Developing and Refining Quantitative Microbial Risk Assessment Using Molecular Viral Fecal Indicators

Exposure to enteric pathogens in polluted recreational water causes millions of illnesses that cost society billions of dollars to treat annually [1]. Most currently established monitoring efforts of environmental waters utilize culturable bacterial fecal indicators, which require long turnaround times, are limited in their ability to represent viral pathogens, and fail to correlate with undesirable health outcomes. Molecular human viral fecal indicators have great potential to reduce the burden of gastrointestinal disease in the United States and worldwide. Efforts to improve water quality indicators can reduce the burden of disease by increasing the ability to detect the presence of human pathogens at lower concentrations. Relating measurements of human viral fecal indicators to health outcomes can be conducted with Quantitative Microbial Risk Assessments (QMRA). In this dissertation work, the proposed indicator crAssphage was quantified in international wastewater and assessed for its ability to predict risk associated with swimming in polluted recreational water. QPCR and PCR methods were used to measure the concentration of crAssphage alongside viral pathogens in legacy Italian wastewater samples collected from 2014-2018. CrAssphage performed well and was consistently detected across Italy at high concentrations and correlated with pathogenic viruses. Additionally, a major limitation of molecular indicators was discovered: different DNA concentration methods significantly impacted results. Following these results, and to connect molecular viral fecal indicators to health outcomes, a stochastic model was developed representing a recreational water body polluted with human wastewater. This model was used to estimate the risk of infection and illness due to a pollution event using crAssphage and pepper mild mottle virus as molecular indicators. This model was also converted into a web application to communicate the method of relating indicator measurements to pathogen concentrations and health outcomes using a ratio approach, which assumes that the ratio of indicators to pathogens remains constant in sewage and in environmental waters. Lastly, the core assumption of the ratio approach was tested in a QMRA model. Decay rates were collected for pathogens and the time at which the assumption causes a significant overestimation of risk was determined. The results of this model showed that the ratio method quickly overestimates risk in a recreational water setting, suggesting that future QMRAs should carefully characterize the pollution event that the health risks are predicted to arise from to avoid misrepresenting the hazard.