Clinical Characteristics and outcomes in HBV carriers with COVID-19 in WuHan, China: a retrospective cohort study

Background: Coronavirus 2019 (COVID-19) is a novel infectious disease that was rst reported in Wuhan, China, but has spread to all parts of the world. At the same time, because China has millions of HBV carriers, HBV infection has become a major public health problem in China. In this study, we aim to describe the clinical features of HBV carriers (AsC) infected with COVID-19 and to assess the factors that may affect the outcome during disease progression. Methods: This retrospective cohort study included 72 patients diagnosed with COVID-19 in Wuhan Jinyintan Hospital. These patients were also diagnosed as HBV carriers. The epidemiological characteristics, demographic features, clinical manifestations, laboratory test, treatment, management and nal outcome were collected and analyzed. Results: The median age of 72 patients is 58.5 years old, of which 55.56% (n=40) are male. 20 (30.56%) patients were severe cases and 50 (69.44%) were non-severe cases. Fever is the most common symptom, followed by cough, chest tightness and sputum. Laboratory test results including hematologic, biochemical, infection and coagulation parameters and several indicators, such as Aspartate Aminotransferase (AST), Total Bilirubin (TBil), Direct Bilirubin (DBil), Indirect Bilirubin (IBil), γ-glutamyl Transferase (GGT) showed difference between their admission and discharge. The level of Prealbumin (PA) and Serum Amyloid A (SAA) in the study showed a signicant trend from high to low, which has statistical signicance. Conclusions: The clinical features of HBV carriers with COVID-19 have obvious systemic symptoms, such as fever, cough, and chest tightness. Compared with liver function data on admission and discharge, SARS-CoV-2 does not directly activate the Hepatitis B virus, and the risk of liver cell damage of HBV carriers with COVID-19 does not increase. Both PA and SAA are sensitive indicators and can be used to evaluate the prognosis and outcome of these patients. ventilation; PCT:Procalcitonin; PLT:platelets; PT:prothrombin PT-Fbg:Fibrinogen; SAA:serum


Background
In December 2019, a group of unexplained pneumonia patients appeared in Wuhan, Hubei Province, China.The clinical manifestations are very similar to viral pneumonia [1].According to the deep sequencing analysis of the lower respiratory tract samples, it was named SARS-CoV-2 [2].According to the interim guidelines of the World Health Organization (WHO), the diagnosis of the disease mainly depends on the positive result of SARS-CoV-2 nucleic acid in respiratory specimens.
Hepatitis B virus carrier (AsC) is de ned as a person who is chronically infected with hepatitis B virus (HBV) and is positive for hepatitis B surface antigen (HBsAg) for more than 6 months.They rarely have liver-related symptoms and signs and liver function is normal.HBV serological marker (HBVM) is a necessary test indicator for diagnosing hepatitis B virus infection.After analyzing the clinical features of 72 hepatitis B virus carriers diagnosed with COVID-19 in Wuhan Jinyintan Hospital, this study aims to clarify the clinical characteristics of HBV carriers of COVID-19 and nd out the possible in uence factors of HBV carriers.

Study design & subjects
This is a retrospective cohort study of 72 patients, diagnosed with COVID-19, admitted in Jinyintan Hospital, Wuhan City, Hubei Province, China.
All patients were HBV carriers and diagnosed with COVID-19, according to the interim guideline of the World Health Organization (WHO) [3].These patients were admitted on January 10, 2020 and February 24, 2020.Epidemiological characteristics, clinical manifestations, laboratory test results, CT scan imaging and clinical treatment were collected from electronic medical records.
Severe cases are de ned by any of the following conditions [4]: 1) Signi cant increase in respiratory rate ≥ 30 times/min; 2) Oxygen Saturation ≤ 93%; 3) Partial pressure of oxygen(PaO2)/fraction of inspired oxygen( FiO2) ≤ 300 mmHg; or 4) Respiratory or other organs failure that requires intensive care unit (ICU) monitoring and treatment or shock.
Most clinical data used in this study was collected from the day since admission to the day before discharge.Written informed consent was waived for the retrospective case series and it would not cause any potential risk to patients.

Statisitical analysis
Descriptive analysis of variables is expressed as median (interquartile range [IQR]) or number (%).When the data are normally distributed, an independent group t-test is used to compare the means of continuous variables.Otherwise, the Mann-Whitney test is used.For the classi ed variables, Chi-squared test (x 2 -test) was conducted.All analysis are performed using SPSS version 23.0, and P values <0.05 is considered statistically signi cant.

baseline characteristics of the study patients
All consecutive HBV carriers with con rmed COVID-19 admitted to Jinyintan Hospital were included in this study (Table 1).Among the 72 patients, the median age was 58.5 years (27-82 yrs), and the

Comparison with the characteristics of the laboratory indicators from admission to discharge
All patients received blood tests, virology test and chest CT examination at the rst visit.Some laboratory results needed to be re-examined before discharge.
Table 2 shows the laboratory test indicators when all patients were admitted and discharged.No signi cant difference showed in blood test, when they were admitted and before discharge, including WBC, NE, LY, EO, PLT.Similarly, the neutral lymphatic ratio (NLR) also showed no difference at admission and discharge.In this study, several serum indicator for liver function, including AST, GGT and LDH showed improve better when patients of the HBV carriers with COVID-19 before discharge.Albumin (ALB) at discharge is signi cantly increase than at admission.A/G has the same trend.Most patients showed a signi cant decrease in PA at admission and elevated to normal range as the condition improved (P < 0.001).SAA and serum ferritin have shown a declining trend in the study (P < 0.05), while other infections and coagulation function indexes did not change much before and after treatment (P > 0.05).The INR of most patients increased slightly (P < 0.05).
The majority of the patients presented mild liver abnormalities.Additionally, 4 patients who had liver injury presented > 3 ULN elevation of ALT, 2 patients presented > 3 ULN elevation of AST and 3 patients presented > 3 ULN elevation of GGT.

Discussion
COVID-19 has become a global public health crisis, with as many as 4 million reported cases of infection, including 84,431 cases in China.Meanwhile, a total of 300 million people in the world have been infected with hepatitis B (HBV) as of March 2018, while the number of HBs-Ag positive people in China is about 80 million [5].
Because the liver is an organ located between the portal vein and the systemic circulation, it is often exposed potentially to viruses, bacteria and in ammatory, sometimes causing liver damage.It is reported that people infected with SARS [6] and the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) [7] have liver damage.A recent epidemiological study in Wuhan showed that of the 99 patients initially infected with 2019-nCoV, 43 had varying degrees of liver dysfunction and ALT or AST were both above the normal range [8].Another retrospective analysis of patients of COVID-19 with impaired liver function.It is also found that abnormal liver function is common in patients with COVID-19 [9].The pathological ndings of the liver biopsy specimen of the patient who died of COVID-19 were moderate microvesicular steatosis and mild lea ets, with portal vein activity, indicating that SARS-CoV-2 may cause liver damage [10] and viral RNA was also detected in liver tissues in concentrations exceeding viremia [11].
For carriers of hepatitis B, it is unclear whether the SARS-CoV-2 will exacerbate liver function damage or not.Few studies have analyzed the clinical characteristics and outcomes of hepatitis B virus carriers complicated by COVID-19.In this study, we included 72 COVID-19 carriers of hepatitis B. Of these patients, 39 (54.2%) patients had abnormal liver examination results, and 9 (12.50%)patients had liver injury upon admission.With the improvement of COVID-19 treatment, these liver function indicators can gradually return to normal range.Similar to a recent retrospective study based on the characteristics of liver function tests in large hospitals, COVID-19 patients also reported that about half of them had abnormal liver function, and 5% of patients had liver injury upon admission [4].It is recommended that among the carriers of hepatitis B, the incidence of liver function damage may not increase.Some studies have shown that patients with abnormal liver function tests will face a higher risk of serious illness [4].However, in our study, we found that few patients with abnormal liver function at admission exacerbated the disease and liver damage.This nding is consistent with the results of a retrospective study showing that despite common liver function abnormalities in patients with COVID-19, liver damage is not the main feature of COVID-19 and may not have serious clinical consequences [9].SARS-CoV-2 can directly attack the liver and cause liver dysfunction, but as far as we are concerned, it should be distinguished from the liver function damage caused by the in ammatory factor storm caused by coronavirus, which may be related to the aggravation of the disease.
Two recent studies have shown that angiotensin converting enzyme 2 (ACE2) is a key receptor for SARS-CoV-2 [12] [13].RNA-seq data in the Human Protein Atlas (HPA) database [14] shows that ACE2 is relatively of low expression in liver and lung which are the main target organs for 2019-nCoV and SARS.A Low-throughput study of ACE2 protein expression in the liver indicates that ACE2 occurs less frequently in cholangiocytes, but not in hepatocytes, Kupffer cells, and endothelial cells [15].A current study [16] found that coronavirus may directly bind to ACE2-positive cholangiocytes, but not necessarily to hepatocytes, indicating that liver abnormalities in SARS and 2019-nCoV patients may not be caused by hepatocyte damage, probably induced by cholangiocyte dysfunction and other causes, such as drug toxicity and systemic in ammatory response.However, after analyzing the indicators of abnormal liver function at the time of admission, we found that there were not only cholangiocyte dysfunction, but also hepatocyte damage, suggesting that for hepatitis B carriers, in the early stage of COVID-19, SARS-CoV-2 may attack hepatocytes directly as well as cholangiocyte.Therefore, in addition to ACE2,whether there are other SARS-CoV-2 binding sites on hepatocytes, such as CD147 [17], DC-SIGN and l-sign [18] remains to be further clari ed, meanwhile, it is not ruled out that the liver dysfunction in these patients is due to coronavirus-induced in ammation or drug toxicity.
In this study, we found sensitive prognostic indicators.The majority of patients had a signi cant decrease in PA when they were admitted and the PA can gradually increase to normal levels before rehabilitation or discharge.There was a statistically signi cant difference between admission and discharge (P < 0.0001).PA is an acute reactive protein and mainly present in the blood and cerebrospinal uid and the plasma half-life is about 1.9 days.PA is synthesized in the liver, when liver damage occurs and intrahepatic synthesis decreases, it can be quickly detected from peripheral blood at an early stage.
Therefore, it is considered to be a sensitive indicator of liver function damage and recovery.Our results show that compared with other liver function indicators, the early dynamic changes of PA can be used as a sensitive indicator to judge the prognosis and outcomes of HBV carriers complicated with COVID-19.
Our research also conducted an exploratory analysis of infection-related indicators.We found that the level of SAA increased at the initial stage and will gradually return to normal levels after recovery.SAA is also an acute phase protein and is commonly used to assess the acute response process.There is a similar expression in serum ferritin.
It is well known that there is a dynamic balance between HBV and the host immune system.When the balance is broken, HBV cccDNA is the main substance that replicates with HBV in hepatocytes, which will speed up the replication speed, increase the number of infected cells, and cause HBV reactivation to a certain extent [19].Therefore, indicators that may impair immune response, such as tumor chemotherapy, immunosuppression or transplantation, may cause HBV reactivation.In patients with COVID-19 is also associated with immune imbalances, while peripheral blood neutrophils are signi cantly increased and lymphocyte counts are signi cantly decreased [20] [21] [22].Therefore, in this study, we tried to explore whether SARS-CoV-2 can cause HBV activation in HBV carriers.Generally, the diagnosis of HBV activation is based on dynamic monitoring of HBV DNA quantitative detection and liver function (especially ALT).
However, due to retrospective analysis and limited laboratory test data, there is no direct evidence that SARS-CoV-2 can cause hepatitis B virus reactivation (such as dynamic monitoring of HBV-DNA) [23].However, from the patient's prognosis and recovery of liver function, SARS-CoV-2 may not directly cause HBV reactivation.In addition, the in ammatory cytokine storm is thought to be related to the severity of the disease, so large doses of glucocorticoids and immunosuppressants will be used during treatment.In this condition, whether it will reactivate HBV and aggravate the occurrence of liver function damage or not remains to be further studied.
Our research also has several limitations.First of all, due to the design of this retrospective study, we have relatively few cases in a single center and not all patients have undergone all laboratory tests, especially liver tests and HBV-DNA quantitative detection.Second, because most patients have a good prognosis and only three patients died in our study, the potential impact of liver abnormalities or injury on mortality can not be assessed.With the emergence of new cases in the world, it is necessary to conduct further large-scale studies among HBV carriers.
In summary, this study describes the clinical features of HBV carriers with COVID-19.We found that more than half of the patients had varying degrees of liver dysfunction in the early stages of pneumonia and these abnormalities may gradually return to normal levels.This phenomenon may be caused by SARS-CoV-2 directly attacking hepatocytes and cholangiocytes.Both PA and SAA are sensitive indicators and can be used to judge the prognosis and outcome of these patients.In addition, in this study, we did not nd direct evidence that SARS-CoV-2 may cause hepatitis B virus reactivation.Further research is still needed to explore the impact of SARS-CoV-2 on hepatitis B carriers.

Conclusions
The clinical features of HBV carriers with COVID-19 have obvious systemic symptoms.Compared with liver function data on admission and discharge, SARS-CoV-2 does not directly activate the Hepatitis B virus, and the risk of liver cell damage of HBV carriers with COVID-19 does not increase.Both PA and SAA are sensitive indicators to evaluate the prognosis and outcome of these patients.
where indicated, data = n/N (%), n is number of patients, where N is the total number of patients with available data.