key: cord-0940500-d1n4dlla
authors: Falcone, Marco; Tiseo, Giusy; Barbieri, Greta; Galfo, Valentina; Russo, Alessandro; Virdis, Agostino; Forfori, Francesco; Corradi, Francesco; Guarracino, Fabio; Carrozzi, Laura; Celi, Alessandro; Santini, Massimo; Monzani, Fabio; De Marco, Salvatore; Pistello, Mauro; Danesi, Romano; Ghiadoni, Lorenzo; Alessio, Farcomeni; Menichetti, Francesco
title: Role of low-molecular weight heparin in hospitalized patients with SARS-CoV-2 pneumonia: a prospective observational study
date: 2020-11-19
journal: Open Forum Infect Dis
DOI: 10.1093/ofid/ofaa563
sha: e91718005de7b6b0aa0e00ac94677737ed32bfc2
doc_id: 940500
cord_uid: d1n4dlla

OBJECTIVES: To evaluate the impact of low molecular weight heparin (LMWH) on the outcome of patients with SARS-CoV-2 pneumonia. METHODS: Prospective observational study including consecutive patients with laboratory confirmed SARS-CoV-2 pneumonia admitted to the University Hospital of Pisa (4th March-30(th) April 2020). Demographic, clinical, and outcome data were collected. The primary endpoint was 30-day mortality. The secondary endpoint was a composite of death or severe ARDS. LMWH, hydroxychloroquine, doxycycline, macrolides, antiretrovirals, remdesivir, baricitinib, tocilizumab, and steroids were evaluated as treatment exposures of interest. First, a Cox-regression analysis, in which treatments were introduced as time-dependent variables, was performed to evaluate the association of exposures and outcomes. Then, a time-dependent Propensity-score (PS) was calculated and a PS-matching performed for each treatment variable. RESULTS: Among 315 patients with SARS-CoV-2 pneumonia, 70 (22.2%) died during hospital stay. The composite endpoint was achieved by 114 (36.2%) patients. Overall, 244 (77.5%) patients received LMWH, 238 (75.5%) hydroxychloroquine, 201 (63.8%) proteases inhibitors, 150 (47.6%) doxycycline, 141 (44.8%) steroids, 42 (13.3%) macrolides, 40 (12.7%) baricitinib, 13 (4.1%) tocilizumab, and 13 (4.1%) remdesivir. At multivariate analysis, LMWH was associated with a reduced risk of 30-day mortality (HR 0.36 [95% CI 0.21-0.6], p<0.001) and composite endpoint (HR 0.61 [95% CI 0.39-0.95], p=0.029). The PS-matched cohort of 55 couples confirmed the same results for both primary and secondary endpoint. CONCLUSIONS: This study suggests that LMWH might reduce the risk of in-hospital mortality and severe ARDS in Covid-19. Randomized controlled trials are warranted to confirm these preliminary findings.

Since its initial detection, the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) rapidly spread worldwide, causing more than 9 million cases of Coronavirus Disease-2019 (Covid-19) and 490000 deaths [1] . The mortality rate of hospitalized patients with Covid-19 ranges from 11.7 to 28.3% in published studies [2] [3] [4] , with the highest mortality reported in patients with acute respiratory distress syndrome (ARDS), septic shock and disseminated intravascular coagulopathy [5] [6] . The dramatic clinical impact of SARS-CoV-2 pneumonia has prompted the medical community to identify effective treatments before a vaccine is developed and made widely available. In the absence of reliable evidence from large-scale randomized clinical trials, there is great uncertainty about the effectiveness of several treatment options in patients with Covid-19. The scientific community interfaced with concerns about consistency and reliability of clinical data that have also lead to retraction of published studies [7] . Dexamethasone is the only therapeutic agent showing a benefit on the outcome of patients with Covid-19 in the RECOVERY randomized clinical trial [8] .

However, the reduction of 28-day mortality is higher in patients who received invasive mechanical ventilation and not confirmed in patients not receiving oxygen therapy.

Recruitment in randomized clinical trials has slowed down in some countries due to a reduction in the pandemic. Awaiting for results from randomized clinical trials, we aimed to explore the impact of different treatments, in particular of low molecular weight heparin (LMWH), on the outcome of hospitalized patients with SARS-CoV-2 pneumonia.

A c c e p t e d M a n u s c r i p t 5

This prospective observational study included patients consecutively admitted to the tertiary-care, University Hospital of Pisa, Italy from March 4 th to April 30 th , 2020. Patients with pneumonia and laboratory confirmed Covid-19, diagnosed by a positive SARS-CoV-2 real time-PCR test on a nasopharyngeal swab were included in the study. Patients' identification was performed in real time: a dedicated staff of research fellows identified patients with SARS-CoV-2 pneumonia as soon as they arrived at the Emergency Department, followed the patients during the hospital stay and collected all data prospectively without interfering with the therapeutic decisions. Epidemiological and demographic information, medical history, comorbidities, information on clinical symptoms at admission, treatments and interventions, including need for oxygen or invasive mechanical ventilation support, received during the hospital course were prospectively collected. Venous and arterial blood samples for standard biochemistry and arterial blood gas analysis were collected at the time of hospitalization and repeated according to clinical practice and depending on the patient clinical conditions. To assess comorbidity burden, the age adjusted Charlson Comorbidity Index was calculated [9] . The severity of disease at hospital admission was estimated by the Sequential Organ Failure Assessment (SOFA) score [10] . Development of moderate to severe ARDS was defined as the acute onset of hypoxemia, manifestations of pneumonia on chest computed tomography (CT) imaging of a non-cardiac origin, and a PaO 2 /FiO 2 ratio of less than 200 mmHg according to the Berlin Definition [11] . Major bleeding was defined as follows: fatal bleeding and/or symptomatic bleeding in a critical area or organ, such as intracranial, intraspinal, intraocular, retroperitoneal, intraarticular, or pericardial or intramuscular with compartment syndrome and/or bleeding causing a fall in hemoglobin level of 2 g/dL or more or leading to transfusion of two or more units of whole blood or red cells [12] . During the study period there was limited evidence for any anti-Covid-19 M a n u s c r i p t 6 treatment. A panel of experts of our hospital elaborated a guide for the management of Covid-19 patients, that was regularly updated according to any new release from scientific literature (Supplementary Material, Figure S1 ). The majority of Covid-19 patients were treated following indications of the above-mentioned guide. Drugs such as LMWH, dexamethasone, tocilizumab, baricitinib, remdesivir, and antivirals were prescribed only in patients who needed hospitalization of at least 1 day. LMWH dosage was classified as prophylactic if subcutaneous enoxaparin 40-60 mg daily was administered, or therapeutic if a subcutaneous enoxaparin dosage of 40-60 mg twice daily was used [13] . The decision to adopt standard prophylactic or therapeutic dosages was decided by the attending physician.

Remdesivir was allowed at our center as compassionate use in patients undergoing invasive mechanical ventilation (NCT04323761). Patients were followed-up until death or 30-day after hospital admission.

The primary objective of the study was to evaluate the impact of different treatments on the outcome of patients with SARS-CoV2 pneumonia. In particular, the research question was to evaluate whether LMWH is effective in reducing the risk of 30-day all-cause mortality (main outcome measure). The research question was framed before the data collection and the database creation. Other variables evaluated as potential confounders were defined a priori. Covariates that could be associated with outcome were chosen based on clinical judgement and on previous published studies [2, 4] : age, male sex, comorbidities assessed by Charlson Comorbidity Index, severity of disease evaluated by the SOFA score at admission, lymphocytes, platelets count, troponin values during the first 48 hours, D-dimer and PiO 2 /FiO 2 ratio on admission were considered as potential confounders and included in the Propensity score (PS) analysis (see below).

Sample size estimation was performed on the basis of previous studies showing a decreased mortality of severe Covid-19 patients with coagulopathy who received anticoagulant treatment [14] .

Assuming that 75% of patients would be treated with LMWH, with an overall mortality of 30% and an HR of 0.5, a sample size of 290 patients would guarantee a power of at least 80% to a Cox regression model when Type I error rate is fixed at 5%.

Continuous variables are reported as mean ± standard deviation and medians and interquartile ranges (IQRs) according to their distribution. The normality of distributions was assessed by the Kolmogorov-Smirnov test. Continuous variables were compared by the Student t-test or the Mann-Whitney U-test, as appropriate. Categorical data were expressed as frequency distributions and the chi-square test or Fisher exact test was used to determine if differences existed between groups.

According with the study objectives, we used two different methods to evaluate the impact of different treatments on both the primary and secondary endpoints. It shall be noted that in our data treatments are not necessarily started at baseline time for all patients, therefore there might be immortal time bias if this is not taken into account. First, we presented results from multivariate Cox regression analyses, where all treatments and interventions which were potentially associated to the outcome on univariate analysis (p < 0.1) were added as covariates in the model. In order to avoid A c c e p t e d M a n u s c r i p t 8 issues with immortal time bias, treatment status was always introduced as a time-dependent covariate. To confirm the study results, a logistic regression analysis was also performed, including all treatments and interventions which were potentially associated to the outcome. More specifically, hydroxychloroquine, LMWH, doxycycline, macrolides, proteases inhibitors, remdesivir, baricitinib, tocilizumab, steroids and non-invasive ventilation were included as covariates of the regression analysis.

Secondly, we performed a PS-matched analysis. The PS method attempts to balance treated and nontreated groups in order to reduce confounding by indication in observational designs, thereby creating a quasi-randomized experiment, according to reporting guidelines on PS analysis [15] . In order to deal with possible immortal time bias, we performed a time-dependent PS analysis according to Lu et al [16] . The PS were calculated with a multivariable Cox regression model for timeto-treatment. Covariates that could be associated with outcome or allocation to each treatment group (clinically relevant based on previous published studies [4] [5] [6] or chosen through univariate analyses) were used to generate the PS: age, male sex, Charlson Comorbidity Index, lymphocytes, platelets count, troponin value during the first 48 hours, PiO 2 /FiO 2 ratio on admission, all treatments including antiretroviral, remdesivir, steroids, hydroxychloroquine, doxycycline, macrolides, LMWH, baricitinib, tocilizumab, excluding the current treatment of interest.

Longitudinally PS-matched cohorts (1:1 matching ratio) were then built: each patient receiving the treatment of interest was matched with a patient among those who at the time of treatment were at risk, eligible, and not treated previously. Matching was based on the nearest-neighbor algorithm, without replacement, with a caliper of 1%. A caliper of 1% was fixed a priori as it was deemed as a compound difference that would not be clinically relevant. In order to assess if adequate matching was achieved, we evaluated standardized differences. We also tested (using two-sample T-tests or

Chi-squared tests, as appropriate) differences in confounders between the two groups after matching. Finally, we performed a Cox regression analysis with the matched cohort to test the A c c e p t e d M a n u s c r i p t 9 association between each treatment and primary and secondary outcome and reported the results as hazard ratio (HR) and 95% CI. As before, treatment was a time-dependent covariate to take into account the possibility of immortal time bias.

The variance inflation factor (VIF) value was calculated to control the influence of collinearity. We assumed lack of multicollinearity if all variables had a VIF value <2. Missing data was deemed missing at random, and dealt with through list-wise exclusion. Since the proportion of excluded patients was moderate (21%), we also performed a multiple imputation analysis alongside the complete case analyses. We used Fully Conditional Specification for Multivariate Imputations by Chained Equations, generating m=10 completed data sets. 

This observational study was conducted according to the principles stated in the Declaration of Helsinki and it conforms to standards currently applied in our country. The study was approved by the Comitato Etico Area Vasta Nord Ovest (Internal Review Board -IRB number 230320). The patient's informed consent was obtained.

Overall, 315 consecutive patients with SARS-CoV-2 pneumonia were included in the study. The Compared to patients who survived, non survivors were older, with higher median Charlson Comorbidity Index and SOFA score on admission ( Table 1) . Among laboratory findings, lymphopenia, thrombocytopenia and an increase in aspartate transaminase >3 upper limit of normality were more frequently detected in non-survivors. Higher levels of D-dimer and troponin were also significantly higher in non survivors ( Table 1) .

We analyzed the following treatment variables Table 2 shows the time (expressed in days) from the start of symptoms to the start of each treatment in survivors versus non survivors. None of the variables included in the multivariate models showed multicollinearity. Table 5 

Among 244 patients treated with LMWH, 187 (76.6%) received a prophylactic dosage and 57 (23.4%) a therapeutic dosage. Eleven patients (4.5%) developed major bleeding episodes: 3 had hematuria, 3 bleeding from the respiratory tract, 2 bleedings from the gastrointestinal tract, 2 hematoma needing embolization and 1 bleeding of the subclavian vein. All patients who developed a major bleeding received therapeutic dosages of LMWH. Three deaths were attributed to a major bleeding episode.

A c c e p t e d M a n u s c r i p t 12

Our study shows that after adjusting for age, sex, baseline comorbidities, degree of respiratory dysfunction, and the receipt of other treatments, LMWH resulted the only factor associated with reduced risk of developing severe ARDS and mortality.

Considering the observational nature of the study, this finding should be considered as hypothesis serving as basis for randomized controlled trials. Our study has some limitations. First, since it was monocentric, the results might be affected by local practice in the management of the Covid-19 infection; moreover, almost all patients (except for 4) were of Caucasian ethnicity, our findings cannot be generalized to other health care settings and/or populations. Thus, external validation of our results is needed. However, it should be considered that all patients underwent similar treatment and interventions, according to the approved internal guide for Covid-19 patients admitted to our hospital. Second, our study was not powered to analyze differences in subgroups of patients receiving treatments other than LMWH, and the limited number of patients assigned to some treatments (e.g. remdesivir, steroids, tocilizumab, baricitinib) might underestimate the role of these therapies in the management of Covid-19. Third, although criteria for ICU admission in our hospital were based on the degree of respiratory impairment expressed by FiO 2 /PO 2 ratio, elderly sick patients with ultimately fatal diseases were excluded from ICU admission and this may impact the interpretation of some interventions. Finally, the analysis on the beneficial effects of treatments should be interpreted cautiously, as it was not conducted on randomized groups and might be therefore affected by several measured and unmeasured confounding factors. Randomized, controlled trials are needed to confirm our preliminary findings.

The potential role of LMWH in patients with Covid-19 seems to be plausible and supported by recent studies. Occlusion and micro-thrombosis formation in pulmonary small vessels have been reported in dead patients with Covid-19, and typical microvascular platelet-rich thrombotic depositions has been also described in other tissues, such as the myocardium [17] . High rates of thromboembolic A c c e p t e d M a n u s c r i p t 13 events have been reported in patients with severe SARS-CoV-2 pneumonia admitted to ICUs [18] . This is in line with the demonstration of platelet activation and artery dysfunction in patients with community-acquired pneumonia [19] [20] [21] , but may also represent a specific pathogenetic finding in patients with SARS-CoV-2 pneumonia. As a matter of fact, the radiological demonstration of contiguity of filling defects to the parenchymal opacities suggests a link between the SARS-CoV-2induced lung inflammation and vascular occlusion [18] . Previous observations suggested the potential role of LMWH in Covid-19. In a retrospective study including 449 patients with severe Covid-19 in China (99 of which received heparin) it was found that 28-day mortality of heparin users was lower than that of non-users in patients with D-dimer levels more than six-fold the upper limit of normal and sepsis-induced coagulopathy score ≥4 [14] ; the major limitations of this report are the small sample size, the retrospective design and the fact that some patients were still hospitalized at the time of manuscript submission (thus outcome cannot be definitively assessed) [14] . More recently, an observational study investigated a large cohort of 2,773 Covid-19 patients hospitalized within the Mount Sinai Health System in New York City: in patients who required mechanical ventilation the in-hospital mortality was 29.1% with a median survival of 21 days for those treated with anticoagulant therapy as compared to 62.7% with a median survival of 9 days in patients who did not receive anticoagulant treatment. In the multivariate model, duration of anticoagulant treatment was associated with a reduced risk of mortality (HR of 0.86 per day; 95% CI 0.82-0.89; p< 0.001) [22] . The International Society of Thrombosis and Haemostasis suggests to use LMWH at prophylactic dosages in all patients with three-to four-fold increase in D-dimer value, prolonged Prothrombin time, fibrinogen levels <2.0 g/L and platelet count <100 x 10 9 /L [23] . Of interest, a recent paper found that among Covid-19 patients the development of clinically significant thrombosis was associated with abnormal thromboelastographic (TEG) parameters [24] . TEG results outside reference ranges were detected in the 62% of thrombosis events, suggesting that TEG may be useful in accurately identifying patients at increased thrombosis risk and thereby necessitating anticoagulation [24] .

A c c e p t e d M a n u s c r i p t 14 Anyway, as for other diseases with high prevalence in older population, the prescription of an anticoagulation treatment should be weighed against the risk of bleeding. This is particularly important because we observed several cases of major bleeding and 3 subsequent deaths were directly attributed to the consequences of anticoagulant therapy. Therefore, it is crucial to identify clinical or laboratory parameters able to select those patients who could receive prophylactic vs therapeutic dosages of anticoagulant therapy, thus minimizing the risk of bleeding. Ongoing multicentre, randomized, controlled trials will be able to address this question (NCT04372589, NCT04367831, NCT04345848, and NCT04366960).

In conclusion, modulating the activation of the coagulopathy pathways, LMWH may be beneficial in patients with Covid-19. Randomized clinical trials are warranted to confirm these preliminary results and identify the most adequate LMWH dosage.

A c c e p t e d M a n u s c r i p t 15 A c c e p t e d M a n u s c r i p t 20 

FM has participated in advisory boards and/or received speaker honoraria from Angelini

Acknowledgments: None Contribution: MF, FM designed the study; GT created the CRF, developed the database, analyzed and interpreted data; GT and GB coordinated the data collection; GB and VG recruited patients and collected clinical data

MF, FM wrote the study; AF performed the statistical analysis and PSmatching

LG revised and contributed to the critical revision of the final manuscript. REFERENCES 1. World Health Organization

Last accessed 01st

Predictors of mortality for patients with COVID-19 pneumonia caused by SARS-CoV-2: a prospective cohort study

Presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with COVID-19 in the New York City area

Prediction for progression risk in patients with COVID-19 pneumonia: the CALL Score

Clinical predictors of mortality due to COVID-19 based on an analysis of data of 150 patients from Wuhan, China

Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in Wuhan, China

Pharmacologic Treatments for Coronavirus Disease 2019 (COVID-19): A Review

Dexamethasone in Hospitalized Patients with Covid-19 -Preliminary Report

A new method of classifying prognostic comorbidity in longitudinal studies: development and validation

The SOFA (Sepsis-related organ failure assessment) score to describe organ dysfunction/failure

Acute respiratory distress syndrome: the Berlin Definition

Subcommittee on control of anticoagulation of the scientific and standardization committee of the International Society on Thrombosis and Haemostasis. Definition of major bleeding in clinical investigations of antihemostatic medicinal products in non-surgical patients

Guidance for the practical management of the heparin anticoagulants in the treatment of the venous thromboembolism

Anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy

Comparing Treatment Effects between propensity scores and randomized controlled trials: improving conduct and reporting

Propensity score matching with time-dependent covariates

Pulmonary and cardiac pathology in African American patients with COVID-19: an autopsy series from New Orleans

High rate of pulmonary thromboembolism in patients with SARS-CoV-2 pneumonia

Platelet activation is associated with myocardial infarction in patients with pneumonia

Cardiovascular complications and short-term mortality risk in community-acquired pneumonia

Lower mortality rate in elderly patients with community-onset pneumonia on treatment with aspirin

Association of treatment dose anticoagulation with in-hospital survival among hospitalized patients with COVID-19

ISTH DIC subcommittee communication on anticoagulation in COVID-19

Thromboelastographic results and Hypercoagulability syndrome in patients with Coronavirus Disease 2019 who are critically ill

A c c e p t e d M a n u s c r i p t 24