Key Takeaways Urban water systems and public well‐being are threatened by factors that include aging infrastructure, population growth and density, and conditions related to climate change. Floods and droughts have been particularly hard on urban areas, with short‐ and long‐term effects running the gamut of environmental, human (physical and emotional), and economic devastation. Cities and organizations around the world are devising innovative ways to achieve water resilience; more can be done through targeted research to raise awareness and understanding.

Environmental surveillance as a part of wastewater-based epidemiology (WBE) of SARS-CoV-2 can provide an early, cost-effective, unbiased community-level indicator of circulating COVID-19 in a population. The objective of this study was to determine how widely SARS-CoV-2 detection in wastewater is being investigated and what methods are used. A survey was developed and distributed, with results showing that methods were rapidly applied to conduct SARS-CoV-2 WBE, primarily to test wastewater influent from large urban wastewater treatment plants. Additionally, most methods utilized small wastewater volumes and the primary concentration methods used were polyethylene glycol precipitation, membrane filtration and centrifugal ultrafiltration followed by nucleic acid extraction and assay for primarily nucleocapsid gene targets (N1, N2, and/or N3). Since this survey was performed, many laboratories have continued to optimize and implement a variety of methods for SARS-CoV-2 WBE. Method comparison studies completed since this survey was conducted will assist in developing WBE as a supplemental tool to support public health and policy decision making responses.

Numerous quantitative PCR assays for microbial fecal source tracking (MST) have been developed and evaluated in recent years. Widespread application has been hindered by a lack of knowledge regarding the geographical stability and hence applicability of such methods beyond the regional level. This study assessed the performance of five previously reported quantitative PCR assays targeting human-, cattle-, or ruminant-associated Bacteroidetes populations on 280 human and animal fecal samples from 16 countries across six continents. The tested cattle-associated markers were shown to be ruminant-associated. The quantitative distributions of marker concentrations in target and nontarget samples proved to be essential for the assessment of assay performance and were used to establish a new metric for quantitative source-specificity. In general, this study demonstrates that stable target populations required for marker-based MST occur around the globe. Ruminant-associated marker concentrations were strongly correlated with total intestinal Bacteroidetes populations and with each other, indicating that the detected ruminant-associated populations seem to be part of the intestinal core microbiome of ruminants worldwide. Consequently tested ruminant-targeted assays appear to be suitable quantitative MST tools beyond the regional level while the targeted human-associated populations seem to be less prevalent and stable, suggesting potential for improvements in human-targeted methods.

Waterborne pathogens (and associated faecal indicator organisms (FIOs)) derived from human and animal faeces are a significant water quality concern in many parts of the world. In the United States of America (USA) “pathogens” (actually FIOs: coliforms and enterococci) are the most frequent cause of “impairment” (i.e. non-compliance) in waters covered by the US Clean Water Act (see: Figure 6.1) (USEPA 2009). The design and implementation of measures to

The U.S. Environmental Protection Agency (EPA) has committed to issuing in 2012 new or revised criteria designed to protect the health of those who use surface waters for recreation. For this purpose, the U.S. EPA has been conducting epidemiologic studies to establish relationships between microbial measures of water quality and adverse health outcomes among swimmers. New methods for testing water quality that would provide same-day results will likely be elements of the new criteria. Although the epidemiologic studies upon which the criteria will be based were conducted at Great Lakes and marine beaches, the new water quality criteria may be extended to inland waters (IWs). Similarities and important differences between coastal waters (CWs) and IWs that should be considered when developing criteria for IWs were the focus of an expert workshop. Here, we summarize the state of knowledge and research needed to base IWs microbial criteria on sound science. Two key differences between CWs and IWs are the sources of indicator bacteria, which may modify the relationship between indicator microbes and health risk, and the relationship between indicators and pathogens, which also may vary within IWs. Monitoring using rapid molecular methods will require the standardization and simplification of analytical methods, as well as greater clarity about their interpretation. Research needs for the short term and longer term are described.

BACKGROUND: Although pathogens such as Cryptosporidium oocysts present in animal fecal deposits on land, have been qualitatively and causally linked to event-related increases in pathogen concentrations in streams and reservoirs, there have been few attempts to quantify the relationship between pathogen dispersion and transport, rainfall, and surface water pathogen loads. Models that estimate sediment and nutrient export from land and predict the effectiveness of improved management practices are available. However, studies undertaken to date have not provided a similar basis for the prediction of pathogen exports. One of the major limitations is the lack of accurate data that is relevant to field conditions.

A detailed literature review was undertaken of pathogen prevalence and concentration in fecal matrices and sewage effluent. Most of the reports described microbial prevalence rather than concentration and the majority of studies were from developed countries in Europe, the Americas, and Australasia. Prevalence varied from nondetected to up to 100% and reported values varied between studies for the same host and matrix. Juvenile animals typically had higher reported pathogen prevalence than adult animals. The reported prevalence of pathogens was usually higher in domestic animals than wildlife. Extensive variation in reported pathogen concentrations was observed covering up to four orders of magnitude for the same matrix. Similar to the trends in prevalence, the reported concentration of pathogens was higher among juvenile animals than among adults and generally higher in domestic animals than in wildlife. The source of variation was highest for reports of concentrations in feces and lowest for reports of the sewage effluent, presumably because the latter is effectively a pooled sample. Reported concentrations of pathogens in similarly treated sewage effluent were reasonably consistent between studies from different parts of the world, although it is noted that the countries from which the reports originated had similar socioeconomic characteristics. The priorities for reducing pathogen presence in water-supply catchments are to adequately treat domestic sewage before discharge and to improve the management of domestic animals to reduce levels of infection.

The pathogen catchment budget (PCB) model (Ferguson et al. 2007) was developed for estimating the pathogen (Cryptosporidium and Giardia) and faecal indicator (E. coli) loads generated within and exported from drinking water catchments. The model uses a mass-balance approach and predicts the total loads generated and the total loads exported from each sub-catchment for the pathogens Cryptosporidium and Giardia and the faecal indicator E. coli. Briefly, the PCB model consists of 5 components: a hydrologic module, a land budget module, an on-site systems module, a sewage treatment plant (STP) module and an in-stream transport module.PCB is an event-based model, representing the likely fluxes from sub-catchments in dry, wet and flood conditions. The model was developed in the Interactive Component Modelling System (ICMS), (Cuddy et al. 2002), and is freely available from the Commonwealth Scientific Information and Resource Organisation (CSIRO). Inputs to the model include GIS land use and hydrologic data as well as catchment specific information such as animal density and the location of on-site systems and sewage treatment plants (STPs) as well information on pathogen concentrations in different species, pathogen inactivation rates and mobilisation of faecal material. The hydrologic module uses the non-linear loss module of the IHACRES rainfall-runoff model described by Croke and Jakeman (2004).The PCB model was initially applied to the Wingecarribee catchment and subsequently to the Sydney Catchment Authority (SCA) area of operations (Ferguson 2005; Ferguson and Croke 2005). As part of a project funded primarily by the American Water Works Association Research Foundation, the model has been applied to drinking water supply catchments in Australia (Googong), the UK (Thirlmere) and the USA (Kensico). This paper describes the application of the existing event-based PCB model to these catchments, and the adaptations to the model needed in each case. The influence of snow in the study catchments in the UK and USA has not been taken into account, so the model results are suitable for summer storm events only.The higher inactivation rate for E. coli leads to only nearby sub-catchments contributing significantly to loads entering the dam in the Googong catchment. In comparison, Cryptosporidium and Giardia do show evidence of transport from the headwater areas in the Googong catchment even under low flow conditions. The study sites in the UK and the USA are significantly smaller, and each sub-catchment is directly connected to the reservoir so that the inactivation rates during transport are not significant. Table 3 shows the sub-catchments which the model predicts will have the largest input into the stream network (i.e. ignoring in-stream processes).The output from the PCB model facilitates identification of those sub-catchments that represent the highest pathogen (and indicator) risk to the quality of raw drinking water supplies. This enables managers to prioritise the implementation of control measures, to inform water supply strategies and target best management practices. The outputs from the model can also be used as input data to hydrodynamic models of pathogen transport in reservoirs.

The dispersion and transport of Cryptosporidium parvum oocysts, Escherichia coli and PRD1 bacteriophage seeded into artificial bovine faecal pats was studied during simulated rainfall events. Experimental soil plots were divided in two, one sub-plot with bare soil and the other with natural vegetation. Simulated rainfall events of 55 mm.h(-1) for 30 min were then applied to the soil plots. Each experimental treatment was performed in duplicate and consisted of three sequential artificial rainfall events ('Runs'): a control run (no faecal pats); a fresh faecal pat run (fresh faecal pats); and an aged faecal pat run (one week aged faecal pats). Transportation efficiency increased with decreasing size of the microorganism studied; Cryptosporidium oocysts were the least mobile followed by E. coli and then PRD1 phage. Rainfall events mobilised 0.5 to 0.9% of the Cryptosporidium oocysts, 1.3-1.4% of E. coli bacteria, and 0.03-0.6% of PRD1 bacteriophages from the fresh faecal pats and transported them a distance of 10 m across the bare soil sub-plots. Subsequent rainfall events applied to aged faecal pats only mobilised 0.01-0.06% of the original Cryptosporidium oocyst load, between 0.04 and 15% of the E. coli load and 0.0006-0.06% of PRD1 bacteriophages, respectively.

In drinking water catchments, reduction of pathogen loads delivered to reservoirs is an important priority for the management of raw source water quality. To assist with the evaluation of management options, a process-based mathematical model (pathogen catchment budgets - PCB) is developed to predict Cryptosporidium, Giardia and E. coli loads generated within and exported from drinking water catchments. The model quantifies the key processes affecting the generation and transport of microorganisms from humans and animals using land use and flow data, and catchment specific information including point sources such as sewage treatment plants and on-site systems. The resultant pathogen catchment budgets (PCB) can be used to prioritize the implementation of control measures for the reduction of pathogen risks to drinking water. The model is applied in the Wingecarribee catchment and used to rank those sub-catchments that would contribute the highest pathogen loads in dry weather, and in intermediate and large wet weather events. A sensitivity analysis of the model identifies that pathogen excretion rates from animals and humans, and manure mobilization rates are significant factors determining the output of the model and thus warrant further investigation.

A workshop titled "Application of Genotyping Methods to Assess Pathogen Risks from Cryptosporidium in Drinking Water Catchments" was held at the International Water Association biennial conference, Marrakech, Morocco, 23 September 2004. The workshop presented and discussed the findings of an interlaboratory trial that compared methods for genotyping Cryptosporidium oocysts isolated from feces. The primary goal of the trial and workshop was to assess the utility of current Cryptosporidium genotyping methods for determining the public health significance of oocysts isolated from feces in potable-water-supply watersheds. An expert panel of 16 watershed managers, public health practitioners, and molecular parasitologists was assembled for the workshop. A subordinate goal of the workshop was to educate watershed management and public health practitioners. An open invitation was extended to all conference delegates to attend the workshop, which drew approximately 50 interested delegates. In this report we summarize the peer consensus emerging from the workshop. Recommendations on the use of current methods by watershed managers and public health practitioners were proposed. Importantly, all the methods that were reported in the trial were mutually supporting and found to be valuable and worthy of further utility and development. Where there were choices as to which method to apply, the small-subunit ribosomal RNA gene was considered to be the optimum genetic locus to target. The single-strand conformational polymorphism method was considered potentially the most valuable for discriminating to the subtype level and where a large number of samples were to be analyzed. A research agenda for protozoan geneticists was proposed to improve the utility of methods into the future. Standardization of methods and nomenclature was promoted.

A study was undertaken to compare the performance of five different molecular methods (available in four different laboratories) for the identification of Cryptosporidium parvum and Cryptosporidium hominis and the detection of genetic variation within each of these species. The same panel of oocyst DNA samples derived from faeces (n=54; coded blindly) was sent for analysis by: (i) DNA sequence analysis of a fragment of the HSP70 gene; (ii) DNA sequence analysis and the ssrRNA gene in laboratory 1; (iii) single-strand conformation polymorphism analysis of part of the ssrRNA; (iv) SSCP analysis of the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA region in laboratory 2; (v) 60 kDa glycoprotein (gp60) gene sequencing with prior species determination using PCR with restriction fragment length polymorphism analysis of the ssrRNA gene in laboratory 3; and (vi) multilocus genotyping at three microsatellite markers in laboratory 4. For detecting variation within C. parvum and C. hominis, SSCP analysis of ITS-2 was considered to have superior utility and determined ‘subgenotypes’ in samples containing DNA from both species. SSCP was also most cost effective in terms of time, cost and consumables. Sequence analysis of gp60 and microsatellite markers ML1, ML2 and ‘gp15’ provided good comparators for the SSCP of ITS-2. However, applicability of these methods to other Cryptosporidium species or genotypes and to environmental samples needs to be evaluated. This trial provided, for the first time, a direct comparison of multiple methods for the genetic characterisation of C. parvum and C. hominis samples. A protocol has been established for the international distribution of samples for the characterisation of Cryptosporidium. This can be applied in further evaluation of molecular methods by investigation of a larger number of unrelated samples to establish sensitivity, typability, reproducibility and discriminatory power based on internationally accepted methods for evaluation of microbial typing schemes.

ABSTRACT A fecal analysis survey was undertaken to quantify animal inputs of pathogenic and indicator microorganisms in the temperate watersheds of Sydney, Australia. The feces from a range of domestic animals and wildlife were analyzed for the indicator bacteria fecal coliforms and Clostridium perfringens spores, the pathogenic protozoa Cryptosporidium and Giardia, and the enteric viruses adenovirus, enterovirus, and reovirus. Pathogen and fecal indicator concentrations were generally higher in domestic animal feces than in wildlife feces. Future studies to quantify potential pathogen risks in drinking-water watersheds should thus focus on quantifying pathogen loads from domestic animals and livestock rather than wildlife.

Outbreaks of water-borne disease via public water supplies continue to be reported in developed countries even though there is increased awareness of, and treatment for, pathogen contamination. Pathogen episodes in lakes and reservoirs are often associated with rain events, and the riverine inflow is considered to be major source of pathogens. Consequently, the behaviour of these inflows is of particular importance in determining pathogen transport and distribution. Inflows are controlled by their density relative to that of the lake, such that warm inflows will flow over the surface of the lake as a buoyant surface flow and cold, dense inflows will sink beneath the lake water where they will flow along the bathymetry towards the deepest point. The fate of pathogens is determined by loss processes including settling and inactivation by temperature, UV and grazing. The general trend is for the insertion timescale to be shortest, followed by sedimentation losses and temperature inactivity. The fate of Cryptosporidium due to UV light inactivation can occur at opposite ends of the scale, depending on the location of the oocysts in the water column and the extinction coefficient for UV light. For this reason, the extinction coefficient for UV light appears to be a vitally important parameter for determining the risk of Cryptosporidium contamination. For risk assessment of pathogens in supply reservoirs, it is important to understand the role of hydrodynamics in determining the timescale of transport to the off-take relative to the timescale of inactivation. The characteristics of the riverine intrusion must also be considered when designing a sampling program for pathogens. A risk management framework is presented that accounts for pathogen fate and transport for reservoirs.

The purpose of this chapter is to discuss microbiological health criteria for Cryptosporidium. In the process of revising the current World Health Organization (WHO) guidelines for drinking water quality, it has become apparent that there is a need for fundamental change in approach. The current format and the national guidelines that draw from them, focus on end product enumeration of Escherichia coli. This system is reactive in the sense that the warning signal generated by the detection of contamination is received at a time when consumers' health is already at risk. Current microbiological guidelines need to be harmonized with a source-to-consumer risk assessment. This paradigm shift in thinking would change the focus of attention from end-water testing to verification that the safeguards in the water supply system are actually in place and effective.

To protect the quality of drinking water in Sydney, the Sydney Water Corporation (SWC) and the recently created Sydney Catchment Authority (SCA) have adopted a catchment to customer risk management approach after the Cryptosporidium water crisis in 1998. In Sydney such an approach involves close collaboration with the New South Wales Department of Health (NSW Health), as well as the consortia managing several of Sydney's large water filtration plants (WFPs) under Build-Own-Operate (BOO) contracts. This chapter presents a paper, which aims to provide an update of the main actions that have been taken since the water crisis in Sydney in 1998. These updates need to be in: the catchments, water treatment plants, distribution system, and analytical laboratories for Cryptosporidium analysis.