key: cord-307044-4czeehkq
authors: Liu, Jiaye; Wang, Tingyan; Cai, Qingxian; Sun, Liqin; Huang, Deliang; Zhou, Guangde; He, Qing; Wang, Fu‐Sheng; Liu, Lei; Chen, Jun
title: Longitudinal Changes of Liver Function and Hepatitis B Reactivation in COVID‐19 Patients with Pre‐existing Chronic HBV Infection
date: 2020-08-06
journal: Hepatol Res
DOI: 10.1111/hepr.13553
sha: 
doc_id: 307044
cord_uid: 4czeehkq

AIM: With pandemic of COVID‐19 currently and high endemic of chronic HBV infection worldwide, it is quite urgent to investigate liver function changes of COVID‐19 patients with chronic HBV infection, and how SARS‐CoV‐2 infection in turn affects the course of chronic HBV infection. METHOD: We conducted a retrospective study based on 347 COVID‐19 patients (21 vs. 326 with vs. without chronic HBV infection). With the PSM method, we yielded 20 and 51 matched patients for HBV group and non‐HBV group, respectively. RESULTS: At the end of follow‐up, all these 71 patients achieved SARS‐CoV‐2 clearance (p=0.1). During the follow‐up, 30% vs. 31.4% in HBV group vs. non‐HBV group progressed to severe COVID‐19 (p=0.97). After PSM, the longitudinal changes of median values for liver biochemistries were no significant difference between two groups. In HBV group vs. non‐HBV‐group, 35% (7/20) vs. 37.25% (19/51) (p = 0.86) had abnormal ALT at least once during hospitalization, while 30% (6/20) vs. 31.37% (16/51) for abnormal AST (p = 0.91), 40% (8/20) vs. 37.25% (19/51) for abnormal GGT (p = 0.83), and 45% (9/20) vs. 39.22% (20/51) for abnormal TBIL (p = 0.91). Moreover, 3 patients in HBV group had hepatitis B reactivation. CONCLUSIONS: Liver dysfunction presented in COVID‐19 patients with/without chronic HBV. Moreover, those COVID‐19 patients coinfected with chronic HBV could had a risk of hepatitis B reactivation. It is necessary to monitor liver function of COVID‐19 patients, as well as HBV DNA levels for those coinfected with HBV during the whole disease course.

Coronavirus disease 2019 (COVID-19), an emerging respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has recently become a pandemic. A total of 14,348,858 confirmed cases and 603,691 deaths were reported globally as of July 20, 2020. 1 Unfortunately, neither targeted drugs nor vaccines are available to date, and the number of infections is growing around the world. For the foreseeable future, COVID-19 could constantly pose a great threat to the people of the world.

COVID-19 is typically characterized by the symptoms of viral pneumonia such as fever, fatigue, dry cough and anosmia, which may evolve to respiratory failure. Unexpectedly, there is increasing evidence that some individuals with COVID-19 have frequent abnormal liver function. [2] [3] [4] It was observed that the elevated liver biochemistries were more common in severe COVID-19 cases than in mild cases. 5 The limited autopsy results of liver of COVID-19 cases showed moderate microvascular steatosis, 6 or hepatocyte degeneration accompanied by lobular focal necrosis and neutrophil infiltration. 7 These histological characteristics of liver were not specific manifestations of liver damage caused by SARS-CoV-2. Therefore, it is unclear currently whether liver damage/dysfunction of COVID-19 patients is mainly due to the SARS-CoV-2 infection or other coexisting conditions. Hepatitis B remains another major worldwide public health problem, with approximately 257 million individuals infected with hepatitis B virus (HBV), and more than 94 million suffer from chronic hepatitis B (CHB). 8 A large cohort study from China reported that 2.1% (21/1 099) of enrolled COVID-19 cases had pre-existing hepatitis B. 9 Given the endemic and high burden of HBV infection, CHB may be one important comorbidity of pre-existing liver diseases affecting the outcome of COVID-19. It is confirmed that HBV infection can cause the damage of innate immune responses and imbalance of adaptive immune responses. 10 Meanwhile, uncontrolled inflammatory innate responses and impaired adaptive immune responses causing by SARS-CoV-2 may lead to harmful tissue damage, both locally and systemically. 11 Therefore, coinfection of SARS-CoV-2 and HBV may increase the damage of immune function and liver.

However, to the best of our knowledge, no studies had been carried out on the impact of chronic HBV infection on the disease progression and liver function changes of COVID-19 patients, and how the SARS-CoV-2 infection in turn affects the course of chronic HBV infection.

More evidence is urgently needed to guide the screening of HBV coinfection and management of comorbidity of CHB during the pandemic of COVID-19. Hence, in this study we aimed to assess the independent effect of HBV infection on the outcomes of COVID-19 as well as the progression of HBV infection.

All diagnosed COVID-19 patients according to WHO interim guidance, with or without chronic HBV infection, who admitted to Shenzhen Third people's Hospital during January 1 to March 1, 2020, were enrolled. Chronic HBV infection was determined on the basis of testing positive for HBsAg and/or HBV DNA at hospital admission and medical history of chronic HBV infection. The clinical outcomes of COVID-19 and dynamics of liver biochemistries were monitored up to April 12, 2020, the final date of follow-up. The inclusion criteria were as follows: (1) subjects diagnosed with COVID-19, (2) the records were well documented, (3) subjects with longitudinal follow-up, i.e., liver function testing, chest computed tomography (CT) scan, or blood gas assay with at least across two days. The exclusion criteria were: (1) subjects without data available at baseline, i.e., blood routine examinations, liver biochemistries, CT score, 12 blood gas assay, (2) subjects coinfected with human immunodeficiency virus, (3) subjects coinfected with hepatitis virus other than HBV, or had liver diseases other than CHB. The process of patients' enrolment was presented in 

Baseline was defined as the first time of hospital admission due to COVID-19. At baseline and during follow-up, all subjects included in this study underwent routine examination, monitoring of liver biochemistries, and SARS-CoV-2 nucleic acid testing with a median follow-up interval of 3 days.

The primary outcome was progression to severe COVID-19, and the secondary outcomes included clearance of SARS-CoV-2, liver injury, and hepatitis B reactivation. The time point of COVID-19 onset was defined as the day of symptoms presence self-reported by patients who were further confirmed with SARS-CoV-2 after hospital admission. The virus clearance was defined by the presence of two consecutive negative results in quantitative PCR detection for SARS-CoV-2 RNA at an interval of 24 hours, and the day of the first one of these two tests were considered as the clearance day. Patients were discharged from hospital after the clearance of SARS-CoV-2. Hepatitis B reactivation was defined as the abrupt reappearance of HBV DNA viremia in a patient with previously inactive or resolved HBV infection, or an sudden and rapid rise of HBV DNA level by at least 2 log10 in those with previously detectable. 13 According to the national guidelines for community-acquired pneumonia, and the diagnosis and treatment plan for the new coronavirus in China, all COVID-19 patients were classified into severe or mild cases based on chest radiography, clinical examinations, and symptoms. 14, 15 We analyzed the dynamics of liver biochemistry indicators (i.e., ALT, AST, total bilirubin

[TBIL], gamma-glutamyl transferase [GGT]) to investigate the liver function changes. The normal range of ALT, AST, GGT and TBIL in this study was 0-45 U/L, 0-45 U/L, 0-49 U/L, and 1.7-21 umol/L, respectively. As one patient coinfected with HBV was underwent liver biopsy, we investigated the pathological characteristics of liver injury of this patient.

All statistical analysis was conducted using R 3.6.1. We performed propensity score matching (PSM) on the selected 347 subjects so that the group coinfected with HBV is comparable to the group without HBV coinfection in terms of observed covariates at baseline. 16 The factors for propensity score calculation include age, gender, body mass index (BMI), time intervals between COVID-19 onset to hospital admission, number of comorbidities except for CHB, liver biochemistries (ALT, AST, GGT, TBIL), PaO2/FIO2 ratio, chest CT score, CRP, lymphocyte count, and platelet count at baseline. The PSM process was conducted by using R package MatchIt, with nearest-neighbour method, 1:3 matching ratio, and a caliper size of 0.1.

For baseline characteristics, we used median (interquartile range [IQR]) for continuous variables and Wilcoxon test for comparison, while we reported count (percentage) for categorical variables and Fisher exact test for comparison.

We used Kaplan-Meier (K-M) method to estimate cumulative probabilities for the clearance of SARS-CoV-2 and progression to severe COVID-19 and compared the probabilities between HBV group and non-HBV group using a log-rank test. We use multivariable Cox proportionalhazards model to compare the risk of progression to severe COVID-19.

To investigate the longitudinal changes over time, we first performed comparison of the median values of liver biochemistries (ALT, AST, GGT, TBIL) over time between groups using Wilcoxon signed-rank test. Then we compared the values of these indicators between groups at each time point (to examine difference between groups) and compared the values of these indicators at each time point to their baseline values within each group (to examine difference within groups) using Wilcoxon test or Kruskal−Wallis test. In addition, we also reported the proportion of patients with abnormal values for liver biochemistries over time to examine the liver function changes, with using χ 2 test or Fisher exact test to compare the proportions between groups.

All significance tests performed were two-sided. P values less than 0.05 were deemed statistically significant and 95% confidence intervals (CIs) were calculated for point estimates.

A total of 347 COVID-19 patients with/without HBV coinfection (21 vs. 326) were analysed before matching. In HBV coinfection group, 20 were diagnosed as HBeAg-negative chronic HBV infection or HBeAg-negative CHB, and one patient had a pre-existing cirrhosis while did not receive any imaging examinations during the hospitalization of COVID-19. We described the details of history of HBV infection and antiviral treatment, virological and serological testing at baseline in Supplementary The propensity score matching of entire study population yielded 20 and 51 matched patients for HBV group and non-HBV group, and the covariates used for matching and not used for matching were comparable between the two groups after matching (Table 1 , all p > 0.1).

At the end of follow-up, all the patients in both groups achieved SARS-CoV-2 clearance and none of them died. The median time to SARS-CoV-2 clearance (21 days, 95% CI: 19-29) in HBV group was longer than that in non-HBV group (14 days, 95%CI: 13-21), however, no significant difference was observed regarding the probability of SARS-CoV-2 clearance over time between the two groups (p=0.1, Figure 1A ).

During the follow-up period, 30% (6/20) and 31.4% (16/51) of patients in HBV group and non-HBV group progressed to severe COVID-19, respectively, and there was no difference between the two groups in the probability of progression to severe COVID-19 over time (p=0.97, Figure   1B ). By multivariate analysis, the risk of progression to severe COVID-19 was not statistically Table 3 ).

In HBV group vs. non-HBV group, 35% (7/20) vs. 37.25% (19/51) had abnormal ALT at least once during hospitalization, respectively (p = 0.86). The proportion of abnormal ALT had a rise-fall trend in HBV group while a constantly increasing in non-HBV group since admission to hospital (Figure 2A) , which was 18.18% and 19.23% at 15 days in the two groups, respectively (p = 0.94). Figure 2D ).

As the median of testing/assessing time intervals and follow-up durations were 3 days and 14 days for liver biochemistries (ALT, AST, GGT, TBIL), we compared the dynamic levels of these indicators within/between the two groups at baseline, 3, 6, 9, 12, 15 days during hospitalization.

The median levels of liver biochemistries over time were no significant difference between two groups ( Figure 3 ; Wilcoxon signed-rank test, ALT: p=0.56, AST: p=0.58, GGT: p=0.43, TBIL: p=0. 16 ). In addition, we found no significant difference in the ALT, AST, GGT and TBIL levels between the two groups at each time point (Supplementary Figure 2A-2D) .

For the 20 COVID-19 patients with chronic HBV infection, 19 of whom had HBV DNA viral load testing at least twice during hospitalization, however, one patient had not any test of HBV DNA viral load. Of the 19 patients, three patients had HBV reactivation, 15 patients had the HBV DNA viral loads maintained at low levels (<300 IU/mL) or undetectable, and two patients'

HBV DNA viral loads were at high levels throughout the hospitalization.

All the three patients with hepatitis B reactivation were HBeAg negative and did not received any antiviral treatment for HBV before admission. We further described the dynamics of HBV DNA viral load and liver biochemistries, and treatment information in Figure 4 :

 Case 1: received methylprednisolone therapies during 3 to 6 days after admission, and interferon α-1b treatment (atomized inhalation) during 5 to 9 days after admission; This article is protected by copyright. All rights reserved.

HBV DNA viremia was undetectable on 9 days, but abruptly measured as 3.30 log10 IU/mL on 30 days, and finally as 4.05 log10 IU/mL when discharged; ALT and AST respectively increased to 387 IU/L (8.6 xULN) and 497 IU/L (11 xULN) on 8 days after admission.

 Case 2: received methylprednisolone therapy during 2 to 5 days after admission, and interferon α-1b treatment (atomized inhalation) during 1 to 25 days after admission;

HBV DNA levels were at 3.5-5 log10 IU/mL in early time of hospitalization but had a rapid increase from 2.26 log10 IU/mL on 29 days to 6.65 log10 IU/mL on 31 days;

ALT and AST were mildly high (< 2xULN) at admission and then restored to normal levels since 3 days after admission.

 Case 3: received interferon α-1b treatment (atomized inhalation) during 1 to 53 days after admission; HBV DNA viremia was undetectable at admission but hepatitis B reactivation (detectable as 1.30 log10 IU/mL) at 9 days after admission; ALT and AST were persistently normal during hospitalization.

One female patient who was 33 years old and whose BMI was 17.8 kg/m 2 at the admission to hospital, had a history of HBV infection over than 20 years but had not received antiviral treatment for CHB previously. In addition, this patient had not any history of alcohol use. The maximum level of ALT and AST of this patient was 31.8 U/L and 35.6 U/L during hospitalization, respectively. The patients discharged from hospital after SARS-CoV-2 clearance but readmitted to the hospital due to hepatalgia and had a liver biopsy 40 days after the clearance of SARS-CoV-2. The detailed information regarding her treatment course and laboratory tests was provided in Supplementary Figure 3 . The results of liver needle biopsy for this patient showed the structure of the hepatic lobules was clear, and the hepatocytes in the interlobular were arranged orderly, with some hepatocytes diffuse swelling (ballooning degeneration), and necrosis of isolated hepatocytes. No canalicular bile plugs and interface hepatitis were seen. Reticulin staining and Sirius Red staining indicated periportal fibrosis. The portal tracts were infiltrated with few inflammatory cells. The immunohistochemical analysis showed positive for HBsAg and negative for HBcAg ( Figure 5 ).

This observational study found no significant difference in probability of SARS-CoV-2 clearance and progression to severe COVID-19 over time in COVID-19 patients with vs.

without chronic HBV infection. We observed similar dynamics and non-significant difference at each time point on liver biochemistries (ALT, AST, GGT, TBIL) between the two groups.

However, we observed a continuous abnormality of ALT and GGT for both groups, which may be due to SARS-CoV-2 infection. More importantly, we identified three patients who underwent hepatitis B reactivation. This study provided preliminary evidence concerning the effect of chronic HBV infection on the outcomes and liver function of COVID-19 patients and added important data to support the management of COVID-19 patients with chronic HBV infection for physicians.

Investigation of SARS-CoV-2 shedding will be the key for determining the risk of transmission and formulating the criteria of releasing from quarantine. For the non-HBV COVID-19 patients, we found the median time of SARS-CoV-2 clearance was 12 days after the onset of COVID-19 symptoms, which was consistent with a study on eight discharged COVID-19 patients from Singapore (median, 14 days). 17 . Despite the confirmed host immune dysfunction resulting from chronic HBV infection, our results reveal that chronic HBV infection could not delay the SARS-CoV-2 shedding for COVID-19 patients, compared to those without chronic HBV. After excluding non-HBV related chronic liver diseases, we explored the independent impact of chronic HBV infection on the progression to severe COVID-19 and found that chronic HBV infection did not increase the risk of progression to severe COVID-19. Together with these comparisons, we tend to conclude that the comorbidity of chronic HBV infection would not increase the risk of poor outcomes related to SARS-CoV-2. It is worth mentioning that only one patient in HBV group were cirrhotic in this study. Patients with HBV related cirrhosis typically have poor immune function compared to those who had chronic HBV infection but without cirrhosis. Therefore, further studies are necessary to examine the impact of HBV related cirrhosis on the outcomes of COVID-19.

Previous studies have found that both hepatocytes and bile duct epithelial cells may also express the angiotensin-converting enzyme 2 (ACE2) receptor, while the latter has a higher concentration. It suggests SARS-CoV-2 might cause the damage of both hepatocytes and bile duct epithelial cells. Current studies showed that 6.2% -36.6% of patients had increased serum AST levels, while 21.3% -28.1% had elevated serum ALT levels. 18 A few studies reported the abnormal proportions of TBIL (4.9% to 10.53%) 4,19,20 and GGT (6.5% to14.8%) 4,19 at baseline.

Our study also identified the abnormality of the above-mentioned liver laboratory tests.

Moreover, with further analysis of longitudinal patterns, we found that the abnormality of AST and TBIL manifested as transient elevation, while high proportions of patients with abnormal ALT and GGT did not achieve the normalization of these two indicators. The dynamics of ALT and GGT suggested that SARS-CoV-2 possibly caused a continuous damage of bile duct epithelial cells and hepatocytes during the disease course. Although chronic HBV infection would not increase the injury of liver compared to non-HBV COVID-19 patients as suggested in this study, liver function monitoring is still essential for both COVID-19 patients with and without chronic HBV infection during the whole disease course.

Glucocorticoids have powerful anti-inflammatory effects, and have been confirmed to alleviate clinical symptoms, shorten treatment course, and improve the absorption of lung infiltrates for severe acute respiratory syndrome (SARS) patients. 21, 22 In our study, six COVID-19 patients with HBV received methylprednisolone, one type of corticosteroids. It is well known that moderate to high dose (≥10 mg) of methylprednisolone can lead to a high risk of hepatitis B reactivation. 23, 24 In our study, two of the three patients developed hepatitis B reactivation, which was possibly caused by methylprednisolone. However, one of the three patients who did not receive any corticosteroid also developed hepatitis B reactivation. Our results suggested that for COVID-19 patients who had chronic HBV infection, whether or not corticosteroids were used, they could have a risk of hepatitis B reactivation, therefore it is necessary to monitor the HBV DNA levels for these patients, and for them physicians should take precautions to hepatitis B reactivation.

This study is no without limitations. Firstly, the number of patients in HBV group was small, although we expected to increase the test efficiency using PSM designed with 1:3 matching ratio, and we validated our results by multivariable Cox proportional-hazards model. Secondly, we were unable to explore if the liver abnormality associated with COVID-19 treatment drugs, as all the 71 patients in two groups had received them (Supplementary Table 2 

Clinical characteristics of Non-ICU hospitalized patients with coronavirus disease 2019 and liver injury: A Retrospective study

Liver injury during highly pathogenic human coronavirus infections

Liver impairment in COVID-19 patients: a retrospective analysis of 115 cases from a single center in Wuhan city, China

COVID-19 and the liver: little cause for concern

Pathological findings of COVID-19 associated with acute respiratory distress syndrome

General anatomy report of novel coronavirus pneumonia patients

Polaris Observatory Collaborators. Global prevalence, treatment, and prevention of hepatitis B virus infection in 2016: a modelling study

Clinical Characteristics of Coronavirus Disease 2019 in China

Diagnosis and Treatment of Adults with Community-acquired Pneumonia. An Official Clinical Practice Guideline of the American Thoracic Society and Infectious Diseases Society of America

Clinical epidemiology: the essentials

Epidemiologic Features and Clinical Course of Patients Infected With SARS-CoV-2 in Singapore

COVID-19 and Liver Dysfunction: Current Insights and Emergent Therapeutic Strategies

Exploring the mechanism of liver enzyme abnormalities in patients with novel coronavirus-infected pneumonia

Hydrocortisone infusion for severe community-acquired pneumonia: a preliminary randomized study

Use of glucocorticoid in treatment of severe acute respiratory syndrome cases

American Gastroenterological Association Institute technical review on prevention and treatment of hepatitis B virus reactivation during immunosuppressive drug therapy

Cord Blood Transplantation Rescued by Medical Treatment