RESUMO
Wastewater-based epidemiology has been described as a valuable tool for monitoring the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a community. However, there is no consensus on the best concentration method to allow reliable detection of SARS-CoV-2 in this matrix, considering different laboratory facilities. This study compares two viral concentration methods, ultracentrifugation (ULT) and skimmed-milk flocculation (SMF), for detecting SARS-CoV-2 in wastewater samples. The analytical sensitivity (limits of detection and quantification [LoD/LoQ]) of both methods was evaluated using a bovine respiratory syncytial virus (BRSV) as a surrogate. Three different approaches were conducted to establish LoD of each method based on the assays on the standard curve (ALoDsc), on the dilution of internal control (ALoDiC), and the processing steps (PLoD). For PLoD, ULT method had the lowest value (1.86 × 103 genome copy/microliter [GC/µL]) when compared to the SMF method (1.26 × 107 GC/µL). The LoQ determination showed a mean value of 1.55 × 105 GC/µL and 3.56 × 108 GC/µL to ULT and SMF, respectively. The detection of SARSCoV-2 in naturally contaminated wastewater revealed 100% (12/12) and 25% (3/12) of detection using ULT and SMF with quantification ranging from 5.2 to 7.2 log10 genome copy/liter (GC/L) and 5.06 to 5.46 log10 GC/L, respectively. The detection success rate of BRSV used as an internal control process was 100% (12/12) for ULT and 67% (8/12) for SMF, with an efficiency recovery rate ranging from 12 to 38% and 0.1 to 5%, respectively. Our data consolidates the importance of assessing the methods used; however, further analysis should be carried out to improve low-cost concentration methodologies, essential for use in low-income and developing countries.
Assuntos
COVID-19 , Vírus , Animais , Bovinos , SARS-CoV-2/genética , COVID-19/diagnóstico , Águas Residuárias , Limite de Detecção , RNA ViralRESUMO
We described the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in stool samples from patients presenting only acute gastroenteritis (AGE) symptoms. From January to July 2020, 121 AGE stool samples were screened by quantitative reverse-transcription polymerase chain reaction. We detected SARS-CoV-2 in 27.5% of samples received during the epidemic period. No infectious viruses were observed in Vero E6 cells.
Assuntos
COVID-19/diagnóstico , COVID-19/virologia , Gastroenterite/virologia , RNA Viral/isolamento & purificação , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Adulto , Brasil/epidemiologia , COVID-19/epidemiologia , Teste para COVID-19 , Fezes/virologia , Feminino , Humanos , Lactente , Masculino , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Adulto JovemRESUMO
BACKGROUND: Rotavirus A (RVA) is one of the leading causes of acute gastroenteritis worldwide; however, few studies assessed RVA genetics with community surveillance. OBJECTIVES: This study aimed to investigate clinical data, genetic diversity, and coinfection patterns of RVA infections in children from 2 to 36 months old with or without community childhood diarrhea in the Brazilian semiarid region during postvaccination era. METHODS: We enrolled and collected socioeconomic/clinical information using a standardized questionnaire and fecal samples from 291 children. Viral RNA samples were extracted and analyzed using quantitative reverse transcription polymerase chain reaction to establish the diagnosis of RVA. Sequencing of VP7 and VP4 (VP8*) regions and phylogenetic analysis were performed. RESULTS: RVA-negative diagnosis was associated with children 24 to 36 months old with complete vaccination schedule. Genotype G1P[8] was the most prevalent (57%), whereas unusual genotypes including G1P[4], G2P[8], and G3P[9] were also detected. G1- and P[8]-positive samples showed high degrees of similarity with the vaccine strain. RVA coinfections were frequently observed, and enteroaggregative Escherichia coli was the most prevalent copathogen. CONCLUSIONS: These results demonstrate that genotype G1P[8] is the most prevalent strain. VP7 and/or VP8* gene segments arising from RV1 vaccine strain were documented in these children, suggesting shedding or herd vaccination. Moreover, our study indicates full vaccination is important for protection against RVA infections.
Assuntos
Diarreia Infantil/complicações , Infecções por Rotavirus/epidemiologia , Rotavirus/imunologia , Brasil/epidemiologia , Pré-Escolar , Clima , Diarreia Infantil/epidemiologia , Diarreia Infantil/virologia , Fezes/virologia , Feminino , Humanos , Lactente , Masculino , Filogenia , RNA Viral/análise , Rotavirus/classificação , Rotavirus/genética , Infecções por Rotavirus/complicações , Infecções por Rotavirus/prevenção & controle , Vacinas contra Rotavirus , Fatores Socioeconômicos , Inquéritos e Questionários , Vacinação , Vacinas AtenuadasRESUMO
BACKGROUND: Brazil introduced the monovalent rotavirus vaccine (Rotarix®) in 2006. This study aimed to assess the epidemiology and genotype distribution of species-A rotavirus (RVA) in Brazil, comparing the pre- and post-vaccination periods. METHODS: Laboratory-based RVA surveillance included 866 municipalities in 22 Brazilian states, over a 21-year period. A total of 16,185 children with diarrheal diseases (DD) aged up to 12 years between 1996 and 2005 (pre-vaccination period, n = 7030) and from 2006 to 2017 (post-vaccination period, n = 9155) were enrolled. RVA was detected using ELISA immune assay and/or polyacrylamide gel electrophoresis and genotyped using nested PCR and/or nucleotide sequencing. RVA-positivity and genotypes detection rates were compared in distinct periods and age groups and Rotarix vaccination status. RESULTS: RVA-positivity in pre- and post-vaccination periods was, respectively: 4-11 months bracket, 33.3% (668/2006) and 16.3% (415/2547) (p < 0.001); 12-24 months, 28.2% (607/2154) and 22.2% (680/3068) (p < 0.001); 25-48 months, 17.4% (215/1235) and 29.4% (505/1720) (p < 0.001). Genotypes distribution in the pre- and post-vaccination periods was, respectively: G1P [8]/G1P[Not Typed], 417/855 (48.8%) and 118/1835 (6.4%) (p < 0.001); G2P [4]/G2P[NT], 47/855 (5.5%) and 838/1835 (45.7%) (p < 0.001); G3P [8]/G3P[NT], 55/855 (6.4%) and 253/1835 (13.8%) (p < 0.001); G9P [8]/G9P[NT], 238/855 (27.8%) and 152/1835 (8.3%) (p < 0.001); G12P [8]/G129P[NT], 0/871 (0%) and 249/1835(13.6%) (p < 0.001). Concerning infants aged 4-11 months, RVA frequency in fully vaccinated and non-vaccinated individuals was 11.9% (125/1052) and 24.5% (58/237) (p < 0.001), respectively. In children aged 12-24 months, RVA detection rate was 18.1% (253/1395) and 29.6% (77/260) (p < 0.001), for the vaccinated and non-vaccinated individuals, respectively (p < 0.001). CONCLUSIONS: RVA infection was significantly less frequent in children aged ≤2 years with DD after implementing vaccination, mainly among vaccinated children. It was also observed a decrease of P [8] circulation and emergence of G2P[4] in 2005, and afterwards in the post-vaccine era, with spreading of G12P[8] in 2014-2015 and of G3P[8] in 2017. Continuous RVA surveillance must be carried out in this scenario.
Assuntos
Infecções por Rotavirus/epidemiologia , Infecções por Rotavirus/prevenção & controle , Vacinas contra Rotavirus , Brasil/epidemiologia , Criança , Pré-Escolar , Genótipo , Humanos , Lactente , Estudos Retrospectivos , Rotavirus/classificação , Rotavirus/genética , Infecções por Rotavirus/virologia , Fatores de Tempo , Cobertura Vacinal , Vacinas AtenuadasRESUMO
Noroviruses are a leading cause of epidemic and pandemic acute gastroenteritis (AGE) worldwide, and contaminated food and water are important routes for its transmission. Raw sewage has been used for viral surveillance to monitor the emergence of new norovirus strains with the potential to cause epidemics. In this study, we investigated norovirus occurrence and norovirus RNA levels in 156 samples collected from May 2013 to May 2014, across three different stages (52 samples each) of a wastewater treatment plant (WWTP) in Rio de Janeiro, Brazil. We also explored norovirus GII diversity in raw sewage samples by next-sequencing generation (NGS). In addition, we examined norovirus prevalence and molecular epidemiology from acute gastroenteritis cases. Using RT-qPCR, norovirus GI and GII was detected in 38.5% and 96.1% of raw sewage samples, 40.4% and 96.1% of primary effluent samples and 1.9% and 5.8% of final effluent samples, respectively. Norovirus RNA levels varied from 4 to 6.2 log10 genome copies per litre (gc L-1) for GI and from 4.4 to 7.3â¯log10â¯gcâ¯L-1 for GII. Using MiSeq NGS, we identified 13 norovirus genotypes over the one-year period, with six dominant capsid genotypes, including GII.4, GII.17, GII.5, GII.2, GII.3 and GII.1. GII.4 noroviruses were the most prevalent in wastewater samples (68.5%), and a similar trend was observed in AGE cases (71%). The emergent GII.17 was the second most prevalent genotype (14.3%) identified in the raw sewage samples, however, it was not detected in clinical cases. Due to the high burden of norovirus outbreaks and the lack of vaccine and antiviral drugs, it is essential to understand the genotypic diversity of norovirus at the population level. Complementary data obtained from both clinical and environmental (sewage) samples proved to be an effective strategy to monitor the circulation and emergence of norovirus epidemic genotypes.
Assuntos
Norovirus/isolamento & purificação , Esgotos/virologia , Brasil , Infecções por Caliciviridae/epidemiologia , Infecções por Caliciviridae/virologia , Surtos de Doenças , Epidemias , Gastroenterite/epidemiologia , Gastroenterite/virologia , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Epidemiologia Molecular , Norovirus/genética , Filogenia , RNA Viral , Reação em Cadeia da Polimerase em Tempo Real , Águas Residuárias/virologiaRESUMO
BACKGROUND AND OBJECTIVE: Norovirus (NoV) infections are known to have high-morbidity and mortality rates and are a major health problem globally. The impact of NoV on child development is, however, poorly understood. We evaluated the distribution of NoV genotypes in children from a low-income Brazilian semiarid region, in relation with their clinical symptoms, nutritional status, and co-pathogens. METHODS: The test population included children aged 2 to 36 months from 6 cities of the Brazilian semiarid region. Fecal samples were collected from each child, along with the information regarding their socioeconomic/clinical conditions using a standardized questionnaire. Detection and quantification of NoV were performed by reverse-transcription quantitative polymerase chain reaction, followed by molecular and phylogenetic analyses. RESULTS: The NoV detection rate was 45.2%. Presence of NoV was associated with lower z scores for weight-for-age (Pâ=â0.03), weight-for-height (Pâ=â0.03), and body mass index-for-age (Pâ=â0.03). NoV infection was associated with more frequent respiratory illnesses (Pâ<â0.01). GII.P7 (polymerase) and GII.3 (capsid) were the most frequent NoV genotypes. Analysis of the open reading frame (ORF)1-2 junction identified recombinant NoV strains in 80% of the sequenced samples. Enteroaggregative Escherichia coli coinfection was the major predictor for diarrhea in NoV-positive samples (Pâ<â0.02). Moreover, Shigella spp was also associated with NoV-positive diagnosis (Pâ=â0.02). CONCLUSIONS: This study highlights the genetic variability of NoV and, associated co-infections and undernutrition in infants from low-income Brazilian semiarid region.
Assuntos
Infecções por Caliciviridae/virologia , Caliciviridae/genética , Transtornos da Nutrição Infantil/virologia , Coinfecção/microbiologia , Variação Genética , Estatura , Índice de Massa Corporal , Peso Corporal , Brasil/epidemiologia , Infecções por Caliciviridae/epidemiologia , Infecções por Caliciviridae/fisiopatologia , Proteínas do Capsídeo/análise , Transtornos da Nutrição Infantil/epidemiologia , Pré-Escolar , Coinfecção/epidemiologia , Diarreia/virologia , Escherichia coli , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/virologia , Fezes/virologia , Feminino , Genótipo , Humanos , Lactente , Masculino , Estado Nutricional , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Shigella , Fatores SocioeconômicosRESUMO
Investigation of major viruses responsible for acute viral gastroenteritis, such as norovirus (NoV), rotavirus species A (RVA) and human adenovirus (HAdV), was conducted in the mountainous region of the state of Rio de Janeiro in a lettuce-producing area. Irrigation water and lettuce samples were collected at different production stages. Viruses were concentrated using an adsorption-elution method and detected by quantitative polymerase chain reaction (qPCR). We detected HAdV in all collection points, although no virus infectivity was shown. The RVA was the most prevalent virus from both water (16.7% [10/60]) and lettuce samples (11.1% [4/36]), with loads ranging from 2.97 × 102 to 6.88 × 103 genomic copies per litre (gc L-1) and 6.24 × 102 to 1.30 × 104 gc per 25 g, respectively. NoV was detected in 8.33% [8/96] in water and lettuce samples, with concentrations ranging from 7.29 × 101 to 1.92 × 103 gc L-1 and from 4.29 × 101 to 2.98 × 103 gc 25 g-1, respectively. Escherichia coli values also demonstrated poor quality of the irrigation and washing water. The presence of at least two different virus strains in all sites reveals the need to improve basic sanitation measures in order to increase food safety.
Assuntos
Países em Desenvolvimento , Microbiologia de Alimentos , Gastroenterite/virologia , Lactuca/virologia , Irrigação Agrícola , Brasil , Infecções por Caliciviridae/transmissão , Infecções por Caliciviridae/virologia , Fezes/virologia , Gastroenterite/prevenção & controle , Humanos , Norovirus/genética , Norovirus/isolamento & purificação , Saúde Pública , RNA Viral/genética , Rotavirus/genética , Rotavirus/isolamento & purificação , Infecções por Rotavirus/transmissão , Infecções por Rotavirus/virologia , Saneamento , Microbiologia da ÁguaRESUMO
A newly GII.17 Kawazaki_2014 variant strain was detected recently in Brazil. Phylogenetic analysis reveals at least four independent introduction events of this lineage into this country that took place throughout 2014, coinciding with FIFA World Cup in Brazil, 2014, and Hong Kong has been identified as the most likely source of introduction. This variant emerged in Asia causing outbreaks and replacing prevalent GII.4. Emergence of GII.P17/GII.17 variant emphasizes the need for active laboratory surveillance for NoV including molecular epidemiology and studies on virus evolution.
Assuntos
Infecções por Caliciviridae/epidemiologia , Gastroenterite/epidemiologia , Norovirus/genética , Filogenia , RNA Viral/genética , Brasil/epidemiologia , Infecções por Caliciviridae/transmissão , Infecções por Caliciviridae/virologia , Monitoramento Epidemiológico , Fezes/virologia , Gastroenterite/virologia , Genótipo , Hong Kong/epidemiologia , Humanos , Epidemiologia Molecular , Norovirus/classificação , Norovirus/isolamento & purificação , PrevalênciaRESUMO
BACKGROUND: Gastroenteritis is one of the most important causes of morbidity and mortality in children and an important etiological agent is norovirus. OBJECTIVE: We describe the occurrence and characteristics of norovirus diarrhoea in children from Sergipe, Northeast-Brazil, over two consecutive periods of three years following rotavirus vaccine introduction. STUDY DESIGN: A cross sectional hospital-based survey conducted from October-2006 to September-2009 and from July-2011 to January-2013. Acute diarrhoea cases had a stool sample collected and tested for norovirus by RT-PCR and positive samples were sequenced. RESULTS: In total 280 (19.6%) of 1432 samples were norovirus positive, including 204 (18.3%) of 1113 samples collected during the first period and 76 (23.9%) of 318 collected during the second period. The proportion of children with norovirus infection increased significantly through the second study period (χ2 for trend=6.7; p=0.009), was more frequent in rotavirus vaccinated and in younger children (p<0.001). Of 280 norovirus-positive specimens, 188 (67.1%) were sequenced. Of these, 12 were genogroup I and 176 genogroup II. The main genotype was GII.4 (149/188, 79.3%), followed by GII.2 (6, 3.2%) and GII.6 (5, 2.6%). CONCLUSION: Norovirus annual detection rates increased over the study period. The detection of norovirus was higher among young children.
Assuntos
Infecções por Caliciviridae/epidemiologia , Diarreia/epidemiologia , Gastroenterite/epidemiologia , Norovirus/isolamento & purificação , Brasil , Pré-Escolar , Estudos Transversais , Diarreia/virologia , Fezes/virologia , Feminino , Gastroenterite/virologia , Genótipo , Humanos , Lactente , Masculino , Norovirus/classificação , Norovirus/genética , Prevalência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
Feline caliciviruses (FCVs) have occasionally been described in cats in association with enteric disease, but an etiological role for these viruses in acute gastroenteritis is still unclear. In this study, molecular characterization of FCV and feline norovirus (FNoV) was undertaken using real-time PCR (RT-PCR) and sequence analysis of the ORF1 region in fecal specimens from 29 diarrheic cats. The specimens were also screened for parvovirus, coronavirus, astrovirus and group A rotavirus. A quantitative one step RT-PCR was also performed to detect and quantitate NoV genogroup IV and the role of these animal caliciviruses in feline gastroenteritis was investigated. This is the first description of enteric FCV and FNoV in South America.