Incidence of hospital acquired pressure injury in critically ill patients with COVID-19 in prone position admitted to the intensive care unit.

Critical patients have conditions that may favor the occurrence of hospital-acquired pressure injury (HAPI). The objective of this study was to identify the incidence and factors associated with the occurrence of HAPI in patients with coronavirus disease 2019 admitted to the intensive care unit (ICU) who used the prone position. Retrospective cohort study carried out in an ICU of a tertiary university hospital. Two hundred four patients with positive real-time polymerase chain reactions were evaluated, of which 84 were placed in the prone position. All patients were sedated and submitted to invasive mechanical ventilation. Of the prone patients, 52 (62%) developed some type of HAPI during hospitalization. The main place of occurrence of HAPI was the sacral region, followed by the gluteus and thorax. Of the patients who developed HAPI, 26 (50%) had this event in places possibly associated with the prone position. The factors associated with the occurrence of HAPI in patients prone to coronavirus disease 2019 were the Braden Scale and the length of stay in the ICU. The incidence of HAPI in prone patients was extremely high (62%), which denotes the need to implement protocols in order to prevent the occurrence of these events.

Inhibition of IL-6 signaling prevents serum-induced umbilical cord artery dysfunction from patients with severe COVID-19.

Coronavirus disease 2019 (COVID-19) infection has a negative impact on the cytokine profile of pregnant women. Increased levels of proinflammatory cytokines seem to be correlated with the severity of the disease, in addition to predisposing to miscarriage or premature birth. Proinflammatory cytokines increase the generation of reactive oxygen species (ROS). It is unclear how interleukin-6 (IL-6) found in the circulation of patients with severe COVID-19 might affect gestational health, particularly concerning umbilical cord function. This study tested the hypothesis that IL-6 present in the circulation of women with severe COVID-19 causes umbilical cord artery dysfunction by increasing ROS generation and activating redox-sensitive proteins. Umbilical cord arteries were incubated with serum from healthy women and women with severe COVID-19. Vascular function was assessed using concentration-effect curves to serotonin in the presence or absence of pharmacological agents, such as tocilizumab (antibody against the IL-6 receptor), tiron (ROS scavenger), ML171 (Nox1 inhibitor), and Y27632 (Rho kinase inhibitor). ROS generation was assessed by the dihydroethidine probe and Rho kinase activity by an enzymatic assay. Umbilical arteries exposed to serum from women with severe COVID-19 were hyperreactive to serotonin. This effect was abolished in the presence of tocilizumab, tiron, ML171, and Y27632. In addition, serum from women with severe COVID-19 increased Nox1-dependent ROS generation and Rho kinase activity. Increased Rho kinase activity was abolished by tocilizumab and tiron. Serum cytokines in women with severe COVID-19 promote umbilical artery dysfunction. IL-6 is key to Nox-linked vascular oxidative stress and activation of the Rho kinase pathway.

Mitochondrial DNA and TLR9 activation contribute to SARS-CoV-2-induced endothelial cell damage.

BACKGROUND AND PURPOSE: Mitochondria play a central role in the host response to viral infection and immunity, being key to antiviral signaling and exacerbating inflammatory processes. Mitochondria and Toll-like receptor (TLR) have been suggested as potential targets in SARS-CoV-2 infection. However, the involvement of TLR9 in SARS-Cov-2-induced endothelial dysfunction and potential contribution to cardiovascular complications in COVID-19 have not been demonstrated. This study determined whether infection of endothelial cells by SARS-CoV-2 affects mitochondrial function and induces mitochondrial DNA (mtDNA) release. We also questioned whether TLR9 signaling mediates the inflammatory responses induced by SARS-CoV-2 in endothelial cells. EXPERIMENTAL APPROACH: Human umbilical vein endothelial cells (HUVECs) were infected by SARS-CoV-2 and immunofluorescence was used to confirm the infection. Mitochondrial function was analyzed by specific probes and mtDNA levels by real-time polymerase chain reaction (RT-PCR). Inflammatory markers were measured by ELISA, protein expression by western blot, intracellular calcium (Ca2+) by FLUOR-4, and vascular reactivity with a myography. KEY RESULTS: SARS-CoV-2 infected HUVECs, which express ACE2 and TMPRSS2 proteins, and promoted mitochondrial dysfunction, i.e. it increased mitochondria-derived superoxide anion, mitochondrial membrane potential, and mtDNA release, leading to activation of TLR9 and NF-kB, and release of cytokines. SARS-CoV-2 also decreased nitric oxide synthase (eNOS) expression and inhibited Ca2+ responses in endothelial cells. TLR9 blockade reduced SARS-CoV-2-induced IL-6 release and prevented decreased eNOS expression. mtDNA increased vascular reactivity to endothelin-1 (ET-1) in arteries from wild type, but not TLR9 knockout mice. These events were recapitulated in serum samples from COVID-19 patients, that exhibited increased levels of mtDNA compared to sex- and age-matched healthy subjects and patients with comorbidities. CONCLUSION AND APPLICATIONS: SARS-CoV-2 infection impairs mitochondrial function and activates TLR9 signaling in endothelial cells. TLR9 triggers inflammatory responses that lead to endothelial cell dysfunction, potentially contributing to the severity of symptoms in COVID-19. Targeting mitochondrial metabolic pathways may help to define novel therapeutic strategies for COVID-19.

Nosocomial Outbreak of Extensively Drug-Resistant (Polymyxin B and Carbapenem) Klebsiella pneumoniae in a Collapsed University Hospital Due to COVID-19 Pandemic.

We correlated clinical, epidemiological, microbiological, and genomic data of an outbreak with polymyxin B (PB)- and carbapenem-resistant Klebsiella pneumoniae during the COVID-19 pandemic. Twenty-six PB- and carbapenem-resistant K. pneumoniae were isolated from patients in the COVID-19 ICU (Intensive Care Unit), non-COVID-19 ICU (Intensive Care Unit), clinical, or surgical ward. Bacterial identification, drug susceptibility tests, and DNA sequencing were performed, followed by in silico resistance genes identification. All isolates showed extensively drug-resistant (XDR) phenotypes. Four different sequence types (ST) were detected: ST16, ST11, ST258, and ST437. Nineteen isolates were responsible for an outbreak in the ICU in September 2020. They belong to ST258 and harbored the 42Kb IncX3plasmid (pKP98M3N42) with the same genomic pattern of two K. pneumoniae identified in 2018. Twenty-four isolates carried bla-KPC-2 gene. No plasmid-mediated colistin (mcr) resistance genes were found. Eight isolates presented mgrB gene mutation. The clonal isolates responsible for the outbreak came from patients submitted to pronation, with high mortality rates in one month. XDR-K. pneumoniae detected during the outbreak presented chromosomal resistance to PB and plasmid-acquired carbapenem resistance due to KPC production in most isolates and 42Kb IncX3(pKP98M3N42) plasmid carrying blaKPC-2 was associated with ST258 isolates. The outbreak followed the collapse of the local healthcare system with high mortality rates.

Timeline Kinetics of Systemic and Airway Immune Mediator Storm for Comprehensive Analysis of Disease Outcome in Critically Ill COVID-19 Patients.

In the present study, the levels of serum and airway soluble chemokines, pro-inflammatory/regulatory cytokines, and growth factors were quantified in critically ill COVID-19 patients (total n=286) at distinct time points (D0, D2-6, D7, D8-13 and D>14-36) upon Intensive Care Unit (ICU) admission. Augmented levels of soluble mediators were observed in serum from COVID-19 patients who progress to death. An opposite profile was observed in tracheal aspirate samples, indicating that systemic and airway microenvironment diverge in their inflammatory milieu. While a bimodal distribution was observed in the serum samples, a unimodal peak around D7 was found for most soluble mediators in tracheal aspirate samples. Systems biology tools further demonstrated that COVID-19 display distinct eccentric soluble mediator networks as compared to controls, with opposite profiles in serum and tracheal aspirates. Regardless the systemic-compartmentalized microenvironment, networks from patients progressing to death were linked to a pro-inflammatory/growth factor-rich, highly integrated center. Conversely, patients evolving to discharge exhibited networks of weak central architecture, with lower number of neighborhood connections and clusters of pro-inflammatory and regulatory cytokines. All in all, this investigation with robust sample size landed a comprehensive snapshot of the systemic and local divergencies composed of distinct immune responses driven by SARS-CoV-2 early on severe COVID-19.

SARS-CoV-2 productively infects primary human immune system cells in vitro and in COVID-19 patients.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with a hyperinflammatory state and lymphocytopenia, a hallmark that appears as both signature and prognosis of disease severity outcome. Although cytokine storm and a sustained inflammatory state are commonly associated with immune cell depletion, it is still unclear whether direct SARS-CoV-2 infection of immune cells could also play a role in this scenario by harboring viral replication. We found that monocytes, as well as both B and T lymphocytes, were susceptible to SARS-CoV-2 infection in vitro, accumulating double-stranded RNA consistent with viral RNA replication and ultimately leading to expressive T cell apoptosis. In addition, flow cytometry and immunofluorescence analysis revealed that SARS-CoV-2 was frequently detected in monocytes and B lymphocytes from coronavirus disease 2019 (COVID-19) patients. The rates of SARS-CoV-2-infected monocytes in peripheral blood mononuclear cells from COVID-19 patients increased over time from symptom onset, with SARS-CoV-2-positive monocytes, B cells, and CD4+ T lymphocytes also detected in postmortem lung tissue. These results indicated that SARS-CoV-2 infection of blood-circulating leukocytes in COVID-19 patients might have important implications for disease pathogenesis and progression, immune dysfunction, and virus spread within the host.

Clinical investigation of intestinal fatty acid-binding protein (I-FABP) as a biomarker of SARS-CoV-2 infection.

OBJECTIVES: SARS-CoV-2 exhibits tropism for the gastrointestinal tract; however, lesions in enterocytes and their correlation with disease severity and patient prognosis are still unknown. METHODS: SARS-CoV-2 patients were enrolled in 5 medical centres in São Paulo, Brazil and their clinical characteristics and laboratory findings recorded. At admission, day 7 and day 14 of hospitalisation, plasma and urine samples were collected, and cytokine levels and intestinal fatty acid-binding protein (I-FABP) concentrations measured. RESULTS: COVID-19 patients displayed ≈48-, 74- and 125-fold increased urinary I-FABP levels at admission (n=283; P<0.001), day 7 (n=142; P<0.01) and day 14 (n=75; P<0.01) of hospitalisation. Critically ill patients and nonsurvivors showed higher I-FABP concentrations compared with patients with less severe illness. At admission, infected patients demonstrated enhanced production of plasma interferon (IFN)-γ and interleukin (IL)-6. The receiver operating characteristic curve suggested I-FABP as a biomarker for COVID-19 disease severity at admission (P<0.0001; Youden index=6.89; area under the curve=0.699). Patients with I-FABP ≥6.89 showed higher IL-6 and C-reactive protein levels (P<0.001) at admission and had a prolonged length of hospital stay. CONCLUSIONS: Our findings revealed damage to enterocytes in SARS-CoV-2 infection, which is associated with illness severity, poor prognosis and exacerbated inflammatory response.

MMP-2 and MMP-9 levels in plasma are altered and associated with mortality in COVID-19 patients.

Respiratory symptoms are one of COVID-19 manifestations, and the metalloproteinases (MMPs) have essential roles in the lung physiology. We sought to characterize the plasmatic levels of matrix metalloproteinase-2 and 9 (MMP-2 and MMP-9) in patients with severe COVID-19 and to investigate an association between plasma MMP-2 and MMP-9 levels and clinical outcomes and mortality. MMP-2 and MMP-9 levels in plasma from patients with COVID-19 treated in the ICU (COVID-19 group) and Control patients were measured with the zymography. The study groups were matched for age, sex, hypertension, diabetes, BMI, and obesity profile. MMP-2 levels were lower and MMP-9 levels were higher in a COVID-19 group (p < 0.0001) compared to Controls. MMP-9 levels in COVID-19 patients were not affected by comorbidity such as hypertension or obesity. MMP-2 levels were affected by hypertension (p < 0.05), but unaffected by obesity status. Notably, hypertensive COVID-19 patients had higher MMP-2 levels compared to the non-hypertensive COVID-19 group, albeit still lower than Controls (p < 0.05). No association between MMP-2 and MMP-9 plasmatic levels and corticosteroid treatment or acute kidney injury was found in COVID-19 patients. The survival analysis showed that COVID-19 mortality was associated with increased MMP-2 and MMP-9 levels. Age, hypertension, BMI, and MMP-2 and MMP-9 were better predictors of mortality during hospitalization than SAPS3 and SOFA scores at hospital admission. In conclusion, a significant association between MMP-2 and MMP-9 levels and COVID-19 was found. Notably, MMP-2 and MMP-9 levels predicted the risk of in-hospital death suggesting possible pathophysiologic and prognostic roles.

Histo-blood group A is a risk factor for severe COVID-19.

OBJECTIVES: Evaluate the impact of ABO histo-blood group type on COVID-19 severity. BACKGROUND: ABO histo-blood type has been associated with different outcomes in infectious diseases. It has also shown a higher proportion of type A patients with SARS-CoV-2. In this observational study, extracted from an ongoing clinical trial on the efficacy of convalescent plasma transfused in COVID-19 patients, we describe the impact of ABO blood type on the risk of developing severe COVID-19. MATERIALS AND METHODS: Seventy-two consecutive patients (37 type A, 23 type O, 11 type B, 1 type AB) with severe (respiratory failure) COVID-19 were included. Control group was composed of 160 individuals randomly selected from the same populational basis. RESULTS: Blood group A was overrepresented (51.39%) in the patient group in relation to the control group (30%), whereas blood group O was less represented (31.94%) in patient than in control group (48%). Odds ratio (A vs. O) was 2.581 (1.381-4.817), CI 95%; p = 0.004. Also, blood group A patients appeared to have more severe disease, given by the scores of the Sequential Organ Failure Assessment and Simplified Acute Physiologic Score 3 (p = 0.036 and p = 0.058, respectively). CONCLUSION: Histo-blood type A is associated with a higher risk of developing severe COVID-19 in relation to blood type O.

Heparin prevents in vitro glycocalyx shedding induced by plasma from COVID-19 patients.

The severe forms and worsened outcomes of COVID-19 (coronavirus disease 19) are closely associated with hypertension and cardiovascular disease. Endothelial cells express Angiotensin-Converting Enzyme 2 (ACE2), which is the entrance door for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The hallmarks of severe illness caused by SARS-CoV-2 infection are increased levels of IL-6, C-reactive protein, D-dimer, ferritin, neutrophilia and lymphopenia, pulmonary intravascular coagulopathy and microthrombi of alveolar capillaries. The endothelial glycocalyx, a proteoglycan- and glycoprotein-rich layer covering the luminal side of endothelial cells, contributes to vascular homeostasis. It regulates vascular tonus and permeability, prevents thrombosis, and modulates leukocyte adhesion and inflammatory response. We hypothesized that cytokine production and reactive oxygen species (ROS) generation associated with COVID-19 leads to glycocalyx degradation. A cohort of 20 hospitalized patients with a confirmed COVID-19 diagnosis and healthy subjects were enrolled in this study. Mechanisms associated with glycocalyx degradation in COVID-19 were investigated. Increased plasma concentrations of IL-6 and IL1-ß, as well as increased lipid peroxidation and glycocalyx components were detected in plasma from COVID-19 patients compared to plasma from healthy subjects. Plasma from COVID-19 patients induced glycocalyx shedding in cultured human umbilical vein endothelial cells (HUVECs) and disrupted redox balance. Treatment of HUVECs with low molecular weight heparin inhibited the glycocalyx perturbation. In conclusion, plasma from COVID-19 patients promotes glycocalyx shedding and redox imbalance in endothelial cells, and heparin treatment potentially inhibits glycocalyx disruption.

Beneficial effects of colchicine for moderate to severe COVID-19: a randomised, double-blinded, placebo-controlled clinical trial.

OBJECTIVE: To evaluate whether the addition of colchicine to standard treatment for COVID-19 results in better outcomes. DESIGN: We present the results of a randomised, double-blinded, placebo-controlled clinical trial of colchicine for the treatment of moderate to severe COVID-19, with 75 patients allocated 1:1 from 11 April to 30 August 2020. Colchicine regimen was 0.5 mg thrice daily for 5 days, then 0.5 mg twice daily for 5 days. The primary endpoints were the need for supplemental oxygen, time of hospitalisation, need for admission and length of stay in intensive care unit and death rate. RESULTS: Seventy-two patients (36 for placebo and 36 for colchicine) completed the study. Median (and IQR) time of need for supplemental oxygen was 4.0 (2.0-6.0) days for the colchicine group and 6.5 (4.0-9.0) days for the placebo group (p<0.001). Median (IQR) time of hospitalisation was 7.0 (5.0-9.0) days for the colchicine group and 9.0 (7.0-12.0) days for the placebo group (p=0.003). At day 2, 67% versus 86% of patients maintained the need for supplemental oxygen, while at day 7, the values were 9% versus 42%, in the colchicine and the placebo groups, respectively (log rank; p=0.001). Two patients died, both in placebo group. Diarrhoea was more frequent in the colchicine group (p=0.26). CONCLUSION: Colchicine reduced the length of both, supplemental oxygen therapy and hospitalisation. The drug was safe and well tolerated. Once death was an uncommon event, it is not possible to ensure that colchicine reduced mortality of COVID-19. TRIAL REGISTRATION NUMBER: RBR-8jyhxh.

Inflammasomes are activated in response to SARS-CoV-2 infection and are associated with COVID-19 severity in patients.

Severe cases of COVID-19 are characterized by a strong inflammatory process that may ultimately lead to organ failure and patient death. The NLRP3 inflammasome is a molecular platform that promotes inflammation via cleavage and activation of key inflammatory molecules including active caspase-1 (Casp1p20), IL-1ß, and IL-18. Although participation of the inflammasome in COVID-19 has been highly speculated, the inflammasome activation and participation in the outcome of the disease are unknown. Here we demonstrate that the NLRP3 inflammasome is activated in response to SARS-CoV-2 infection and is active in COVID-19 patients. Studying moderate and severe COVID-19 patients, we found active NLRP3 inflammasome in PBMCs and tissues of postmortem patients upon autopsy. Inflammasome-derived products such as Casp1p20 and IL-18 in the sera correlated with the markers of COVID-19 severity, including IL-6 and LDH. Moreover, higher levels of IL-18 and Casp1p20 are associated with disease severity and poor clinical outcome. Our results suggest that inflammasomes participate in the pathophysiology of the disease, indicating that these platforms might be a marker of disease severity and a potential therapeutic target for COVID-19.

Complement C3 vs C5 inhibition in severe COVID-19: Early clinical findings reveal differential biological efficacy.

Growing clinical evidence has implicated complement as a pivotal driver of COVID-19 immunopathology. Deregulated complement activation may fuel cytokine-driven hyper-inflammation, thrombotic microangiopathy and NET-driven immunothrombosis, thereby leading to multi-organ failure. Complement therapeutics have gained traction as candidate drugs for countering the detrimental consequences of SARS-CoV-2 infection. Whether blockade of terminal complement effectors (C5, C5a, or C5aR1) may elicit similar outcomes to upstream intervention at the level of C3 remains debated. Here we compare the efficacy of the C5-targeting monoclonal antibody eculizumab with that of the compstatin-based C3-targeted drug candidate AMY-101 in small independent cohorts of severe COVID-19 patients. Our exploratory study indicates that therapeutic complement inhibition abrogates COVID-19 hyper-inflammation. Both C3 and C5 inhibitors elicit a robust anti-inflammatory response, reflected by a steep decline in C-reactive protein and IL-6 levels, marked lung function improvement, and resolution of SARS-CoV-2-associated acute respiratory distress syndrome (ARDS). C3 inhibition afforded broader therapeutic control in COVID-19 patients by attenuating both C3a and sC5b-9 generation and preventing FB consumption. This broader inhibitory profile was associated with a more robust decline of neutrophil counts, attenuated neutrophil extracellular trap (NET) release, faster serum LDH decline, and more prominent lymphocyte recovery. These early clinical results offer important insights into the differential mechanistic basis and underlying biology of C3 and C5 inhibition in COVID-19 and point to a broader pathogenic involvement of C3-mediated pathways in thromboinflammation. They also support the evaluation of these complement-targeting agents as COVID-19 therapeutics in large prospective trials.

SARS-CoV-2-triggered neutrophil extracellular traps mediate COVID-19 pathology.

Severe COVID-19 patients develop acute respiratory distress syndrome that may progress to cytokine storm syndrome, organ dysfunction, and death. Considering that neutrophil extracellular traps (NETs) have been described as important mediators of tissue damage in inflammatory diseases, we investigated whether NETs would be involved in COVID-19 pathophysiology. A cohort of 32 hospitalized patients with a confirmed diagnosis of COVID-19 and healthy controls were enrolled. The concentration of NETs was augmented in plasma, tracheal aspirate, and lung autopsies tissues from COVID-19 patients, and their neutrophils released higher levels of NETs. Notably, we found that viable SARS-CoV-2 can directly induce the release of NETs by healthy neutrophils. Mechanistically, NETs triggered by SARS-CoV-2 depend on angiotensin-converting enzyme 2, serine protease, virus replication, and PAD-4. Finally, NETs released by SARS-CoV-2-activated neutrophils promote lung epithelial cell death in vitro. These results unravel a possible detrimental role of NETs in the pathophysiology of COVID-19. Therefore, the inhibition of NETs represents a potential therapeutic target for COVID-19.