Quantification and molecular characterization of Cryptosporidium spp. from raw surface water and raw sewage for the monitoring of public water supplies in the metropolitan region of São Paulo, Brazil

Abstract

Waterborne diseases have emerged as a major public health problem. Cryptosporidium is an important parasite associated with cases of waterborne outbreaks in the world. The complex epidemiology of this protozoan increases the interest for sensitive and rapid methods for the detection of species responsible for infection in humans. In this study, 50 samples were collected from two public water supplies, São Lourenço River (P1A) and Guarapiranga Dam (P2A), and 50 samples of raw sewage were collected from São Lourenço da Serra’s (P1E) and Taboão da Serra’s vertical wells (P2E), between January and December of 2013. The isolation of oocysts in water was carried out by the USEPA Method 1623 (2005) and raw sewage samples were processed by the centrifugation-filtration method. Genotyping occurred by nested PCR, cloning and sequencing based on 18S rRNA gene. Real time PCR assays (qPCR) were carried out for detection and quantification of oocysts in water samples. According to the results, Cryptosporidium was detected in 12% (3/25) of samples at P1A and 16% (4/25) at P2A in the water by nested PCR. In sewage, the parasite was detected in 20% (5/25) of samples at P1E and 24% (6/25) in P2E vertical well. Quantitative PCR detected 0.79 to 1.85 oocysts/L (52%) at P1A and 0.72 to 1.4 oocysts/L (64%) at P2A. Molecular characterization allowed the identification of C. parvum and C. hominis in water, and C. hominis, C. parvumand C. muris in raw sewage by nested PCR. Cryptosporidium species identified herein belong to a group of organisms of significant relevance in waterborne diseases. Therefore, concentration and characterization methodologies applied in our analyses showed to be useful for environmental surveillance studies. The detection of C. muris, which is associated with anthroponotic species, was helpful in the investigation of likely contamination sources in the environment, confirming the need of expansion in effective measures to protect these water supplies.