rotavirus-associated hospitalization was 85 percent (P<0.001 for the comparison with placebo) and reached 100 percent against more severe rotavirus gastroenteritis. Hospitalization for diarrhea of any cause was reduced by 42 percent (95 percent confidence interval, 29 to 53 percent; P<0.001). During the 31-day window after each dose, six vaccine recipients and seven placebo recipients had definite intussusception (difference in risk, -0.32 per 10,000 infants; 95 percent confidence interval, -2.91 to 2.18; P=0.78). CONCLUSIONS: Two oral doses of the live attenuated G1P[8] HRV vaccine were highly efficacious in protecting infants against severe rotavirus gastroenteritis, significantly reduced the rate of severe gastroenteritis from any cause, and were not associated with an increased risk of intussusception. (ClinicalTrials.gov numbers, NCT00139347 and NCT00263666.)

Development of a microtiter plate hybridization-based PCR-enzyme-linked immunosorbent assay for identification of clinically relevant human group A rotavirus G and P genotypes.

A microtiter plate hybridization-based PCR-enzyme-linked immunosorbent assay (PCR-ELISA) has been used for the detection and identification of a variety of microorganisms. Here, we report the development of a PCR-ELISA for the identification of clinically relevant human rotavirus VP7 (G1 to G6, G8 to G10, and G12) and VP4 (P[4], P[6], P[8], P[9], and P[14]) genotypes. The G and P types of reference human and animal rotavirus strains for which specific probes were available were correctly identified by the PCR-ELISA. In addition, reference strains bearing G or P genotypes for which specific probes were unavailable, such as G11, G14, P[3], P[10], and P[11], did not display any cross-reactivity to the probes. The usefulness of the assay was further evaluated by analyzing a total of 396 rotavirus-positive stool samples collected in four countries: Brazil, Ghana, Japan, and the United States. The results of this study showed that the PCR-ELISA was sensitive and easy to perform without the use of any expensive and sophisticated equipment, the reagents used are easy to obtain commercially and advantageous over multiplex PCR since more than one type-specific probe is used and the selection of probes is more flexible.

Evidence for zoonotic transmission of group C rotaviruses among children in Belém, Brazil.

The prevalence and potential zoonotic transmission of group C rotavirus (RVC) were examined by testing fecal samples collected from children during a longitudinal study that was carried out in the outskirts of Belém, Brazil, from December 1982 to March 1986. The study involved a group of 30 children who were followed from birth to 3 years. Of the 77 samples tested from 29 children, 5 (6.5%) were positive for human and 3 (4%) for porcine RVC by using nested PCR assay with primers specific for VP6 gene of human or porcine RVC and by Southern hybridization using a probe specific for VP6 gene of both human and porcine RVC. In addition, a total of 59 fecal specimens from the 30th child were tested, 1 (1.7%) and 14 (23.7%) were positive for human and porcine RVC, respectively. Partial nucleotide sequences of VP6 gene demonstrated that the six human strains detected in Brazil were homologous with other human RVC, and 14 of the 17 porcine RVC strains examined showed a complete homology among themselves but differed slightly from the porcine Cowden strain, suggesting that a single porcine RVC strain was circulating in Belém. This study is the first to provide evidence for transmission of RVC from swine to human. They also indicate that both human and porcine RVC were endemic in Belém.

Molecular characterization of human erythrovirus B19 strains obtained from patients with several clinical presentations in the Amazon region of Brazil.

BACKGROUND: Human erythrovirus B19, endemic in the Amazon region since 1990, is associated with a wide spectrum of clinical presentations. OBJECTIVES: To assess the prevalence of erythrovirus B19 infection and the relative frequency of erythrovirus B19 genotypes in patients in the Amazon region with various clinical presentations. STUDY DESIGN: A total of 487 clinical samples obtained from patients with symptoms suggestive of erythrovirus infection were tested using specific IgM and IgG antibody assays (ELISA) and PCR for viral DNA detection. Partial VP1 and VP2 regions were sequenced and genotyped by phylogenetic reconstruction. RESULTS: B19 DNA was detected in 117 (24%) of 487 samples. Of these, 106 (91%) isolates were genotype 1 and 11 (9%) were genotype 3. No genotype 2 was found. Genotype 1 had three clusters (A1, A2 and B) and all genotype 3 sequences were subtype 3b. All patients with hematological disorders within cluster B of genotype 1 were infected by the same B19 lineage, suggesting that this lineage of B19 may have been transmitted via transfusion of blood products. CONCLUSION: We reported two genotypes, 1 and 3b, with three genotype 1 clusters co-circulating in the Amazon region during the past 10 years.