Molecular detection of hepatitis E virus in feces and slurry from swine farms, Rio Grande do Sul, Southern Brazil / Detecção molecular do vírus da hepatite E em fezes e chorume de propriedades suinícolas, Rio Grande do Sul, Sul do Brasil

Hepatitis E virus (HEV) is highly disseminated among swine herds worldwide. HEV is also a threat to public health, since particularly genotypes 3 and 4 may cause acute hepatitis in human beings. No previous studies were done on the occurrence of HEV in environmental samples in Rio Grande do Sul, Brazil. In the present study, reverse transcriptase-polymerase chain reaction (RT-PCR) was employed to detect the presence of HEV in swine feces and in effluents from slurry lagoons in farms located in the municipality of Teutônia, inside the area of swine husbandry in the state. Pooled fecal samples from the floor of pig barns from 9 wean-to-finish farms and liquid manure samples were collected from the slurry lagoons from 8 of these farms. From the pooled fecal samples, 8/9 were positive for the HEV ORF1 gene by RT-PCR; all the slurry lagoon samples were positive for HEV RNA (100%). The identity of the HEV ORF1 amplicons was confirmed by sequencing belonging to HEV genotype 3, which was previously shown to be circulating in South America.

O vírus da hepatite E (HEV) é altamente disseminado entre rebanhos suínos no mundo todo. O HEV é também uma ameaça à saúde pública, já que os genótipos 3 e 4 podem causar hepatite aguda em seres humanos. Não há estudos anteriores sobre a ocorrência de HEV em amostras ambientais no Rio Grande do Sul. No presente estudo, empregou-se transcrição reversa e reação em cadeia da polimerase (RT-PCR) para detectar a presença de HEV em fezes de suínos e efluentes de lagoas de chorume em fazendas localizadas no município de Teutônia, representativo da região de maior produção de suínos no estado. Pools de amostras fecais foram coletadas a partir do chão de galpões de suínos provenientes de 9 propriedades de terminação; outra amostra de esterco líquido foi coletada das lagoas de chorume de 8 dessas fazendas. A partir das amostras fecais reunidas, 8/9 foram positivas para o gene ORF1 de HEV por PCR convencional; todas as amostras de lagoas de chorume foram positivas para RNA de HEV (100%). A identificação dos produtos de amplificação de HEV ORF1 foi confirmada por sequenciamento pertencente ao HEV genótipo 3, o qual foi previamente detectado na América do Sul.

In vitro effect of Acanthospermum australe (Asteraceae) extracts on Acanthamoeba polyphaga trophozoites / Efeito in vitro de extratos de Acanthospermum australe (Asteraceae) sobre trofozoitos de Acanthamoeba polyphaga

Acanthamoeba is a free-living protozoan widely distributed in the environment, occurring in vegetative trophozoite and resistance cyst stages during its life cycle. It constitutes an etiological agent of Acanthamoeba keratitis, a disease that may cause severe ocular inflammation and blindness. New drugs can be developed from molecules found in plants and thus help in its difficult treatment. Acanthospermum australe (Asteraceae), a plant used in folk medicine, had its effect tested on Acanthamoeba polyphaga. Aqueous and ethanolic extracts of A. austral were obtained from aerial parts for infusion and static maceration, respectively. Concentrations of 10, 5, 2.5, 1.25 and 0.625 mg/ml of the extract were tested against Acanthamoeba polyphaga trophozoites. The cytotoxic effect of the extracts was tested in mammalian cells using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. RESULTS: The 10 mg/ml concentration of ethanolic extract was lethal to 100% of the A. polyphaga trophozoites in 24 h and both extracts presented cytotoxic effect against mammalian cells. These findings suggest that the A. austral ethanolic extract may have compounds with relevance to the development of new amoebicidal drugs.

Acanthamoeba é um protozoário de vida livre amplamente distribuído no ambiente, ocorrendo sob a forma trofozoítica (metabolicamente ativa) e cística (de resistência), durante seu ciclo de vida. O protozoário constitui um agente etiológico da Ceratite Amebiana, uma doença que pode causar inflamação ocular severa e cegueira. Novos fármacos podem ser desenvolvidos a partir de moléculas encontradas em plantas e assim ajudar em seu difícil tratamento. Aqui, Acanthospermum australe (Asteraceae), uma planta utilizada na medicina popular, teve seu efeito sobre trofozoítos de Acanthamoeba polyphaga testado. O extrato aquoso e etanólico de A. australe foram obtidos das partes aéreas por infusão e maceração estática, respectivamente. As concentrações 10, 5, 2,5, 1,25 e 0,625 mg/ml dos extratos foram testadas contra trofozoítos do protozoário. O efeito citotóxico dos extratos foi testado em células de mamífero utilizando o ensaio de brometo de 3-[4,5-dimetiltiazol-2-il]-2,5-difeniltetrazólio (MTT). A concentração de 10 mg/ml do extrato etanólico foi letal a 100% dos trofozoítos de A. polyphaga em 24 h e ambos os extratos apresentaram efeito citotóxico contra as células de mamífero. Estes resultados sugerem que o extrato etanólico de A. australe pode ter componentes com relevância para o desenvolvimento de novos fármacos amebicidas.

Assessment of the antimicrobial effect of three plants used for therapy of community-acquired urinary tract infection in Rio Grande do Sul (Brazil).

ETHNOPHARMACOLOGICAL RELEVANCE: The urinary tract infections (UTI) are among the infectious diseases of bacterial origin most commonly diagnosed, and may result in a significant mortality. The microorganisms most frequently involved in community-acquired infections are Escherichia coli and Klebsiella pneumoniae, both Gram-negative bacillus of the Enterobacteriaceae family. In southern Brazil, it is common the treatment of UTI using herbs and teas, a tradition inherited from the ancient indigenous people. Nevertheless, there is still poor scientific information about the action of these plants. MATERIALS AND METHODS: Tests included the determination of minimum inhibitory concentration (MIC) by microdilution method at concentrations of 5000-156.25 µg/µL, and evaluation of antimicrobial activity by agar disc diffusion method at concentrations 500-12.5 µg/µL, against the microorganisms Escherichia coli and Klebsiella pneumoniae. RESULTS: In the tests for MIC and antimicrobial activity by disc diffusion method in agar, the growth of Escherichia coli and Klebsiella pneumoniae isolates were not inhibited by extracts from Piptochaetium montevidense, Bulbostylis capillaris and Juncus capillaceus employed in the study. CONCLUSIONS: Despite the popular use of the plants used in this study, there is no relation between the use and the expected antimicrobial activity. Prolonged use of these plants may worsen the disease and lead to kidney involvement, and in severe cases, lead to death.

Epidemiology of meningococcal disease in southern Brazil from 1995 to 2003, and molecular characterization of Neisseria meningitidis using multilocus sequence typing.

OBJECTIVE: To describe the epidemiology of meningococcal disease (MD) in southern Brazil. METHODS: Retrospective cohort study among 2215 MD cases reported from 1995 to 2003 in Rio Grande do Sul (RS) State. RESULTS: The overall incidence fell by 50%; the case-fatality rate during this period was 22%. Even so, the incidence of MD remained high after the epidemic period ended in 1999. Together, the age groups of 1-4 years and infants accounted for 54.1% of reported cases with incidences of 11.3/100 000 and 31.3/100 000, respectively; 69.8% of cases were caused by Neisseria meningitidis serogroup B, which increased significantly. There was a significant decrease in serogroup C cases in the whole period. The phenotypes B:4,7:P1.19,15, B:15:P1.7,16 and B:NT:P1.3 caused almost 50% of all serotyped cases. Fifty-six isolates obtained from RS patients during the first non-epidemic year 2000 plus 20 isolates from other southern Brazilian states (Santa Catarina and Paraná), Denmark and France were typed by multilocus sequence typing. Twenty sequence types (STs) were identified, eight of them found only in RS. ST-33 (27%) and ST-259 (18%) were the most frequent; both belong to the ST-32/ET-5 complex. ST-259 cases showed a trend towards higher risk of fatal outcome. ST-259 isolates were not detected among geographic controls or in other studies in Brazil. CONCLUSION: Our data suggest that ST-33 and ST-259 clones and the emergence of the ST-103 isolates contributed to the continued high incidence of MD in RS.

Direct-test PCR for detection of meningococcal DNA and its serogroup characterization: standardization and adaptation for use in a public health laboratory.

A direct PCR test (DT-PCR) was established to detect Neisseria meningitidis DNA in clinical samples from patients with suspected bacterial meningitis. Specific primers for the 16S rDNA of N. meningitidis were designed to amplify a 600 bp DNA fragment. One hundred and ninety-three clinical samples were analysed, corresponding to 114 samples from patients diagnosed as positive and 79 as negative for infection by N. meningitidis using conventional methods (culture, latex agglutination and counterimmunoelectrophoresis). These samples were submitted to PCR by two different clinical sample preparation approaches (with and without DNA extraction and purification) and submitted to different PCR protocols to improve the results. In agarose gel detection, the sensitivity value for DT-PCR was 88.5 % and, using dot-blot DNA detection, the sensitivity increased to 96.4 %. The detection limit for meningococcus in cerebrospinal fluid was 2x10(2) c.f.u. ml(-1). Serogroup prediction was done using a multiplex PCR protocol and the sensitivity was 83 % for agarose gel DNA detection and 96.4 % using dot-blot DNA detection.