Viral load of human bocavirus-1 in stools from children with viral diarrhoea in Paraguay.

Since their discovery, four species of human bocavirus (HBoV) have been described in patients with respiratory and gastrointestinal diseases. However, a clear causal association between HBoV-1 and gastroenteritis has not been demonstrated. In this study, we describe the detection and quantification of HBoV-1 in stools from children with acute non-bacterial gastroenteritis using quantitative polymerase chain reaction. HBoV-1 genome was detected in 10.6% of stools with frequent association with rotavirus and norovirus. The median of HBoV-1 viral load was 1.88 × 104 genome/ml, lower than previously shown in secretions of patients with respiratory infections, without any obvious association between high viral load and presence of HBoV as single agent. Thus, although HBoV-1 was frequently detected in these patients, there is no clear causal association of this agent with diarrhoea. Indeed, HBoV-1 DNA in stools of patients with gastroenteritis without respiratory symptoms may be a remnant of previous infections or associated with prolonged shedding of virus in the respiratory or digestive tracts.

Detección del virus influenza pandémica A (H1N1) en Paraguay 2009, y amplificación de genes hemaglutinina y neuraminidasa / Detection of pandemic influenza A (H1N1) virus in Paraguay in 2009, and amplification of the hemagglutinin and neuraminidase genes

Introducción: El virus de influenza pandémica A (H1N1), cuya circulación se inició en abril del año 2009 en México y Estados Unidos, se constituyó en el último virus pandémico desde los casos detectados en Hong Kong en 1968. El genoma del virus de influenza A está formado por 8 segmentos ARN de cadena simple (polaridad negativa), que codifican para 10 proteínas. Los genes hemaglutinina y neuraminidasa codifican para dos proteínas de superficie y son los utilizados en los análisis de variabilidad genética. Objetivos: a) Detectar la circulación del virus pandémico en pacientes con sospecha clínica de infección por influenza, y b) Diseñar una estrategia para amplificar de forma completa los genes hemaglutinina y neuraminidasa. Materiales y Métodos: Fueron analizados por Real-Time RT-PCR (transcripción reversa y reacción en cadena de la polimerasa en tiempo real) un total de 181 muestras de hisopado faríngeo, colectadas o remitidas al Hospital de Clínicas, del 6 de agosto al 11 de octubre de 2009. Para el diseño de amplificación de los genes hemaglutinina y neuraminidasa, se han utilizado herramientas bioinformáticas y reacción en cadena de la polimerasa. Resultados: Del total de muestras analizadas, 27 (14.9 %) dieron resultado positivo para el nuevo virus pandémico. Por otra parte, la amplificación completa de ambos genes proporcionó los resultados esperados: 1678-pares de bases (pb) para la hemaglutinina, y 1427-pb para la neuraminidasa. Conclusiones: La implementación de esta tecnología de amplificación permitirá posteriormente la secuenciación de estos genes a fin de determinar las variaciones genéticas del virus que podrían tener un impacto en la salud humana.

Introduction: The pandemic influenza A (H1N1) virus, whose circulation was detected in April 2009 in Mexico and the United States, is the latest pandemic virus since the cases reported in Hong Kong in 1968. The genome of the influenza A virus consists of 8 segments of single-stranded RNA of negative polarity, coding for 10 proteins. The hemagglutinin and neuraminidase genes encode for two surface proteins and are used in the analysis of genetic variability. Objectives: a) to detect circulation of the pandemic virus in patients with clinical suspicion of influenza infection and b) design a strategy to fully amplify the hemagglutinin and neuraminidase genes.Materials and Methods: A total of 181 pharyngeal swabs were collected and sent to the Hospital de Clínicas for analysis using Real-Time RT-PCR (reverse transcription and polymerase chain reaction in real time) between 6 August and 11 October 2009. To design the amplification of hemagglutinin and neuraminidase genes, we used bioinformatic tools and polimerase chain reaction. Results: Of the samples analyzed, 27 (14.9%) were positive for the new pandemic virus. Moreover, the complete amplification of both genes provided the expected results: 1678-base pairs (bp) for the hemagglutinin, and 1427-bp for neuraminidase. Conclusions: The use of this technology for amplification will eventually allow sequencing to identify genetic variations of the virus that could have an impact on human health.

Sensibilidad y especificidad del método inmunocromatográfico utilizado para el diagnóstico de rotavirus / Sensibility and specificity of the immunochromatographic method used for rotavirus diagnosis

El rotavirus (RV) es el principal agente viral causante de diarrea aguda en niños menores de 5 años y es responsable de aproximadamente el 6% de las muertes en este grupo etáreo, lo que conlleva la necesidad de utilizar métodos de diagnósticos rápidos y confiables. El objetivo del estudio es evaluar la sensibilidad y especificidad del método inmunocromatográfico (ICG), en el que se basan muchos kits comerciales utilizados para el diagnóstico de rotavirus grupo A, tomando como referencia el método de electroforesis en gel de poliacrilamida (PAGE). Se seleccionaron muestras de heces de 317 pacientes con diarrea aguda que concurrieron a un laboratorio privado de mayo a noviembre del 2006, con pedido de análisis de rotavirus y todos los datos de los pacientes fueron manejados de manera confidencial Se utilizaron kits de las marcas comerciales Operón simple (n=154) o SD Bioline rotavirus (n=163), siguiendo estrictamente las instrucciones del fabricante. Las muestras fueron conservadas en frío y remitidas al IICS para la realización de los estudios moleculares. La sensibilidad obtenida por el método ICG fue de 97,8% y la especificidad de 84%. La concordancia absoluta fue del 90%. Las muestras que dieron resultados discrepantes entre ICG y PAGE, fueron confirmadas por nRT-PCR, resultados que coincidieron con los obtenidos por PAGE. La sensibilidad del método ICG es muy buena, si bien la especificidad es moderada el método puede ser utilizado como screening para el diagnóstico rápido de rotavirus y sería aconsejable utilizar métodos más específicos como los moleculares para estudios epidemiológicos.

The rotavirus (RV) is the major causative agent of acute viral diarrhea in children under 5 years old and is responsible for approximately 6% of the deaths in this age group making necessary the use of quick and reliable diagnosis methods. The aim of this study is to evaluate the sensibility and specificity of the immunochromatographic method (ICG) on which many commercial kits used for the diagnosis of A rotavirus group are based taking as a reference the polyacrylamide gel electrophoresis (PAGE). Feces samples were collected from 317 patients with acute that attended a private laboratory from May to November, 2006 for rotavirus analysis. All the information of the patients was managed confidentially. Kits of the commercial brands simple Operón (n=154) or SD Bioline rotavirus (n=163) were used strictly following the instructions of the manufacturers. Samples were maintained in cold and sent to the IICS for the performance of the molecular studies. The sensibility obtained by the method ICG was 97.8%, the specificity was 84% and the absolute concordance was 90 %. The samples that gave discrepant results between ICG and PAGE were confirmed by nRT-PCR that provided results similar to those obtained by PAGE. The sensibility of the ICG method is very good and though the specificity is moderated it can be used as screening for the quick diagnosis of rotavirus and it would be advisable to use more specific methods, as the molecular ones, for epidemiological studies.

Sequence and phylogenetic analysis of the VP4 gene of human rotaviruses isolated in Paraguay.

Nucleotide and amino acid analyzes of the VP4 gene of human rotaviruses isolated both in Paraguay and worldwide were carried out in order to increase our knowledge about the complex pattern of evolution of this virus in nature. Paraguayan strains bearing the P[8] genotype were grouped in the lineages P[8]-1, P[8]-2, and P[8]-3. Regardless of the year of detection, all of the G4 and G9 strains were related to lineage P[8]-3, whereas the G1 strains were related to the three lineages detected in Paraguay; this fact reinforces the notion of the existence of constraints within specific populations of rotavirus strains except for the G1 strains. In addition, we propose a phylogenetic classification for the P[4] strains in five different lineages (i.e. P[4]-1 to P[4]-5). The findings presented in this paper reinforce the importance of a continuous surveillance of rotavirus strains in order to predict the possible variants that will circulate in a country, and ultimately improve current vaccination programs.

Differences in central and peripheral responses to oxotremorine in young and aged rats.

Few studies have investigated the pharmacological response of agents that act on the cholinergic system from the point of view of age. The present article investigated central responses (tremor) and peripheral responses (chromodacryorrhea) subsequent to the administration of oxotremorine to young (3-6 months of age) and aged rats (24-30 months of age). The aged rats presented greater duration and intensity of tremor in three doses utilized (0.25, 0.5, and 1.0 mg/kg) compared to young rats. These two groups of animals did differ in latency for the onset of the tremor. The aged rats presented more intense chromodacryorrhea than the young rats in all utilized doses. These data are indicative that both responses--central and peripheral--are affected by aging, possibly as a result of pharmacokinetic alterations and/or alterations in functionality of the cholinergic system in aged rats.

Guarana (Paullinia cupana): toxic behavioral effects in laboratory animals and antioxidants activity in vitro.

The effects on toxic and behavioral levels of guarana (Paullinia cupana) were assessed in rats and mice subsequent to acute and chronic administrations and were compared to those produced by Ginseng (Panax ginseng). Experimental parameters included tests for antioxidant capacity in vitro and measured in vivo, toxicological screening, progress in weight, motor activity, death rate, and histopathological examination of the viscera. Guarana showed an antioxidant effect because, even at low concentrations (1.2 microg/ml), it inhibited the process of lipid peroxidation. In high doses of 1000-2000 mg/kg (i.p. and p.o.) it did not induce significant alterations in parameters for toxicological screening. No effects on motor activity were observed, neither did guarana alter the hypnotic effect of pentobarbital. Ginseng (250-1000 mg/kg i.p.), however, elicited reductions in motor activity, eyelid ptosis and bristling fur. Consumption of liquids containing guarana or ginseng and progress in weight of the animals remained at levels similar to the controls, even after prolonged administration. The percentage mortality was equivalent in control and in treated groups. The absence of toxicity of guarana was also demonstrated by histopathological examination, with no alteration being detected in heart, lungs, stomach, small and large intestine, liver, pancreas, kidneys, bladder and spleen.

Pharmacological activity of Guarana (Paullinia cupana Mart.) in laboratory animals.

Mice that ingested a suspension of guarana (Paullinia cupana, Sapindaceae) in a dose of 0.3 mg/ml showed a significant increase in physical capacity when subjected to a stressful situation such as forced swimming after 100 and 200 days of treatment. Such an effect, however, was not obtained with a concentration of 3.0 mg/ml, nor with the ingestion of a suspension of ginseng 5.0 mg/ml, nor of a solution of caffeine 0.1 mg/ml. Guarana, both after a single (3.0 and 30 mg/kg) or chronic administrations (0.3 mg/ml), was able to partially reverse the amnesic effect of scopolamine as measured through a passive avoidance test in mice and rats, indicating a positive effect on memory acquisition. However, no effect was observed when an active avoidance task was used in rats, even after 20 days of guarana administration. There was also a tendency of rats treated with 0.3 mg/ml of guarana to better maintain the memory of a Lashley III maze path. The animals had the same average lifespan, indicating a low toxicity of guarana, even after 23 months of treatment.