SARS-CoV-2 Nucleocapsid Protein Mutations Found in Switzerland Disrupt N-Gene Amplification in Commonly Used Multiplex RT-PCR Assay

At the end of 2021, we observed an increase in N-gene target failures (NGTF) with the TaqPathTM COVID-19 CE-IVD RT-PCR Kit from Thermo Fisher Scientific (TaqPath). We subsequently used whole-genome sequencing (Oxford Nanopore Technology) to identify potential issues with N-gene PCR efficacy. Among 168,101 positive samples with a cycle threshold (CT) value <30 from August 2021 to May 2022, 194 specimens without N-gene amplification by PCR were identified (0.12%). Most NGTF samples originated from a wave of infection attributable to the Delta variant (B.1.617.2) and its sublineages. Sequencing revealed the nucleotide substitution G28922T (A217S) in 151 samples (88.8%). The substitution G215C, a hallmark mutation for Delta lineages, was concurrently present in all of these samples. Ten samples (5.9%) carried the deletion 28,913-28,918 (del214/215), eight samples (4.7%) the deletion 28,913-28,915 (del214) and one sample (0.6%) the deletion 28,892-28,930 (del207-219). Samples showing intact N-gene amplification by PCR lacked these specific mutations, but delayed-type amplification (i.e., partial or pNGTF) was attributable to the exclusive presence of A217S. As the N gene is a common target in many RT-PCR methods for SARS-CoV-2, an in-depth analysis of single-target failures using a combination with viral whole genome sequencing may allow for the identification of diagnostic flaws and eventual new variants

Flattening the curve in 52 days: characterisation of the COVID-19 pandemic in the Principality of Liechtenstein - an observational study.

BACKGROUND The principality of Liechtenstein had its first COVID-19 case at the beginning of March 2020. After exponential growth, the pandemic&rsquo;s first wave was contained, with the last case being diagnosed 52 days after the initial occurrence. AIM To characterise the COVID-19 pandemic in Liechtenstein. METHODS All patients diagnosed in Liechtenstein were followed up until recovery and again 6&ndash;8 weeks after symptom onset. They were contacted every 2 days to record their clinical status until the resolution of their symptoms. The diagnosis of COVID-19 was based on clinical symptoms and molecular testing. Household and close workplace contacts were included in the follow-up, which also comprised antibody testing. In addition, public health measures installed during the pandemic in Liechtenstein are summarised. RESULTS During the first wave, 5% of the population obtained a reverse transcriptase polymerase chain reaction test. A total of 95 patients (median age 39 years) were diagnosed with COVID-19 (82 who resided in Liechtenstein), resulting in an incidence in Liechtenstein of 0.211%. One patient, aged 94, died (mortality rate 1%). Only 62% of patients could retrospectively identify a potential source of infection. Testing the patients&rsquo; household and close workplace contacts (n = 170) with antibody tests revealed that 25% of those tested were additional COVID-19 cases, a quarter of whom were asymptomatic. Those households which adhered to strict isolation measures had a significantly lower rate of affected household members than those who didn&rsquo;t follow such measures. The national public health measures never restricted free movement of residents. Masks were only mandatory in healthcare settings. The use of home working for the general workforce was promoted. Gatherings were prohibited. Schools, universities, certain public spaces (like sports facilities and playgrounds), childcare facilities, nonessential shops, restaurants and bars were closed. Social distancing, hygienic measures, solidarity and supporting individuals who were at risk were the main pillars of the public health campaigns. CONCLUSION The close collaboration of all relevant stakeholders allowed for the complete workup of all COVID-19 patients nationwide. A multitude of factors (e.g., young age of the patients, low-threshold access to testing, close monitoring of cases, high alertness and adherence to public health measures by the population) led to the early containment of the first wave of the pandemic, with a very low rate of serious outcomes. Antibody testing for SARS-CoV-2 revealed a substantial proportion of undiagnosed COVID-19 cases among close contacts of the patients

Frequency of serological non-responders and false-negative RT-PCR results in SARS-CoV-2 testing: a population-based study.

Objectives The sensitivity of molecular and serological methods for COVID-19 testing in an epidemiological setting is not well described. The aim of the study was to determine the frequency of negative RT-PCR results at first clinical presentation as well as negative serological results after a follow-up of at least 3 weeks. Methods Among all patients seen for suspected COVID-19 in Liechtenstein (n=1921), we included initially RT-PCR positive index patients (n=85) as well as initially RT-PCR negative (n=66) for follow-up with SARS-CoV-2 antibody testing. Antibodies were detected with seven different commercially available immunoassays. Frequencies of negative RT-PCR and serology results in individuals with COVID-19 were determined and compared to those observed in a validation cohort of Swiss patients (n=211). Results Among COVID-19 patients in Liechtenstein, false-negative RT-PCR at initial presentation was seen in 18% (12/66), whereas negative serology in COVID-19 patients was 4% (3/85). The validation cohort showed similar frequencies: 2/66 (3%) for negative serology, and 16/155 (10%) for false negative RT-PCR. COVID-19 patients with negative follow-up serology tended to have a longer disease duration (p=0.05) and more clinical symptoms than other patients with COVID-19 (p<0.05). The antibody titer from quantitative immunoassays was positively associated with the number of disease symptoms and disease duration (p<0.001). Conclusions RT-PCR at initial presentation in patients with suspected COVID-19 can miss infected patients. Antibody titers of SARS-CoV-2 assays are linked to the number of disease symptoms and the duration of disease. One in 25 patients with RT-PCR-positive COVID-19 does not develop antibodies detectable with frequently employed and commercially available immunoassays

Characterization of a Pan-Immunoglobulin Assay Quantifying Antibodies Directed against the Receptor Binding Domain of the SARS-CoV-2 S1-Subunit of the Spike Protein: A Population-Based Study.

Pan-immunoglobulin assays can simultaneously detect IgG, IgM and IgA directed against the receptor binding domain (RBD) of the S1 subunit of the spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 S1-RBD Ig). In this work, we aim to evaluate a quantitative SARS-CoV-2 S1-RBD Ig electrochemiluminescence immunoassay (ECLIA) regarding analytical, diagnostic, operational and clinical characteristics. Our work takes the form of a population-based study in the principality of Liechtenstein, including 125 cases with clinically well-described and laboratory confirmed SARS-CoV-2 infection and 1159 individuals without evidence of coronavirus disease 2019 (COVID-19). SARS-CoV-2 cases were tested for antibodies in sera taken with a median of 48 days (interquartile range, IQR, 43-52) and 139 days (IQR, 129-144) after symptom onset. Sera were also tested with other assays targeting antibodies against non-RBD-S1 and -S1/S2 epitopes. Sensitivity was 97.6% (95% confidence interval, CI, 93.2-99.1), whereas specificity was 99.8% (95% CI, 99.4-99.9). Antibody levels linearly decreased from hospitalized patients to symptomatic outpatients and SARS-CoV-2 infection without symptoms (p < 0.001). Among cases with SARS-CoV-2 infection, smokers had lower antibody levels than non-smokers (p = 0.04), and patients with fever had higher antibody levels than patients without fever (p = 0.001). Pan-SARS-CoV-2 S1-RBD Ig in SARS-CoV-2 infection cases significantly increased from first to second follow-up (p < 0.001). A substantial proportion of individuals without evidence of past SARS-CoV-2 infection displayed non-S1-RBD antibody reactivities (248/1159, i.e., 21.4%, 95% CI, 19.1-23.4). In conclusion, a quantitative SARS-CoV-2 S1-RBD Ig assay offers favorable and sustained assay characteristics allowing the determination of quantitative associations between clinical characteristics (e.g., disease severity, smoking or fever) and antibody levels. The assay could also help to identify individuals with antibodies of non-S1-RBD specificity with potential clinical cross-reactivity to SARS-CoV-2

Investigation of the use of a sensor bracelet for the presymptomatic detection of changes in physiological parameters related to COVID-19: an interim analysis of a prospective cohort study (COVI-GAPP).

OBJECTIVES We investigated machinelearningbased identification of presymptomatic COVID-19 and detection of infection-related changes in physiology using a wearable device. DESIGN Interim analysis of a prospective cohort study. SETTING, PARTICIPANTS AND INTERVENTIONS Participants from a national cohort study in Liechtenstein were included. Nightly they wore the Ava-bracelet that measured respiratory rate (RR), heart rate (HR), HR variability (HRV), wrist-skin temperature (WST) and skin perfusion. SARS-CoV-2 infection was diagnosed by molecular and/or serological assays. RESULTS A total of 1.5 million hours of physiological data were recorded from 1163 participants (mean age 44±5.5 years). COVID-19 was confirmed in 127 participants of which, 66 (52%) had worn their device from baseline to symptom onset (SO) and were included in this analysis. Multi-level modelling revealed significant changes in five (RR, HR, HRV, HRV ratio and WST) device-measured physiological parameters during the incubation, presymptomatic, symptomatic and recovery periods of COVID-19 compared with baseline. The training set represented an 8-day long instance extracted from day 10 to day 2 before SO. The training set consisted of 40 days measurements from 66 participants. Based on a random split, the test set included 30% of participants and 70% were selected for the training set. The developed long short-term memory (LSTM) based recurrent neural network (RNN) algorithm had a recall (sensitivity) of 0.73 in the training set and 0.68 in the testing set when detecting COVID-19 up to 2 days prior to SO. CONCLUSION Wearable sensor technology can enable COVID-19 detection during the presymptomatic period. Our proposed RNN algorithm identified 68% of COVID-19 positive participants 2 days prior to SO and will be further trained and validated in a randomised, single-blinded, two-period, two-sequence crossover trial. Trial registration number ISRCTN51255782; Pre-results

SARS-CoV-2 N501Y Introductions and Transmissions in Switzerland from Beginning of October 2020 to February 2021—Implementation of Swiss-Wide Diagnostic Screening and Whole Genome Sequencing

The rapid spread of the SARS-CoV-2 lineages B.1.1.7 (N501Y.V1) throughout the UK, B.1.351 (N501Y.V2) in South Africa, and P.1 (B.1.1.28.1; N501Y.V3) in Brazil has led to the definition of variants of concern (VoCs) and recommendations for lineage specific surveillance. In Switzerland, during the last weeks of December 2020, we established a nationwide screening protocol across multiple laboratories, focusing first on epidemiological and microbiological definitions. In January 2021, we validated and implemented an N501Y-specific PCR to rapidly screen for VoCs, which are then confirmed using amplicon sequencing or whole genome sequencing (WGS). A total of 13,387 VoCs have been identified since the detection of the first Swiss case in October 2020, with 4194 being B.1.1.7, 172 B.1.351, and 7 P.1. The remaining 9014 cases of VoCs have been described without further lineage specification. Overall, all diagnostic centers reported a rapid increase of the percentage of detected VOCs, with a range of 6 to 46% between 25 to 31 of January 2021 increasing towards 41 to 82% between 22 to 28 of February. A total of 739 N501Y positive genomes were analysed and show a broad range of introduction events to Switzerland. In this paper, we describe the nationwide coordination and implementation process across laboratories, public health institutions, and researchers, the first results of our N501Y-specific variant screening, and the phylogenetic analysis of all available WGS data in Switzerland, that together identified the early introduction events and subsequent community spreading of the VoCs

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Third-Generation-Cephalosporin-Resistant Klebsiella pneumoniae Isolates from Humans and Companion Animals in Switzerland: Spread of a DHA-Producing Sequence Type 11 Clone in a Veterinary Setting.

Characterization of third-generation-cephalosporin-resistant Klebsiella pneumoniae isolates originating mainly from one human hospital (n = 22) and one companion animal hospital (n = 25) in Bern (Switzerland) revealed the absence of epidemiological links between human and animal isolates. Human infections were not associated with the spread of any specific clone, while the majority of animal infections were due to K. pneumoniae sequence type 11 isolates producing plasmidic DHA AmpC. This clonal dissemination within the veterinary hospital emphasizes the need for effective infection control practices