Characterisation of human astrovirus in a diarrhoea outbreak using nanopore and Sanger sequencing protocols.

Human astroviruses (HAstV) are etiologic agents of acute gastroenteritis that most often afflict young children and elderly adults. Most studies of HAstV have focused on epidemiology. In this study, we collected 10 stool samples from a diarrhea outbreak from a diarrhea sentinel surveillance hospital in Beijing. Samples were evaluated immediately using parallel multiplex RT-qPCR and nanopore sequencing, and were then amplified by designed primers and Sanger sequencing to obtain whole genome sequences. Six isolates were categorized as HAstV-5 and subjected to whole genome analysis to characterize their genetic variation and evolution. Full genome analysis revealed low genetic variation (99.38-100% identity) among isolates. Phylogenetic analysis showed that all isolates were closely related to domestic strains Yu/1-CHN and 2013/Fuzhou/85. The recombination breakpoint of the six isolates was located at 2741 bp in the overlap region of ORF1a and ORF1b, similar to those of Yu/1-CHN and 2013/Fuzhou/85. Overall, our study highlights the combined use of RT-qPCR and sequencing as an important tool in rapid diagnosis and acquisition of whole genome sequences of HAstV.

Escherichia coli O8:H8 Carrying a Novel Variant of the Heat-Labile Enterotoxin LT2 Gene Caused Outbreaks of Diarrhea.

No outbreaks caused by Escherichia coli-producing heat-labile enterotoxin LT2 have been reported to date. Here, we revealed that the E. coli O8:H8 strains isolated from patients in 2 independent diarrhea outbreaks were negative for any known virulence determinants in routine microbiological tests, were very closely related, and carried a prophage-encoded gene for a novel LT2 variant (LT2d) and the genes for colonization factor antigen III. We also showed that LT2d has a cytotonic activity similar to LT1. These data indicate the importance of E. coli strains producing LT2d as a human pathogen.

Molecular Characterization of a New G (VP7) Genotype in Group B Porcine Rotavirus.

Rotaviruses (RVs), a common cause of viral gastroenteritis in humans and animals, are classified into 9 established groups/species (RVA-RVI). Although RVB has been found in several countries, genetic variation among RVB field strains remains poorly characterized. RVB strains can be classified into G genotypes based on a nucleotide (nt) homology that exceeds a cutoff value of 80% for the gene that encodes the structural protein VP7. In this study, we determined the VP7 nt and deduced amino acid sequences of one RVB strain (RB62) identified in a diarrheic fecal sample obtained from a piglet in Brazil in 2012. Comparative analysis of this strain and the strains of the other 21 previously identified VP7 ge-notypes showed that the highest nt identity (71.2%) was found with the porcine PB-70-H5 strain within the G4 genotype. However, when compared with the nonclassified Vietnamese RVB G genotype 14177_18 strain, the nt sequence identity was of 82.9%. These results led us to conclude that the Brazilian strain BR62 and the Vietnamese strain 14177_18 belong to a novel G genotype (G22).

Diarrhea outbreak in Pernambuco, Brazil, associated with a heat-stable cytotoxic enterotoxin produced by Aeromonas caviae / O surto de diarreia em Pernambuco, Brasil é associado com uma enterotoxina citotóxica termo-estável produzida por Aeromonas caviae

SUMMARYIn the present study enterotoxic and cytotoxic activities of twenty Aeromonas caviaestrains were examined. They originated from fecal specimens of patients with acute diarrhea during an outbreak in Brazil in 2004. Culture supernatants of fourteen strains (70%) caused fluid accumulation in rabbit ileal intestinal loops and in suckling mice assays, and also showed a cytotoxic activity in Vero and Caco-2 cells. The enterotoxic and cytotoxic factors were heat-stable after culture supernatants treatment at 100 ºC. The results revealed that A. caviaestrains produce a putative diarrheagenic virulence factor, a heat-stable cytotoxic enterotoxin that could be linked to the diarrhea outbreak that took place in Brazil.

RESUMOEm 2004 ocorreu um surto de diarreia aguda no Estado de Pernambuco, Brasil. Setenta por cento (14 dos 20) dos sobrenadantes de cultura de Aeromonas caviae,isoladas neste episódio induziram acúmulo de líquido em testes de alça ligada de intestino de coelhos, assim como em teste em camundongos recém-nascidos. Os mesmos sobrenadantes mostraram também atividade citotóxica em células de Vero e Caco-2, mas não em células HeLa e HEp2. As atividades enterotóxicas e citotóxicas mantiveram-se mesmo após o aquecimento a 100 ºC dos sobrenadantes de cultura. Este trabalho revela a expressão de um provável fator diarreiogênico: uma enterotoxina-citotóxica termo-estável, produzida por A. caviaeque pode ser associada ao surto de diarreia ocorrido no Brasil. Atualmente estamos purificando esta enterotoxina termo-estável, com o objetivo de elucidar seu papel como fator de virulência na diarreia causada por A. caviae.

Brote de diarrea debida a intoxicación por alimentos en una empresa X: San José, Costa Rica, del 8 al 9 de julio 2003 / Outbreak of diarrhea due to food poisoning at a company for San José, Costa Rica, from 8 to 9 July 2003

El 9 de julio del 2003, se informó al Ministerio de Salud, la existencia de un brote de diarrea en una empresa X, en San José. Se investigó el brote, con el objetivo de verificar su mangnitud, determinar factores de riesgo y mecanismo de transmisión, también recomendar medidas de prevención y control, a las autoridades de salud. Se revisaron 40 expedientes clínicos de pacientes atendidos por diarrea, en el consultorio de la empresa. Se definió como caso a todo funcionario, que presentó más de 2 deposiciones semilíquidas, y que consumió alimentos del comedor de la empresa, el 8 julio del 2003. Se tomaron corpocultivos, muestras de alimentos consumidos, y se enviaron a un laboratorio privado para el análisis. Se realizó un estudio de cohorte retrospectivo, y se aplicó un cuestionario. Se calcularon los riesgos relativos, RR, con intervalos de confianza del 95 por ciento, las tasas de ataque y porcentajes para los alimentos servidos. El 100 por ciento de los casos presentaron diarrea, sin fiebre. A los coprocultivos y a las muestras de alimentos, no se les buscó Clostridium perfringens. Las muestras de agua no reportaron contaminación fecal. El alimento sospechoso: cerdo con piña, presentó un RR igual 1,94; IC: 95 por ciento, 1, 26-2,96. El período de incubación y la descripción de la enfermedad coincidieron con una intoxicación por Clostridium perfringens, por lo que, se asoció el brote al alimento sospechoso. Como en la empresa X se desconocían los procedimientos a seguir ante un brote de diarrea, causado por alimentos, se recomendó a los encargados de epidemiología del Área Rectora de Salud, capacitar en el abordaje de las intoxicaciones causadas por alimentos; a las empresas bajo su responsabilidad, que cuentan con establecimientos de alimentación.

On July the 9th., 2003, an outbreak in company X was notified to the Ministry of Public Health in San José. This outbreak was investigated with thepurposes of verifying its magnitude, determining risk factors, and mechanisms of transmission as well asrecommending prevention and control measures to the Local Health authorities. Forty clinical chartsbelonging to diarrhea patients seen at the company´s infirmary, were reviewed. The case definition was anyworker with more than 2 semi-liquid bowel movements and who ate in the worker´s dining room on the 8th.of July, 2003. Stool and food cultures were taken and sent for analysis to a private lab. A retrospective cohort study was carried out and a questionnaire wasadministered to the employees. Relative Risks (with 95% confidence intervals) as well as attack rates and percentages were calculated for the foodstuffs served. All cases showed diarrhea but without fever. Clostridium perfringens was not searched in either thestool or the food cultures. Water samples didn´t report fecal contamination. The likely source of contamination, namely pineapple pork, showed a relative risk of 1.94 with a confidence interval between 1.26 and 2.96. The incubation period as well as the clinical characteristics of the illness were consistent with an intoxication dueto Clostridium perfringens. Since the employees were unaware of the procedures to be followed duringa foodborne outbreak, training was offered to the Local Health Area which in turn, educated the companies under its supervision regarding protocols to be followed during any future similar problems.