Maria Lúcia Rácz

Perfil etiológico das diarréias agudas de crianças atendidas em São Paulo

Objetivo: determinar o perfil etiologico das diarreias agudas de um grupo de criancas de baixo nivel socioeconomico atendidas em um servico regional de pronto-atendimento pediatrico. Metodo: durante dois anos, as criancas com diarreia aguda atendidas durante um horario pre-estabelecido do dia e da semana foram incluidas no estudo. Os outros criterios seletivos eram: a) idade inferior a 5 anos; b) nao utilizacao de antibiotico no mes precedente; c) ausencia de viagem para fora da cidade no mes precedente. Foram pesquisados nas fezes: a) rotavirus (imunofluorescencia e contra-imunoeletroforese); b) bacterias - cultura em agar MacConkey, agar SS, agar Columbia, verde brilhante, soroaglutinacao, deteccao de toxinas - INV, LT,ST,SLT I, SLT II, teste de Sereny, deteccao de fatores de virulencia -- EAF, eae ,BFP; c) protozoarios (Hoffman e Faust). No mesmo periodo, um grupo controle sem diarreia foi tambem avaliado para os mesmos patogenos fecais. Resultados: no periodo de marco de 1994 a junho de 1996, foram selecionadas para o estudo 154 criancas com diarreia aguda (GDA) e 42 criancas sem diarreia (GSDA). Foram detectados agentes enteropatogenicos em 112 casos (72,8%) do GDA, e em 9 (21,5%) do GSDA. A associacao de dois ou mais enteropatogenos ocorreu em 47 (30,5%) casos do GDA, e em 3 (7,1%) do GSDA. Os patogenos encontrados por caso, do GDA, foram: rotavirus 32 (20,8%), bacterias 53 (34,4%), ambos 25 (16,2%), e 2 (1,4%) com Giardia lamblia (em um caso associada a rotavirus e noutro a bacteria). No GSDA, foram detectadas bacterias em 8 casos (19,1%), e bacteria associada a Giardia lamblia em 1 (2,4%) caso. Das 105 bacterias isoladas no GDA, 90 eram Escherichia coli (EPEC 27, DAEC 24, ETEC 21, EAEC 18), 12 eram Shigella sp, 2 eram Salmonella sp, e uma era Yersinia sp. As criancas com infeccao mista - viral e bacteriana - apresentaram maior ocorrencia de vomitos repetidos, desidratacao e internacao.Conclusoes: as bacterias foram os enteropatogenos mais detectados nos casos de diarreia aguda, sendo a Escherichia coli a mais frequente. Na maior parte, as cepas de Escherichia coli eram de biovariedade nao-EPEC, habitualmente nao investigadas nos laboratorios de patologia clinica. O rotavirus foi encontrado em grande parcela dos casos, muitas vezes em associacao com as bacterias. Os protozoarios tiveram importância reduzida.

Etiologic profile of acute diarrhea in children in São Paulo

OBJECTIVE: To evaluate the etiologic profile of acute diarrhea in socioeconomically deprived children assisted at a regional pediatric emergency care service. METHODS: During two years all children with acute diarrhea assisted at a previously established day and week time schedule were included in the study. Other selective criteria were: a) age less than 5 years; b) nonuse of antibiotics in the previous month; and c) no travel outside the city in the previous month. Stool examination was used for the detection of the following microorganisms: a) rotavirus (immunofluorescence and counterimmunoelectrophoresis); b) bacteria - culture in MacConkey agar, SS agar, Columbia agar, bright green, serotyping, detection of toxins - INV, LT,ST,SLT I, SLT II, Sereny test, detection of virulence factors- EAF, eae, BFP; and c) protozoa (Hoffman and Faust). In the same period, a control group without diarrhea was also evaluated for the same fecal pathogens. RESULTS: Between March 1994 and June 1996, 154 children with acute diarrhea (AD) and 42 control children (WAD), that is, without acute diarrhea, were selected. In the AD group, intestinal pathogens were detected in 112 (72.8%) cases, and in 9 (21.5%) cases in the WAD group. The association of two or more intestinal pathogens occurred in 47 (30.5%) cases in the AD group, and in 3 (7.1%) cases in the WAD group. The pathogens identified in the AD cases were: Rotavirus: 32 (20.8%), bacteria: 53 (34.4%), both: 25 (16.2%), and 2 (1.4%) with Giardia lamblia (in one case associated with Rotavirus and in another one associated with bacteria). In the WAD group, only bacteria were detected in 8 (19.1%) cases, and bacteria associated with Giardia lamblia in 1 (2.4%) case. Altogether, there were 105 bacteria isolated in the AD group: 90 were Escherichia coli (EPEC 27, DAEC 24, ETEC 21, EAEC 18), 12 were Shigella sp, 2 were Salmonella sp, and one was Yersinia sp. Children with mixed infections (viral and bacterial) had increased incidence of severe vomiting, dehydration and hospitalization. CONCLUSIONS: Bacteria were the most frequent pathogens detected in acute diarrhea cases, among which Escherichia coli was highly predominant. The majority of Escherichia coli strains belong to non-EPEC varieties, strains that are not routinely evaluated in clinical laboratories of pathology. Rotavirus was found in a great number of diarrhea cases, often associated with bacteria. Protozoa showed reduced importance.

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.

Characterization of mixed infections with different strains of bovine rotavirus in an outbreak of diarrhea in dairy herds in Goiás, Brazil

Ten faecal samples of bovine rotavirus from calves less than 30 days old from an outbreak of diarrhea in Hidrolândia, Goias, Brazil were submitted to serological and molecular characterization, using enzyme immunoassay for subgrouping and serotyping, PAGE for determination of electropherotypes and PCR for genome typing. Nine samples belonged to group A/subgroup I rotavirus and one sample was group A / subgroup non-I/non-II. Four samples were characterized as G10P[11] (B223-like), four samples showed a mixture of two rotavirus strains (G6G10 and P[5]P[11]), one sample was characterized as G6P[11] and one sample was characterized only by G serotyping/genotyping, and did not react with any P primer used. Two electropherotypes were detected and both were present in the same animal. This study demonstrates that two different electropherotypes and/or serotypes of bovine rotavirus can circulate in the same outbreak.

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.

Molecular characterization of G and P-types bovine rotavirus strains from Goiás, Brazil: high frequency of mixed P-type infections

In this study, 331 samples from calves less than one month old from a dairy herd in the district of Piracanjuba, state of Goiás, Brazil were tested for rotavirus. Thirty-three samples (9.9%) tested positive for rotavirus. Out of those, 31 were submitted to G and P characterization by reverse transcription followed by semi-nested polymerase chain reaction. Two samples were characterized as G6P[1], three as G10P[11] and five as G6P[11]. The majority of the samples (51.6%) displayed multiple P genotypes (P-genotype mixtures), including typical human genotypes P[4] and P[6M], suggesting the occurrence of co-infections and genetic reassortment. Also, the detection of human genotypes in bovine samples may be considered evidence of the zoonotic potential of rotaviruses. To our knowledge, this is the first report of such a high frequency of P genotype mixtures in bovine rotavirus samples. It also increases data on G and P rotavirus genotypes circulating in dairy herds in Brazil and can help in the development of more efficient immunization approaches, thereby controlling infection and reducing economical losses.

G and P rotavirus genotypes in stool samples from children in Teresina, State of Piauí

A total of 123 stool specimens collected in Teresina, Piauí between 1994 and 1996, from 0 to 2-year-old children with diarrhea, were used for this study. Molecular characterization of the G and P rotavirus genotypes was performed using the reverse transcriptase polymerase chain reaction. The following results were obtained for the P genotypes: P[8] (17. 1%), P[1] (4. 9%), P[4] (3. 3%), P[6, M37] (2. 4%) and mixtures (27. 6%). The P[1]+P[8] mixture was found in 19. 5% of the samples. For the G genotypes, the results were: G1 (25. 2%), G5 (13. 8%), G2 (2. 5%), G4 (2. 5%), G9 (0. 8%) and mixtures (41. 5%). G1+G5 was the mixture most frequently found (12. 1%). Our results showed unusual combinations such as P[1]G5 and P[1]+P[8]G5. The high percentage of mixtures and unusual combinations containing mixtures of human and animal rotavirus genotypes strongly suggests the possibility of gene reassortment and interspecies transmission.

Projecting the effectiveness of RotaTeq® against rotavirus-related hospitalisations in Brazil

RotaTeq® (Merck & Company, Inc, Whitehouse Station, NJ, USA) is an oral pentavalent rotavirus vaccine (RV5) that has shown high and consistent efficacy in preventing rotavirus gastroenteritis (RGE) in randomised clinical trials previously conducted in industrialised countries with high medical care resources. To date, the efficacy and effectiveness data for RV5 are available in some Latin American countries, but not Brazil. In this analysis, we projected the effectiveness of RV5 in terms of the percentage reduction in RGE-related hospitalisations among children less than five years of age in four regions of Brazil, using a previously validated mathematical model. The model inputs included hospital-based rotavirus surveillance data from Goiânia, Porto Alegre, Salvador and São Paulo from 2005-2006, which provided the proportions of rotavirus attributable to serotypes G1, G2, G3, G4 and G9, and published rotavirus serotype-specific efficacy from the Rotavirus Efficacy and Safety Trial. The model projected an overall percentage reduction of 93% in RGE-related hospitalisations, with an estimated annual reduction in RGE-related hospitalisations between 42,991-77,383 in the four combined regions of Brazil. These results suggest that RV5 could substantially prevent RGE-related hospitalisations in Brazil.

Detection of rotavirus in dogs with diarrhea in Brazil

Rotavirus was detected by the enzyme-linked immunosorbent assay (ELISA) in the faeces of a diarrheic dog. Virus particles with morphology typical of rotavirus were visualized by direct electron microscopy. This sample was subsequently tested for the four main human serotypes (G1-G4), by ELISA with monoclonal antibodies. G genotyping was attempted by RT-PCR using G1-G6 and G8-G11 primers but no positive results could be yielded. Also using RT-PCR it was possible to characterize this canine strain as belonging to P[ 3] genotype. This is the first canine rotavirus detected in Brazil.

Molecular characterisation of the NSP4 gene of group A human rotavirus G2P[4] strains circulating in Sao Paulo, Brazil, from 1994 and 2006 to 2010

Group A human rotaviruses (HuRVA) are causative agents of acute gastroenteritis. Six viral structural proteins (VPs) and six nonstructural proteins (NSPs) are produced in RV-infected cells. NSP4 is a diarrhoea-inducing viral enterotoxin and NSP4 gene analysis revealed at least 15 (E1-E15) genotypes. This study analysed the NSP4 genetic diversity of HuRVA G2P[4] strains collected in the state of São Paulo (SP) from 1994 and 2006-2010 using reverse transcription-polymerase chain reaction, sequencing and phylogenetic analysis. Forty (97.6%) G2P[4] strains displayed genotype E2; one strain (2.4%) displayed genotype E1. These results are consistent with the proposed linkage between VP4/VP7 (G2P[4]) and the NSP4 (E2) genotype of HuRVA. NSP4 phylogenetic analysis showed distinct clusters, with grouping of most strains by their genotype and collection year, and most strains from SP were clustered together with strains from other Brazilian states. A deduced amino acid sequence alignment for E2 showed many variations in the C-terminal region, including the VP4-binding domain. Considering the ability of NSP4 to generate host immunity, monitoring NSP4 variations, along with those in the VP4 or VP7 protein, is important for evaluating the circulation and pathogenesis of RV. Finally, the presence of one G2P[4]E1 strain reinforces the idea that new genotype combinations emerge through reassortment and independent segregation.