Could SP-A and SP-D Serum Levels Predict COVID-19 Severity?

Given the various clinical manifestations that characterize Coronavirus Disease 2019 (COVID-19), the scientific community is constantly searching for biomarkers with prognostic value. Surfactant proteins A (SP-A) and D (SP-D) are collectins that play a crucial role in ensuring proper alveolar function and an alteration of their serum levels was reported in several pulmonary diseases characterized by Acute Respiratory Distress Syndrome (ARDS) and pulmonary fibrosis. Considering that such clinical manifestations can also occur during Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, we wondered if these collectins could act as prognostic markers. In this regard, serum levels of SP-A and SP-D were measured by enzyme immunoassay in patients with SARS-CoV-2 infection (n = 51) at admission (T0) and after seven days (T1) and compared with healthy donors (n = 11). SP-D increased in COVID-19 patients compared to healthy controls during the early phases of infection, while a significant reduction was observed at T1. Stratifying SARS-CoV-2 patients according to disease severity, increased serum SP-D levels were observed in severe compared to mild patients. In light of these results, SP-D, but not SP-A, seems to be an eligible marker of COVID-19 pneumonia, and the early detection of SP-D serum levels could be crucial for preventive clinical management.

Postoperative pulmonary complications and mortality after major abdominal surgery. An observational multicenter prospective study.

BACKGROUND: Postoperative pulmonary complications (PPCs) significantly contribute to postoperative morbidity and mortality. We conducted a study to determine the incidence of PPCs after major elective abdominal surgery and their association with early and 1-year mortality in patient without pre-existing respiratory disease. METHODS: We conducted a multicenter observational prospective clinical study in 40 Italian centers. 1542 patients undergoing elective major abdominal surgery were recruited in a time period of 14 days and clinically managed according to local protocol. The primary outcome was to determine the incidence of PPCs. Further, we aimed to identify independent predictors for PPCs and examine the association between PPCs and mortality. RESULTS: PPCs occurred in 12.6% (95% CI 11.1-14.4%) of patients with significant differences among general (18.3%, 95% CI 15.7-21.0%), gynecological (3.7%, 95% CI 2.1-6.0%) and urological surgery (9.0%, 95% CI 6.0-12.8%). PPCs development was associated with known pre- and intraoperative risk factors. Patients who developed PPCs had longer length of hospital stay, higher risk of 30-days hospital readmission, and increased in-hospital and one-year mortality (OR 3.078, 95% CI 1.825-5.191; P<0.001). CONCLUSIONS: The incidence of PPCs in patients without pre-existing respiratory disease undergoing elective abdominal surgery is high and associated with worse clinical outcome at one year after surgery. General surgery is associated with higher incidence of PPCs and mortality compared to gynecological and urological surgery.

Case Report: Severe cutaneous adverse event associated with checkpoint inhibition in the setting of CAR T-cell therapy: beyond CRS.

Anti-CD19 chimeric antigen receptor (CAR) T cell therapy actually represents the standard of care for multiple relapsed or refractory primary mediastinal B-cell lymphoma (r/r PMBCL). Checkpoint inhibitors, such as pembrolizumab, appear to be a safe and effective treatment strategy for patients who are ineligible for or resistant to autologous stem cell transplantation. Although preclinical studies suggested that checkpoint inhibitors may enhance the vitality and anti-tumor activity of CAR T cells, there are no substantial/robust clinical data about the immune-mediated toxicity of their association. We describe a case of a severe cutaneous adverse event arising immediately after Cytokine Release Syndrome (CRS) on day +6 from CAR T cells infusion in a young r/r PMBCL patient who previously received pembrolizumab. These skin lesions were interpreted as an immune mediated adverse event, considering their prompt improvement and fully recovering achieved with the addition of immunoglobulin infusion to systemic steroid therapy. This case of life-threatening cutaneous adverse event calls for further investigations about off-target immune-related adverse events deriving from the combination of CAR T cell therapy and checkpoint inhibition, whose synergic therapeutic effect is promising.

Cellular Immune Profiling of Lung and Blood Compartments in Patients with SARS-CoV-2 Infection.

BACKGROUND: SARS-CoV-2 related immunopathology may be the driving cause underlying severe COVID-19. Through an immunophenotyping analysis on paired bronchoalveolar lavage fluid (BALF) and blood samples collected from mechanically ventilated patients with COVID-19-associated Acute Respiratory Distress Syndrome (CARDS), this study aimed to evaluate the cellular immune responses in survivors and non-survivors of COVID-19. METHODS: A total of 36 paired clinical samples of bronchoalveolar lavage fluid (BALF) mononuclear cells (BALF-MC) and peripheral blood mononuclear cells (PBMC) were collected from 18 SARS-CoV-2-infected subjects admitted to the intensive care unit (ICU) of the Policlinico Umberto I, Sapienza University Hospital in Rome (Italy) for severe interstitial pneumonia. The frequencies of monocytes (total, classical, intermediate and non-classical) and Natural Killer (NK) cell subsets (total, CD56bright and CD56dim), as well as CD4+ and CD8+ T cell subsets [naïve, central memory (TCM) and effector memory (TEM)], and those expressing CD38 and/or HLADR were evaluated by multiparametric flow cytometry. RESULTS: Survivors with CARDS exhibited higher frequencies of classical monocytes in blood compared to non-survivors (p < 0.05), while no differences in the frequencies of the other monocytes, NK cell and T cell subsets were recorded between these two groups of patients (p > 0.05). The only exception was for peripheral naïve CD4+ T cells levels that were reduced in non-survivors (p = 0.04). An increase in the levels of CD56bright (p = 0.012) and a decrease in CD56dim (p = 0.002) NK cell frequencies was also observed in BALF-MC samples compared to PBMC in deceased COVID-19 patients. Total CD4+ and CD8+ T cell levels in the lung compartment were lower compared to blood (p = 0.002 and p < 0.01, respectively) among non-survivors. Moreover, CD38 and HLA-DR were differentially expressed by CD4+ and CD8+ T cell subsets in BALF-MC and in PBMC among SARS-CoV-2-infected patients who died from COVID-19 (p < 0.05). CONCLUSIONS: These results show that the immune cellular profile in blood and pulmonary compartments was similar in survivors and non-survivors of COVID-19. T lymphocyte levels were reduced, but resulted highly immune-activated in the lung compartment of patients who faced a fatal outcome.

Breakthrough infections after COVID-19 vaccinations do not elicit platelet hyperactivation and are associated with high platelet-lymphocyte and low platelet-neutrophil aggregates.

Background: Severe COVID-19 is associated with an excessive immunothrombotic response and thromboinflammatory complications. Vaccinations effectively reduce the risk of severe clinical outcomes in patients with COVID-19, but their impact on platelet activation and immunothrombosis during breakthrough infections is not known. Objectives: To investigate how preemptive vaccinations modify the platelet-immune crosstalk during COVID-19 infections. Methods: Cross-sectional flow cytometry study of the phenotype and interactions of platelets circulating in vaccinated (n = 21) and unvaccinated patients with COVID-19, either admitted to the intensive care unit (ICU, n = 36) or not (non-ICU, n = 38), in comparison to matched SARS-CoV-2-negative patients (n = 48), was performed. Results: In the circulation of unvaccinated non-ICU patients with COVID-19, we detected hyperactive and hyperresponsive platelets and platelet aggregates with adaptive and innate immune cells. In unvaccinated ICU patients with COVID-19, most of whom had severe acute respiratory distress syndrome, platelets had high P-selectin and phosphatidylserine exposure but low capacity to activate integrin αIIbß3, dysfunctional mitochondria, and reduced surface glycoproteins. In addition, in the circulation of ICU patients, we detected microthrombi and platelet aggregates with innate, but not with adaptive, immune cells. In vaccinated patients with COVID-19, who had no acute respiratory distress syndrome, platelets had surface receptor levels comparable to those in controls and did not form microthrombi or platelet-granulocyte aggregates but aggregated avidly with adaptive immune cells. Conclusion: Our study provides evidence that vaccinated patients with COVID-19 are not associated with platelet hyperactivation and are characterized by platelet-leukocyte aggregates that foster immune protection but not excessive immunothrombosis. These findings advocate for the importance of vaccination in preventing severe COVID-19.

Clinical Impact of Colonization with Carbapenem-Resistant Gram-Negative Bacteria in Critically Ill Patients Admitted for Severe Trauma.

Multidrug-resistant (MDR) Gram-negative bacteria (GNB) have raised concerns as common, frequent etiologic agents of nosocomial infections, and patients admitted to intensive care units (ICUs) present the highest risk for colonization and infection. The incidence of colonization and infection in trauma patients remains poorly investigated. The aim of this study was to assess the risk factors for Carbapenem-resistant (CR)-GNB colonization and the clinical impact of colonization acquisition in patients with severe trauma admitted to the ICU in a CR-GNB hyperendemic country. This is a retrospective observational study; clinical and laboratory data were extracted from the nosocomial infection surveillance system database. Among 54 severe trauma patients enrolled in the study, 28 patients were colonized by CR-GNB; 7 (12.96%) patients were already colonized at ICU admission; and 21 (38.89%) patients developed a new colonization during their ICU stay. Risk factors for colonization were the length of stay in the ICU (not colonized, 14.81 days ± 9.1 vs. colonized, 38.19 days ± 27.9; p-value = 0.001) and days of mechanical ventilation (not colonized, 8.46 days ± 7.67 vs. colonized, 22.19 days ± 15.09; p-value < 0.001). There was a strong statistical association between previous colonization and subsequent development of infection (OR = 80.6, 95% CI 4.5−1458.6, p-value < 0.001). Factors associated with the risk of infection in colonized patients also included a higher Charlson comorbidity index, a longer length of stay in the ICU, a longer duration of mechanical ventilation, and a longer duration of treatment with carbapenem and vasopressors (not infected vs. infected: 0(0−4) vs. 1(0−3), p = 0.012; 24.82 ± 16.77 vs. 47 ± 28.51, p = 0.016; 13.54 ± 15.84 vs. 31.7 ± 16.22, p = 0.008; 1.09 ± 1.14 vs. 7.82 ± 9.15, p = 0.008). The adoption of MDR-GNB colonization prevention strategies in critically ill patients with severe trauma is required to improve the quality of care and reduce nosocomial infections, length of hospital stay and mortality.

A new ex-situ machine perfusion device. A preliminary evaluation using a model of donors after circulatory death pig livers.

We herein describe a new ex-situ machine perfusion device as a "technology spotlight" using a model of donors after circulatory death liver grafts procured from slaughterhouse pigs. Fourteen pig liver grafts were included. The device allowed stable perfusion in both hypothermic (n = 6) and normothermic (n = 8) conditions and no technical failure was observed. During perfusion, perfusate and bile samples were collected to assess liver metabolism and viability. An integrated adsorption device showed efficient removal of inflammatory cytokines during treatment. This preliminary experience represents the starting point for further investigations on the potential clinical benefits of cytokines and other inflammatory mediators adsorption during machine perfusion.

The role of hypothermic machine perfusion in selecting renal grafts with advanced histological score.

BACKGROUND: Few studies explored the role of hypothermic machine perfusion (HMP) in the sub-group of non-standard renal grafts with a biopsy-proven advanced histological impairment. This study aimed to investigate the role of HMP in grafts with a Karpinski Score >3 in terms of the need for dialysis, creatinine reduction ratio at day-7 (CRR7), and 3-year graft survival. METHODS: Twenty-three perfused grafts with Karpinski Score >3 evaluated between November 2017 and December 2018 were retrospectively analyzed and compared with a control group of 32 non-perfused grafts transplanted between January 2014 and October 2017. RESULTS: After transplantation, perfused grafts had fewer cases requiring dialysis (8.7% vs. 34.4%; p = 0.051), a better reduction in serum creatinine (median at 7 days: 2.2 vs. 4.3 mg/dl; p = 0.045), and shorter length of hospital stay (median 11 vs. 15 days; p = 0.01). Three-year death-censored graft survival was better in the perfused cases (91.3% vs. 77.0%; p = 0.16). In perfused grafts, initial renal resistance (RR) had the best predictive value for renal function recovery after the first week, as defined by CRR7 ≤ 70% (AUC = 0.83; p = 0.02). A cut-off value of 0.5 mm Hg/ml/min showed a sensitivity of 82.4%, a specificity of 83.3%, and diagnostic odds ratio = 23.4. After dividing the entire population into a Low-RR (n = 8) and a High-RR Group (n = 15), more cases with CRR7 ≤ 70% were reported in the latter group (86.7 vs. 13.3%; p = 0.03). CONCLUSION: HMP yielded promising results in kidneys with Karpinski Score >3. Initial RR should be of interest in selecting non-standard organs for single kidney transplantation even in impaired histology.

Intraoperative use of extracorporeal CO2 removal (ECCO2R) and emergency ECMO requirement in patients undergoing lung transplant: a case-matched cohort retrospective study.

BACKGROUND: The use of extracorporeal carbon dioxide removal (ECCO2R) is less invasive than extracorporeal membrane oxygenation (ECMO), and intraoperative control of gas exchange could be feasible. The aim of this study in intermediate intraoperative severity patients undergoing LT was to assess the role of intraoperative ECCO2R on emergency ECMO requirement in patients. METHODS: Thirty-eight consecutive patients undergoing lung transplantation (LT) with "intermediate" intraoperative severity in the intervals 2007 to 2010 or 2011 to 2014 were analyzed as historical comparison of case-matched cohort retrospective study. The "intermediate" intraoperative severity was defined as the development of intraoperative severe respiratory acidosis with maintained oxygenation function (i.e., pH <7.25, PaCO2 >60 mmHg, and PaO2/FiO2 >150), not associated with hemodynamic instability. Of these 38 patients, twenty-three patients were treated in the 2007-2010 interval by receiving "standard intraoperative treatment," while 15 patients were treated in the 2011-2014 interval by receiving "standard intraoperative treatment + ECCO2R." RESULTS: ECMO requirement was more frequent among patients that received "standard intraoperative treatment" alone than in those treated with "standard intraoperative treatment + ECCO2R" (17/23 vs. 3/15; p = 0.004). The use of ECCO2R improved pH and PaCO2 while mean pulmonary artery pressure (mPAP) decreased. CONCLUSION: In intermediate intraoperative severity patients, the use of ECCO2R reduces the ECMO requirement.

Cardiac involvement in consecutive unselected hospitalized COVID-19 population: In-hospital evaluation and one-year follow-up.

BACKGROUND: Cardiovascular disease (CVD) can occur in COVID-19 and has impact on clinical course. Data on CVD prevalence in hospitalized COVID-19 patients and sequelae in survivors is limited. Aim of this prospective study carried out on consecutive unselected COVID-19 population, was to assess: 1) CVD occurrence among hospitalized COVID-19 patients, 2) persistence or new onset of CVD at one-month and one-year follow-up. METHODS: Over 30 days n = 152 COVID-19 patients underwent cardiovascular evaluation. Standard electrocardiogram (ECG), Troponin and echocardiography were integrated by further tests when indicated. Medical history, arterial blood gas, blood tests, chest computed tomography and treatment were recorded. CVD was defined as the occurrence of a new condition during the hospitalization for COVID-19. Survivors attended a one-month follow-up visit and a one-year telephone follow-up. RESULTS: Forty-two patients (28%) experienced a wide spectrum of CVD with acute myocarditis being the most frequent. Death occurred in 32 patients (21%) and more frequently in patients who developed CVD (p = 0.032). After adjustment for confounders, CVD was independently associated with death occurrence. At one-month follow-up visit, 7 patients (9%) presented persistent or delayed CVD. At one-year telephone follow-up, 57 patients (48%) reported persistent symptoms. CONCLUSION: Cardiovascular evaluation in COVID-19 patients is crucial since the occurrence of CVD in hospitalized COVID-19 patients is common (28%), requires specific treatment and increases the risk of in-hospital mortality. Persistence or delayed presentation of CVD at 1-month (9%) and persistent symptoms at 1-year follow-up (48%) suggest the need for monitoring COVID-19 survivors.

Arterial and venous thrombosis in coronavirus 2019 disease (Covid-19): relationship with mortality.

BACKGROUND: Patients with coronavirus disease 2019 (Covid-19) may experience venous thrombosis while data regarding arterial thrombosis are sparse. METHODS: Prospective multicenter study in 5 hospitals including 373 patients with Covid-19-related pneumonia. Demographic data, laboratory findings including coagulation tests and comorbidities were reported. During the follow-up any arterial or venous thrombotic events and death were registered. RESULTS: Among 373 patients, 75 (20%) had a thrombotic event and 75 (20%) died. Thrombotic events included 41 venous thromboembolism and 34 arterial thrombosis. Age, cardiovascular disease, intensive care unit treatment, white blood cells, D-dimer, albumin and troponin blood levels were associated with thrombotic events. In a multivariable regression logistic model, intensive care unit treatment (Odds Ratio [OR]: 6.0; 95% Confidence Interval [CI] 2.8-12.6; p < 0.001); coronary artery disease (OR: 2.4; 95% CI 1.4-5.0; p = 0.022); and albumin levels (OR: 0.49; 95% CI 0.28-0.87; p = 0.014) were associated with ischemic events. Age, sex, chronic obstructive pulmonary disease, diabetes, heart failure, coronary heart disease, intensive care unit treatment, in-hospital thrombotic events, D-dimer, C-reactive protein, troponin, and albumin levels were associated with mortality. A multivariable Cox regression analysis showed that in-hospital thrombotic events (hazard ratio [HR]: 2.72; 95% CI 1.59-4.65; p < 0.001), age (HR: 1.035; 95% CI 1.014-1.057; p = 0.001), and albumin (HR: 0.447; 95% CI 0.277-0.723; p = 0.001) predicted morality. CONCLUSIONS: Covid-19 patients experience an equipollent rate of venous and arterial thrombotic events, that are associated with poor survival. Early identification and appropriate treatment of Covid-19 patients at risk of thrombosis may improve prognosis.

The role of teicoplanin in the treatment of SARS-CoV-2 infection: A retrospective study in critically ill COVID-19 patients (Tei-COVID study).

Teicoplanin has a potential antiviral activity expressed against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and was suggested as a complementary option to treat coronavirus disease 2019 (COVID-19) patients. In this multicentric, retrospective, observational research the aim was to evaluate the impact of teicoplanin on the course of COVID-19 in critically ill patients. Fifty-five patients with severe COVID-19, hospitalized in the intensive care units (ICUs) and treated with best available therapy were retrospectively analysed. Among them 34 patients were also treated with teicoplanin (Tei-COVID group), while 21 without teicoplanin (control group). Crude in-hospital Day-30 mortality was lower in Tei-COVID group (35.2%) than in control group (42.8%), however not reaching statistical significance (p = .654). No statistically significant differences in length of stay in the ICU were observed between Tei-COVID group and control group (p = .248). On Day 14 from the ICU hospitalization, viral clearance was achieved in 64.7% patients of Tei-COVID group and 57.1% of control group, without statistical difference. Serum C-reactive protein level was significantly reduced in Tei-COVID group compared to control group, but not other biochemical parameters. Finally, Gram-positive were the causative pathogens for 25% of BSIs in Tei-COVID group and for 70.6% in controls. No side effects related to teicoplanin use were observed. Despite several limitations require further research, in this study the use of teicoplanin is not associated with a significant improvement in outcomes analysed. The antiviral activity of teicoplanin against SARS-CoV-2, previously documented, is probably more effective at early clinical stages.

Malaria in an asylum seeker paediatric liver transplant recipient: diagnostic challenges for migrant population.

Transplanted patients are particularly exposed to a major risk of infectious diseases due to prolonged immunosuppressive treatment. Over the last decade, the growing migration flows and the transplant tourism have led to increasing infections caused by geographically restricted organisms. Malaria is an unusual event in organ transplant recipients than can be acquired primarily or reactivation following immunosuppression, by transfusion of blood products or through the transplanted organ. We report a rare case of Plasmodium falciparum infection in a liver transplanted two years-old African boy who presented to one Italian Asylum Seeker Center on May 2019. We outlined hereby diagnostic challenges, possible aetiologies of post-transplantation malaria and finally we summarized potential drug interactions between immunosuppressive agents and antimalarials. This report aims to increase the attention to newly arrived migrants, carefully evaluating patients coming from tropical areas and taking into consideration also rare tropical infections not endemic in final destination countries.

Acute Kidney Injury and COVID-19: A Picture from an Intensive Care Unit.

INTRODUCTION: Acute kidney injury (AKI) is a frequent complication in coronavirus disease 2019 (COVID-19) patients admitted to intensive care unit (ICU) for severe respiratory failure. The aim is to evaluate the rate of AKI, defined according to Kidney Disease: Improving Global Outcome guidelines, in a series of critical COVID-19 patients admitted to the ICU of a single tertiary teaching hospital. METHODS: From April to May 2020, all consecutive critically ill COVID-19 patients admitted to the ICU who did not meet exclusion criteria (length of ICU stay <48 h, ESRD requiring dialysis, and patients still hospitalized in ICU at the time of data analysis) were enrolled in this study. Patients were stratified according to the highest AKI stage attained during ICU stay. RESULTS: Sixty-one patients were included in the analysis. AKI was observed in 35/61 patients (57.4%): 25/35 episodes (71.4%) were observed within the first 7 days. AKI was classified as follows: 17.1% stage 1, 25.7% stage 2, and 57.2% stage 3. Fourteen out of 20 stage-3 patients required continuous renal replacement therapy (CRRT), mostly related to persistent oliguria. The overall ICU mortality was 68.9%, and it was higher in patients developing AKI if compared to no-AKI patients (p = 0.006). Renal function recovery of any grade was observed in 14 out of 35 AKI patients (40%). Among patients undergoing CRRT, 13 patients were still dialysis dependent at the time of death. CONCLUSION: In critical COVID-19 patients, ICU mortality is particularly high, especially in patients developing AKI. An accurate monitoring of renal function in early phases of respiratory failure should be ensured in order to timely apply any strategy aimed at limiting renal complications during ICU stay.

Von Willebrand factor with increased binding capacity is associated with reduced platelet aggregation but enhanced agglutination in COVID-19 patients: another COVID-19 paradox?

Patients with Coronavirus-associated disease-2019 (COVID-19) display alterations of the hemostatic system and the presence of a prothrombotic status frequently leading to vascular complications. However, the impact of COVID-19 on platelet activity, aggregation and agglutination still needs to be clarified. We measured total levels of von Willebrand factor (vWF) and vWF binding to the platelet glycoprotein (Gp) complex (GPIb-IX-V), in a cohort of COVID-19 patients admitted to the intensive care unit of our Institution. Moreover, we evaluated platelet aggregation in response to agonists (ADP, collagen, arachidonic acid) and platelet agglutination in response to ristocetin. We found that levels of vWF antigen and the active form of vWF binding to platelets (vWF:RCo), were markedly increased in these patients. These results were associated with higher agglutination rates induced by ristocetin, thereby indirectly indicating an increased capability of vWF to bind to platelets. Conversely, we found that platelet aggregation in response to both ADP and collagen was lower in COVID-19 patients compared to healthy volunteers. This study shows that COVID-19 is associated with increased vWF-induced platelet agglutination but reduced platelet responsivity to aggregation stimuli. Our findings have translational relevance since platelet adhesion to vWF may represent a marker to predict possible complications and better delineate therapeutic strategies in COVID-19 patients.

Ozone as adjuvant support in the treatment of COVID-19: A preliminary report of probiozovid trial.

The evaluation of new therapeutic resources against coronavirus disease 2019 (COVID-19) represents a priority in clinical research considering the minimal options currently available. To evaluate the adjuvant use of systemic oxygen-ozone administration in the early control of disease progression in patients with COVID-19 pneumonia. PROBIOZOVID is an ongoing, interventional, randomized, prospective, and double-arm trial enrolling patient with COVID-19 pneumonia. From a total of 85 patients screened, 28 were recruited. Patients were randomly divided into ozone-autohemotherapy group (14) and control group (14). The procedure consisted in a daily double-treatment with systemic Oxygen-ozone administration for 7 days. All patients were treated with ad interim best available therapy. The primary outcome was delta in the number of patients requiring orotracheal-intubation despite treatment. Secondary outcome was the difference of mortality between the two groups. Moreover, hematological parameters were compared before and after treatment. No differences in the characteristics between groups were observed at baseline. As a preliminary report we have observed that one patient for each group needed intubation and was transferred to ITU. No deaths were observed at 7-14 days of follow up. Thirty-day mortality was 8.3% for ozone group and 10% for controls. Ozone therapy did not significantly influence inflammation markers, hematology profile, and lymphocyte subpopulations of patients treated. Ozone therapy had an impact on the need for the ventilatory support, although did not reach statistical significance. Finally, no adverse events related to the use of ozone-autohemotherapy were reported. Preliminary results, although not showing statistically significant benefits of ozone on COVID-19, did not report any toxicity.