Resumo Giardia spp. e Cryptosporidium spp. são agentes etiológicos responsáveis por uma série de epidemias de gastroenterites ocorridas, principalmente, após o consumo de água contaminada. Os cistos e oocistos destes protozoários são resistentes às variações ambientais, bem como a maioria dos processos físicos, químicos e microbiológicos utilizados nas estações de tratamento de água e esgoto. Deste modo, este estudo teve como objetivo detectar e avaliar a remoção de cistos de Giardia spp. e oocistos de Cryptosporidium spp. no sistema de tratamento combinado anaeróbio + aeróbio da ETE Garcia em Blumenau, SC. Para efetuar a detecção de cistos e oocistos, as amostras de efluentes e lodos provenientes da ETE foram concentradas através de filtração em membranas de ésteres de celulose e centrifugação, seguida por reação de imunofluorescência direta (RID) utilizando o Kit diagnóstico - Merifluor(. Para a obtenção dos parâmetros físicos, químicos e microbiológicos, as análises seguiram os padrões preconizados em APHA (2012). Concentrações elevadas de cistos de Giardia spp. (máx. 900.000 cistos L-1) foram detectadas em 83,3% dos pontos analisados. Oocistos de Cryptosporidium spp. também foram detectados em elevadas concentrações (máx. 300.000 oocistos L-1) em 66,6% dos pontos analisados. Não foram detectados cistos e oocistos nas amostras do efluente tratado, deste modo o sistema combinado da ETE Garcia apresentou uma eficiência >99,9% para a remoção das formas resistentes destes patógenos, contribuindo para a redução da contaminação ambiental por protozoários patogênicos presentes no esgoto doméstico do município de Blumenau, SC, Brasil.

Abstract Giardia spp. and Cryptosporidium spp. are responsible for a number of outbreaks of gastroenteritis, particularly associated with the consumption of contaminated water. The cysts and oocysts of these protozoa are resistant to environmental variations, as well as to most of the physical, chemical and microbiological processes used in water treatment and sewage plants. This study therefore aimed to detect and evaluate the removal of Giardia spp. and Cryptosporidium spp. in a combined system anaerobic / aerobic Waste Water Treatment Plant (WWTP) located in the city of Blumenau, SC, Brazil. For the study of cysts and oocysts, samples of effluents and sludge from the WWTP were concentrated by filtration using a membrane of cellulose esters and by centrifugation, followed by direct immuno-fluorescence assay (RID) using the Merifluor( diagnostic kit. The analysis followed the standards published in APHA (2012) to obtain the physical, chemical and microbiological parameters. High concentrations of Giardia spp. (Max. 900,000 L-1 cysts) were detected in 83.3% of the analyzed points. Oocysts Cryptosporium spp. were detected at high concentrations (max. 300,000 oocysts L-1) in 66.6% of the analyzed points. Neither cysts nor oocysts were detected in treated effluent samples. Thus, the combined system of the Garcia WWTP showed a 99.9% efficiency rate for the removal of resistant forms of pathogens, contributing to the reduction of environmental contamination by these pathogens present in the domestic sewage of Blumenau, SC.

BACKGROUND Enterocytozoon bieneusi are the most common microsporidia associated with different clinical manifestations such as diarrhoea, respiratory tract inflammation and acalculous cholecystitis, especially in immunocompromised patients. Infection usually occurs by ingestion of food and water contaminated with spores, but can also result from direct contact with spores through broken skin, eye lesions, and sexual transmission, depending on the microsporidian species. Although there are reports of E. bieneusi found in humans and animals in Brazil, there are no published studies of environmental samples examined by molecular methods. OBJECTIVES The purpose of this study was to verify the presence of E. bieneusi in raw sewage and treated effluent from a combined system by molecular methods. METHODS Raw sewage and treated effluent samples collected from a combined system were analysed for the presence of E. bieneusi using the internal transcriber spacer (ITS) region of E. bieneusi by nested polymerase chain reaction. FINDINGS The analysis revealed E. bieneusi presence and a novel genotype (EbRB) in one raw sewage sample and one treated effluent. MAIN CONCLUSIONS The presence of E. bieneusi in final effluent indicates that the combined system may not remove microsporidian spores. This study is the first report of E. bieneusi in environmental samples in Brazil.

Resumo Giardia spp. e Cryptosporidium spp. são protozoários patogênicos de transmissão fecal-oral de veiculação hídrica, que causam vários problemas de saúde, como doenças gastrointestinais associados com consumo de água contaminada. Devido à escassez de dados sobre a ocorrência destes protozoários em águas superficiais no Sul do Brasil, este estudo teve como objetivo analisar a presença de cistos e oocistos na água bruta das estações de tratamento de água no município de Blumenau, SC, Brasil. Para a pesquisa dos protozoários foi utilizada a metodologia de filtração em membranas de ésteres mistos de celulose, seguida por reação de imunofluorescência utilizando o kit Merifluor(r). Para as análises microbiológicas, empregou-se a metodologia do "Standard Methods for the Examination of Water and Wastewater"; para as análises físico-químicas foi utilizada sonda multiparâmetros. Cistos de Giardia spp. foram encontrados em 23,19% das amostras e oocistos de Cryptosporidium spp. em 7,24% do total de amostras analisadas (n=67). Todas as amostras analisadas apresentaram contaminação por Escherichia coli e 11,76% apresentaram valores de turbidez da água bruta acima do recomendado. Nas análises da turbidez da água tratada 23,52% apresentaram valores acima do preconizado pela Portaria 2914/2011. A detecção de protozoários patogênicos na água bruta das estações de tratamento de água aponta para a importância de adoção de medidas preventivas, como a proteção de áreas de mananciais e tratamento adequado do esgoto doméstico objetivando reduzir os riscos de transmissão de protozoários por meio da água de consumo humano na região de Blumenau, SC, Brasil.

Abstract Giardia spp. and Cryptosporidium spp. are water-borne pathogenic protozoans spread through fecal-oral transmission which cause several health problems, to include gastrointestinal diseases associated with the consumption of contaminated water. There are limited data regarding the occurrence of these protozoans in surface waters in southern Brazil. The aim of this study was therefore to investigate the presence of cysts and oocysts in untreated water collected from water treatment plants in Blumenau, Santa Catarina State, Brazil. The methodology employed to study the protozoans was filtration through mixed cellulose ester membranes followed by the immunofluorescence reaction using the Merifluor(r) kit. Microbiological analysis was conducted using the Standard Methods for the Examination of Water and Wastewater and multi-parameter probes were used for the physico-chemical analysis. Cysts of Giardia spp. were found in 23.19% of the samples and oocysts of Cryptosporidium spp. in 7.24% of the samples (n=67). All of the samples analyzed were contaminated with Escherichia coli and 11.76% of the untreated water samples had turbidity values higher than the recommended limit. In the case of the treated water samples, 23.52% had turbidity values above the limit established by legislation (Portaria MS 2914/2011). The detection of pathogenic protozoans in the untreated water at the treatment plants highlights the importance of adopting preventative measures, such as the protection of areas where water is present and adequate treatment of domestic sewage, in order to reducing the risk of protozoan transmission via potable water.

After a gastroenteritis outbreak of unknown etiology in the municipality of Sebastião da Grama, SãoPaulo, Brazil, we conducted a parasitological survey to establish the epidemiological profile of enteroparasitosis in children and staff members attending the public urban schools in operation in town. The cross-sectional study evaluated 172 children aged 11 months to 6 years old and 33 staff members aged 19 to 58 years old. Overall, 96 (55.81%) children and 20 (60.61%) staff members were mono-parasitized, while 58 (33.72%) children and 4 (12.12%) workers were poly-parasitized. Protozoa (88.37%; 72.73%) was more prevalent than helminthes (3.48%; 0%) in children and staff members respectively. Blastocystis spp. was the most prevalent parasite in children (86.63%) and staff members (66.67%). The age of 1 year old or less was found to be associated with increased prevalence of giardiasis [OR = 13.04; 95%CI 2.89-58.91; p = 0.00] and public garbage collection was identified as a protective factor against intestinal helminth infections [OR = 0.06; 95%CI 0.00-0.79; p = 0.03]. Although most of the children tested positive for Blastocystis spp. and also presented clinical signs/symptoms (62.2%), this association was not statistically significant [OR = 1.35; 95%CI 0.53-3.44; p = 0.51]. Intestinal parasites still represent a public health concern and this study underscores the importance of further investigations to better understand the pathogenic role of Blastocystis spp.

No presente trabalho foi avaliado um processo oxidativo avançado (POA), peróxido de hidrogênio assistido por radiação ultravioleta (H2O2/UV), e um processo oxidativo, peroxidação (H2O2), na inativação de cistos de Giardia duodenalis. Foram utilizadas concentrações de peróxido de hidrogênio de 15 mg.L-1 e 6x103 mg.L-1 e dose de UV de 44, 2.736 e 5.472 mW s.cm-2. A eficiência dos processos foi aferida com base na observação de danos à parede dos cistos por meio da reação de imunofluorescência direta (RID) e da microscopia eletrônica de varredura (MEV). Quando utilizada a menor dose de radiação e concentração de oxidante, o POA se mostrou mais eficiente que a peroxidação tanto em causar dano na parede de G. duodenalis quanto em reduzir o número de cistos. Com a aplicação do POA e da peroxidação houve uma redução média de 45,2 e 22,6% do número de cistos de G. duodenalis, sendo que, em média, 42 e 29,4% dos cistos restantes apresentaram danos, respectivamente.

Performance of an advanced oxidative process (AOP) with the application of hydrogen peroxide and UV radiation (H2O2/UV) has proven effective to inactivate Giardia duodenalis cysts. Hydrogen peroxide concentrations of 15 mg.L-1 and 6x103 mg.L-1 and 44 UV doses of 2,736 and 5,472 mW s.cm-2 were used in this study. The process effectiveness was assessed observing the occurrence of damage to the cyst's wall using direct immunofluorescence reaction (IFA) and scanning electron microscopy (SEM). When using the lower UV radiation dose and the lower oxidant concentration the AOP has shown higher effectiveness both in causing damage to the Giardia duodenalis wall and in reducing the number of cysts inoculated at the beginning. The average reduction AOP and peroxidation processes reduced 45.2 and 22.6% in the number of G. duodenalis cysts and 42 and 29.4% of the remaining cysts presented damage, respectively.

Neste trabalho, avaliou-se a eficiência dos métodos centrífugo-concentração e filtração em membrana, na detecção de oocistos de Cryptosporidium spp. e cistos de Giardia spp. em amostras de esgoto bruto e tratado, provenientes de um sistema de lodos ativados (estação de tratamento de esgoto, Samambaia, Campinas, em São Paulo). As amostras foram coletadas quinzenalmente por dois anos: 53 amostras de esgoto bruto (AFL), 53 de efluente tratado sem desinfecção por luz ultravioleta (EFL) e 38 de efluente tratado e desinfetado por luz ultravioleta (EFL+UV). Cistos de Giardia spp. foram encontrados em 90,5% das amostras AFL; em 96,2%, de EFL; e em 94,7%, de EFL+UV. Oocistos de Cryptosporidium spp. foram detectados em 6,4% das amostras AFL e em 2,6 % de EFL+UV. Ambos os métodos mostraram-se eficientes na detecção destes protozoários em todos os tipos de amostras, além de apresentarem baixo custo por análise.

In this study, the efficiency of centrifuge-concentration and membrane filtrated methods was evaluated in the detection of Cryptosporidium spp. oocysts and Giardia spp. cysts in raw or treated wastewater samples, from activated sludge systems (ETE - Samambaia, Campinas, in São Paulo). The samples were collected once a fortnight for two years: 53 samples of influent (AFL), 53 samples of treated effluent without ultraviolet disinfection (EFL), and 38 samples of treated effluent with ultraviolet disinfection (EFL+UV). Giardia spp. cysts were found in 90.5% of the AFL samples; in 96.2% of the samples, EFL; and in 94.7%, EFL+UV. Cryptosporidium spp. oocysts were detected in 6.4% of AFL samples and 2.6% of EFL+UV. Both methods showed efficiency when detecting protozoa in all types of samples, besides having low costs by analysis.

Giardia e Cryptosporidium causaram vários surtos epidêmicos de gastroenterite, associados à água potável. Efluentes de esgoto contaminados foram incriminados como uma fonte potencial de cistos e oocistos. Uma investigação foi conduzida para verificar a presença de cistos de Giardia e oocistos de Cryptosporidium em amostras de lodo ativado de uma Estação de Tratamento de Esgoto. Para isto as amostras foram submetidas: i) a concentração pelo processo de clarificação com éter (ECP) e ii) método de purificação por flutuação em sacarose (SFM) e, as alíquotas dos sedimentos foram examinadas por imunofluorescência. Cistos de Giardia estiveram presentes em todas as amostras avaliadas (100,0%; n = 8) quando utilizado ECP e kit 1, enquanto o kit 2 resultou em 6 amostras positivas (85,7%; n = 7). Para SFM, cistos de Giardia foram detectados em 75,0% e 100,0% destas amostras (para kit 1 e 2 respectivamente). Considerando os oocistos de Cryptosporidium, duas amostras (25,0%; kit 1 e 28,5% kit 2) foram positivas usando-se ECP enquanto para SFM, apenas uma amostra (examinada pelo kit 1) foi positiva (12,5%). Os resultados do experimento-controle revelaram que as taxas de recuperação para Giardia e Cryptosporidium, quando utilizado ECP foi de 54,5% e 9,6% e para SFM, foi de 10,5% e 3,2%, respectivamente. Considerando a detecção de alta concentração desses protozoários, é recomendada a avaliação prévia do lodo ativado antes de sua aplicação na agricultura e, com alguma melhora, ECP pode ser uma técnica apropriada e simples para a detecção de protozoários em amostras de esgoto.

Giardia and Cryptosporidium have caused several outbreaks of gastroenteritis in humans associated with drinking water. Contaminated sewage effluents are recognized as a potential source of waterborne protozoa. Due to the lack of studies about the occurrence of these parasites in sewage samples in Brazil, we compared the efficiency of two procedures for concentrating cysts and oocysts in activated sludge samples of one sewage treatment plant. For this, the samples were submitted to i) concentration by the ether clarification procedure (ECP) and to ii) purification by sucrose flotation method (SFM) and aliquots of the pellets were examined by immunofluorescence. Giardia cysts were present in all samples (100.0%; n = 8) when using ECP and kit 1 reagents, while kit 2 resulted in six positive samples (85.7%; n = 7). As for SFM, cysts were detected in 75.0% and 100.0% of these samples (for kit 1 and 2, respectively). Regarding Cryptosporidium, two samples (25.0%; kit 1 and 28.5% for kit 2) were detected positive by using ECP, while for SFM, only one sample (examined by kit 1) was positive (12.5%). The results of the control trial revealed Giardia and Cryptosporidium recovery efficiency rates for ECP of 54.5% and 9.6%, while SFM was 10.5% and 3.2%, respectively. Considering the high concentration detected, a previous evaluation of the activated sludge before its application in agriculture is recommended and with some improvement, ECP would be an appropriate simple technique for protozoa detection in sewage samples.

Cryptosporidium parvum e Giardia duodenalis são parasitos transmitidos pela água e têm causado muitos surtos de doenças gastrointestinais associados à ingestão de água. Devido à falta de estudo sobre a ocorrência destes protozoários na água, no Sudeste do Brasil, uma investigação foi conduzida para verificar a presença de cistos e oocistos na água superficial do rio Atibaia. As amostras de água foram submetidas à filtração em membrana e a eluição foi processada por: (1) lavagem e raspagem da membrana (RM) e (2) dissolução em acetona (ADM). Parâmetros microbiológicos e químicos foram analisados. Alíquotas do sedimento foram examinadas por imunofluorescência. Todas as amostras de água foram positivas, a despeito da alta turbidez. Maior taxa de recuperação ocorreu em amostras tratadas pelo RM que por ADM. A meta em futuros trabalhos é determinar a viabilidade de cistos e oocistos para se obter o significado deste achado em Saúde Pública.

Cryptosporidium parvum and Giardia duodenalis are waterborne parasites that have caused several outbreaks of gastrointestinal disease associated with drinking water. Due to the lack of studies about the occurrence of these protozoa in water in the Southeast of Brazil, an investigation was conducted to verify the presence of cysts and oocysts in superficial raw water of the Atibaia River. The water samples were submitted to membrane filtration (3.0 mum) and elution was processed by (1) scraping and rinsing of membrane (RM method) and (2) acetone-dissolution (ADM method). Microbiologic and chemical parameters were analyzed. Aliquots of the pellets were examined by immunofluorescence (Merifluor, Meridian Diagnostics, Cincinnati, Ohio). All water samples were positive for Cryptosporidium and Giardia, in spite of the high turbidity. Higher recovery rates occurred in samples treated by the RM method than by the ADM technique. The goal for future work is the assessment of viability of cysts and oocysts to determine the public health significance of this finding.