Quantitative fecal source characterization of urban municipal storm sewer system outfall 'wet' and 'dry' weather discharges.

Urban areas are built environments containing substantial amounts of impervious surfaces (e.g., streets, sidewalks, roof tops). These areas often include elaborately engineered drainage networks designed to collect, transport, and discharge untreated stormwater into local surface waters. When left uncontrolled, these discharges may contain unsafe levels of fecal waste from sources such as sanitary sewage and wildlife even under dry weather conditions. This study evaluates paired measurements of host-associated genetic markers (log10 copies per reaction) indicative of human (HF183/BacR287 and HumM2), ruminant (Rum2Bac), canine (DG3), and avian (GFD) fecal sources, 12-hour cumulative precipitation (mm), four catchment land use metrics determined by global information system (GIS) mapping, and Escherichia coli (MPN/100 ml) from seven municipal separate storm sewer system outfall locations situated at the southern portion of the Anacostia River Watershed (District of Columbia, U.S.A.). A total of 231 discharge samples were collected twice per month (n = 24 sampling days) and after rain events (n = 9) over a 13-month period. Approximately 50 % of samples (n = 116) were impaired, exceeding the local E. coli single sample maximum of 2.613 log10 MPN/100 ml. Genetic quality controls indicated the absence of amplification inhibition in 97.8 % of samples, however 14.7 % (n = 34) samples showed bias in DNA recovery. Of eligible samples, quantifiable levels were observed for avian (84.1 %), human (57.4 % for HF183/BacR287 and 40 % for HumM2), canine (46.7 %), and ruminant (15.9 %) host-associated genetic markers. Potential links between paired measurements are explored with a recently developed Bayesian qPCR censored data analysis approach. Findings indicate that human, pet, and urban wildlife all contribute to storm outfall discharge water quality in the District of Columbia, but pollutant source contributions vary based on 'wet' and 'dry' conditions and catchment land use, demonstrating that genetic-based fecal source identification methods combined with GIS land use mapping can complement routine E. coli monitoring to improve stormwater management in urban areas.

Occurrence of recreational water quality monitoring general fecal indicator bacteria and fecal source identification genetic markers in gray seal scat.

The number of gray seals (Halichoerus grypus) observed along the United States Northwest Atlantic region has been increasing for decades. These colonial animals often haul-out on beaches seasonally in numbers ranging from a few individuals to several thousands. While these larger aggregations are an important part of gray seal behavior, there is public concern that haul-outs could lead to large amounts of fecal waste in recreational areas, potentially resulting in beach closures. Yet, data to confirm whether these animals contribute to beach closures is lacking and minimal information is available on the occurrence of key water quality monitoring genetic markers in gray seal scat. This study evaluates the concentration of E. coli (EC23S857), enterococci (Entero1a), and fecal Bacteroidetes (GenBac3) as well as six fecal source identification genetic markers (HF183/BacR287, HumM2, CPQ_056, Rum2Bac, DG3, and GFD) measured by qPCR in 48 wild gray seal scat samples collected from two haul-out areas in Cape Cod (Massachusetts, U.S.A.). Findings indicate that FIB genetic markers are shed in gray seal scat at significantly different concentrations with the Entero1a genetic marker exhibiting the lowest average concentration (-0.73 log10 estimated mean copies per nanogram of DNA). In addition, systematic testing of scat samples demonstrated that qPCR assays targeting host-associated genetic markers indicative of human, ruminant, and canine fecal pollution sources remain highly specific in waters frequented by gray seals (>97 % specificity).

A fecal score approximation model for analysis of real-time quantitative PCR fecal source identification measurements.

Numerous qPCR-based methods are available to estimate the concentration of fecal pollution sources in surface waters. However, qPCR fecal source identification data sets often include a high proportion of non-detections (reactions failing to attain a prespecified minimal signal intensity for detection) and measurements below the assay lower limit of quantification (minimal signal intensity required to estimate target concentration), making it challenging to interpret results in a quantitative manner while accounting for error. In response, a Bayesian statistic based Fecal Score (FS) approach was developed that estimates the weighted average concentration of a fecal source identification genetic marker across a defined group of samples, mathematically incorporating qPCR measurements from all samples. Yet, implementation is technically demanding and computationally intensive requiring specialized training, the use of expert software, and access to high performance computing. To address these limitations, this study reports a novel approximation model for FS determination based on a frequentist approach. The performance of the Bayesian and Frequentist models are compared using fecal source identification qPCR data representative of different 'censored' data scenarios from a recently published study focusing on the impact of stormwater discharge in urban streams. In addition, data set eligibility recommendations for the responsible use of these models are presented. Findings indicate that the Frequentist model can generate similar average concentrations and uncertainty estimates for FS, compared to the original Bayesian approach. The Frequentist model should make calculations less computationally and technically intensive, allowing for the development of easier to use data analysis tools for fecal source identification applications.

Physiologic effects of surgical masking in children versus adults.

Background: Surgical masks remain a focal part of the CDC guidelines to decrease COVID-19 transmission. Evidence refuting significant effects of masking on ventilation is mostly limited to small studies, with a paucity of studies on children, and none comparing children to adults. Methods: A total of 119 subjects were enrolled (71 adults, 49 children) in a prospective interventional study with each subject serving as their own mask-free control. End tidal CO2 (ETCO2), inspired CO2 (ICO2), and respiratory rate were measured by nasal cannula attached to an anesthesia machine D-fend module. Pulse oximetry and heart rate were also followed. After the mask-free period, an ASTM Level 3 disposable surgical mask was donned and 15 min of mask-worn data were collected. Results: A steady state was confirmed for ETCO2 and ICO2 over the masked period, and mean ICO2 levels rose significantly (p < 0.001) after masking in all age groups. The increase in ICO2 for the 2- to 7-year-old group of 4.11 mmHg (3.23-4.99), was significantly higher (p < 0.001) than the final ΔICO2 levels for both the 7- to 14-year-old group, 2.45 mmHg (1.79-3.12), and adults, 1.47 mmHg (1.18-1.76). For the pediatric group there was a negative, significant correlation between age and ΔICO2, r = -0.49, p < 0.001. Masking resulted in a statistically significant (p < 0.01) rise in ETCO2 levels of 1.30 mmHg in adults and 1.36 mmHg in children. The final respective ETCO2 levels, 34.35 (33.55-35.15) and 35.07 (34.13-36.01), remained within normal limits. Pulse oximetry, heart rate, and respiratory rate were not significantly affected. Discussion: The physiology of mechanical dead space is discussed, including the inverse relationship of subject age vs ICO2. The methodology and results are compared to previously published studies which detracted from the physiologic safety of surgical masking. Conclusions: The wearing of a surgical mask results in a statistically significant rise in ICO2 and a smaller rise in ETCO2. Because ETCO2 and other variables remain well within normal limits, these changes are clinically insignificant.

Genetic fecal source identification in urban streams impacted by municipal separate storm sewer system discharges.

Municipal stormwater systems are designed to collect, transport, and discharge precipitation from a defined catchment area into local surface waters. However, these discharges may contain unsafe levels of fecal waste. Paired measurements of Escherichia coli, precipitation, three land use metrics determined by geographic information system (GIS) mapping, and host-associated genetic markers indicative of human (HF183/BacR287 and HumM2), ruminant (Rum2Bac), dog (DG3), and avian (GFD) fecal sources were assessed in 231 urban stream samples impacted by two or more municipal stormwater outfalls. Receiving water samples were collected twice per month (n = 24) and after rain events (n = 9) from seven headwaters of the Anacostia River in the District of Columbia (United States) exhibiting a gradient of impervious surface, residential, and park surface areas. Almost 50% of stream samples (n = 103) were impaired, exceeding the local E. coli single sample maximum assessment level (410 MPN/100 ml). Fecal scores (average log10 copies per 100 ml) were determined to prioritize sites by pollution source and to evaluate potential links with land use, rainfall, and E. coli levels using a recently developed censored data analysis approach. Dog, ruminant, and avian fecal scores were almost always significantly increased after rain or when E. coli levels exceeded the local benchmark. Human fecal pollution trends showed the greatest variability with detections ranging from 9.1% to 96.7% across sites. Avian fecal scores exhibited the closest connection to land use, significantly increasing in catchments with larger residential areas after rain events (p = 0.038; R2 = 0.62). Overall, results demonstrate that combining genetic fecal source identification methods with GIS mapping complements routine E. coli monitoring to improve management of urban streams impacted by stormwater outfalls.

Interlaboratory performance and quantitative PCR data acceptance metrics for NIST SRM® 2917.

Surface water quality quantitative polymerase chain reaction (qPCR) technologies are expanding from a subject of research to routine environmental and public health laboratory testing. Readily available, reliable reference material is needed to interpret qPCR measurements, particularly across laboratories. Standard Reference Material® 2917 (NIST SRM® 2917) is a DNA plasmid construct that functions with multiple water quality qPCR assays allowing for estimation of total fecal pollution and identification of key fecal sources. This study investigates SRM 2917 interlaboratory performance based on repeated measures of 12 qPCR assays by 14 laboratories (n = 1008 instrument runs). Using a Bayesian approach, single-instrument run data are combined to generate assay-specific global calibration models allowing for characterization of within- and between-lab variability. Comparable data sets generated by two additional laboratories are used to assess new SRM 2917 data acceptance metrics. SRM 2917 allows for reproducible single-instrument run calibration models across laboratories, regardless of qPCR assay. In addition, global models offer multiple data acceptance metric options that future users can employ to minimize variability, improve comparability of data across laboratories, and increase confidence in qPCR measurements.

Performance of NIST SRM® 2917 with 13 recreational water quality monitoring qPCR assays.

Fecal pollution remains a significant challenge for recreational water quality management worldwide. In response, there is a growing interest in the use of real-time quantitative PCR (qPCR) methods to achieve same-day notification of recreational water quality and associated public health risk as well as to characterize fecal pollution sources for targeted mitigation. However, successful widespread implementation of these technologies requires the development of and access to a high-quality standard control material. Here, we report a single laboratory qPCR performance assessment of the National Institute of Standards and Technology Standard Reference Material 2917 (NIST SRM® 2917), a linearized plasmid DNA construct that functions with 13 recreational water quality qPCR assays. Performance experiments indicate the generation of standard curves with amplification efficiencies ranging from 0.95 ± 0.006 to 0.99 ± 0.008 and coefficient of determination values (R2) ≥ 0.980. Regardless of qPCR assay, variability in repeated measurements at each dilution level were very low (quantification threshold standard deviations ≤ 0.657) and exhibited a heteroscedastic trend characteristic of qPCR standard curves. The influence of a yeast carrier tRNA added to the standard control material buffer was also investigated. Findings demonstrated that NIST SRM® 2917 functions with all qPCR methods and suggests that the future use of this control material by scientists and water quality managers should help reduce variability in concentration estimates and make results more consistent between laboratories.

Engaging the Voices of Children: A Scoping Review of How Children and Adolescents Are Involved in the Development of Quality-of-Life-Related Measures.

OBJECTIVES: Patient-reported outcomes are increasingly recommended to guide patient care, develop and evaluate interventions, and modify health systems. However, not enough is known about whether and how children and adolescents, as "experts" in their own health and quality of life (QoL), are being engaged in the development of instruments. Our goals in this review were (1) to identify all QoL-related instruments that have included children and/or adolescents in the development of questionnaire content, including identification of themes and items; and (2) to report how this was done; and (3) to highlight those that used qualitative methods. METHODS: MEDLINE and Embase were searched for child- or adolescent-completed QoL-related instruments, supplemented by hand-searching of relevant reviews until 2020. Original development papers were identified and retrieved when possible, from which instrument characteristics and details of qualitative development methods were extracted. RESULTS: We identified 445 instruments, of which 88 used qualitative methods for content development. Interviews and focus groups were the most common methods. A variety of play techniques were used to engage the child and adolescent participants. The specific criteria for the inclusion of children and adolescents (age, developmental stage, duration, and nonclinical location) varied considerably. CONCLUSIONS: Researchers frequently involve children and adolescents in qualitative methods when developing QoL-related measures; however, there is little information about the methods used. Better reporting of methodology, improved dissemination of methods guidelines, and research into optimal ways of including children and adolescents in the process of instrument development would be useful.

Genomic Survey of E. coli From the Bladders of Women With and Without Lower Urinary Tract Symptoms.

Urinary tract infections (UTIs) are one of the most common human bacterial infections. While UTIs are commonly associated with colonization by Escherichia coli, members of this species also have been found within the bladder of individuals with no lower urinary tract symptoms (no LUTS), also known as asymptomatic bacteriuria. Prior studies have found that both uropathogenic E. coli (UPEC) strains and E. coli isolates that are not associated with UTIs encode for virulence factors. Thus, the reason(s) why E. coli sometimes causes UTI-like symptoms remain(s) elusive. In this study, the genomes of 66 E. coli isolates from adult female bladders were sequenced. These isolates were collected from four cohorts, including women: (1) without lower urinary tract symptoms, (2) overactive bladder symptoms, (3) urgency urinary incontinence, and (4) a clinical diagnosis of UTI. Comparative genomic analyses were conducted, including core and accessory genome analyses, virulence and motility gene analyses, and antibiotic resistance prediction and testing. We found that the genomic content of these 66 E. coli isolates does not correspond with the participant's symptom status. We thus looked beyond the E. coli genomes to the composition of the entire urobiome and found that the presence of E. coli alone was not sufficient to distinguish between the urobiomes of individuals with UTI and those with no LUTS. Because E. coli presence, abundance, and genomic content appear to be weak predictors of UTI status, we hypothesize that UTI symptoms associated with detection of E. coli are more likely the result of urobiome composition.

Pellino-1 Regulates Immune Responses to Haemophilus influenzae in Models of Inflammatory Lung Disease.

Non-typeable Haemophilus influenzae (NTHi) is a frequent cause of lower respiratory tract infection in people with chronic obstructive pulmonary disease (COPD). Pellino proteins are a family of E3 ubiquitin ligases that are critical regulators of TLR signaling and inflammation. The aim of this study was to identify a role for Pellino-1 in airway defense against NTHi in the context of COPD. Pellino-1 is rapidly upregulated by LPS and NTHi in monocyte-derived macrophages (MDMs) isolated from individuals with COPD and healthy control subjects, in a TLR4 dependent manner. C57BL/6 Peli1-/- and wild-type (WT) mice were subjected to acute (single LPS challenge) or chronic (repeated LPS and elastase challenge) airway inflammation followed by NTHi infection. Both WT and Peli1-/- mice develop airway inflammation in acute and chronic airway inflammation models. Peli1-/- animals recruit significantly more neutrophils to the airway following NTHi infection which is associated with an increase in the neutrophil chemokine, KC, in bronchoalveolar lavage fluid as well as enhanced clearance of NTHi from the lung. These data suggest that therapeutic inhibition of Pellino-1 may augment immune responses in the airway and enhance bacterial clearance in individuals with COPD.

Youth with epilepsy: Their insight into participating in enhanced physical activity study.

PURPOSE: We aimed to explore (i) the impact that a motivated walking program had on youth with epilepsy and (ii) the facilitators and barriers to implementing and sustaining the program. METHODS: Data were gathered using semi-structured interviews with the intervention group of a randomized controlled trial to study the effect of enhanced physical activity on youth with epilepsy. Participants had active epilepsy and were 8-14 years at recruitment. All wore an activity tracker for a year and received coaching via phone calls during the first six months to encourage reaching a step goal, which they then attempted to maintain independently for the following six months. Nine participants and one parent per child were separately interviewed at six months. Eleven participants including the original nine, and one parent per youth, were interviewed at twelve months. Data collection and analysis used a phenomenological research framework and coded the data according to the International Classification of Functioning, Disability and Health (the ICF). This was done in order to capture all relevant impacts of the intervention. RESULTS: Physical activity was associated with benefits in all components of the ICF. Changes in personal factors such as improved attitude, confidence, and insight into activity were emphasized. Parents also experienced personal benefits as a result of their child engaging in the program. CONCLUSIONS: Physical activity had positive effects in many aspects of the lives of the participants and their parents. The ICF was a relatively useful and effective system for coding qualitative research. These findings should assist in development of future exercise programs.

Static tank depuration and chronic short-term experimental contamination of Eastern oysters (Crassostrea virginica) with Giardia duodenalis cysts.

Cysts of the protozoan parasite Giardia have been detected in many bivalve shellfish species worldwide. The detection of zoonotic Giardia duodenalis assemblages A and B is of public health concern, yet there is limited data available demonstrating the bioaccumulation and elimination of Giardia cysts in bivalve shellfish. This study quantified G. duodenalis cysts that were filtered and retained by oysters (Crassostrea virginica) over a one week chronic exposure period, or 24 hour exposure followed by a 6 day depuration period, using static tank systems containing 10 L of 29 ppt water inoculated with 1000 or 10,000 cysts. Under chronic exposure, each oyster retained a mean of 13.4 and 87.4 cysts during the first 24h of exposure at low and high doses, respectively, and the cysts bioaccumulated at a rate of 1.2 and 6.8 cysts/oyster/day, respectively, for the remaining duration of the trials. In acute exposure trials, oysters retained 13.8 cysts or 78.9 cysts at low and high doses, respectively, during the initial 24 hour exposure and naturally depurated cysts at a rate of -0.92 cysts/oyster/day and -2.2 cysts/oyster/day, respectively, after transfer. Although most G. duodenalis cysts were eliminated within the first 24h via pseudofeces and feces, detection of some cysts in the fecal material on day 7 of acute exposure trials was indicative of cysts which passed through the digestive tract and released in feces. Only 48-53% of the initial tank inocula were recovered and may indicate that some cysts were selectively filtered by oysters but degraded through digestion.

Bioaccumulation and elimination of Cryptosporidium parvum oocysts in experimentally exposed Eastern oysters (Crassostrea virginica) held in static tank aquaria.

A variety of human enteropathogens, including viruses, bacteria, and parasites, have been shown to bioaccumulate in suspension-feeding bivalve shellfish. Cryptosporidium parvum is a zoonotic protozoan parasite that has been detected in many shellfish species within both fecally contaminated and clean oyster growing areas across the globe. For this study, C. parvum oocysts (1000 and 10,000) were spiked into 10 L of water in static tank systems housing Crassostrea virginica. Oysters were either held in the contaminated aquaria for 7 days of exposure or were exposed for 24h and subsequently placed in a clean static tank system for the remainder of the trial. Individual oysters, fecal material, and tank water were analyzed for oocysts up to 7 days post-exposure via direct immunofluorescence. Oysters held under chronic exposure conditions gradually accumulated oocysts (1.5 or 34.4 oocysts/oyster/day for low or high dose exposure groups, respectively) between days 1 and 7, with an exponential uptake in oocysts observed within the first 24h post-exposure (mean uptake of 29.6 or 241.9 oocysts/oyster, respectively). Oysters that were transferred to clean water after 24h were capable of slowly depurating oocysts, following a linear trend. During chronic exposure trials 48-49% of the total spiked inoculum was recovered from oyster tissue, whereas 4.8-5.9% and 38-40% was recovered from tank water and from fecal material at day 7, respectively. In acute exposure trials, 30-31% of the total tank inoculum was found in oysters, suggesting that chronically exposed oysters were likely re-filtering some oocysts. Examinations of oyster fecal material from acute exposures revealed that 72-82% of oocysts recovered were already excreted at the time of oyster transfer (day 1), with only 18-28% being excreted during the static depuration phase. These data support that although most C. parvum oocysts are removed by C. virginica oysters within 24h, elimination after this point occurs slowly. Additionally, chronic exposures demonstrate that wild or cultured oysters in saline environments that are frequently exposed to sources of Cryptosporidium may be unable to eliminate the parasites at a rate that balances initial uptake.