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Enterococci target sequence density estimates from analyses of diluted river water DNA extracts by EPA Methods 1611 and 1609 and estimates with lower detection limits from undiluted DNA extracts by Method 1609 were indistinguishable. These methods should be equally suitable for comparison with U.S. EPA 2012 Recreational Water Quality Criteria values., (Published by Elsevier B.V.)

Quantitative polymerase chain reaction (qPCR) is increasingly being used for the quantitative detection of fecal indicator bacteria in beach water. QPCR allows for same-day health warnings, and its application is being considered as an option for recreational water quality testing in the United States (USEPA, 2011. EPA-OW-2011-0466, FRL-9609-3, Notice of Availability of Draft Recreational Water Quality Criteria and Request for Scientific Views). However, transition of qPCR from a research tool to routine water quality testing requires information on how various method variations affect target enumeration. Here we compared qPCR performance and enumeration of enterococci in spiked and environmental water samples using three qPCR platforms (Applied Biosystem StepOnePlus™, the BioRad iQ™5 and the Cepheid SmartCycler(®) II), two reference materials (lyophilized cells and frozen cells on filters) and two comparative CT quantification models (ΔCT and ΔΔCT). Reference materials exerted the biggest influence, consistently affecting results by approximately 0.5 log(10) unit. Platform had the smallest effect, generally exerting <0.1 log(10) unit difference in final results. Quantification model led to small differences (0.04-0.2 log(10) unit) in this study with relatively uninhibited samples, but has the potential to cause as much as 8-fold (0.9 log(10) unit) difference in potentially inhibitory samples. Our findings indicate the need for a certified and centralized source of reference materials and additional studies to assess applicability of the quantification models in analyses of PCR inhibitory samples., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A quantitative polymerase chain reaction (qPCR) method for the detection of enterococci fecal indicator bacteria has been shown to be generally applicable for the analysis of temperate fresh (Great Lakes) and marine coastal waters and for providing risk-based determinations of water quality at recreational beaches. In this study we further examined the applicability of the method for analyses of diverse inland waters as well as tropical marine waters from Puerto Rico based on the frequencies of samples showing presumptive PCR interference. Interference was assessed by salmon DNA sample processing control (SPC) and internal amplification control (IAC) assay analysis results and pre-established acceptance criteria of <3.0 and <1.5 cycle threshold (Ct) offsets from control samples, respectively. SPC assay results were accepted in analyses of 93% of the inland water samples whereas the criterion was met at frequencies of 60% and 97% in analyses of samples from Puerto Rico in two different years of sampling. The functionality of the control assays and their acceptance criteria was assessed on the basis of relative recovery estimates of spiked enterococci target organisms extracted in the presence of water sample filters and sample-free control filters and was supported by observations that recovery estimates from the water sample and control filters were substantially different for samples that failed these criteria. Through the combined use of the SPC and IAC assays, two presumptive types of interference were identified. One type, observed in the tropical marine water samples, appeared to primarily affect the availability of the DNA templates for detection. The second type, observed in river water samples, appeared to primarily affect PCR amplification efficiency. In the presence of DNA template interference, adjustments from SPC assay results by the ΔΔCt comparative Ct calculation method decreased the variability of spiked enterococci recovery estimates and increased the similarity with control filters as compared to unadjusted recovery estimates obtained by the ΔCt calculation method. Use of a higher salmon DNA concentration in the extraction buffer also reduced this type of interference. The effects of amplification interference were largely reversed by dilution of the DNA extracts and even more effectively by the use of an alternative, commercial PCR reagent, designed for the analysis of environmental samples., (Published by Elsevier Ltd.)

Several studies have examined how fecal indicator bacteria (FIB) measurements compare between quantitative PCR (qPCR) and the culture methods it is intended to replace. Here, we extend those studies by examining the stability of that relationship within a beach, as affected by time of day and seasonal variations in source. Enterococcus spp. were quantified at three southern California beaches in the morning and afternoon using two qPCR assays, membrane filtration, and defined-substrate testing. While qPCR and culture-based measurements were consistently and significantly correlated, strength of the correlation varied both among and within beaches. Correlations were higher in the morning (0.45 < ρ < 0.74 [P < 0.002]) than in the afternoon (0.18 < ρ < 0.45 [P < 0.021]) and higher when the fecal contamination was concentrated (0.38 < ρ < 0.83 [P < 0.001]) than when it was diffuse (0.19 < ρ < 0.34 [P < 0.003]). The ratios of culture-based and qPCR results (CFU or most probable number [MPN] per calibrator cell equivalents [CCE]) also varied spatially and temporally. Ratios ranged between 0.04 and 0.85 CFU or MPN per CCE and were lowest at the beach affected by diffuse pollution. Patterns in the ratios over the course of the day were dissimilar across beaches, increasing with time at one beach and decreasing at another. The spatial and temporal variability we observed indicate that the empirical relationship between culture-based and qPCR results is not universal, even within a beach.

Molecular methods for quantifying defined Bacteroidales species from the human gastrointestinal tract may have important clinical and environmental applications, ranging from diagnosis of infections to fecal source tracking in surface waters. In this study, sequences from the V2 region of the small subunit ribosomal RNA gene were targeted in the development of qPCR assays to quantify DNA from six Bacteroides and one Prevotella species. In silico and experimental analyses suggested that each of the assays was highly discriminatory in detecting DNA from the intended species. Analytical sensitivity, precision and ranges of quantification were demonstrated for each assay by coefficients of variation of less than 2% for cycle threshold measurements over a range from 10 to 4×10(4) target sequence copies. The assays were applied to assess the occurrence and relative abundance of their target sequences in feces from humans and five animal groups as well as in 14 sewage samples from 13 different treatment facilities. Sequences from each of the species were detected at high levels (>10(3)copies/ng total extracted DNA) in human wastes. Sequences were also detected by each assay in all sewage samples and, with exception of the Prevotella sequences, showed highly correlated (R(2)≥0.7) variations in concentrations between samples. In contrast, the occurrence and relative abundance profiles of these sequences differed substantially in the fecal samples from each of the animal groups. These results suggest that analyses for multiple individual Bacteroidales species may be useful in identifying human fecal pollution in environmental waters., (Published by Elsevier GmbH.)

Real-time PCR absolute quantification applications are becoming more common in the recreational and drinking water quality industries. Many methods rely on the use of standard curves to make estimates of DNA target concentrations in unknown samples. Traditional absolute quantification approaches dictate that a standard curve must accompany each experimental run. However, the generation of a standard curve for each qPCR experiment set-up can be expensive and time consuming, especially for studies with large numbers of unknown samples. As a result, many researchers have adopted a master calibration strategy where a single curve is derived from DNA standard measurements generated from multiple instrument runs. However, a master curve can inflate uncertainty associated with intercept and slope parameters and decrease the accuracy of unknown sample DNA target concentration estimates. Here we report two alternative strategies termed 'pooled' and 'mixed' for the generation of calibration equations from absolute standard curves which can help reduce the cost and time of laboratory testing, as well as the uncertainty in calibration model parameter estimates. In this study, four different strategies for generating calibration models were compared based on a series of repeated experiments for two different qPCR assays using a Monte Carlo Markov Chain method. The hierarchical Bayesian approach allowed for the comparison of uncertainty in intercept and slope model parameters and the optimization of experiment design. Data suggests that the 'pooled' model can reduce uncertainty in both slope and intercept parameter estimates compared to the traditional single curve approach. In addition, the 'mixed' model achieved uncertainty estimates similar to the 'single' model while increasing the number of available reaction wells per instrument run., (Published by Elsevier Ltd.)

There are numerous PCR-based assays available to characterize human fecal pollution in ambient waters. Each assay employs distinct oligonucleotides and many target different genes and microorganisms leading to potential variations in assay performance. Performance comparisons utilizing feces and raw sewage samples are needed to determine which assays are best suited for expensive and time-consuming field validation, fate, transport, and epidemiology studies. We report the assessment of five end-point PCR and 10 real-time quantitative PCR (qPCR) assays that target genes from presumptive Bacteroidales microorganisms reported to be associated with human feces. Each assay was tested against a reference collection of 54 primary influent sewage samples collected from different geographical locations across the United States and 174 fecal DNA extracts from 23 different animal sources. Experiments indicate that human-associated genetic markers are distributed across a broad range of human populations but show substantial differences in specificity for human feces suggesting that particular assays may be more suitable than others depending on the abundance of genetic marker required for detection and the animal sources impacting a particular watershed or beach of interest.

The quantitative polymerase chain reaction (qPCR) method provides rapid estimates of fecal indicator bacteria densities that have been indicated to be useful in the assessment of water quality. Primarily because this method provides faster results than standard culture-based methods, the U.S. Environmental Protection Agency is currently considering its use as a basis for revised ambient water quality criteria. In anticipation of this possibility, we sought to examine the relationship between qPCR-based and culture-based estimates of enterococci in surface waters. Using data from several research groups, we compared enterococci estimates by the two methods in water samples collected from 37 sites across the United States. A consistent linear pattern in the relationship between cell equivalents (CCE), based on the qPCR method, and colony-forming units (CFU), based on the traditional culturable method, was significant (P < 0.05) at most sites. A linearly decreasing variance of CCE with increasing CFU levels was significant (P < 0.05) or evident for all sites. Both marine and freshwater sites under continuous influence of point-source contamination tended to reveal a relatively constant proportion of CCE to CFU. The consistency in the mean and variance patterns of CCE versus CFU indicates that the relationship of results based on these two methods is more predictable at high CFU levels (e.g., log(10)CFU > 2.0/100 mL) while uncertainty increases at lower CFU values. It was further noted that the relative error in replicated qPCR estimates was generally higher than that in replicated culture counts even at relatively high target levels, suggesting a greater need for replicated analyses in the qPCR method to reduce relative error. Further studies evaluating the relationship between culture and qPCR should take into account analytical uncertainty as well as potential differences in results of these methods that may arise from sample variability, different sources of pollution, and environmental factors.

There are numerous PCR-based assays available to characterize bovine fecal pollution in ambient waters. The determination of which approaches are most suitable for field applications can be difficult because each assay targets a different gene, in many cases from different microorganisms, leading to variation in assay performance. We describe a performance evaluation of seven end-point PCR and real-time quantitative PCR (qPCR) assays reported to be associated with either ruminant or bovine feces. Each assay was tested against a reference collection of DNA extracts from 247 individual bovine fecal samples representing 11 different populations and 175 fecal DNA extracts from 24 different animal species. Bovine-associated genetic markers were broadly distributed among individual bovine samples ranging from 39 to 93%. Specificity levels of the assays spanned 47.4% to 100%. End-point PCR sensitivity also varied between assays and among different bovine populations. For qPCR assays, the abundance of each host-associated genetic marker was measured within each bovine population and compared to results of a qPCR assay targeting 16S rRNA gene sequences from Bacteroidales. Experiments indicate large discrepancies in the performance of bovine-associated assays across different bovine populations. Variability in assay performance between host populations suggests that the use of bovine microbial source-tracking applications will require a priori characterization at each watershed of interest.

Assessment of health risk and fecal bacterial loads associated with human fecal pollution requires reliable host-specific analytical methods and a rapid quantification approach. We report the development of quantitative PCR assays for quantification of two recently described human-specific genetic markers targeting Bacteroidales-like cell surface-associated genes. Each assay exhibited a range of quantification from 10 to 1 x 10(6) copies of target DNA. For each assay, internal amplification controls were developed to detect the presence or absence of amplification inhibitors. The assays predominantly detected human fecal specimens and exhibited specificity levels greater than 97% when tested against 265 fecal DNA extracts from 22 different animal species. The abundance of each human-specific genetic marker in primary effluent wastewater samples collected from 20 geographically distinct locations was measured and compared to quantities estimated by real-time PCR assays specific for rRNA gene sequences from total Bacteroidales and enterococcal fecal microorganisms. Assay performances combined with the prevalence of DNA targets in sewage samples provide experimental evidence supporting the potential application of these quantitative methods for monitoring fecal pollution in ambient environmental waters.

Accurate assessment of health risks associated with bovine (cattle) fecal pollution requires a reliable host-specific genetic marker and a rapid quantification method. We report the development of quantitative PCR assays for the detection of two recently described bovine feces-specific genetic markers and a method for the enumeration of these markers using a Markov chain Monte Carlo approach. Both assays exhibited a range of quantification from 25 to 2 x 10(6) copies of target DNA, with a coefficient of variation of <2.1%. One of these assays can be multiplexed with an internal amplification control to simultaneously detect the bovine-specific genetic target and presence of amplification inhibitors. The assays detected only cattle fecal specimens when tested against 204 fecal DNA extracts from 16 different animal species and also demonstrated a broad distribution among individual bovine samples (98 to 100%) collected from five geographically distinct locations. The abundance of each bovine-specific genetic marker was measured in 48 individual samples and compared to quantitative PCR-enumerated quantities of rRNA gene sequences representing total Bacteroidetes, Bacteroides thetaiotaomicron, and enterococci in the same specimens. Acceptable assay performance combined with the prevalence of DNA targets across different cattle populations provides experimental evidence that these quantitative assays will be useful in monitoring bovine fecal pollution in ambient waters.

Mold specific quantitative PCR (MSQPCR) was used to measure the concentrations of the 36 mold species in indoor and outdoor air samples that were taken simultaneously for 48 h in and around 17 homes in Cincinnati, Ohio. The total spore concentrations of 353 per m(3) of indoor air and 827 per m(3) of outdoor air samples were significantly different (por=0.5). These results suggest that interpretation of the meaning of short-term (<48 h) mold measurements in indoor and outdoor air samples must be made with caution.

Indoor mold concentrations were measured in the dust of moldy homes (MH) and reference homes (RH) by quantitative PCR (QPCR) assays for 82 species or related groups of species (assay groups). About 70% of the species and groups were never or only rarely detected. The ratios (MH geometric mean : RH geometric mean) for 6 commonly detected species (Aspergillus ochraceus, A. penicillioides, A. unguis, A. versicolor, Eurotium group, and Cladosporium sphaerospermum) were >1 (Group I). Logistic regression analysis of the sum of the logs of the concentrations of Group I species resulted in a 95% probability for separating MH from RH. These results suggest that it may be possible to evaluate whether a home has an abnormal mold condition by quantifying a limited number of mold species in a dust sample. Also, four common species of Aspergillus were quantified by standard culturing procedures and their concentrations compared to QPCR results. Culturing underestimated the concentrations of these four species by 2 to 3 orders of magnitude compared to QPCR.

Monitoring for pathogenic Aspergillus species using a rapid, highly sensitive, quantitative polymerase chain reaction technique during carpet removal in a burn unit provided data that allowed patients to be safely returned to the refloored area sooner than if only conventional culture monitoring had been used.

A total of 65 quantitative PCR (QPCR) assays, incorporating fluorigenic 5' nuclease (TaqMan) chemistry and directed at the nuclear ribosomal RNA operon, internal transcribed spacer regions (ITS1 or ITS2) was developed and tested for the detection of selected Aspergillus, Penicillium and Paecilomyces species. The assays varied in specificity from species or subspecies to closely related species groups, subject to the amount of nucleotide sequence variation in the different organisms. A generic assay for all target species of Aspergillus, Penicillium and Paecilomyces was also developed and tested. Using a previously reported DNA extraction method, estimated conidia detection limits for target species ranged from less than one to several hundred per sample for the different assays. Conidia detection limits for non-target species were at least 1,000 fold higher in nearly all instances. The assays were used to analyze ten HVAC dust samples from different sources around the US. Total quantities of Aspergillus, Penicillium and Paecilomyces conidia in the samples, determined by the generic assay and the summed totals from the specific assays, were in general agreement, suggesting that all of the numerically dominant species in the samples were accounted for by the specific assays. QPCR analyses of these samples after spiking them with selected target organisms indicated that the enumeration results were within approximately a one-half log range of the expected values 95% of the time. Evidence is provided that the commonly used practices of enumerating Aspergillus and Penicillium as a single group or only by genus can be misleading in understanding the indoor populations of these organisms and their potential health risks.

Quantitative PCR (QPCR) technology, incorporating fluorigenic 5' nuclease (TaqMan) chemistry, was utilized for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C. lusitaniae) in water. Known numbers of target cells were added to distilled and tap water samples, filtered, and disrupted directly on the membranes for recovery of DNA for QPCR analysis. The assay's sensitivities were between one and three cells per filter. The accuracy of the cell estimates was between 50 and 200% of their true value (95% confidence level). In similar tests with surface water samples, the presence of PCR inhibitory compounds necessitated further purification and/or dilution of the DNA extracts, with resultant reductions in sensitivity but generally not in quantitative accuracy. Analyses of a series of freshwater samples collected from a recreational beach showed positive correlations between the QPCR results and colony counts of the corresponding target species. Positive correlations were also seen between the cell quantities of the target Candida species detected in these analyses and colony counts of Enterococcus organisms. With a combined sample processing and analysis time of less than 4 h, this method shows great promise as a tool for rapidly assessing potential exposures to waterborne pathogenic Candida species from drinking and recreational waters and may have applications in the detection of fecal pollution.

A 16S rDNA real-time PCR method was developed to detect Enterococcus faecalis in water samples. The dynamic range for cell detection spanned five logs and the detection limit was determined to be 6 cfu/reaction. The assay was capable of detecting E. faecalis cells added to biofilms from a simulator of a water distribution system and in freshwater samples. Nucleic acid extraction was not required, permitting the detection of E. faecalis cells in less than 3 h.

Three comparatively rapid methods for the extraction of DNA from fungal conidia and yeast cells in environmental (air, water and dust) samples were evaluated for use in real-time PCR (TaqMan) analyses. A simple bead milling method was developed to provide sensitive, accurate and precise quantification of target organisms in air and water (tap and surface) samples. However, quantitative analysis of dust samples required further purification of the extracted DNA by a streamlined silica adsorption procedure.

Quantitative polymerase chain reaction (qPCR) has become a frequently used technique for quantifying enterococci in recreational surface waters, but there are several methodological options. Here we evaluated how three method permutations, type of mastermix, sample extract dilution and use of controls in results calculation, affect method reliability among multiple laboratories with respect to sample interference. Multiple samples from each of 22 sites representing an array of habitat types were analyzed using EPA Method 1611 and 1609 reagents with full strength and five-fold diluted extracts. The presence of interference was assessed three ways: using sample processing and PCR amplifications controls; consistency of results across extract dilutions; and relative recovery of target genes from spiked enterococci in water sample compared to control matrices with acceptable recovery defined as 50 to 200%. Method 1609, which is based on an environmental mastermix, was found to be superior to Method 1611, which is based on a universal mastermix. Method 1611 had over a 40% control assay failure rate with undiluted extracts and a 6% failure rate with diluted extracts. Method 1609 failed in only 11% and 3% of undiluted and diluted extracts analyses. Use of sample processing control assay results in the delta–delta Ct method for calculating relative target gene recoveries increased the number of acceptable recovery results. Delta–delta tended to bias recoveries from apparent partially inhibitory samples on the high side which could help in avoiding potential underestimates of enterococci — an important consideration in a public health context. Control assay and delta–delta recovery results were largely consistent across the range of habitats sampled, and among laboratories. The methodological option that best balanced acceptable estimated target gene recoveries with method sensitivity and avoidance of underestimated enterococci densities was Method 1609 without extract dilution and using the delta–delta calculation method. The applicability of this method can be extended by the analysis of diluted extracts to sites where interference is indicated but, particularly in these instances, should be confirmed by augmenting the control assays with analyses for target gene recoveries from spiked target organisms. [ABSTRACT FROM AUTHOR]

Mycobacterium avium (MA), Mycobacterium intracellulare (MI), and Mycobacterium avium subsp. paratuberculosis (MAP) are difficult to culture due to their slow growing nature. A quantitative polymerase chain reaction (qPCR) method for the rapid detection of MA, MI, and MAP can be used to provide data supporting drinking water biofilms as potential sources of human exposure. The aim of this study was to characterize two qPCR assays targeting partial 16S rRNA gene sequences of MA and MI and use these assays, along with two previously reported MAP qPCR assays (IS900 and Target 251), to investigate Mycobacterium occurrence in kitchen faucet biofilms. MA and MI qPCR assays demonstrated 100% specificity and sensitivity when evaluated against 18 non-MA complex, 76 MA, and 17 MI isolates. Both assays detected approximately 1,000 cells from a diluted cell stock inoculated on a sampling swab 100% of the time. DNA analysis by qPCR indicated that 35.3, 56.9 and 11.8% of the 51 kitchen faucet biofilm samples collected contained MA, MI, and MAP, respectively. This study introduces novel qPCR assays designed to specifically detect MA and MI in biofilm. Results support the use of qPCR as an alternative to culture for detection and enumeration of MA, MI, and MAP in microbiologically complex samples. [ABSTRACT FROM AUTHOR]

The U.S. EPA has published recommendations for calibrator cell equivalent (CCE) densities of enterococci in recreational waters determined by a qPCR method in its 2012 recreational water quality criteria (RWQC). The CCE quantification unit stems from the calibration model used to estimate enterococci densities in recreational beach waters in the EPA National Epidemiological and Environmental Assessment of Recreational (NEEAR) Water Study and directly informed the derivation of the RWQC recommendations. Recent studies have demonstrated that CCE estimates from the method can vary when using different cultured Enterococcus cell preparations in calibrator samples. These differences have been attributed to differences in the quantities of targeted gene copies (target sequences) that are recovered per nominal calibrator cell by DNA extraction. Standardization of results from the calibration model will require the estimation of target sequence recoveries from the calibrator and water samples. In addition, comparisons of water sample results with the RWQC values will require a knowledge of target sequence recoveries from the NEEAR study calibrator samples. In this study recoveries of target sequences and the mean target sequence/cell ratio for the NEEAR study calibrator samples were retrospectively estimated with a corroborated standard curve. A modification of the calibration model was then used to estimate enterococci target sequence quantities in water samples from eight midwestern U.S. rivers. CCE estimates were obtained by dividing these target sequence quantities by the mean NEEAR study target sequence/cell ratio. This target sequence-based quantification approach resulted in a high degree of agreement in beach action decisions (determinations of whether bacterial fecal indicator densities are above or below RWQC-recommended values) from CCE results of the qPCR method and from culture dependent enumeration of both enterococci and Eschericia coli in the corresponding water samples. [ABSTRACT FROM AUTHOR]

The US Environmental Protection Agency has proposed the use of quantitative polymerase chain reaction (qPCR) as a rapid alternative analytical method for monitoring recreational water quality at beaches. For qPCR to be considered for other Clean Water Act purposes, such as inclusion in discharge permits and use in Total Maximum Daily Load calculations, it is necessary to understand how qPCR detectable genetic markers are influenced by wastewater disinfection. This study investigated genetic markers for Escherichia coli, Enterococcus, Clostridium spp., Bacteroides, total Bacteroidales, as well as the human-associated Bacteroides markers, HF183 and HumM2, to determine which, if any, were influenced by disinfection (chlorination or ultraviolet light) of effluents from secondary wastewater treatment in different seasons. The effects of disinfection on culturable enterococci, E. coli, Bacteroides, and C. perfringens were also compared to their associated genetic markers. Disinfection of secondary treatment effluents significantly reduced culturable fecal indicator bacteria (FIB) but not genetic marker densities. No significant differences were observed in the responses of FIB culture and genetic marker densities to type of disinfection (chlorination vs UV) or season. Results of this study provide evidence that qPCR may not be suitable for monitoring efficacy of wastewater disinfection on the inactivation of bacterial pathogens. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Introduction: In the United States and elsewhere, recreational water quality is monitored for fecal indicator bacteria to help prevent swimming-associated illnesses. Standard methods to measure these bacteria take at least 24 hours to obtain results. Molecular approaches such as quantitative polymerase chain reaction (qPCR) can estimate these bacteria faster, in under 3 hours. Previously, we demonstrated that measurements of the fecal indicator bacteria Enterococcus using qPCR were associated with gastrointestinal (GI) illness among swimmers at freshwater beaches. In this paper, we report on results from three marine beach sites. Methods: We interviewed beach-goers and collected water samples at marine beaches affected by treated sewage discharges in Mississippi in 2005, and Rhode Island and Alabama in 2007. Ten to twelve days later, we obtained information about gastrointestinal, respiratory, eye, ear and skin symptoms by telephone. We tested water samples for fecal indicator organisms using qPCR and other methods. Results: We enrolled 6,350 beach-goers. The occurrence of GI illness among swimmers was associated with a log10-increase in exposure to qPCR-determined estimates of fecal indicator organisms in the genus Enterococcus (AOR = 2.6, 95% CI 1.3-5.1) and order Bacteroidales (AOR = 1.9, 95% CI 1.3-2.9). Estimates of organisms related to Clostridium perfringens and a subgroup of organisms in the genus Bacteroides were also determined by qPCR in 2007, as was F+ coliphage, but relationships between these indicators and illness were not statistically significant. Conclusions: This study provides the first evidence of a relationship between gastrointestinal illness and estimates of fecal indicator organisms determined by qPCR at marine beaches. [ABSTRACT FROM AUTHOR]

Background: In real-time quantitative PCR studies using absolute plasmid DNA standards, a calibration curve is developed to estimate an unknown DNA concentration. However, potential differences in the amplification performance of plasmid DNA compared to genomic DNA standards are often ignored in calibration calculations and in some cases impossible to characterize. A flexible statistical method that can account for uncertainty between plasmid and genomic DNA targets, replicate testing, and experiment-to-experiment variability is needed to estimate calibration curve parameters such as intercept and slope. Here we report the use of a Bayesian approach to generate calibration curves for the enumeration of target DNA from genomic DNA samples using absolute plasmid DNA standards. Results: Instead of the two traditional methods (classical and inverse), a Monte Carlo Markov Chain (MCMC) estimation was used to generate single, master, and modified calibration curves. The mean and the percentiles of the posterior distribution were used as point and interval estimates of unknown parameters such as intercepts, slopes and DNA concentrations. The software WinBUGS was used to perform all simulations and to generate the posterior distributions of all the unknown parameters of interest. Conclusion: The Bayesian approach defined in this study allowed for the estimation of DNA concentrations from environmental samples using absolute standard curves generated by real-time qPCR. The approach accounted for uncertainty from multiple sources such as experiment-to-experiment variation, variability between replicate measurements, as well as uncertainty introduced when employing calibration curves generated from absolute plasmid DNA standards. [ABSTRACT FROM AUTHOR]

Draft method C is a standardized method for quantifying E. coli densities in recreational waters using quantitative polymerase chain reaction (qPCR). The method includes a Microsoft Excel workbook that automatically screens for poor-quality data using a set of previously proposed acceptance criteria, generates weighted linear regression (WLR) composite standard curves, and calculates E. coli target gene copies in test samples. We compared standard curve parameter values and test sample results calculated with the WLR model to those from a Bayesian master standard curve (MSC) model using data from a previous multi-lab study. The two models' mean intercept and slope estimates from twenty labs' standard curves were within each other's 95% credible or confidence intervals for all labs. E. coli gene copy estimates of six water samples analyzed by eight labs were highly overlapping among labs when quantified with the WLR and MSC models. Finally, we compared multiple labs' 2016–2018 composite curves, comprised of data from individual curves where acceptance criteria were not used, to their corresponding composite curves with passing acceptance criteria. Composite curves developed from passing individual curves had intercept and slope 95% confidence intervals that were often narrower than without screening and an analysis of covariance test was passed more often. The Excel workbook WLR calculation and acceptance criteria will help laboratories implement draft method C for recreational water analysis in an efficient, cost-effective, and reliable manner. [ABSTRACT FROM AUTHOR]

Abstract: The U.S. Environmental Protection Agency will be recommending a quantitative polymerase chain reaction (qPCR) method targeting Enterococcus spp. as an option for monitoring recreational beach water quality. A practical consideration for implementation of this and other qPCR methods is whether the results are comparable on different PCR instruments. In this study, quantitative estimates of Enterococcus densities from marine and freshwater samples were determined by the qPCR method from cycle threshold (Ct) measurements obtained on Applied Biosystems StepOnePlus and Cepheid SmartCycler instruments. Three variations of a comparative Ct model, differing in their sources of calibration data, were used in the estimations. Both traditional and Bayesian statistical modeling approaches were examined in the instrument comparisons. The traditional analysis of variance (ANOVA) approach indicated no significant differences (p >0.05) between mean density estimates from the instruments in two of the three model variations. The Bayesian approach indicated that the 95% Bayesian credible intervals of density estimates from the instruments overlapped in all models; however, the uncertainty of the estimates varied depending on the model. These results support the interchangeable use of the two instruments in the method and also illustrate the importance of defining the source of calibration data used in the comparative Ct model. [Copyright &y& Elsevier]

Sieved vacuum bag dust from the homes of 143 children in Detroit was analyzed by mold specific quantitative PCR (MSQPCR) and the Environmental Relative Moldiness Index (ERMIsm) was calculated for each home. Children living in these homes were grouped as non-asthmatic (n =83), moderately asthmatic (n =28) and severely asthmatic (n =32) based on prescription medication usage for their asthma management (none, occasional and daily, respectively). The mean ERMI for each group of homes was 6.2 for non-asthmatic, 6.3 for moderately asthmatic and 8.2 for severely asthmatic children. The ERMI values in the homes of severely asthmatic children were significantly greater compared to the non-asthmatics (p =0.04 in Wilcoxon Rank-sum test). Aspergillus niger and Aspergillus unguis were the primary mold species that distinguished severely asthmatic children''s homes and non-asthmatic children''s homes (p <0.05; Wilcoxon Rank-sum test). The determination of the home''s ERMI values may aid in prioritizing home remediation efforts, particularly in those children who are at increased risk for asthma exacerbation. [Copyright &y& Elsevier]

There is growing interest in the application of rapid quantitative polymerase chain reaction (qPCR) and other PCR-based methods for recreational water quality monitoring and management programs. This interest has strengthened given the publication of U.S. Environmental Protection Agency (EPA)-validated qPCR methods for enterococci fecal indicator bacteria (FIB) and has extended to similar methods for Escherichia coli (E. coli) FIB. Implementation of qPCR-based methods in monitoring programs can be facilitated by confidence in the quality of the data produced by these methods. Data quality can be determined through the establishment of a series of specifications that should reflect good laboratory practice. Ideally, these specifications will also account for the typical variability of data coming from multiple users of the method. This study developed proposed standardized data quality acceptance criteria that were established for important calibration model parameters and/or controls from a new qPCR method for E. coli (EPA Draft Method C) based upon data that was generated by 21 laboratories. Each laboratory followed a standardized protocol utilizing the same prescribed reagents and reference and control materials. After removal of outliers, statistical modeling based on a hierarchical Bayesian method was used to establish metrics for assay standard curve slope, intercept and lower limit of quantification that included between-laboratory, replicate testing within laboratory, and random error variability. A nested analysis of variance (ANOVA) was used to establish metrics for calibrator/positive control, negative control, and replicate sample analysis data. These data acceptance criteria should help those who may evaluate the technical quality of future findings from the method, as well as those who might use the method in the future. Furthermore, these benchmarks and the approaches described for determining them may be helpful to method users seeking to establish comparable laboratory-specific criteria if changes in the reference and/or control materials must be made. Image 1 • Data QA criteria were established for an EPA E. coli qPCR method (Draft Method C). • QA parameters were slope, intercept for standard curve, LLOQ, Ct values of controls. • Data QA was based on use of prescribed reference and control materials by 21 labs. • The study also provides guidance for labs to establish QA with their own materials. • Polymerase reagent lots should be checked for E. coli signal before use in Method C. [ABSTRACT FROM AUTHOR]

There is interest in the application of rapid quantitative polymerase chain reaction (qPCR) methods for recreational freshwater quality monitoring of the fecal indicator bacteria Escherichia coli (E. coli). In this study we determined the performance of 21 laboratories in meeting proposed, standardized data quality acceptance (QA) criteria and the variability of target gene copy estimates from these laboratories in analyses of 18 shared surface water samples by a draft qPCR method developed by the U.S. Environmental Protection Agency (EPA) for E. coli. The participating laboratories ranged from academic and government laboratories with more extensive qPCR experience to "new" water quality and public health laboratories with relatively little previous experience in most cases. Failures to meet QA criteria for the method were observed in 24% of the total 376 test sample analyses. Of these failures, 39% came from two of the "new" laboratories. Likely factors contributing to QA failures included deviations in recommended procedures for the storage and preparation of reference and control materials. A master standard curve calibration model was also found to give lower overall variability in log 10 target gene copy estimates than the delta-delta Ct (ΔΔCt) calibration model used in previous EPA qPCR methods. However, differences between the mean estimates from the two models were not significant and variability between laboratories was the greatest contributor to overall method variability in either case. Study findings demonstrate the technical feasibility of multiple laboratories implementing this or other qPCR water quality monitoring methods with similar data quality acceptance criteria but suggest that additional practice and/or assistance may be valuable, even for some more generally experienced qPCR laboratories. Special attention should be placed on providing and following explicit guidance on the preparation, storage and handling of reference and control materials. Image 1 • Variability in E. coli gene copy estimates from an EPA qPCR method was assessed. • Alternative calibration models affected variability but not overall mean estimates. • Failures to meet proposed data quality acceptance criteria excluded 24% of analyses. • Outlier gene copy estimates were not always associated with criteria failures. • Lab experience and their handling of control materials affected method performance. [ABSTRACT FROM AUTHOR]

Abstract An obstacle to establishing widely useful data acceptance criteria for U.S. Environmental Protection Agency (EPA) qPCR methods has been the unavailability of standardized reference materials. Earlier versions of EPA Methods 1609 and 1611 for enterococci used cellular reference materials for quantifying enterococci in unknown test samples, however, EPA updates to these fundamentally DNA-based analysis methods have shifted toward the use of DNA standards. This report describes the application of droplet digital PCR (ddPCR) analysis for the quantification of a set of synthetic plasmid DNA standards that have been made available for updated EPA Methods 1609.1 and 1611.1 as well as for EPA Draft Method C for Escherichia coli. To obtain the most accurate concentration estimates possible, part of this effort was to develop a data analysis model for determining the fluorescence thresholds that distinguish positive from negative droplets produced by the ddPCR reactions. Versions of this model are described for applications with individual reactions, multiple reactions within a ddPCR system run, and multiple reactions within and across different system runs. The latter version was applied toward determinations of error in the concentration estimates of the standards from replicate analyses of each standard in multiple ddPCR system runs. Mean concentration estimates for the five standards from the ddPCR analyses were 4.356, 3.381, 2.371, 1.641 and 1.071 log 10 copies/5 μL with associated standard deviations of 0.074, 0.082, 0.108, 0.131 and 0.188, respectively. These estimates contrasted with expected log 10 concentrations of 4.6, 3.6, 2.6, 1.9 and 1.3 copies/5 μL, respectively, based on the yield of the plasmid reported by the vendor and spectrophotometric analysis of the initial stock solution of this material. These results illustrate how the analyses of original stocks may lead to potential bias(es) in the concentration estimates of final DNA standards and subsequently in the estimates of unknown test samples determined from these standards in qPCR analyses. Highlights • Plasmid DNA standards were prepared as reference materials for EPA qPCR methods. • The main basis of standards quantification was droplet digital PCR (ddPCR) analysis. • Instrument software & a novel Mixture Model were used to identify positive droplets. • Error estimates from the Mixture Model were used to assess standards stability. • Results illustrate the value of quantifying actual standards over undiluted stocks. [ABSTRACT FROM AUTHOR]

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 (notemplate 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. [ABSTRACT FROM AUTHOR]

Abstract: Enterococci target sequence density estimates from analyses of diluted river water DNA extracts by EPA Methods 1611 and 1609 and estimates with lower detection limits from undiluted DNA extracts by Method 1609 were indistinguishable. These methods should be equally suitable for comparison with U.S. EPA 2012 Recreational Water Quality Criteria values. [Copyright &y& Elsevier]

Gulls are often cited as important contributors of fecal contamination to surface waters, and some recreational beaches have used gull control measures to improve microbial water quality. In this study, gulls were chased from a Lake Michigan beach using specially trained dogs, and water quality improvements were quantified. Fecal indicator bacteria and potentially pathogenic bacteria were measured before and during gull control using culture methods and quantitative polymerase chain reaction (qPCR). Harassment by dogs was an effective method of gull control: average daily gull populations fell from 665 before to 17 during intervention; and a significant reduction in the density of a gull-associated marker was observed (p < 0.001). Enterococcus spp. and Escherichia coli densities were also significantly reduced during gull control (p < 0.001 and p = 0.012, respectively for culture methods; p = 0.012 and p = 0.034, respectively for qPCR). Linear regression results indicate that a 50% reduction in gulls was associated with a 38% and 29% decrease in Enterococcus spp. and E. coli densities, respectively. Potentially human pathogenic bacteria were detected on 64% of days prior to gull control and absent during gull intervention, a significant reduction (p = 0.005). This study demonstrates that gull removal can be a highly successful beach remedial action to improve microbial water quality. [ABSTRACT FROM AUTHOR]

Abstract: Background: Traditional fecal indicator bacteria (FIB) measurement is too slow (>18 h) for timely swimmer warnings. Objectives: Assess relationship of rapid indicator methods (qPCR) to illness at a marine beach impacted by urban runoff. Methods: We measured baseline and two-week health in 9525 individuals visiting Doheny Beach 2007–08. Illness rates were compared (swimmers vs. non-swimmers). FIB measured by traditional (Enterococcus spp. by EPA Method 1600 or Enterolert™, fecal coliforms, total coliforms) and three rapid qPCR assays for Enterococcus spp. (Taqman, Scorpion-1, Scorpion-2) were compared to health. Primary bacterial source was a creek flowing untreated into ocean; the creek did not reach the ocean when a sand berm formed. This provided a natural experiment for examining FIB-health relationships under varying conditions. Results: We observed significant increases in diarrhea (OR 1.90, 95% CI 1.29–2.80 for swallowing water) and other outcomes in swimmers compared to non-swimmers. Exposure (body immersion, head immersion, swallowed water) was associated with increasing risk of gastrointestinal illness (GI). Daily GI incidence patterns were different: swimmers (2-day peak) and non-swimmers (no peak). With berm-open, we observed associations between GI and traditional and rapid methods for Enterococcus; fewer associations occurred when berm status was not considered. Conclusions: We found increased risk of GI at this urban runoff beach. When FIB source flowed freely (berm-open), several traditional and rapid indicators were related to illness. When FIB source was weak (berm-closed) fewer illness associations were seen. These different relationships under different conditions at a single beach demonstrate the difficulties using these indicators to predict health risk. [Copyright &y& Elsevier]

Abstract: DNA extracts from enumerated target organism cells (calibrator samples) have been used for estimating Enterococcus cell equivalent densities in surface waters by a comparative cycle threshold (Ct) qPCR analysis method. To compare surface water Enterococcus density estimates from different studies by this approach, either a consistent source of calibrator cells must be used or the estimates must account for any differences in target sequence recoveries from different sources of calibrator cells. In this report we describe two methods for estimating target sequence recoveries from whole cell calibrator samples based on qPCR analyses of their serially diluted DNA extracts and most probable number (MPN) calculation. The first method employed a traditional MPN calculation approach. The second method employed a Bayesian hierarchical statistical modeling approach and a Monte Carlo Markov Chain (MCMC) simulation method to account for the uncertainty in these estimates associated with different individual samples of the cell preparations, different dilutions of the DNA extracts and different qPCR analytical runs. The two methods were applied to estimate mean target sequence recoveries per cell from two different lots of a commercially available source of enumerated Enterococcus cell preparations. The mean target sequence recovery estimates (and standard errors) per cell from Lot A and B cell preparations by the Bayesian method were 22.73 (3.4) and 11.76 (2.4), respectively, when the data were adjusted for potential false positive results. Means were similar for the traditional MPN approach which cannot comparably assess uncertainty in the estimates. Cell numbers and estimates of recoverable target sequences in calibrator samples prepared from the two cell sources were also used to estimate cell equivalent and target sequence quantities recovered from surface water samples in a comparative Ct method. Our results illustrate the utility of the Bayesian method in accounting for uncertainty, the high degree of precision attainable by the MPN approach and the need to account for the differences in target sequence recoveries from different calibrator sample cell sources when they are used in the comparative Ct method. [Copyright &y& Elsevier]

The article presents the results of the study which determines whether specific molds were found in significantly higher concentrations in the water-damaged homes of asthmatic children compared to the homes with no visible water damage in Cleveland, Ohio. The researchers used the mold-specific quantitative polymerase chain reaction to measure the mold concentrations in the asthmatic children's bedrooms located in water-damaged and control homes. It was discovered that the molds Scopulariopsis brevicaulis and Trichoderma viride had higher concentrations in the dwellings of asthmatic kids.