Identification of environmental and methodological factors driving variability of Pepper Mild Mottle Virus (PMMoV) across three wastewater treatment plants in the City of Toronto.

PMMoV has been widely used to normalize the concentration of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, influenza, and respiratory syncytial virus (RSV) to account for variations in the fecal content of wastewater. PMMoV is also used as an internal RNA recovery control for wastewater-based epidemiology (WBE) tests. While potentially useful for the interpretation of WBE data, previous studies have suggested that PMMoV concentration can be affected by various physico-chemical characteristics of wastewater. There is also the possibility that laboratory methods, particularly the variability in centrifugation steps to remove supernatant from pellets can cause PMMoV variability. The goal of this study is to improve our understanding of the main drivers of PMMoV variability by assessing the relationship between PMMoV concentration, the physico-chemical characteristics of wastewater, and the methodological approach for concentrating wastewater samples. We analyzed 24-hour composite wastewater samples collected from the influent stream of three wastewater treatment plants (WWTPs) located in the City of Toronto, Ontario, Canada. Samples were collected 3 to 5 times per week starting from the beginning of March 2021 to mid-July 2023. The influent flow rate was used to partition the data into wet and dry weather conditions. Physico-chemical characteristics (e.g., total suspended solids (TSS), biological oxygen demand (BOD), alkalinity, electrical conductivity (EC), and ammonia (NH3)) of the raw wastewater were measured, and PMMoV was quantified. Spatial and temporal variability of PMMoV was observed throughout the study period. PMMoV concentration was significantly higher during dry weather conditions. Multiple linear regression analysis demonstrates that the number and type of physico-chemical parameters that drive PMMoV variability are site-specific, but overall BOD and alkalinity were the most important predictors. Differences in PMMoV concentration for a single WWTP between two different laboratory methods, along with a weak correlation between pellet mass and TSS using one method may indicate that differences in sample concentration and subjective subsampling bias could alter viral recovery and introduce variability to the data.

Underestimation of SARS-CoV-2 in wastewater due to single or double mutations in the N1 qPCR probe binding region.

Wastewater surveillance using RT-qPCR has now been widely adopted to track circulating levels of SARS-CoV-2 virus in many sewersheds. The CDC qPCR assays targeting two regions (N1 and N2) within the N gene are commonly used, but a discrepancy between the two biomarkers has been noticed by independent studies using these methods since late 2021. The reason is presumed to be due to mutations in regions targeted by the N1 qPCR probe. In this study, we systematically investigated and unequivocally confirmed that the underlying reason for this discrepancy was mutations in the N1 probe target, and that a single mutation could cause a significant drop in signal. We first confirmed the proportion of related mutations in wastewater samples (Jan 2021-Dec 2022) using nested PCR and LC-MS. Based on relative proportions of N1 alleles, we separated the wastewater data into four time periods corresponding to different variant waves: Period I (Alpha and Delta waves with 0 mutation), Period II (BA.1/BA.2 waves with a single mutation found in all Omicron strains), Period III (BA.5.2* wave with two mutations), and Period IV (BQ.1* wave with two mutations). Significantly lower N1 copies relative to N2 copies in samples from Periods II-IV compared to those from Period I was observed in wastewater. To further pinpoint the extent to which each mutation impacted N1 quantification, we compared the qPCR response among different synthetic oligomers with corresponding mutations. This study highlighted the impact of even just one or two mutations on qPCR-based wastewater surveillance of SARS-CoV-2.

Detection of a Human Adenovirus Outbreak, Including Some Critical Infections, Using Multipathogen Testing at a Large University, September 2022-January 2023.

Background: Human adenoviruses (HAdVs) can cause outbreaks of flu-like illness in university settings. Most infections in healthy young adults are mild; severe illnesses rarely occur. In Fall 2022, an adenovirus outbreak was identified in university students. Methods: HAdV cases were defined as university students 17-26 years old who presented to the University Health Service or nearby emergency department with flu-like symptoms (eg, fever, cough, headache, myalgia, nausea) and had confirmed adenovirus infections by polymerase chain reaction (PCR). Demographic and clinical characteristics were abstracted from electronic medical records; clinical severity was categorized as mild, moderate, severe, or critical. We performed contact investigations among critical cases. A subset of specimens was sequenced to confirm the HAdV type. Results: From 28 September 2022 to 30 January 2023, 90 PCR-confirmed cases were identified (51% female; mean age, 19.6 years). Most cases (88.9%) had mild illness. Seven cases required hospitalization, including 2 critical cases that required intensive care. Contact investigation identified 44 close contacts; 6 (14%) were confirmed HAdV cases and 8 (18%) reported symptoms but never sought care. All typed HAdV-positive specimens (n = 36) were type 4. Conclusions: While most students with confirmed HAdV had mild illness, 7 otherwise healthy students had severe or critical illness. Between the relatively high number of hospitalizations and proportion of close contacts with symptoms who did not seek care, the true number of HAdV cases was likely higher. Our findings illustrate the need to consider a wide range of pathogens, even when other viruses are known to be circulating.

Inhibitory Control Training for Anxiety and Math Achievement in Primary School Children: Protocol for a Proof-of-Concept Study.

BACKGROUND: Cognitive control training (CCT) has shown potential to reduce emotional vulnerability in adults and adolescents. However, there is scant literature testing the efficacy of CCT for the reduction of anxiety and transferring the effects to educational outcomes in children. Building on the evidence that a greater ability to suppress a prepotent response (inhibitory control) is associated with higher math achievement in children, it is plausible that training inhibitory processes using a CCT paradigm may be beneficial for reducing anxiety, improving inhibitory control, and in turn increasing math achievement. OBJECTIVE: This proof-of-concept study aims to investigate the efficacy of 15 sessions of inhibitory control training for reduction in anxiety and improvement in math achievement in primary school children. METHODS: We will use a 2 (group: CCT, adaptive Go/No-Go vs active control, low-load task) multiplied by 4 (time: pre- vs posttraining vs 1-month vs 3-month follow-up) randomized design in a nonselected sample of 100 children aged 8-10 years. Both groups will complete 10 minutes of daily training for 3 weeks at school. The dependent variables will be anxiety and correlates (Spence Children's Anxiety Scale, Penn State Worry Questionnaire for Children, Revised Children's Anxiety and Depression Scale, Child Response Style Questionnaire, and Modified Abbreviated Math Anxiety Scale), inhibitory control (Go/No-Go task), shifting (color-shape shifting task), updating (n-back task), and math achievement (Applied Problems, Calculation, and Math Facts Fluency subtests from the Woodcock-Johnson IV Tests of Achievement). RESULTS: We opened enrollment in September 2023. The initial results are expected to be published in late 2024. We predict that children in the CCT group will show a reduction in emotional symptoms; improvements in inhibition, shifting, and updating performance; and advances in math achievement from pre- to posttraining, and that these effects will be maintained at 1- and 3-month follow-ups, compared to children in the active control group. CONCLUSIONS: The CCT paradigm used in our study will provide a greater understanding of the emotional and cognitive transfer effects on children and inform future work. Specifically, the findings will advance the knowledge of deploying inhibitory control training with children and provide valuable insights into its use for reducing anxiety and advancing math achievement. TRIAL REGISTRATION: Open Science Framework ofs.io/de2qa; https://doi.org/10.17605/OSF.IO/DE2QA. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/52929.

The Effect of Varying Fatty Acid Composition on Postprandial Airway Inflammation, Pulmonary Function, and Airway Resistance in Healthy, Young Adults.

OBJECTIVE: To examine the effect of varying fatty acid composition in a HFM on eNO, pulmonary function, and airway resistance. METHODS: Fifteen individuals [6 M/9 F; 21.9 ± 1.5 years old] each completed three HFM conditions {SF, O6FA, and O3FA; 12 kcal/kg body weight, 63% total fat, and 0.72 g/kg sugar smoothies} in random order separated by at least 48 h. Airway inflammation assessed via eNO, pulmonary function measured using the maximum flow volume loop (MFVL) and airway resistance measured using impulse oscillometry (iOS) were taken at baseline, 2h and 4h postprandially. RESULTS: There was no difference in eNO or iOS across time in any condition or between conditions (p > 0.05). There was a significant time by condition effect for FEV1 post-HFM in the SF and O6FA conditions (p < 0.05). CONCLUSION: Different fatty acid compositions do not increase eNO or iOS in healthy, college-aged participants after consumption of a HFM, though the minimally processed meals with fruit added may contribute to these findings.

Functional screening pipeline to uncover laccase-like multicopper oxidase enzymes that transform industrial lignins.

Laccase-like multicopper oxidases are recognized for their potential to alter the reactivity of lignins for application in value-added products. Typically, model compounds are employed to discover such enzymes; however, they do not represent the complexity of industrial lignin substrates. In this work, a screening pipeline was developed to test enzymes simultaneously on model compounds and industrial lignins. A total of 12 lignin-active fungal multicopper oxidases were discovered, including 9 enzymes active under alkaline conditions (pH 11.0). Principal component analysis revealed the poor ability of model compounds to predict enzyme performance on industrial lignins. Additionally, sequence similarity analyses grouped these enzymes with Auxiliary Activity-1 sub-families with few previously characterized members, underscoring their taxonomic novelty. Correlation between the lignin-activity of these enzymes and their taxonomic origin, however, was not observed. These are critical insights to bridge the gap between enzyme discovery and application for industrial lignin valorization.

Metagenomes and metagenome-assembled genomes from a sequentially fed anaerobic digester treating solid organic municipal waste.

We present a data set of four metagenomes and 281 metagenome-assembled genomes describing the microbial community of a laboratory-scale high solids anaerobic digester. Our objective was to obtain information on the coding potential of the microbial community and draft genomes of the most abundant organisms in the digester.

Depression Moderates the Relationship between Trait Anxiety, Worry and Attentional Control in Melanoma Survivors.

Cancer survivors commonly contend with concurrent cognitive difficulties such as problems with attention and concentration, and psychological distress, including anxiety and depression. However, the associations between attentional and emotional difficulties within the specific context of melanoma survivors remain relatively unexplored. Premised on attentional control theory, the current study employed a cross-sectional design to explore the interplay among trait anxiety (dispositional) and situational anxiety (cancer-related worry), depression and attentional control (ability to inhibit distractors and flexibly shift within and between tasks) in a sample of 187 melanoma survivors aged 18 to 58 years (Mage = 36.83 years, SDage = 5.44 years; 93% female). Data were analyzed using a moderated multiple regression, with anxiety, cancer worry and depression as predictors, and attentional control as the criterion variable. After statistically controlling for the variance of chemotherapy, we found that individuals with higher trait anxiety and higher cancer-related worry reported greater attentional control at low levels of depression, yet poorer attentional control at high depression, relative to individuals with low anxiety. Our findings suggest that anxiety and depression are differentially related to attentional control in melanoma survivors. The results provide a marker for clinicians addressing anxiety and depression in this population. Implications for primary healthcare are discussed.

A Multifunctional Dehalobacter? Tandem Chloroform and Dichloromethane Degradation in a Mixed Microbial Culture.

Chloroform (CF) and dichloromethane (DCM) contaminate groundwater sites around the world but can be cleaned up through bioremediation. Although several strains of Dehalobacter restrictus can reduce CF to DCM and multiple Peptococcaceae can ferment DCM, these processes cannot typically happen simultaneously due to CF sensitivity in the known DCM-degraders or electron donor competition. Here, we present a mixed microbial culture that can simultaneously metabolize CF and DCM and create an additional enrichment culture fed only DCM. Through genus-specific quantitative polymerase chain reaction, we find that Dehalobacter grows while either CF alone or DCM alone is converted, indicating its involvement in both metabolic steps. Additionally, the culture was maintained for over 1400 days without the addition of an exogenous electron donor, and through electron balance calculations, we show that DCM metabolism would produce sufficient reducing equivalents (likely hydrogen) for CF respiration. Together, these results suggest intraspecies electron transfer could occur to continually reduce CF in the culture. Minimizing the addition of electron donor reduces the cost of bioremediation, and "self-feeding" could prolong bioremediation activity long after donor addition ends. Overall, understanding this mechanism informs strategies for culture maintenance and scale-up and benefits contaminated sites where the culture is employed for remediation worldwide.

Excessive Smartphone Use is Associated with Depression, Anxiety, Stress, and Sleep Quality of Australian Adults.

Problematic smartphone use has been associated with poorer mental health in different population groups; however, little is known about how levels of smartphone use were associated with mental health outcomes of adults in Australia. Using data from a cross-sectional survey among Australian adults aged 18-59 years (n = 655, Mean = 24.55 [SD = 5.59] years; 66% female), the current study aimed to examine association between problematic smartphone use and different psychological outcomes. Participants completed measures of problematic smartphone use with Mobile Phone Problem Use Scale (MPPUS), mental health outcomes with Depression, Anxiety, and Stress Scale (DASS-21), and Pittsburgh Sleep Quality Index (PSQI), in addition to some socio-demographics. Smartphone use was categorised into three groups: low-moderate, moderate-high, and high-severe. A total of 160 adults (24.4%) reported high-severe smartphone use. Multivariable linear regression analyses showed that smartphone use was inversely associated with psychological outcomes in a dose-dependent manner with high-severe smartphone uses having the most adverse effects. Compared to low-moderate use, average depression score was 3.5 points higher for moderate-high smartphone use (ß = 3.51, 95% CI: 1.63-5.40) and 6.9 points higher for high-severe smartphone use (ß = 6.91, 95% CI: 4.74-9.07). Similarly, average stress score was 3.4 points higher for moderate-high smartphone use (ß = 3.40, 95% CI: 1.75-5.06) and 7.0 points higher for high-severe smartphone use (ß = 7.02, 95% CI: 5.11-8.93). Similar association estimates were found for anxiety and sleep quality. Reducing smartphone use has the potential to optimise depression, anxiety, stress, and sleep quality; however, longitudinal research is warranted to establish directionality of the association.

Body composition measured by multi-frequency bioelectrical impedance following creatine supplementation.

BACKGROUND: Acute fluid ingestion increases estimated body fat percentage (BF%) measurements by single frequency (SF-BIA) and multi-frequency bioelectrical impedance (MF-BIA). It is unknown if MF-BIA accurately measures total BF% and total body water (TBW) after creatine supplementation, which causes fluid retention, and resultant increases in fat-free mass and TBW. The purpose of this study was to analyze the effect of creatine supplementation on body composition and TBW measured through a popular MF-BIA device (InBody 770). METHODS: Thirteen male and 14 female subjects (18-22 years) completed one week of creatine monohydrate (0.3 g/kg body weight) or maltodextrin. Pre- and post-supplementation body composition measurements included dual-energy X-ray absorptiometry (DEXA), SF-BIA measured by an Omron HBF-306C device, and MF-BIA measured by an InBody 770 device to measure BF%, fat free mass (FFM), and fat mass (FM). Additionally, intracellular water (ICW), extracellular water (ECW), and TBW were estimated by MF- BIA. RESULTS: FFM increased more in the creatine group than the placebo group measured by all body composition modes (1.2 kg, 1.9 kg, and 1.1 kg increase for SF-BIA, MF-BIA, and DEXA respectively, P<0.05). Creatine supplementation resulted in a 2% increase (P<0.05) in TBW measured by MF-BIA (40.4±9.5 to 41.2±9.6 kg). CONCLUSIONS: One week of creatine supplementation increased TBW as detected by the InBody 770 device. Changes in body composition that occurred due to the increase in TBW were detected as an increase in FFM measured by SF-BIA, MF-BIA, and DEXA.

ß-Cell function during a high-fat meal in young versus old adults: role of exercise.

The acute effect of exercise on ß-cell function during a high-fat meal (HFM) in young adults (YA) versus old adults (OA) is unclear. In this randomized crossover trial, YA (n = 5 M/7 F, 23.3 ± 3.9 yr) and OA (n = 8 M/4 F, 67.7 ± 6.0 yr) underwent a 180-min HFM (12 kcal/kg body wt; 57% fat, 37% CHO) after a rest or exercise [∼65% heart rate peak (HRpeak)] condition ∼12 h earlier. After an overnight fast, plasma lipids, glucose, insulin, and free fatty acid (FFA) were determined to estimate peripheral, or skeletal muscle, insulin sensitivity (Matsuda index) as well as hepatic [homeostatic model assessment of insulin resistance (HOMA-IR)] and adipose insulin resistance (adipose-IR). ß-Cell function was derived from C-peptide and defined as early-phase (0-30 min) and total-phase (0-180 min) disposition index [DI, glucose-stimulated insulin secretion (GSIS) adjusted for insulin sensitivity/resistance]. Hepatic insulin extraction (HIE), body composition [dual-energy X-ray absorptiometry (DXA)], and peak oxygen consumption (V̇o2peak) were also assessed. OA had higher total cholesterol (TC), LDL, HIE, and DI across organs as well as lower adipose-IR (all, P < 0.05) and V̇o2peak (P = 0.056) despite similar body composition and glucose tolerance. Exercise lowered early-phase TC and LDL in OA versus YA (P < 0.05). However, C-peptide area under the curve (AUC), total phase GSIS, and adipose-IR were reduced postexercise in YA versus OA (P < 0.05). Skeletal muscle DI increased in YA and OA after exercise (P < 0.05), whereas adipose DI tended to decline in OA (P = 0.06 and P = 0.08). Exercise-induced skeletal muscle insulin sensitivity (r = -0.44, P = 0.02) and total-phase DI (r = -0.65, P = 0.005) correlated with reduced glucose AUC180min. Together, exercise improved skeletal muscle insulin sensitivity/DI in relation to glucose tolerance in YA and OA, but only raised adipose-IR and reduced adipose-DI in OA.NEW & NOTEWORTHY High-fat diets may induce ß-cell dysfunction. This study compared how young and older adults responded to a high-fat meal with regard to ß-cell function and whether exercise comparably impacted glucose regulation. Older adults secreted more insulin during the high-fat meal than younger adults. Although exercise increased ß-cell function adjusted for skeletal muscle insulin sensitivity in relation to glucose tolerance, it raised adipose insulin resistance and reduced pancreatic ß-cell function relative to adipose tissue in older adults. Additional work is needed to discern nutrient-exercise interactions across age to mitigate chronic disease risk.

Risk factors for infection with influenza A(H3N2) virus on a US university campus, October-November 2021.

BACKGROUND: Knowledge of the specific dynamics of influenza introduction and spread in university settings is limited. METHODS: Persons with acute respiratory illness symptoms received influenza testing by molecular assay during October 6-November 23, 2022. Viral sequencing and phylogenetic analysis were conducted on nasal swab samples from case-patients. Case-control analysis of a voluntary survey of persons tested was used to identify factors associated with influenza; logistic regression was conducted to calculate odds ratios and 95% CIs. A subset of case-patients tested during the first month of the outbreak was interviewed to identify sources of introduction and early spread. RESULTS: Among 3268 persons tested, 788 (24.1%) tested positive for influenza; 744 (22.8%) were included in the survey analysis. All 380 sequenced specimens were influenza A (H3N2) virus clade 3C.2a1b.2a.2, suggesting rapid transmission. Influenza (OR [95% CI]) was associated with indoor congregate dining (1.43 [1.002-2.03]), attending large gatherings indoors (1.83 [1.26-2.66]) or outdoors (2.33 [1.64-3.31]), and varied by residence type (apartment with ≥1 roommate: 2.93 [1.21-7.11], residence hall room alone: 4.18 [1.31-13.31], or with roommate: 6.09 [2.46-15.06], or fraternity/sorority house: 15.13 [4.30-53.21], all compared with single-dwelling apartment). Odds of influenza were lower among persons who left campus for ≥1 day during the week before their influenza test (0.49 [0.32-0.75]). Almost all early cases reported attending large events. CONCLUSIONS: Congregate living and activity settings on university campuses can lead to rapid spread of influenza following introduction. Isolating following a positive influenza test or administering antiviral medications to exposed persons may help mitigate outbreaks.

Redox and Nucleophilic Reactions of Naphthoquinones with Small Thiols and Their Effects on Oxidization of H2S to Inorganic and Organic Hydropolysulfides and Thiosulfate.

Naphthoquinone (1,4-NQ) and its derivatives (NQs, juglone, plumbagin, 2-methoxy-1,4-NQ, and menadione) have a variety of therapeutic applications, many of which are attributed to redox cycling and the production of reactive oxygen species (ROS). We previously demonstrated that NQs also oxidize hydrogen sulfide (H2S) to reactive sulfur species (RSS), potentially conveying identical benefits. Here we use RSS-specific fluorophores, mass spectroscopy, EPR and UV-Vis spectrometry, and oxygen-sensitive optodes to examine the effects of thiols and thiol-NQ adducts on H2S-NQ reactions. In the presence of glutathione (GSH) and cysteine (Cys), 1,4-NQ oxidizes H2S to both inorganic and organic hydroper-/hydropolysulfides (R2Sn, R=H, Cys, GSH; n = 2-4) and organic sulfoxides (GSnOH, n = 1, 2). These reactions reduce NQs and consume oxygen via a semiquinone intermediate. NQs are also reduced as they form adducts with GSH, Cys, protein thiols, and amines. Thiol, but not amine, adducts may increase or decrease H2S oxidation in reactions that are both NQ- and thiol-specific. Amine adducts also inhibit the formation of thiol adducts. These results suggest that NQs may react with endogenous thiols, including GSH, Cys, and protein Cys, and that these adducts may affect both thiol reactions as well as RSS production from H2S.

Metagenomic and Genomic Sequences from a Methanogenic Benzene-Degrading Consortium.

Draft and complete metagenome assembled genomes (MAGs) were created from multiple metagenomic assemblies of DGG-B, a strictly anaerobic, stable mixed microbial consortium that degrades benzene completely to methane and CO2. Our objective was to obtain closed genome sequences of benzene-fermenting bacteria to enable the elucidation of their elusive anaerobic benzene degradation pathway.

"Candidatus Nealsonbacteria" Are Likely Biomass Recycling Ectosymbionts of Methanogenic Archaea in a Stable Benzene-Degrading Enrichment Culture.

The Candidate Phyla Radiation (CPR), also referred to as superphylum Patescibacteria, is a very large group of bacteria with no pure culture representatives discovered by 16S rRNA sequencing or genome-resolved metagenomic analyses of environmental samples. Within the CPR, candidate phylum Parcubacteria, previously referred to as OD1, is prevalent in anoxic sediments and groundwater. Previously, we had identified a specific member of the Parcubacteria (referred to as DGGOD1a) as an important member of a methanogenic benzene-degrading consortium. Phylogenetic analyses herein place DGGOD1a within the clade "Candidatus Nealsonbacteria." Because of its persistence over many years, we hypothesized that "Ca. Nealsonbacteria" DGGOD1a must play an important role in sustaining anaerobic benzene metabolism in the consortium. To try to identify its growth substrate, we amended the culture with a variety of defined compounds (pyruvate, acetate, hydrogen, DNA, and phospholipid), as well as crude culture lysate and three subfractions thereof. We observed the greatest (10-fold) increase in the absolute abundance of "Ca. Nealsonbacteria" DGGOD1a only when the consortium was amended with crude cell lysate. These results implicate "Ca. Nealsonbacteria" in biomass recycling. Fluorescence in situ hybridization and cryogenic transmission electron microscope images revealed that "Ca. Nealsonbacteria" DGGOD1a cells were attached to larger archaeal Methanothrix cells. This apparent epibiont lifestyle was supported by metabolic predictions from a manually curated complete genome. This is one of the first examples of bacterial-archaeal episymbiosis and may be a feature of other "Ca. Nealsonbacteria" found in anoxic environments. IMPORTANCE An anaerobic microbial enrichment culture was used to study members of candidate phyla that are difficult to grow in the lab. We were able to visualize tiny "Candidatus Nealsonbacteria" cells attached to a large Methanothrix cell, revealing a novel episymbiosis.

An improved method for determining frequency of multiple variants of SARS-CoV-2 in wastewater using qPCR assays.

Wastewater-based surveillance has become an effective tool around the globe for indirect monitoring of COVID-19 in communities. Variants of Concern (VOCs) have been detected in wastewater by use of reverse transcription polymerase chain reaction (RT-PCR) or whole genome sequencing (WGS). Rapid, reliable RT-PCR assays continue to be needed to determine the relative frequencies of VOCs and sub-lineages in wastewater-based surveillance programs. The presence of multiple mutations in a single region of the N-gene allowed for the design of a single amplicon, multiple probe assay, that can distinguish among several VOCs in wastewater RNA extracts. This approach which multiplexes probes designed to target mutations associated with specific VOC's along with an intra-amplicon universal probe (non-mutated region) was validated in singleplex and multiplex. The prevalence of each mutation (i.e. VOC) is estimated by comparing the abundance of the targeted mutation with a non-mutated and highly conserved region within the same amplicon. This is advantageous for the accurate and rapid estimation of variant frequencies in wastewater. The N200 assay was applied to monitor frequencies of VOCs in wastewater extracts from several communities in Ontario, Canada in near real time from November 28, 2021 to January 4, 2022. This includes the period of the rapid replacement of the Delta variant with the introduction of the Omicron variant in these Ontario communities in early December 2021. The frequency estimates using this assay were highly reflective of clinical WGS estimates for the same communities. This style of qPCR assay, which simultaneously measures signal from a non-mutated comparator probe and multiple mutation-specific probes contained within a single qPCR amplicon, can be applied to future assay development for rapid and accurate estimations of variant frequencies.

Informed consent in episiotomy: Co-analysis with midwives and distillation of best practice.

BACKGROUND: Performing an episiotomy where clinically indicated is a key intervention in the Obstetric Anal Sphincter Injury Care Bundle (OASI-CB) implemented across England and Wales to reduce the risk and increase the detection of severe perineal trauma after birth. Standards of consent provided to people in maternity care generally and for episiotomy specifically have been reported as suboptimal. Compromising birthing people's personal autonomy or sense of control has been linked to a dissatisfying birth experience, negative psychological sequelae, and litigation. METHODS: This study explored experienced midwives' practice of informed consent for episiotomy during a midwife-led birth. We sampled 43 midwives across eight NHS Trusts in England and Wales using online focus groups and telephone interviews about their experience of consent in episiotomy. Using qualitative content analysis and art-based co-analysis methods with eight midwives from across the research sites, we co-analyzed and co-constructed three themes and four practice recommendations from the data. RESULTS: Three themes were constructed from the data: Assent rather than consent, Change in culture to support best practice, and Standardized information. These themes informed the shaping of four recommendations for best practice in episiotomy informed consent. CONCLUSION: This study has shown how variations in midwifery practice and culture may impact birthing people's experience of informed consent in episiotomy. Midwives may not have the knowledge or skills to conduct a detailed consent conversation, leading to variation in practice and messages for birthing people. The use of antenatal discussion aids can offer women the opportunity to become informed and fully participate in the decision-making process.