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

Smoking and High-Sensitivity Troponin I Levels in Young and Healthy Adults from the General Population

Lower troponin concentrations measured in smokers in a healthy population raise the question of whether a lower troponin threshold should be considered for tobacco users. We aim to evaluate differences in troponin levels according to the smoking status in healthy young adults. Participants aged 25–41 years were enrolled in a population-based observational study. The smoking status was self-assessed, and participants were classified as never-, past-, and current smokers. Pack-years of smoking were calculated. High-sensitivity cardiac troponin I (hs-cTnI) concentrations were measured from thawed blood samples, and associations were assessed using multivariable linear regression analyses. We included 2155 subjects in this analysis. The mean (SD) age was 35.4 ± 5.22 years; 53% were women. The median hs-cTnI levels across smoking status categories were 0.70 (interquartile range 0.43–1.23) ng/L in never smokers (n = 1174), 0.69 (interquartile range 0.43–1.28) ng/L in past smokers (n = 503), and 0.67 (interquartile range 0.41–1.04) ng/L in current smokers (n = 478), p = 0.04. The troponin levels remained significantly lower in current smokers after adjustment for potential confounders (β-coefficient [95%CI] of −0.08 [−0.25; −0.08], p < 0.001). Our results confirm that current smokers have lower hs-cTnI levels than past or never smokers, with a significant dose–response relationship among current smokers. The absolute differences in hs-cTnI levels were small

Preparation data of the bromodomains BRD3(1), BRD3(2), BRD4(1), and BRPF1B and crystallization of BRD4(1)-inhibitor complexes

AbstractThis article presents detailed purification procedures for the bromodomains BRD3(1), BRD3(2), BRD4(1), and BRPF1B. In addition we provide crystallization protocols for apo BRD4(1) and BRD4(1) in complex with numerous inhibitors. The protocols described here were successfully applied to obtain affinity data by isothermal titration calorimetry (ITC) and by differential scanning fluorimetry (DSF) as well as structural characterizations of BRD4(1) inhibitor complexes (PDB codes: PDB: 4LYI, PDB: 4LZS, PDB: 4LYW, PDB: 4LZR, PDB: 4LYS, PDB: 5D24, PDB: 5D25, PDB: 5D26, PDB: 5D3H, PDB: 5D3J, PDB: 5D3L, PDB: 5D3N, PDB: 5D3P, PDB: 5D3R, PDB: 5D3S, PDB: 5D3T). These data have been reported previously and are discussed in more detail elsewhere [1,2]

Concentrations of Serum Brain Injury Biomarkers Following SARS-CoV-2 Infection in Individuals with and without Long-COVID-Results from the Prospective Population-Based COVI-GAPP Study.

It is unknown whether neurological symptoms are associated with brain injury after SARS-CoV-2 infections and whether brain injury and related symptoms also emerge in Long-COVID patients. Biomarkers such as serum neurofilament light chain (sNfL) and glial fibrillary acidic protein (sGFAP) can be used to elucidate neuro-axonal and astroglial injuries. We investigated whether these biomarkers are associated with COVID-19 infection status, associated symptoms and Long-COVID. From 146 individuals of the general population with a post-acute, mild-to-moderate SARS-CoV-2 infection, sNfL and sGFAP were measured before, during and after (five and ten months) the infection. Individual symptoms and Long-COVID status were assessed using questionnaires. Neurological associated symptoms were described for individuals after a mild and moderate COVID-19 infection; however, sNfL (p = 0.74) and sGFAP (p = 0.24) did not change and were not associated with headache (p = 0.51), fatigue (p = 0.93), anosmia (p = 0.77) or ageusia (p = 0.47). In Long-COVID patients, sGFAP (p = 0.038), but not sNfL (p = 0.58), significantly increased but was not associated with neurological associated symptoms. Long-COVID status, but not post-acute SARS-CoV-2 infections, may be associated with astroglial injury/activation, even if neurological associated symptoms were not correlated

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

COX7A2L genetic variants determine cardiorespiratory fitness in mice and human

Benegiamo et al. identify genetic variants of the mitochondrial supercomplex assembly factor COX7A2L in the skeletal muscle of mice and humans that promote cardiorespiratory fitness.Mitochondrial respiratory complexes form superassembled structures called supercomplexes. COX7A2L is a supercomplex-specific assembly factor in mammals, although its implication for supercomplex formation and cellular metabolism remains controversial. Here we identify a role for COX7A2L for mitochondrial supercomplex formation in humans. By using human cis-expression quantitative trait loci data, we highlight genetic variants in the COX7A2L gene that affect its skeletal muscle expression specifically. The most significant cis-expression quantitative trait locus is a 10-bp insertion in the COX7A2L 3 ' untranslated region that increases messenger RNA stability and expression. Human myotubes harboring this insertion have more supercomplexes and increased respiration. Notably, increased COX7A2L expression in the muscle is associated with lower body fat and improved cardiorespiratory fitness in humans. Accordingly, specific reconstitution of Cox7a2l expression in C57BL/6J mice leads to higher maximal oxygen consumption, increased lean mass and increased energy expenditure. Furthermore, Cox7a2l expression in mice is induced specifically in the muscle upon exercise. These findings elucidate the genetic basis of mitochondrial supercomplex formation and function in humans and show that COX7A2L plays an important role in cardiorespiratory fitness, which could have broad therapeutic implications in reducing cardiovascular mortality.Peer reviewe

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