SARS-CoV-2 E-PROTEIN VIROPORIN INHIBITOR BIT225 ACTIVE IN hACE2 TRANSGENIC MICE

Background: The CoV-2 envelope (E) protein plays an important role in virus assembly, budding, immunopathogenesis and disease severity. E protein has ion channel activity, is located in Golgi and ER membranes of infected cells and is associated with inflammasome activation and immune dysregulation. Here we report that BIT225, an investigational HIV clinical compound, inhibits E ion channel activity and prevents body weight loss and mortality and reduces inflammation in lethally infected K18-hACE2 transgenic mice. BIT225 efficacy was observed when dosing was initiated before or 24 h or 48 h after infection. Method(s): SARS-CoV-2 E protein ion channel activity and Xenopus TMEM16A were measured in Xenopus oocytes. K18-hACE2 transgenic mice were infected intranasally with 104 pfu SARS CoV 2 (US-WA1/2020) and dosed orally twice daily with BIT225 for up to 12 Days. Dosing was initiated 12 h pre-infection or 24 h or 48 h post-infection. Disease parameters measured were survival, body weight, viral RNA by qPCR and infectious virus titre (plaque assay) in lung tissue homogenates and serum. In addition, levels of pro-inflammatory cytokines (IL-6, IL-1alpha, IL-1beta, TNFalpha & TGFbeta, MCP-1) were measured in lung and serum samples. Result(s): BIT225 inhibited ion channel activity of E-protein, but not that of TMEM16A in Xenopus oocytes. BIT225 dosed at 300mg/kg BID for 12 days starting 12 h pre-infection completely prevented body weight loss and mortality in SARS-CoV-2 infected K18 mice (n=12), while all vehicle-dosed animals reached a mortality endpoint by day 9 across two studies (n=12). Figure 1 shows results from a time of addition study: When treatment with BIT225 started at 24 h post-infection, body weight loss and mortality was also prevented (100% survival, n=5). In the group of mice where treatment started at 48 h after infection, body weight loss and mortality were prevented in 4 of 5 mice. Treatment efficacy was associated with significant reduction in lung viral load (3.5 log10), virus titer (4000 pfu/ml) and lung and serum cytokine levels. Conclusion(s): BIT225 is an inhibitor of SARS-CoV-2 E-protein viroporin activity. In the K18 model BIT225 protected mice from weight loss and death, inhibited virus replication and reduced inflammation. These effects were noted when treatment with BIT225 was initiated before or 24-48 hours after infection and validate viroporin E as a viable antiviral target and support the clinical study of BIT225 in treatment of SARS-CoV-2.

Inactivation of SARS-CoV-2 and Other Enveloped and Non-Enveloped Viruses with Non-Thermal Plasma for Hospital Disinfection.

As recently highlighted by the SARS-CoV-2 pandemic, viruses have become an increasing burden for health, global economy, and environment. The control of transmission by contact with contaminated materials represents a major challenge, particularly in hospital environments. However, the current disinfection methods in hospital settings suffer from numerous drawbacks. As a result, several medical supplies that cannot be properly disinfected are not reused, leading to severe shortages and increasing amounts of waste, thus prompting the search for alternative solutions. In this work, we report that non-thermal plasma (NTP) can effectively inactivate SARS-CoV-2 from non-porous and porous materials commonly found in healthcare facilities. We demonstrated that 5 min treatment with a dielectric barrier discharge NTP can inactivate 100% of SARS-CoV-2 (Wuhan and Omicron strains) from plastic material. Using porcine respiratory coronavirus (surrogate for SARS-CoV-2) and coxsackievirus B3 (highly resistant non-enveloped virus), we tested the NTP virucidal activity on hospital materials and obtained complete inactivation after 5 and 10 min, respectively. We hypothesize that the produced reactive species and local acidification contribute to the overall virucidal effect of NTP. Our results demonstrate the potential of dielectric barrier discharge NTPs for the rapid, efficient, and low-cost disinfection of healthcare materials.

The Emergence of Travel-related Infections in Critical Care Units

Several tropical or geographically confined infectious diseases may lead to organ failure requiring management in an intensive care unit (ICU), both in endemic low- and middle-income countries where ICU facilities are increasingly being developed and in (nonendemic) high-income countries through an increase in international travel and migration. The ICU physician must know which of these diseases may be encountered and how to recognize, differentiate, and treat them. The four historically most prevalent "tropical” diseases (malaria, enteric fever, dengue, and rickettsiosis) can present with single or multiple organ failure in a very similar manner, which makes differentiation based solely on clinical signs very difficult. Specific but frequently subtle symptoms should be considered and related to the travel history of the patient, the geographic distribution of these diseases, and the incubation period. In the future, ICU physicians may also be more frequently confronted with rare but frequently lethal diseases, such as Ebola and other viral hemorrhagic fevers, leptospirosis, and yellow fever. No one could have foreseen the worldwide 2019–up to now coronavirus disease 2019 (COVID-19) crisis caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which was initially spread by travel too. In addition, the actual pandemic due to SARS-CoV-2 reminds us of the actual and potential threat of (re)-emerging pathogens. If left untreated or when treated with a delay, many travel-related diseases remain an important cause of morbidity and even mortality, even when high-quality critical care is provided. Awareness and a high index of suspicion of these diseases is a key skill for the ICU physicians of today and tomorrow to develop.

The Emergence of Travel-related Infections in Critical Care Units.

Several tropical or geographically confined infectious diseases may lead to organ failure requiring management in an intensive care unit (ICU), both in endemic low- and middle-income countries where ICU facilities are increasingly being developed and in (nonendemic) high-income countries through an increase in international travel and migration. The ICU physician must know which of these diseases may be encountered and how to recognize, differentiate, and treat them. The four historically most prevalent "tropical" diseases (malaria, enteric fever, dengue, and rickettsiosis) can present with single or multiple organ failure in a very similar manner, which makes differentiation based solely on clinical signs very difficult. Specific but frequently subtle symptoms should be considered and related to the travel history of the patient, the geographic distribution of these diseases, and the incubation period. In the future, ICU physicians may also be more frequently confronted with rare but frequently lethal diseases, such as Ebola and other viral hemorrhagic fevers, leptospirosis, and yellow fever. No one could have foreseen the worldwide 2019-up to now coronavirus disease 2019 (COVID-19) crisis caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which was initially spread by travel too. In addition, the actual pandemic due to SARS-CoV-2 reminds us of the actual and potential threat of (re)-emerging pathogens. If left untreated or when treated with a delay, many travel-related diseases remain an important cause of morbidity and even mortality, even when high-quality critical care is provided. Awareness and a high index of suspicion of these diseases is a key skill for the ICU physicians of today and tomorrow to develop.

Anti-C5a antibody (vilobelimab) therapy for critically ill, invasively mechanically ventilated patients with COVID-19 (PANAMO): a multicentre, double-blind, randomised, placebo-controlled, phase 3 trial.

BACKGROUND: Vilobelimab, an anti-C5a monoclonal antibody, was shown to be safe in a phase 2 trial of invasively mechanically ventilated patients with COVID-19. Here, we aimed to determine whether vilobelimab in addition to standard of care improves survival outcomes in this patient population. METHODS: This randomised, double-blind, placebo-controlled, multicentre phase 3 trial was performed at 46 hospitals in the Netherlands, Germany, France, Belgium, Russia, Brazil, Peru, Mexico, and South Africa. Participants aged 18 years or older who were receiving invasive mechanical ventilation, but not more than 48 h after intubation at time of first infusion, had a PaO2/FiO2 ratio of 60-200 mm Hg, and a confirmed SARS-CoV-2 infection with any variant in the past 14 days were eligible for this study. Eligible patients were randomly assigned (1:1) to receive standard of care and vilobelimab at a dose of 800 mg intravenously for a maximum of six doses (days 1, 2, 4, 8, 15, and 22) or standard of care and a matching placebo using permuted block randomisation. Treatment was not continued after hospital discharge. Participants, caregivers, and assessors were masked to group assignment. The primary outcome was defined as all-cause mortality at 28 days in the full analysis set (defined as all randomly assigned participants regardless of whether a patient started treatment, excluding patients randomly assigned in error) and measured using Kaplan-Meier analysis. Safety analyses included all patients who had received at least one infusion of either vilobelimab or placebo. This study is registered with ClinicalTrials.gov, NCT04333420. FINDINGS: From Oct 1, 2020, to Oct 4, 2021, we included 368 patients in the ITT analysis (full analysis set; 177 in the vilobelimab group and 191 in the placebo group). One patient in the vilobelimab group was excluded from the primary analysis due to random assignment in error without treatment. At least one dose of study treatment was given to 364 (99%) patients (safety analysis set). 54 patients (31%) of 177 in the vilobelimab group and 77 patients (40%) of 191 in the placebo group died in the first 28 days. The all-cause mortality rate at 28 days was 32% (95% CI 25-39) in the vilobelimab group and 42% (35-49) in the placebo group (hazard ratio 0·73, 95% CI 0·50-1·06; p=0·094). In the predefined analysis without site-stratification, vilobelimab significantly reduced all-cause mortality at 28 days (HR 0·67, 95% CI 0·48-0·96; p=0·027). The most common TEAEs were acute kidney injury (35 [20%] of 175 in the vilobelimab group vs 40 [21%] of 189 in the placebo), pneumonia (38 [22%] vs 26 [14%]), and septic shock (24 [14%] vs 31 [16%]). Serious treatment-emergent adverse events were reported in 103 (59%) of 175 patients in the vilobelimab group versus 120 (63%) of 189 in the placebo group. INTERPRETATION: In addition to standard of care, vilobelimab improves survival of invasive mechanically ventilated patients with COVID-19 and leads to a significant decrease in mortality. Vilobelimab could be considered as an additional therapy for patients in this setting and further research is needed on the role of vilobelimab and C5a in other acute respiratory distress syndrome-causing viral infections. FUNDING: InflaRx and the German Federal Government.

Flow-controlled ventilation in moderate acute respiratory distress syndrome due to COVID-19: an open-label repeated-measures controlled trial.

BACKGROUND: Flow-controlled ventilation (FCV), a novel mode of mechanical ventilation characterised by constant flow during active expiration, may result in more efficient alveolar gas exchange, better lung recruitment and might be useful in limiting ventilator-induced lung injury. However, data regarding FCV in mechanically ventilated patients with acute lung injury or acute respiratory distress syndrome (ARDS) are scarce. OBJECTIVES: We hypothesised that the use of FCV is feasible and would improve oxygenation in moderate COVID-19 ARDS compared to conventional ventilation. DESIGN: Open-label repeated-measures controlled trial. SETTING: From February to April 2021, patients with moderate COVID-19 ARDS were recruited in a tertiary referral intensive care unit. PATIENTS: Patients with moderate ARDS (PaO2/FIO2 ratio 100-200 mmHg, SpO2 88-94% and PaO2 60-80 mmHg) were considered eligible. Exclusion criteria were: extremes of age (< 18 years, > 80 years), obesity (body mass index > 40 kg/m2), prone positioning at the time of intervention, mechanical ventilation for more than 10 days and extracorporeal membrane oxygenation. Eleven patients were recruited. INTERVENTION: Participants were ventilated in FCV mode for 30 min, and subsequently in volume-control mode (VCV) for 30 min. MAIN OUTCOME MEASURES: Feasibility of FCV to maintain oxygenation was assessed by the PaO2/FiO2 ratio (mmHg) as a primary outcome parameter. Secondary outcomes included ventilator parameters, PaCO2 and haemodynamic data. All adverse events were recorded. RESULTS: FCV was feasible in all patients and no adverse events were observed. There was no difference in the PaO2/FIO2 ratio after 30 min of ventilation in FCV mode (169 mmHg) compared to 30 min of ventilation in VCV mode subsequently (168 mmHg, 95% CI of pseudo-medians (- 10.5, 3.6), p = 0.56). The tidal volumes (p < 0.01) and minute ventilation were lower during FCV (p = 0.01) while PaCO2 was similar at the end of the 30-min ventilation periods (p = 0.31). Mean arterial pressure during FCV was comparable to baseline. CONCLUSIONS: Thirty minutes of FCV in patients with moderate COVID-19 ARDS receiving neuromuscular blocking agents resulted in similar oxygenation, compared to VCV. FCV was feasible and did not result in adverse events. TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT04894214.

COVID-19-Associated cardiac pathology at the postmortem evaluation: a collaborative systematic review.

BACKGROUND: Many postmortem studies address the cardiovascular effects of COVID-19 and provide valuable information, but are limited by their small sample size. OBJECTIVES: The aim of this systematic review is to better understand the various aspects of the cardiovascular complications of COVID-19 by pooling data from a large number of autopsy studies. DATA SOURCES: We searched the online databases Ovid EBM Reviews, Ovid Embase, Ovid Medline, Scopus, and Web of Science for concepts of autopsy or histopathology combined with COVID-19, published between database inception and February 2021. We also searched for unpublished manuscripts using the medRxiv services operated by Cold Spring Harbor Laboratory. STUDY ELIGIBILITY CRITERIA: Articles were considered eligible for inclusion if they reported human postmortem cardiovascular findings among individuals with a confirmed SARS coronavirus type 2 (CoV-2) infection. PARTICIPANTS: Confirmed COVID-19 patients with post-mortem cardiovascular findings. INTERVENTIONS: None. METHODS: Studies were individually assessed for risk of selection, detection, and reporting biases. The median prevalence of different autopsy findings with associated interquartile ranges (IQRs). RESULTS: This review cohort contained 50 studies including 548 hearts. The median age of the deceased was 69 years. The most prevalent acute cardiovascular findings were myocardial necrosis (median: 100.0%; IQR, 20%-100%; number of studies = 9; number of patients = 64) and myocardial oedema (median: 55.5%; IQR, 19.5%-92.5%; number of studies = 4; number of patients = 46). The median reported prevalence of extensive, focal active, and multifocal myocarditis were all 0.0%. The most prevalent chronic changes were myocyte hypertrophy (median: 69.0%; IQR, 46.8%-92.1%) and fibrosis (median: 35.0%; IQR, 35.0%-90.5%). SARS-CoV-2 was detected in the myocardium with median prevalence of 60.8% (IQR 40.4-95.6%). CONCLUSIONS: Our systematic review confirmed the high prevalence of acute and chronic cardiac pathologies in COVID-19 and SARS-CoV-2 cardiac tropism, as well as the low prevalence of myocarditis in COVID-19.

Angiotensin Receptor-Neprilysin Inhibition in Acute Myocardial Infarction.

BACKGROUND: In patients with symptomatic heart failure, sacubitril-valsartan has been found to reduce the risk of hospitalization and death from cardiovascular causes more effectively than an angiotensin-converting-enzyme inhibitor. Trials comparing the effects of these drugs in patients with acute myocardial infarction have been lacking. METHODS: We randomly assigned patients with myocardial infarction complicated by a reduced left ventricular ejection fraction, pulmonary congestion, or both to receive either sacubitril-valsartan (97 mg of sacubitril and 103 mg of valsartan twice daily) or ramipril (5 mg twice daily) in addition to recommended therapy. The primary outcome was death from cardiovascular causes or incident heart failure (outpatient symptomatic heart failure or heart failure leading to hospitalization), whichever occurred first. RESULTS: A total of 5661 patients underwent randomization; 2830 were assigned to receive sacubitril-valsartan and 2831 to receive ramipril. Over a median of 22 months, a primary-outcome event occurred in 338 patients (11.9%) in the sacubitril-valsartan group and in 373 patients (13.2%) in the ramipril group (hazard ratio, 0.90; 95% confidence interval [CI], 0.78 to 1.04; P = 0.17). Death from cardiovascular causes or hospitalization for heart failure occurred in 308 patients (10.9%) in the sacubitril-valsartan group and in 335 patients (11.8%) in the ramipril group (hazard ratio, 0.91; 95% CI, 0.78 to 1.07); death from cardiovascular causes in 168 (5.9%) and 191 (6.7%), respectively (hazard ratio, 0.87; 95% CI, 0.71 to 1.08); and death from any cause in 213 (7.5%) and 242 (8.5%), respectively (hazard ratio, 0.88; 95% CI, 0.73 to 1.05). Treatment was discontinued because of an adverse event in 357 patients (12.6%) in the sacubitril-valsartan group and 379 patients (13.4%) in the ramipril group. CONCLUSIONS: Sacubitril-valsartan was not associated with a significantly lower incidence of death from cardiovascular causes or incident heart failure than ramipril among patients with acute myocardial infarction. (Funded by Novartis; PARADISE-MI ClinicalTrials.gov number, NCT02924727.).

A dynamic mucin mRNA signature associates with COVID-19 disease presentation and severity.

BACKGROUNDSARS-CoV-2 infection induces mucin overexpression, further promoting disease. Given that mucins are critical components of innate immunity, unraveling their expression profiles that dictate the course of disease could greatly enhance our understanding and management of COVID-19.METHODSUsing validated RT-PCR assays, we assessed mucin mRNA expression in the blood of patients with symptomatic COVID-19 compared with symptomatic patients without COVID-19 and healthy controls and correlated the data with clinical outcome parameters. Additionally, we analyzed mucin expression in mucus and lung tissue from patients with COVID-19 and investigated the effect of drugs for COVID-19 treatment on SARS-CoV-2-induced mucin expression in pulmonary epithelial cells.RESULTSWe identified a dynamic blood mucin mRNA signature that clearly distinguished patients with symptomatic COVID-19 from patients without COVID-19 based on expression of MUC1, MUC2, MUC4, MUC6, MUC13, MUC16, and MUC20 (AUCROC of 91.8%; sensitivity and specificity of 90.6% and 93.3%, respectively) and that discriminated between mild and critical COVID-19 based on the expression of MUC16, MUC20, and MUC21 (AUCROC of 89.1%; sensitivity and specificity of 90.0% and 85.7%, respectively). Differences in the transcriptional landscape of mucins in critical cases compared with mild cases identified associations with COVID-19 symptoms, respiratory support, organ failure, secondary infections, and mortality. Furthermore, we identified different mucins in the mucus and lung tissue of critically ill COVID-19 patients and showed the ability of baricitinib, tocilizumab, favipiravir, and remdesivir to suppress expression of SARS-CoV-2-induced mucins.CONCLUSIONThis multifaceted blood mucin mRNA signature showed the potential role of mucin profiling in diagnosing, estimating severity, and guiding treatment options in patients with COVID-19.FUNDINGThe Antwerp University Research and the Research Foundation Flanders COVID-19 funds.

Cross-sectional cycle threshold values reflect epidemic dynamics of COVID-19 in Madagascar (preprint)

As the national reference laboratory for febrile illness in Madagascar, we processed samples from the first epidemic wave of COVID-19, between March and September 2020. We fit generalized additive models to cycle threshold (Ct) value data from our RT-qPCR platform, demonstrating a peak in high viral load, low-Ct value infections temporally coincident with peak epidemic growth rates estimated in real time from publicly-reported incidence data and retrospectively from our own laboratory testing data across three administrative regions. We additionally demonstrate a statistically significant effect of duration of time since infection onset on Ct value, suggesting that Ct value can be used as a biomarker of the stage at which an individual is sampled in the course of an infection trajectory. As an extension, the population level Ct distribution at a given timepoint can be used to estimate population-level epidemiological dynamics. We illustrate this concept by adopting a recently-developed, nested modeling approach, embedding a within-host viral kinetics model within a population-level Susceptible-Exposed-Infectious-Recovered (SEIR) framework, to mechanistically estimate epidemic growth rates from cross-sectional Ct distributions across three regions in Madagascar. We find that Ct-derived epidemic growth estimates slightly precede those derived from incidence data across the first epidemic wave, suggesting delays in surveillance and case reporting. Our findings indicate that public reporting of Ct values could offer an important resource for epidemiological inference in low surveillance settings, enabling forecasts of impending incidence peaks in regions with limited case reporting.

Fatal lymphocytic cardiac damage in coronavirus disease 2019 (COVID-19): autopsy reveals a ferroptosis signature.

AIMS: Cardiovascular complications, including myocarditis, are observed in coronavirus disease 2019 (COVID-19). Major cardiac involvement is a potentially lethal feature in severe cases. We sought to describe the underlying pathophysiological mechanism in COVID-19 lethal cardiogenic shock. METHODS AND RESULTS: We report on a 48-year-old male COVID-19 patient with cardiogenic shock; despite extracorporeal life support, dialysis, and massive pharmacological support, this rescue therapy was not successful. Severe acute respiratory syndrome coronavirus 2 RNA was detected at autopsy in the lungs and myocardium. Histopathological examination revealed diffuse alveolar damage, proliferation of type II pneumocytes, lymphocytes in the lung interstitium, and pulmonary microemboli. Moreover, patchy muscular, sometimes perivascular, interstitial mononuclear inflammatory infiltrates, dominated by lymphocytes, were seen in the cardiac tissue. The lymphocytes 'interlocked' the myocytes, resulting in myocyte degeneration and necrosis. Predominantly, T-cell lymphocytes with a CD4:CD8 ratio of 1.7 infiltrated the interstitial myocardium, reflecting true myocarditis. The myocardial tissue was examined for markers of ferroptosis, an iron-catalysed form of regulated cell death that occurs through excessive peroxidation of polyunsaturated fatty acids. Immunohistochemical staining with E06, a monoclonal antibody binding to oxidized phosphatidylcholine (reflecting lipid peroxidation during ferroptosis), was positive in morphologically degenerating and necrotic cardiomyocytes adjacent to the infiltrate of lymphocytes, near arteries, in the epicardium and myocardium. A similar ferroptosis signature was present in the myocardium of a COVID-19 subject without myocarditis. In a case of sudden death due to viral myocarditis of unknown aetiology, however, immunohistochemical staining with E06 was negative. The renal proximal tubuli stained positively for E06 and also hydroxynonenal (4-HNE), a reactive breakdown product of the lipid peroxides that execute ferroptosis. In the case of myocarditis of other aetiology, the renal tissue displayed no positivity for E06 or 4-HNE. CONCLUSIONS: The findings in this case are unique as this is the first report on accumulated oxidized phospholipids (or their breakdown products) in myocardial and renal tissue in COVID-19. This highlights ferroptosis, proposed to detrimentally contribute to some forms of ischaemia-reperfusion injury, as a detrimental factor in COVID-19 cardiac damage and multiple organ failure.