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.

Blood Transcriptomes of Anti-SARS-CoV-2 Antibody-Positive Healthy Individuals Who Experienced Asymptomatic Versus Clinical Infection.

The reasons behind the clinical variability of SARS-CoV-2 infection, ranging from asymptomatic infection to lethal disease, are still unclear. We performed genome-wide transcriptional whole-blood RNA sequencing, bioinformatics analysis and PCR validation to test the hypothesis that immune response-related gene signatures reflecting baseline may differ between healthy individuals, with an equally robust antibody response, who experienced an entirely asymptomatic (n=17) versus clinical SARS-CoV-2 infection (n=15) in the past months (mean of 14 weeks). Among 12.789 protein-coding genes analysed, we identified six and nine genes with significantly decreased or increased expression, respectively, in those with prior asymptomatic infection relatively to those with clinical infection. All six genes with decreased expression (IFIT3, IFI44L, RSAD2, FOLR3, PI3, ALOX15), are involved in innate immune response while the first two are interferon-induced proteins. Among genes with increased expression six are involved in immune response (GZMH, CLEC1B, CLEC12A), viral mRNA translation (GCAT), energy metabolism (CACNA2D2) and oxidative stress response (ENC1). Notably, 8/15 differentially expressed genes are regulated by interferons. Our results suggest that subtle differences at baseline expression of innate immunity-related genes may be associated with an asymptomatic disease course in SARS-CoV-2 infection. Whether a certain gene signature predicts, or not, those who will develop a more efficient immune response upon exposure to SARS-CoV-2, with implications for prioritization for vaccination, warrant further study.

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.

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.