Giardia Cysts in Wastewater Treatment Plants in Italy

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Abstract
Reductions in annual rainfall in some regions and increased human consumption have caused a shortage of water resources at the global level. The recycling of treated wastewaters has been suggested for certain domestic, industrial, and agricultural activities. The importance of microbiological and parasitological criteria for recycled water has been repeatedly emphasized. Among water-borne pathogens, protozoa of the genera Giardia and Cryptosporidium are known to be highly resistant to water treatment procedures and to cause outbreaks through contaminated raw or treated water. We conducted an investigation in four wastewater treatment plants in Italy by sampling wastewater at each stage of the treatment process over the course of 1 year. The presence of the parasites was assessed by immunofluorescence with monoclonal antibodies. While Cryptosporidium oocysts were rarely observed, Giardia cysts were detected in all samples throughout the year, with peaks observed in autumn and winter. The overall removal efficiency of cysts in the treatment plants ranged from 87.0 to 98.4%. The removal efficiency in the number of cysts was significantly higher when the secondary treatment consisted of active oxidation with O2 and sedimentation instead of activated sludge and sedimentation (94.5% versus 72.1 to 88.0%; P = 0.05, analysis of variance). To characterize the cysts at the molecular level, the β-giardin gene was PCR amplified, and the products were sequenced or analyzed by restriction. Cysts were typed as assemblage A or B, both of which are human pathogens, stressing the potential risk associated with the reuse of wastewater. At the global level, there has been a growing shortage of freshwater reserves, mainly those of good quality, as a result of increasing human consumption and, in some regions, decreases in the annual rainfall or annual rainfall consisting mostly of heavy rain, which is poorly absorbed by the soil (19, 20, 21). To address this problem, dual water networks in which treated wastewater can be used for domestic, industrial, and agricultural purposes, for which the use of water with a low level of chemical or microbiological contaminants would not represent a threat to human health have been proposed (18). The importance of microbiological and parasitological criteria for controlling the contamination of recycled water has been repeatedly emphasized. In industrialized countries, the most common human parasitic protozoa transmitted by water belong to the genera Giardia and Cryptosporidium (34). Giardiasis and cryptosporidiosis are also common infections of domestic and wild animals, which shed a large number of cysts and oocysts in the environment. These cysts are insensitive to disinfectants at the concentration commonly used in water treatment plants to reduce bacterial contamination, although it has been shown that at higher concentrations of chlorine and ozone, Giardia cysts are less resistant than Cryptosporidium oocysts (35). Moreover, Giardia cysts have been shown to survive in water for up to 2 months at temperatures as low as 8°C (26), and Cryptosporidium oocysts can survive for up to 1 year at 4°C in artificial seawater (36). Furthermore, the infectious dose has been estimated to be as low as 10 cysts for Giardia (1) and 30 oocysts for Cryptosporidium (12). Numerous water-borne outbreaks of giardiasis and cryptosporidiosis have been documented in the past several decades, mainly in the United States, Europe, and Australia (33, 34). This has led the U.S. Environmental Protection Agency (EPA) to regulate the level of Giardia cysts and Cryptosporidium oocysts allowed in drinking water (40). To detect oocysts, many methods have been developed based on the filtration of large volumes of water, followed by centrifugation, clarification (either by density gradients or immunomagnetic separation), and microscopic screening of the sample after staining with monoclonal antibodies (28). Although these methods have proven to be very useful for determining whether or not waters are contaminated with parasites, they cannot distinguish among the different species or genotypes. To this end, PCR assays have been developed (33), yet the efficiency of amplification techniques is often reduced by the presence of inhibitory substances in water samples, such as humic and fulvic acids (37, 42). In Italy, there are few published data on the prevalence of Giardia and Cryptosporidium in wastewaters. The objectives of the present study were to evaluate the prevalence of these parasites in four wastewater treatment plants, to estimate the efficiency of treatment plants in removing these parasites, to develop a reliable method for DNA extraction from concentrated water samples, and to determine the species and genotype of these parasites by means of a molecular assay.