Reactogenicity and antigenicity of rhesus rotavirus vaccine (MMU-18006) in newborn infants in Venezuela.

The reactogenicity and antigenicity of the rhesus rotavirus vaccine, strain MMU18006, developed at the Laboratory of Infectious Diseases (National Institute of Allergy and Infectious Diseases, National Institutes of Health) were examined in a double blind, placebo-controlled study of 40 newborn infants in Caracas, Venezuela. The children were observed for the first few days after birth in the hospital nursery and by home visits for 10 days after vaccination to detect any adverse reactions. No reactions could be attributed to the vaccine. Serologic responses to the vaccine were evaluated in paired sera obtained at birth (cord blood) and 4 weeks after vaccination. Serologic responses to the vaccine were not observed by complement fixation, neutralization or a rhesus rotavirus VP7 epitope-specific competition assay. However, such responses were found in 9 of 14 tested infants by an immunoglobulin A-specific enzyme-linked immunosorbent assay. Seventeen of the 20 vaccinees also shed rhesus rotavirus vaccine in stool during the postvaccination period.

Rotavirus-associated medical visits and hospitalizations in South America: a prospective study at three large sentinel hospitals.

BACKGROUND: Knowledge of the impact of rotavirus-associated disease on the health care systems of South America can aid in defining strategies for diagnosis, management and prevention. Up to date information on the impact of rotavirus disease in South America is scarce. AIM: To determine prospectively the impact of rotavirus disease as a cause of medical visits and hospitalizations at three large sentinel pediatric hospitals in Argentina, Chile and Venezuela. METHODS: A 2-year prospective surveillance for rotavirus-associated medical visits and hospitalizations was conducted during 1997 through 1998 at three large sentinel public hospitals, one each in Argentina, Chile and Venezuela. A common surveillance protocol was implemented at the three sites, and a representative number of nonbloody diarrhea stool samples from children <36 months of age were tested for rotavirus by enzyme-linked immunosorbent assay. RESULTS: For our target age group, acute diarrhea-associated medical visits/hospitalizations represented 41%/2%, 5%/6% and 9%/13% of all medical visits/all hospitalizations at the Argentinean, Chilean and Venezuelan sites, respectively (P < 0.001 for difference among the three sites). Rotavirus detection rates among a total of 5,801/1,256 medical visit/hospitalization diarrhea stool samples tested were 39%/71% in Argentina, 34%/47% in Chile and 29%/38% in Venezuela (P < 0.01 by chi square for difference among the three sites). Rotavirus was associated with a mean of 1.5, 1.8 and 3% of total medical visits and 1.6, 2.8 and 5% of hospitalizations among children <36 months of age at the Argentinean, Chilean and Venezuelan sites, respectively. Seasonality was evident for medical visits at all three sites (although less striking in Chile) with peak activity occurring between November and May. Rotavirus-associated hospitalizations had a marked peak in Venezuela, represented largely by short stays, but not in Argentina and Chile. CONCLUSIONS: Rotavirus was a significant cause of medical visits at all three sentinel sites. Rotavirus caused less hospitalizations than previously reported in Argentina and Chile. On the basis of our findings we estimate that approximately 106,000/ 21,000, 48,000/8,000 and 98,000/31,000 rotavirus-associated medical visits/hospitalizations occur yearly in Argentina, Chile and Venezuela, respectively.

Jennerian and modified Jennerian approach to vaccination against rotavirus diarrhea using a quadrivalent rhesus rotavirus (RRV) and human-RRV reassortant vaccine.

Rotaviruses are the single most important cause of severe diarrhea of infants and young children world-wide. Deaths from rotavirus diarrhea occur infrequently in developed countries; however, in developing countries, rotaviruses are estimated to cause over 870000 deaths in the under five-year age group. There is, therefore, a vital need for a vaccine to prevent severe rotavirus diarrhea in infants and young children. The most extensively evaluated strategy for rotavirus vaccination has been the "Jennerian" approach in which an antigenically related rotavirus strain from an animal host (bovine or simian [rhesus monkey]) is used as the immunogen to induce protection against the four epidemiologically important group A human rotavirus serotypes. These orally administered vaccines were safe and immunogenic but had only limited success because serotype-specific immunity was not induced consistently in the under six-month age group. Therefore, a modified "Jennerian" approach was adopted with the goal of attaining broader antigenic coverage. In this approach four serotypes are combined to form a quadrivalent vaccine comprised of (i) rhesus rotavirus (RRV) which provides coverage for VP7 serotype 3, and (ii) three human-RRV reassortants each with ten RRV genes and a single human rotavirus gene that encodes VP7 serotype 1, 2, or 4 specificity. This modified "Jennerian" approach appears to be quite promising in preventing severe diarrhea in field trials. However, if this approach fails to yield an optimal level of protection consistently, additional modified "Jennerian" strategic, are under development that consider not only human rotavirus VP7 but also human rotavirus VP4, the other outer capsid protein. In addition, a non-"Jennerian" approach includes the development of cold-adapted human rotavirus strains or cold-adapted human rotavirus reassortants as vaccine candidates.

Cryptosporidiosis in Venezuelan children with acute diarrhea.

Thirteen of 120 Venezuelan children with acute diarrhea were found to be excreting Cryptosporidium oocysts in their stools. This confirms that Cryptosporidium can infect immunocompetent children, and the relatively high frequency found suggests that this protozoan may be an important cause of diarrhea in Venezuela.

Enumeration of selected leukocytes in the small intestine of BALB/c mice infected with Cryptosporidium parvum.

Neonatal, suckling BALB/c mice inoculated with Cryptosporidium parvum produce an infection characterized by continuous shedding of oocysts that spontaneously clears by the time the animals are three weeks of age. Neonatal mice were used to characterize the leukocyte subgroups present in Peyer's patches from the ileum and jejunum of Cryptosporidium-infected and healthy mice. After infection, ileal Peyer's patches showed a predominant CD8+ response, with abundant monocytes-macrophages (MOMA-2+) and nonlymphoid dendritic cells (NLDC-145+ cells). In contrast, jejunal Peyer's patches showed more T lymphocytes than ileal patches, with a predominance of CD4+ cells and many dendritic NLDC-145+ cells and MOMA-2+ cells. The present results showed that ileal and jejunal Peyer's patches are functionally different in response to Cryptosporidium parasites. These findings suggest a preferential involvement of jejunal Peyer's patches in T cell-dependent immunity against the parasite, whereas ileal patches may be associated with B cell expansion and maturation.

Immunocytochemical detection of Entamoeba histolytica.

Human anti-Entamoeba histolytica immunoglobulin was used to detect Entamoeba histolytica in 74 positive samples from several different sources, using an indirect immunoperoxidase method. In 73 samples, the protozoan was easily identified. Trophozoites and cysts of all cultured Entamoeba strains examined were strongly stained, and as few as 3 trophozoites per microscope slide could be detected. In addition, 51 negative control samples were also tested and non-specific reactions were not observed. These preliminary results show that this method is both sensitive and specific, and can easily detect trophozoites and cysts of different E. histolytica strains.

Characterization of rotaviruses isolated from pigs with diarrhoea in Venezuela.

The prevalence of porcine rotavirus infection was studied in 15 different herds located in the north-western region of Venezuela. The presence of rotavirus was studied by direct electron microscopy (EM) and by an enzyme-linked immunosorbent assay (ELISA). From 136 samples analyzed during the six months of the study (September 1983-February 1984), 38 (27.9%) were found to be positive for rotaviruses, with infection more common in animals that were 4-6 weeks old. Atypical rotaviruses were not detected in any of the samples examined. Most rotavirus positive specimens were subgrouped using specific monoclonal antibodies in an ELISA test. The majority of the samples (26 out of 38) were found to exhibit Subgroup I antigenicity. Only two specimens, collected from the same herd in two consecutive months, were found to belong to Subgroup II. To characterize further the circulating rotaviruses, electrophoretic analysis of the RNA genome was performed on samples selected from nine different herds. Great variability in the RNA electropherotypes was observed. No correlation was found between subgroup specificity and the migration of the two smaller segments (Genes 10 and 11), as has been described for human rotaviruses.

[Descriptive study of 43 nutritional education programs in Venezuela]. / Un estudio descriptivo de 43 programas de educación nutricional en Venezuela.

This study aimed to determine the existing resources and specific characteristics of non-formal nutrition education programs in Venezuela. Of the 174 institutions initially contacted, 43 were involved in these activities-22 official and 21 private institutions. The bulk of the programs fell under the official health sector and the private food industry. While objectives of the former were to motivate and educate the audience, the latter's goal was basically commercial. Forty-three per cent of the program content dealt with basic nutrition concepts; only 19% of programs included information on breast feeding, and maternal and child nutrition. There is a significantly greater use of interpersonal communication (65%) as compared to mass media (35%), irrespective of the type of institution. In the majority of programs, the nutritionist-dietitians and medical doctors carry out their activities, primarily oriented towards homemakers and school-age children. The official health sector gears its programs toward the poor, while the main interest of private industry are the middle and upper class communities. A total of 46% of the official programs and 55% of the private institutions do not include an evaluation component. It is concluded that the nutrition education programs in the country appear to follow traditional concepts as far as objectives, program content, media and target groups are concerned. This could have contributed to the apparent lack of impact of non-formal nutrition education in the country; thus the re-planning of existing programs, is suggested. This should be based on well-defined and measurable objectives: include messages geared toward the practical solution of specific nutrition problems, and the use of combined techniques of social communication with as much participation of the community as possible, not only in program implementation but also in its conception and planning.

A short report on highlights of world-wide development of RIX4414: a Latin American experience.

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.

The impact of rotavirus disease in Venezuela.

Information concerning the disease burden of rotavirus, particularly in developing countries, has important implications for the use and for monitoring the impact of rotavirus vaccines. Although rotavirus has been recognized as the most frequent cause of hospitalization in the world, national estimates and specific information about the incidence of hospitalization for rotavirus gastroenteritis are very limited. Consequently, estimates of the incidence of hospitalization among children during the first 2 years of life in Venezuela were determined by extrapolation of data from a community-based study carried out in Caracas.

Stimulation of intestinal adenylate cyclase by cholera toxin in malnourished rats.

The stimulation of intestinal adenylate cyclase by cholera toxin (CT) was studied in normal and malnourished rats 4 to 24 hr after a 30-min incubation of intestinal loops with the toxin. Whereas in control rats the enzyme activity returned to basal levels after 12 hr of incubation, in malnourished rats the activity of the enzyme remained significantly elevated even after 24 hr of the initial incubation. Malnourished animals had a reduced turnover rate of intestinal cells as determined by thymidine kinase activity. The delayed turnover of intoxicated cells may account for continuous activation of mucosal adenylate cyclase and possibly for prolongation of diarrhea in malnutrition.

Heterogeneous plasmid population from enterotoxigenic Escherichia coli strains isolated in Venezuelan children with acute diarrhea.

Thirty eight enterotoxigenic Escherichia coli (ETEC) isolated from children with acute diarrhea were analyzed in order to assess the possible associations among enterotoxigenicity, antibiotic resistance and other plasmid-mediated virulence properties such as CoIV, Hly and CFA/I. Eighty four percent of ETEC strains were multiresistant. Twenty strains (52.63%) were able to transfer one or more properties studied and 92.68% of the transconjugants were multiresistant. The simultaneous transfer of genes encoding ST enterotoxin and CoIV, Hly or CFA/I was very low (1.82%). The plasmid analysis revealed the presence of a heterogeneous enterotoxigenic (Ent) plasmid population. Additionally, the existence of a conjugative plasmid of approximately 31 megadaltons (Md) of molecular weight encoding for ST and resistance to ampicillin, kanamycin and streptomycin was found. However, this plasmid was not present in all isolates. These results show a diversity of Ent plasmid population which is probably a consequence of the indiscriminate use of antibiotics and the molecular mechanism of transposition of ST and drug-resistance in the evolution of bacterial strains.

Serological response to rotavirus infection in newborn infants.

We report the identification of rotavirus in stools of newborn infants at the "Hospital Materno Infantil de Caricuao" (HMIC) as well as the infants' serological responses to various rotavirus strains. The serological responses of another group of rotavirus-positive neonates studied previously at the "Maternidad Concepcion Palacios" (MCP) hospital was also evaluated. Fifty-four of 266 (20%) newborns examined at HMIC shed rotavirus. The infection rate was higher among infants admitted to the nursery (75%) than in those "rooming in" with their mothers (7%) (P < .01). Eleven of the 54 neonates (20%) had diarrhea; seven of them experienced mild, short-lived episodes, whereas five had frequent diarrhea bouts or diarrhea lasting for over 3 days; the remaining 43 infants were asymptomatic. Twenty-seven of 28 rotavirus specimens tested at HMIC had VP7 serotype 4 specificity and one belonged to VP7 serotype 1; VP4 typing performed on 24 of the viruses by RNA hybridization showed these viruses to be similar to the M37 strain, a rotavirus previously associated with asymptomatic infections in newborns at MCP. IgA seroresponses were detected in eight of 11 infants born at HMIC (73%), but most failed to developed neutralization responses to homologous or heterologous strains. Newborn infants who had shed the M37 rotavirus strain at MCP reacted similarly: 16 of 24 (67%) developed a rotavirus IgA rise, but only 29% developed a neutralization response.

Seroepidemiology of Cryptosporidium infection in two Latin American populations.

An enzyme-linked immunosorbent assay was used to examine randomly selected sera from 389 children and adults in Lima, Peru, and 84 children in Maracaibo and Caracas, Venezuela, for IgM or IgG antibody to Cryptosporidium. In Peru and Venezuela, 19.8% and 15.5% of the study populations, respectively, were positive for both specific IgG and IgM antibodies, a result consistent with active or recent infection and representing a larger percentage than normally reported from stool examinations of individuals seeking medical attention. Sixty-four percent of subjects from each country had detectable levels of specific IgG, indicating infection sometime in life. Detection of specific IgG increased in the two- to three-year-old age-group, a result suggesting that this is a common age for infection. Persistence of IgG and, less often, IgM antibody response over 12 mo occurred in some subjects, although the significance of this finding is uncertain. Our findings suggest that Cryptosporidium infections are endemic in the communities surveyed and that most residents have been infected.

Age-specific prevalence of Escherichia coli with localized and aggregative adherence in Venezuelan infants with acute diarrhea.

To evaluate the epidemiological significance of HEp-2 cell-adherent Escherichia coli isolates in diarrheal disease, we performed a study with 513 Venezuelan infants with diarrhea and 241 age-matched controls to determine the prevalence of enteropathogenic E. coli (enteroadherent E. coli, enterotoxigenic E. coli, enteroinvasive E. coli, and enterohemorrhagic E. coli) and their correlation with O:H serotypes. E. coli isolates exhibiting localized and aggregative adherence in the HEp-2 cell assay were significantly more frequently isolated from the patients (8.5 and 26.9%, respectively) than from the controls (1.7 and 15%, respectively). This difference was significant for the group 0 to 2 months of age but for older infants. Regardless of age, E. coli isolates with diffuse adherence were found at similar frequencies in both the patients and the controls. A striking correlation between classic O serogroups and localized adherence was also observed. These findings confirm the pathogenic role of E. coli with localized and aggregative adherence in diarrheal disease, as well as the epidemiological importance of O:H serotyping for characterizing localized-adhering E. coli.

Efficacy of a quadrivalent rhesus rotavirus-based human rotavirus vaccine aimed at preventing severe rotavirus diarrhea in infants and young children.

The most extensively explored strategy for rotavirus vaccination has been the Jennerian approach, which uses an antigenically related rotavirus strain from an animal host as the immunogen to induce protection against the 4 epidemiologically important group A rotavirus VP7 serotypes. Because this approach has shown limited efficacy, a modified Jennerian approach was developed with the goal of achieving broader antigenic coverage. Four VP7 serotypes were incorporated into a quadrivalent vaccine comprised of three rhesus-human rotavirus reassortants, each with 10 rhesus rotavirus genes and 1 human rotavirus gene that encodes VP7 serotype 1, 2, or 4 specificity; the rhesus rotavirus itself provides coverage for VP7 serotype 3. This approach appears quite promising for preventing severe rotavirus diarrhea, including those episodes that lead to dehydration. Additional strategies under development stress the role not only of human rotavirus VP7 but also of human rotavirus VP4, the other outer capsid protein that also induces neutralizing antibodies.

Homotypic immune response to primary infection with rotavirus serotype G1.

Some aspects of rotavirus humoral immunity were assessed on the basis of distinguishing serotype-specific specificities (VP4/VP7) by using rotavirus reassortants, human and animal strains in neutralization assays in serum samples obtained during the acute phase, and 1, 6 and 12 months after primary natural infection. In this study, all the infecting virus strains were characterized as G type and some also as P type. Primary natural infection induces a significantly greater homotypic neutralization response than heterotypic response. In addition, there was no significant difference in the number of homotypic or heterotypic responses following reinfection. Transplacentally acquired homotypic antibodies were associated with protection against dehydration during rotavirus gastroenteritis.

[Etiologic, clinical and socio-democratic characteristics of acute diarrhea in Venezuela]. / Características etiológicas, clínicas y sociodemográficas de la diarrea aguda en Venezuela.

In four cities of Venezuela a study was carried out to evaluate the epidemiological, clinical, and etiological characteristics of acute diarrhea in children under 5 years of age. The study was done between June 1993 and May 1995 and involved children who were seen in a hospital, 2,552 with diarrhea and 793 controls. The Fisher exact test was used for the statistical analysis of the results. Rotaviruses were the most important agents, both in terms of their frequency (30%) and their association with dehydration (58%). Following in importance were Campylobacter spp. (13%) and Escherichia coli classical O serogroups (9%), but their association with diarrhea was only statistically significant among children less than 3 months old, a fact that is particularly important from the standpoint of treatment. The importance of age was confirmed as a determining factor in the prevalence and severity of diarrhea.