Distributions of waterborne pathogens in raw wastewater based on a 14-month, multi-site monitoring campaign.

The California State Water Resources Control Board is the first regulatory body in the United States to develop statewide regulations for direct potable reuse (DPR). To support this effort, a pathogen monitoring campaign was undertaken to develop and implement an optimized standard operating protocol to better characterize the concentration of human pathogens in raw wastewater. Methods to detect relevant viral and protozoan pathogens in raw wastewater were optimized and implemented during a 14-month monitoring campaign. Over 120 samples were collected from five wastewater treatment plants treating a quarter of California's population. Samples were analyzed for two protozoa (Cryptosporidium and Giardia) using microscopy methods, three enteric viruses (enterovirus, adenovirus, and norovirus) using culture and/or molecular methods, and male-specific coliphage using culture methods. The method recovery efficiency was measured in every protozoa sample and every other virus sample to confirm minimum recovery efficiencies were achieved and to correct the concentrations for pathogen losses during sample processing. The results from this study provide the industry with a large, high-quality dataset as demonstrated by the high degree of method sensitivity, method recovery, and QA/QC steps. Such high-quality data on pathogen concentrations in raw wastewater are critical for confirming the level of treatment needed to reduce pathogen concentrations down to acceptable levels for potable water in DPR projects.

A real-time RT-PCR method to detect viable Giardia lamblia cysts in environmental waters.

Currently, USEPA Method 1623 is the standard assay used for simultaneous detection of Giardia cysts and Cryptosporidium oocysts in various water matrices. However, the method is unable to distinguish between species, genotype, or to assess viability. Therefore, the objective of the present study was to address the shortcomings of USEPA Method 1623 by developing a novel molecular-based method that can assess viability of Giardia cysts in environmental waters and identify genotypes that pose a human health threat (assemblage groups A and B). Primers and TaqMan(®) probes were designed to target the beta-giardin gene in order to discriminate among species and assemblages. Viability was determined by detection of de-novo mRNA synthesis after heat induction. The beta-giardin primer/probe sets were able to detect and differentiate between Giardia lamblia assemblages A and B, and did not detect Giardia muris (mouse species) or G. lamblia assemblages C, D, E and F (non-human), with the exception of Probe A which did detect G. lamblia assemblage F DNA. Additionally, DNA or cDNA of other waterborne organisms were not detected, suggesting that the method is specific to Giardia assemblages. Assay applicability was demonstrated by detection of viable G. lamblia cysts in spiked (assemblage B) and unspiked (assemblage A and B) reclaimed water samples.

Comparison of tissue culture and animal models for assessment of Cryptospridium parvum infection.

The current increased interest for using tissue culture as a surrogate for mouse infection to assess Cryptospridium viability suggests that a comparison of the two models is essential for data interpretation. Therefore, a need remains for a statistical comparison that can demonstrate if infection and inactivation predicted by new tissue culture models are comparable with those predicted by animal models. Data from a total of 31 dose-response trials using both tissue culture and mouse models to assess C. parvum infectivity were compared. The dose needed to infect 50% of the tissue cultures (ID(50)) was also compared to each ID(50) in mice. Average ID(50)s developed using the logit dose-response method for tissue culture and mice were 8 and 107, respectively, suggesting that tissue culture was more sensitive to infection. However, correlation (r) between tissue culture and mouse infectivity was statistically significant (0.9167 [95% CI=0.8428 to 0.9594, p<0.0001]). Comparison of oocyst disinfection by UV and chlorine dioxide showed no significant difference between inactivation predicted by tissue culture and mouse models (p=0.8893; t=0.0141; n=21). These results demonstrate that tissue culture can successfully be used to measure C. parvum infection and can be used for determining inactivation in disinfection studies.