Veridiana Munford

Hospital-based surveillance to evaluate the impact of rotavirus vaccination in São Paulo, Brazil.

Background: Brazil implemented routine immunization with the human rotavirus vaccine, Rotarix, in 2006 and vaccination coverage reached 81% in 2008 in São Paulo. Our aim was to assess the impact of immunization on the incidence of severe rotavirus acute gastroenteritis (AGE). Methods: We performed a 5-year (2004–2008) prospective surveillance at a sentinel hospital in São Paulo, with routine testing for rotavirus in all children less than 5 years of age hospitalized with AGE. Genotypes of positive samples were determined by reverse transcription polymerase chain reaction. Results: During the study, 655 children hospitalized with AGE were enrolled; of whom 169 (25.8%) were positive for rotavirus. In the postvaccine period, a 59% reduction in the number of hospitalizations of rotavirus AGE and a 42.2% (95% confidence interval [CI], 18.6%–59.0%; P = 0.001) reduction in the proportion of rotavirus-positive results among children younger than 5 years were observed, with the greatest decline among infants (69.2%; 95% CI, 24.7%–87.4%; P = 0.004). Furthermore, the number of all-cause hospitalizations for AGE was reduced by 29% among children aged <5 years. The onset and peak incidences of rotavirus AGE occurred 3 months later in the 2007 and 2008 seasons compared with previous years. Genotype G2 accounted for 15%, 70%, and 100% of all cases identified, respectively, in 2006, 2007, and 2008. Conclusions: After vaccine implementation, a marked decline in rotavirus AGE hospitalizations was demonstrated among children younger than 5 years of age, with the greatest reduction in the age groups targeted for vaccination. The predominance of genotype G2P[4] highlights the need of continued postlicensure surveillance studies.

Detection, Subgroup Specificity, and Genotype Diversity of Rotavirus Strains in Children with Acute Diarrhea in Paraguay

ABSTRACT Of a total of 220 stool specimens from children with acute diarrhea, mostly under the age of 3 years, collected in Paraguay between January 1999 and March 2000, 70 (31.8%) were found positive for rotaviruses (RV). Positive samples were characterized by electropherotyping and subgrouping. Sixty-one (87.1%) were classified as group A, subgroup II; one (1.4%) was classified as group A, subgroup I; six (8.6%) were group A, non-I non-II; and two (2.9%) were not tested. RV strains were G and P genotyped by reverse transcription-PCR. The following G types were detected: G4 (34.3%), G1 (21.4%), G2 (1.4%), and G9 (5.7%). Mixtures of human and animal genotypes were detected in 15 (21.4%) samples, and 11 samples (15.7%) were nontypeable. The following P types were detected: P[8] (48.6%), P[4] (1.4%), and P[1] (1.4%). A mixed type was found in 10% of samples, and an unexpectedly high percentage (38.6%) of nontypeable samples was found. The common human G- and P-type combinations P[8], G4 (15.7%) and P[8], G1 (14.2%) were detected. Mixed human and animal genotypes were observed as the following combinations: G4 + G5, G4 + G5 + G10, and G1 + G10 for G types and P[8]-P[1] for P types. The emerging G9 genotype was detected in four samples. These results show for the first time the diversity of RV circulating among children in Paraguay and contribute to the knowledge of this pathogen required to devise strategies to prevent diarrheal illness in this country. The finding of mixed genotypes may indicate interspecies transmission of RV between humans and animals.  

Genetic Diversity of Norovirus among Children with Gastroenteritis in São Paulo State, Brazil

ABSTRACT Norovirus (NoV) is one of the most common causes of acute gastroenteritis in children and adults. To study the prevalence and genetic variability of NoV in children with acute gastroenteritis in São Paulo State, Brazil, we examined 234 stool samples from children with or without gastroenteritis during a 5-year period (1995 to 1999). NoV RNA was detected by reverse transcription-PCR and confirmed by DNA sequence analysis. We used two different oligonucleotide primer sets targeting the 3′ end of the RNA polymerase gene (region B), as well a partial capsid region at the 3′ end of the VP1 gene (region D). A total of 78 (33.3%) of the samples tested positive for NoV, and in region B, of the 66 strains sequenced, 4 (6.1%) belonged to GI, 52 (78.7%) belonged to GII, and five samples (7.6%) contained a mixture of the GI and GII genotypes. Phylogenetic analysis showed that the majority (40 of 66 [60.6%]) of the strains belonged to genotype GII.4. The nucleotide sequence identity of three strains was lower than 77.9% compared to a region B reference sequence database but showed 85.3 to 88.8% identity with GII.2 Melksham strain in region D, indicating the circulation of a possible recombinant NoV strain. One sample (GII.3) was sequenced only in region D. In conclusion, we have a total of 67 sequenced strains. This is the first report that describes the predominance of GII.4 NoV strains in children visiting the ambulatory of different hospitals in São Paulo State, Brazil, and we show that mixtures of different strains can be found in individual samples, including some possible new recombinant strains.

DNA repair diseases: What do they tell us about cancer and aging?

The discovery of DNA repair defects in human syndromes, initially in xeroderma pigmentosum (XP) but later in many others, led to striking observations on the association of molecular defects and patients’ clinical phenotypes. For example, patients with syndromes resulting from defective nucleotide excision repair (NER) or translesion synthesis (TLS) present high levels of skin cancer in areas exposed to sunlight. However, some defects in NER also lead to more severe symptoms, such as developmental and neurological impairment and signs of premature aging. Skin cancer in XP patients is clearly associated with increased mutagenesis and genomic instability, reflecting the defective repair of DNA lesions. By analogy, more severe symptoms observed in NER-defective patients have also been associated with defective repair, likely involving cell death after transcription blockage of damaged templates. Endogenously induced DNA lesions, particularly through oxidative stress, have been identified as responsible for these severe pathologies. However, this association is not that clear and alternative explanations have been proposed. Despite high levels of exposure to intense sunlight, patients from tropical countries receive little attention or care, which likely also reflects the lack of understanding of how DNA damage causes cancer and premature aging.

Epidemiological features of rotavirus infection in Goiânia, Goiás, Brazil, from 1986 to 2000

A total of 2,605 faecal specimens from children up to 10 years old with or without diarrhoea were collected. Samples were obtained from 1986 to 2000 in hospitals, outpatient clinics and day-care centers in Goiânia, Goiás. Two methodologies for viral detection were utilized: a combined enzyme immunoassay for rotavirus and adenovirus and polyacrylamide gel electrophoresis. Results showed 374 (14.4%) faecal specimens positive for Rotavirus A, most of them collected from hospitalized children. A significant detection rate of rotavirus during the period from April to August, dry season in Goiânia, and different frequencies of viral detection throughout the years of study were also observed. Rotavirus was significantly related to hospitalization and to diarrhoeal illness in children up to 24 months old. This study reinforces the importance of rotavirus as a cause of diarrhoea in children and may be important in regards to the implementation of rotavirus vaccination strategies in our country.

Rotavirus Gastroenteritis in Children in 4 Regions in Brazil: A Hospital-Based Surveillance Study

BACKGROUND Rotavirus is a major cause of gastroenteritis in children. Knowledge of rotavirus genotypes is important for vaccination strategies. METHODS During 2005-2006, rotavirus surveillance studies were conducted in São Paulo, Salvador, Goiânia, and Porto Alegre, Brazil. Stool samples were collected from children <5 years of age who had diarrhea and were screened by the Rotaclone Enzyme Immunoassay for the presence of rotavirus. Confirmed rotavirus-positive samples were characterized for P and G genotypes by reverse-transcriptase polymerase chain reaction. RESULTS A total of 510 stool samples were collected. Of these, 221 (43.3%) were positive for rotavirus. Overall, G9 was the predominant G type, followed by G2, and G1; P[4] and P[8] were the predominant P types. The most frequent G/P genotype combination detected was G2P[4], followed by G9P[8], G9P[4], and G1P[8]. G2P[4] was the predominant type in Goiânia and Salvador; G9P[8] and G1P[8] were predominant in São Paulo and Porto Alegre, respectively. CONCLUSIONS The prevalence, seasonality, and genotype distribution of rotavirus infection varied in different regions in Brazil. With immunization programs, continuous monitoring of rotavirus types is important to detect novel and emerging strains.

Novel XPG (ERCC5) Mutations Affect DNA Repair and Cell Survival after Ultraviolet but not Oxidative Stress

Nucleotide excision repair (NER) is the most flexible of all known DNA‐repair mechanisms, and XPG is a 3′‐endonuclease that participates in NER. Mutations in this gene (ERCC5) may result in the human syndrome xeroderma pigmentosum (XP) and, in some cases, in the complex phenotype of Cockayne syndrome (CS). Two Brazilian XP siblings, who were mildly affected, were investigated and classified into the XP‐G group. The cells from these patients were highly ultraviolet (UV) sensitive but not sensitive to photosensitized methylene blue, an agent that causes oxidative stress. This phenotype is in contrast to XP‐G/CS cells, which are highly sensitive to this oxidative agent. Sequencing revealed a compound heterozygous genotype with two novel missense mutations: c.83C>A (p.Ala28Asp) and c.2904G>C (p.Trp968Cys). The first mutation maps to the catalytic site of the XPG protein, whereas the second may compromise binding to DNA. Functional assays indicated that the mutated alleles were unable to perform the complete repair of UV‐irradiated plasmids; however, full correction was observed for oxidatively damaged plasmids. Therefore, the XP phenotype of these patients is caused by novel missense mutations that specifically affect DNA repair for UV‐ but not oxidative‐stress‐induced DNA damage, and implications for XP versus XP/CS phenotype are discussed.

Molecular and Serological Characterization of Group A Rotavirus Isolates Obtained from Hospitalized Children in Goiânia, Brazil, 1998–2000

Fecal samples positive for rotavirus group A (n=120) were analyzed by enzyme immunoassay-monoclonal antibody (EIA-MAb) serotyping and/or reverse transcription/multiplex polymerase chain reaction (PCR) amplification to determine the prevalence of the [P] and G genotypes. The most prevalent G genotype/serotype detected was G1 (76.7%), followed by G2 (5.0%). Six samples were characterized as G9 by multiplex PCR, and one sample was characterized as G3 by EIA-MAb. The combinations of [P] and G genotypes found were P[8] and G1 (20.8%), P[6] and G1 (10.8%), P[6] and G9 (4.2%), P[8] and G2 (1.7%), and P[6] and G2 (0.8%). The diversity of rotavirus group A [P] and G genotypes/serotypes reinforces the need for continuous characterization of rotaviruses circulating in populations in Brazil.

Molecular characterization of astrovirus in stool samples from children in São Paulo, Brazil

The purpose of this study was to characterize astrovirus in faecal samples collected from children with and without diarrhea in São Paulo, Brazil, grouped into two sets: EPM and HU. Detection and genotyping were carried out using reverse transcription nested polymerase chain reaction (RT-PCR) with specific primers directed towards the genome open reading frame 2 (ORF2). Results for EPM set showed that 66/234 (28.2%) were positive: 28/94 (29.7%) from children with acute diarrhea, 14/45 (31.1%) with persistent diarrhea, and 9/55 (16.3%) from control individuals. No data was available for 15/40 (37.5%) of samples. Mixed infections with other viruses were found in 33 samples. In the HU, 18/187 (9.6%) were positive: 12/158 (7.6%) from individuals with acute diarrhea and 6/29 (20.7%) from control children. Four samples were mixed with other viruses. Out of 66 astrovirus positive EPM samples, 18 (27.2%) were characterized as human astrovirus type-1 (HAstV-1), two (3.0%) as HAstV-2, two (3.0%) as HAstV-3, and three (4.5%) as HAstV-8. Among 18 astrovirus positive HU samples, one (5.5%) was characterized as HAstV-1, six (33.3%) as HAstV-2, and one (5.5%) as HAstV-8. Two HAstV-8 genotyped samples were further confirmed by nucleotide sequencing. Our results shows that astroviruses are circulating in a constant manner in the population, with multiple serotypes, in higher frequency than it was described for other Brazilian regions. For the first time in Sao Paulo, Brazil, it was shown that astroviruses play an important role in children gastroenteritis, as described for most locations where they were detected.

Serological and molecular diversity of human rotavirus in São Paulo, Brazil

From a total of 187 fecal samples from children with ages between 0 and 5 years, collected in the Hospital Universitario -USP, Brazil, from 1994 to 1996, 54 (28.9%) were positive for rotavirus. Positive samples were characterized by electropherotyping, subgrouping, G serotype and genotype and P genotype. Rotavirus electropherotypes were characterized in four different long genome patterns (38.9%), one short genome pattern (34.0%) and 18.0% were characterized as an unusual pattern. Subgroup I was found in 38.9% strains, subgroup II in 50.0% and 7.7% was subgroup nonI-nonII. For G serotypes, G2 was found in 59.3%, G1 was identified in 33.3% of strains, two samples showed mixtures of G1+G2 and one sample was G1+G3. Ten samples characterized as serotype G2 showed a long eletropherotype. Genotype G2 was the most frequently and was found in 37 (44.0%) samples (23 samples as a single genotype and 14 as mixtures of genotypes). G1 was found in 15 samples. G3 and G4 was detected mainly in mixtures of genotypes and G5, G6 and G9 were identified only in mixtures. A total of 20 (38.5%) samples were characterized as G genotype mixtures and P mixtures were found in 16 (29.6%) samples. P[4] was found in 55.6% of samples, P[8] in 51.9% and P[6-M37 like] in 22.3% of cases. P[6-Gottfried like] and P[11] were detected only in mixtures. One sample with G6 specificity, mixed with a G2 rotavirus and a P[11] strain, mixed with P[4] and P[8]strain was described for the first time in Latin America.