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.

Delta SARS-CoV-2 variant is entirely substituted by the omicron variant during the fifth COVID-19 wave in Attica region.

Wastewater analysis is the most attractive alternative way for the quantification and variant profiling of SARS-CoV-2. Infection dynamics can be monitored by RT-qPCR assays while NGS can provide evidence for the presence of existing or new emerging SARS-CoV-2 variants. Herein, apart from the infection dynamic in Attica since June 1st, 2021, the monitoring of 9 mutations of the omicron and 4 mutations of the delta SARS-CoV-2 variants, utilizing both novel Nested-Seq and RT-PCR, is reported and the substitution of the delta variant (B.1.617.2) by the omicron variant (B.1.1.529) in Attica, Greece within approximately one month is highlighted. The key difference between the two methodologies is discovery power. RT-PCR can only detect known sequences cost-effectively, while NGS is a hypothesis-free approach that does not require prior knowledge to detect novel genes. Overall, the potential of wastewater genomic surveillance for the early discovery and monitoring of variants important for disease management at the community level is underlined. This is the first study, reporting the SARS-CoV-2 infection dynamic for an extended time period and the first attempt to monitor two of the most severe variants with two different methodologies in Greece.

Development and Analytical Validation of a One-Step Five-Plex RT-ddPCR Assay for the Quantification of SARS-CoV-2 Transcripts in Clinical Samples.

Highly sensitive methodologies for SARS-CoV-2 detection are essential for the control of COVID-19 pandemic. We developed and analytically validated a highly sensitive and specific five-plex one-step RT-ddPCR assay for SARS-CoV-2. We first designed in-silico novel primers and probes for the simultaneous absolute quantification of three different regions of the nucleoprotein (N) gene of SARS-CoV-2 (N1, N2, N3), a synthetic RNA as an external control (RNA-EC), and Beta-2-Microglobulin (B2M) as an endogenous RNA internal control (RNA-IC). The developed assay was analytically validated using synthetic DNA and RNA calibrator standards and then was applied to 100 clinical specimens previously analyzed with a commercially available CE-IVD RT-qPCR assay. The analytical validation of the developed assay resulted in very good performance characteristics in terms of analytical sensitivity, linearity, analytical specificity, and reproducibility and recovery rates even at very low viral concentrations. The simultaneous absolute quantification of the RNA-EC and RNA-IC provides the necessary metrics for quality control assessment. Direct comparison of the developed one-step five-plex RT-ddPCR assay with a CE-IVD RT-qPCR kit revealed a very high concordance and a higher sensitivity [concordance: 99/100 (99.0%, Spearman's correlation coefficient: -0.850, p < 0.001)]. The developed assay is highly sensitive, specific, and reproducible and has a broad linear dynamic range, providing absolute quantification of SARS-COV-2 transcripts. The inclusion of two RNA quality controls, an external and an internal, is highly important for standardization of SARS-COV-2 molecular testing in clinical and wastewater samples.

SARS-CoV-2 wastewater surveillance data can predict hospitalizations and ICU admissions.

We measured SARS-CoV-2 RNA load in raw wastewater in Attica, Greece, by RT-qPCR for the environmental surveillance of COVID-19 for 6 months. The lag between RNA load and pandemic indicators (COVID-19 hospital and intensive care unit (ICU) admissions) was calculated using a grid search. Our results showed that RNA load in raw wastewater is a leading indicator of positive COVID-19 cases, new hospitalization and admission into ICUs by 5, 8 and 9 days, respectively. Modelling techniques based on distributed/fixed lag modelling, linear regression and artificial neural networks were utilized to build relationships between SARS-CoV-2 RNA load in wastewater and pandemic health indicators. SARS-CoV-2 mutation analysis in wastewater during the third pandemic wave revealed that the alpha-variant was dominant. Our results demonstrate that clinical and environmental surveillance data can be combined to create robust models to study the on-going COVID-19 infection dynamics and provide an early warning for increased hospital admissions.

Novel Nested-Seq Approach for SARS-CoV-2 Real-Time Epidemiology and In-Depth Mutational Profiling in Wastewater.

Considering the lack of effective treatments against COVID-19, wastewater-based epidemiology (WBE) is emerging as a cost-effective approach for real-time population-wide SARS-CoV-2 monitoring. Here, we report novel molecular assays for sensitive detection and mutational/variant analysis of SARS-CoV-2 in wastewater. Highly stable regions of SARS-CoV-2 RNA were identified by RNA stability analysis and targeted for the development of novel nested PCR assays. Targeted DNA sequencing (DNA-seq) was applied for the analysis and quantification of SARS-CoV-2 mutations/variants, following hexamers-based reverse transcription and nested PCR-based amplification of targeted regions. Three-dimensional (3D) structure models were generated to examine the predicted structural modification caused by genomic variants. WBE of SARS-CoV-2 revealed to be assay dependent, and significantly improved sensitivity achieved by assay combination (94%) vs. single-assay screening (30%-60%). Targeted DNA-seq allowed the quantification of SARS-CoV-2 mutations/variants in wastewater, which agreed with COVID-19 patients' sequencing data. A mutational analysis indicated the prevalence of D614G (S) and P323L (RdRP) variants, as well as of the Β.1.1.7/alpha variant of concern, in agreement with the frequency of Β.1.1.7/alpha variant in clinical samples of the same period of the third pandemic wave at the national level. Our assays provide an innovative cost-effective platform for real-time monitoring and early-identification of SARS-CoV-2 variants at community/population levels.

Change in the chemical content of untreated wastewater of Athens, Greece under COVID-19 pandemic.

COVID-19 pandemic spread rapidly worldwide with unanticipated effects on mental health, lifestyle, stability of economies and societies. Although many research groups have already reported SARS-CoV-2 surveillance in untreated wastewater, only few studies evaluated the implications of the pandemic on the use of chemicals by influent wastewater analysis. Wide-scope target and suspect screening were used to monitor the effects of the pandemic on the Greek population through wastewater-based epidemiology. Composite 24 h influent wastewater samples were collected from the wastewater treatment plant of Athens during the first lockdown and analyzed by liquid chromatography mass spectrometry. A wide range of compounds was investigated (11,286), including antipsychotic drugs, illicit drugs, tobacco compounds, food additives, pesticides, biocides, surfactants and industrial chemicals. Mass loads of chemical markers were estimated and compared with the data obtained under non-COVID-19 conditions (campaign 2019). The findings revealed increases in surfactants (+196%), biocides (+152%), cationic quaternary ammonium surfactants (used as surfactants and biocides) (+331%), whereas the most important decreases were estimated for tobacco (-33%) and industrial chemicals (-52%). The introduction of social-restriction measures by the government affected all aspects of life.

Patterns of pharmaceuticals use during the first wave of COVID-19 pandemic in Athens, Greece as revealed by wastewater-based epidemiology.

Since 2019, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), impaired public health with considerable morbidity and mortality due to the lack of vaccines and effective treatment. The severe disease mainly harmed adults with predisposing medical comorbidities (such as heart disease, hypertension, chronic lung disease), while it can occur in healthy individuals that may be asymptomatic. Wastewater-based Epidemiology (WBE), a non-invasive, objective, chemical tool was used to monitor and estimate the changes in drug's consumption and prescription patterns under normal conditions (2019) and under COVID-19 pandemic conditions (2020). NSAIDs, antihypertensives, diuretics, antiepileptics, antilipidemics, antibiotics, analgesics, antivirals, anticancer drugs, contrast iodinated drugs, antidiabetics, antiallergic drugs, antiulcers and other pharmaceuticals were studied in wastewater and revealed the application of various treatments during the first wave of the pandemic in Athens, Greece. Data were correlated with COVID-19 infection therapeutical plans. The result of the analysis revealed a remarkable increase for antiviral drugs (170%), hydroxychloroquine (387%), and antibiotics (57%), which were the most applied treatments against COVID-19 during the first wave in Greece. In agreement with related authorities urge, NSAIDs presented decrease (27%) during the first lockdown, while paracetamol demonstrated a remarkable increase (198%). The use levels for Angiotensin II receptor blockers such as valsartan, and co-administrated diuretics, such as hydrochlorothiazide, were reduced during 2020, by 32% and 26% respectively.

SARS-CoV-2 Infection Is Asymptomatic in Nearly Half of Adults with Robust Anti-Spike Protein Receptor-Binding Domain Antibody Response.

Between June and November 2020, we assessed plasma antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein in 4996 participants (aged 18-82 years, 34.5% men) from the National and Kapodistrian University of Athens. The weighted overall prevalence was 1.6% and monthly prevalence correlated with viral RNA-confirmed SARS-CoV-2 infections in Greece, in the same period. Notably, 49% of seropositive cases reported no history of SARS-CoV-2 infection-related clinical symptoms and 33% were unsuspected of their previous infection. Additionally, levels of anti-SARS-CoV-2 antibodies against the spike-protein receptor-binding domain were similar between symptomatic and asymptomatic individuals, irrespective of age and gender. Using Food and Drug Administration Emergency Use Authorization-approved assays, these results support the need for such studies on pandemic evaluation and highlight the development of robust humoral immune responses even among asymptomatic individuals. The high percentage of unsuspected/asymptomatic active cases, which may contribute to community transmission for more days than that of cases who are aware and self-isolate, underscores the necessity of measures across the population for the efficient control of the pandemic.

A review of the impact of weather and climate variables to COVID-19: In the absence of public health measures high temperatures cannot probably mitigate outbreaks.

The new severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) pandemic was first recognized at the end of 2019 and has caused one of the most serious global public health crises in the last years. In this paper, we review current literature on the effect of weather (temperature, humidity, precipitation, wind, etc.) and climate (temperature as an essential climate variable, solar radiation in the ultraviolet, sunshine duration) variables on SARS-CoV-2 and discuss their impact to the COVID-19 pandemic; the review also refers to respective effect of urban parameters and air pollution. Most studies suggest that a negative correlation exists between ambient temperature and humidity on the one hand and the number of COVID-19 cases on the other, while there have been studies which support the absence of any correlation or even a positive one. The urban environment and specifically the air ventilation rate, as well as air pollution, can probably affect, also, the transmission dynamics and the case fatality rate of COVID-19. Due to the inherent limitations in previously published studies, it remains unclear if the magnitude of the effect of temperature or humidity on COVID-19 is confounded by the public health measures implemented widely during the first pandemic wave. The effect of weather and climate variables, as suggested previously for other viruses, cannot be excluded, however, under the conditions of the first pandemic wave, it might be difficult to be uncovered. The increase in the number of cases observed during summertime in the Northern hemisphere, and especially in countries with high average ambient temperatures, demonstrates that weather and climate variables, in the absence of public health interventions, cannot mitigate the resurgence of COVID-19 outbreaks.

Analytical methodologies for the detection of SARS-CoV-2 in wastewater: Protocols and future perspectives.

In March 2020 the World Health Organization announced a pandemic outbreak. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative pathogen for the coronavirus disease-19 (COVID-19) pandemic. The authorities worldwide use clinical science to identify infected people, but this approach is not able to track all symptomatic and asymptomatic cases due to limited sampling capacity of the testing laboratories. This drawback is eliminated by the Wastewater-Based Epidemiology (WBE) approach. In this review, we summarized the peer-reviewed published literature (available as of September 28, 2020), in the field of WBE. The commonly used steps (sampling, storage, concentration, isolation, detection) of the analytical protocols were identified. The potential limitations of each stage of the protocols and good practices were discussed. Finally, new methods for the efficient detection of SARS-CoV-2 were proposed.

Seroprevalence of Antibodies against SARS-CoV-2 among the Personnel and Students of the National and Kapodistrian University of Athens, Greece: A Preliminary Report

Due to early implementation of public health measures, Greece had low number of SARS-CoV-2 infections and COVID-19 severe incidents in hospitalized patients. The National and Kapodistrian University of Athens (NKUA), especially its health-care/medical personnel, has been actively involved in the first line of state responses to COVID-19. To estimate the prevalence of antibodies (Igs) against SARS-CoV-2 among NKUA members, we designed a five consecutive monthly serosurvey among randomly selected NKUA consenting volunteers. Here, we present the results from the first 2500 plasma samples collected during June-July 2020. Twenty-five donors were tested positive for anti-SARS-CoV-2 Igs;thus, the overall seroprevalence was 1.00%. The weighted overall seroprevalence was 0.93% (95% CI: 0.27, 2.09) and varied between males [1.05% (95% CI: 0.18, 2.92)] and females [0.84% (95% CI: 0.13, 2.49)], age-groups and different categories (higher in participants from the School of Health Sciences and in scientific affiliates/faculty members/laboratory assistants), but no statistical differences were detected. Although focused on the specific population of NKUA members, our study shows that the prevalence of anti-SARS-CoV-2 Igs for the period June-July 2020 remained low and provides knowledge of public health importance for the NKUA members. Given that approximately one in three infections was asymptomatic, continuous monitoring of the progression of the pandemic by assessing Ig seroprevalence is needed.