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Aims: Widespread adoption of the new U.S. Environmental Protection Agency (USEPA) Method 1642 for enumeration of coliphage in recreational water requires demonstration that laboratories consistently meet internal method performance goals and yield results that are consistent across laboratories., Methods and Results: Here we assess the performance of six laboratories processing a series of blind wastewater- and coliphage-spiked samples along with laboratory blanks. All laboratories met the method-defined recovery requirements when performance was averaged across samples, with the few failures on individual samples mostly occurring for less-experienced laboratories on the initial samples processed. Failures that occurred on later samples were generally attributed to easily correctable activities. Failure rates were higher for somatic vs. F+ coliphage, attributable to the more stringent performance criteria associated with somatic coliphage. There was no difference in failure rate between samples prepared in a marine water matrix compared to that in phosphate-buffered saline., Conclusions: Variation among laboratories was similar to that previously reported for enterococci, the current bacterial indicator used for evaluating beach water quality for public health protection., Significance and Impact of the Study: These findings suggest that laboratory performance is not an inhibitor to the adoption of coliphage as a new indicator for assessing recreational health risk., (© 2022 Society for Applied Microbiology.)

Urban beaches are frequently impacted from multiple sources of fecal contamination. This along with high beach usage underscores the importance of appropriate management that protects swimmer health. The USEPA has enabled the use of QMRA as a tool for quantifying swimmer health risk and setting site-specific water quality objectives. This study illustrates the challenges associated with human and non-human source identification and how these challenges influence the decision of whether QMRA at typical urban beaches for water quality management is appropriate. In this study, a similar and correlated spatial relationship with elevated Enterococcus and avian-specific markers was observed, suggesting shorebirds as a primary source of FIB. However, human-associated markers were also detected frequently but at low concentrations. Ultimately, a QMRA was not conducted because pathogen loading from potential human sources could not be confidently quantified, having consequences for health risk in receiving waters where recreational contact occurs., (Published by Elsevier Ltd.)

Human fecal pollution of recreational waters remains a public health concern worldwide. As a result, there is a growing interest in the application of human-associated fecal source identification quantitative real-time PCR (qPCR) technologies for water quality research and management. However, there are currently no standardized approaches for field implementation and interpretation of qPCR data. In this study, a standardized HF183/BacR287 qPCR method was combined with a water sampling strategy and a novel Bayesian weighted average approach to establish a human fecal contamination score (HFS) that can be used to prioritize sampling sites for remediation based on measured human waste levels. The HFS was then used to investigate 975 study design scenarios utilizing different combinations of sites with varying sampling intensities (daily to once per week) and number of qPCR replicates per sample (2-14 replicates). Findings demonstrate that site prioritization with HFS is feasible and that both sampling intensity and number of qPCR replicates influence reliability of HFS estimates. The novel data analysis strategy presented here provides a prescribed approach for the implementation and interpretation of human-associated HF183/BacR287 qPCR data with the goal of site prioritization based on human fecal pollution levels. In addition, information is provided for future users to customize study designs for optimal HFS performance., (Published by Elsevier Ltd.)

qPCR has become increasingly popular for microbial water quality testing because it is faster, more specific, and more flexible than culture-based methods. However, qPCR method limitations such as quantification bias introduced by reliance on standards and susceptibility to PCR inhibitors are major obstacles for implementation in water testing. This is because water testing requires accurate quantification of rare targets and because environmental waters often contain PCR inhibitors. Digital PCR offers the opportunity to maintain qPCR's advantages over culture-based methods while ameliorating two of qPCR's major limitations: the necessity to run standard curves and high susceptibility to inhibition. Here we describe a complete method for simultaneous testing for a general microbial water quality indicator (Enterococcus spp.) and a human-associated fecal marker in environmental waters. The complete method includes water sampling and filtration to capture bacteria, DNA extraction from bacteria captured on the filter, and droplet digital PCR to quantify the genetic markers from bacteria indicative of general and human-associated fecal contamination.

We modeled the risk of gastrointestinal (GI) illness associated with recreational exposures to marine water following storm events in San Diego County, California. We estimated GI illness risks via quantitative microbial risk assessment (QMRA) techniques by consolidating site specific pathogen monitoring data of stormwater, site specific dilution estimates, literature-based water ingestion data, and literature based pathogen dose-response and morbidity information. Our water quality results indicated that human sources of contamination contribute viral and bacterial pathogens to streams draining an urban watershed during wet weather that then enter the ocean and affect nearshore water quality. We evaluated a series of approaches to account for uncertainty in the norovirus dose-response model selection and compared our model results to those from a concurrently conducted epidemiological study that provided empirical estimates for illness risk following ocean exposure. The preferred norovirus dose-response approach yielded median risk estimates for water recreation-associated illness (15 GI illnesses per 1000 recreation events) that closely matched the reported epidemiological results (12 excess GI illnesses per 1000 wet weather recreation events). The results are consistent with norovirus, or other pathogens associated with norovirus, as an important cause of gastrointestinal illness among surfers in this setting. This study demonstrates the applicability of QMRA for recreational water risk estimation, even under wet weather conditions and describes a process that might be useful in developing site-specific water quality criteria in this and other locations., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Coliphages have been proposed as indicators of fecal contamination in recreational waters because they better mimic the persistence of pathogenic viruses in the environment and wastewater treatment than fecal indicator bacteria. We estimated the association between coliphages and gastrointestinal illness and compared it with the association with culturable enterococci., Methods: We pooled data from six prospective cohort studies that enrolled coastal beachgoers in California, Alabama, and Rhode Island. Water samples were collected and gastrointestinal illness within 10 days of the beach visit was recorded. Samples were tested for enterococci and male-specific and somatic coliphages. We estimated cumulative incidence ratios (CIR) for the association between swimming in water with detectable coliphage and gastrointestinal illness when human fecal pollution was likely present, not likely present, and under all conditions combined. The reference group was unexposed swimmers. We defined continuous and threshold-based exposures (coliphage present/absent, enterococci >35 vs. ≤35 CFU/100 ml)., Results: Under all conditions combined, there was no association between gastrointestinal illness and swimming in water with detectable coliphage or enterococci. When human fecal pollution was likely present, coliphage and enterococci were associated with increased gastrointestinal illness, and there was an association between male-specific coliphage level and illness that was somewhat stronger than the association between enterococci and illness. There were no substantial differences between male-specific and somatic coliphage., Conclusions: Somatic coliphage and enterococci had similar associations with gastrointestinal illness; there was some evidence that male-specific coliphage had a stronger association with illness than enterococci in marine waters with human fecal contamination.

Objectives: To provide summary estimates of gastroenteritis risks and illness burden associated with recreational water exposure and determine whether children have higher risks and burden., Methods: We combined individual participant data from 13 prospective cohorts at marine and freshwater beaches throughout the United States (n = 84 411). We measured incident outcomes within 10 days of exposure: diarrhea, gastrointestinal illness, missed daily activity (work, school, vacation), and medical visits. We estimated the relationship between outcomes and 2 exposures: body immersion swimming and Enterococcus spp. fecal indicator bacteria levels in the water. We also estimated the population-attributable risk associated with these exposures., Results: Water exposure accounted for 21% of diarrhea episodes and 9% of missed daily activities but was unassociated with gastroenteritis leading to medical consultation. Children aged 0 to 4 and 5 to 10 years had the most water exposure, exhibited stronger associations between levels of water quality and illness, and accounted for the largest attributable illness burden., Conclusions: The higher gastroenteritis risk and associated burden in young children presents important new information to inform future recreational water quality guidelines designed to protect public health.

Introduction: Advances in molecular methods provide new opportunities for directly measuring pathogens or host-associated markers of fecal pollution instead of relying on fecal indicator bacteria (FIB) alone for beach water quality monitoring. Adoption of new indicators depends on identifying relationships between either the presence or concentration of the indicators and illness among swimmers. Here we present results from three epidemiologic studies in which a broad range of bacterial and viral indicators of fecal contamination were measured simultaneously by either culture or molecular methods along with Enterococcus to assess whether they provide better health risk prediction than current microbial indicators of recreational water quality., Methods: We conducted prospective cohort studies at three California beaches -- Avalon Bay (Avalon), Doheny State Beach (Doheny), Surfrider State Beach (Malibu) -- during the summers of 2007, 2008 and 2009. The studies enrolled 10,785 swimmers across the beaches and recorded each swimmer's water exposure. Water and sand samples were collected several times per day at multiple locations at each beach and analyzed for up to 41 target indicators using 67 different methodologies. Interviewers contacted participants by phone 10-14 days later and recorded symptoms of gastrointestinal illness occurring after their beach visit. Regression models were used to evaluate the association between water quality indicators and gastrointestinal illness among swimmers at each beach., Results: F+ coliphage (measured using EPA Method 1602) exhibited a stronger association with GI illness than did EPA Method 1600 at the two beaches where it was measured, while a molecular method, F+ RNA Coliphage Genotype II, was the only indicator significantly associated with GI illness at Malibu. MRSA, a known pathogen, had the strongest association with GI illness of any microbe measured at Avalon. There were two methods targeting human-associated fecal anaerobic bacteria that were more strongly associated with GI illness than EPA Method 1600, but only at Avalon. No indicator combinations consistently had a higher odds ratio than EPA Method 1600, but one composite indicator, based on the number of pathogens detected at a beach, was significantly associated with gastrointestinal illness at both Avalon and Doheny when freshwater flow was high., Discussion: While EPA Method1600 performed adequately at two beaches based on its consistency of association with gastrointestinal illness and the precision of its estimated associations, F+ coliphage measured by EPA Method 1602 had a stronger association with GI illness under high risk conditions at the two beaches where it was measured. One indicator, F+ Coliphage Genotype II was the only indicator significantly associated with GI illness at Malibu. Several indicators, particularly those targeting human associated bacteria, exhibited relationships with GI illness that were equal to or greater than that of EPA Method 1600 at Avalon, which has a focused human fecal source. Our results suggest that site-specific conditions at each beach determine which indicator or indicators best predict GI illness., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

This manuscript describes a duplex digital PCR assay (EntHF183 dPCR) for simultaneous quantification of Enterococcus spp. and the human fecal-associated HF183 marker. The EntHF183 duplex dPCR (referred as EntHF183 dPCR hereon) assay uses the same primer and probe sequences as its published individual quantitative PCR (qPCR) counterparts. Likewise, the same water filtration and DNA extraction procedures as performed prior to qPCR are followed prior to running dPCR. However, the duplex dPCR assay has several advantages over the qPCR assays. Most important, the dPCR assay eliminates the need for running a standard curve and hence, the associated bias and variability, by direct quantification of its targets. In addition, while duplexing (i.e., simultaneous quantification) Enterococcus and HF183 in qPCR often leads to severe underestimation of the less abundant target in a sample, dPCR provides consistent quantification of both targets, whether quantified individually or simultaneously in the same reaction. The dPCR assay is also able to tolerate PCR inhibitor concentrations that are one to two orders of magnitude higher than those tolerated by qPCR. These advantages make the EntHF183 dPCR assay particularly attractive because it simultaneously provides accurate and repeatable information on both general and human-associated fecal contamination in environmental waters without the need to run two separate qPCR assays. Despite its advantages over qPCR, the upper quantification limit of the dPCR assay with currently available instrumentation is approximately four orders of magnitude lower than that achievable by qPCR. Consequently, dilution is needed for measurement of high concentrations of target organisms such as those typically observed following sewage spills.

Waterborne enteric viruses may pose disease risks to bather health but occurrence of these viruses has been difficult to characterize at recreational beaches. The aim of this study was to evaluate water for human virus occurrence at two Southern California recreational beaches with a history of beach closures. Human enteric viruses (adenovirus and norovirus) and viral indicators (F+ and somatic coliphages) were measured in water samples over a 4-month period from Avalon Beach, Catalina Island (n = 324) and Doheny Beach, Orange County (n = 112). Human viruses were concentrated from 40 L samples and detected by nested reverse transcriptase polymerase chain reaction (PCR). Detection frequencies at Doheny Beach were 25.5% (adenovirus) and 22.3% (norovirus), and at Avalon Beach were 9.3% (adenovirus) and 0.7% (norovirus). Positive associations between adenoviruses and fecal coliforms were observed at Doheny (p = 0.02) and Avalon (p = 0.01) Beaches. Human viruses were present at both beaches at higher frequencies than previously detected in the region, suggesting that the virus detection methods presented here may better measure potential health risks to bathers. These virus recovery, concentration, and molecular detection methods are advancing practices so that analysis of enteric viruses can become more effective and routine for recreational water quality monitoring.

Aims: To determine the extent to which discrepancies between qPCR and culture-based results in beach water quality monitoring can be attributed to: (i) within-method variability, (ii) between-method difference within each method class (qPCR or culture) and (iii) between-class difference., Methods and Results: We analysed 306 samples using two culture-based (EPA1600 and Enterolert) and two qPCR (Taqman and Scorpion) methods, each in duplicate. Both qPCR methods correlated with EPA1600, but regression analyses indicated approximately 0·8 log10 unit overestimation by qPCR compared to culture methods. Differences between methods within a class were less than half of this and were minimal for between-replicate within a method. Using the 104 Enterococcus per 100 ml management decision threshold, Taqman qPCR indicated the same decisions as EPA1600 for 87% of the samples, but indicated beach posting for unhealthful water when EPA1600 did not for 12% of the samples. After accounting for within-method and within-class variability, 8% of the samples exhibited true between-class discrepancy where both qPCR methods indicated beach posting while both culture methods did not., Conclusion: Measurement target difference (DNA vs growth) accounted for the majority of the qPCR-vs-culture discrepancy, but its influence on monitoring application is outweighed by frequent incorrect posting with culture methods due to incubation time delay., Significance and Impact of the Study: This is the first study to quantify the frequency with which culture-vs-qPCR discrepancies can be attributed to target difference - vs - method variability., (© 2013 The Society for Applied Microbiology.)

The State of California has mandated the preparation of a guidance document on the application of fecal source identification methods for recreational water quality management. California contains the fifth highest population of cattle in the United States, making the inclusion of cow-associated methods a logical choice. Because the performance of these methods has been shown to change based on geography and/or local animal feeding practices, laboratory comparisons are needed to determine which assays are best suited for implementation. We describe the performance characterization of two end-point PCR assays (CF128 and CF193) and five real-time quantitative PCR (qPCR) assays (Rum2Bac, BacR, BacCow, CowM2, and CowM3) reported to be associated with either ruminant or cattle feces. Each assay was tested against a blinded set of 38 reference challenge filters (19 duplicate samples) containing fecal pollution from 12 different sources suspected to impact water quality. The abundance of each host-associated genetic marker was measured for qPCR-based assays in both target and non-target animals and compared to quantities of total DNA mass, wet mass of fecal material, as well as Bacteroidales, and enterococci determined by 16S rRNA qPCR and culture-based approaches (enterococci only). Ruminant- and cow-associated genetic markers were detected in all filters containing a cattle fecal source. However, some assays cross-reacted with non-target pollution sources. A large amount of variability was evident across laboratories when protocols were not fixed suggesting that protocol standardization will be necessary for widespread implementation. Finally, performance metrics indicate that the cattle-associated CowM2 qPCR method combined with either the BacR or Rum2Bac ruminant-associated methods are most suitable for implementation., (Published by Elsevier Ltd.)

Microbial source tracking (MST) methods were evaluated in the Source Identification Protocol Project (SIPP), in which 27 laboratories compared methods to identify host sources of fecal pollution from blinded water samples containing either one or two different fecal types collected from California. This paper details lessons learned from the SIPP study and makes recommendations to further advance the field of MST. Overall, results from the SIPP study demonstrated that methods are available that can correctly identify whether particular host sources including humans, cows and birds have contributed to contamination in a body of water. However, differences between laboratory protocols and data processing affected results and complicated interpretation of MST method performance in some cases. This was an issue particularly for samples that tested positive (non-zero Ct values) but below the limits of quantification or detection of a PCR assay. Although false positives were observed, such samples in the SIPP study often contained the fecal pollution source that was being targeted, i.e., the samples were true positives. Given these results, and the fact that MST often requires detection of targets present in low concentrations, we propose that such samples be reported and identified in a unique category to facilitate data analysis and method comparisons. Important data can be lost when such samples are simply reported as positive or negative. Actionable thresholds were not derived in the SIPP study due to limitations that included geographic scope, age of samples, and difficulties interpreting low concentrations of target in environmental samples. Nevertheless, the results of the study support the use of MST for water management, especially to prioritize impaired waters in need of remediation. Future integration of MST data into quantitative microbial risk assessments and other models could allow managers to more efficiently protect public health based on site conditions., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

An inter-laboratory study of the accuracy of microbial source tracking (MST) methods was conducted using challenge fecal and sewage samples that were spiked into artificial freshwater and provided as unknowns (blind test samples) to the laboratories. The results of the Source Identification Protocol Project (SIPP) are presented in a series of papers that cover 41 MST methods. This contribution details the results of the virus and bacteriophage methods targeting human fecal or sewage contamination. Human viruses used as source identifiers included adenoviruses (HAdV), enteroviruses (EV), norovirus Groups I and II (NoVI and NoVII), and polyomaviruses (HPyVs). Bacteriophages were also employed, including somatic coliphages and F-specific RNA bacteriophages (FRNAPH) as general indicators of fecal contamination. Bacteriophage methods targeting human fecal sources included genotyping of FRNAPH isolates and plaque formation on bacterial hosts Enterococcus faecium MB-55, Bacteroides HB-73 and Bacteroides GB-124. The use of small sample volumes (≤50 ml) resulted in relatively insensitive theoretical limits of detection (10-50 gene copies or plaques × 50 ml(-1)) which, coupled with low virus concentrations in samples, resulted in high false-negative rates, low sensitivity, and low negative predictive values. On the other hand, the specificity of the human virus methods was generally close to 100% and positive predictive values were ∼40-70% with the exception of NoVs, which were not detected. The bacteriophage methods were generally much less specific toward human sewage than virus methods, although FRNAPH II genotyping was relatively successful, with 18% sensitivity and 85% specificity. While the specificity of the human virus methods engenders great confidence in a positive result, better concentration methods and larger sample volumes must be utilized for greater accuracy of negative results, i.e. the prediction that a human contamination source is absent., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The contribution of fecal pollution from dogs in urbanized areas can be significant and is an often underestimated problem. Microbial source tracking methods (MST) utilizing quantitative PCR of dog-associated gene sequences encoding 16S rRNA of Bacteroidales are a useful tool to estimate these contributions. However, data about the performance of available assays are scarce. The results of a multi-laboratory study testing two assays for the determination of dog-associated Bacteroidales (DogBact and BacCan-UCD) on 64 single and mixed fecal source samples created from pooled fecal samples collected in California are presented here. Standardization of qPCR data treatment lowered inter-laboratory variability of sensitivity and specificity results. Both assays exhibited 100% sensitivity. Normalization methods are presented that eliminated random and confirmed non-target responses. The combination of standardized qPCR data treatment, use of normalization via a non-target specific Bacteroidales assay (GenBac3), and application of threshold criteria improved the calculated specificity significantly for both assays. Such measures would reasonably improve MST data interpretation not only for canine-associated assays, but for all qPCR assays used in identifying and monitoring fecal pollution in the environment., (Copyright © 2013. Published by Elsevier Ltd.)

A number of PCR-based methods for detecting human fecal material in environmental waters have been developed over the past decade, but these methods have rarely received independent comparative testing in large multi-laboratory studies. Here, we evaluated ten of these methods (BacH, BacHum-UCD, Bacteroides thetaiotaomicron (BtH), BsteriF1, gyrB, HF183 endpoint, HF183 SYBR, HF183 Taqman(®), HumM2, and Methanobrevibacter smithii nifH (Mnif)) using 64 blind samples prepared in one laboratory. The blind samples contained either one or two fecal sources from human, wastewater or non-human sources. The assay results were assessed for presence/absence of the human markers and also quantitatively while varying the following: 1) classification of samples that were detected but not quantifiable (DNQ) as positive or negative; 2) reference fecal sample concentration unit of measure (such as culturable indicator bacteria, wet mass, total DNA, etc); and 3) human fecal source type (stool, sewage or septage). Assay performance using presence/absence metrics was found to depend on the classification of DNQ samples. The assays that performed best quantitatively varied based on the fecal concentration unit of measure and laboratory protocol. All methods were consistently more sensitive to human stools compared to sewage or septage in both the presence/absence and quantitative analysis. Overall, HF183 Taqman(®) was found to be the most effective marker of human fecal contamination in this California-based study., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Molecular microbial community analyses provide information on thousands of microorganisms simultaneously, and integrate biotic and abiotic perturbations caused by fecal contamination entering water bodies. A few studies have explored community methods as emerging approaches for microbial source tracking (MST), however, an evaluation of the current state of this approach is lacking. Here, we utilized three types of community-based methods with 64 blind, single- or dual-source, challenge samples generated from 12 sources, including: humans (feces), sewage, septage, dogs, pigs, deer, horses, cows, chickens, gulls, pigeons, and geese. Each source was a composite from multiple donors from four representative geographical regions in California. Methods evaluated included terminal restriction fragment polymorphism (TRFLP), phylogenetic microarray (PhyloChip), and next generation (Illumina) sequencing. These methods correctly identified dominant (or sole) sources in over 90% of the challenge samples, and exhibited excellent specificity regardless of source, rarely detecting a source that was not present in the challenge sample. Sensitivity, however, varied with source and community analysis method. All three methods distinguished septage from human feces and sewage, and identified deer and horse with 100% sensitivity and 100% specificity. Method performance improved if the composition of blind dual-source reference samples were defined by DNA contribution of each single source within the mixture, instead of by Enterococcus colony forming units. Data analysis approach also influenced method performance, indicating the need to standardize data interpretation. Overall, results of this study indicate that community analysis methods hold great promise as they may be used to identify any source, and they are particularly useful for sources that currently do not have, and may never have, a source-specific single marker gene., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The last decade has seen development of numerous new microbial source tracking (MST) methodologies, but many of these have been tested in just a few laboratories with a limited number of fecal samples. This method evaluation study examined the specificity and sensitivity of 41 MST methodologies by analyzing data generated in 27 laboratories. MST methodologies that targeted human, cow, ruminant, dog, gull, pig, horse, and sheep were tested against sewage, septage, human, cow, dog, deer, pig, chicken, pigeon, gull, horse, and goose fecal samples. Each laboratory received 64 blind samples containing a single source (singletons) or two sources (doubletons), as well as diluted singleton samples to assess method sensitivity. Laboratories utilized their own protocols when performing the methods and data were deposited in a central database before samples were unblinded. Between one and seven laboratories tested each method. The most sensitive and specific assays, based on an analysis of presence/absence of each marker in target and non-target fecal samples, were HF183 endpoint and HF183SYBR (human), CF193 and Rum2Bac (ruminant), CowM2 and CowM3 (cow), BacCan (dog), Gull2SYBR and LeeSeaGull (gull), PF163 and pigmtDNA (pig), HoF597 (horse), PhyloChip (pig, horse, chicken, deer), Universal 16S TRFLP (deer), and Bacteroidales 16S TRFLP (pig, horse, chicken, deer); all had sensitivity and specificity higher than 80% in all or the majority of laboratories. When the abundance of MST markers in target and non-target fecal samples was examined, some assays that performed well in the binary analysis were found to not be sensitive enough as median concentrations fell below a minimum abundance criterion (set at 50 copies per colony forming units of enterococci) in target fecal samples. Similarly, some assays that cross-reacted with non-target fecal sources in the binary analysis were found to perform well in a quantitative analysis because the cross-reaction occurred at very low levels. Based on a quantitative analysis, the best performing methods were HF183Taqman and BacH (human), Rum2Bac and BacR (ruminant), LeeSeaGull (gull), and Pig2Bac (pig); no cow or dog-specific assay met the quantitative specificity and sensitivity criteria. Some of the best performing assays in the study were run by just one laboratory so further testing of assay portability is needed. While this study evaluated the marker performance in defined samples, further field testing as well as development of frameworks for fecal source allocation and risk assessment are needed., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Many PCR-based methods for microbial source tracking (MST) have been developed and validated within individual research laboratories. Inter-laboratory validation of these methods, however, has been minimal, and the effects of protocol standardization regimes have not been thoroughly evaluated. Knowledge of factors influencing PCR in different laboratories is vital to future technology transfer for use of MST methods as a tool for water quality management. In this study, a blinded set of 64 filters (containing 32 duplicate samples generated from 12 composite fecal sources) were analyzed by three to five core laboratories with a suite of PCR-based methods utilizing standardized reagents and protocols. Repeatability (intra-laboratory variability) and reproducibility (inter-laboratory variability) of observed results were assessed. When standardized methodologies were used, intra- and inter-laboratory %CVs were generally low (median %CV 0.1-3.3% and 1.9-7.1%, respectively) and comparable to those observed in similar inter-laboratory validation studies performed on other methods of quantifying fecal indicator bacteria (FIB) in environmental samples. ANOVA of %CV values found three human-associated methods (BsteriF1, BacHum, and HF183Taqman) to be similarly reproducible (p > 0.05) and significantly more reproducible (p < 0.05) than HumM2. This was attributed to the increased variability associated with low target concentrations detected by HumM2 (approximately 1-2 log10copies/filter lower) compared to other human-associated methods. Cow-associated methods (BacCow and CowM2) were similarly reproducible (p > 0.05). When using standardized protocols, variance component analysis indicated sample type (fecal source and concentration) to be the major contributor to total variability with that from replicate filters and inter-laboratory analysis to be within the same order of magnitude but larger than inherent intra-laboratory variability. However, when reagents and protocols were not standardized, inter-laboratory %CV generally increased with a corresponding decline in reproducibility. Overall, these findings verify the repeatability and reproducibility of these MST methods and highlight the need for standardization of protocols and consumables prior to implementation of larger scale MST studies involving multiple laboratories., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Studies of health risks associated with recreational water exposure require investigators to make choices about water quality indicator averaging techniques, exposure definitions, follow-up periods, and model specifications; however, investigators seldom describe the impact of these choices on reported results. Our objectives are to report illness risk from swimming at a marine beach affected by nonpoint sources of urban runoff, measure associations between fecal indicator bacteria levels and subsequent illness among swimmers, and investigate the sensitivity of results to a range of exposure and outcome definitions., Methods: In 2009, we enrolled 5674 people in a prospective cohort at Malibu Beach, a coastal marine beach in California, and measured daily health symptoms 10-19 days later. Concurrent water quality samples were analyzed for indicator bacteria using culture and molecular methods. We compared illness risk between nonswimmers and swimmers, and among swimmers exposed to various levels of fecal indicator bacteria., Results: Diarrhea was more common among swimmers than nonswimmers (adjusted odds ratio = 1.88 [95% confidence interval = 1.09-3.24]) within 3 days of the beach visit. Water quality was generally good (fecal indicator bacteria levels exceeded water quality guidelines for only 7% of study samples). Fecal indicator bacteria levels were not consistently associated with swimmer illness. Sensitivity analyses demonstrated that overall inference was not substantially affected by the choice of exposure and outcome definitions., Conclusions: This study suggests that the 3 days following a beach visit may be the most relevant period for health outcome measurement in recreational water studies. Under the water quality conditions observed in this study, fecal indicator bacteria levels were not associated with swimmer illness.

Aims:   Draft criteria for the optional use of qPCR for recreational water quality monitoring have been published in the United States. One concern is that inhibition of the qPCR assay can lead to false-negative results and potentially inadequate public health protection. We evaluate the effectiveness of strategies for minimizing the impact of inhibition., Methods and Results:   Five qPCR method permutations for measuring Enterococcus were challenged with 133 potentially inhibitory fresh and marine water samples. Serial dilutions were conducted to assess Enterococcus target assay inhibition, to which inhibition identified using four internal controls (IC) was compared. The frequency and magnitude of inhibition varied considerably among qPCR methods, with the permutation using an environmental master mix performing substantially better. Fivefold dilution was also effective at reducing inhibition in most samples (>78%). ICs were variable and somewhat ineffective, with 54-85% agreement between ICs and serial dilution., Conclusions:   The current IC methods appear to not accurately predict Enterococcus inhibition and should be used with caution; fivefold dilution and the use of reagents designed for environmental sample analysis (i.e. more robust qPCR chemistry) may be preferable., Significance and Impact of the Study:   Suitable approaches for defining, detecting and reducing inhibition will improve implementation of qPCR for water monitoring., (© 2012 The Authors. Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.)

Norovirus is associated commonly with human sewage and is responsible for numerous cases of waterborne and foodborne gastroenteritis every year. Assays using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) have been developed for norovirus, however, accurate detection and quantitation remain problematic owing to a lack of suitable positive controls. To improve enumeration of norovirus genomes from water, a synthetic norovirus genogroup II quantitation standard and competitive internal positive control were developed. The quantitation standard demonstrates identical amplification efficiency as wildtype norovirus and can be used as a viral surrogate in labs with restricted access to norovirus. The internal control quantifies sample inhibition, allowing for accurate quantitation of norovirus from complex environmental samples. Seawater samples spiked with sewage or bird guano were evaluated using the norovirus assay as part of a methods comparison study. Inhibition was detected in nine of 36 (25%) samples, two of which proved to be positive upon re-analysis. Results support the specificity of this assay for human-source (sewage) fecal contamination. Overall, use of this quantitation standard and internal control signify a great advance over traditional positive controls and suggest that molecular techniques for viral analysis could become standardized for routine water quality monitoring., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Recreational water quality is currently monitored using culture-based methods that require 18 to 96 h for results. Quantitative PCR (QPCR) methods that can be completed in less than 2 h have been developed, but they could yield different results than the conventional methods. We present two studies in which samples were processed simultaneously for Enterococcus spp. and Escherichia coli using two culture-based methods (EPA method 1600 and Enterolert/Colilert-18) and QPCR. The proprietary QPCR assays targeted the 23S rRNA (Enterococcus spp.) and uidA (E. coli) genes and were conducted using lyophilized beads containing all reagents. In the first study, the QPCR method developers processed 54 blind samples that were inoculated with sewage or pure cultures or were ambient beach samples. The second study involved 163 samples processed by water quality personnel. The correlation between results of QPCR and EPA 1600 during the first study (r²) was 0.69 for Enterococcus spp., which was less than that observed between the culture-based methods (r², 0.87). During the second study, the correlations were similar. No false positives occurred in either study when QPCR-based assays were used with blank samples. Levels of reproducibility measured through coefficients of variation were similar for results by Enterococcus QPCR and culture-based methods during both studies but were higher for E. coli QPCR results in the first study. Regarding the concentration at which beach management decisions are issued in the State of California, the agreement between results of Enterococcus QPCR and EPA method 1600 was 88%, compared to 94% agreement between EPA method 1600 and Enterolert. The beach management decision agreement between E. coli QPCR and Colilert-18 was 94%. The samples showing disagreement suggested an underestimation bias for QPCR.

Contamination of recreational waters with Escherichia coli and Enterococcus sp. is a widespread problem resulting in beach closures and loss of recreational activity. While E. coli is frequently used as an indicator of fecal contamination, and has been extensively measured in waterways, few studies have examined the presence of potentially pathogenic E. coli strains in beach waters. In this study, a combination of high-throughput, robot-assisted colony hybridization and PCR-based analyses were used to determine the genomic composition and frequency of virulence genes present in E. coli isolated from beach water in Avalon Bay, Santa Catalina Island, CA. A total of 24,493 E. coli isolates were collected from two sites at a popular swimming beach between August through September 2007 and from July through August 2008. All isolates were examined for the presence of shiga-like toxins (stx1/stx2), intimin (eaeA), and enterotoxins (ST/LT). Of the 24,493 isolates examined, 3.6% contained the eaeA gene, indicating that these isolates were potential EPEC strains. On five dates, however, greater than 10% of the strains were potential EPEC, suggesting that incidence of virulence genes at this beach has a strong temporal component. No STEC or ETEC isolates were detected, and only eight (<1.0%) of the potential EPEC isolates were found to carry the EAF plasmid. The potential EPEC isolates mainly belonged to E. coli phylogenetic groups B1 or B2, and carried the β intimin subtype. DNA fingerprint analyses of the potential EPEC strains indicated that the isolates belonged to several genetically diverse groups, although clonal isolates were frequently detected. While the presence of virulence genes alone cannot be used to determine the pathogenicity of strains, results from this study show that potential EPEC strains can be found in marine beach water and their presence needs to be considered as one of the factors used in decisions concerning beach closures., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Aims: Developing a rapid method for detection of faecal pollution is among the critical goals set forth by the Environmental Protection Agency in its revision of water quality criteria. The purpose of this study is to devise and test covalently linked antibody-bead complexes for faecal indicator bacteria (FIB), specifically Escherichia coli or Enterococcus spp., in measuring water quality in freshwater and marine systems., Methods and Results: Covalently linked complexes were 58-89% more robust than antibody-bead complexes used in previous studies. Freshwater and marine water samples analysed using covalently linked immunomagnetic separation/adenosine triphosphate quantification technique (Cov-IMS/ATP) and culture-based methods yielded good correlations for E. coli (R = 0·87) and Enterococcus spp. (R = 0·94), with method detection limits below EPA recreational water quality health standards for single standard exceedances (E. coli- 38 cells per 100 ml; Enterococcus spp. - 25 cells per 100 ml). Cov-IMS/ATP correctly classified 87% of E. coli and 94% of Enterococcus spp. samples based on these water quality standards. Cov-IMS/ATP was also used as a field method to rapidly distinguish differential loading of E. coli between two stream channels to their confluence., Conclusions: Cov-IMS/ATP is a robust, in-field detection method for determining water quality of both fresh and marine water systems as well as differential loading of FIB from two converging channels., Significance and Impact of the Study: To our knowledge, this is the first work to present a viable rapid, in-field assay for measuring FIB concentrations in marine water environments. Cov-IMS/ATP is a potential alternative detection method, particularly in areas with limited laboratory support and resources, because of its increased economy and portability., (© 2010 The Authors. Journal compilation © 2010 The Society for Applied Microbiology.)

Although urban wet weather discharges may have elevated concentrations of fecal indicator bacteria impacting water quality at swimming beaches, not all of these bacteria may arise from human sources. In this study, the contribution of non-human fecal indicator bacteria was quantified by sampling coastal reference beaches in southern California. Samples were collected at beaches near stormwater discharges from undeveloped watersheds and analyzed for total coliform, Escherichia coli, and enterococci. Surfzone samples exceeded water quality thresholds >10 times more frequently during wet weather than dry weather. Exceedences were greatest <24h following rainfall, then steadily declined on successive days. Early season storms exceeded thresholds more frequently, and by greater magnitude, compared to late season storms. Large storms exceeded thresholds more frequently than smaller-sized storms, partly due to the breaching of sand berms. When discharges did reach the surf zone, bacterial concentrations in the wave wash were correlated with watershed bacterial flux., ((c) 2009 Elsevier Ltd. All rights reserved.)

More than 30 laboratories routinely monitor water along southern California's beaches for bacterial indicators of fecal contamination. Data from these efforts frequently are combined and compared even though three different methods (membrane filtration (MF), multiple tube fermentation (MTF), and chromogenic substrate (CS) methods) are used. To assess data comparability and quantify variability within method and across laboratories, 26 laboratories participated in an intercalibration exercise. Each laboratory processed three replicates from eight ambient water samples employing the method or methods they routinely use for water quality monitoring. Verification analyses also were conducted on a subset of wells from the CS analysis to confirm or exclude the presence of the target organism. Enterococci results were generally comparable across methods. Confirmation revealed a 9% false positive rate and a 4% false negative rate in the CS method for enterococci, though these errors were small in the context of within- and among-laboratory variability. Fecal coliforms also were comparable across all methods, though CS underestimated the other methods by about 10%, probably because it measures only E. coli, rather than the larger fecal coliform group measured by MF and MTF. CS overestimated total coliforms relative to the other methods by several fold and was found to have a 40% false positive rate in verification. Across-laboratory variability was small relative to within- and among-method variability, but only after data entry errors were corrected. One fifth of the laboratories committed data entry errors that were much larger than any method-related errors. These errors are particularly significant because these data were submitted in a test situation where laboratories were aware they would be under increased scrutiny. Under normal circumstances, it is unlikely that these errors would have been detected and managers would have been obliged to issue beach water quality warnings.

The ubiquity of fecal indicator bacteria such as Escherichia coli and Enterococcus spp. in urban environments makes tracking of fecal contamination extremely challenging. A multitiered approach was used to assess sources of fecal pollution in Ballona Creek, an urban watershed that drains to the Santa Monica Bay (SMB) near Los Angeles, Calif. A mass-based design at six main-stem sites and four major tributaries over a 6-h period was used (i) to assess the flux of Enterococcus spp. and E. coli by using culture-based methods (tier 1); (ii) to assess levels of Enterococcus spp. by using quantitative PCR and to detect and/or quantify additional markers of human fecal contamination, including a human-specific Bacteroides sp. marker and enterovirus, using quantitative reverse transcriptase PCR (tier 2); and (iii) to assess the specific types of enterovirus genomes found via sequence analysis (tier 3). Sources of fecal indicator bacteria were ubiquitous, and concentrations were high, throughout Ballona Creek, with no single tributary dominating fecal inputs. The flux of Enterococcus spp. and E. coli averaged 10(9) to 10(10) cells h(-1) and was as high at the head of the watershed as at the mouth prior to discharge into the SMB. In addition, a signal for the human-specific Bacteroides marker was consistently detected: 86% of the samples taken over the extent during the study period tested positive. Enteroviruses were quantifiable in 14 of 36 samples (39%), with the highest concentrations at the site furthest upstream (Cochran). These results indicated the power of using multiple approaches to assess and quantify fecal contamination in freshwater conduits to high-use, high-priority recreational swimming areas.

Microbiological source tracking (MST) methods are increasingly being used to identify fecal contamination sources in surface waters, but these methods have been subjected to limited comparative testing. In this study, 22 researchers employing 12 different methods were provided sets of identically prepared blind water samples. Each sample contained one to three of five possible fecal sources (human, dog, cattle, seagull or sewage). Researchers were also provided with portions of the fecal material used to inoculate the blind water samples for use as library material. No MST method that was tested predicted the source material in the blind samples perfectly. Host-specific PCR performed best at differentiating between human and non-human sources, but primers are not yet available for differentiating between all of the non-human sources. Virus and F+ coliphage methods reliably identified sewage, but were unable to identify fecal contamination from individual humans. Library-based isolate methods correctly identified the dominant source in most samples, but also had frequent false positives in which fecal sources not in the samples were incorrectly identified as being present. Among the library-based methods, genotypic methods generally performed better than phenotypic methods.