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The association between rotavirus serotypes and severity is not well established. Analysis of a clinical trial conducted in Latin America points at more-severe disease associated with serotype G9. Thus, demonstration of efficacy against G9 will be an important asset of any rotavirus vaccine to be introduced into a Latin American country or any country where G9 has been shown to be prevalent.
An oral, human-derived monovalent (G1P1A) rotavirus vaccine, strain RIX4414, has been developed by GlaxoSmithKline, Rixensart, Belgium. The safety, immunogenicity and efficacy of this vaccine were evaluated in a randomized, double-blind, placebo-controlled, phase IIb trial conducted in Brazil, Mexico and Venezuela. Healthy infants were given two doses of vaccine (104.7, 105.2 or 105.8 ffu) or placebo at age 2 and 4 months, with routine DTPw-HBV and Hib vaccines. OPV was given separately, at least 2 weeks before or after administration of the study vaccine. A total of 2155 infants were enrolled, of whom 1618 received one of the three vaccine viral concentrations and 537 were given placebo. Analysis of efficacy included diarrheal episodes occurring from 2 weeks after second dose until one year of age. Efficacy rates against any rotavirus gastroenteritis, severe rotavirus gastroenteritis and hospitalizations for rotavirus disease were as high as 70% (46-84%; 95%CI), 86% (63-96%; 95%CI), and 93% (54-100%; 95%CI), respectively. For non-G1 (mainly G9) serotypes, RIX4414 vaccine conferred protection as high as 83% (40-97%; 95%CI) against severe gastroenteritis. A decrease was noted in the incidence of severe rotavirus-related gastroenteritis after first dose. It is demonstrated that two doses of RIX4414 are highly efficacious against severe rotavirus gastroenteritis and hospitalization, including disease caused by non-G1 strains, namely G9 serotypes.
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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.
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.
Human herpesvirus 8 (HHV-8) epidemiology in Brazilian Amerindians was studied. Use of an immunofluorescence (IFA) test for latent antibody demonstrated that the prevalence of HHV-8 in 781 Amerindians of diverse tribes (overall, 53% prevalence) was not related to language group or sex but rather increased gradually from 41% in children <10 years of age to 65% in adults >/=30 years of age. In IFA-positive subjects, HHV-8 DNA was detected in 3 (16%) of 19 mononuclear cell samples from peripheral blood and in 1 of 16 saliva samples. The sequences of conserved ORF22 and K6 genes were typical of HHV-8, but the variable K1 gene sequences were only 70%-75% identical to other known HHV-8 strains. Thus, a new HHV-8 subtype, E, is hyperendemic in Brazilian Amerindians, although Kaposi's sarcoma has not been reported. Transmission is probably oral rather than sexual. The limited genetic pool in isolated groups may permit more frequent transmission of a virus with a low prevalence in heterogeneous populations.
Worldwide, rotaviruses account for more than 125 million cases of infantile gastroenteritis and nearly 1 million deaths per year, mainly in developing countries. Rather than other control measures, vaccination is most likely to have a major impact on rotavirus disease incidence. The peak incidence of rotavirus diarrhea occurs between 6 and 24 months of age. In developing countries, however, cases are not uncommon among children younger than 6 months. G serotypes 1 to 4 are responsible for most disease, but there are indications that in Brazil that G type 5 is of emerging epidemiological importance. Both homotypic and heterotypic responses are elicited during natural rotavirus infection, and the immunological response at the intestinal mucosal surface is probably the more consistent predictor of clinical immunity. With the primary objective of protecting children against life-threatening dehydrating diarrhea, many approaches to rotavirus vaccine development have been attempted. One vaccine, the tetravalent rhesus-human reassortant rotavirus vaccine (RRV-TV), was given licensing approval in the United States of America, introduced to the market, and later withdrawn. A number of studies have found better efficacy of RRV-TV in developed countries than in developing ones. Field trials with a 4 x 10(4) plaque-forming units (PFU) preparation of RRV-TV have been carried out in two countries in Latin America, Brazil and Peru. Those trials yielded protective efficacy rates against all rotavirus diarrhea ranging from 18% to 35%. Data from a large catchment trial in Venezuela with a higher RRV-TV dose, of 4 x 10(5) PFU/dose, indicated an efficacy rate of 48% against all rotavirus diarrhea and 88% against severe rotavirus diarrhea. It appears that breast-feeding does not compromise the efficacy of RRV-TV if three doses of the vaccine are administered. Similarly, possible interference of oral poliovirus vaccine with the "take" of the rotavirus vaccine can be overcome by giving three doses of the rotavirus vaccine or by using a higher-titer formulation of it. Wild enteroviruses, however, may cause primary rotavirus vaccine failure in developing countries. Studies in Peru with RRV-TV have shown a trend towards higher vaccine efficacy rates against "pure" (rotavirus-only) diarrheal episodes. Economic analyses made in the United States indicate that a vaccine that costs less than US$ 9 per dose would lead to a net savings in medical costs. To date, however, cost-benefit studies have not been done in developing countries. In the future, it is possible that some Latin American countries might adapt their polio production facilities to the preparation of rotavirus vaccines for human use. A year after RRV-TV was licensed for vaccination of infants in the United States, the occurrence of intussusception as an adverse event led to the vaccine's withdrawal from the market. The implications of that action, particularly for Latin America, will be addressed in this article, including the need to explore alternative rotavirus candidate vaccines, particularly through the conduct of parallel clinical trials in both developed and developing countries.
From November 1992 to November 1994 stool samples were obtained from 237 children admitted to a public hospital in Belém. Rotaviruses were detected in 19.3 per cent (60/310) of faecal samples. Of these, 32.1 per cent (18/56), 20.9 per cent (38/181), and 5.4 per cent (4/73) were recorded in cases of nosocomial diarrhoea, community-acquired diarrhoea, and controls, respectively. Fifty-two (86.7 per cent) of the 60 rotavirus-positive specimens were subgrouped and the G serotypes of 55 (91.7 per cent) of them were determined. Subgroups I and II were detected in 50 per cent each of the 52 subgrouped strains. G type 2 was present in 46 (83.6 per cent) of the 55 serotyped samples; serotypes G1 and (mixed) G1 and G4 were found in 14.5 per cent and 1.8 per cent, respectively, of these specimens. Viral RNA electrophoresis showed 14 distinct patterns, including 56.7 per cent (34/60) and 43.3 per cent (26/60) of long and short profiles, respectively. In 40 (66.6 per cent) of the 60 rotavirus-positive faecal samples no enteropathogens other than rotavirus were detected. There was an increased incidence of rotavirus infection from July 1993 to February 1994. The rotavirus-related episodes of diarrhoea were more severe than those of other aetiology and greater clinical severity was not related to a specific G type, subgroup, or electrophoretype.
OBJECTIVES AND METHODS: With the purpose of better understanding the efficacy of the lower titer [4 x 10(4) plaque-forming units (pfu)] tetravalent rhesus-human reassortant rotavirus vaccine (RRV-TV) against diarrheal episodes of different severities, the Peruvian and Brazilian efficacy data were reanalyzed with a 20-point scoring system. Mild, moderate/severe and very severe rotavirus diarrhea were scored as 0 to 8, 9 to 14 and >14, respectively. RESULTS: In the Peruvian study one dose of vaccine yielded 64% (P = 0.04) protection against pure cases of rotavirus disease (i.e. those in which no other enteropathogen was found) with clinical scores ranging from 9 to 14. Protective efficacy against very severe rotavirus gastroenteritis could not be assessed because of the small number of cases. In Brazil there was a trend in preventing "all" and "pure" cases of rotavirus diarrhea scored 9 to 14 (44%, P = 0.06, and 45%, P = 0.08, respectively) and the vaccine was 75% (P = 0.02) protective against pure rotavirus diarrhea scored >14. No protection was observed for mild rotavirus diarrhea (scores <9). These data were compared with those from trials in Venezuela (4 x 10(5) pfu/dose), US (4 x 10(4) pfu/dose and 4 x 10(5) pfu/dose) and Finland (4 x 10(5) pfu/dose). Combining the Peruvian (one dose, pure cases) and Brazilian studies together, the levels of protection against 9- to 14-scored rotavirus diarrhea are comparable with those from the Venezuelan (47%) and American (57, 57 and 65%) efficacy trials. In Brazil the level of protection (75%) against pure, >14-scored rotavirus diarrhea is similar to the efficacy rates yielded in the three US trials (82, 80 and 69%) and the Finnish trial (100%) for episodes of the same severity. CONCLUSIONS: Our reanalysis provides evidence that, at least against moderate/severe rotavirus gastroenteritis, RRV-TV, 4 x 10(4) pfu/dose is potentially as efficacious as RRV-TV, 4 x 10(5) pfu/dose, even in settings with very high rotavirus disease burden. The reanalysis of the Peruvian data suggests that one and three vaccine doses may yield similar efficacy rates. It is also suggested that vaccine efficacy against most severe episodes in Peru and Brazil was not evident because of the trial design used in those studies (i.e. prospective, active home surveillance rather than a catchment trial), resulting in too few cases of severe disease even in the placebo group. To confirm these findings, future trials with this vaccine are necessary in developing countries with high diarrhea morbidity rates. These trials should use catchment designs and focus on the evaluation of the efficacy of one or three doses of RRV-TV against moderate to severe/very severe rotavirus diarrhea.
OBJECTIVES: This report was prepared with the main objective of making an extensive review of both clinical features and prevention of rotavirus gastroenteritis. In addition, it provides an evaluation about the potential for introduction of the tetravalent rhesus-human reassortant rotavirus vaccine (RRV-TV) in the developing countries. METHODS: The main source of information was the most relevant articles published in both national and international journals, as well as selected official reports from the World Health Organization. With regard to rotavirus vaccines, particular emphasis has been placed on the results available from studies carried out during the past five years. Notice we have stressed the epidemiological features of rotavirus infections in Brazil. RESULTS: As a background for the main subjects of this report - clinical features and prevention - available general information on rotavirus infections are briefly discussed in the Introduction. This includes an overview of the etiological agent, epidemiology, immunity and laboratory diagnosis. A detailed description of the typical symptomatic syndrome is made, as well as of other (unusual) clinical manifestations of rotavirus illness. We also discuss the rotavirus candidate vaccines that have been evaluated to date, highlighting the most significant observations which resulted from field trials with the RRV-TV in several countries. The potential for large-scale use of RRV-TV in developing countries is also discussed, focusing mainly on the epidemiological characteristics of rotavirus disease in these regions. CONCLUSION: Rotaviruses are the leading cause of severe gastroenteritis in infants and young children in both industrialized and lessdeveloped countries; in the latter regions, rotavirus diarrhoeal disease represents a major cause of mortality. There is currently a consensus that attempts at prevention need to be directed toward the development of an effective rotavirus vaccine, for large-scale use, that would primarily protect children (aged 0 to 2 years) against severe rotavirus gastroenteritis. In this regard, results from a recent reevaluation of the Peruvian and Brazilian lower-titer RRV-TV efficacy data are promising. Although the RRV-TV ("Rotashield(R)") (recently licensed for general use in U.S.A.) seems to be currently the most promising rotavirus vaccine, it has been suggested that further trials with this vaccine should be conducted in Africa and Asia (efficacy studies) and Latin America [immunogenicity and effectiveness (efficacy under real conditions in a given setting)]. In addition, it is of paramount importance to establish in these regions a surveillance system to monitor the circulating rotavirus strains.
In August 1993, an outbreak of group C rotavirus-associated gastroenteritis occurred among children attending a day-care centre in Belém, Brazil. Of the 64 children, 21 (33%) became ill. Group C rotavirus was identified in faecal specimens from 8 (38%) children with diarrhoea by electron microscopy (EM) and an enzyme immunoassay (EIA), using antibodies specific to the Cowden strain of porcine group C rotavirus. By polyacrylamide gel electrophoresis (PAGE), a pattern similar to that of group C rotavirus was observed in 5 (62.5%) of the 8 EM- and EIA-positive samples. These 5 faecal samples were confirmed to be positive for group C rotavirus by reverse transcriptase-polymerase chain reaction, using specific VP6 and VP7 primers. This is the first report of an outbreak of diarrhoea in North Brazil associated with group C rotavirus. These findings suggest that group C rotavirus may be an important aetiological agent of diarrhoea in this region, which requires further study.
In our study, 300 pregnant women were screened for the presence of human parvovirus B19 IgG and IgM antibodies by an enzyme-linked immunosorbent assay (ELISA). Overall, 253 (84.3%) were found to be IgG-positive and IgM-negative (IgG+gM ), 42 (14%) had neither IgG nor IgM antibodies (IgG /IgM ) and 5 (1.7%) were both IgM- and IgG-positive (IgG+/IgM+). Maternal serology was performed routinely for cytomegalovirus, rubella, toxoplasmosis and syphilis. All IgG /IgM and IgG+/IgM+ women were followed up till the time of delivery, venous blood sample being taken monthly from each one; one IgG /IgM mother seroconverted to IgG+/IgM and B19 DNA was detected by nested polymerase chain reaction technique (PCR) in her serum. All babies born to IgG+/IgM+ mothers (and from that who seroconverted) were IgG+IgM , no B19 DNA could be detected in their sera and no adverse effects were documented either by ultrasonographic examination or the detection of maternal serum alpha-fetoprotein. While 5 of these mothers delivered normal children at term, one gave birth to a premature (low-weight) baby who developed severe anemia and had convulsions; this mother was found to have toxoplasma-specific IgM. As based on serial testing of sera, it is notable that B19 IgM may last up to six months. Our data indicates a low incidence rate of B19 infection in pregnancy in our region, at least during interepidemic periods. In addition, it suggests that recent B19 infection represents a low risk for the development of adverse fetal outcomes.
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.
A large placebo-controlled efficacy trial of the rhesus tetravalent (RRV-TV) and serotype G1 monovalent (RRV-S1) rotavirus vaccines was conducted in 1991-1992 at 24 sites across the United States. Protection was 49% and 54% against all diarrhea but 80% and 69% against very severe gastroenteritis for the two vaccines, respectively. Post-vaccination neutralizing antibody titers to the G1 Wa strain, whose VP7 protein is nearly identical to that of the D strain of rotavirus contained in both vaccines, did not correlate with protection against subsequent illness with G1 strains. This result raised the possibility that in infants who developed post-vaccination neutralizing antibody to Wa, breakthrough (i.e., vaccine failure-the occurrence of rotavirus diarrhea after immunization) may have been due to infection by G1 strains that were sufficiently antigenically distinct from the vaccine strain to evade the neutralizing antibodies elicited by vaccination. To test this hypothesis, we initially compared post-vaccination neutralizing antibody titers of vaccinees against Wa and G1 breakthrough strains using sera from subjects who experienced breakthrough. Post-immunization neutralizing antibody titers to Wa elicited by vaccination were significantly (P < 0.001) greater than to the breakthrough strains subsequently obtained from these subjects. This difference did not, however, correlate with lack of protection since similar differences in titer to Wa and breakthrough strains were found using post-vaccination sera from vaccinees who either experienced asymptomatic rotavirus infections or no infections. To determine the genetic basis for these differences, we compared the VP7 gene sequences of Wa with vaccine strain D, 12 G1 breakthrough strains, and 3 G1 control strains isolated during the same trial from placebo recipients. All breakthrough strains were distinct from Wa and D in antigenically important regions throughout the VP7 protein, but these differences were conserved between breakthrough and placebo strains. Furthermore, a comparative analysis of the deduced amino sequences form VP7 genes of G1 rotaviruses from 12 countries indicated that four distinct lineages have evolved. All breakthrough and control strains from the U.S. vaccine trial were in a lineage different from strain D, the serotype G1 vaccine strain. Although the overall results do not support our original hypothesis that immune selection of antigenically distinct escape mutants led to vaccine breakthrough in subjects with a neutralization response to Wa, it cannot be excluded that breakthrough could be partially due to antigenic differences in the VP7 proteins of currently circulating G1 strains.
A tetravalent rhesus-human reassortant rotavirus (RRV-TV) vaccine (4 x 10(4) plaque-forming units/dose) was evaluated for safety, immunogenicity and efficacy in a prospective, randomized, double-blind, placebo-controlled trial involving 540 Brazilian infants. Doses of vaccine or placebo were given at ages 1, 3 and 5 months. No significant differences were noted in the occurrence of diarrhoea or vomiting in vaccine and placebo recipients following each dose. Low-grade fever occurred on days 3-5 in 2-3% of vaccinees after the first dose, but not after the second or third doses of vaccine. An IgA antibody response to rhesus rotavirus (RRV) occurred in 58% of vaccinees and 33% of placebo recipients. Neutralizing antibody responses to individual serotypes did not exceed 20% when measured by fluorescent focus reduction, but exceeded 40% when assayed by plaque reduction neutralization. There were 91 cases of rotavirus diarrhoea among the 3-dose (vaccine or placebo) recipients during two years of follow-up, 36 of them among children given the vaccine. Overall vaccine efficacy was 8% (P = 0.005) against any diarrhoea and 35% (P = 0.03) against any rotavirus diarrhoea. Protection during the first year of follow-up, when G serotype 1 rotavirus predominated, was 57% (P = 0.008), but fell to 12% in the second year. Similar results were obtained when analysis was restricted to episodes in which rotavirus was the only identified pathogen. There was a tendency for enhanced protection by vaccine against illness associated with an average of 6 or more stools per day. These results are sufficiently encouraging to warrant further studies of this vaccine in developing countries using a higher dosage in an attempt to improve its immunogenicity and efficacy.
We attempted to assess the role of enteroviruses in the etiology of myocarditis (MC), pericarditis (PC) and dilated cardiomyopathy (DCM) among 15 in-patients at a public hospital in Belém, Brazil, from November 1992 to December 1993. We obtained stool specimens and throat swabs from each patient (particularly acute cases) and, when possible, acute and convalescent serum samples for both isolation and serological procedures. MC, PC and DCM occurred in 10, 2 and 3 patients, respectively, mostly in the 0- to 10-year age group. Neutralizing antibody seroconversions were detected as follows: one for Coxsackievirus (Cox) B2 in one patient suffering from MC, and two for Cox B4, in patients with DCM and MC. In addition, antibody titers of 1/320 against Cox B2 and Cox B4 were noted in two other patients, one suffering from PC and the other presenting MC. Isolation of echovirus (ECHO) serotype 1 was recorded in a patient with MC, without either seroconversion or high antibody levels for Cox B 1 to 6. These results indicate that enteroviruses may be involved in the etiology of MC, PC and DCM in the Amazon region.
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.
We have characterized the neutralizing antibody immune response to six human rotavirus serotypes (G1 to G4, G8, and G9) in Brazilian children with primary and secondary rotavirus infections and correlated the response with the G serotype of the infecting rotavirus strain. Twenty-five children were studied: 17 had a single rotavirus infection, 4 were reinfected once, and 4 experienced three infections. Two of the reinfections were by non-group A rotaviruses. Among the 25 primary infections, we observed homotypic as well as heterotypic responses; the serotype G1 viruses, which accounted for 13 of these infections, induced mostly a homotypic response, while infections by serotype G2 and G4 viruses induced, in addition to the homotypic, a heterotypic response directed primarily to serotype G1. Two of the primary infections induced heterotypic antibodies to 69M, a serotype G8 virus that by RNA electrophoresis analysis was found not to circulate in the population during the time of the study. The specificity of the neutralizing antibody immune response induced by a virus of a given serotype was the same in primary as well as secondary infections. These results indicate that the heterotypic immune response induced in a primary rotavirus infection is an intrinsic property of the virus strain, and although there seem to be general patterns of serotype-specific seroconversion, these may vary from serotype to serotype and from strain to strain within a serotype.