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.

Adverse effects of COVID-19 mRNA vaccines: the spike hypothesis.

Vaccination is a major tool for mitigating the coronavirus disease 2019 (COVID-19) pandemic, and mRNA vaccines are central to the ongoing vaccination campaign that is undoubtedly saving thousands of lives. However, adverse effects (AEs) following vaccination have been noted which may relate to a proinflammatory action of the lipid nanoparticles used or the delivered mRNA (i.e., the vaccine formulation), as well as to the unique nature, expression pattern, binding profile, and proinflammatory effects of the produced antigens - spike (S) protein and/or its subunits/peptide fragments - in human tissues or organs. Current knowledge on this topic originates mostly from cell-based assays or from model organisms; further research on the cellular/molecular basis of the mRNA vaccine-induced AEs will therefore promise safety, maintain trust, and direct health policies.

Comparative kinetics of SARS-CoV-2 anti-spike protein RBD IgGs and neutralizing antibodies in convalescent and naïve recipients of the BNT162b2 mRNA vaccine versus COVID-19 patients.

BACKGROUND: Coronavirus SARS-CoV-2, the causative agent of COVID-19, has caused a still evolving global pandemic. Given the worldwide vaccination campaign, the understanding of the vaccine-induced versus COVID-19-induced immunity will contribute to adjusting vaccine dosing strategies and speeding-up vaccination efforts. METHODS: Anti-spike-RBD IgGs and neutralizing antibodies (NAbs) titers were measured in BNT162b2 mRNA vaccinated participants (n = 250); we also investigated humoral and cellular immune responses in vaccinated individuals (n = 21) of this cohort 5 months post-vaccination and assayed NAbs levels in COVID-19 hospitalized patients (n = 60) with moderate or severe disease, as well as in COVID-19 recovered patients (n = 34). RESULTS: We found that one (boosting) dose of the BNT162b2 vaccine triggers robust immune (i.e., anti-spike-RBD IgGs and NAbs) responses in COVID-19 convalescent healthy recipients, while naïve recipients require both priming and boosting shots to acquire high antibody titers. Severe COVID-19 triggers an earlier and more intense (versus moderate disease) immune response in hospitalized patients; in all cases, however, antibody titers remain at high levels in COVID-19 recovered patients. Although virus infection promotes an earlier and more intense, versus priming vaccination, immune response, boosting vaccination induces antibody titers significantly higher and likely more durable versus COVID-19. In support, high anti-spike-RBD IgGs/NAbs titers along with spike (vaccine encoded antigen) specific T cell clones were found in the serum and peripheral blood mononuclear cells, respectively, of vaccinated individuals 5 months post-vaccination. CONCLUSIONS: These findings support vaccination efficacy, also suggesting that vaccination likely offers more protection than natural infection.

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.

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.

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.