key: cord-1029737-l12ukn0u
authors: Belli, Luca S.; Fondevila, Constantino; Cortesi, Paolo A.; Conti, Sara; Karam, Vincent; Adam, Rene; Coilly, Audrey; Ericzon, Bo Goran; Loinaz, Carmelo; Cuervas-Mons, Valentin; Zambelli, Marco; Llado, Laura; Diaz, Fernando; Invernizzi, Federica; Patrono, Damiano; Faitot, Francois; Bhooori, Sherrie; Pirenne, Jacques; Perricone, Giovanni; Magini, Giulia; Castells, LLuis; Detry, Oliver; Cruchaga, Pablo Mart; Colmenero, Jordi; Berrevoet, Frederick; Rodriguez, Gonzalo; Ysebaert, Dirk; Radenne, Sylvie; Metselaar, Herold; Morelli, Cristina; De Carlis, Luciano; Polak, Wojciech G.; Duvoux, Christophe
title: Protective role of tacrolimus, deleterious role of age and comorbidities in liver transplant recipients with Covid-19: results from the ELITA/ELTR multi-center European study.
date: 2020-12-09
journal: Gastroenterology
DOI: 10.1053/j.gastro.2020.11.045
sha: a8c237fc919818f36811c81853bdc3f98dcf4f59
doc_id: 1029737
cord_uid: l12ukn0u

Background and aims Despite concerns that liver transplant (LT) recipients may be at increased risk of unfavorable outcomes from COVID-19 due the high prevalence of co-morbidities, immunosuppression and ageing, a detailed analysis of their effects in large studies is lacking Methods Data from adult LT recipients with laboratory confirmed SARS-CoV2 infection were collected across Europe. All consecutive patients with symptoms were included in the analysis, Results Between March 1st and June 27th2020, data from 243 adult symptomatic cases from 36 centers and 9 countries were collected. Thirty-nine (16%) were managed as outpatients while 204 (84%) required hospitalization including admission to the ICU (39/204, 19.1%). Forty-nine (20.2%) patients died after a median of 13.5 (10-23) days, respiratory failure was the major cause. After multivariable Cox regression analysis, age > 70 (HR 4.16; 95%CI 1.78-9.73) had a negative effect and tacrolimus (TAC) use (HR 0.55; 95%CI 0.31-0.99) had a positive independent effect on survival. The role of co-morbidities was strongly influenced by the dominant effect of age where comorbidities increased with the increasing age of the recipients. In a second model excluding age, both diabetes (HR 1.95; 95%CI 1.06 - 3.58) and chronic kidney disease (HR 1.97; 95%CI 1.05 - 3.67) emerged as associated with death Conclusions Twenty-five per cent of patients requiring hospitalization for Covid-19 died, the risk being higher in patients older than 70 and with medical co-morbidities, such as impaired renal function and diabetes. Conversely, the use of TAC was associated with a better survival thus encouraging clinicians to keep TAC at the usual dose.

The current COVID-19 pandemic has presented unforeseen challenges to health care systems worldwide with several issues remaining unmet. To date, firm knowledge on disease evolution, risk factors and optimal management in specific categories of patients is lacking. All transplant recipients are potentially vulnerable to SARS-CoV-2 infection with immune suppression, aging and metabolic or cardiovascular co-morbidities likely being risk factors for symptomatic disease and its severe complications (2) . Liver Transplant (LT) patients in particular, represent one of the largest immunosuppressed cohorts in Europe with 102.116 alive recipients being reported in the European Liver Transplant Registry (ELTR), 42 .432 (41.6%) of whom are in their sixties and 12.669 in J o u r n a l P r e -p r o o f their seventies or older (3) . At present, available data related to COVID-19 in LT patients is limited to a small number of case series (4) (5) (6) , to preliminary reports from 2 international registries (7) (8) (9) and to a single international prospective cohort on 57 cases (10) . All authors agreed that greater case numbers were urgently required to accurately improve our understanding of individual risk in LT recipients. Thus, a large-scale collaborative study promoted by the European Liver Transplant Association (ELITA) and European Liver Transplant Registry (ELTR) was performed, the main aim being the search for risk factors associated with mortality during the COVID-19 pandemic and with a specific focus on comorbidities and immunosuppression

ELITA called for a COVID-19 study which was circulated on March 30, 2020 among 149 LT centres affiliated to ELTR) and located in 30 European countries. All centres that reported at least one case were provided with a database and instructions on how to record structured data. Data collection was managed by ELTR. One hundred and fourteen centres (76.5%) responded, with 56 centres (38%) having observed COVID-19 cases in adult LT recipients between March 1st and May 19th, 2020. All patients with symptoms and having SARS-CoV-2 infection confirmed by a positive result on a reverse-transcriptase-polymerase-chain-reaction (RT-PCR) assay of a specimen collected on a nasopharyngeal swab or on broncho alveolar lavage, were included in the study.

Demographic and clinical data, including clinical symptoms or signs at presentation, laboratory and radiologic results during COVID-19 management as well as administered antiviral therapies and anti-thrombotic prophylaxis were retrospectively collected. All laboratory tests and radiologic assessments were performed on the discretion of the treating physician discretion. Serum creatinine was converted to mg/dl for analysis. Information on baseline immunosuppression and on changes during Covid-19, namely reduction or discontinuation, were also obtained. Obesity was defined as a given BMI of >30 kg/m2. Liver injury during Covid-19 was defined as alanine amino-transferase (ALT) level > 30 IU/L for male and 19 IU/L for female in those patients with normal ALT levels at last outpatient visit. 14 Hepatic flare was defined as ALT level ≥5 x upper limit of normality. The time on study started at occurrence of COVID symptoms. All submitted files from each centre were manually reviewed to assess for data quality, completeness and inconsistencies. In addition, submitting clinicians were contacted and asked to provide corrections or data integration whenever needed.

J o u r n a l P r e -p r o o f Data was collected in accordance with General Data Protection Regulation (GDPR), the European Union legislation and the ELTR privacy policy.

Analysis was led by the Research Centre on Public Health (CESP), University of Milan-Bicocca, Monza, Italy. A descriptive analysis of the cohort was carried out on the overall population and after stratifying the population by site of management: at home, in general wards or in intensive care units (ICU). Categorical variables were summarized through percentages, while continuous variables through median, first quartile (Q1) and third quartile (Q3). Categorical variables were compared using the χ2 or the Fisher's exact tests; continuous variables were compared using the Mann-Whitney U-test or the Kruskall-Wallis test, when appropriate. All tests were twosided and used a significance level of 0.05.

The rates of missing data for each variable were reported. For each patient, the time between the date of COVID symptoms and death or end of follow-up was computed, and the association between mortality and baseline patients' characteristics was evaluated through univariate Cox proportional hazard models. All characteristic analyzed in univariate model were included in a stepwise selection process that identified the best multivariate model. The same process was repeated after excluding age from potential predictors. Given the exploratory nature of the study and the limited sample size, a 0.1 significance level was established to retain predictors in the final multivariate models possibly favoring the tracing of borderline significant associations that could be the basis for further studies on wider samples. All statistical analyses were conducted using SAS version 9.4 (The SAS institute, Cary, NC) and R version 4.0.0 (R Core Team, Vienna, Austria). The map was drawn using QGIS software version 3.10 (QGIS Development Team).

The COVID-19 pandemic was experienced not uniformly in Europe, with large areas being spared. This explains why of the 111 centers responding to the ELITA/ELTR call, only 36 centers from nine European countries observed at least one patient with PCR confirmed SARS-CoV-2 infection (Fig 1 and Fig 2) . Of the 29.981 alive patients in regular follow up at the participating centers, 258 (0.9%) have been consecutively reported in the Registry. Eleven of them (4.3%) were asymptomatic at the time of diagnosis, the PCR test being performed according to surveillance protocols in case of contact with a SARS-CoV-2 positive subject; these patients were excluded from the study. Four additional patients were excluded because aged < 18 years. The remaining 243 symptomatic cases were considered for statistical analysis with 39 patients (16%) receiving homecare, the remaining 204 J o u r n a l P r e -p r o o f requiring hospitalization (Fig 2) . Of these, 167 (68.7%) patients were treated in a general ward and 37 in intensive care units. Baseline patient characteristics are reported in Table 1 . Thirty-two LT recipients with Covid-10 analyzed in this study were also included in the report from Becchetti et al (10) .

One hundred-eleven (45.7%) patients had arterial hypertension, 94 (38.7%) diabetes mellitus, 49 (20.2%) chronic kidney disease with a creatinine > 2mg/dL and 25 (10.3%) chronic lung diseases. Concurrent comorbidities were frequent with 107 (44%) patients having two or more ( Table 1 ). The prevalence of at least 2 co-morbidities increased with age being observed in 25.3%, 53.4% and 64.2% in recipient aged < 60, from 60 to 70 or > 70 years, respectively.

Tacrolimus (TAC) and cyclosporinne (CsA) were considered as the main immunosuppressive drugs. Since, some of the patients were off CNI, the proportion of patients receiving each immunosuppressive drug or combination of drugs were also obtained. At the time of analysis, 162 (66.7%) patients were on tacrolimus (TAC), alone or in combination, 29 (11.9%) on Cyclosporine A (CsA) alone or in combination, 119 (49.0%) on mycofenolatemofetil (MMF) alone or in combination and 37 (15.2%) on mTOR inhibitors alone or in combination. (Table 1) .

At the time of diagnosis, the most commonly self-reported symptoms included fever (190 patients, 78.2%), cough (143 patients, 58.8%), dyspnea (82 patients, 33.7%), muscle pain or asthenia (90 patients, 37.0%), anosmia or dysgeusia (21 patients, 8.6%) and diarrhea (55 patients, 22.6%). Radiological findings, either on CT scan or on chest radiography, showed typical ground glass opacities in 145 cases (59.7%) ( Table 2 ). Overall 137 (56.4%) patients required respiratory support during hospitalization with 26 requiring non-invasive ventilation and 25 mechanical ventilation ( Table 2 ). One hundred forty-nine patients received specific anti-SARS-CoV-2 treatment: 116 (47.7%) patients were treated with hydroxy-chloroquine either alone or in combination, 41 (16.9%) with lopinavir-ritonavir; 34 (14.0%) with high doses of corticosteroids and 15 (6.2%) with tocilizumab.

Thrombo-prophylaxis, mainly with low molecular weight heparin, was started on COVID-19 diagnosis in 117 patients (48.2%). Seven hospitalized patients (7/204=3.4%) experienced thrombotic events, 3 pulmonary embolism, 2 deep vein thrombosis and 2 strokes. An acute liver injury was observed in 56 patients with previous persistently normal ALT, being in the flare range in 10 cases Three patients were reported as having acute rejection.

Notably, CNI had been withdrawn in 2 cases and the dose of TOR dose had been halved in the third case. Baseline patients-characteristics of patients with less severe symptoms who could be treated at home and those with more severe symptoms requiring hospitalization in general wards and ICU are reported in Table 2 . Patients treated at home were younger, had less co-morbidities and were more frequently receiving TAC as primary immunosuppressant. KM survival after stratification by place of management, at home, general ward or ICU is provided in Figure 3 : patients managed at home survived, while the probability of survival at 30 days was 93.1% (95% CI: 86.7 -96.5) and 57.0% (95% CI: 37.6 -72.4) respectively for patients in ward and in ICU, and it declined at 89.8% (95% CI: 82.1 -94.3) and 46.6% (95% CI: 26.2 -64.6) at 90 days. Notably, 12 patients with advanced Covid-19 disease were not admitted to ICU, 8 because deemed too sick for ICU due to a combination of advanced age and severe co-morbidities and four because ICU were overwhelmed.

Factors significantly associated with death by univariable analysis were the following: increased age of the recipient, time from LT, diabetes, chronic kidney disease, number of comorbidities and use of TAC (Table. 3). After multivariable analysis, advanced age (>70 yrs vs < 60yrs) remained independently associated with an increased mortality risk (HR 4.16; 95CI 1.78-9.73) while use of TAC was confirmed independently associated with a reduced mortality risk (HR 0.55; 95CI 0.31-0.99). The Kaplan-Meier survival curves stratified by age >70 or <70, and type of immunosuppressant, TAC vs non TAC, may be helpful for the clinician to better understand the individual risk. (supplementary Fig.1 ). Since the number of co-morbidities increased with the increasing age of the recipient, a second model excluding age was constructed. This allowed diabetes and chronic renal failure to emerge as predictors of mortality, their effect having been shadowed in the first model by the dominant effect of age (supplementary Table 1 ). The interplay among age of the recipient, primary immunosuppressant and chronic renal failure is shown in supplementary Table 2 Table 3 J o u r n a l P r e -p r o o f patients receiving TAC based vs non-TAC based regimens are compared with respect to some relevant clinical variables such as age, time from transplant, chronic renal failure, concurrent exposure to ACE, or ARB and presence of HCC. In fact, patients receiving TAC were younger and had less co-morbidities, these variables being potentially associated with a better outcome. Conversely patients on TAC were much less frequently treated with ACE or ARB inhibitors, this therapy being associated with a better outcome. All these variables were included in the multivariable analysis which confirmed the independent protective role of TAC.

As more than 200 countries world-wide are still struggling with the COVID-19 pandemic, all solid organ transplant recipients are at risk of infection and poor outcome due to chronic immunosuppression, high rates of comorbidities, advanced age and frequent hospitalization. We have analyzed the characteristics, management and outcome of a large multinational European cohort of liver transplant recipients with symptomatic SARS-

Rates of hospitalization and death in the current study were 85% and 20.2%, confirming what has already shown in our preliminary report on the first 103 cases (8) where some patients were still experiencing their disease course. These findings concur with the 23% mortality risk reported by Webb et al (7), however compare unfavorably with the 12% mortality risk observed by Becchetti et al (10) , possibly due to the lower percentage of patients requiring hospitalization in this latter study. Our study confirmed that abdominal symptoms and more specifically diarrhea is at least twice more frequent than in the general population (10) and it is possibly associated to MMF. This hypothesis is supported by the fact that almost 50% of the 26 patients maintained on MMF as primary immunosuppressant had diarrhea as presenting symptom. Clinicians should therefore be vigilant and consider SARS-CoV2-testing in transplanted patients presenting with diarrhea particularly if using MMF.

However, the main finding of the present study is the significant variation in mortality risk with both age of the recipients and use of TAC as immunosuppressant. The role of advanced age confirms what has been extensively observed in the general population, with patients older than 70 having an increased four-fold mortality risk (1, (11) (12) (13) . The lower risk of death for patients maintained on TAC was unexpected and had not been previously reported. In particular Becchetti et al. (10) could not explore this association in their prospective cohort of 57 LT recipients with Covid-19, as the great majority of their patients were receiving tacrolimus. Notably, in our analysis, the beneficial impact of TAC was robust and persisted after controlling for various confounders. The biologi-J o u r n a l P r e -p r o o f cal explanation of the potential favorable role of TAC is unknown but may be dual, inhibition of the viral replication and interaction with the immune response. Some studies have shown that Coronavirus replication (CoV), depends on active immunophyllin pathways and TAC is capable to strongly inhibit the growth of some human coronavirus, notably SARS COV1, probably by binding the immunophyllin FKBP although not specifically SARS -CoV-2 (14) (15) (16) . Another potential driver of the TAC protective effect could be related to the immunosuppressive property of this CNI (17) . By inhibiting calcineurin and suppressing the early phase of T-cell activation, TAC reduces the production of many cytokines, notably pro inflammatory cytokines, as TNFα and IFNγ, and possibly The role of co-morbidities as relevant risk factors for mortality has been clearly demonstrated in the general population with Covid-19 (18) . Despite being highly prevalent among liver transplant recipients (19) , neither a specific comorbidity nor their combination, emerged as independently associated with outcome. This is at least in part explained by the dominant effect of age as comorbidities increased with the increasing age of the recipients. Nevertheless, in our exploratory analysis, chronic renal failure defined by a serum creatinine greater than 2 mg/dL, maintained a trend of significance (p <0.1) even if shadowed by the dominant effect of increasing age.

Notably, the negative impact of renal failure on survival was particularly relevant in patients who are not receiving Tacrolimus, once again pointing to its possible protective role against Covid-19, at least in liver transplant recipients. Some limitations are also to be acknowledged. Firstly, although we attempted to collect data on major covariables there remains the possibility of missing confounders. Secondly, we focused on symptomatic cases with confirmed positive SARS-CoV-2 PCR test despite test sensitivity below 80%. Thus, some cases were excluded.

In conclusion, this study including more than 240 liver transplant recipients confirmed that 25% of patients requiring hospitalization for Covid-19 died, the mortality risk being greater in patients older than 70 and with medical co-morbidities, such as impaired renal function and diabetes. Conversely, the use of TAC was associated with an increased survival probability. Although the biological explanation of this latter finding is currently unknown, our preliminary evidence should encourage clinicians to keep TAC at the usual dose as it may be beneficial when treating COVID-19. A more precise estimate of the protective effect of TAC requires studies on larger cohorts of transplants. 

BACKGROUND AND CONTEXT Few studies have analyzed the impact of Cocid-19 in liver transplant recipients and the association of co-morbidities, immunosuppression and ageing on the mortality risk.

NEW FINDINGS Age > 70 and tacrolimus use had respectively a negative and a positive independent effect on survival. The role of co-morbidities was strongly influenced by the dominant effect of age as the number of comorbidities increased with the increasing age of the recipients.

Although we attempted to collect data on major co-variables there remains the possibility of missing confounders.

Thees findings should encourage clinicians to keep Tacrolimus at the usual dose as it may be beneficial when treating COVID-19.

In liver transplant recipients with Covid-19, tacrolimus use had a positive independent effect on survival. This novel finding should encourage clinicians to keep Tacrolimus at the usual dose as it may be beneficial when treating COVID-19.

J o u r n a l P r e -p r o o f Tables   Table 1. J o u r n a l P r e -p r o o f 

Coronavirus Disease (COVID-19) Pandemic. World Health 284 Organization

Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: A retrospective cohort study

European Liver Transplant Registry (ELTR)

COVID-19 in long-term liver transplant patients: preliminary experience from an Italian transplant centre in Lombardy

COVID-19 in solid organ transplant recipients: a singlecenter case series from Spain

COVID-19 in solid organ transplant recipients: initial report from the US EpiCenter

Determining risk factors for mortality in liver transplant patients with COVID-19

COVID-19 in liver transplant recipients:317 preliminary data from the ELITA/ELTR registry

Impact of COVID-19 on liver transplantation in Europe: alert from an early survey of European Liver and Intestine Transplantation Association and European Liver Transplant Registry

COVID-19 in an international European liver transplant recipient cohort

Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China

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

Risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease 2019 pneumonia in Wuhan, China

Replication of human coronaviruses SARS-CoV, HCoVNL63 and HCoV-229E is inhibited by the drug FK506

Associations between immune-suppressive and stimulating drugs and novel COVID-19-a systematic review of current evidence. Ecancer medical science

Suppression of coronavirus replication by cyclophilin inhibitors

COVID-19 and Calcineurin Inhibitors: Should They Get Left Out in the Storm?

Comorbidity and its impact on 1590 patients with COVID-19 in China: a nationwide analysis

Time varying mpact of comorbidities on mortality after liver transplantation: a national cohort study using linked clinical and administrative data

Dexamethasone in Hospitalized Patients with Covid-19 -Preliminary

Remdesivir for the Treatment of Covid-19 -Preliminary Report

Anne-Catherine Saouli 10. University Hospital Copenhagen, Department for Surgery and Transplantation Rigshospitalet

Emmanuel Gonzales 17. The Queen Elizabeth Hospital

Department of Hepatology, Hepato-pancreaticbiliary Surgery and Liver Transplantation

Stefania Petruccelli 26

Sonia Pascual 31. Hospital Clinic I Provincial De Barcelona

Liver Unit (Lluís Castells, Isabel Campos-Varela) and Liver Transplant Unit