Desarrollo de un método de transcripción inversa seguida de reacción en cadena de la polimerasa para la detección del virus de la fiebre amarilla / Development of a reverse transcription polymerase chain reaction method for yellow fever virus detection

Introduction. Yellow fever is considered a re-emerging disease and is endemic in tropical regions of Africa and South America. At present, there are no standardized or commercialized kits available for yellow fever virus detection. Therefore, diagnosis must be made by time-consuming routine techniques, and sometimes, the virus or its proteins are not detected. Furthermore, co-circulation with other flaviviruses, including dengue virus, increases the difficulty of diagnosis. Objective. To develop a specific reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR-based assay to improve the detection and diagnosis of yellow fever virus using both serum and fresh tissue samples. Materials and methods. RT-PCR primers were designed to amplify a short fragment of all yellow fever virus genotypes reported. A second set of primers was used in a nested PCR to increase sensitivity. Thirty-three clinical samples were tested with the standardized reaction. Results. The expected amplicon was obtained in 25 out of 33 samples analyzed using this approach, and 2 more samples tested positive after a subsequent nested PCR approach. Conclusion. This improved technique not only ensures the specific detection of a wide range of yellow fever virus genotypes but also may increase the sensitivity of detection by introducing a second round of amplification, allowing a rapid differential diagnosis between dengue and yellow fever infection, which is required for effective surveillance and opportune epidemiologic measures.

Introducción. La fiebre amarilla se considera una enfermedad reemergente y endémica en regiones tropicales de África y Suramérica. Actualmente, no existen estuches estandarizados o comerciales disponibles para la detección del virus de la fiebre amarilla y, por lo tanto, el diagnóstico debe hacerse mediante técnicas de rutina que consumen mucho tiempo y algunas veces no garantizan la detección del virus o de sus proteínas. Además, la cocirculación con otros flavivirus, incluyendo el del dengue, hacen el diagnóstico más complicado. Objetivo. Desarrollar un ensayo específico de amplificación basado en transcripción inversa seguida de reacción en cadena de la polimerasa, con el fin de mejorar la detección y el diagnóstico de la fiebre amarilla, tanto a partir de suero como de tejido fresco. Materiales y métodos. Se diseñaron iniciadores específicos para amplificar un fragmento conservado del virus de la fiebre amarilla. Un segundo par de iniciadores se usó en una reacción de amplificación anidada para incrementar la sensibilidad. Se probaron 33 muestras clínicas con la técnica estandarizada. Resultados. El amplímero esperado se obtuvo en 25 de las 33 muestras analizadas usando este método y 2 más resultaron positivas después de la reacción anidada. Conclusión. Esta técnica mejorada garantiza la detección de todos los genotipos virales de fiebre amarilla y puede incrementar la sensibilidad del ensayo introduciendo una segunda etapa de amplificación, lo cual permite el diagnóstico diferencial con infección por dengue y otros flavivirus, lo cual es de gran importancia para la vigilancia y la toma de medidas epidemiológicas oportunas.

Development of a reverse transcription polymerase chain reaction method for yellow fever virus detection.

INTRODUCTION: Yellow fever is considered a re-emerging disease and is endemic in tropical regions of Africa and South America. At present, there are no standardized or commercialized kits available for yellow fever virus detection. Therefore, diagnosis must be made by time-consuming routine techniques, and sometimes, the virus or its proteins are not detected. Furthermore, co-circulation with other flaviviruses, including dengue virus, increases the difficulty of diagnosis. OBJECTIVE: To develop a specific reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR-based assay to improve the detection and diagnosis of yellow fever virus using both serum and fresh tissue samples. MATERIALS AND METHODS: RT-PCR primers were designed to amplify a short fragment of all yellow fever virus genotypes reported. A second set of primers was used in a nested PCR to increase sensitivity. Thirty-three clinical samples were tested with the standardized reaction. RESULTS: The expected amplicon was obtained in 25 out of 33 samples analyzed using this approach, and 2 more samples tested positive after a subsequent nested PCR approach. CONCLUSION: This improved technique not only ensures the specific detection of a wide range of yellow fever virus genotypes but also may increase the sensitivity of detection by introducing a second round of amplification, allowing a rapid differential diagnosis between dengue and yellow fever infection, which is required for effective surveillance and opportune epidemiologic measures.

Childbearing during adolescence and offspring mortality: findings from three population-based cohorts in southern Brazil.

BACKGROUND: The role of young maternal age as a determinant of adverse child health outcomes is controversial, with existing studies providing conflicting results. This work assessed the association between adolescent childbearing and early offspring mortality in three birth cohort studies from the city of Pelotas in Southern Brazil. METHODS: All hospital births from 1982 (6,011), 1993 (5,304), and 2004 (4,287) were identified and these infants were followed up. Deaths were monitored through vital registration, visits to hospitals and cemeteries. The analyses were restricted to women younger than 30 years who delivered singletons (72%, 70% and 67% of the original cohorts, respectively). Maternal age was categorized into three groups (< 16, 16-19, and 20-29 years). Further analyses compared mothers aged 12-19 and 20-29 years. The outcome variables included fetal, perinatal, neonatal, postneonatal and infant mortality. Crude and adjusted odds ratios (ORs) were estimated with logistic regression models. RESULTS: There were no interactions between maternal age and cohort year. After adjustment for confounding, pooled ORs for mothers aged 12-19 years were 0.6 (95% CI = 0.4; 1.0) for fetal death, 0.9 (0.6; 1.3) for perinatal death, 1.0 (0.7; 1.6) for early neonatal death, 1.6 (0.7; 3.4) for late neonatal death, 1.8 (1.1; 2.9) for postneonatal death, and 1.6 (1.2; 2.1) for infant death, when compared to mothers aged 20-29 years. Further adjustment for mediating variables led to the disappearance of the excess of postneonatal mortality. The number of mothers younger than 16 years was not sufficient for most analyses. CONCLUSION: The slightly increased odds of postneonatal mortality among children of adolescent mothers suggest that social and environmental factors may be more important than maternal biologic immaturity.

Axonal degeneration in sheep caused by the ingestion of Halimium brasiliense.

Nervous system disease is reported in sheep from 2 farms in southern Brazil and in 33 farms in Uruguay. The illness was seasonal, occurring from May to November, during the growing season of Halimium brasiliense, and primarily affected sheep older than 3 years of age. Clinical signs included transient seizures that occurred mainly when sheep were disturbed or frightened. Most affected sheep recovered when removed to other pastures. Feeding trials produced clinical signs in 1 sheep after the ingestion of 2,117 g/kg of body weight of H. brasiliense over 142 days. Two sheep that had previously recovered from spontaneous toxicosis developed clinical signs after the ingestion of 263 g and 565 g of H. brasiliense per kg body weight given over 36 and 31 days, respectively. The main histologic lesion was vacuolation of the brain and spinal cord, with rare axonal spheroid formation. Transmission electron microscopy revealed segmental axonal swelling with degeneration and disappearance of the axonal organelles and vacuolation of the axoplasm. A pigment identified as ceroid was also present in neurons, astrocytes, and macrophages. These lesions suggested a novel morphologic manifestation of a toxic axonopathy.

Cetose em vacas de corte em gestaçäo / Ketosis in pregnant beef cows

Descreveram-se 6 surtos de cetose em bovinos de corte que ocorreram durante o inverno, em vacas gordas ou em bom estado nos últimos 3 meses de gestaçäo, submetidas a períodos variáveis de carência alimentar. Os animais apresentaram constipaçäo, incoordenaçäo, tremores musculares e hiperexcitabilidade, com posterior decúbito e morte após uma evoluçäo de 3 a 7 dias. Nas necrópsias a única alteraçäo significativa foi a degeneraçäo graxa do fígado. Os surtos foram controlados mediante uma alimentaçäo adequada

Intoxicaçäo crônica por cobre em ovinos no Rio Grande do Sul / Chronic copper poisoning in sheep in Rio Grande do Sul, southern Brazil

Descreve-se intoxicaçäo crônica por cobre (Cu) em ovinos estabulados em 4 estabelecimentos no Rio Grande do Sul. Os sinais clínicos e as lesöes macroscópicas e histológicas foram características da intoxicaçäo. Os níveis de Cu em fígado e rins de 5 ovinos afetados variaram de 489 a 1760 ppm e 60 a 470 ppm na matéria seca, respectivamente. Os níveis de Cu em 28 raçöes, provenientes de 8 diferentes fabricantes, foram de 23,2 ñ 6,73 (X ñ S). Conclui-se que a intoxicaçäo ocorre devido aos altos níveis de Cu das raçöes para ovinos utilizadas no Rio Grande do Sul. Recomenda-se näo adicionar cobre na formulaçäo de raçöes para essa espécie