TiSA: TimeSeriesAnalysis-a pipeline for the analysis of longitudinal transcriptomics data.

Improved transcriptomic sequencing technologies now make it possible to perform longitudinal experiments, thus generating a large amount of data. Currently, there are no dedicated or comprehensive methods for the analysis of these experiments. In this article, we describe our TimeSeries Analysis pipeline (TiSA) which combines differential gene expression, clustering based on recursive thresholding, and a functional enrichment analysis. Differential gene expression is performed for both the temporal and conditional axes. Clustering is performed on the identified differentially expressed genes, with each cluster being evaluated using a functional enrichment analysis. We show that TiSA can be used to analyse longitudinal transcriptomic data from both microarrays and RNA-seq, as well as small, large, and/or datasets with missing data points. The tested datasets ranged in complexity, some originating from cell lines while another was from a longitudinal experiment of severity in COVID-19 patients. We have also included custom figures to aid with the biological interpretation of the data, these plots include Principal Component Analyses, Multi Dimensional Scaling plots, functional enrichment dotplots, trajectory plots, and complex heatmaps showing the broad overview of results. To date, TiSA is the first pipeline to provide an easy solution to the analysis of longitudinal transcriptomics experiments.

Corticosteroids and superinfections in COVID-19 patients on invasive mechanical ventilation.

OBJECTIVES: To determine the incidence and characteristics of superinfections in mechanically ventilated COVID-19 patients, and the impact of dexamethasone as standard therapy. METHODS: This multicentre, observational, retrospective study included patients ≥ 18 years admitted from March 1st 2020 to January 31st 2021 with COVID-19 infection who received mechanical ventilation. Patient characteristics, clinical characteristics, therapy and survival were examined. RESULTS: 155/156 patients (115 men, mean age 62 years, range 26-84 years) were included. 67 patients (43%) had 90 superinfections, pneumonia dominated (78%). Superinfections were associated with receiving dexamethasone (66% vs 32%, p<0.0001), autoimmune disease (18% vs 5.7%, p<0.016) and with longer ICU stays (26 vs 17 days, p<0,001). Invasive fungal infections were reported exclusively in dexamethasone-treated patients [8/67 (12%) vs 0/88 (0%), p<0.0001]. Unadjusted 90-day survival did not differ between patients with or without superinfections (64% vs 73%, p=0.25), but was lower in patients receiving dexamethasone versus not (58% vs 78%, p=0.007). In multiple regression analysis, superinfection was associated with dexamethasone use [OR 3.7 (1.80-7.61), p<0.001], pre-existing autoimmune disease [OR 3.82 (1.13-12.9), p=0.031] and length of ICU stay [OR 1.05 p<0.001]. CONCLUSIONS: In critically ill COVID-19 patients, dexamethasone as standard of care was strongly and independently associated with superinfections.

Soluble ST2 concentrations associate with in-hospital mortality and need for mechanical ventilation in unselected patients with COVID-19.

OBJECTIVE: Soluble ST2 (sST2) reflects inflammation, endothelial dysfunction and myocardial fibrosis, is produced in the lungs and is an established biomarker in heart failure. We sought to determine the role of sST2 in COVID-19 by assessing pathophysiological correlates and its association to in-hospital outcomes. METHODS: We enrolled 123 consecutive, hospitalised patients with COVID-19 in the prospective, observational COVID-19 MECH study. Biobank samples were collected at baseline, day 3 and day 9. The key exposure variable was sST2, and the outcome was ICU treatment with mechanical ventilation or in-hospital death. RESULTS: Concentrations of sST2 at baseline was median 48 (IQR 37-67) ng/mL, and 74% had elevated concentrations (>37.9 ng/mL). Higher baseline sST2 concentrations were associated with older age, male sex, white race, smoking, diabetes, hypertension and chronic kidney disease. Baseline sST2 also associated with the presence of SARS-CoV-2 viraemia, lower oxygen saturation, higher respiratory rate and increasing concentrations of biomarkers reflecting inflammation, thrombosis and cardiovascular disease. During the hospitalisation, 8 (7%) patients died and 27 (22%) survivors received intensive care unit (ICU) treatment. Baseline sST2 concentrations demonstrated a graded association with disease severity (median, IQR): medical ward 43 (36-59) ng/mL; ICU 67 (39-104) ng/mL and non-survivors 107 (72-116) ng/mL (p<0.001 for all comparisons). These associations persisted at day 3 and day 9 . CONCLUSIONS: sST2 concentrations associate with SARS-CoV-2 viraemia, hypoxaemia and concentrations of inflammatory and cardiovascular biomarkers. There was a robust association between baseline sST2 and disease severity that was independent of, and superior to, established risk factors. sST2 reflects key pathophysiology and may be a promising biomarker in COVID-19. TRIAL REGISTRATION NUMBER: NCT04314232.

Cardiac pathology 6 months after hospitalization for COVID-19 and association with the acute disease severity.

BACKGROUND: Coronavirus disease 2019 (COVID-19) may cause myocardial injury and myocarditis, and reports of persistent cardiac pathology after COVID-19 have raised concerns of long-term cardiac consequences. We aimed to assess the presence of abnormal cardiovascular resonance imaging (CMR) findings in patients recovered from moderate-to-severe COVID-19, and its association with markers of disease severity in the acute phase. METHODS: Fifty-eight (49%) survivors from the prospective COVID MECH study, underwent CMR median 175 [IQR 105-217] days after COVID-19 hospitalization. Abnormal CMR was defined as left ventricular ejection fraction (LVEF) <50% or myocardial scar by late gadolinium enhancement. CMR indices were compared to healthy controls (n = 32), and to circulating biomarkers measured during the index hospitalization. RESULTS: Abnormal CMR was present in 12 (21%) patients, of whom 3 were classified with major pathology (scar and LVEF <50% or LVEF <40%). There was no difference in the need of mechanical ventilation, length of hospital stay, and vital signs between patients with vs without abnormal CMR after 6 months. Severe acute respiratory syndrome coronavirus 2 viremia and concentrations of inflammatory biomarkers during the index hospitalization were not associated with persistent CMR pathology. Cardiac troponin T and N-terminal pro-B-type natriuretic peptide concentrations on admission, were higher in patients with CMR pathology, but these associations were not significant after adjusting for demographics and established cardiovascular disease. CONCLUSIONS: CMR pathology 6 months after moderate-to-severe COVID-19 was present in 21% of patients and did not correlate with severity of the disease. Cardiovascular biomarkers during COVID-19 were higher in patients with CMR pathology, but with no significant association after adjusting for confounders. TRIAL REGISTRATION: COVID MECH Study ClinicalTrials.gov Identifier: NCT04314232.

Severe Acute Respiratory Syndrome Coronavirus 2 RNA in Plasma Is Associated With Intensive Care Unit Admission and Mortality in Patients Hospitalized With Coronavirus Disease 2019.

The clinical significance of severe acute respiratory syndrome coronavirus 2 RNA in the circulation is unknown. In this prospective cohort study, we detected viral RNA in the plasma of 58 of 123 (47%) patients hospitalized with coronavirus disease 2019. RNA was detected more frequently, and levels were higher, in patients who were admitted to the intensive care unit and/or died.

Comparison of SARS-CoV-2 infections in healthcare workers with high and low exposures to Covid-19 patients in a Norwegian University Hospital.

INTRODUCTION: A year into the pandemic, the knowledge of SARS-CoV-2 infection risks among healthcare workers remains limited. In this cross-sectional study, we examined whether healthcare workers with high exposure to Covid-19 patients had a higher risk of SARS-CoV-2 infection than other healthcare workers in a Norwegian University Hospital. We also investigated the prevalence of asymptomatic healthcare workers in a ward with a SARS-CoV-2 outbreak. METHODS: Healthcare workers from five wards at Akershus University Hospital were included between May 11 and June 11, 2020. Blood samples were analyzed for SARS-CoV-2 antibodies and seroprevalences compared between participants with high and low exposure to Covid-19 patients. Demographic data and SARS-CoV-2 infection risk factors were recorded in a questionnaire. Naso-/oropharyngeal swabs from participants from the outbreak ward were analyzed by reverse transcriptase-polymerase chain reaction. RESULTS: 360/436 (82.6%) healthcare workers participated. 9/262 (3.4%) participants from wards with high exposure to Covid-19 patients were SARS-CoV-2 seropositive versus 3/98 (3.1%) from wards with low exposure (OR 1.13; 95%CI 0.3-4.26, p = .861). SARS-CoV-2 antibodies were found in 11/263 (4.2%) participants who had worked one or more shifts caring for Covid-19 patients versus in 1/85 (1.2%) without any known occupational Covid-19 exposure (OR 3.67; 95%CI 0.46-29.06, p = .187). SARS-CoV-2 was detected in naso-/oropharyngeal swabs from 2/78 (2.6%) participants. CONCLUSION: We found no significantly increased risk of SARS-CoV-2 infection in healthcare workers with high exposure to COVID-19 patients. Five healthcare workers had either serologic or molecular evidence of past or present unrecognized SARS-CoV-2 infection.

SARS-CoV-2 Viremia is Associated With Inflammatory, But Not Cardiovascular Biomarkers, in Patients Hospitalized for COVID-19.

Background COVID-19 may present with a variety of cardiovascular manifestations, and elevations of biomarkers reflecting myocardial injury and stress are prevalent. SARS-CoV-2 has been found in cardiac tissue, and myocardial dysfunction post-COVID-19 may occur. However, the association between SARS-CoV-2 RNA in plasma and cardiovascular biomarkers remains unknown. Methods and Results COVID MECH (COVID-19 Mechanisms) was a prospective, observational study enrolling consecutive, hospitalized patients with laboratory-confirmed infection with SARS-CoV-2 and symptoms of COVID-19. Biobank plasma samples used to measure SARS-CoV-2 RNA and cardiovascular and inflammatory biomarkers were collected in 123 patients at baseline, and in 96 patients (78%) at day 3. Patients were aged 60±15 (mean ± SD) years, 71 (58%) were men, 68 (55%) were White, and 31 (25%) received mechanical ventilation during hospitalization. SARS-CoV-2 RNA was detected in plasma from 48 (39%) patients at baseline. Patients with viremia were more frequently men, had more diabetes mellitus, and lower oxygen saturation. Patients with viremia had higher concentrations of interleukin-6, C-reactive protein, procalcitonin, and ferritin (all <0.001), but comparable levels of cTnT (cardiac troponin T; P=0.09), NT-proBNP (N-terminal pro-B-type natriuretic peptide; P=0.27) and D-dimer (P=0.67) to patients without viremia. SARS-CoV-2 RNA was present in plasma at either baseline or day 3 in 50 (52%) patients, and these patients experienced increase from baseline to day 3 in NT-proBNP and D-dimer concentrations, while there was no change in cTnT. Conclusions SARS-CoV-2 viremia was associated with increased concentrations of inflammatory, but not cardiovascular biomarkers. NT-proBNP and D-dimer, but not cTnT, increased from baseline to day 3 in patients with viremia. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT04314232.

Growth Differentiation Factor 15 Provides Prognostic Information Superior to Established Cardiovascular and Inflammatory Biomarkers in Unselected Patients Hospitalized With COVID-19.

BACKGROUND: Growth differentiation factor 15 (GDF-15) is a strong prognostic marker in sepsis and cardiovascular disease (CVD). The prognostic value of GDF-15 in coronavirus disease 2019 (COVID-19) is unknown. METHODS: Consecutive, hospitalized patients with laboratory-confirmed infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and symptoms of COVID-19 were enrolled in the prospective, observational COVID Mechanisms Study. Biobank samples were collected at baseline, day 3 and day 9. The primary end point was admission to the intensive care unit or death during hospitalization, and the prognostic performance of baseline and serial GDF-15 concentrations were compared with that of established infectious disease and cardiovascular biomarkers. RESULTS: Of the 123 patients enrolled, 35 (28%) reached the primary end point; these patients were older, more often had diabetes, and had lower oxygen saturations and higher National Early Warning Scores on baseline. Baseline GDF-15 concentrations were elevated (>95th percentile in age-stratified healthy individuals) in 97 (79%), and higher concentrations were associated with detectable SARS-CoV-2 viremia and hypoxemia (both P<0.001). Patients reaching the primary end point had higher concentrations of GDF-15 (median, 4225 [IQR, 3197-5972] pg/mL versus median, 2187 [IQR, 1344-3620] pg/mL, P<0.001). The area under the receiver operating curve was 0.78 (95% CI, 0.70-0.86). The association between GDF-15 and the primary end point persisted after adjusting for age, sex, race, body mass index, estimated glomerular filtration rate, previous myocardial infarction, heart failure, and atrial fibrillation (P<0.001) and was superior and incremental to interleukin-6, C-reactive protein, procalcitonin, ferritin, D-dimer, cardiac troponin T, and N-terminal pro-B-type natriuretic peptide. Increase in GDF-15 from baseline to day 3 was also greater in patients reaching the primary end point (median, 1208 [IQR, 0-4305] pg/mL versus median, -86 [IQR, -322 to 491] pg/mL, P<0.001). CONCLUSIONS: GDF-15 is elevated in the majority of patients hospitalized with COVID-19, and higher concentrations are associated with SARS-CoV-2 viremia, hypoxemia, and worse outcome. The prognostic value of GDF-15 was additional and superior to established cardiovascular and inflammatory biomarkers. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT04314232.

A single-centre, prospective cohort study of COVID-19 patients admitted to ICU for mechanical ventilatory support.

BACKGROUND: Mortality rates in COVID-19 patients in need of mechanical ventilation are high, with wide variations between countries. Most studies were retrospective, and results may not be generalizable due to differences in demographics, healthcare organization and surge capacity. We present a cohort of mechanically ventilated COVID-19 patients from a resource-rich, publicly financed healthcare system. METHODS: Prospective study from a tertiary hospital. Consecutive SARS-CoV-2 positive adult patients admitted to the ICU for mechanical ventilation from 10 March 2020 to 04 May 2020 were included. Triage and treatment were protocolized. High-dose dalteparin was adjusted by D-dimer. Demographics, treatments and high-resolution physiological variables were collected. Outcomes were 30-day and hospital mortality. Data are medians (quartiles). RESULTS: Of the 1484 persons in the hospital catchment area testing positive for SARS-CoV-2, 201 (13.5%) were hospitalized. Thirty-eight (19%) patients were mechanically ventilated, of whom five (13%) died. Of the 163 patients treated with supplemental oxygen, eight (5%) died. In ventilated patients (75% males, age 61 (53-70) years), severe, moderate and mild ARDS was present in 25%, 70% and 5%. Tidal volume ≤8 mL/kg ideal bodyweight was achieved in 34 (94%) patients. Proning and neuromuscular blockers were used in 19 (54%) and 20 (61%) patients. Duration of ventilation was 12 days (8-23). D-dimer peaked at 3.8 mg/L (2.1-5.3), and maximum dalteparin dose was 15 000 IU/24 h (10 000-15 000). Despite organizational changes, a high degree of adherence to treatment protocols was achieved. CONCLUSION: In a prospective cohort study of mechanically ventilated COVID-19 patients treated in a resource-rich, publicly financed healthcare system, mortality was considerably lower than previously reported in retrospective studies.

A pragmatic randomized controlled trial reports lack of efficacy of hydroxychloroquine on coronavirus disease 2019 viral kinetics.

Here, we randomized 53 patients hospitalized with coronavirus disease 2019 (COVID-19) to hydroxychloroquine therapy (at a dose of 400 mg twice daily for seven days) in addition to standard care or standard care alone (ClinicalTrials.gov Identifier, NCT04316377). All severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positive patients 18 years of age or older were eligible for study inclusion if they had moderately severe COVID-19 at admission. Treatment with hydroxychloroquine did not result in a significantly greater rate of decline in SARS-CoV-2 oropharyngeal viral load compared to standard care alone during the first five days. Our results suggest no important antiviral effect of hydroxychloroquine in humans infected with SARS-CoV-2.

Norwegian Coronavirus Disease 2019 (NO COVID-19) Pragmatic Open label Study to assess early use of hydroxychloroquine sulphate in moderately severe hospitalised patients with coronavirus disease 2019: A structured summary of a study protocol for a randomised controlled trial.

OBJECTIVES: The hypothesis of the study is that treatment with hydroxychloroquine sulphate in hospitalised patients with coronavirus disease 2019 (Covid-19) is safe and will accelerate the virological clearance rate for patients with moderately severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) when compared to standard care. Furthermore, we hypothesize that early treatment with hydroxychloroquine sulphate is associated with more rapid resolve of clinical symptoms as assessed by the National Early Warning Score 2 (NEWS2), decreased admission rate to intensive care units and mortality, and improvement in protein biomarker profiles (C-reactive protein, markers of renal and hepatic injury, and established cardiac biomarkers like cardiac troponin and B-type natriuretic peptide). TRIAL DESIGN: The study is a two-arm, open label, pragmatic randomised controlled group sequential adaptive trial designed to assess the effect on viral loads and clinical outcome of hydroxychloroquine sulphate therapy in addition to standard care compared to standard care alone in patients with established Covid-19. By utilizing resources already paid for by the hospitals (physicians and nurses in daily clinical practice), this pragmatic trial can include a larger number of patients over a short period of time and at a lower cost than studies utilizing traditional randomized controlled trial designs with an external study organization. The pragmatic approach will enable swift initiation of randomisation and allocation to treatment. PARTICIPANTS: Patients will be recruited from all inpatients at Akershus University Hospital, Lørenskog, Norway. Electronic real-time surveillance of laboratory reports from the Department of Microbiology will be examined regularly for SARS-CoV-2 positive subjects. All of the following conditions must apply to the prospective patient at screening prior to inclusion: (1) Hospitalisation; (2) Adults 18 years or older; (3) Moderately severe Covid-19 disease (NEWS2 of 6 or less); (4) SARS-CoV-2 positive nasopharyngeal swab; (5) Expected time of hospitalisation > 48 hours; and (6) Signed informed consent must be obtained and documented according to Good Clinical Practice guidelines of the International Conference on Harmonization, and national/local regulations. Patients will be excluded from participation in the study if they meet any of the following criteria: (1) Requiring intensive care unit admission at screening; (2) History of psoriasis; (3) Known adverse reaction to hydroxychloroquine sulphate; (4) Pregnancy; or (5) Prolonged corrected QT interval (>450 ms). Clinical data, including standard hospital biochemistry, medical therapy, vital signs, NEWS2, and microbiology results (including blood culture results and reverse transcriptase polymerase chain reaction [RT-PCR] for other upper airway viruses), will be automatically extracted from the hospital electronic records and merged with the study specific database. INTERVENTION AND COMPARATOR: Included patients will be randomised in a 1:1 ratio to (1) standard care with the addition of 400 mg hydroxychloroquine sulphate (PlaquenilTM) twice daily for seven days or (2) standard care alone. MAIN OUTCOMES: The primary endpoint of the study is the rate of decline in SARS-CoV-2 viral load in oropharyngeal samples as assessed by RT-PCR in samples collected at baseline, 48 and 96 hours after randomization and administration of drug for the intervention arm. Secondary endpoints include change in NEWS2 at 96 hours after randomisation, admission to intensive care unit, mortality (in-hospital, and at 30 and 90 days), duration of hospital admission, clinical status on a 7-point ordinal scale 14 days after randomization ([1] Death [2] Hospitalised, on invasive mechanical ventilation or extracorporeal membrane oxygenation [3] Hospitalised, on non-invasive ventilation or high flow oxygen devices [4] Hospitalized, requiring supplemental oxygen [5] Hospitalised, not requiring supplemental oxygen [6] Not hospitalized, but unable to resume normal activities [7] Not hospitalised, with resumption of normal activities), and improvement in protein biomarker profiles (C-reactive protein, markers of renal and hepatic injury, and established cardiac biomarkers like cardiac troponin and B-type natriuretic peptide) at 96 hours after randomization. RANDOMISATION: Eligible patients will be allocated in a 1:1 ratio, using a computer randomisation procedure. The allocation sequence has been prepared by an independent statistician. BLINDING (MASKING): Open label randomised controlled pragmatic trial without blinding, no active or placebo control. The virologist assessing viral load in the oropharyngeal samples and the statistician responsible for analysis of the data will be blinded to the treatment allocation for the statistical analyses. NUMBERS TO BE RANDOMIZED (SAMPLE SIZE): This is a group sequential adaptive trial where analyses are planned after 51, 101, 151 and 202 completed patients, with a maximum sample size of 202 patients (101 patients allocated to intervention and standard care and 101 patients allocated to standard care alone). TRIAL STATUS: Protocol version 1.3 (March 26, 2020). Recruitment of first patient on March 26, 2020, and 51 patients were included as per April 28, 2020. Study recruitment is anticipated to be completed by July 2020. TRIAL REGISTRATION: ClinicalTrials.gov number, NCT04316377. Trial registered March 20, 2020. FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.