Usefulness of monocyte distribution width and presepsin for early assessment of disease severity in COVID-19 patients.

Early predictors of severe coronavirus disease 2019 (COVID-19) would identify patients requiring intensive care. Recently, the monocyte distribution width (MDW) and presepsin level have been used for the early diagnosis of sepsis. Here, we assessed the utility of MDW and presepsin for the early assessment of COVID-19 severity. Eighty-seven inpatients with confirmed COVID-19 were enrolled and divided into 3 groups by the type of respiratory support: (1) mechanical ventilation or high-flow nasal cannula oxygen therapy (MVHF-OT), (2) conventional oxygen therapy, and (3) no oxygen therapy. We measured the complete blood count; MDW; erythrocyte sedimentation rate; and the levels of presepsin, C-reactive protein, procalcitonin, lactate dehydrogenase, ferritin, Krebs von den Lungen-6 (KL-6), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibody. Thirteen (14.9%) patients on MVHF-OT exhibited a significantly higher mortality and a longer hospital stay than did the others. The MDW and presepsin levels were significantly elevated on admission, and correlated with COVID-19 severity (both P < .001). Notably, only the MDW correlated significantly with symptoms in the no oxygen therapy group (P < .012). In the first week after admission, the MDW fell and no longer differed among the groups. The KL-6 level did not differ by disease severity at any time. Neutralizing antibodies were detected in 74 patients (91.4%) and the level of neutralization correlated significantly with COVID-19 severity (P < .001). The MDW and presepsin are useful indicators for early assessment of disease severity in COVID-19 patients.

Inhibitory effects and mechanisms of the anti-covid-19 traditional Chinese prescription, Keguan-1, on acute lung injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Keguan-1, a new traditional Chinese medicine (TCM) prescription contained seven Chinese herbs, is developed to treat coronavirus disease 19 (COVID-19). The first internationally registered COVID-19 randomised clinical trial on integrated therapy demonstrated that Keguan-1 significantly reduced the incidence of ARDS and inhibited the severe progression of COVID-19. AIM OF THE STUDY: To investigate the protective mechanism of Keguan-1 on ARDS, a lipopolysaccharide (LPS)-induced acute lung injury (ALI) model was used to simulate the pathological state of ARDS in patients with COVID-19, focusing on its effect and mechanism on ALI. MATERIALS AND METHODS: Mice were challenged with LPS (2 mg/kg) by intratracheal instillation (i.t.) and were orally administered Keguan-1 (low dose, 1.25 g/kg; medium dose, 2.5 g/kg; high dose, 5 g/kg) after 2 h. Bronchoalveolar lavage fluid (BALF) and lung tissue were collected 6 h and 24 h after i.t. administration of LPS. The levels of inflammatory factors tumour necrosis factor alpha (TNF-α), interleukin (IL)-6, IL-1ß, keratinocyte-derived chemokine (KC or mCXCL1), macrophage inflammatory protein 2 (MIP2 or mCXCL2), angiotensin II (Ang II), and endothelial cell junction-associated proteins were analysed using ELISA or western blotting. RESULTS: Keguan-1 improved the survival rate, respiratory condition, and pathological lung injury; decreased the production of proinflammatory factors (TNF-α, IL-6, IL-1ß, KC, and MIP2) in BALF and the number of neutrophils in the lung tissues; and ameliorated inflammatory injury in the lung tissues of the mice with LPS-induced ALI. Keguan-1 also reduced the expression of Ang II and the adhesion molecule ICAM-1; increased tight junction proteins (JAM-1 and claudin-5) and VE-cadherin expression; and alleviated pulmonary vascular endothelial injury in LPS-induced ALI. CONCLUSION: These results demonstrate that Keguan-1 can improve LPS-induced ALI by reducing inflammation and pulmonary vascular endothelial injury, providing scientific support for the clinical treatment of patients with COVID-19. Moreover, it also provides a theoretical basis and technical support for the scientific use of TCMs in emerging infectious diseases.

Rapid and sensitive detection of SARS-CoV-2 infection using quantitative peptide enrichment LC-MS analysis.

Reliable, robust, large-scale molecular testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for monitoring the ongoing coronavirus disease 2019 (COVID-19) pandemic. We have developed a scalable analytical approach to detect viral proteins based on peptide immuno-affinity enrichment combined with liquid chromatography-mass spectrometry (LC-MS). This is a multiplexed strategy, based on targeted proteomics analysis and read-out by LC-MS, capable of precisely quantifying and confirming the presence of SARS-CoV-2 in phosphate-buffered saline (PBS) swab media from combined throat/nasopharynx/saliva samples. The results reveal that the levels of SARS-CoV-2 measured by LC-MS correlate well with their correspondingreal-time polymerase chain reaction (RT-PCR) read-out (r = 0.79). The analytical workflow shows similar turnaround times as regular RT-PCR instrumentation with a quantitative read-out of viral proteins corresponding to cycle thresholds (Ct) equivalents ranging from 21 to 34. Using RT-PCR as a reference, we demonstrate that the LC-MS-based method has 100% negative percent agreement (estimated specificity) and 95% positive percent agreement (estimated sensitivity) when analyzing clinical samples collected from asymptomatic individuals with a Ct within the limit of detection of the mass spectrometer (Ct ≤ 30). These results suggest that a scalable analytical method based on LC-MS has a place in future pandemic preparedness centers to complement current virus detection technologies.

N-Terminomics for the Identification of In Vitro Substrates and Cleavage Site Specificity of the SARS-CoV-2 Main Protease.

The genome of coronaviruses, including SARS-CoV-2, encodes for two proteases, a papain like (PLpro ) protease and the so-called main protease (Mpro ), a chymotrypsin-like cysteine protease, also named 3CLpro or non-structural protein 5 (nsp5). Mpro is activated by autoproteolysis and is the main protease responsible for cutting the viral polyprotein into functional units. Aside from this, it is described that Mpro proteases are also capable of processing host proteins, including those involved in the host innate immune response. To identify substrates of the three main proteases from SARS-CoV, SARS-CoV-2, and hCoV-NL63 coronviruses, an LC-MS based N-terminomics in vitro analysis is performed using recombinantly expressed proteases and lung epithelial and endothelial cell lysates as substrate pools. For SARS-CoV-2 Mpro , 445 cleavage events from more than 300 proteins are identified, while 151 and 331 Mpro derived cleavage events are identified for SARS-CoV and hCoV-NL63, respectively. These data enable to better understand the cleavage site specificity of the viral proteases and will help to identify novel substrates in vivo. All data are available via ProteomeXchange with identifier PXD021406.

No difference in biomarkers of ischemic heart injury and heart failure in patients with COVID-19 who received treatment with chloroquine phosphate and those who did not.

BACKGROUND: Chloroquine was promoted as a COVID-19 therapeutic early in the pandemic. Most countries have since discontinued the use of chloroquine due to lack of evidence of any benefit and the risk of severe adverse events. The primary aim of this study was to examine if administering chloroquine during COVID-19 imposed an increased risk of ischemic heart injury or heart failure. METHODS: Medical records, laboratory findings, and electrocardiograms of patients with COVID-19 who were treated with 500 mg chloroquine phosphate daily and controls not treated with chloroquine were reviewed retrospectively. Controls were matched in age and severity of disease. RESULTS: We included 20 patients receiving chloroquine (500 mg twice daily) for an average of five days, and 40 controls. The groups were comparable regarding demographics and biochemical analyses including C-reactive protein, thrombocytes, and creatinine. There were no statistically significant differences in cardiac biomarkers or in electrocardiograms. Median troponin T was 10,8 ng/L in the study group and 17.9 ng/L in the control group, whereas median NT-proBNP was 399 ng/L in patients receiving chloroquine and 349 ng/L in the controls. CONCLUSIONS: We found no increased risk of ischemic heart injury or heart failure as a result of administering chloroquine. However, the use of chloroquine to treat COVID-19 outside of clinical trials is not recommended, considering the lack of evidence of its effectiveness, as well as the elevated risk of fatal arrythmias.

Validation of an admission coagulation panel for risk stratification of COVID-19 patients.

BACKGROUND: There is limited data on the markers of coagulation and hemostatic activation (MOCHA) profile in Coronavirus disease 2019 (COVID-19) and its ability to identify COVID-19 patients at risk for thrombotic events and other complications. METHODS: Hospitalized patients with confirmed SARS-COV-2 from four Atlanta hospitals were included in this observational cohort study and underwent admission testing of MOCHA parameters (plasma d-dimer, prothrombin fragment 1.2, thrombin-antithrombin complex, fibrin monomer). Clinical outcomes included deep vein thrombosis, pulmonary embolism, myocardial infarction, ischemic stroke, access line thrombosis, ICU admission, intubation and mortality. MAIN RESULTS: Of 276 patients (mean age 59 ± 6.4 years, 47% female, 62% African American), 45 (16%) had a thrombotic endpoint. Each MOCHA parameter was independently associated with a thrombotic event (p<0.05) and ≥ 2 abnormalities was associated with thrombotic endpoints (OR 3.3, 95% CI 1.2-8.8) as were admission D-dimer ≥ 2000 ng/mL (OR 3.1, 95% CI 1.5-6.6) and ≥ 3000 ng/mL (OR 3.6, 95% CI 1.6-7.9). However, only ≥ 2 MOCHA abnormalities were associated with ICU admission (OR 3.0, 95% CI 1.7-5.2) and intubation (OR 3.2, 95% CI 1.6-6.4). MOCHA and D-dimer cutoffs were not associated with mortality. MOCHA with <2 abnormalities (26% of the cohort) had 89% sensitivity and 93% negative predictive value for a thrombotic endpoint. CONCLUSIONS: An admission MOCHA profile is useful to risk-stratify COVID-19 patients for thrombotic complications and more effective than isolated d-dimer for predicting risk of ICU admission and intubation.

Prognostic value of cardiac biomarkers in COVID-19 infection.

Multiple Biomarkers have recently been shown to be elevated in COVID-19, a respiratory infection with multi-organ dysfunction; however, information regarding the prognostic value of cardiac biomarkers as it relates to disease severity and cardiac injury are inconsistent. The goal of this meta-analysis was to summarize the evidence regarding the prognostic relevance of cardiac biomarkers from data available in published reports. PubMed, Embase and Web of Science were searched from inception through April 2020 for studies comparing median values of cardiac biomarkers in critically ill versus non-critically ill COVID-19 patients, or patients who died versus those who survived. The weighted mean differences (WMD) and 95% confidence interval (CI) between the groups were calculated for each study and combined using a random effects meta-analysis model. The odds ratio (OR) for mortality based on cardiac injury was combined from studies reporting it. Troponin levels were significantly higher in COVID-19 patients who died or were critically ill versus those who were alive or not critically ill (WMD 0.57, 95% CI 0.43-0.70, p < 0.001). Additionally, BNP levels were also significantly higher in patients who died or were critically ill (WMD 0.45, 95% CI - 0.21-0.69, p < 0.001). Cardiac injury was independently associated with significantly increased odds of mortality (OR 6.641, 95% CI 1.26-35.1, p = 0.03). A significant difference in levels of D-dimer was seen in those who died or were critically ill. CK levels were only significantly higher in those who died versus those who were alive (WMD 0.79, 95% CI 0.25-1.33, p = 0.004). Cardiac biomarkers add prognostic value to the determination of the severity of COVID-19 and can predict mortality.

Baseline characteristics and changes of biomarkers in disease course predict prognosis of patients with COVID-19.

The outbreak of coronavirus disease (COVID-19) has brought great challenges to the world. The objectives of this study were to describe the baseline characteristics and changes of biomarkers of these COVID-19 patients and identify predictive value of the above markers for patient death. Using patient death as the observational endpoints, clinical data of inpatients in a special ward for COVID-19 in Wuhan, China were retrospectively collected. Univariate and multivariate Cox regression analyses were used to evaluate prognostic value of baseline characteristics and laboratory data changes. This study included clinical data of 75 patients. Age, c-reactive protein (CRP) and interleukin-6 levels were independent predictors of patient death. Survivors were characterized as having declining neutrophil counts, D-dimer, N-terminal pronatriuretic peptide, troponin I (TnI) and c-reactive protein levels, while counts of lymphocyte gradually came back. Non-survivors were characterized with increasing white blood cell counts (WBC) and neutrophil counts. Changes of WBC, TnI and interleukin-6 were also independently associated with patient death. Older age, baseline CRP and IL-6 levels may be used as meaningful predictors to identify patients with poor prognosis. Changes of biomarkers should be closely monitored in the management of patients with COVID-19, while constantly increasing levels of WBC, TnI and interleukin-6 in the disease course also predict patient death.

Mass-Spectrometric Detection of SARS-CoV-2 Virus in Scrapings of the Epithelium of the Nasopharynx of Infected Patients via Nucleocapsid N Protein.

The detection of viral RNA by polymerase chain reaction (PCR) is currently the main diagnostic tool for COVID-19 ( Eurosurveillance 2019, 25 (3), 1). The PCR-based test, however, shows limited sensitivity, especially in the early and late stages of disease development ( Nature 2020, 581, 465-469; J. Formosan Med. Assoc. 2020, 119 (6) 1123), and is relatively time-consuming. Fast and reliable complementary methods for detecting the viral infection would be of help in the current pandemic conditions. Mass spectrometry is one of such possibilities. We have developed a mass-spectrometry-based method for the detection of the SARS CoV-2 virus in nasopharynx epithelial swabs based on the detection of the viral nucleocapsid N protein. Our approach shows confident identification of the N protein in patient samples, even those with the lowest viral loads, and a much simpler preparation procedure. Our main protocol consists of virus inactivation by heating and the addition of isopropanol and tryptic digestion of the proteins sedimented from the swabs followed by MS analysis. A set of unique peptides, produced as a result of proteolysis of the nucleocapsid phosphoprotein of SARS-CoV-2, is detected. The obtained results can further be used to create fast parallel mass-spectrometric approaches for the detection of the virus in the nasopharyngeal mucosa, saliva, sputum and other physiological fluids.

Mass Spectrometric Identification of SARS-CoV-2 Proteins from Gargle Solution Samples of COVID-19 Patients.

Mass spectrometry (MS) can deliver valuable diagnostic data that complement genomic information and allow us to increase our current knowledge of the COVID-19 disease caused by the SARS-CoV-2 virus. We developed a simple, MS-based method to specifically detect SARS-CoV-2 proteins from gargle solution samples of COVID-19 patients. The protocol consists of an acetone precipitation and tryptic digestion of proteins contained within the gargle solution, followed by a targeted MS analysis. Our methodology identifies unique peptides originating from SARS-CoV-2 nucleoprotein. Building on these promising initial results, faster MS protocols can now be developed as routine diagnostic tools for COVID-19 patients. Data are available via ProteomeXchange with identifier PXD019423.

Prognostic value of NT-proBNP in patients with severe COVID-19.

BACKGROUND: The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in China has been declared a public health emergency of international concern. The cardiac injury is a common condition among the hospitalized patients with COVID-19. However, whether N terminal pro B type natriuretic peptide (NT-proBNP) predicted outcome of severe COVID-19 patients was unknown. METHODS: The study initially enrolled 102 patients with severe COVID-19 from a continuous sample. After screening out the ineligible cases, 54 patients were analyzed in this study. The primary outcome was in-hospital death defined as the case fatality rate. Research information and following-up data were obtained from their medical records. RESULTS: The best cut-off value of NT-proBNP for predicting in-hospital death was 88.64 pg/mL with the sensitivity for 100% and the specificity for 66.67%. Patients with high NT-proBNP values (> 88.64 pg/mL) had a significantly increased risk of death during the days of following-up compared with those with low values (≤88.64 pg/mL). After adjustment for potential risk factors, NT-proBNP was independently correlated with in-hospital death. CONCLUSION: NT-proBNP might be an independent risk factor for in-hospital death in patients with severe COVID-19. TRIAL REGISTRATION: ClinicalTrials, NCT04292964. Registered 03 March 2020.