Human health risk assessment for (re)emerging protozoan parasites in surface water used for public supply and recreational activities.

Gastrointestinal diseases caused by protozoan parasites remain a major challenge in developing countries and ingestion of contaminated surface water represents one of the main sources by which these diseases are contracted. This study assessed the risk of infection and diseases caused by Cryptosporidium spp. and Giardia sp. due to ingestion of surface water used for public supply and recreational activities, focusing on the southeastern Brazilian Pardo River and applying the USEPA 1623 method to quantify (oo)cyst concentrations. Infection and disease probabilities due to ingestion of drinking water or during recreational activities were estimated using the Quantitative Microbial Risk Assessment (QMRA) approach. Mean concentrations of Cryptosporidium spp. and Giardia sp. in surface water ranged from 0.2 to 0.4 oocysts L-1 and 0.2 to 4.4 cysts L-1, respectively. Considering public water supply, annual infection probabilities were higher for adults than children and exceeded the USEPA limit; also, disease probabilities were higher for adults than children. For recreational activities, annual infection and disease probabilities were higher for children, followed by men and women. The occurrence of both parasites likely reflects raw sewage discharge, effluent from sewage treatment plants, and diffuse sources of pollution, such as runoff from pasture lands and deforested riparian forest corridors. Our results highlight substantial infection risks by both parasite types after conventional treatment of water used for public supply and also call for careful monitoring of water bodies used for recreational purposes.

Real-time PCR detection of Toxoplasma gondii in surface water samples in São Paulo, Brazil.

Water is considered an important vehicle for the spread of human toxoplasmosis in several countries. Toxoplasma gondii oocysts can persist in the environment for long periods, being highly resistant to the various chemical inactivation processes commonly used by water supply systems, distinctly from simple filtration and flocculation that are efficient in removing oocysts from drinking water. The existing methodologies for identification and quantification of this parasite in water samples are not standardized and have limitations. This study aimed to evaluate the presence of T. gondii oocysts in surface water samples used as a source for the production of drinking water in the State of São Paulo, through the implementation of a specific methodology using real-time PCR technique (qPCR). Volumes of 20 L of the sample were concentrated by filtration in Envirocheck® HV capsules. For DNA extraction, the PowerSoil DNA isolation® kit (currently DNeasy PowerSoil®) was used. The target sequence selected for qPCR was a 62-base-pair fragment of the B1 gene. In the initial recovery evaluation of the method in four replicates of reverse osmosis water, the mean recovery was 48.5% (SD ± 11.5), while the mean recovery for method performance in matrices was 3.2% (SD ± 3.2) (rainy season) and 62.0% (SD ± 6.2) (dry period), suggesting that the characteristics of the samples and the climatic conditions interfere in the recovery efficiency. Of the 39 samples analyzed (May to December 2015), 7.7% (3/39) were positive for T. gondii, and among the ten sources studied; the occurrence of the oocysts was detected in 30% (3/10).

Environmental surveillance of Cryptosporidium and Giardia in surface supply water and treated sewage intended for reuse from an urban area in Brazil.

Environmental monitoring of protozoa, with the potential to trigger diseases, is essential for decision-making by managing authorities and for the control of water surveillance. This study aimed to detect and quantify Cryptosporidium oocysts and Giardia cysts in surface water for drinking water supply and treated sewage for reuse in the city of São Paulo. Samples collected bimonthly for one year were concentrated using the USEPA 1623.1 and 1693 methods for surface water and treated effluents, respectively. Immunofluorescence and nucleic acid amplification techniques were used to detect and quantify (oo)cysts. The cloning technique followed by sequencing and phylogenetic analyses were performed to characterize species and genotypes. The immunofluorescence detected Cryptosporidium spp. and Giardia spp. in 69.2% (9/13) and 100% (13/13) of the surface water samples (0.1-41 oocysts/L and 7.2-354 cysts/L, respectively). In the reuse samples, 85.7% (12/14) were positive for both protozoa and the concentrations varied from 0.4 to 100.6 oocysts/L and 1.2 and 93.5 cysts/L. qPCR assays showed that 100% of surface water (0.1-14.6 oocysts/L and 0.3-639.8 cysts/L) and reused samples (0.1-26.6 oocysts/L and 0.3-92.5 cysts/L) were positive for both protozoa. Species C. parvum, C. hominis, and C. muris were identified using the 18S rRNA gene, demonstrating anthroponotic and zoonotic species in the samples. Multilocus SSU rRNAanalyses of the SSU rRNA, tpi, and gdh genes from Giardia intestinalis identified the AII, BII, and BIV assemblages, revealing that contamination in the different matrices comes from human isolates. The study showed the circulation of these protozoa in the São Paulo city area and the impairment of surface water supply in metropolitan regions impacted by the discharge of untreated or inadequately treated sewage regarding the removal of protozoa, emphasizing the need to implement policies for water safety, to prevent the spread of these protozoa in the population.

Detection and molecular characterization of Cryptosporidium species and Giardia assemblages in two watersheds in the metropolitan region of São Paulo, Brazil.

Cryptosporidium and Giardia are associated with cases of water and foodborne outbreaks in the world. This study included 50 samples of surface raw water collected from two watersheds in the state of São Paulo, Brazil. The isolation of (oo)cysts was performed in accordance with the U.S. Environmental Protection Agency's methods 1623 and genotypic characterization and quantification were carried out by Nested PCR and qPCR assays based on 18S rRNA and gdh genes, respectively. U.S. EPA 1623 method showed the presence of (oo)cysts in 40% ([Formula: see text] = 0.10 oocysts/L) and 100% ([Formula: see text] = 7.6 cysts/L) of samples from São Lourenço River, respectively, and 24% ([Formula: see text] = 0.8 oocysts/L) and 60% ([Formula: see text] = 1.64 cysts/L) of Guarapiranga Reservoir, respectively. The qPCR assay detected C. hominis/parvum in 52% (0.06 to 1.85 oocysts/L) of São Lourenço River and 64% (0.09 to 1.4 oocysts/L) of Guarapiranga Reservoir samples. Presence/absence test for Giardia intestinalis was positive in 92% of São Lourenço River and 8% of Guarapiranga Reservoir samples. The assemblage A was detected in 16% (0.58 to 2.67 cysts/L) in São Lourenço River and no positive samples were obtained for assemblage B in both water bodies. The characterization of anthroponotic species C. parvum/hominis, G. intestinalis, and assemblage A was valuable in the investigation of possible sources of contamination in the watersheds studied confirming the need of expanding environmental monitoring measures for protection of these water sources in our country.

Assessing the infection risk of Giardia and Cryptosporidium in public drinking water delivered by surface water systems in Sao Paulo State, Brazil.

A survey of Giardia and Cryptosporidium was conducted in surface water used as drinking water sources by public water systems in four densely urbanized regions of Sao Paulo State, Brazil. A Quantitative Microbial Risk Assessment, based on protozoa concentrations, was performed to estimate the probability of protozoa infection associated with drinking water ingestion. A total of 206 source water samples were analyzed over a 24 month period using the USEPA Method 1623. The risk of infection was estimated using an exponential dose response model, children and adults exposure and a gamma distribution for (oo)cyst concentrations with three scenarios for treating censored data. Giardia was detected in 102 of the samples, and 19 of them were also positive for Cryptosporidium, with maximum concentrations of 97.0 cysts/L and 6.0 oocysts/L, respectively. Risk distributions were similar for the three scenarios. In the four regions, the estimated risk of Giardia infection per year, for adults and children, ranged from 0.29% to 2.47% and from 0.08% to 0.70%, respectively. Cryptosporidium risk infection varied from 0.15% to 0.29% for adults and from 0.04% to 0.08% for children. In both cases, the calculated risk surpassed the risk of infection of 10(-4) (1:10,000) defined as tolerable by USEPA for a yearly exposure. The probability of Giardia infection was very close to the rates of acute diarrheic disease for adults (1% to 3%) but lower for children (2% to 7%). The daily consumption of drinking water was an important contributing factor for these differences. The Microbiological Risk Assessment carried out in this study provides an indication of infection risks by Giardia and Cryptosporidium in the population served by these source waters. Strategies for source water protection and performance targets for the water treatment should be established to achieve the required level of public health risk.