Antimicrobials and antimicrobial resistance genes in the shadow of COVID-19 pandemic: A wastewater-based epidemiology perspective.

Higher usage of antimicrobial agents in both healthcare facilities and the communities has resulted in an increased spread of resistant bacteria. However, the improved infection prevention and control practices may also contribute to decreasing antimicrobial resistance (AMR). In the present study, wastewater-based epidemiology (WBE) approach was applied to explore the link between COVID-19 and the community usage of antimicrobials, as well as the prevalence of resistance genes. Longitudinal study has been conducted to monitor the levels of 50 antimicrobial agents (AAs), 24 metabolites, 5 antibiotic resistance genes (ARGs) and class 1 integrons (intI 1) in wastewater influents in 4 towns/cities over two years (April 2020 - March 2022) in the South-West of England (a total of 1,180 samples collected with 87,320 individual AA measurements and 8,148 ARG measurements). Results suggested higher loads of AAs and ARGs in 2021-22 than 2020-21, with beta-lactams, quinolones, macrolides and most ARGs showing statistical differences. In particular, the intI 1 gene (a proxy of environmental ARG pollution) showed a significant increase after the ease of the third national lockdown in England. Positive correlations for all quantifiable parent AAs and metabolites were observed, and consumption vs direct disposal of unused AAs has been identified via WBE. This work can help establish baselines for AMR status in communities, providing community-wide surveillance and evidence for informing public health interventions. Overall, studies focused on AMR from the start of the pandemic to the present, especially in the context of environmental settings, are of great importance to further understand the long-term impact of the pandemic on AMR.

Understanding treatment of pain during SARS-CoV-2 pandemic in a two-year intercity longitudinal study using wastewater-based epidemiology.

SARS-CoV-2 pandemic had a significant impact on the society, economy, and health of people around the world with consequences that need to be better understood for future pandemic preparedness. This manuscript provides insights into the usage of pharmaceuticals for pain treatment management throughout SARS-CoV-2 pandemic. Four towns and cities with a total population of > 1 million people covering an area of 2000 km2 in South West England were monitored for twenty-four months. Results showed different patterns in pain pharma usage, with small towns having higher population normalised daily loads (PNDLs) than big cities for majority of pain killers studied. This is likely due to demographics of these cities with smaller cities having older population. Per capita consumption of non-steroidal anti-inflammatory drugs (NSAIDs) increased compared to pre-pandemic usage in line with SARS-CoV-2 infections (ibuprofen and acetaminophen), while body pain drugs (diclofenac and naproxen) decreased in line with restrictions and closure of sports facilities. Changes in population normalised daily intake (PNDI) of pain killers were particularly apparent during the 1st and 3rd national lockdown. Comparison of PNDIs with prescriptions highlighted differences related to medication availability (OTC drugs) and patients' nonadherence (prescribed drugs). In addition, several instances of direct disposal events across the catchments were observed which raises an issue of lack of pharma compliance and general understanding of potential environmental impacts from pharma usage.

Assessment of restriction measures implemented during COVID pandemics on community lifestyle choices via wastewater-based epidemiology.

SARS-CoV-2 pandemic affected lifestyle habits, and the mental health and wellbeing of people around the world. In this manuscript, two towns (Paulton and Radstock) and two cities (Bath and Bristol) in Southwest England (> 1 million people) were monitored for two years using Wastewater-Based Epidemiology to assess impacts of COVID (including management measures such as lockdowns and movement restrictions) on community lifestyle choices: illicit drugs, legal stimulants, abused pharmaceuticals and pain pharma usage. Results were triangulated with key dates captured during the pandemic (national lockdowns, restrictions and social distancing measures, etc.). This highlighted a reduction in cocaine intake (as benzoylecgonine) (community average: -36 %) during the first lockdown and an increase in illicit drugs usage after the 3rd national lockdown (community averages for amphetamine: +8 %, cocaine/benzoylecgonine: +39 %, ketamine:+70 %) when restrictions were removed, and social interaction and recreational activities increased. There was a reduction in the intake of caffeine (as 1,7-dimethylxanthine) (community average:-39 %) after 3rd national lockdown coinciding with pubs reopening while nicotine intake (via cotinine) remained stable indicating lack of impact of COVID on smoking habits. Pain pharma often used in pain management resulting from injuries linked with sport activities (naproxen and diclofenac) showed decrease in usage due to lockdown restrictions in physical exercise and access to gyms/sport facilities.

An integrated One Health framework for holistic evaluation of risks from antifungal agents in a large-scale multi-city study.

A new framework for retrospective mass spectral data mining for antifungal agents (AFs) and Wastewater-Based Epidemiology (WBE) was developed as part of One Health framework to tackle risks from AFs. A large scale, multi-city study was undertaken in South-West England. Key drivers of AFs in the catchment were identified with communal wastewater discharges being the main driver for human AFs (fluconazole, ketoconazole) and agricultural runoff being the main driver for pesticide AFs (prochloraz, prothioconazole and tebuconazole). Average WBE-estimated human used fluconazole and ketoconazole PNDIs (population normalised daily intake) exceeded 300 mg day-1 1000 inh-1 and 2000 mg day-1 1000 inh-1. This is much higher than PNDPs (population normalised daily prescriptions <40 mg day-1 1000 inh-1 and <80 mg day-1 1000 inh-1 for fluconazole and ketoconazole respectively). This was expected due to both prescription and over-the-counter usage, and both oral and topical applications. Pesticide AF, prothioconazole had PNDIs <40,000 mg day-1 1000 inh -1, which gave intake: 0.43, 0.26, 0.07 mg kg-1 in City A, B, and C, likely due to accounting for external/non-human sources. This is higher than the acceptable daily intake (ADI) of 0.01 mg kg-1bw day-1, which warrants further study. Intake per kg of body weight estimated using tebuconazole was 0.86, 1.39, 0.12, 0.13, and 2.7 mg kg-1 in City A-E respectively and is likely due to external/non-human sources. Intake calculated using its metabolite was 0.02 and 0.01 mg kg-1 in City B and C respectively, which aligned with ADI (0.03 mg kg-1bw day-1). The environmental risk assessment of AFs indicated low/medium risk from fluconazole, prochloraz, and tebuconazole, medium risk from epoxiconazole, prothioconazole's metabolite, and tebuconazole, and high risk for prothioconazole in river water. High risk was estimated from fluconazole, epoxiconazole, prothioconazole and its metabolite, tebuconazole, ketoconazole in wastewater samples, which is important during raw sewage discharge events via sewer overflows.

Antimicrobials and antimicrobial resistance genes in a one-year city metabolism longitudinal study using wastewater-based epidemiology.

This longitudinal study tests correlations between antimicrobial agents (AA) and corresponding antimicrobial resistance genes (ARGs) generated by a community of >100 k people inhabiting one city (Bath) over a 13 month randomised monitoring programme of community wastewater. Several AAs experienced seasonal fluctuations, such as the macrolides erythromycin and clarithromycin that were found in higher loads in winter, whilst other AA levels, including sulfamethoxazole and sulfapyridine, stayed consistent over the study period. Interestingly, and as opposed to AAs, ARGs prevalence was found to be less variable, which indicates that fluctuations in AA usage might either not directly affect ARG levels or this process spans beyond the 13-month monitoring period. However, it is important to note that weekly positive correlations between individual associated AAs and ARGs were observed where seasonal variability in AA use was reported: ermB and macrolides CLR-clarithromycin and dmCLR-N-desmethyl clarithromycin, aSPY- N-acetyl sulfapyridine and sul1, and OFX-ofloxacin and qnrS. Furthermore, ARG loads normalised to 16S rRNA (gene load per microorganism) were positively correlated to the ARG loads normalised to the human population (gene load per capita), which indicates that the abundance of microorganisms is proportional to the size of human population and that the community size, and not AA levels, is a major driver of ARG levels in wastewater. Comparison of hospital and community wastewater showed higher number of AAs and their metabolites, their frequency of occurrence and concentrations in hospital wastewater. Examples include: LZD-linezolid (used only in severe bacterial infections) and AMX-amoxicillin (widely used, also in community but with very low wastewater stability) that were found only in hospital wastewater. CIP-ciprofloxacin, SMX-sulfamethoxazole, TMP-trimethoprim, MTZ-metronidazole and macrolides were found at much higher concentrations in hospital wastewater while TET-tetracycline and OTC-oxytetracycline, as well as antiretrovirals, had an opposite trend. In contrast, comparable concentrations of resistant genes were observed in both community and hospital wastewater. This supports the hypothesis that AMR levels are more of an endemic nature, developing over time in individual communities. Both hospital and community wastewater had AAs that exceeded PNEC values (e.g. CLR-clarithromycin, CIP-ciprofloxacin). In general, though, hospital effluents had a greater number of quantifiable AAs exceeding PNECs (e.g. SMX-sulfamethoxazole, ERY-erythromycin, TMP-trimethoprim). Hospitals are therefore an important consideration in AMR surveillance as could be high risk areas for AMR.

Community infectious disease treatment with antimicrobial agents - A longitudinal one year study of antimicrobials in two cities via wastewater-based epidemiology.

Antimicrobial resistance (AMR) is one of the most significant global health threats. Inappropriate and over-usage of antimicrobial agents (AAs) is a major driver for AMR. Wastewater-based epidemiology (WBE) is a promising tool for monitoring AA usage in communities which is, for the first time, explored in this large scale, longitudinal study. Two contrasting urban catchment areas have been investigated: one city and one small town in the Southwest of the UK over a 13-month period in 2018-2019. Per capita daily intake of 17 AAs and metabolites has been estimated and obtained estimates were triangulated with catchment specific AA prescription data to understand AA usage patterns (both seasons driven prescription and AA prescription compliance). Results have demonstrated positive correlations for all quantifiable parent AAs and metabolites in wastewater, and spatial variability in AA usage was observed even in neighbouring urban areas. WBE and catchment specific prescription data showed similar seasonal trends but with low correlation in intake. The reasons might be variable prescribing patterns, prescription/intake outside the studied catchment, and/or lack of patient compliance. WBE proved useful in differentiating between consumption vs topical usage and/or direct disposal of unused AA. WBE is considered superior to prescription data as it provides information on AAs prescribed outside of the monitoring catchment, e.g. HIV antivirals and TB drugs. However, data triangulation, of both prescription data and wastewater data, provides the most comprehensive approach to understanding AA usage in communities.

Wastewater-based epidemiology for comprehensive community health diagnostics in a national surveillance study: Mining biochemical markers in wastewater.

This manuscript showcases results from a large scale and comprehensive wastewater-based epidemiology (WBE) study focussed on multi-biomarker suite analysis of both chemical and biological determinants in 10 cities and towns across England equating to a population of ∼7 million people. Multi-biomarker suite analysis, describing city metabolism, can provide a holistic understanding to encompass all of human, and human-derived, activities of a city in a single model: from lifestyle choices (e.g. caffeine intake, nicotine) through to health status (e.g. prevalence of pathogenic organisms, usage of pharmaceuticals as proxy for non-communicable disease, NCD, conditions or infectious disease status), and exposure to harmful chemicals due to environmental and industrial sources (e.g. pesticide intake via contaminated food and industrial exposure). Population normalised daily loads (PNDLs) of many chemical markers were found, to a large extent, driven by the size of population contributing to wastewater (especially NCDs). However, there are several exceptions providing insights into chemical intake that can inform either disease status in various communities or unintentional exposure to hazardous chemicals: e.g. very high PNDLs of ibuprofen in Hull resulting from its direct disposal (confirmed by ibuprofen/2-hydroxyibuprofen ratios) and bisphenol A (BPA) in Hull, Lancaster and Portsmouth likely related to industrial discharge. An importance for tracking endogenous health markers such as 4-hydroxy-2-nonenal-mercapturic acid (HNE-MA, an oxidative stress marker) as a generic marker of health status in communities was observed due to increased levels of HNE-MA seen at Barnoldswick wastewater treatment plant that coincided with higher-than-average paracetamol usage and SARS-CoV-2 prevalence in this community. PNDLs of virus markers were found to be highly variable. Being very prevalent in communities nationwide during sampling, SARS-CoV-2 presence in wastewater was to a large extent community driven. The same applies to the fecal marker virus, crAssphage, which is very prevalent in urban communities. In contrast, norovirus and enterovirus showed much higher variability in prevalence across all sites investigated, with clear cases of localized outbreaks in some cities while maintaining low prevalence in other locations. In conclusion, this study clearly demonstrates the potential for WBE to provide an integrated assessment of community health which can help target and validate policy interventions aimed at improving public health and wellbeing.

PERK: An R/Shiny application to predict and visualise concentrations of pharmaceuticals in the aqueous environment.

Predicting the concentration of active pharmaceuticals ingredients (API) in the environment using modelling approaches is an important aspect in the assessment of their environmental risk, especially for the API with no or limited analytical detection methods. However, handling, validating, and incorporating diverse datasets, including API prescription/consumption data, metabolism, flow data, removal efficiency during wastewater treatment, and dilution factor for the modelling is often laborious and time-consuming. The aim of this manuscript is to evaluate R/Shiny based tool, PERK, to facilitate automated modelling and reporting predicted environmental concentration (PEC) of a comprehensive set of API in different environmental matrices. PERK helped to calculate PEC in wastewater influent, effluent, and river, and compare with measured environmental concentrations (MEC) for five catchments located in England. Prediction accuracy (PA), the ratio between PEC and MEC, can be also generated with the tool. PERK provides consistent interactive user-interface, enabling user to visualise the results with limited programming knowledge.

The burden of city's pain treatment - A longitudinal one year study of two cities via wastewater-based epidemiology.

This paper explores Wastewater-Based Epidemiology (WBE) as a tool enabling understanding of city's pain treatment in an intercity longitudinal study. An intensive 13-month monitoring programme was undertaken in two adjacent urban areas in South-West England: a small commuter town Keynsham and the city of Bath (>180 samples collected). The study has shown a great potential of using triangulated WBE and National health Service (NHS) prescription data in understanding pain treatment in two contrasting communities with strong apparent seasonal patterns of short pain medications vs chronic pain treatment as well as the type of treatment used (e.g. oral vs topical). Community-wide usage of Non-Steroidal Anti-inflammatory Drugs (NSAIDs) and paracetamol in the intercity study is population size and season driven with the highest usage recorded in winter months. This contrasts with other pain pharmaceuticals, especially those used for chronic pain, where no/limited seasonal usage was recorded. Unmetabolized NSAIDs are, to a large extent, directly disposed of into the sewerage system bypassing metabolism due to their topical application. This is particularly apparent in winter months with naproxen showing the highest seasonal variability. Pharma/met (ratio of pharmaceutical and its metabolite concentration) analysis allows for tracking topical (non-metabolic) application/down-the-drain disposal of pharmaceuticals with frequent instances of direct disposal of NSAIDs into the sewerage system observed. Normalisation of pharma markers to population size shows comparable estimates of pharma usage in the two cities confirming population as the main driver of pharma loads in wastewater. Variable application patterns of pain pharmaceuticals make back-calculation of intake more convoluted. Intake calculated using percentage excretion of parent NSAIDs will likely lead to overestimation, as it is assumed that NSAIDs are subject to extensive metabolism (this is not the case for topical applications). Intake calculated using percentage excretion of metabolites (or parent compound) as consumption markers leads to underestimation of NSAIDs usage due to contributions from topical application not being accounted for. Prescription data indicates cumulative internal and topical usage, but the data ignores large proportion of over-the-counter usage. Therefore, we have proposed a combined approach allowing for estimation of total usage including, and differentiating between, topical application and oral administration.

Quantifying community-wide antimicrobials usage via wastewater-based epidemiology.

Increasing usage of antimicrobials is a significant contributor to the emergence and dissemination of antimicrobial resistance. Wastewater-based epidemiology is a useful tool for evaluating public health, via the monitoring of chemical and biological markers in wastewater influent, such as antibiotics. Sixteen antimicrobials and their metabolites were studied: sulfonamides, trimethoprim, metronidazole, quinolones, nitrofurantoin, cyclines, and antiretrovirals. Correction factors (CFs) for human drug excretion, for various drug forms, were determined via a systematic literature review of pharmacokinetic research. Analyte stability was examined over a 24 h study. The estimation of community-wide drug intake was evaluated using the corresponding catchment prescription data. Overall, antimicrobials excreted in an unchanged form were often observed to over-estimate daily intake. This could be attributed to biotransformation, e.g., via glucuronide cleavage, or direct disposal of unused drugs. Acetyl-sulfonamides, trimethoprim, hydroxy-metronidazole, clarithromycin, ciprofloxacin, ofloxacin, tetracycline, and oxytetracycline generally performed well in the estimation of drug intake, relative to prescription records. The low prevalence of quinolone and trimethoprim metabolites, and the low stability of nitrofurantoin, limited the ability to evaluate these metabolites and their respective CFs.

Human population as a key driver of biochemical burden in an inter-city system: Implications for One Health concept.

This paper tests the hypothesis that human population and city function are key drivers of biochemical burden in an inter-city system, which can be used to inform One Health actions as it enables a holistic understanding of city's metabolism encompassing all of the activities of a city in a single model: from lifestyle choices, through to health status and exposure to harmful chemicals as well as effectiveness of implemented management strategies. Chemical mining of wastewater for biophysico-chemical indicators (BCIs) was undertaken to understand speciation of BCIs in the context of geographical as well as community-wide socioeconomic factors. Spatiotemporal variabilities in chemical and biological target groups in the studied inter-city system were observed. A linear relationship (R2 > 0.99) and a strong positive correlation between most BCIs and population size (r > 0.998, p < 0.001) were observed which provides a strong evidence for the population size as a driver of BCI burden. BCI groups that are strongly correlated with population size and are intrinsic to humans' function include mostly high usage pharmaceuticals that are linked with long term non-communicable conditions (NSAIDs, analgesics, cardiovascular, mental health and antiepileptics) and lifestyle chemicals. These BCIs can be used as population size markers. BCIs groups that are produced as a result of a specific city's function (e.g. industry presence and occupational exposure or agriculture) and as such are not correlated with population size include: pesticides, PCPs and industrial chemicals. These BCIs can be used to assess city's function, such as occupational exposure, environmental or food exposure, and as a proxy of community-wide health. This study confirmed a strong positive correlation between antibiotics (ABs), population size and antibiotic resistance genes (ARGs). This confirms the population size and AB usage as the main driver of AB and ARG levels and provides an opportunity for interventions aimed at the reduction of AB usage to reduce AMR. Holistic evaluation of biophysicochemical fingerprints (BCI burden) of the environment and data triangulation with socioeconomic fingerprints (indices) of tested communities are required to fully embrace One Health concept.

PrAna: an R package to calculate and visualize England NHS primary care prescribing data.

BACKGROUND: The objective of this work to calculate prescribed quantity of an active pharmaceutical ingredient (API) in prescription medications for human use, to facilitate research on the prediction of amount of API released to the environment and create an open-data tool to facilitate spatiotemporal and long-term prescription trends for wider usage. DESIGN: We have developed an R package, PrAna to calculate the prescribed quantity (in kg) of an APIs by postcode using England's national level prescription data provided by National Health Service, for the years 2015-2018. Datasets generated using PrAna can be visualized in a real-time interactive web-based tool, PrAnaViz to explore spatiotemporal and long-term trends. The visualisations can be customised by selecting month, year, API, and region. RESULTS: PrAnaViz's targeted API approach is demonstrated with the visualisation of prescribed quantities of 14 APIs in the Bath and North East Somerset (BANES) region during 2018. Once the APIs list is loaded, the back end retrieves relevant data and populates the graphs based on user-defined data features in real-time. These plots include the prescribed quantity of APIs over a year, by month, and individual API by month, general practice, postcode, and medicinal form. The non-targeted API approach is demonstrated with the visualisation of clarithromycin prescribed quantities at different postcodes in the BANES region. CONCLUSION: PrAna and PrAnaViz enables the analysis of spatio-temporal and long-term trends with prescribed quantities of different APIs by postcode. This can be used as a support tool for policymakers, academics and researchers in public healthcare, and environmental scientist to monitor different group of pharmaceuticals emitted to the environment and for prospective risk assessment of pharmaceuticals in the environment.

Diagnosing Down-the-Drain Disposal of Unused Pharmaceuticals at a River Catchment Level: Unrecognized Sources of Environmental Contamination That Require Nontechnological Solutions.

Down-the-drain disposal of pharmaceuticals remains an overlooked and unrecognized source of environmental contamination that requires nontechnological "at-source" solutions. Monitoring of 31 pharmaceuticals over 7 days in five wastewater treatment plants (WWTPs) serving five cities in South-West UK revealed down-the-drain codisposal of six pharmaceuticals to three WWTPs (carbamazepine and propranolol in city A, sildenafil in city B, and diltiazem, capecitabine, and sertraline in city D), with a one-off record codisposal of estimated 253 pills = 40 g of carbamazepine and estimated 96 pills = 4 g of propranolol in city A accounting for their 10- and 3-fold respective increases in wastewater daily loads. Direct disposal of pharmaceuticals was found to affect the efficiency of wastewater treatment with much higher pharmaceutical removal (decrease in daily load) during "down-the-drain disposal" days. This is due to lack of conjugated glucuronide metabolites that are cleaved during "consumption-only" days, with the release of a parent pharmaceutical counterbalancing its removal. Higher removal of pharmaceuticals during down-the-drain disposal days reduced pharmaceutical loads reaching receiving environment, albeit with significant levels remaining. The estimated daily loads in receiving water downstream from a discharge point accounted for 13.8 ± 3.4 and 2.1 ± 0.2 g day-1 of carbamazepine and propranolol, respectively, during consumption-only days and peaked at 20.9 g day-1 (carbamazepine) and 4.6 g day-1 (propranolol) during down-the-drain disposal days. Actions are needed to reduce down-the-drain disposal of pharmaceuticals. Our recent work indicated that down-the-drain disposal of pharmaceuticals doubled since the last study in 2005, which may be due to the lack of information and messaging that informs people to dispose of unused medicines at pharmacies. Media campaigns that inform the public of how to safely dispose of medicines are key to improving rates of return and reducing pharmaceutical waste in the environment. The environment is a key motivator for returning unused medicines to a pharmacy and so messaging should highlight environmental risks associated with improper disposal.

A high prevalence of blaOXA-48 in Klebsiella (Raoultella) ornithinolytica and related species in hospital wastewater in South West England.

Klebsiella species occupy a wide range of environmental and animal niches, and occasionally cause opportunistic infections that are resistant to multiple antibiotics. In particular, Klebsiella pneumoniae (Kpne) has gained notoriety as a major nosocomial pathogen, due principally to the rise in non-susceptibility to carbapenems and other beta-lactam antibiotics. Whilst it has been proposed that the urban water cycle facilitates transmission of pathogens between clinical settings and the environment, the level of risk posed by resistant Klebsiella strains in hospital wastewater remains unclear. We used whole genome sequencing (WGS) to compare Klebsiella species in contemporaneous samples of wastewater from an English hospital and influent to the associated wastewater treatment plant (WWTP). As we aimed to characterize representative samples of Klebsiella communities, we did not actively select for antibiotic resistance (other than for ampicillin), nor for specific Klebsiella species. Two species, Kpne and K. (Raoultella) ornithinolytica (Korn), were of equal dominance in the hospital wastewater, and four other Klebsiella species were present in low abundance in this sample. In contrast, despite being the species most closely associated with healthcare settings, Kpne was the dominant species within the WWTP influent. In total, 29 % of all isolates harboured the blaOXA-48 gene on a pOXA-48-like plasmid, and these isolates were almost exclusively recovered from the hospital wastewater. This gene was far more common in Korn (68 % of isolates) than in Kpne (3.4 % of isolates). In general plasmid-borne, but not chromosomal, resistance genes were significantly enriched in the hospital wastewater sample. These data implicate hospital wastewater as an important reservoir for antibiotic-resistant Klebsiella, and point to an unsuspected role of species within the Raoultella group in the maintenance and dissemination of plasmid-borne blaOXA-48. This article contains data hosted by Microreact.

Estimation of community-wide multi-chemical exposure via water-based chemical mining: Key research gaps drawn from a comprehensive multi-biomarker multi-city dataset.

This paper explores the strong potential of chemical mining of wastewater for markers of community-wide intake of wide-ranging harmful chemicals belonging to several usage groups: industrial chemicals, personal care products, pesticides, illicit drugs, lifestyle chemicals and prescription pharmaceuticals as a proxy for multi-chemical community-wide exposure. An estimation of chemical intake in five contrasting town/cities based in the Avon River catchment in the South-West UK was undertaken. High-resolution spatiotemporal pharmaceutical prescription databases were used for system calibration, both in terms of biomarker selection and its correction factor, as well as for the overall system performance evaluation, both spatially and temporality. Only metabolism data accounting for phase two metabolism provided correct estimates of pharma intake. Using parent compounds as XCRs (xenobiotic compounds residue) was found to overestimate exposure due to an inclusion of directly disposed (unused) drugs. Spatiotemporal trends in XC intake were observed as a result of occupational exposure (higher bisphenol A (BPA) intake during weekday), and lifestyle choices (higher cocaine and pyrethroid pesticides intake during weekend). WBE is not intended to estimate individual exposure to chemicals. It can however provide estimates at a community level, and as a result, it has the potential to be developed into an early warning system, a powerful tool for large scale screening studies identifying communities at risk and in need of high resolution individual testing at a localised scale.

Wastewater-based epidemiology combined with local prescription analysis as a tool for temporalmonitoring of drugs trends - A UK perspective.

This paper reports the application of wastewater-based epidemiology (WBE) for the monitoring of one city in the UK in years 2014-2018 as a means of 1) exploring relative temporal changes of illicit drug usage trends across 5 sampling weeks in 5 years, (2) assessing policy impact in reducing drug consumption, focussing particularly on mephedrone, which was classified as a class B drug in the UK in 2010, and the effects of subsequent regulation such as the novel psychoactive substances (NPS) bill of 2016, (3) investigating temporal changes in consumption of prescription pharmaceuticals vs illicit drug usage, and (4) comparing consumption of prescription drugs with WBE to enable more accurate verification of prescription drugs with abuse potential. Mephedrone was quantified only for the first two years of the study, 2014-2015, and remained undetected for the next three years of the study. This shows that given enough time changes in drug policy can have an effect on drug consumption. However, after the introduction of the 2016 NPS bill, between the third and fourth study years, there was an observable increase in the consumption of "classic" drugs of abuse such as cocaine, MDMA and ketamine suggesting a shift away from novel psychoactives. The unique prescription dataset allowed for a more accurate calculation of heroin consumption using morphine by examining other sources morphine. Additionally, for compounds with controlled prescription like methadone, trends in consumption estimated by wastewater and trends in prescription correlated. Wastewater-based epidemiology is a powerful tool for examining whole populations and determining the efficacy and direction of government actions on health, as it can, alongside prescription and wider monitoring data, provide a clear insight into what is being consumed by a population and what action is needed to meet required goals.

Solid-phase extraction of the alcohol abuse biomarker phosphatidylethanol using newly synthesized polymeric sorbent materials containing quaternary heterocyclic groups.

Phosphatidylethanol (PEth) is an interesting biomarker finding increased use for detecting long term alcohol abuse with high specificity and sensitivity. Prior to detection, sample preparation is an unavoidable step in the work-flow of PEth analysis and new protocols may facilitate it. Solid-phase extraction (SPE) is a versatile sample preparation method widely spread in biomedical laboratories due to its simplicity of use and the possibility of automation. In this work, SPE was used for the first time to directly extract PEth from spiked human plasma and spiked human blood. A library of polymeric SPE materials with different surface functionalities was screened for PEth extraction in order to identify the surface characteristics that control PEth retention and recovery. The plasma samples were diluted 1:10 (v/v) in water and spiked at different concentrations ranging from 0.3 to 5µM. The library of SPE materials was then evaluated using the proposed SPE method and detection was done by LC-MS/MS. One SPE material efficiently retained and recovered PEth from spiked human plasma. With this insight, four new SPE materials were formulated and synthesized based on the surface characteristics of the best SPE material found in the first screening. These new materials were tested with spiked human blood, to better mimic a real clinical sample. All the newly synthetized materials outperformed the pre-existing commercially available materials. Recovery values for the new SPE materials were found between 29.5% and 48.6% for the extraction of PEth in spiked blood. A material based on quaternized 1-vinylimidazole with a poly(trimethylolpropane trimethacrylate) backbone was found suitable for PEth extraction in spiked blood showing the highest analyte recovery in this experiment, 48.6%±6.4%.

MALDIViz: A Comprehensive Informatics Tool for MALDI-MS Data Visualization and Analysis.

Recently, mass spectrometry (MS) has emerged as an important tool for high-throughput screening (HTS) providing a direct and label-free detection method, complementing traditional fluorescent and colorimetric methodologies. Among the various MS techniques used for HTS, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) provides many of the characteristics required for high-throughput analyses, such as low cost, speed, and automation. However, visualization and analysis of the large datasets generated by HTS MALDI-MS can pose significant challenges, especially for multiparametric experiments. The datasets can be generated fast, and the complexity of the experimental data (e.g., screening many different sorbent phases, the sorbent mass, and the load, wash, and elution conditions) makes manual data analysis difficult. To address these challenges, a comprehensive informatics tool called MALDIViz was developed. This tool is an R-Shiny-based web application, accessible independently of the operating system and without the need to install any program locally. It has been designed to facilitate easy analysis and visualization of MALDI-MS datasets, comparison of multiplex experiments, and export of the analysis results to high-quality images.

Filter Plate-Based Screening of MIP SPE Materials for Capture of the Biomarker Pro-Gastrin-Releasing Peptide.

Affinity-based solid-phase extraction (SPE) is an attractive low-cost sample preparation strategy for biomarker analysis. Molecularly imprinted polymers (MIPs) as affinity sorbents offer unique opportunities for affinity SPE, due to their low manufacturing cost and high robustness. A limitation is the prediction of their affinity; therefore, screening of analyte recovery and specificity within a large range of SPE conditions is important in order to ensure high-sensitivity detection and assay reproducibility. Here, a µ-SPE method for screening of the MIP-SPE materials using a commercial 384-well filter plate is presented. The method allows for rapid and automated screening using 10-30 µL of packed SPE sorbent per well and sample volumes in the range of 10-70 µL. This enables screening of many different SPE sorbents while simultaneously identifying optimal SPE conditions. In addition, the 384-well format also facilitates detection with a multitude of analytical platforms. Performance of the µ-MIP-SPE method was investigated using a series of MIPs designed to capture pro-gastrin-releasing peptide (ProGRP). Fractions coming from sample load, cartridge wash, and elution were collected and analyzed using mass spectrometry (MS). The top-performing MIPs were identified, together with proper SPE conditions.

Catalytic Formation of Disulfide Bonds in Peptides by Molecularly Imprinted Microgels at Oil/Water Interfaces.

This work describes the preparation and investigation of molecularly imprinted polymer (MIP) microgel (MG) stabilized Pickering emulsions (PEs) for their ability to catalyze the formation of disulfide bonds in peptides at the O/W interface. The MIP MGs were synthesized via precipitation polymerization and a programmed initiator change strategy. The MIP MGs were characterized using DLS analysis, SEM measurement, and optical microscopy analysis. The dry and wet MIP MGs showed a hydrodynamic diameter of 100 and 280 nm, respectively. A template rebinding experiment showed that the MIP MGs bound over two times more template (24 mg g-1) compared to the uptake displayed by a nonimprinted reference polymer (NIP) MG (10 mg g-1) at saturation. Using the MIP MGs as stabilizers, catalytic oxidation systems were prepared by emulsifying the oil phase and water phase in the presence of different oxidizing agents. During the cyclization, the isolation of the thiol precursors and the oxidizing reagents nonselectively decreased the formation of the byproducts, while the imprinted cavities on the MIP MGs selectively promoted the intramolecular cyclization of peptides. When I2 was used as the oxidizing agent, the MIP-PE-I2 system showed a product yield of 50%, corresponding to a nearly 2-fold increase compared to that of the nonimprinted polymer NIP-PE-I2 system (26%). We believe the interfacial catalysis system presented in this work may offer significant benefits in synthetic peptide chemistry by raising productivity while suppressing the formation of byproducts.