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.

Experimental verification of real-time gamma-ray energy spectrum and dose monitor.

The purpose of this study is to develop a portable monitor that can measure the energy spectrum and dose of gamma-rays simultaneously in real time for the benefit of medical staff who must work in clinical radiation environments. For this purpose, we have developed a prototype monitor using a CsI (Tl) scintillator combined with a multi-pixel photon counter (MPPC). For real-time measurement, we employed an improved sequential Bayesian estimation (k-α method) to convert the measured pulse height spectrum into an energy spectrum. Then we confirmed that reconstruction of the energy spectrum and dose estimation could simultaneously be carried out in real time by the k-α method in a radiation field composed of mixed standard gamma-ray sources. In this study, we carried out measurements in a background gamma-ray field to confirm applicability of the prototype monitor to the weakest type of radiation field. In addition, we conducted measurements in front of a nuclear fuel storage room (∼2 µSv/h) in the authors' laboratory to evaluate practicality of the monitor for measuring fields with a complex energy spectrum. As a result, it was found that the dose could be estimated in about 20 s after start of measurements even in the background field. For the energy spectrum, it was instantly reconstructed within 60 s in front of the fuel storage room. On the other hand, it could successfully be estimated within 10 min in the background gamma-ray field. Currently, the convergence of the energy spectrum is determined visually from time dependent change of the spectrum and dose. As a next step, we will attempt to develop a more quantitative procedure for determining the convergence.

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.

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 (ΝΚUA), 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.

A complicated case of primary hypophysitis with bilateral intracavernous carotid artery occlusion.

UNLABELLED: Primary hypophysitis (PH) is a rare clinical entity characterized by inflammatory infiltration of the pituitary gland with various degrees of pituitary dysfunction. OBJECTIVE: To present a complicated case of aggressive PH with bilateral cavernous sinuses infiltration, successfully treated with azathioprine after failure of corticosteroid treatment. METHODS AND RESULTS: A 48-year-old woman presented with episodes of recurrent headache and progressively worsening muscle weakness. Magnetic resonance imaging (MRI) identified an intrasellar pituitary lesion with thickened pituitary stalk extending to the cavernous sinuses and causing asymptomatic occlusion of both internal carotid arteries (ICAs). Hormonal investigation showed severe anterior pituitary deficiency. The diagnosis of PH, and more specifically of lymphocytic hypophysitis (LYH), was suspected and glucocorticoid treatment was initiated. Because of the patient's susceptibility to infections, the attempt to gradually reduce glucocorticoid dosage induced a relapse of PH. Immunosuppressive therapy with azathioprine was administered. Significant pituitary mass reduction with regression of the inflammation to the cavernous sinuses was documented. At follow-up the pituitary function was normal, while the patient was on the minimum dose of azathioprine. Thereafter, azathioprine was discontinued without any clinical/biochemical or radiological evidence of PH except for the permanent ICA occlusion. CONCLUSIONS: Despite its rarity, PH should be included in the differential diagnosis of pituitary masses and involvement of ICAs occlusion should not be underestimated. Azathioprine, applied as an alternative treatment, was shown to result in remarkable PH improvement.