Three years of wastewater surveillance for new psychoactive substances from 16 countries.

The proliferation of new psychoactive substances (NPS) over recent years has made their surveillance complex. The analysis of raw municipal influent wastewater can allow a broader insight into community consumption patterns of NPS. This study examines data from an international wastewater surveillance program that collected and analysed influent wastewater samples from up to 47 sites in 16 countries between 2019 and 2022. Influent wastewater samples were collected over the New Year period and analysed using validated liquid chromatography - mass spectrometry methods. Over the three years, a total of 18 NPS were found in at least one site. Synthetic cathinones were the most found class followed by phenethylamines and designer benzodiazepines. Furthermore, two ketamine analogues, one plant based NPS (mitragynine) and methiopropamine were also quantified across the three years. This work demonstrates that NPS are used across different continents and countries with the use of some more evident in particular regions. For example, mitragynine has highest mass loads in sites in the United States, while eutylone and 3-methylmethcathinone increased considerably in New Zealand and in several European countries, respectively. Moreover, 2F-deschloroketamine, an analogue of ketamine, has emerged more recently and could be quantified in several sites, including one in China, where it is considered as one of the drugs of most concern. Finally, some NPS were detected in specific regions during the initial sampling campaigns and spread to additional sites by the third campaign. Hence, wastewater surveillance can provide an insight into temporal and spatial trends of NPS use.

Do the lockdown-imposed changes in a wastewater treatment plant catchment's socio-demographics impact longitudinal temporal trends in psychoactive pharmaceutical use?

Wastewater-based epidemiology (WBE) includes the analysis of human metabolic biomarkers of xenobiotics in influent wastewater. WBE complements existing drug utilization approaches and provides objective, spatio-temporal information on the consumption of pharmaceuticals in the general population. This approach was applied to 24-h composite influent wastewater samples from Leuven, Belgium. Daily samples were analysed from September 2019 to December 2019 (n = 76), and on three days of the week (Monday, Wednesday, Saturday) from January 2020 to April 2022 (n = 367). Sample analysis consisted of 96-well solid-phase extraction and liquid chromatography coupled to tandem mass spectrometry. Measured concentrations of 21 biomarkers for antidepressant and opioid use were converted to population-normalized mass loads (PNML) by considering the flow rate and catchment population. To capture population movements, mobile phone data was used. Amitriptyline, hydroxy-bupropion, norcitalopram, citalopram, normirtazapine, trazodone, O-desmethylvenlafaxine, codeine, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), methadone, morphine, O-desmethyltramadol, and tramadol were included in the temporal assessment since concentrations were above the lower limit of quantification. The PNML of most biomarkers increased (with 3-119 %) throughout the sampling period. The population disruption during the COVID-19 pandemic led to a major change in the socio-demographics of the catchment area, resulting in temporal differences in the PNML of the different biomarkers. As such, higher PNML were observed during the different lockdown phases, which were characterized by the outflow of university students and a decreasing commuting in and out the catchment area. The effects of the fluctuating socio-demographics of the catchment population were further evidenced by the different week-weekend pattern of PNMLs over the course of the sampling campaign. Mean parent/metabolite ratios (i.e., citalopram/norcitalopram, tramadol/O-desmethyltramadol, venlafaxine/O-desmethylvenlafaxine, and methadone/EDDP) remained relatively stable throughout the entire sampling campaign (RSD% below 25 % for all ratios, except for methadone/EDDP) and therefore were not affected by this population change.

Evaluating the impact of COVID-19 countermeasures on alcohol consumption through wastewater-based epidemiology: A case study in Belgium.

Wastewater-based epidemiology (WBE) is a complementary approach to monitor alcohol consumption in the general population. This method measures concentrations of xenobiotic biomarkers (e.g., ethyl sulphate) in influent wastewater (IWW) and converts these to population-normalized mass loads (PNML, in g/day/1000 inhabitants) by multiplying with the flow rate and dividing by the catchment population. The aims of this case study were to: (i) investigate temporal trends in alcohol use during the COVID-19 pandemic; and (ii) measure the effect of policy measures on alcohol consumption. Daily 24-h composite IWW samples (n = 735) were collected in the wastewater treatment plant of the university city of Leuven (Belgium) starting from September 2019 to September 2021. This is the first study that investigates alcohol use through WBE for a continuous period of two years on a daily basis. Mobile phone data was used to accurately capture population fluxes in the catchment area. Data was evaluated using a time series based statistical framework to graphically and quantitatively assess temporal differences in the measured PNML. Different WBE studies observed temporal changes in alcohol use during the COVID-19 pandemic. In this study, the PNML of ethyl sulphate decreased during the first lockdown phase, potentially indicating that less alcohol was consumed at the Leuven area during home confinement. Contrastingly, alcohol use increased after the re-opening of the catering industry. Additionally, a decrease in alcohol use was observed during the exam periods at the University of Leuven and an increase during the holiday periods. The present study shows the potential of WBE to rapidly assess the impact of some policy measures on alcohol consumption in Belgium. This study also indicates that WBE could be employed as a complementary data source to fill in some of the current knowledge gaps linked to lifestyle behavior.

Temporal monitoring of pharmaceutical consumption using a wastewater-based epidemiologic approach

Aim Temporal monitoring of antidepressants, opioids consumption through influent wastewater, before and during the COVID-19 pandemic in Leuven, Belgium. Introduction: Conventional epidemiologic data sources on pharmaceutical consumption include surveys, prescription, sales, and dispensing data. However, prescribed medication may not always be dispensed, and surveys are affected by bias (e.g., response, reporting). Furthermore, data on pharmaceutical dispensing in Belgium only covers public pharmacies, and only records reimbursed medication (e.g., over-the-counter medication, some opioids are unrecorded). In addition, patients may not take the dispensed drugs as prescribed, or incorrectly. There is also a long lag time such as three years for survey data, and over three months for dispensing data. Wastewater-based epidemiology (WBE) centres on the analysis of biomarkers, i.e., human metabolic excretion products of xenobiotics in influent wastewater. WBE complements existing drug utilisation approaches and provides valuable, objective, spatio-temporal information on the consumption of pharmaceuticals in the general population that may not be measured in other datasets. Method WBE was applied to 24-h composite influent wastewater samples from Leuven, Belgium. Daily samples were analysed from Sept 2019 to Dec 2019 (n=63), and on each Monday, Wednesday, and Saturday of the week from Jan 2020 to Dec 2021 (n=165). The sampled period also included several governmental restrictions (e.g., stay at home measures) to contain the spread of SARS-CoV-2. Sample preparation and analysis consisted of preconcentration with solid-phase extraction and liquid chromatography coupled to tandem mass spectrometry, respectively. Measured concentrations (ng/L) of 21 pharmaceutical biomarkers, i.e., parent compounds and metabolites, were converted to population normalised mass loads (PNML) by considering the flow rate and catchment population. To better capture population movements, mobile phone data was used in the back-calculations. Results Concentrations of bupropion, hydromorphone, melitracen, noroxycodone, oxycodone, and tilidine were negligible or below limit of quantification and therefore excluded from further analysis. The pharmaceuticals, amitriptyline, hydroxy-bupropion, N-desmethylcitalopram, citalopram, N-desmethyl-mirtazapine, mirtazapine, trazodone, O-desmethylvenlafaxine, codeine, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine, methadone, morphine, nortilidine, O-desmethyltramadol, and tramadol were detected and included in the temporal assessment. The PNML of most psychoactive pharmaceuticals remained stable throughout the entire sampling period. Highest median PNML levels by pharmaceutical class, respectively opioid and antidepressant, were obtained for tramadol (median 508mg/day/1000 people) and O-desmethylvenlafaxine (median 392mg/day/1000 people). Governmental measures appear to have minimal effect on the consumption. Several studies have described the psychological distress (e.g., anxiety, stress) of these measures on mental health (COVID-19 Impact, Sciensano;Mental Health Considerations during COVID-19, WHO). However, there is a long lag time between onset of mental distress, help seeking, and the actual start of pharmaceutical therapy. The sampled period may need to be extended to detect this. It remains important to monitor psychoactive pharmaceuticals with addiction potential (e.g., opioids), as they have led to health crisis in other countries. Throughout the sampled period, a small difference in week/weekend PNML is observed for all analysed pharmaceuticals. Antidepressants and opioids are indicated to be used consistently, and long-term in case of antidepressants. We hypothesize that this change may be explained due to a change in population demographics. Leuven has a large commuting student population, approx. 45% of census population, although it should be noted that the catchment area also encompasses nearby areas. Conclusion This study shows the potential of WBE to monitor consumption trends of pharmaceuticals with high temporal resolution, may not be captured by other data sources. Furthermore, our findings demonstrate that care should be taken when interpreting WBE results in case of large population disruption, such as a large commuting population or COVID-19 governmental measures.

Temporal monitoring of stimulants during the COVID-19 pandemic in Belgium through the analysis of influent wastewater.

BACKGROUND AND AIMS: Wastewater-based epidemiology (WBE) is a complementary epidemiological data source to monitor stimulant consumption. The aims were to: (i) study intra- and inter-year temporal changes in stimulant use in Belgium during the first wave of the COVID-19 pandemic; and (ii) evaluate the effect of COVID-19 restrictive measures on stimulant consumption. METHODS: The study population corresponded to the catchments of four wastewater treatment plants corresponding with four Belgian cities (i.e., Antwerp-Zuid, Boom, Brussels, Leuven). Daily 24-h composite influent wastewater samples collected over one week in September 2019 and March through June 2020 during the first wave of the COVID-19 pandemic were analyzed for biomarkers of amphetamine, cocaine, methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA). Measured concentrations were converted to population-normalized mass loads by considering the daily flow rate and the catchment population size. Mobile network data was used to accurately capture population movements in the different catchment areas. Temporal changes were assessed with multiple linear regression models, and the effect of the COVID-19 interventions on stimulant consumption were investigated. RESULTS: An increase in amphetamine use was observed in three cities during governmental restrictions, with highest consumption predominantly during lockdown. Similarly, cocaine consumption was higher after the pandemic started, with highest consumption noted during the lockdown period in Boom and Leuven. Consumption of MDMA was similar in Antwerp-Zuid, Brussels and Leuven throughout the entire sampled period. In Boom, the highest consumption was observed during the full lockdown period. CONCLUSIONS: The present study shows the potential of WBE to assess the impact of stringent lockdown measures on stimulant use in Belgium. This paper shows that strong restrictive measures did not have a profound effect on stimulant consumption.

Optimization and Application of a Multiplex Digital PCR Assay for the Detection of SARS-CoV-2 Variants of Concern in Belgian Influent Wastewater.

Since the beginning of the COVID-19 pandemic, the wastewater-based epidemiology (WBE) of SARS-CoV-2 has been used as a complementary indicator to follow up on the trends in the COVID-19 spread in Belgium and in many other countries. To further develop the use of WBE, a multiplex digital polymerase chain reaction (dPCR) assay was optimized, validated and applied for the measurement of emerging SARS-CoV-2 variants of concern (VOC) in influent wastewater (IWW) samples. Key mutations were targeted in the different VOC strains, including SΔ69/70 deletion, N501Y, SΔ241 and SΔ157. The presented bioanalytical method was able to distinguish between SARS-CoV-2 RNA originating from the wild-type and B.1.1.7, B.1.351 and B.1.617.2 variants. The dPCR assay proved to be sensitive enough to detect low concentrations of SARS-CoV-2 RNA in IWW since the limit of detection of the different targets ranged between 0.3 and 2.9 copies/µL. This developed WBE approach was applied to IWW samples originating from different Belgian locations and was able to monitor spatio-temporal changes in the presence of targeted VOC strains in the investigated communities. The present dPCR assay developments were realized to bring added-value to the current national WBE of COVID-19 by also having the spatio-temporal proportions of the VoC in presence in the wastewaters.

Factors influencing SARS-CoV-2 RNA concentrations in wastewater up to the sampling stage: A systematic review.

Wastewater-based surveillance (WBS) for SARS-CoV-2 RNA is a promising complementary approach to monitor community viral circulation. A myriad of factors, however, can influence RNA concentrations in wastewater, impeding its epidemiological value. This article aims to provide an overview and discussion of factors up to the sampling stage that impact SARS-CoV-2 RNA concentration estimates in wastewater. To this end, a systematic review was performed in three databases (MEDLINE, Web of Science and Embase) and two preprint servers (MedRxiv and BioRxiv). Two authors independently screened and selected articles published between January 1, 2019 and May 4, 2021. A total of 22 eligible articles were included in this systematic review. The following factors up to sampling were identified to have an influence on SARS-CoV-2 RNA concentrations in wastewater and its interpretation: (i) shedding-related factors, including faecal shedding parameters (i.e. shedding pattern, recovery, rate, and load distribution), (ii) population size, (iii) in-sewer factors, including solid particles, organic load, travel time, flow rate, wastewater pH and temperature, and (iv) sampling strategy. In conclusion, factors influencing SARS-CoV-2 RNA concentration estimates in wastewater were identified and research gaps were discussed. The identification of these factors supports the need for further research on WBS for COVID-19.

An alternative approach for bioanalytical assay optimization for wastewater-based epidemiology of SARS-CoV-2.

Wastewater-based epidemiology of SARS-CoV-2 could play a role in monitoring the spread of the virus in the population and controlling possible outbreaks. However, sensitive sample preparation and detection methods are necessary to detect trace levels of SARS-CoV-2 RNA in influent wastewater (IWW). Unlike predecessors, method optimization of a SARS-CoV-2 RNA concentration and detection procedure was performed with IWW samples with high viral SARS-CoV-2 RNA loads. This is of importance since the SARS-CoV-2 genome in IWW might have already been subject to in-sewer degradation into smaller genome fragments or might be present in a different form (e.g. cell debris, …). Centricon Plus-70 (100 kDa) centrifugal filter devices resulted in the lowest and most reproducible Ct-values for SARS-CoV-2 RNA. Lowering the molecular weight cut-off did not improve our limit of detection and quantification (approximately 100 copies/µL for all genes). Quantitative polymerase chain reaction (qPCR) was employed for the amplification of the N1, N2, N3 and E-gene fragments. This is one of the first studies to apply digital polymerase chain reaction (dPCR) for the detection of SARS-CoV-2 RNA in IWW. dPCR showed high variability at low concentration levels (100 copies/µL), indicating that variability in bioanalytical methods for wastewater-based epidemiology of SARS-CoV-2 might be substantial. dPCR results in IWW were in line with the results found with qPCR. On average, the N2-gene fragment showed high in-sample stability in IWW for 10 days of storage at 4 °C. Between-sample variability was substantial due to the low native concentrations in IWW. Additionally, the E-gene fragment proved to be less stable compared to the N2-gene fragment and showed higher variability. Freezing the IWW samples resulted in a 10-fold decay of loads of the N2- and E-gene fragment in IWW.

Changes in drug use in European cities during early COVID-19 lockdowns - A snapshot from wastewater analysis.

The COVID-19 outbreak has forced countries to introduce severe restrictive measures to contain its spread. In particular, physical distancing and restriction of movement have had important consequences on human behaviour and potentially also on illicit drug use and supply. These changes can be associated with additional risks for users, in particular due to reduced access to prevention and harm reduction activities. Furthermore, there have been limitations in the amount of data about drug use which can be collected due to restrictions. To goal of this study was to obtain information about potential changes in illicit drug use impacted by COVID-19 restrictions. Wastewater samples were collected in seven cities in the Netherlands, Belgium, Spain and Italy at the beginning of lockdowns (March-May 2020). Using previously established and validated methods, levels of amphetamine (AMP), methamphetamine (METH), MDMA, benzoylecgonine (BE, the main metabolite of cocaine) and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH, main metabolite of tetrahydrocannabinol (THC)) were measured and compared with findings from previous years. Important differences in levels of consumed drugs were observed across the considered countries. Whilst for some substances and locations, marked decreases in consumption could be observed (e.g., 50% decrease in MDMA levels compared to previous years). In some cases, similar or even higher levels compared to previous years could be found. Changes in weekly patterns were also observed, however these were not clearly defined for all locations and/or substances. Findings confirm that the current situation is highly heterogeneous and that it remains very difficult to explain and/or predict the effect that the present pandemic has on illicit drug use and availability. However, given the current difficulty in obtaining data due to restrictions, wastewater analysis can provide relevant information about the situation at the local level, which would be hard to obtain otherwise.