Reproducibility and sensitivity of 36 methods to quantify the SARS-CoV-2 genetic signal in raw wastewater: findings from an interlaboratory methods evaluation in the U.S.

In response to COVID-19, the international water community rapidly developed methods to quantify the SARS-CoV-2 genetic signal in untreated wastewater. Wastewater surveillance using such methods has the potential to complement clinical testing in assessing community health. This interlaboratory assessment evaluated the reproducibility and sensitivity of 36 standard operating procedures (SOPs), divided into eight method groups based on sample concentration approach and whether solids were removed. Two raw wastewater samples were collected in August 2020, amended with a matrix spike (betacoronavirus OC43), and distributed to 32 laboratories across the U.S. Replicate samples analyzed in accordance with the project's quality assurance plan showed high reproducibility across the 36 SOPs: 80% of the recovery-corrected results fell within a band of ±1.15 log10 genome copies per L with higher reproducibility observed within a single SOP (standard deviation of 0.13 log10). The inclusion of a solids removal step and the selection of a concentration method did not show a clear, systematic impact on the recovery-corrected results. Other methodological variations (e.g., pasteurization, primer set selection, and use of RT-qPCR or RT-dPCR platforms) generally resulted in small differences compared to other sources of variability. These findings suggest that a variety of methods are capable of producing reproducible results, though the same SOP or laboratory should be selected to track SARS-CoV-2 trends at a given facility. The methods showed a 7 log10 range of recovery efficiency and limit of detection highlighting the importance of recovery correction and the need to consider method sensitivity when selecting methods for wastewater surveillance.

Survey of rapid development of environmental surveillance methods for SARS-CoV-2 detection in wastewater.

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.

Performance characteristics of qPCR assays targeting human- and ruminant-associated bacteroidetes for microbial source tracking across sixteen countries on six continents.

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.

Meeting report: knowledge and gaps in developing microbial criteria for inland recreational waters.

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.

Development of a process-based model to predict pathogen budgets for the Sydney drinking water catchment.

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.

Field scale quantification of microbial transport from bovine faeces under simulated rainfall events.

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.

Meeting report: Application of genotyping methods to assess risks from cryptosporidium in watersheds.

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.

Relative value of surrogate indicators for detecting pathogens in lakes and reservoirs.

This study investigated the relative behavior of pathogens, fecal indicator organisms, and particles of varying size during transport through a reservoir following a storm event inflow in Myponga Reservoir, South Australia. During the inflow, samples were collected from the river and at various locations within the reservoir to determine the fate and transport of microroganisms as they progressed through the water body. Microbiological analysis included the indicator organisms Escherichia coli, enterococci, Clostridium perfringens, aerobic spores, and somatic coliphages, the protozoan pathogens Cryptosporidium spp. and Giardia spp., and the potential physical surrogates of pathogen contamination including particle size and turbidity. Of the microbial indicator groups, C. perfringens spores were the most highly correlated with Cryptosporidium spp. concentrations (Spearman Rho = 0.58), closely followed by enterococci (Spearman Rho = 0.57). Cryptosporidium spp. oocysts were predominantly associated with small sized particles (range of 14.3-27.7 microm). All of the microbial indicator groups tested were associated with larger sized particle ranges (> 63.3 microm) except C. perfringens spores which were associated with particles in the size range of 45.5-63.3 microm. Although indicators may rank correlate with Cryptosporidium spp., the variation in settling rates of different microorganisms has significant implications for the use of surrogates to estimate pathogen attenuation within reservoirs. For example, concentrations of Cryptosporidium spp. oocysts were reduced by a factor of 3 on reaching the dam wall, whereas enterococci were reduced by a factor of 10.

Concentrations of pathogens and indicators in animal feces in the Sydney watershed.

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.

Direct comparison of selected methods for genetic categorisation of Cryptosporidium parvum and Cryptosporidium hominis species.

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.

Fate and transport of pathogens in lakes and reservoirs.

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.

Dispersion and transport of Cryptosporidium Oocysts from fecal pats under simulated rainfall events.

The dispersion and initial transport of Cryptosporidium oocysts from fecal pats were investigated during artificial rainfall events on intact soil blocks (1,500 by 900 by 300 mm). Rainfall events of 55 mm h(-1) for 30 min and 25 mm h(-1) for 180 min were applied to soil plots with artificial fecal pats seeded with approximately 10(7) oocysts. The soil plots were divided in two, with one side devoid of vegetation and the other left with natural vegetation cover. Each combination of event intensity and duration, vegetation status, and degree of slope (5 degrees and 10 degrees ) was evaluated twice. Generally, a fivefold increase (P < 0.05) in runoff volume was generated on bare soil compared to vegetated soil, and significantly more infiltration, although highly variable, occurred through the vegetated soil blocks (P < 0.05). Runoff volume, event conditions (intensity and duration), vegetation status, degree of slope, and their interactions significantly affected the load of oocysts in the runoff. Surface runoff transported from 10(0.2) oocysts from vegetated loam soil (25-mm h(-1), 180-min event on 10 degrees slope) to up to 10(4.5) oocysts from unvegetated soil (55-mm h(-1), 30-min event on 10 degrees slope) over a 1-m distance. Surface soil samples downhill of the fecal pat contained significantly higher concentrations of oocysts on devegetated blocks than on vegetated blocks. Based on these results, there is a need to account for surface soil vegetation coverage as well as slope and rainfall runoff in future assessments of Cryptosporidium transport and when managing pathogen loads from stock grazing near streams within drinking water watersheds.

Comparison of methods for the concentration of Cryptosporidium oocysts and Giardia cysts from raw waters.

This study compared the recovery of Cryptosporidium oocysts and Giardia cysts ((oo)cysts) from raw waters using 4 different concentration-elution methods: flatbed membranes, FiltaMax foam, Envirochek HV capsules, and Hemoflow ultrafilters. The recovery efficiency of the combined immunomagnetic separation and staining steps was also determined. Analysis of variance of arcsine-transformed data demonstrated that recovery of Cryptosporidium oocysts by 2 of the methods was statistically equivalent (flatbed filtration 26.7% and Hemoflow 28.3%), with FiltaMax and Envirochek HV recoveries significantly lower (18.9% and 18.4%). Recovery of Giardia cysts was significantly higher using flatbed membrane filtration (42.2%) compared with the other 3 methods (Envirochek HV 29.3%, FiltaMax 29.0%, and Hemoflow 20.9%). All methods were generally acceptable and are suitable for laboratory use; 2 of the methods are also suitable for field use (FiltaMax and Envirochek HV). In conclusion, with recoveries generally being statistically equivalent or similar, practical considerations become important in determining which filters to use for particular circumstances. The results indicate that while low-turbidity or "finished" waters can be processed with consistently high recovery efficiencies, recoveries from raw water samples differ significantly with variations in raw water quality. The use of an internal control with each raw water sample is therefore highly recommended.