Polymerase chain reaction and nested-PCR approaches for detecting Cryptosporidium in water catchments of water treatment plants in Curitiba, State of Paraná, Brazil

Introduction Cryptosporidium is an important protozoan cause of waterborne disease worldwide of concern to public health authorities. To prevent outbreaks of cryptosporidiosis, the monitoring of this parasite in drinking water is necessary. In the present work, the polymerase chain reaction (PCR) and nested-PCR techniques were used to detect Cryptosporidium in raw water from catchment points of four water treatment plants (WTP) in Curitiba, Paraná, Brazil. Methods First, DNA extraction techniques were tested in samples containing decreasing amount of oocysts in reagent water, and PCR and nested-PCR with specific primers for 18SSU rDNA of Cryptosporidium were conducted to determine their sensitivity. In reagent water, a commercial extraction kit provided the best analytical sensitivity, and PCR and nested-PCR allowed the detection of five and two oocysts, respectively, with the primers XIAOR/XIAOF and XIAO1F/XIAO2R. Results In the spiking experiments, only the PCR with the primers AWA995F/AWA1206R was successful at detecting concentrations of 0.1 oocysts/mL. Two catchments samples of raw water and/or water sludge from four WTPs were contaminated with Cryptosporidium. Conclusions The application of the techniques to monitor Cryptosporidium in water and detect contamination in water catchments of WTPs in Curitiba are discussed in the present work

Ocorrência de oocistos de Cryptosporidium spp. em praias artificiais do Lago Itaipu, Paraná, Brasil

Cryptosporidiosis is an emerging zoonosis that causes many outbreaks worldwide. Its main route of transmission is the consumption of contaminated water or food. In Brazil, data on to the occurrence of cryptosporidiosis in animals and in humans are scarce; thus, it is important to determine the occurrence of Cryptosporidium sp. and to identify possible sources of water and food contamination in order to prevent the disease and, consequently, preserve of human health. From July/2011 to March/2012, five liters of water were collected in 10 different locations from eight artificial beaches of Itaipu Lake, Paraná, in a total of 40 samples. In two other small beaches of the lake, water was also collected from two different sites of each beach, given their size and because they were made up of water from different sources. Samples were analyzed by the modified Ziehl-Neelsen technique and nested-PCR (nPCR). All samples were negative in the modified Ziehl-Neelsen; only one (2.25%) sample was positive in nPCR. These results indicate that PCR is a more sensitive technique for the identification of Cryptosporidium in water samples. In this study, it was not possible to identify the species involved and, thus, the source of infection. Water contamination by Cryptosporidium oocytes can be caused by livestock kept near waterways, and by the people who use the place for recreational purposes. These results demonstrate risk for people who use the lake. Care should be determined to prevent the transmission of this important zoonosis.A criptosporidiose é uma zoonose emergente responsável por vários surtos no mundo todo. Sua principal via de transmissão ocorre pelo consumo de água ou alimentos contaminados. Os dados de ocorrência de criptosporidiose tanto em animais como em seres humanos no Brasil são bastante escassos, dessa forma, torna-se importante a pesquisa da ocorrência de oocistos de Cryptosporidium sp. e a identificação de possíveis fontes de contaminação, visando a prevenção da contaminação da água e consequente preservação da saúde humana. Durante o período de jul/2011 a mar/2012 foram coletados cinco litros de água em 10 pontos de oito praias artificiais do Lago da Itaipu, Paraná, resultando em 40 amostras. Em duas prainhas foram coletadas água em dois locais devido ao tamanho das mesmas e por apresentarem fontes de água diferentes. As amostras foram analisadas pelo método de Ziehl-Neelsen modificado e nested-PCR. Das 40 amostras analisadas, todas foram negativas no método de Ziehl-Neelsen modificado e uma (1) apresentou fragmento de 819-825 bp (2,25%) na técnica de nPCR, o que sugere que a PCR é uma técnica mais sensível para a pesquisa de Cryptosporidium em água. No presente trabalho não foi possível identificar a espécie envolvida, e dessa forma a fonte de infecção, ou espécie responsável pela contaminação da água não foi conhecida. A contaminação da água por oocistos de Cryptosporidium pode ser decorrente da criação animal nas proximidades dos cursos d’água e também pelas próprias pessoas que frequentam e utilizam o local. Esses resultados demonstram um risco para as pessoas que frequentam o local, devendo haver uma orientação quanto aos cuidados para evitar a transmissão dessa importante zoonose