Insights into the lung microenvironment after severe COVID-19

The SARS-CoV-2 is the betacoronavirus responsible for the coronavirus disease 2019 (COVID-19) pandemic. Severe COVID-19 affects approximately 10-15% of patients and results in prolonged morbidity and mortality. Little is known about the immunophenotypic changes of the lung parenchyma driven by the viral infection in patients who die of severe COVID-19. Ultrasound-guided lung biopsies (LB) were collected (IRB approval#1561/21) within few hours from death in 15 severe COVID-19 patients between November 2020 and January 2021, in two patients who underwent lung transplantation after COVID-19 and in one patient who had surgery for bacterial superinfection during COVID-19 disease. All samples underwent histologic and immunohistochemistry evaluation and molecular profiling using the nCounter Host Response and Coronavirus Panel plus. As controls, lungs from end-stage usual interstitial pneumonia (UIP;n=9) and from lobectomy for lung cancer (Norm;n=5) were used. Eleven lungs (61%) were positive for SARS-CoV-2 RNA. Signs of diffuse alveolar damage (DAD) were observed in 6 patients (30%). COVID-19 lungs showed a marked macrophage infiltration with M2 polarization compared with controls. Globally, COVID-19 lungs showed distinct molecular profiles from UIP or Norm lungs. Specifically, a marked upregulation of interferon-genes that was directly correlated with SARS-CoV-2 genes was seen in COVID-19 lungs. COVID-19-specific genes signatures (Log2FC >1.5;adj p<0.05) obtained using VENN diagram showed impairment of the STAT3-pathway accompanied by the upregulation of the NFkB signaling. Results herein provide new insights into lung alterations induced by severe COVID-19 and suggest novel potential targets for therapeutic intervention.

COVID-19 vaccination in patients with hemophilia: Experience from a single center

Background: Coronavirus disease-2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2), has resulted in an ongoing world-wide pandemic. Vaccination is the key countermeasure of the COVID-19 pandemic. Data on the efficacy, safety and immunogenicity of COVID-19 vaccination in patients with hemophilia -and in particular in those with HIV -are still scarce. Aim(s): The aim of our study was to characterize the immunogenicity and biomarkers of coagulation and endothelial perturbation after mRNA-COVID- 19 vaccination in HIV-positive hemophilic patients. Method(s): We collected blood from 24 adult HIV-positive hemophilic patients followed at our centre (19 with hemophilia A, 5 with hemophilia B) before and two weeks after the administration of the complete vaccination schedule with mRNA-1273 (Moderna Biotech). Most patients had severe hemophilia (n = 21). We measured antibodies to SARS-CoV- 2 spike protein by Elecsys (Roche) to assess immunogenicity and we evaluated protein C, VWF, D-dimer plasma levels as biomarkers of coagulation and endothelial perturbation. Anti-Platelet Factor 4 (PF4) antibodies were also measured. Result(s): Before vaccination, three patients out of 24 showed anti-Spike IgG levels >0.8 U/ml (cut-off value). Two weeks after completing the vaccination schedule, all patients had high values of anti-Spike IgG (min-max 2,387-12,500 U/ml). Mean (standard deviation) basal values of protein C, VWF and D-dimer (106 +/- 21%, 171 +/- 45%, 593 +/- 692 ng/ml respectively) were not significantly different from values measured two weeks after the second dose of vaccine (103 +/- 20%, 162 +/- 43%, 583 +/- 531 ng/ml). Anti-PF4 antibodies were detected in three patients with no associated clinical manifestations. None of the patients reported bleeding in the site of inoculation nor serious adverse events after the vaccination. Conclusion(s): Since immune abnormalities can occur in HIV-positive patients, it is important to collect data on COVID-19 vaccination immunogenicity. We demonstrated that hemophilic HIV-positive patients have a normal antibody response against SARS-CoV- 2 spike protein. In addition, mRNA-1273 had no effect on coagulation and endothelial perturbation.

Evaluation of D-dimer concentrations measured with three methods in patients with COVID-19

Background: COVID-19 is associated with an increased risk of venous thrombosis, even when patients are on standard-dose antithrombotic prophylaxis. Hence, the identification of biomarkers of thrombosis helps tailoring dosage of antithrombotic prophylaxis. D-dimer has been extensively employed as a biomarker and cut-off values at hospital admission have been proposed to stratify the risk of thrombosis and make decision on prophylaxis. However, D-dimer measurement is not standardized, and it is unknown if the cut-off values used for decision making can be used interchangeably between methods. Aim(s): To assess for concordance of results obtained with different commercially available laboratory methods measuring D-dimer. Method(s): Plasma samples collected from COVID-19 patients at the Hospital of Cremona were evaluated for D-dimer with three widely used immunoturbidimetric methods (Liatest D-di, Stago, Asnieres, France;D-dimer HS 500, Werfen, Orangeburg, NY;Innovance D-dimer, Siemens, Marburg, Germany). Result(s): A total of 87 COVID-19 patients [54 male and 33 female, median age of 73 years (range 28-98)] were enrolled in the study. No significant differences were found between mean D-dimer concentrations obtained with the three methods even when stratifying D-dimer levels in 4 groups (<1000, 1000-2000, 2000-5000, >5000 ng/mL) (Figure 1). The three methods showed substantial result agreement [Stago-vs- Werfen and Siemens-vs- Stago (Cohen's kappa coefficient of 0.760 and 0.699, respectively)] to an almost perfect agreement [Siemens-vs- Werfen (Cohen's kappa coefficient of 0.811)], with a p-value < 0.001. Results from the three methods showed a good linear correlation (Rho = 0.94) (Figure 2). Conclusion(s): The relatively good concordance of D-dimer results among the three investigated methods indicates that D-dimer cut-off values could be used interchangeably regardless of the method used for testing. The results pave the way to clinical trials aimed to assess the value of D-dimer as a biomarker to make decision on the intensity of antithrombotic prophylaxis in COVID-19 patients. (Figure Presented).

Involvement of the contact pathway in COVID-19 coagulopathy

Background: A novel acquired coagulopathy characterized by a severe procoagulant imbalance is common in COVID-19 patients and is associated with the clinical severity of the disease. Aim(s): Our study aims to elucidate the underlying mechanisms of coagulation activation in COVID-19 patients. Method(s): Symptomatic COVID-19 patients during Milan first wave were consecutively enrolled and stratified into 3 groups based on the intensity of care: Low, requiring only high-flow oxygen by nasal cannula;intermediate, requiring continuous positive airway pressure;high, requiring mechanical ventilation. Blood samples were tested for markers of activation of the intrinsic pathway (FXIa, FXIIa) together with its physiologic inhibitor (C1-inhibitor), of the extrinsic pathway (FVIIa), of global activation of the coagulation cascade (D-dimer, FDP, FM) and of fibrinolysis (plasminogen, t-PA, alpha2-antiplasmin, PAI-1). Result(s): 111 patients were included: 26 at low, 42 intermediate and 43 high care-intensity. Median age was 59 +/- 12 (34 patients >65 years);32 patients (29%) developed a venous thrombosis and 12 (11%) died (Table). Median D-dimer, FDP and FM plasma levels were higher in COVID-19 patients compared to controls, with a gradient of increase across the three care intensities, while all the fibrinolytic pathway parameters were in the normal range. Median plasma levels of FVIIa were lower in COVID-19 patients (27.5 mU/ml) than in controls (40.1 mU/ml) while median plasma levels of FXIIa and FXIa were higher in COVID-19 patients (11.2 and 11.3 mU/ml) than in controls (7.2 and 5.5 mU/ml), with a gradient of increase across the three care intensities. C1-inhibitor plasma levels were above the normal range in all the 3 COVID-19 patients' groups (Figure). Conclusion(s): Our study showed a prevalent activation of the contact pathway over the extrinsic pathway of the coagulation cascade in COVID-19 patients, which is proportional to the clinical severity of the infection, opening the possibility for targeted anticoagulant therapies. (Table Presented).

Impact of SARS-CoV- 2 vaccination in thrombotic thrombocytopenic purpura patients

Background: Thrombotic thrombocytopenic purpura (TTP) has occasionally been described after vaccination. Since the availability of anti-SARS- CoV- 2 vaccines, 12 cases have been described on a possible association with TTP onset. Aim(s): This study aims to evaluate the relapse rates in patients affected by TTP undergoing anti-SARS- CoV- 2 vaccination. Method(s): All consecutive TTP patients undergoing anti-SARS- CoV- 2 vaccination from March to May 2021 were enrolled. Blood samples were collected before vaccination (T0), 2 weeks after the first (T1) and the second dose (T2) to evaluate ADAMTS13 activity and anti-ADAMTS13 antibody titer. Result(s): A total of 49 TTP patients were enrolled (48 acquired and 1 congenital), all vaccinated with an mRNA vaccine. No patients had a clinical TTP relapse, with an ADAMTS13 relapse rate of 1.36% per month. Mean levels of ADAMTS13 activity were stable among the three timepoints (Figure). In only two patients a significant drop in ADAMTS13 levels occurred after the first dose (from 28% to <3% and from 101% to 82%), and both remained stable after the second dose, with negative anti-ADAMTS13 antibodies. Due to a stable undetectable ADAMTS13, the first patient was treated with 4 doses of weekly 375 mg/m2 rituximab with a rapid ADAMTS13 response. One patient had positive basal anti-ADAMTS13 antibodies with a titer remaining stable after the two vaccine doses, while in another patient anti-ADAMTS13 antibodies became detectable after the first dose, with no corresponding drop in ADAMTS13 levels and a stable titer after the second dose. Conclusion(s): The result of our study prospectively evaluating the effect of anti-SARS- CoV- 2 vaccination on the risk of relapse in a large cohort of patients with TTP in Milan showed a lower than reported relapse rate (1.36% vs 2.6%) with an observed to expected incidence rate ratio of 0.52, confirming the safety of mRNA-based anti-SARS- CoV- 2 vaccination in TTP patients.

Reduced circulating FABP2 in patients with moderate to severe COVID-19 may indicate enterocyte functional change rather than cell death.

The gut is of importance in the pathology of COVID-19 both as a route of infection, and gut dysfunction influencing the severity of disease. Systemic changes caused by SARS-CoV-2 gut infection include alterations in circulating levels of metabolites, nutrients and microbial products which alter immune and inflammatory responses. Circulating plasma markers for gut inflammation and damage such as zonulin, lipopolysaccharide and ß-glycan increase in plasma along with severity of disease. However, Intestinal Fatty Acid Binding Protein / Fatty Acid Binding Protein 2 (I-FABP/FABP2), a widely used biomarker for gut cell death, has paradoxically been shown to be reduced in moderate to severe COVID-19. We also found this pattern in a pilot cohort of mild (n = 18) and moderately severe (n = 19) COVID-19 patients in Milan from March to June 2020. These patients were part of the first phase of COVID-19 in Europe and were therefore all unvaccinated. After exclusion of outliers, patients with more severe vs milder disease showed reduced FABP2 levels (median [IQR]) (124 [368] vs. 274 [558] pg/mL, P < 0.01). A reduction in NMR measured plasma relative lipid-CH3 levels approached significance (median [IQR]) (0.081 [0.011] vs. 0.073 [0.024], P = 0.06). Changes in circulating lipid levels are another feature commonly observed in severe COVID-19 and a weak positive correlation was observed in the more severe group between reduced FABP2 and reduced relative lipid-CH3 and lipid-CH2 levels. FABP2 is a key regulator of enterocyte lipid import, a process which is inhibited by gut SARS-CoV-2 infection. We propose that the reduced circulating FABP2 in moderate to severe COVID-19 is a marker of infected enterocyte functional change rather than gut damage, which could also contribute to the development of hypolipidemia in patients with more severe disease.

Managing hematological cancer patients during the COVID-19 pandemic: an ESMO-EHA Interdisciplinary Expert Consensus.

BACKGROUND: The COVID-19 pandemic has created enormous challenges for the clinical management of patients with hematological malignancies (HMs), raising questions about the optimal care of this patient group. METHODS: This consensus manuscript aims at discussing clinical evidence and providing expert advice on statements related to the management of HMs in the COVID-19 pandemic. For this purpose, an international consortium was established including a steering committee, which prepared six working packages addressing significant clinical questions from the COVID-19 diagnosis, treatment, and mitigation strategies to specific HMs management in the pandemic. During a virtual consensus meeting, including global experts and lead by the European Society for Medical Oncology and the European Hematology Association, statements were discussed and voted upon. When a consensus could not be reached, the panel revised statements to develop consensual clinical guidance. RESULTS AND CONCLUSION: The expert panel agreed on 33 statements, reflecting a consensus, which will guide clinical decision making for patients with hematological neoplasms during the COVID-19 pandemic.

Acute liver necrosis in a SARS-CoV-2 positive patient with triple positive antiphospholipid syndrome

Background : Several thrombotic manifestations have been reported with SARS-Cov-2 infection including liver vascular involvement. Aims : We present a dramatic case of acute liver necrosis in a 36-year-old SARS-Cov-2 positive Italian woman with no respiratory symptoms and triple positive antiphospholipid syndrome (APS). Methods : The patient was referred to our University Hospital for acute hypertransaminasemia and liver failure (Figure). She had systemic lupus erythematosus (positivity for: ANA, anti-dsDNA, complement activation, Coombs;thrombocytopenia, previous arthritis). Anti-phopspholipid antibodies (aPL) were detected for the first time in 2015 during routine pregnancy screening and chronically treated with aspirin. Apparently, no venous/arterial nor obstetric events were recorded up to the recent hospitalization. FIGURE 1 Results : At arrival, US-Doppler and CT-scan were consistent with signs of chronic liver disease and occlusion of the three hepatic veins defining a Budd-Chiari syndrome. We opted for a stepwise approach considering anticoagulation (clexane 100 UI/Kg b.i.d) the first line of therapy before any invasive intervention. Dexamethasone 6 mg/ day b.i.d., 6 sessions of plasma-exchange, i.v.-immunoglobulin were sequentially planned to revert the liver damage sustained by aPL. After 5-days, two hepatic-veins resulted recanalized in association with amelioration of liver-enzyme/function and aPL quantification. Then we performed hepatic vein catheterization and transjugular liver biopsy. The histology showed multiple areas of necrosis associated with liver cirrhosis. Unexpectedly, no signs of acute Budd-Chiari were observed (e.g. intraparenchymal hemorrhages, centrilobular congestion, sinusoidal dilation). Other etiologies were also excluded and we hypothesized the involvement of small arteries of the liver in a triple positive APS in a patients with SLE. We finally addressed the patient to a liver transplant program and a tight multispecialistic follow-up. Conclusions : Thrombosis of arterial/venous vessels or microcirculation causes liver damage in some patients with aPL. Our report suggests that SARS-Cov-2 can exacerbate this prothrombotic condition determining a life-threatening complication such as acute liver failure.

Adoption of emicizumab for hemophilia A in europe: Data from the 2020 european association for haemophilia and allied disorders survey

Background : Emicizumab is a bispecific monoclonal antibody, mimicking coagulation factor (F) VIII by linking factor IXa and X and thereby restoring steady-state hemostasis in patients with hemophilia A. It is administered subcutaneously and has been approved for use in hemophilia A patients with and without inhibitors. Aims : To assess availability, acceptance and safety of emicizumab for hemophilia A in Europe. Methods : An online questionnaire was sent to 144 European Haemophilia Comprehensive Care Centers (EHCCC) and European Haemophilia Treatment Centers (EHTC). The survey was open from November 17, 2020 to January 31, 2021. Descriptive statistics were performed using SPSS version 27. Results : Forty-six physicians from 21 countries responded. Emicizumab is reimbursed for all patients with inhibitors and the majority of patients without inhibitors. Reducing treatment burden was the main reason to switch both inhibitor and non-inhibitor patients to emicizumab. An annualized bleeding rate (ABR) of zero could be achieved in most patients with inhibitors on emicizumab (72.9%). Inhibitor titers were regularly monitored in 78.4% of inhibitor patients on emicizumab and chromogenic FVIII assay was not available in 27.0% of centers. Hemostasis was satisfactory in the majority of minor (93.7%) and major (90.7%) surgical procedures performed while on emicizumab. In the 35 centers that responded to the question, a total of four patients on emicizumab have died to date, although none of these deaths were directly linked to emicizumab. The Covid-19 pandemic did not have a considerable impact on the adoption of emicizumab in most centers (64.9%). Conclusions : Three years after its rollout in Europe, emicizumab is widely available for both inhibitor and non-inhibitor patients. Satisfaction of physicians with emicizumab is very good. Unmet needs include availability of laboratory assays and guidelines for cotreatment during surgery and in case of breakthrough bleeding.

D-dimer as a predictive marker for mortality in hospitalized COVID-19 patients

Background : The novel coronavirus disease 2019 (COVID-19) presents an important and urgent threat to global health. Identifying strong predictors of mortality could assist medical staff in treating patients and allocating limited healthcare resources. Aims : The primary aim of this paper was to study the effect of ddimer levels at admission as a predictive marker for in-hospital mortality. Methods : This was a retrospective cohort study evaluating hospitalized patients (age > 18 years), who were positive for COVID-19 based on real-time PCR at one of nine COVID-19 units during the period of the first COVID-19 wave in Lombardy, Italy. The primary endpoint was in-hospital mortality. Information was obtained from patient records. Statistical analyses were performed using a Fine-Gray competing risk survival model. Predictive power was assessed using Harrell's C-index. Results : Out of 1049 patients that were admitted to the emergency department and subsequently hospitalized, 501 patients had evaluable data for d-dimer. Of these 501 patients, 96 did not survive. Cumulative incidence of in-hospital mortality within 30 days was 20%, and the majority of deaths occurred within the first 10 days. (Figure 1) When compared to patients in the lowest quartile of d-dimer blood concentration, the hazard ratio of in-hospital mortality for patients in the 2 nd , 3 rd and 4 th quartile was 3.9 (95CI: 1.5-10.0), 5.8 (95CI: 2.3-14.7), and 4.6 (95CI: 1.8-11.5) respectively, after multivariable adjustment for age, sex and number of comorbidities. The C-statistic of d-dimer for in-hospital mortality was 0.67 (95CI: 0.62-0.71). (Table 2) Conclusions : Higher d-dimer levels were strongly associated with inhospital mortality. However, the predictive power of d-dimer alone was not high enough to be useful as a risk prediction score. Future research should focus on the added value of d-dimer as part of a larger risk prediction score.

Anticoagulant response to tailored low molecular weight heparin regimens in patients with COVID-19 infection

Background : Covid-19 infection is associated with a widespread global activation of coagulation and affected patients are at an increased risk of thrombosis. Aims : Heparin therapy is effective in various setting in preventing thromboembolic complications and aim of this study was to assess heparin response in COVID-19 patients through anti-FXa test. Methods : In 52 patients, M:F ratio 59:41, median age 59 years old, admitted in different intensity of care units of our hospital, treated with different regimens of heparin (100 U/kg every 24 h in low intensity care, 70 U/kg every 12 h in intermediate intensity care and 100 U/kg every 12 h in intensive care unit), anti-FXa levels were measured immediately before and 3 h after subcutaneous enoxaparin administration. On the same samples thrombin generation tests were performed. Results : Patients treated with 100 U/kg every 24 h and 70 U/ kg every 12 h had median anti-FXa basal levels in the prophylactic range, respectively 0.18 and 0.22 U/ml, while patients treated with 100 U/kg every 12 h were in the anticoagulant range (0.37 U/ ml). Despite heparin therapy thrombin generation was elevated in COVID-19 patients, indicating a high level of coagulation activation. Conclusions : In conclusion we demonstrated that the biological response to enoxaparin in COVID-19 patients is in the expected range using anti-FXa assay and patients are not resistant to heparin therapy.

Metabolic and liver alterations impact on the severity of SARS-COV2 disease

Background: SARS-COV2 is a threatful viral disease which can evolve into respiratory failure but identification of risk factors for progression towards severe forms is still ongoing During infection impairment of liver function tests has been frequently reported and evidence of the negative impact of metabolic alterations on the clinical course are emerging, mainly evaluated in Asiatic populations Aim: to define the prognostic role of metabolic disease and liver damage on SARS-COV2 severity in a cohort of Italian patients Methods: All patients with confirmed COVID-19 infection admitted to low-intensity care COVID Units between March and April 2020 were enrolled Severe SARS-COV2 infection was defined according to International consensus requiring intensive respiratory support (CPAP/orotracheal intubation) All data were collected at admission Results: 382 patients were enrolled, mean age was 65±17 ys and 60% were male Mean BMI was 27±5 kg/m2 (61% overweight and 25% obese), prevalence of T2DM 17%, hypertension 44%, dyslipidemia 29% At admission 39% and 40% of patients had increased ALT and GGT levels, respectively Median hospitalization stay was 14 days (IQR 8-24), with 30% of patients experiencing severe SARS-COV2 infection and mortality in 14% of overall cases Subjects with severe infection presented higher prevalence of male sex (68% vs 56%, p=0 03), T2DM (25% vs 14%, p=0 01), dyslipidemia (36% vs 26%, p=0 05) and increased ALT (53% vs 32%, p<0 001) and GGT (53% vs 34%, p=0 01) levels at admission In multivariate analysis (adjusted for age, sex, T2DM, dyslipidemia, increased ALT and GGT at admission and SARS-COV2 therapy), T2DM (OR 3 1, 95%CI 1 6-6 0;p=0 001), dyslipidemia (OR 1 9, 95%CI 1 1-3 3;p=0 03), and increased ALT (OR 4 7, 95%CI 2 5- 9 0;p<0 001) and GGT (OR 2 0, 95%CI 1 2-3-3;p=0 009), resulted associated with high risk of more severe SARSCOV2 disease Interestingly, in patients with both T2DM and dyslipidemia, this risk was further increased (OR 5 2, 95%CI 2 1-12 6;p<0 001) Conclusion: We confirm that liver involvement is common during SARS-COV2 infection and it is a negative prognostic factor although it is not clear whether it precedes or follows, the severity of clinical course In addition, the risk of severe form of respiratory distress is increased by metabolic alterations, and the more numerous the alterations, the higher the risk.