First detection of a human astrovirus type 8 in a child with diarrhea in Belém, Brazil: comparison with other strains worldwide and identification of possible three lineages

This study describes the genetic relationships of the first human astrovirus type-8 (HAstV-8) detected in Belém-Brazil, during a public hospital-based study. This strain was compared with other HAstV-8 strains identified elsewhere which have sequences available at GeneBank. The regions ORF1a (primers Mon348/Mon340) and ORF2 (primers Mon269/Mon270) were analyzed by nucleotide sequencing and a high similarity rate was observed among the Belém strain and other HAstV-8 strains. In ORF1a, homology values of 93-100% were detected, and in ORF2 96-99%. Considering the sequence variation (7%) observed in ORF2 region, it was suggested that HAstV-8 strains could be divided in three different lineages.

Rotavirus A genotype G1P[8]: a novel method to distinguish wild-type strains from the Rotarix® vaccine strain

Rotaviruses are important enteric pathogens for humans and animals. Group A rotaviruses (RV-A) are the most common agents of severe gastroenteritis in infants and young children and vaccination is the most effective method to reduce RV-A-associated diseases. G1P[8], the most prevalent RV-A genotype worldwide, is included in the RV-A vaccine Rotarix®. The discrimination between wild-type G1P[8] and vaccine G1P[8] strains is an important topic in the study of RV-A epidemiology to manage outbreaks and to define control measures for vaccinated children. In this study, we developed a novel method to segregate the wild-type and vaccine strains using restriction endonucleases. The dsRNA from the Rotarix® vaccine was sequenced and the NSP3 gene was selected as the target gene. The vaccine strain has a restriction pattern that is different than that of wild-type RV-A G1P[8] isolates after digestion with the restriction endonuclease BspHI. This pattern could be used as a marker for the differentiation of wild-type G1P[8] strains from the vaccine strain.

ROTAVIRUS GENOTYPES CIRCULATING IN BRAZIL, 2007-2012: IMPLICATIONS FOR THE VACCINE PROGRAM

SUMMARY Regarding public health in Brazil, a new scenario emerged with the establishment of universal rotavirus (RV) vaccination programs. Herein, the data from the five years of surveillance (2007-2012) of G- and P-type RV strains isolated from individuals with acute gastroenteritis in Brazil are reported. A total of 6,196 fecal specimens were investigated by ELISA and RT-PCR. RVs were detected in 19.1% (1,181/6,196). The peak of RV incidence moved from June-August to September. RV was detected less frequently (19.5%) among children ≤ 5 years than in older children and adolescents (6-18 years) (40.6%). Genotype distribution showed a different profile for each year: G2P[4] strains were most prevalent during 2007-2010, G9P[8] in 2011, and G12P[8] in 2012. Mixed infections (G1+G2P[4], G2+G3P[4]+P[8], G2+G12P[8]), unusual combinations (G1P[4], G2P[6]), and rare strains (G3P[3]) were also identified throughout the study period. Widespread vaccination may alter the RV seasonal pattern. The finding of RV disease affecting older children and adolescents after vaccine implementation has been reported worldwide. G2P[4] emergence most likely follows a global trend seemingly unrelated to vaccination, and G12, apparently, is emerging in the Brazilian population. The rapidly changing RV genotype patterns detected during this study illustrate a dynamic population of co-circulating wildtype RVs in Brazil.

Group A rotavirus genotypes and the ongoing Brazilian experience: a review

Brazil was the first Latin American country to introduce universal group A rotavirus (RV-A) vaccination in March 2006, resulting in a unique epidemiological scenario. Since RV-A first identification in Brazil, 2,691 RV-A-positive stool samples, collected between 1982- 2007, were typed by independent research groups throughout the country. In the pre-vaccination era, 2,492 RV-A-positive samples collected from 1982-2005 were successfully typed, while 199 samples were analyzed from 2006-2007. According to the reviewed studies, there were two important times in the pre-vaccination era: (i) the period from 1982-1995, during which the detection of G5P[8] RV-A, in addition to the classical genotypes G1-4, challenged vaccine development programs; and (ii) the period from 1996-2005, during which genotype G9P[8] emerged, following a global trend. The rate of G2P[4] RV-A detection decreased from 26% (173/653) during 1982-1995 to 2% (43/1,839) during 1996-2005. The overall detection rate of RV-A genotypes from 1982-2005 was as follows: 43% (n = 1,079) G1P[8]/G1P[not typed (NT)]; 20% (n = 488) G9P[8]/G9P[NT]; 9% (n = 216) G2P[4]/G2P[NT]; 6% (n = 151) G3P[8]/G3P[NT]; 4% (n = 103) G4P[8]/G4P[NT]; and 4% (n = 94) G5P[8]/G5P[NT]. Mixed infections accounted for 189 (7%) of the positive samples, while atypical G/P combinations or other genotypes, including G6, G8, G10 and G12, were identified in 172 (7%) samples. The initial surveillance studies carried out in several Brazilian states with RV-A-positive samples collected in 2006 and 2007 show a predominance of G2P[4] strains (148/199 or 74%). Herein, we review RV-A typing studies carried out since the 1980s in Brazil, highlighting the dynamics of RV-A strain circulation profiles before and early after universal use of RV-A vaccine in Brazil.

Rotavírus A em crianças de até três anos de idade, hospitalizadas com gastroenterite aguda em Campo Grande, Estado do Mato Grosso do Sul

Através da eletroforese em gel de poliacrilamida e do ensaio imunenzimático combinado para rotavírus e adenovirus, foram analisadas 380 amostras fecais de crianças com até 3 anos, hospitalizadas com diarréia aguda, entre maio de 2000 e janeiro de 2004, em Campo Grande, MS. Do total de amostras, 88 (23,2%) foram positivas para Rotavirus A. Dentre essas, 81 (92%) tiveram padrão eletroferotípico definido, sendo 77 (87,5%) de padrão longo e quatro (4,5%) de padrão curto. A caracterização genotípica G e P foi feita por RT-Nested-PCR para 85 amostras, sendo 56 (65,9%) genotipáveis para genótipo G. Dentre essas, 49 (87,5%) foram G1, cinco (8,9%) G4, uma (1,8%) G3 e uma (1,8%) G9. Considerando a genotipagem P, 37 (43,5%) foram genotipáveis e todas eram P[8]. A associação G e P mais observada foi G1P[8], 33 (89,2%) amostras; seguida de G4P[8], duas (5,4%) amostras; G3P[8], uma (2,7%) amostra; e G9P[8], uma (2,7%) amostra.

Hospitalization due to norovirus and genotypes of rotavirus in pediatric patients, state of Espírito Santo

Viruses are the leading cause for hospitalization due to gastroenteritis worldwide. Group A rotaviruses (RV) are the most prevalent and are assorted in glycoproteins (G) and protease sensitive (P) dual genotypes based on polymorphic genes that encode the external VP7 and VP4 capsid proteins, respectively. Noroviruses (NoV) have increasingly answered by sporadic gastroenteritis. This study aimed to determine the prevalence of NoV and RV in 68 hospitalized children, between July 2004 and November 2006, at a pediatric hospital in Vitória city, state of Espírito Santo, Southeastern Brazil. Nucleic acid was extracted from fecal suspension following the guanidine-silica procedure. Reverse transcriptase-polymerase chain reaction (RT-PCR) and polyacrylamide gel electrophoresis were employed for NoV and RV detection, respectively. RV genotyping was accomplished using RT-PCR followed by heminested multiplex PCR with specific primers for the most prevalent types of G and P. Fecal samples were positive for NoV and RV in 39.7% (27/68) and 20.5% (14/68), respectively and together were responsible for 60% (41/68) of the cases. RV genotypes were: 50% G9P[8], 28.7% G2P[4], 7.1% G1P[8], G2P[8] and G?P[8]. Vomit was a prominent manifestation observed in 92% and 85% of the NoV and RV cases, respectively. The median hospitalization was 5 and 5.5 days for the patients infected with NoV and RV, respectively. The data showed that NoV prevailed over RV and it also corroborated the emergence of RV G9 genotype followed by G2P[4], reinforcing the need for RV genotype surveillance.

Detection of caliciviruses associated with acute infantile gastroenteritis in Salvador, an urban center in Northeast Brazil

Acute gastroenteritis caused by viruses is one of the leading causes of infantile morbidity. The aim of the present study was to investigate the presence of human caliciviruses of the genera norovirus and sapovirus in children up to 3 years of age with acute gastroenteritis from low-income communities in the city of Salvador, Brazil. This study is an extension of previous work carried out to establish the profile of the most prevalent enteric pathogens present in these communities. In this report, 139 fecal samples, collected from July 2001 to January 2002 were analyzed by RT-PCR and 13 (9%) were positive for human caliciviruses. By sequencing, seven isolates were characterized as norovirus genogroup GII and one as sapovirus genotype GII/1. Sequencing of the previously detected group-A rotaviruses and human astroviruses was also performed and revealed the circulation of rotavirus group A genotypes G1P[8] and G9P[8], and human astrovirus genotypes 6, 7, and 8. No mixed infection was observed. Community-based studies provide geographically representative information on disease burden. However, there are only a few reports in developing countries concerning the genotypes of the most important gastroenteric viruses detected in such communities. The present findings demonstrate the wide diversity of genotypes of the most important viruses responsible for acute gastroenteritis circulating in low-income communities.

Oral live attenuated human rotavirus vaccine (RotarixTM) offers sustained high protection against severe G9P[8] rotavirus gastroenteritis during the first two years of life in Brazilian children

In a large Phase III trial conducted in 10 Latin American countries, the safety and efficacy of the live attenuated monovalent rotavirus vaccine RIX4414 was evaluated in 15,183 healthy infants followed up during the first two years of life. Belém was the only site in Brazil included in this multicentre trial. The study in Belém included a subset of 653 infants who were followed up until 24 months of age for protection against severe rotavirus gastroenteritis. These subjects were randomly assigned in a 1:1 ratio to receive two doses of vaccine (n = 328) or two doses of placebo (n = 325) at approximately two and four months of age. Of the 653 enrolled infants, 23 dropped out during the study period. For the combined two-year period, the efficacy of RIX4414 was 72.3% [95% confidence interval (CI) 37.5-89.1%] against severe rotavirus-related gastroenteritis, reaching a protection rate of 81.8% (95% CI 36.4-96.6%) against circulating wild-type G9 rotavirus strains. It is concluded that two doses of RIX4414 are highly efficacious against severe rotavirus gastroenteritis in Belém during the first two years of life and provide high protection against the worldwide emergence and spread of G9P[8] strains.

Immunoelectrophoretic study on common antigens of São Lourenço da Mata and Belo Horizonte strains of Schistosoma mansoni adult worms and Biomphalaria snails

Immunoelectrophoretic studies on common antigens were carried out by using rabbits sera immunized against São Lourenço da Mata and Belo Horizonte strains of Schistosoma mansoni adult worms and antigens of Biomphalaria glabrata pigmented (Jaboatão - PE); B. glabrata albino (Belo Horizonte - MG) and B. straminea (São Lourenço da Mata, PE). Furthermore, the reverse approach was proceeded, namely, sera anti Biomphalaria snails produced in rabbits were tested against both strains of Schistosoma adult worm antigens. The analysis of the common antigens between the SLM strains of S. mansoni adult worm and B. glabrata pigmented showed 8 to 9 precipitin bands, 3 bands with B. glabrata albino and only 1 band with B. straminea crude extracts. On the other hand, the BH strain of S. mansoni adult worm antisera produced 6 to 7 bands with B. glabrata pigmented, 5 bands with B. glabrata albino and 1 band with B. straminea antigenic extract. Biomphalaria snails crude extracts were fractionated by Sephadex G-100 column and three fractions were collected from each snail strain. The fractions were tested with anti SLM and BH strains of S. mansoni adult worm sera by immunoelectrophoresis. The common antigens fractionated from Biomphalaria snails crude extracts and those found for both strains of S. mansoni adult worm mostly existed in the first fraction and they were estimated to have molecular weight over 158,000 daltons. In our laboratory, it was found a relationship between the antigenic similarities and experimental infection rates of S. mansoni towards Biomphalaria snails so that more bands were seen with increasing infection rates of S. mansoni.

Progression of Salmonella Enteritidis phage type 4 strains in São Paulo State, Brazil

A total of 574 S. Enteritidis strains (383 from human sources and 191 from non-human sources) isolated between 1975-95, in São Paulo State, Brazil, were phagetyped. Among the strains isolated during the period of 1975-92, 80.9% of them belonged to phage type 8 (PT-8), but in 1993 strains of PT-4 accounted for 65.2% of all the S. Enteritidis isolates. In the following years, PT-4 strains accounted for 99.7% and 98.4% of phagetyped S. Enteritidis strains. The results obtained suggested that the current epidemic of S. Enteritidis in São Paulo State is clearly associated with the progression of PT-4 strains.

Serotype, mating type and ploidy of Cryptococcus neoformans strains isolated from patients in Brazil

Serotype, mating type and ploidy of 84 strains of Cryptococcus neoformans isolated from 61 AIDS and 23 non-AIDS patients admitted in a tertiary teaching hospital in São Paulo, Brazil were examined. Among 61 strains isolated from AIDS patients, 60 strains were var. grubii (serotype A). Only one strain was var. gattii (serotype B). No var. neoformans (serotype D) was found. Among 23 strains isolated from non-AIDS patients, 15 were var. grubii (serotype A) and the remaining 8 were var. gattii, all of which were serotype B. Seventy-three of the 75 serotype A strains were the heterothallic alpha type (MATalpha) and the remaining 2 were untypable (asexual). Most of the MATalpha strains (69/73) were haploid and the remaining 4 strains were diploid. Similarly, both of the 2 asexual strains among the 75 serotype A strains were haploid. There were no alpha-mating type (MATalpha) strains among the 84 isolates. All of the 8 var. gattii strains were serotype B and haploid. Among a total of 84 strains tested, neither serotype AD nor serotype D were found. Neither triploid nor tetraploid were found. These results suggest that the serological, sexual and ploidy characteristics in C. neoformans strains isolated from AIDS patients in São Paulo were rather simple, whereas strains isolated from non-AIDS patients presented serotype A and B with predominance of serotype A.

Conventional and molecular typing of Salmonella typhi strains from Brazil

Phenotypic and genotypic characteristics of Salmonella Typhi were studied in 30 strains, isolated in different years, from some areas in Brazil. Conventional typing methods were performed by biochemical tests, Vi phage-typing scheme, and antimicrobial susceptibility test. Molecular typing methods were performed by analysis of plasmid DNA and by random amplified polymorphic DNA (RAPD-PCR). For the latter, an optimization step was performed to ensure the reproducibility of the process in genetic characterization of S. Typhi. The predominance of 76.7% of biotype I (xylose +, arabinose -) was noticed in all studied areas. Three phage types were recognized, with prominence for the phage types A (73.3%) and I+IV (23.3%). All the strains were susceptible to the drugs used. However, 36.7% of the strains contained plasmids, with predominance of the 105 Kb plasmid. RAPD was capable of grouping the strains in 8 genotypic patterns using primer 784, in 6, using primer 787 and in 7, using primer 797. Conventional phenotypic typing methods, as well as the DNA plasmid analysis, presented nonsignificant discriminatory power; however, RAPD-PCR analysis showed discriminatory power, reproducibility, easy interpretation and performance, being considered as a promising alternative typing method for S. Typhi.

Phenotypic and molecular characterization of Salmonella Enteritidis strains isolated in São Paulo, Brazil

In São Paulo State, Brazil, the epidemic increase in isolation of Salmonella Enteritidis has been observed since 1994. A total of 105 S. Enteritidis strains (72 from human and 33 from non-human sources) isolated during the period 1975-1995, previously characterized by phage typing, was analyzed by antimicrobial susceptibility, plasmid profile, and ribotyping. Over 70% of the strains were susceptible to all antimicrobial agents tested, however, multiple resistance to antimicrobials was observed among the studied strains, mainly those from hospitalized patients. Phage type 8 (PT-8) was predominant among the strains isolated during the period of 1975-1992, but in the following years, PT-4 was the most frequent phage type identified. Seven different plasmid profiles were detected and 96% of the isolates harbored a plasmid of approximately 36 MDa. Ribotyping discriminated fourteen ribotypes (R1 to R14) among the strains examined. By analysis of dendrogram the strains were included in three groups with similarity level of 60%. The obtained results indicate that, a single ribotype (R11), determined for PT-4 strains isolated from 1993, characterizes the epidemic clone of S. Enteritidis in our region.

Serotyping and evaluation of the virulence in mice of Streptococcus suis strains isolated from diseased pigs

A total of 110 strains of Streptococcus suis, isolated from diseased pigs in Brazil were serotyped and analyzed for virulence. Serotyping of the strains resulted in the following classification: 42 strains of serotype 2 (38.2%), 10 strains of serotype 14 (9.1%), seven strains of serotype 9 (6.4%), three strains each of serotype 7 and 11 (2.7%), two strains each of serotype 1 and 8 (1.8%) and one strain each of serotypes ½, 3, 5, 6 and 10 (0.9%). Cross reactions among serotypes 1, 14 and 7 were observed in 21 strains (19.1%). Only 41.9% of the strains were lethal for mice using the pathogenicity test.

Distribution of virulence markers in clinical and environmental Vibrio cholerae non-O1/non-O139 strains isolated in Brazil from 1991 to 2000

One hundred seventy nine Vibrio cholerae non-O1/non-O139 strains from clinical and different environmental sources isolated in Brazil from 1991 to 2000 were serogrouped and screened for the presence of four different virulence factors. The Random Amplification of Polymorphic DNA (RAPD) technique was used to evaluate the genetic relatedness among strains. Fifty-four different serogroups were identified and V. cholerae O26 was the most common (7.8%). PCR analysis for three genes (ctxA, zot, ace) located of the CTX genetic element and one gene (tcpA) located on the VPI pathogenicity island showed that 27 strains harbored one or more of these genes. Eight (4.5%) strains possessed the complete set of CTX element genes and all but one of these belonged to the O26 serogroup suggesting that V. cholerae O26 has the potential to be an epidemic strain. The RAPD profiles revealed a wide variability among strains and no genetic correlation was observed.