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.

Rotavirus serotype G9 is associated with more-severe disease in Latin America.

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.

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.

Reappraisal of the Peruvian and Brazilian lower titer tetravalent rhesus-human reassortant rotavirus vaccine efficacy trials: analysis by severity of diarrhea.

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.

Prevalence of human herpesvirus 6 antibody in the population of Belém, Pará, northern Brazil.

A total of 497 serum samples obtained from residents of 2 neighbourhoods (Terra-Firme and Guamá) in Belém, Pará, Brazil was tested for the presence of antibody to human herpesvirus 6 (HHV6), using an indirect immunofluorescence assay. The overall seroprevalence was 90%, with seropositivity rates ranging from 75% to 100% in the different age groups and sexes. There was a significant difference between the antibody prevalences in Terra-Firme and Guamá, 94% and 87%, respectively (P = 0.01). The geometric mean titres (GMT) of antibody to HHV6 declined from the first (< or = 2 years) to the sixth (31-40 years) age groups, and slightly increased (GMT > 50) among individuals older than 40 years.

Prevalence of human herpesvirus 6 antibody among isolated Amazonian Amerindian communities in Brazil.

Human herpesvirus 6 (HHV6) antibody was detected by the anticomplement immunofluorescence test in 65 of 592 serum samples (10.9%) obtained from Amerindians belonging to 4 remote communities inhabiting the Amazon Region of Brazil. Seropositivity rates ranged from 5.4% in the Oyampí to 14.9% in the Tucano. Among the Mekranoiti and Tiriyo, the seroprevalence rates were 12.5% and 11.8%, respectively. In children aged 0-10 years, the prevalences of HHV6 antibody ranged from 5.2% to 24.2%, among the Oyampí and Mekranoiti tribes, respectively. In the 11-20 and > 20 years old age groups, percentages of positivity ranged, in the former group, from 9.5% to 17.5% in the Oyampí and Tucano, respectively and, in the latter group, from 1.3% in the Oyampí to 14.8% in the Tucano. The prevalences of HHV6 antibody decreased regularly through the first 3 age-groups in the Mekranoiti and Tiriyo. In addition, frequencies of seropositivity were consistently higher in males than in females in the Mekranoiti (P < 0.05), Oyampí and Tiriyo tribes.

Rotavirus infection in wild marsupials (Didelphis marsupialis) of the Amazon region.

Rotavirus was detected by enzyme-linked immunosorbent assay in faecal specimens collected from two (1.35%) of 148 marsupials trapped in the Amazon jungle environment. The positive samples were both from the "common opossum", Didelphis marsupialis. No infections were found in the stools of 198 animals belonging to other mammalian species: the latter included small rodents, chiropterans and primates. Electron microscopic examination of one (MA 5928) rotavirus-positive specimen showed a large number of empty particles. However, both rotavirus strains grew when inoculated in MA 104 cells (foetal Rhesus monkey kidney cells) producing clear cytopathogenic effect; indirect immunofluorescence technique of these cells showed a typical granular cytoplasmic fluorescence. The electrophoretic profile of strain MA 5928 showed a high grade of homology with that of SA 11, but also showed minor differences.

Acute diarrhoea associated with rotavirus among children living in Belém, Brazil.

Between January 1979 and December 1980, rotaviruses were detected in faecal samples from 122 (33.1%) of 369 diarrhoeic children less than six years old, living in Belém, Brazil. In 55 (45.1%) of the 122 rotavirus-positive specimens, no bacteria or parasite associated with gastro-enteritis was found. Salmonella typhimurium, Shigella and Escherichia coli were found in 92 (35%) of the 263 faecal specimens examined for bacterial pathogens. Rotaviruses were readily detected throughout the year, which may indicate no seasonal pattern of incidence in the Belém region. A comparison of the excretion of rotavirus was made between children of two socio-economic groups. Of 144 children from the higher level group, 63 (47.3%) excreted rotavirus, whereas only 12 (16.7%) of those from the lower level did so. Vomiting (68.0%) and fever (65.6%) were the main symptoms, other than diarrhoea, exhibited by the 122 rotavirus-positive children.

Enteroviruses as a possible cause of myocarditis, pericarditis and dilated cardiomyopathy in Belém, Brazil.

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.

[Biosafety in the laboratory]. / Biossegurança no laboratório.

The occurrence of laboratory-acquired infections have elicited in the last ten years an intense interest in methods and procedures for the safe handling of microbiological material. The major laboratory safety problem is aerial transmission, however, protection against airborne hazards is efficiently achieved by the use of microbiological safety cabinets. Biosafety rules should be strictly followed by all members of a laboratory. Evaluation of these procedures should be effectively performed by an independent biosafety committee. The upsurge of AIDS should stimulate the adoption of safe working procedures in the laboratory.

Detection of enteroviruses in cases of neurological disorders in the State of Pará, Brazil.

Eighty-one cerebrospinal fluid (CSF) samples mainly from cases of aseptic meningitis and motor deficiency syndrome were sent to the Virology Section of Evandro Chagas Institute, Belém Pará, in the period of January 1995 to January 1996 in order to isolate viruses. All samples were inoculated onto HEp-2 cell culture and newborn mice, with negative results. The probability of isolating viruses by these methods is reduced because of the low concentration of viral particles in these specimens. In order to obtain more information about the etiology of these cases, a group of 23 samples were selected to be tested by a more sensitive technique than the virus isolation - the reverse transcription polymerase chain reaction (RT-PCR). Specific primers directed to conserved regions in the enterovirus genome were used, considering that this group of viruses is frequently associated with these neurological disorder. The age of the patients ranged from 1 to 55 years and nearly all of them lived in Belém, State of Pará, North of Brazil. Of 15 samples analyzed by RT PCR nine (60%) were positive; of these, 6 (66.6%) had motor deficiency and 3 (33.3%) developed aseptic meningitis. These results show that it is important to investigate enterovirus as cause of these syndromes.

Outbreaks of human-herpes virus 6 (HHV-6) infection in day-care centers in Belém, Pará, Brazil.

A total of 730 children aged less than 7 years, attending 8 day-care centers (DCCs) in Belém, Brazil were followed-up from January to December 1997 to investigate the occurrence of human-herpes virus 6 (HHV-6) infection in these institutional settings. Between October and December 1997 there have been outbreaks of a febrile- and -exanthematous disease, affecting at least 15-20% of children in each of the DCCs. Both serum- and- plasma samples were obtained from 401 (55%) of the 730 participating children for the detection of HHV-6 antibodies by enzyme-linked immunosorbent assay (ELISA), and viral DNA amplification through the nested-PCR. Recent HHV-6 infection was diagnosed in 63.8% (256/401) of them, as defined by the presence of both IgM and IgG-specific antibodies (IgM+/IgG+); of these, 114 (44.5%) were symptomatic and 142 (55.5%) had no symptoms (p = 0.03). A subgroup of 123 (30.7%) children were found to be IgM-/IgG+, whereas the remaining 22 (5.5%) children had neither IgM nor IgG HHV-6- antibodies (IgM-/IgG-). Of the 118 children reacting strongly IgM-positive (> or = 30 PANBIO units), 26 (22.0%) were found to harbour the HHV-6 DNA, as demonstrated by nested-PCR. Taken the ELISA-IgM- and- nested PCR-positive results together, HHV-6 infection was shown to have occurred in 5 of the 8 DCCs under follow-up. Serological evidence of recent infections by Epstein-Barr virus (EBV) and parvovirus B19 were identified in 2.0% (8/401) and 1. 5% (6/401) of the children, respectively. Our data provide strong evidence that HHV-6 is a common cause of outbreaks of febrile/exanthematous diseases among children attending DCCs in the Belém area.

Fifth disease in children living in Belém, Brazil.

Acute sera from two children suffering from an illness with an erythematous rash were positive for B 19 virus specific IgM antibody, as tested by a capture radioimmunoassay. The first patient, a two year old boy, presented with a cutaneous rash of six days duration, the second was a four year old girl, sister of the first patient, who was examined at the same time and had a three day history of cutaneous rash.

Association of human herpesvirus 6 infection with exanthem subitum in Belem, Brazil.

Recent human herpesvirus 6 (HHV-6) infection was detected in cases of exanthem subitum (ES) involving four children, aged 10 to 24 months, between April and August 1994, in Belém, Brazil. By using the indirect immunofluorescence antibody assay (IFA), significant increases (at least eight times) in antibody concentrations were noted from the acute to the convalescent serum samples, with titers ranging from < 1:10/1:80 to < 1:10/1:640 (patients 3 and 2, respectively). All children had high fever (over 39 degrees C) for three days, followed by generalized, maculo-papular skin rash. A physical examination of the children also revealed concomitant, cervical lymph node swelling and tonsillar pharyngitis in two of them.

Prevalence of HTLV-I antibody among two distinct ethnic groups inhabiting the Amazon region of Brazil.

HTLV-I seroprevalences of 3.63% (02/55), 12.19% (10/82) and 13.88% (10/72) were demonstrated among Tiryio, Mekranoiti and Xicrin Amazonian Indians, respectively, by the Western blotting enzyme assay (WBEI). By indirect immunoelectron microscopy (IIEM), 2 Tiriyo, 9 Mekranoiti and 6 Xicrin Amerindians were reactive. Of 44 serum samples from Japanese immigrants, none reacted by any of the techniques before mentioned. One, 8 and 6 serum samples from Tiryio, Mekranoiti and Xicrin Indians, respectively, were both WBEI and IIEM positive. Our results strongly suggest that HTLV-I and/or an HTLV-I antigenic variant circulate (s) among populations living in the Amazon region of Brazil.

Antibody to human T-lymphotropic virus in a patient with Guillain-Barré syndrome (case report)

Serum sample obtained from a male, 12 year old patient suffering from Guillain-Barré syndrome (GBS) was positive for human T-lymphotropic virus (HTLV-I) antibody by the enzyme-linked immunosorbent assay (ELISA) and the Western Blot analysis (WB). Attempts to isolate enteroviruses (including poliovirus) from faecal material in both tissue culture and suckling mice were unsuccessful; in addition, acute and convalescent paired serum samples did not show any evidence of recent poliovirus infection when tested against the three serotypes. Specific tests for detection of Epstein-Barr virus infection were not performed; however, the Paul-Bunnel test yielded negative results. ELISA for detection of anti-cytomegalovirus IgM was also negative. The concomitant occurrence of either adult T cell leukemia (ATL) or lymphoma was not recorded in this case.

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.

Hepatitis B virus surface antigen (HBsAg) and antibody (anti-HBs) forming immune complexes in fulminant hepatitis.

This paper reports an unusual pattern of serological HBV markers and the presence of HBsAg/anti-HBs immune complexes in serum samples from two patients with fulminant hepatitis from the Brazilian Western Amazon Basin. The diagnosis was made by both serologic tests and demonstration of antigen/antibody complexes by transmission electron microscopy. Concurrent Delta virus superinfection is also discussed.

Detection of enteroviral sequence in endomyocardial tissues from patients with cardiac diseases in northern Brazil.

In the present report we describe the results from a pilot study aimed at detecting enterovirus sequence in cardiac tissues, obtained through endomyocardial biopsies, from patients suffering from cardiac diseases in the Amazon region. Six samples that were collected from three patients were analysed by RT-PCR showing 3 positive and 3 negative results. These preliminary findings suggest the participation of enteroviruses in the etiology of cardiac diseases, mainly myocarditis, and warrant further and broader local studies on this subject.