Increased disparities in waitlist and post-heart transplantation outcomes according to socioeconomic status with the new heart transplant allocation system.

BACKGROUND: The study objective was to assess disparities in outcomes in the waitlist and post-heart transplantation (HT) according to socioeconomic status (SES) in the old and new U.S. HT allocation systems. METHODS: Adult HT candidates in the United Network for Organ Sharing database from 2014 through 2021 were included. Old or new system classification was according to listing before or after October 18, 2018. SES was stratified by patient ZIP code and median household income via U.S. Census Bureau and classified into terciles. Competing waitlist outcomes and post-transplantation survival were compared between systems. RESULTS: In total, 26,450 patients were included. Waitlisted candidates with low SES were more frequently younger, female, African American, and with higher body mass index. Reduced cumulative incidence (CI) of HT in the old system occurred in low SES (53.5%) compared to middle (55.7%, p = 0.046), and high (57.9%, p < 0.001). In the new system, the CI of HT was 65.3% in the low SES vs middle (67.6%, p = 0.002) and high (70.2%, p < 0.001), and SES remained significant in the adjusted analysis. In the old system, CI of death/delisting was similar across SES. In the new system, low SES had increased CI of death/delisting (7.4%) vs middle (6%, p = 0.012) and high (5.4%, p = 0.002). The old system showed similar 1-year survival across SES. In the new system, recipients with low SES had decreased 1-year survival (p = 0.041). CONCLUSIONS: SES affects waitlist and post-transplant outcomes. In the new system, all SES had increased access to HT; however, low SES had increased death/delisting due to worsening clinical status and decreased post-transplant survival.

Non-Medical Characteristics Affect Referral for Advanced Heart Failure Services: a Retrospective Review.

BACKGROUND: Patients with advanced heart failure (AHF) are extensively evaluated before heart transplantation or left ventricular assist device (LVAD) eligibility. Patients are assessed for medical need and psychosocial or economic factors that may affect success post-treatment. For patients to be evaluated, however, they first must be referred. This study investigated social and economic factors affecting AHF referral, specialist visits, or treatment. METHODS: Patients with heart failure (n = 24,258) were reviewed at one large hospital system over 4 years. Independent variables age, sex, marital status, race/ethnicity, preferred language, smoking, and insurance status were assessed for the outcomes of referral, clinic visit, and treatment by Chi-square and ANOVA. In-house and 1-year mortality were evaluated by logistic regression, and time-to-event was assessed by the Cox proportional hazards model. RESULTS: Younger (HR 0.934, 95% CI 0.925-0.943), male (HR 2.216, 95% CI 1.544-3.181), and publicly insured (HR 1.298 [95% CI 1.038, 1.623]) patients were more likely to be referred, while unmarried (HR 0.665, 95% CI 0.488-0.905) and smoking (HR 0.549, 95% CI 0.389-0.776) patients had fewer referrals. Younger, married, and nonsmoking patients were more likely to have a clinic visit. Younger age, White race, and Hispanic/Latino ethnicity were associated with receiving a heart transplant, and LVAD recipients were more likely Hispanic/Latino ethnicity. Advanced age, Hispanic/Latino ethnicity, and smoking were associated with 1-year mortality after heart failure diagnosis. CONCLUSIONS: Disparities in access exist before evaluation for AHF therapies. Improving access at the levels of referral and evaluation is a necessary step toward achieving equity in organ allocation.

Veno-arterial extracorporeal membrane oxygenation versus standard medical management for massive pulmonary embolism.

PURPOSE: There is limited research on the use and outcomes of veno-arterial extracorporeal membrane oxygenation (VA-ECMO) treatment for massive pulmonary embolism (PE). This study compared VA-ECMO treatment for massive PE versus patients treated medically. MATERIALS AND METHODS: Patients diagnosed with massive PE at one hospital system were reviewed. VA-ECMO and non-ECMO groups were compared by t test and Chi-square. Mortality risk factors were identified by logistic regression. Survival was assessed by Kaplan Meier and propensity matching of groups. RESULTS: Ninety-two patients were included (22 VA-ECMO and 70 non-ECMO). Age (OR 1.08, 95% CI 1.03-1.13), arterial SBP (OR 0.97, 95% CI 0.94-0.99), albumin (OR 0.3, 95% CI 0.1-0.8), and phosphorus (OR 2.0, 95% CI 1.4-3.17) were independently associated with 30-day mortality. Alkaline phosphate (OR 1.03, 95% CI 1.01-1.05) and SOFA score (OR 1.3, 95% CI 1.06-1.51) were associated with 1-year mortality. Propensity matching showed no difference in 30-day (59% VA-ECMO versus 72% non-ECMO, p = 0.363) or 1-year survival (50% VA-ECMO versus 64% non-ECMO, p = 0.355). CONCLUSIONS: Patients treated with VA-ECMO for massive PE and medically treated patients have similar short- and long-term survival. Further research is needed to define clinical recommendations and benefits of intensive therapy such as VA-ECMO in this critically ill population.

Variable fecal source prioritization in recreational waters routinely monitored with viral and bacterial general indicators.

Somatic and F+ coliphage methods are under consideration as potential routine surface water quality monitoring tools to identify unsafe levels of fecal pollution in recreational waters. However, little is known about the cooccurrence of these virus-based fecal indicators and host-associated genetic markers used to prioritize key pollution sources for remediation. In this study, paired measurements of cultivated coliphage (somatic and F+) and bacterial (E. coli and enterococci) general fecal indicators and genetic markers indicative of human (HF183/BacR287 and HumM2), ruminant (Rum2Bac), canine (DG3), and avian (GFD) fecal pollution sources were assessed in 365 water samples collected from six Great Lakes Basin beach and river sites over a 15-week recreational season. Water samples were organized into groups based on defined viral and bacterial fecal indicator water quality thresholds and average log10 host-associated genetic marker fecal score ratios were estimated to compare pollutant source inferences based on variable routine water quality monitoring practices. Eligible log10 fecal score ratios ranged from -0.051 (F+ coliphage, GFD) to 2.08 (enterococci, Rum2Bac). Using a fecal score ratio approach, findings suggest that general fecal indicator selection for routine water quality monitoring can influence the interpretation of host-associated genetic marker measurements, in some cases, prioritizing different pollutant sources for remediation. Variable trends were also observed between Great Lake beach and river sites suggesting disparate management practices may be useful for each water type.

Fecal pollution source characterization at non-point source impacted beaches under dry and wet weather conditions.

Though Lake Michigan beaches in Chicago are not impacted by stormwater or wastewater outfalls, several of those beaches often exceed USEPA Beach Action Values (BAVs). We investigated the role of microbial source tracking (MST) as a complement to routine beach monitoring at Chicago beaches. In summer 2016, water samples from nine Chicago beaches were analyzed for E. coli by culture and enterococci by qPCR. A total of 195 archived samples were then tested for human (HF183/BacR287, HumM2), canine (DG3, DG37), and avian (GFD) microbial source tracking (MST) markers. Associations between MST and general fecal indicator bacteria (FIB) measures were evaluated and stratified based on wet and dry weather definitions. Among the 195 samples, HF183/BacR287 was quantifiable in 4%, HumM2 in 1%, DG3 in 6%, DG37 in 2%, and GFD in 23%. The one beach with a dog area was far more likely to have DG3 present in the quantifiable range than other beaches. Exceedance of general FIB BAVs increased the odds of human, dog and avian marker detection. MST marker weighted-average fecal scores for DG3 was 2.4 times, DG37 was 2.1 times, and GFD was 1.6 times higher during wet compared to dry weather conditions. HF183/BacR287 weighted-average fecal scores were not associated with precipitation. Associations between FIB BAV exceedance and MST marker detection were generally stronger in wet weather. Incorporating MST testing into routine beach water monitoring can provide information that beach managers can use when developing protection plans for beaches not impacted by point sources.

Viral and Bacterial Fecal Indicators in Untreated Wastewater across the Contiguous United States Exhibit Geospatial Trends.

Cultivated fecal indicator bacteria such as Escherichia coli and enterococci are typically used to assess the sanitary quality of recreational waters. However, these indicators suffer from several limitations, such as the length of time needed to obtain results and the fact that they are commensal inhabitants of the gastrointestinal tract of many animals and have fate and transport characteristics dissimilar to pathogenic viruses. Numerous emerging technologies that offer same-day water quality results or pollution source information or that more closely mimic persistence patterns of disease-causing pathogens that may improve water quality management are now available, but data detailing geospatial trends in wastewater across the United States are sparse. We report geospatial trends of cultivated bacteriophage (somatic, F+, and total coliphages and GB-124 phage), as well as genetic markers targeting polyomavirus, enterococci, E. coli, Bacteroidetes, and human-associated Bacteroides spp. (HF183/BacR287 and HumM2) in 49 primary influent sewage samples collected from facilities across the contiguous United States. Samples were selected from rural and urban facilities spanning broad latitude, longitude, elevation, and air temperature gradients by using a geographic information system stratified random site selection procedure. Most indicators in sewage demonstrated a remarkable similarity in concentration regardless of location. However, some exhibited predictable shifts in concentration based on either facility elevation or local air temperature. Geospatial patterns identified in this study, or the absence of such patterns, may have several impacts on the direction of future water quality management research, as well as the selection of alternative metrics to estimate sewage pollution on a national scale.IMPORTANCE This study provides multiple insights to consider for the application of bacterial and viral indicators in sewage to surface water quality monitoring across the contiguous United States, ranging from method selection considerations to future research directions. Systematic testing of a large collection of sewage samples confirmed that crAssphage genetic markers occur at a higher average concentration than key human-associated Bacteroides spp. on a national scale. Geospatial testing also suggested that some methods may be more suitable than others for widespread implementation. Nationwide characterization of indicator geospatial trends in untreated sewage represents an important step toward the validation of these newer methods for future water quality monitoring applications. In addition, the large paired-measurement data set reported here affords the opportunity to conduct a range of secondary analyses, such as the generation of new or updated quantitative microbial risk assessment models used to estimate public health risk.

Large-scale implementation of standardized quantitative real-time PCR fecal source identification procedures in the Tillamook Bay Watershed.

Fecal pollution management remains one of the biggest challenges for water quality authorities worldwide. Advanced fecal pollution source identification technologies are now available that can provide quantitative information from many animal groups. As public interest in these methodologies grows, it is vital to use standardized procedures with clearly defined data acceptance metrics and conduct field studies demonstrating the use of these techniques to help resolve real-world water quality challenges. Here we apply recently standardized human-associated qPCR methods with custom data acceptance metrics (HF183/BacR287 and HumM2), along with established procedures for ruminant (Rum2Bac), cattle (CowM2 and CowM3), canine (DG3 and DG37), and avian (GFD) fecal pollution sources to (i) demonstrate the feasibility of implementing standardized qPCR procedures in a large-scale field study, and (ii) characterize trends in fecal pollution sources in the research area. A total of 602 water samples were collected over a one-year period at 29 sites along the Trask, Kilchis, and Tillamook rivers and tributaries in the Tillamook Bay Watershed (OR, USA). Host-associated qPCR results were combined with high-resolution geographic information system (GIS) land use and general indicator bacteria (E. coli) measurements to elucidate water quality fecal pollution trends. Results demonstrate the feasibility of implementing standardized fecal source identification qPCR methods with established data acceptance metrics in a large-scale field study leading to new investigative leads suggesting that elevated E. coli levels may be linked to specific pollution sources and land use activities in the Tillamook Bay Watershed.

Incidence of somatic and F+ coliphage in Great Lake Basin recreational waters.

There is a growing interest for the use of coliphage as an alternative indicator to assess fecal pollution in recreational waters. Coliphage are a group of viruses that infect Escherichia coli and are considered as potential surrogates to infer the likely presence of enteric viral pathogens. We report the use of a dead-end hollow fiber ultrafiltration single agar layer method to enumerate F+ and somatic coliphage from surface waters collected from three Great Lake areas. At each location, three sites (two beaches; one river) were sampled five days a week over the 2015 beach season (n = 609 total samples). In addition, culturable E. coli and enterococci concentrations, as well as 16 water quality and recreational area parameters were assessed such as rainfall, turbidity, dissolved oxygen, pH, and ultra violet absorbance. Overall, somatic coliphage levels ranged from non-detectable to 4.39 log10 plaque forming units per liter and were consistently higher compared to F+ (non-detectable to 3.15 log10 PFU/L), regardless of sampling site. Coliphage concentrations weakly correlated with cultivated fecal indicator bacteria levels (E. coli and enterococci) at 75% of beach sites tested in study (r = 0.28 to 0.40). In addition, ultraviolet light absorption and water temperature were closely associated with coliphage concentrations, but not fecal indicator bacteria levels suggesting different persistence trends in Great Lake waters between indicator types (bacteria versus virus). Finally, implications for coliphage water quality management and future research directions are discussed.

Evidence of Genetic Fecal Marker Interactions between Water Column and Periphyton in Artificial Streams.

Periphyton is a complex mixture of algae, microbes, inorganic sediment, and organic matter that is attached to submerged surfaces in most flowing freshwater systems. This natural community is known to absorb pollutants from the water column, resulting in improved water quality. However, the role of periphyton in the fate and transport of genetic fecal markers suspended in the water column remains unclear. As application of genetic-based methodologies continues to increase in freshwater settings, it is important to identify any interactions that could potentially confound water quality interpretations. A 16 week indoor mesocosm study was conducted to simultaneously measure genetic fecal markers in the water column and in the associated periphyton when subject to wastewater source loading. Treated wastewater effluent was pumped directly from a treatment facility adjacent to the experimental stream facility. Inflow and outflow surface water grabs were paired with the collection of periphyton samples taken from the mesocosm substrates on a weekly basis. Samples were analyzed with three genetic fecal indicator quantitative real-time polymerase chain reaction assays targeting Escherichia coli (EC23S857), enterococci (Entero1), and Bacteroidales (GenBac3), as well as, two human host-associated fecal pollution markers (HF183 and HumM2). In addition, periphyton dry mass was measured. During wastewater effluent loading, genetic markers were detected in periphyton at frequencies up to 100% (EC23S857, Entero1, and GenBac3), 59.4% (HF183), and 21.9% (HumM2) confirming sequestration from the water column. Mean net-flux shifts in water column inflow and outflow genetic indicator concentrations further supported interactions between the periphyton and water column. In addition, positive correlations were observed between periphyton dry mass and genetic marker concentrations ranging from r = 0.693 (Entero1) to r = 0.911 (GenBac3). Overall, findings support the notion that genetic markers suspended in the water column can be trapped by periphyton, further suggesting that the benthic environment in flowing freshwater systems may be an important factor to consider for water quality management with molecular methods.

A human fecal contamination score for ranking recreational sites using the HF183/BacR287 quantitative real-time PCR method.

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.

Quantitative CrAssphage PCR Assays for Human Fecal Pollution Measurement.

Environmental waters are monitored for fecal pollution to protect public health and water resources. Traditionally, general fecal-indicator bacteria are used; however, they cannot distinguish human fecal waste from other animal pollution sources. Recently, a novel bacteriophage, crAssphage, was discovered by metagenomic data mining and reported to be abundant in and closely associated with human fecal waste. To confirm bioinformatic predictions, 384 primer sets were designed along the length of the crAssphage genome. Based on initial screening, two novel crAssphage qPCR assays (CPQ_056 and CPQ_064) were designed and evaluated in reference fecal samples and water matrices. The assays exhibited high specificities (98.6%) when tested against an animal fecal reference library, and crAssphage genetic markers were highly abundant in raw sewage and sewage-impacted water samples. In addition, CPQ_056 and CPQ_064 performance was compared to HF183/BacR287 and HumM2 assays in paired experiments. Findings confirm that viral crAssphage qPCR assays perform at a similar level to well-established bacterial human-associated fecal-source-identification approaches. These new viral-based assays could become important water quality management and research tools.

Integrated preservation and sample clean up procedures for studying water ingestion by recreational swimmers via urinary biomarker determination.

The use of cyanuric acid as a biomarker for ingestion of swimming pool water may lead to quantitative knowledge of the volume of water ingested during swimming, contributing to a better understanding of disease resulting from ingestion of environmental contaminants. When swimming pool water containing chlorinated cyanurates is inadvertently ingested, cyanuric acid is excreted quantitatively within 24 h as a urinary biomarker of ingestion. Because the volume of water ingested can be quantitatively estimated by calculation from the concentration of cyanuric acid in 24 h urine samples, a procedure for preservation, cleanup, and analysis of cyanuric acid was developed to meet the logistical demands of large scale studies. From a practical stand point, urine collected from swimmers cannot be analyzed immediately, given requirements of sample collection, shipping, handling, etc. Thus, to maintain quality control to allow confidence in the results, it is necessary to preserve the samples in a manner that ensures as quantitative analysis as possible. The preservation and clean-up of cyanuric acid in urine is complicated because typical approaches often are incompatible with the keto-enol tautomerization of cyanuric acid, interfering with cyanuric acid sample preparation, chromatography, and detection. Therefore, this paper presents a novel integration of sample preservation, clean-up, chromatography, and detection to determine cyanuric acid in 24 h urine samples. Fortification of urine with cyanuric acid (0.3-3.0 mg/L) demonstrated accuracy (86-93% recovery) and high reproducibility (RSD < 7%). Holding time studies in unpreserved urine suggested sufficient cyanuric acid stability for sample collection procedures, while longer holding times suggested instability of the unpreserved urine. Preserved urine exhibited a loss of around 0.5% after 22 days at refrigerated storage conditions of 4 °C.

Differential decomposition of bacterial and viral fecal indicators in common human pollution types.

Understanding the decomposition of microorganisms associated with different human fecal pollution types is necessary for proper implementation of many water quality management practices, as well as predicting associated public health risks. Here, the decomposition of select cultivated and molecular indicators of fecal pollution originating from fresh human feces, septage, and primary effluent sewage in a subtropical marine environment was assessed over a six day period with an emphasis on the influence of ambient sunlight and indigenous microbiota. Ambient water mixed with each fecal pollution type was placed in dialysis bags and incubated in situ in a submersible aquatic mesocosm. Genetic and cultivated fecal indicators including fecal indicator bacteria (enterococci, E. coli, and Bacteroidales), coliphage (somatic and F+), Bacteroides fragilis phage (GB-124), and human-associated genetic indicators (HF183/BacR287 and HumM2) were measured in each sample. Simple linear regression assessing treatment trends in each pollution type over time showed significant decay (p ≤ 0.05) in most treatments for feces and sewage (27/28 and 32/40, respectively), compared to septage (6/26). A two-way analysis of variance of log10 reduction values for sewage and feces experiments indicated that treatments differentially impact survival of cultivated bacteria, cultivated phage, and genetic indicators. Findings suggest that sunlight is critical for phage decay, and indigenous microbiota play a lesser role. For bacterial cultivated and genetic indicators, the influence of indigenous microbiota varied by pollution type. This study offers new insights on the decomposition of common human fecal pollution types in a subtropical marine environment with important implications for water quality management applications.

Data Acceptance Criteria for Standardized Human-Associated Fecal Source Identification Quantitative Real-Time PCR Methods.

There is growing interest in the application of human-associated fecal source identification quantitative real-time PCR (qPCR) technologies for water quality management. The transition from a research tool to a standardized protocol requires a high degree of confidence in data quality across laboratories. Data quality is typically determined through a series of specifications that ensure good experimental practice and the absence of bias in the results due to DNA isolation and amplification interferences. However, there is currently a lack of consensus on how best to evaluate and interpret human fecal source identification qPCR experiments. This is, in part, due to the lack of standardized protocols and information on interlaboratory variability under conditions for data acceptance. The aim of this study is to provide users and reviewers with a complete series of conditions for data acceptance derived from a multiple laboratory data set using standardized procedures. To establish these benchmarks, data from HF183/BacR287 and HumM2 human-associated qPCR methods were generated across 14 laboratories. Each laboratory followed a standardized protocol utilizing the same lot of reference DNA materials, DNA isolation kits, amplification reagents, and test samples to generate comparable data. After removal of outliers, a nested analysis of variance (ANOVA) was used to establish proficiency metrics that include lab-to-lab, replicate testing within a lab, and random error for amplification inhibition and sample processing controls. Other data acceptance measurements included extraneous DNA contamination assessments (no-template and extraction blank controls) and calibration model performance (correlation coefficient, amplification efficiency, and lower limit of quantification). To demonstrate the implementation of the proposed standardized protocols and data acceptance criteria, comparable data from two additional laboratories were reviewed. The data acceptance criteria proposed in this study should help scientists, managers, reviewers, and the public evaluate the technical quality of future findings against an established benchmark.

Occurrence of Host-Associated Fecal Markers on Child Hands, Household Soil, and Drinking Water in Rural Bangladeshi Households.

We evaluated whether provision and promotion of improved sanitation hardware (toilets and child feces management tools) reduced rotavirus and human fecal contamination of drinking water, child hands, and soil among rural Bangladeshi compounds enrolled in a cluster-randomized trial. We also measured host-associated genetic markers of ruminant and avian feces. We found evidence of widespread ruminant and avian fecal contamination in the compound environment; non-human fecal marker occurrence scaled with animal ownership. Strategies for controlling non- human fecal waste should be considered when designing interventions to reduce exposure to fecal contamination in low-income settings. Detection of a human- associated fecal marker and rotavirus was rare and unchanged by provision and promotion of improved sanitation to intervention compounds. The sanitation intervention reduced ruminant fecal contamination in drinking water and general (non-host specific) fecal contamination in soil but overall had limited effects on reducing fecal contamination in the household environment.

Factors affecting the presence of human-associated and fecal indicator real-time quantitative PCR genetic markers in urban-impacted recreational beaches.

Urban runoff can carry a variety of pollutants into recreational beaches, often including bacterial pathogens and indicators of fecal contamination. To develop complete recreational criteria and risk assessments, it is necessary to understand conditions under which human contamination could be present at beaches solely impacted by urban runoff. Accurately estimating risk requires understanding sources, concentrations, and transport mechanisms of microbial contaminants in these environments. By applying microbial source tracking methods and empirical modeling, we assessed the presence and level of human contamination at urban runoff impacted recreational beaches. We also identified environmental parameters and pollution sources that can influence the concentration and transport of culturable and molecular fecal indicator bacteria (FIB) in systems impacted solely by urban runoff. Water samples and physico-chemical parameters were collected from shoreline locations from three South Carolina (SC) beaches (five locations per beach) and two Florida (FL) beaches (three locations per beach). Each SC beach was directly impacted by swashes or tidal creeks receiving stormwater runoff from the urbanized area and therefore were designated as swash drain associated (SDA) beaches, while FL beaches were designated as non-swash drain associated (NSDA). Sampling in swash drains (SD; three sites per SD) directly impacting each SC beach was also conducted. Results indicate that although culturable (enterococci) and real-time quantitative polymerase chain reaction (qPCR) (EC23S857, Entero1, and GenBac3) FIB concentrations were, on average, higher at SD locations, SDA beaches did not have consistently higher molecular FIB signals compared to NSDA beaches. Both human-associated markers (HF183 and HumM2) were concomitantly found only at SDA beaches. Bacteroidales species-specific qPCR markers (BsteriF1 and BuniF2) identified differences in the Bacteroidales community, depending on beach type. The marker for general Bacteroidales was most abundant at SD locations and exhibited a high correlation with both culturable and other molecular markers. Combining molecular information with predictive modeling allowed us to identify both alongshore movement of currents and SD outflow as significant influences on the concentration of molecular and culturable indicators in the bathing zone. Data also suggests that combining methodologies is a useful and cost effective approach to help understand transport dynamics of fecal contamination and identify potential sources of contamination at marine beaches.

Improved HF183 quantitative real-time PCR assay for characterization of human fecal pollution in ambient surface water samples.

Quantitative real-time PCR (qPCR) assays that target the human-associated HF183 bacterial cluster within members of the genus Bacteroides are among the most widely used methods for the characterization of human fecal pollution in ambient surface waters. In this study, we show that a current TaqMan HF183 qPCR assay (HF183/BFDrev) routinely forms nonspecific amplification products and introduce a modified TaqMan assay (HF183/BacR287) that alleviates this problem. The performance of each qPCR assay was compared in head-to-head experiments investigating limits of detection, analytical precision, predicted hybridization to 16S rRNA gene sequences from a reference database, and relative marker concentrations in fecal and sewage samples. The performance of the modified HF183/BacR287 assay is equal to or improves upon that of the original HF183/BFDrev assay. In addition, a qPCR chemistry designed to combat amplification inhibition and a multiplexed internal amplification control are included. In light of the expanding use of PCR-based methods that rely on the detection of extremely low concentrations of DNA template, such as qPCR and digital PCR, the new TaqMan HF183/BacR287 assay should provide more accurate estimations of human-derived fecal contaminants in ambient surface waters.

Age-related shifts in the density and distribution of genetic marker water quality indicators in cow and calf feces.

Calves make up about 16% of the current bovine population in the United States and can excrete high levels of human pathogens in their feces. We describe the density and distribution of genetic markers from 9 PCR- and real-time quantitative PCR-based assays, including CF128, CF193, CowM2, CowM3, GenBac3, Entero1, EC23S857, CampF2, and ttr-6, commonly used to help assess ambient surface water quality. Each assay was tested against a collection of 381 individual bovine fecal samples representing 31 mother and calf pairings collected over a 10-month time period from time of birth through weaning. Genetic markers reported to be associated with ruminant and/or bovine fecal pollution were virtually undetected in calves for up to 115 days from birth, suggesting that physiological changes in calf ruminant function impact host-associated genetic marker shedding. In addition, general fecal indicator markers for Bacteroidales, Escherichia coli, and Enterococcus spp. exhibited three separate trends across time, indicating that these bacteria respond differently to age-related physiological and dietary changes during calf development. The results of this study suggest that currently available PCR-based water quality indicator technologies can under- or overestimate fecal pollution originating from calves and identify a need for novel calf-associated source identification methods.

Evaluation of the repeatability and reproducibility of a suite of qPCR-based microbial source tracking methods.

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.

Characterization of fecal concentrations in human and other animal sources by physical, culture-based, and quantitative real-time PCR methods.

The characteristics of fecal sources, and the ways in which they are measured, can profoundly influence the interpretation of which sources are contaminating a body of water. Although feces from various hosts are known to differ in mass and composition, it is not well understood how those differences compare across fecal sources and how differences depend on characterization methods. This study investigated how nine different fecal characterization methods provide different measures of fecal concentration in water, and how results varied across twelve different fecal pollution sources. Sources investigated included chicken, cow, deer, dog, goose, gull, horse, human, pig, pigeon, septage and sewage. A composite fecal slurry was prepared for each source by mixing feces from 6 to 22 individual samples with artificial freshwater. Fecal concentrations were estimated by physical (wet fecal mass added and total DNA mass extracted), culture-based (Escherichia coli and enterococci by membrane filtration and defined substrate), and quantitative real-time PCR (Bacteroidales, E. coli, and enterococci) characterization methods. The characteristics of each composite fecal slurry and the relationships between physical, culture-based and qPCR-based characteristics varied within and among different fecal sources. An in silico exercise was performed to assess how different characterization methods can impact identification of the dominant fecal pollution source in a mixed source sample. A comparison of simulated 10:90 mixtures based on enterococci by defined substrate predicted a source reversal in 27% of all possible combinations, while mixtures based on E. coli membrane filtration resulted in a reversal 29% of the time. This potential for disagreement in minor or dominant source identification based on different methods of measurement represents an important challenge for water quality managers and researchers.