ABSTRACT Hepatitis A virus (HAV), a member of Picornaviridae family, is the main causative agent of acute viral hepatitis in the world, mainly in developing countries. HAV may be present in contaminated water and food and its presence is often associated to a lesser extent with socioeconomic factors and environmental quality. The main goals in the present study were to standardize a cell culture combined to a polymerase chain reaction protocol for the detection and quantification of viral viability and analyze whether the virus could be found in water samples collected in four urban streams of Sinos River watershed. Virus recovery was assayed from known virus concentrations measured in experimentally contaminated raw and ultrapure water (MilliQ®). Recovery rates ranged from 270% in raw water to 15,000% in ultrapure water. In a second step, a qPCR coupled to a previous passage in cells, demonstrated more analytical sensitivity when compared to samples assayed without a previous passage in cell cultures. HAV genome was detected in only 1 of 84 samples analyzed, pointing to a very low occurrence of HAV in water samples in the studied region. These findings are remarkable, since no more than 5% of the domestic sewage in this area is treated pointing to a low occurrence of HAV in the population living nearby during the study period.

Resumo A destilação natural da água pode destruir e/ou inativar microorganismos que são sensíveis ao calor e a radiação ultravioleta (UV). Este método é utilizado, atualmente, para fornecer água potável em navios e para dessalinização de água salobra. Para o desenvolvimento desta pesquisa, um destilador solar em escala piloto foi construído e instalado na região sul do Brasil de forma a se verificar a eficiência do equipamento na desinfecção de água. A verificação de eficiência do equipamento foi baseada na determinação do número mais provável (NMP) de coliformes totais e Escherichia coli bem como no número de cópias de DNA do adenovírus humano tipo 5 (HAdV-5) em amostras de água antes e após a destilação. Os resultados demonstraram que houve 100% de remoção de coliformes totais e E. coli e 4,5 log (99,997%) de remoção de HAdV-5 após a destilação, o que está de acordo com o padrão microbiológico de potabilidade determinado pela legislação brasileira, pela USEPA e pela HEALTH CANADA.

Abstract Natural water distillation can destroy and/or inactivate microorganisms that are sensitive to heat and ultraviolet radiation (UV). This method is currently used to provide fresh water in ships and in the desalination of brackish water. For the development of this research, a pilot-scale solar still was built and installed in the southern region of Brazil, in order to assess its efficiency in water disinfection, which was based on the most probable number (MPN) of total coliforms and Escherichia coli, in addition to the DNA copy number of human adenovirus type 5 (HAdV-5) in raw, undistilled samples and in treated distilled water. Results showed that the distillation process removed 100% of total coliform and Escherichia coli and 4.5 log (99.997%) of HAdV-5, which meets the microbiological standards for drinking water according to national Brazilian regulations, as well as USEPA and HEALTH CANADA requirements.

Resumo O abastecimento de água do município de Ivoti provém unicamente de origem subterrânea. A potabilidade da água subterrânea do município foi avaliada através da análise de marcadores bacterianos (Escherichia coli e coliformes totais) e virais (adenovírus, enterovírus e rotavírus) de contaminação. Vinte e sete amostras de água provenientes de poços artesianos, poços cavados e fontes foram coletadas entre fevereiro e abril de 2011. A determinação do número mais provável (NMP) de E. coli e coliformes totais em 100 mL foi realizada pelo método dos tubos múltiplos. Os marcadores virais foram analisados em alíquotas de 500 mL por reação em cadeia da polimerase (PCR). Coliformes totais foram encontrados em 66,6% das amostras. A contaminação encontrada para coliformes termotolerantes foi de 25,9%, 40,7% para adenovírus e 22,2% para rotavírus. Nenhuma amostra apresentou enterovírus. A presença de coliformes e de vírus nas amostras indica contaminação fecal da água subterrânea no local de estudo, situado na porção aflorante do Aquífero Guarani e alerta para a necessidade de precaução quanto ao uso destes mananciais para abastecimento público, captação individual e cultivo de hortaliças.

Abstract The water supply of the municipality of Ivoti comes solely from an underground source. The potability of groundwater in the municipality was evaluated by analyzing bacterial (Escherichia coli and total coliforms) and viral (adenovirus, enterovirus and rotavirus) markers for contamination. Twenty-seven water samples from artesian wells, dug wells and springs were collected between February and April 2011. The determination of the most probable number (MPN) of E. coli and total coliforms in 100 mL was performed by the multiple-tube method. Viral markers were analyzed in 500 mL aliquots by polymerase chain reaction (PCR). Total coliforms were found in 66.6% of samples. Contamination found for E. coli was 25.9%, 40.7% for adenovirus and 22.2% for rotavirus. Enterovirus was not found. The presence of coliform bacteria and viruses in the samples indicates fecal contamination of groundwater at the study site, located in the recharge portion of the Guarani Aquifer and suggests the need of caution regarding the use of these water sources for public water supply, individual water catchment and farming.

RESUMO Acanthamoeba é a mais comum ameba de vida livre no ambiente, pode atuar como um importante veículo para vários micro-organismos que vivem no mesmo ambiente, tais como vírus, e pode ser patogênica para humanos. Este estudo objetivou detectar e quantificar adenovírus humano (HAdV) em Acanthamoebas isoladas de amostras de águas coletadas de piscinas na cidade de Porto Alegre, no sul do Brasil. Amebas de vida livre do gênero Acanthamoeba foram isolados de amostras de água, e os isolados (n = 16) foram usados para investigar a ocorrência de HAdVs. Detecção de HAdV foi realizada usando reação em cadeia de polimerase quantitativa (qPCR) em tempo real. HAdVs foram detectados em 62.5% (10/16) dos isolados de Acanthamoeba, variando de 3.24 x 103 a 5.14 x 105 cópias de DNA por mililitro de isolado. As cargas virais de HAdV encontradas nesse estudo não são negligenciáveis, especialmente porque infecções por HAdV são associadas com diferentes doenças humanas, incluindo gastrenterites, doenças respiratórias e oculares. Este estudo reforça o conceito que Acanthamoeba poderia atuar com um reservatório e promover a transmissão de HAdV através da água.

ABSTRACT Acanthamoeba is the most common free-living environmental amoeba, it may serve as an important vehicle for various microorganisms living in the same environment, such as viruses, being pathogenic to humans. This study aimed to detect and quantify human adenoviruses (HAdV) in Acanthamoebas isolated from water samples collected from swimming pools in the city of Porto Alegre, Southern Brazil. Free-living amoebae of the genus Acanthamoeba were isolated from water samples, and isolates (n=16) were used to investigate the occurrence of HAdVs. HAdV detection was performed by quantitative real-time polymerase chain reaction (qPCR). HAdVs were detected in 62.5% (10/16) of Acanthamoeba isolates, ranging from 3.24x103 to 5.14x105 DNA copies per milliliter of isolate. HAdV viral loads found in this study are not negligible, especially because HAdV infections are associated with several human diseases, including gastroenteritis, respiratory distress, and ocular diseases. These findings reinforce the concept that Acanthamoeba may act as a reservoir and promote HAdV transmission through water.

Human adenoviruses (HAdV), members of the Adenoviridae family, are excreted through the fecal route and may be present in the feces of humans consuming contaminated food or water. The presence of HAdV from different serotypes in the feces of healthy individuals was already reported using conventional polymerase chain reaction; however, real-time PCR (qPCR) may reveal not only the rates of detection as well as demonstrate the viral loads excreted by healthy persons. Aiming to identify and characterize the presence of adenoviruses in stool samples, 147 fecal samples from patients with no records of diarrhea were analyzed (74 from winter season and 73 from summer) by Real-Time PCR (qPCR) assay and conventional PCR. HAdV genome was present in 43.8% (32/73) of stools samples collected during summer season and 21.6% (16/74) during winter. The rate of detection of genomic copies (gc) ranged from 4.04×102 to 6.72×105gc/g of feces among the 147 samples analyzed, of which the ranged of genomic copies of DNA HAdV was major in summer. All samples were negative when tested for rotaviruses (RV) and noroviruses (NoV) by PCR conventional and qPCR respectively. HAdV is excreted constantly by infected individuals in the absence of clinical signs and the occurrence may vary seasonally.

Adenoviruses are among the most promising viral markers of fecal contamination. They are frequently found in the water, sediment and soil of regions impacted by human activity. Studies of the bioaccumulation of enteric viruses in shrimp are scarce. The cities located in the northern coast of the lake systems in Southern Brazil have high urbanization and intensive farming rates, and poor sewage collection and treatment. One hundred (n = 100) Farfantepenaeus paulensis pink-shrimp specimens and 48 water samples were collected from coastal lagoons between June 2012 and May 2013. Water samples were concentrated and the shrimp, mashed. After DNA extraction, samples were analyzed by real time polymerase chain reaction (qPCR) in order to detect and quantify viral genomes. Thirty-five percent of shrimp samples were positive for contamination, predominantly by avian adenoviruses. A total of 91.7% of water samples contained adenoviruses DNA, with the human form being the most frequent. Our results provided evidence of significant bioaccumulation of adenoviruses in shrimp, showing the extent of the impact of fecal pollution on aquatic ecosystems.

RESUMO Os adenovírus humanos (HAdV) são notavelmente resistentes ao ambiente. Estes agentes podem servir como indicadores efetivos de contaminação fecal, tanto quanto podem atuar como agentes causadores de diferentes doenças em seres humanos. A reação em cadeia da polimerase (PCR) e mais recentemente a PCR quantitativa (qPCR) são amplamente usadas para detecção de agentes virais em matrizes ambientais. No presente estudo, PCR e SYBR(r)Green qPCR foram comparadas para a detecção de HAdV em amostras de água (55) e sedimento (20) provenientes de nascentes, poços, açudes e arroios coletadas em propriedades leiteiras. A metodologia quantitativa detectou HAdV em 87,3% das amostras de água e 80% dos sedimentos, enquanto por PCR convencional a detecção foi de 47,3% e 35%, respectivamente.

SUMMARY Human Adenoviruses (HAdV) are notably resistant in the environment. These agents may serve as effective indicators of fecal contamination, and may act as causative agents of a number of different diseases in human beings. Conventional polymerase chain reaction (PCR) and, more recently, quantitative PCR (qPCR) are widely used for detection of viral agents in environmental matrices. In the present study PCR and SYBR(r)Green qPCR assays were compared for detection of HAdV in water (55) and sediments (20) samples of spring and artesian wells, ponds and streams, collected from dairy farms. By the quantitative methodology HAdV were detected in 87.3% of the water samples and 80% of the sediments, while by the conventional PCR 47.3% and 35% were detected in water samples and sediments, respectively.

Um novo teste baseado na reação em cadeia da polimerase em tempo real (qPCR) com SYBR ® Green foi desenvolvido para detectar duas espécies de Bartonella, B. henselae e B. clarridgeiae, diretamente em amostras de sangue. Este teste foi utilizado em amostras de sangue obtidas de gatos que vivem em abrigos de animais do sul do Brasil. Os resultados foram comparados aos obtidos pelo PCR convencional utilizado para a detecção de Bartonella spp. Das 47 amostras analisadas, oito foram positivas no PCR convencional e 12 foram positivas para qPCR. A reação de qPCR, permitiu a detecção da presença simultânea de B. henselae e B. clarridgeiae em duas destas amostras. Os resultados mostram que a qPCR aqui descrita pode ser uma ferramenta confiável para a detecção e diferenciação de duas espécies importantes de Bartonella spp.

A novel SYBR® green-real time polymerase chain reaction (qPCR) was developed to detect two Bartonellaspecies, B. henselae and B. clarridgeiae, directly from blood samples. The test was used in blood samples obtained from cats living in animal shelters in Southern Brazil. Results were compared with those obtained by conventional PCR targeting Bartonella spp. Among the 47 samples analyzed, eight were positive using the conventional PCR and 12 were positive using qPCR. Importantly, the new qPCR detected the presence of both B. henselae and B. clarridgeiae in two samples. The results show that the qPCR described here may be a reliable tool for the screening and differentiation of two important Bartonella species.

Bartonella spp are the causative agent of cat scratch disease in humans. Cats are the natural reservoir of these bacteria and may infect humans through scratches, bites or fleas. Blood samples from 47 cats aged up to 12 months were collected for this study. All animals were lodged in municipal animal shelters in the Vale do Sinos region, Rio Grande do Sul, Brazil. Bartonella spp were detected by genus-specific polymerase chain reaction (PCR) and when the PCR was positive, the species were determined by DNA sequencing. A Giemsa-stained blood smear was also examined for the presence of intraerythrocytic elements suggestive of Bartonella spp infection. Phylogenetic analysis was also performed for all positive samples. Using molecular detection methods, Bartonella spp were detected in 17.02% (8/47) of the samples. In seven out of eight samples confirmed to be positive for Bartonella spp, blood smear examination revealed the presence of intraerythrocytic elements suggestive of Bartonella spp. Phylogenetic analysis characterized positive samples as Bartonella henselae (5) or Bartonella clarridgeiae (3). To the best of our knowledge, this is the first molecular study demonstrating the presence of Bartonella spp in cats from the Southern Region of Brazil.