Scaphoid Fractures in Children: Do We Need to X-ray? A Retrospective Chart Review of 144 Wrists.

OBJECTIVES: The purpose of this study is to determine the predictive value of clinical suspicion for scaphoid fracture in children aged 4 to 11 years, to look at the efficiency and practicality of current management of children presenting to the emergency department, and to help quantify the burden of the treatment strategy of immobilization for 10 to 14 days on clinical grounds despite negative or equivocal x-rays on presentation. METHODS: We performed a retrospective chart review study of a consecutive sample of all children aged 4 to 11 years old who presented to a tertiary pediatric emergency department from January 1, 2009, to December 31, 2013, within 24 hours of a wrist injury, with a clinical suspicion sufficient to order a scaphoid x-ray. Our primary outcome of interest was the positive predictive value of clinical suspicion of a scaphoid fracture. RESULTS: There were 142 patients (144 wrists) included in the study. Of these, 110 had a normal initial x-ray. Initial x-rays showed evidence of nonscaphoid fractures in 30 wrists and equivocal scaphoid fractures in 4 wrists. Of the 110 wrists with normal initial x-rays, 69 were immobilized and 52 of those had a follow-up x-ray (47 normal, 1 scaphoid fracture, 1 nonscaphoid fracture, and 3 equivocal evidence of a scaphoid fracture). The positive predictive value was found to be 2.1% to 12.5%. CONCLUSIONS: This study suggests that children aged 4 to 11 years with clinical suspicion for scaphoid fractures do not necessarily need to be x-rayed or immobilized. A larger multicentered prospective clinical trial would confirm this.

The Inhibition and Variability of Two Different RT-qPCR Assays Used for Quantifying SARS-CoV-2 RNA in Wastewater.

Faecal shedding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its subsequent detection in wastewater turned the spotlight onto wastewater-based epidemiology (WBE) for monitoring the coronavirus-disease 2019 (COVID-19) pandemic. WBE for SARS-CoV-2 has been deployed in 70 countries, providing insights into disease prevalence, forecasting and the spatiotemporal tracking and emergence of SARS-CoV-2 variants. Wastewater, however, is a complex sample matrix containing numerous reverse transcription quantitative PCR (RT-qPCR) inhibitors whose concentration and diversity are influenced by factors including population size, surrounding industry and agriculture and climate. Such differences in the RT-qPCR inhibitor profile are likely to impact the quality of data produced by WBE and potentially produce erroneous results.To help determine the possible impact of RT-qPCR assay on data quality, two assays employed by different laboratories within the UK's SARS-CoV-2 wastewater monitoring programme were assessed in the Cefas laboratory in Weymouth, UK. The assays were based on Fast Virus (FV) and qScript (qS) chemistries using the same primers and probes, but at different concentrations and under different cycling conditions. Bovine serum albumin and MgSO4 were also added to the FV assay reaction mixture. Two-hundred and eighty-six samples were analysed, and an external control RNA (EC RNA)-based method was used to measure RT-qPCR inhibition. Compared with qS, FV showed a 40.5% reduction in mean inhibition and a 57.0% reduction in inter-sample inhibition variability. A 4.1-fold increase in SARS-CoV-2 quantification was seen for FV relative to qS; partially due (1.5-fold) to differences in reverse transcription efficiency and the use of a dsDNA standard. Analytical variability was reduced by 51.2% using FV while qS increased the number of SARS-CoV-2 negative samples by 2.6-fold. This study indicates the importance of thorough method optimisation for RT-qPCR-based WBE which should be performed using a selection of samples which are representative of the physiochemical properties of wastewater. Furthermore, RT-qPCR inhibition, analytical variability and reverse transcription efficiency should be key considerations during assay optimisation. A standardised framework for the optimisation and validation of WBE procedures should be formed including concessions for emergency response situations that would allow flexibility in the process to address the difficult balance between the urgency of providing data and the availability of resources.

Comparison of metagenomic and targeted methods for sequencing human pathogenic viruses from wastewater.

Wastewater-based epidemiology is a powerful tool for monitoring the emergence and spread of viral pathogens at the population scale. Typical polymerase chain reaction (PCR)-based methods of quantitative and genomic monitoring of viruses in wastewater provide high sensitivity and specificity. However, these methods are limited to the surveillance of target viruses in a single assay and require prior knowledge of the target genome(s). Metagenomic sequencing methods may represent a target-agnostic approach to viral wastewater monitoring, allowing for the detection of a broad range of target viruses, including potentially novel and emerging pathogens. In this study, targeted and untargeted metagenomic sequencing methods were compared with tiled-PCR sequencing for the detection and genotyping of viral pathogens in wastewater samples. Deep shotgun metagenomic sequencing was unable to generate sufficient genome coverage of human pathogenic viruses for robust genomic epidemiology, with samples dominated by bacteria. Hybrid-capture enrichment of shotgun libraries for respiratory viruses led to significant increases in genome coverage for a range of targets. Tiled-PCR sequencing led to further improvements in genome coverage compared to hybrid capture for severe acute respiratory syndrome coronavirus 2, enterovirus D68, norovirus GII, and human adenovirus F41 in wastewater samples. In conclusion, untargeted shotgun sequencing was unsuitable for genomic monitoring of the low virus concentrations in wastewater samples analyzed in this study. Hybrid-capture enrichment represented a viable method for simultaneous genomic epidemiology of a range of viral pathogens, while tiled-PCR sequencing provided the optimal genome coverage for individual viruses with the minimum sequencing depth. IMPORTANCE Most public health initiatives that monitor viruses in wastewater have utilized quantitative polymerase chain reaction (PCR) and whole genome PCR sequencing, mirroring techniques used for viral epidemiology in individuals. These techniques require prior knowledge of the target viral genome and are limited to monitoring individual or small groups of viruses. Metagenomic sequencing may offer an alternative strategy for monitoring a broad spectrum of viruses in wastewater, including novel and emerging pathogens. In this study, while amplicon sequencing gave high viral genome coverage, untargeted shotgun sequencing of total nucleic acid samples was unable to detect human pathogenic viruses with enough sensitivity for use in genomic epidemiology. Enrichment of shotgun libraries for respiratory viruses using hybrid-capture technology provided genotypic information on a range of viruses simultaneously, indicating strong potential for wastewater surveillance. This type of targeted metagenomics could be used for monitoring diverse targets, such as pathogens or antimicrobial resistance genes, in environmental samples.

Utility of wastewater genomic surveillance compared to clinical surveillance to track the spread of the SARS-CoV-2 Omicron variant across England.

The world has moved into a new stage of managing the SARS-CoV-2 pandemic with minimal restrictions and reduced testing in the population, leading to reduced genomic surveillance of virus variants in individuals. Wastewater-based epidemiology (WBE) can provide an alternative means of tracking virus variants in the population but decision-makers require confidence that it can be applied to a national scale and is comparable to individual testing data. We analysed 19,911 samples from 524 wastewater sites across England at least twice a week between November 2021 and February 2022, capturing sewage from >70% of the English population. We used amplicon-based sequencing and the phylogeny based de-mixing tool Freyja to estimate SARS-CoV-2 variant frequencies and compared these to the variant dynamics observed in individual testing data from clinical and community settings. We show that wastewater data can reconstruct the spread of the Omicron variant across England since November 2021 in close detail and aligns closely with epidemiological estimates from individual testing data. We also show the temporal and spatial spread of Omicron within London. Our wastewater data further reliably track the transition between Omicron subvariants BA1 and BA2 in February 2022 at regional and national levels. Our demonstration that WBE can track the fast-paced dynamics of SARS-CoV-2 variant frequencies at a national scale and closely match individual testing data in time shows that WBE can reliably fill the monitoring gap left by reduced individual testing in a more affordable way.

Tips for improving consistency of thyroid nodule interpretation with ACR TI-RADS.

Thyroid nodules are very common in the general population. Most are benign and even those that are malignant are typically slow-growing and do not require treatment. Overdiagnosis and overtreatment of thyroid nodules has resulted in significant healthcare costs. ACR TI-RADS was developed to address these concerns, and reduce the number of unnecessary biopsies and follow-up intervals. ACR TI-RADS offers a point-based risk stratification system centered on five sonographic features: consistency, echogenicity, shape, margins and echogenic foci. While the system has noticeable benefits and comparable accuracy with other available risk stratification systems (ATA, EU-TIRADS and K-TIRADS), there are inherent challenges relating to suboptimal inter-reader agreement. In this article, we include 10 educational tips that may be helpful to the ultrasound practitioner for improving the consistency of nodule interpretation with ACR TI-RADS.

Effect of training on resident inter-reader agreement with American College of Radiology Thyroid Imaging Reporting and Data System.

BACKGROUND: The American College of Radiology Thyroid Imaging Reporting and Data System (ACR TI-RADS) was introduced to standardize the ultrasound characterization of thyroid nodules. Studies have shown that ACR-TIRADS reduces unnecessary biopsies and improves consistency of imaging recommendations. Despite its widespread adoption, there are few studies to date assessing the inter-reader agreement amongst radiology trainees with limited ultrasound experience. We hypothesize that in PGY-4 radiology residents with no prior exposure to ACR TI-RADS, a statistically significant improvement in inter-reader reliability can be achieved with a one hour training session. AIM: To evaluate the inter-reader agreement of radiology residents in using ACR TI-RADS before and after training. METHODS: A single center retrospective cohort study evaluating 50 thyroid nodules in 40 patients of varying TI-RADS levels was performed. Reference standard TI-RADS scores were established through a consensus panel of three fellowship-trained staff radiologists with between 1 and 14 years of clinical experience each. Three PGY-4 radiology residents (trainees) were selected as blinded readers for this study. Each trainee had between 4 to 5 mo of designated ultrasound training. No trainee had received specialized TI-RADS training prior to this study. Each of the readers independently reviewed the 50 testing cases and assigned a TI-RADS score to each case before and after TI-RADS training performed 6 wk apart. Fleiss kappa was used to measure the pooled inter-reader agreement. The relative diagnostic performance of readers, pre- and post-training, when compared against the reference standard. RESULTS: There were 33 females and 7 males with a mean age of 56.6 ± 13.6 years. The mean nodule size was 19 ± 14 mm (range from 5 to 63 mm). A statistically significant superior inter-reader agreement was found on the post-training assessment compared to the pre-training assessment for the following variables: 1. "Shape" (k of 0.09 [slight] pre-training vs 0.67 [substantial] post-training, P < 0.001), 2. "Echogenic foci" (k of 0.28 [fair] pre-training vs 0.45 [moderate] post-training, P = 0.004), 3. 'TI-RADS level' (k of 0.14 [slight] pre-training vs 0.36 [fair] post-training, P < 0.001) and 4. 'Recommendations' (k of 0.36 [fair] pre-training vs 0.50 [moderate] post-training, P = 0.02). No significant differences between the pre- and post-training assessments were found for the variables 'composition', 'echogenicity' and 'margins'. There was a general trend towards improved pooled sensitivity with TI-RADS levels 1 to 4 for the post-training assessment while the pooled specificity was relatively high (76.6%-96.8%) for all TI-RADS level. CONCLUSION: Statistically significant improvement in inter-reader agreement in the assigning TI-RADS level and recommendations after training is observed. Our study supports the use of dedicated ACR TI-RADS training in radiology residents.

A comparison of precipitation and filtration-based SARS-CoV-2 recovery methods and the influence of temperature, turbidity, and surfactant load in urban wastewater.

Wastewater-based epidemiology (WBE) has become a complimentary surveillance tool during the SARS-CoV-2 pandemic. Viral concentration methods from wastewater are still being optimised and compared, whilst viral recovery under different wastewater characteristics and storage temperatures remains poorly understood. Using urban wastewater samples, we tested three viral concentration methods; polyethylene glycol precipitation (PEG), ammonium sulphate precipitation (AS), and CP select™ InnovaPrep® (IP) ultrafiltration. We found no major difference in SARS-CoV-2 and faecal indicator virus (crAssphage) recovery from wastewater samples (n = 46) using these methods, PEG slightly (albeit non-significantly), outperformed AS and IP for SARS-CoV-2 detection, as a higher genome copies per litre (gc/l) was recorded for a larger proportion of samples. Next generation sequencing of 8 paired samples revealed non-significant differences in the quality of data between AS and IP, though IP data quality was slightly better and less variable. A controlled experiment assessed the impact of wastewater suspended solids (turbidity; 0-400 NTU), surfactant load (0-200 mg/l), and storage temperature (5-20 °C) on viral recovery using the AS and IP methods. SARS-CoV-2 recoveries were >20% with AS and <10% with IP in turbid samples, whilst viral recoveries for samples with additional surfactant were between 0-18% for AS and 0-5% for IP. Turbidity and sample storage temperature combined had no significant effect on SARS-CoV-2 recovery (p > 0.05), whilst surfactant and storage temperature combined were significant negative correlates (p < 0.001 and p < 0.05, respectively). In conclusion, our results show that choice of methodology had small effect on viral recovery of SARS-CoV-2 and crAssphage in wastewater samples within this study. In contrast, sample turbidity, storage temperature, and surfactant load did affect viral recovery, highlighting the need for careful consideration of the viral concentration methodology used when working with wastewater samples.

An analysis of 45 large-scale wastewater sites in England to estimate SARS-CoV-2 community prevalence.

Accurate surveillance of the COVID-19 pandemic can be weakened by under-reporting of cases, particularly due to asymptomatic or pre-symptomatic infections, resulting in bias. Quantification of SARS-CoV-2 RNA in wastewater can be used to infer infection prevalence, but uncertainty in sensitivity and considerable variability has meant that accurate measurement remains elusive. Here, we use data from 45 sewage sites in England, covering 31% of the population, and estimate SARS-CoV-2 prevalence to within 1.1% of estimates from representative prevalence surveys (with 95% confidence). Using machine learning and phenomenological models, we show that differences between sampled sites, particularly the wastewater flow rate, influence prevalence estimation and require careful interpretation. We find that SARS-CoV-2 signals in wastewater appear 4-5 days earlier in comparison to clinical testing data but are coincident with prevalence surveys suggesting that wastewater surveillance can be a leading indicator for symptomatic viral infections. Surveillance for viruses in wastewater complements and strengthens clinical surveillance, with significant implications for public health.

Genome evolution and the emergence of pathogenicity in avian Escherichia coli.

Chickens are the most common birds on Earth and colibacillosis is among the most common diseases affecting them. This major threat to animal welfare and safe sustainable food production is difficult to combat because the etiological agent, avian pathogenic Escherichia coli (APEC), emerges from ubiquitous commensal gut bacteria, with no single virulence gene present in all disease-causing isolates. Here, we address the underlying evolutionary mechanisms of extraintestinal spread and systemic infection in poultry. Combining population scale comparative genomics and pangenome-wide association studies, we compare E. coli from commensal carriage and systemic infections. We identify phylogroup-specific and species-wide genetic elements that are enriched in APEC, including pathogenicity-associated variation in 143 genes that have diverse functions, including genes involved in metabolism, lipopolysaccharide synthesis, heat shock response, antimicrobial resistance and toxicity. We find that horizontal gene transfer spreads pathogenicity elements, allowing divergent clones to cause infection. Finally, a Random Forest model prediction of disease status (carriage vs. disease) identifies pathogenic strains in the emergent ST-117 poultry-associated lineage with 73% accuracy, demonstrating the potential for early identification of emergent APEC in healthy flocks.

The transcriptional programme of Salmonella enterica serovar Typhimurium reveals a key role for tryptophan metabolism in biofilms.

BACKGROUND: Biofilm formation enhances the capacity of pathogenic Salmonella bacteria to survive stresses that are commonly encountered within food processing and during host infection. The persistence of Salmonella within the food chain has become a major health concern, as biofilms can serve as a reservoir for the contamination of food products. While the molecular mechanisms required for the survival of bacteria on surfaces are not fully understood, transcriptional studies of other bacteria have demonstrated that biofilm growth triggers the expression of specific sets of genes, compared with planktonic cells. Until now, most gene expression studies of Salmonella have focused on the effect of infection-relevant stressors on virulence or the comparison of mutant and wild-type bacteria. However little is known about the physiological responses taking place inside a Salmonella biofilm. RESULTS: We have determined the transcriptomic and proteomic profiles of biofilms of Salmonella enterica serovar Typhimurium. We discovered that 124 detectable proteins were differentially expressed in the biofilm compared with planktonic cells, and that 10% of the S. Typhimurium genome (433 genes) showed a 2-fold or more change in the biofilm compared with planktonic cells. The genes that were significantly up-regulated implicated certain cellular processes in biofilm development including amino acid metabolism, cell motility, global regulation and tolerance to stress. We found that the most highly down-regulated genes in the biofilm were located on Salmonella Pathogenicity Island 2 (SPI2), and that a functional SPI2 secretion system regulator (ssrA) was required for S. Typhimurium biofilm formation. We identified STM0341 as a gene of unknown function that was needed for biofilm growth. Genes involved in tryptophan (trp) biosynthesis and transport were up-regulated in the biofilm. Deletion of trpE led to decreased bacterial attachment and this biofilm defect was restored by exogenous tryptophan or indole. CONCLUSIONS: Biofilm growth of S. Typhimurium causes distinct changes in gene and protein expression. Our results show that aromatic amino acids make an important contribution to biofilm formation and reveal a link between SPI2 expression and surface-associated growth in S. Typhimurium.

Validation of host-specific Bacteriodales 16S rRNA genes as markers to determine the origin of faecal pollution in Atlantic Rim countries of the European Union.

The recent implementation of the Revised Bathing Water Directive in the European Union has highlighted the need for development of effective methods to differentiate between sources of faecal contamination. It had previously been shown that amplification of 16S rRNA genes of host-specific Bacteriodales species using the HF183F and CF128F primers could be used as markers for human and bovine faecal contamination in the United States. This paper determined the sensitivity and specificity of these markers in four Atlantic Rim countries (France, Ireland, Portugal and the United Kingdom) to evaluate their usefulness in determining the origin of faecal contamination. It was shown that the HF183F marker displayed high sensitivity (80-100%) and specificity (91-100%), and is reliable as an indication of human faecal contamination. The CF128F marker displayed 100% sensitivity in all four countries. However, strong regional variations in specificity (41-96%) were observed, highlighting the need for local validation before this marker is employed in source tracking of faecal contamination.

Structural basis for DNA recognition by the transcription regulator MetR.

MetR, a LysR-type transcriptional regulator (LTTR), has been extensively studied owing to its role in the control of methionine biosynthesis in proteobacteria. A MetR homodimer binds to a 24-base-pair operator region of the met genes and specifically recognizes the interrupted palindromic sequence 5'-TGAA-N5-TTCA-3'. Mechanistic details underlying the interaction of MetR with its target DNA at the molecular level remain unknown. In this work, the crystal structure of the DNA-binding domain (DBD) of MetR was determined at 2.16 Šresolution. MetR-DBD adopts a winged-helix-turn-helix (wHTH) motif and shares significant fold similarity with the DBD of the LTTR protein BenM. Furthermore, a data-driven macromolecular-docking strategy was used to model the structure of MetR-DBD bound to DNA, which revealed that a bent conformation of DNA is required for the recognition helix α3 and the wing loop of the wHTH motif to interact with the major and minor grooves, respectively. Comparison of the MetR-DBD-DNA complex with the crystal structures of other LTTR-DBD-DNA complexes revealed residues that may confer operator-sequence binding specificity for MetR. Taken together, the results show that MetR-DBD uses a combination of direct base-specific interactions and indirect shape recognition of the promoter to regulate the transcription of met genes.

The Use of Biochip Array Technology for Rapid Multimycotoxin Screening.

The main known groups of mycotoxins are aflatoxins, fumonisins, ochratoxins, type A trichothecenes (T-2 toxin and HT-2 toxin), type B trichothecenes (deoxynivalenol), and zearalenones. They are harmful to humans, domestic animals, and livestock. In Europe, maximum permitted limits for aflatoxin B1 are set, and guidance levels are recommended for the other mycotoxins. This study applied biochip array technology to semiquantitative multimycotoxin screening at different levels to facilitate the verification of the compliance of feed material with acceptable safety standards. This application was developed and validated based on European Commission Decision No. 2002/657/EC. After a single generic sample-preparation method, simultaneous competitive chemiluminescent immunoassays were used and applied to the Evidence Investigator analyzer. The r and within-laboratory R values showed low overall CVs (10.6 and 11.6%, respectively). Low matrix effect and, consequently, low decision limits and detection capabilities proved the high sensitivity of the technology. The overall average recovery was 104%. Samples (n = 16) investigated within the Food Analysis Performance Assessment Scheme (FAPAS) program showed excellent correlation to assigned values. FAPAS proficiency-testing feed samples (n = 10) were within the schemes' z-score ±2 range. The authentic feed samples survey showed excellent correlation with LC-MS/MS. This application is, therefore, reliable and represents an innovative, cost-effective, and multianalytical tool for mycotoxin screening.

Molecular tools for bathing water assessment in Europe: Balancing social science research with a rapidly developing environmental science evidence-base.

The use of molecular tools, principally qPCR, versus traditional culture-based methods for quantifying microbial parameters (e.g., Fecal Indicator Organisms) in bathing waters generates considerable ongoing debate at the science-policy interface. Advances in science have allowed the development and application of molecular biological methods for rapid (~2 h) quantification of microbial pollution in bathing and recreational waters. In contrast, culture-based methods can take between 18 and 96 h for sample processing. Thus, molecular tools offer an opportunity to provide a more meaningful statement of microbial risk to water-users by providing near-real-time information enabling potentially more informed decision-making with regard to water-based activities. However, complementary studies concerning the potential costs and benefits of adopting rapid methods as a regulatory tool are in short supply. We report on findings from an international Working Group that examined the breadth of social impacts, challenges, and research opportunities associated with the application of molecular tools to bathing water regulations.

Rains, drains and active strains: towards online assessment of wastewater bacterial communities.

Wastewater treatment is one of the largest scale and arguably the most commercially important biotechnological process in the world. Bacterial breakdown of waste materials facilitates the safe disposal of effluents into receiving water bodies. Given this significance, research has focused on identifying the keystone species on which efficient treatment is based. However, unravelling the microbial diversity within such systems has proven difficult. This is highlighted by our lack of detailed knowledge of the microbial interactions within these complex populations, limiting our ability to fully exploit bacterial degradative abilities. Even with the incorporation of new emerging molecular techniques, there have been no investigations linking genetic sequence to microbial function and successful treatment operation. To reach this goal, researchers need the ability to identify, enumerate and monitor the metabolic functions of subpopulations within these complex bacterial communities. Flow cytometry (FCM) combined with fluorescence-based molecular identification techniques provides a method for such studies. Moreover, single-cell sorting provides a unique opportunity to identify and remove individual cells of interest. Laboratory culture of sorted cells is often possible and permits the use of more traditional microbiological techniques to backup molecular investigations. Utilising this approach will advance our understanding of wastewater treatment processes and help maintain and enhance plant operation to improve efficiency.

Opportunities and limitations of molecular methods for quantifying microbial compliance parameters in EU bathing waters.

The debate over the suitability of molecular biological methods for the enumeration of regulatory microbial parameters (e.g. Faecal Indicator Organisms [FIOs]) in bathing waters versus the use of traditional culture-based methods is of current interest to regulators and the science community. Culture-based methods require a 24-48hour turn-around time from receipt at the laboratory to reporting, whilst quantitative molecular tools provide a more rapid assay (approximately 2-3h). Traditional culturing methods are therefore often viewed as slow and 'out-dated', although they still deliver an internationally 'accepted' evidence-base. In contrast, molecular tools have the potential for rapid analysis and their operational utility and associated limitations and uncertainties should be assessed in light of their use for regulatory monitoring. Here we report on the recommendations from a series of international workshops, chaired by a UK Working Group (WG) comprised of scientists, regulators, policy makers and other stakeholders, which explored and interrogated both molecular (principally quantitative polymerase chain reaction [qPCR]) and culture-based tools for FIO monitoring under the European Bathing Water Directive. Through detailed analysis of policy implications, regulatory barriers, stakeholder engagement, and the needs of the end-user, the WG identified a series of key concerns that require critical appraisal before a potential shift from culture-based approaches to the employment of molecular biological methods for bathing water regulation could be justified.

Evaluating short-term changes in recreational water quality during a hydrograph event using a combination of microbial tracers, environmental microbiology, microbial source tracking and hydrological techniques: a case study in Southwest Wales, UK.

Quantitative assessment of multiple sources to short-term variations in recreational water quality, as indexed by faecal indicator organism (FIO) concentrations, is becoming increasingly important with adoption of modern water quality standards and catchment-based water quality management requirements (e.g. the EU Water Framework Directive, Article 11 'Programmes of Measures' and the US Clean Water Act, 'Total Maximum Daily Loads'). This paper describes a study combining microbial tracers, intensive FIO measurement, open channel hydrology and molecular microbial source tracking (MST) to enhance understanding of recreational water quality at Amroth in southwest Wales, UK. Microbial tracers were released from four stream inputs during a moderate hydrograph event. Tracers from two local streams impacted simultaneously with a period of maximum FIO concentrations at the near-shore compliance monitoring site. Connection between these inputs and this site were rapid (9-33 min). Water quality impairment from a more remote stream input followed, 12.85 h after tracer release, sustaining FIO concentrations above desired compliance levels. MST analysis showed dominance of ruminant Bacteroidales genetic markers, associated with agricultural pollution. This integration of tracers and MST offers additional information on the movement and individual sources causing water quality impairment.

Evaluating the operational utility of a Bacteroidales quantitative PCR-based MST approach in determining the source of faecal indicator organisms at a UK bathing water.

Microbial source tracking techniques are used in the UK to provide an evidence-base to guide major expenditure decisions and/or regulatory action relating to sewage disposal. Consequently, it is imperative that the techniques used robustly index faecal indicator organisms (FIOs) that are the regulatory parameters for bathing and shellfish harvesting areas. This study reports a 'field-scale' test of microbial source tracking (MST) based on the quantitative PCR analyses of Bacteroidales 16S rRNA genetic marker sequences. The project acquired data to test the operational utility of quantitative Bacteroidales MST data, comparing it with FIO concentrations in streams, effluents and bathing waters. Overall, the data did not exhibit a consistent pattern of significant correlations between Bacteroidales MST parameters and FIOs within the different sample matrices (i.e. rivers, bathing waters and/or effluents). Consequently, there was little evidence from this study that reported concentrations and/or percentages of human and/or ruminant faecal loadings (that are based on Bacteroidales MST gene copy numbers) offer a credible evidence-base describing FIO contributions to receiving water 'non-compliance'. The study also showed (i) there was no significant attenuation of the Bacteroidales gene copy number 'signal' through the UV disinfection process; and (ii) single non-compliant samples submitted for Bacteroidales MST analysis, do not reliably characterise the balance of faecal loadings due to the high variability in the MST signal observed. At this stage in the development of the MST tool deployed, it would be imprudent to use the percentage human and/or ruminant contributions (i.e. as indicated by MST data acquired at a bathing water) as the sole or principal element in the evidence-base used to guide major expenditure decisions and/or regulatory action.