Toxoplasma gondii em amostras de água de reuso: proposta de método de detecção e quantificação por PCR quantitativo em tempo real (qPCR) / Toxoplasma gondii in water samples for reuse: proposal for a method of detection and quantification by quantitative real-time PCR (qPCR)

Resumo - O reuso de água contribui nos âmbitos ambientais, sociais e econômicos. Entretanto a presença de parasitos em amostras hídricas tem sido evidenciada por diferentes estudos, entre eles Toxoplasma gondii. A ausência de uma metodologia padronizada para detecção de oocistos em amostras hídricas, dificulta a caracterização das mesmas, comprometendo a elaboração de uma legislação. Tendo em vista a importância do reuso da água na mitigação da escassez, do stress hídrico e diante dos desafios expostos é que o presente trabalho se insere. Objetivos - Propor técnica de concentração e recuperação de oocistos em água de reuso; propor método de extração de DNA; detectar e quantificar oocistos por qPCR. Método - Utilizando o método 1693/2014 USEPA, foram concentrados 30 L de água de reuso de 20 amostras provenientes de duas ETE's da cidade de São Paulo, Brasil. Os 15 mL resultantes desse processo foram concentrados em membrana 0,45µm. O material filtrado na membrana foi recuperado por raspagem com alça bacteriológica de 10 µL. Para extração de DNA foram empregadas duas técnicas: o kit comercial DNeasy PowerSoil Kit® otimizado com a enzima Zymolyase® e choque térmico. A quantificação de DNA foi realizada com a sequência alvo B1. Resultados - Na extração por choque-térmico não foi detectado DNA nas vintes amostras realizadas. Das 16 amostras submetidas à extração enzimática, quatro foram consideradas positivas. Discussão - A extração de DNA consistiu na etapa essencial para detecção e quantificação de oocisto, dado a natureza resistente das paredes. Conclusão - O reuso de água pode ser comprometido na ausência de um protocolo padronizado de detecção e quantificação de oocisto de T. gondii, que possibilite a determinação de parâmetros parasitológicos para elaboração de uma legislação.

Abstract - The wastewater reuse contributes in the environmental, social and economic spheres. However, the presence of parasites in water samples has been evidenced by different studies, including Toxoplasma gondii. The absence of a standardized methodology for detecting oocysts in water samples makes it difficult to characterize them, compromising the drafting of legislation. In view of the importance of wastewater reuse in mitigating water scarcity and stress and in view of the challenges presented, this work is inserted. Objectives - To propose oocyst concentration and recovery techniques in wastewater reuse; propose DNA extraction method; detect and quantify oocysts by qPCR. Method - Using method 1693/2014 USEPA, 30 L of wastewater reuse from 20 samples from two WWTPs in the city of São Paulo, Brazil, were concentrated. The 15 mL resulting from this process were concentrated in a 0.45 µm membrane. The material filtered on the membrane was recovered by scraping with a 10 µL bacteriological loop. For DNA extraction, two techniques were used: the commercial kit DNeasy PowerSoil Kit® optimized with the enzyme Zymolyase® and freeze - thaw. DNA quantification was performed with the target sequence B1. Results - In freeze - thaw extraction, no DNA was detected in the 20 samples performed. Of the 16 samples submitted to enzymatic extraction, four were considered positive. Discussion - DNA extraction was the essential step for oocyst detection and quantification, given the resistant nature of the walls. Conclusion - Wastewater reuse can be compromised in the absence of a standardized protocol for the detection and quantification of T. gondii oocysts, that allows the determination of parasitological parameters for the elaboration of legislation.

Detection and quantification of viable Ascaris sp. and other helminth eggs in sewage sludge.

Some epidemiologic studies conducted in developing countries demonstrated a high incidence of enteroparasites, Ascaris sp. being the most prevalent. Therefore, the use of sewage sludge in agriculture may pose risks to human health. In order to protect public health, a Brazilian regulation has established standards regarding its use in rural areas. The objective of this study was to quantify Ascaris sp. and other helminth eggs in sewage sludge from five wastewater treatment plants from a dense metropolitan region, and also to check compliance with the law. The analysis was carried according to USEPA 2003 . A rich parasitological fauna was found, with a prevalence of the eggs of Ascaris sp. (9.55%). The samples analyzed presented a large variety of helminth eggs, and Ascaris sp. proved to be the most prevalent which put in evidence that its application poses public health concerns.

Detecção e quantificação de ovos viáveis de Ascaris sp e outros ovos de helmintos em lodo de esgoto / Detection and quantification of Ascaris sp and other helminth eggs in sewage sludge

Introdução O lodo gerado em estações de tratamento de esgotos é um resíduo de composição variável e com potencial poluidor dependendo de sua origem, tipo de tratamento e sazonalidade; pode conter patógenos dentre os quais os parasitas. Porém, quando tratado adequadamente, o lodo de esgoto pode ser aplicado na agricultura como fertilizante ou condicionador de solo. Em países em desenvolvimento, estudos têm demonstrado que a incidência de enteroparasitoses é elevada, sendo Ascaris sp um dos mais prevalentes. Portanto, o uso agrícola do lodo de esgoto pode oferecer riscos à saúde humana. Com intuito de salvaguardar a saúde da população entrou em vigor a Resolução CONAMA nº375/06, que estabelece critérios e procedimentos para o uso do lodo em áreas agrícolas. Objetivo - Determinar e quantificar ovos de helmintos e de Ascaris sp em lodos provenientes de três ETEs de uma região metropolitana, verificando o atendimento ao padrão parasitológico estabelecido pela CONAMA nº375/06. Método Utilizou-se o método descrito no apêndice I da norma CFR 503 USEPA de 2003. Resultados Os lodos analisados no período do estudo apresentaram rica fauna parasitária onde foram observados ovos de Ancylostoma sp, Ascaris sp, Capillaria sp, Enterobius vermicularis, Fasciola hepatica, Hymenolepis sp, Taenia sp, Toxocara sp e Trichuris sp. Observou-se que ovos de Ascaris sp foram os mais prevalentes com 67,71 por cento , seguido por Toxocara sp (13,62 por cento ). Ovos viáveis de Ascaris sp estavam presentes em 10,16 por cento das amostras. Conclusão Pode-se concluir que o lodo gerado nas ETEs estudadas apresentaram amplo espectro de ovos de helmintos, sendo Ascaris sp o mais prevalente. Das três ETEs analisadas, nenhuma atendia os parâmetros parasitológicos para lodo classe A, segundo a Resolução CONAMA nº375/06.

Quality of water sources used as drinking water in a Brazilian peri-urban area

The objective of this paper was to assess bacteriological quality of drinking water in a peri-urban area located in the Metropolitan Region of São Paulo, Brazil. A total of 89 water samples were collected from community plastic tanks and 177 water samples from wells were collected bimonthly, from September 2007 to November 2008, for evaluating bacteriological parameters including: Escherichia coli, Enterococcus and heterotrophic plate count (HPC). Clostridium perfringens was investigated in a subsample (40 samples from community plastic tank and 40 from wells). E. coli was present in 5 (5.6 percent) samples from community plastic tanks (2.0 - 5.1x10(4) MPN/100mL) and in 70 (39.5 percent) well samples (2.0 - 8.6x10(4) MPN/100mL). Thus, these samples were not in accordance with the Brazilian Regulation. Enterococcus was detected in 20 (22.5 percent) samples of the community plastic tanks (1 to 79 NC/100mL) and in 142 (80.2 percent) well samples (1 to >200 NC/100mL). C. perfringens was detected in 5 (12.5 percent) community plastic tanks samples and in 35 (87.5 percent) wells samples (2.2 to >16 MPN/100mL). HPC were above 500 CFU/mL in 5 (5.6 percent) waters from community plastic tanks. In wells samples, the HPC ranged from <1 to 1.6x10(4) CFU/mL. The residual chlorine did not attend the standard established in the drinking water legislation (0.2 mg/L), except in 20 (22.5 percent) samples. These results confirm the vulnerability of the water supply systems in this peri-urban area what is clearly a public health concern.

Quality of water sources used as drinking water in a Brazilian peri-urban area.

The objective of this paper was to assess bacteriological quality of drinking water in a peri-urban area located in the Metropolitan Region of São Paulo, Brazil. A total of 89 water samples were collected from community plastic tanks and 177 water samples from wells were collected bimonthly, from September 2007 to November 2008, for evaluating bacteriological parameters including: Escherichia coli, Enterococcus and heterotrophic plate count (HPC). Clostridium perfringens was investigated in a subsample (40 samples from community plastic tank and 40 from wells). E. coli was present in 5 (5.6%) samples from community plastic tanks (2.0 - 5.1x10(4) MPN/100mL) and in 70 (39.5%) well samples (2.0 - 8.6x10(4) MPN/100mL). Thus, these samples were not in accordance with the Brazilian Regulation. Enterococcus was detected in 20 (22.5%) samples of the community plastic tanks (1 to 79 NC/100mL) and in 142 (80.2%) well samples (1 to >200 NC/100mL). C. perfringens was detected in 5 (12.5%) community plastic tanks samples and in 35 (87.5%) wells samples (2.2 to >16 MPN/100mL). HPC were above 500 CFU/mL in 5 (5.6%) waters from community plastic tanks. In wells samples, the HPC ranged from <1 to 1.6x10(4) CFU/mL. The residual chlorine did not attend the standard established in the drinking water legislation (0.2 mg/L), except in 20 (22.5%) samples. These results confirm the vulnerability of the water supply systems in this peri-urban area what is clearly a public health concern.

Detection of Giardia and Cryptosporidium cysts/oocysts in watersheds and drinking water sources in Brazil urban areas.

The protozoan parasites Giardia and Cryptosporidium have been described as important waterborne disease pathogens, and are associated with severe gastrointestinal illnesses. The objective of this paper was to investigate the presence of Giardia cysts and Cryptosporidium oocysts in samples from watershed catchments and treated water sources. A total of 25 water samples were collected and examined according to the US EPA--Method 1623, 2005, consisting of 12 from drinking water and 13 from raw water. Positive samples from raw water for Giardia cysts and Cryptosporidium oocysts were 46.1 and 7.6%, respectively. In finished water, positive samples were 41.7% for Giardia cysts and 25.0% for Cryptosporidium oocysts. Concentrations of Giardia cysts found in raw water samples ranged from "not detected" to 3.4 cysts/L, whereas concentrations of Cryptoporidium oocysts ranged from "not detected" to 0.1 oocysts/L. In finished water, Giardia concentrations ranged from "not detected" to 0.06 cysts/L, and Cryptosporidium, from "not detected" to 0.01 oocysts/L. Concentrations of Giardia cysts and Cryptosporidium oocysts were not high in the samples analyzed. Nevertheless, the results of this study highlight the need to monitor these organisms in both raw and drinking water.