Epstein-Barr virus infection is associated with a higher Child-Pugh score and may predict poor prognoses for patients with liver cirrhosis

This study was performed in a retrospective manner. The databases of patients with liver cirrhosis admitted to the First Affiliated Hospital (College of Medicine, Zhejiang University) between 1 January 2014 and 31 December 2016 were reviewed. Patients who were or were not infected with EBV were enrolled in this study. The demographic, clinical characteristics, and some experimental results (liver function, coagulation function, and EBV deoxyribonucleic acid (DNA) level) and ultrasonic scanning results (mainly included liver size and spleen size) during hospitalization were reviewed by a trained team of physicians and entered into a computerized system in duplicate. We also collected patient mortality data (28-, 90-, and 180-day transplant-free mortality) by reviewing medical documents or telephone follow-ups. Mortality was assessed during the hospital stay and after hospital discharge.

Patients who were diagnosed with liver cirrhosis and had EBV DNA assessed were selected.

The diagnosis of cirrhosis was based on the following, as previously described [27, 28]: liver biopsy; clinical evidence of decompensation or varices; radiological evidence of liver nodularity; and intra-abdominal variations in a patient with chronic liver disease.

Patients with the following criteria were excluded: (1) patients in whom the level of EBV DNA was not determined; (2) patients < 18 years of age; (3) patients with acquired immune deficiency syndrome (AIDS); (4) patients with other types of tumors besides hepatocellular carcinoma; (5) a history of liver transplantation; (6) patients who require long-time corticosteroids treatments (prolonged use of corticosteroids at a mean minimum dose of 0.3 mg/kg/day of prednisone equivalent for > 3 weeks) [29]; (7) patients who require immunosuppressive therapy (mainly including recognized T cell immunosuppressants, such as cyclosporine, TNF-αblockers, specific monoclonal antibodies) [29].

Bacterial infections were defined as follows [27, 28]: (1) pneumonia was defined as a new pulmonary infiltrate with fever, respiratory symptoms (cough, sputum production, dyspnea, and pleuritic pain), findings on auscultation (rales or crepitation), and/or a leukocyte count > 10,000/mm3 or < 4000/mm3; (2) spontaneous bacterial peritonitis was defined as ascitic fluid polymorphonuclear cells > 250/μL with/without a positive fluid culture; (3) urinary tract infections were defined as urine white blood cell (WBC) > 15/high power field with positive culture and symptoms of urinary irritation; (4) skin and soft tissue infections were defined as a fever and cellulitis associated with leukocytosis; and (5) undetermined infections were defined as bacterial infections, but without positive cultures or evidence of organ involvement.

EBV infection was referred to as EBV DNA detected in whole blood samples by a quantitative polymerase chain reaction assay.

Liver size referred to the measurement of the inferior oblique diameter of rib margin of right liver. Spleen size referred to the measurement of the thickness of spleen. Within liver was referred as inferior oblique diameter of rib margin of right liver<10.0 cm and splenomegaly was referred as the thickness of spleen > 4.0 cm, which were measured by ultrasonic scanning.

According to the manufacturer’s instructions, EBV DNA was extracted from blood samples using DNA extraction kit (Daan Gene Co.Ltd., Zhongshan University, China). Quantitative polymerase chain reaction (PCR) was detected on a real-time PCR system (Stratagene MX3000P; Agilent Technologies, Santa Clara, CA, USA), just as our previous reported [30].

Liver function, including albumin, the albumin-to-globulin ratio (A/G), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TB), direct bilirubin (DB), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (GGT), and cholinesterase (CHE), was reviewed.

The Child-Pugh score [23] and MELD score [24] are used to accurately assess liver function and prognosis of patients with liver cirrhosis. We calculated the Child-Pugh and MELD scores of the patients according to the collected data.

Diagnostic criteria and grading of ACLF based on the chronic liver failure-sequential organ failure assessment (CLIF-SOFA score) [31]. And CLIF-C Score [32] was also assessed.

In this study, the 28-, 90-, and 180-day mortality rates were determined from the first day of admission to the hospital to 28, 90, and 180 days.

According to previous studies on mortality risk factors for patients with liver cirrhosis [16‐22, 25, 26], we analyzed the 28-, 90-, and 180-day mortality rates by univariate and multivariate logistic regression, including the Child-Pugh score, MELD score, ACLF, age, ascites, hepatic encephalopathy, gastrointestinal bleeding, hepatocellular carcinoma, hepatorenal syndrome, bacterial infection, hypoalbuminemia, EBV infection, international normalized ratio (INR), and serum creatinine (Scr).

We identified 3901 hospitalized patients who were diagnosed with liver cirrhosis from 1 January 2014 to 31 December 2016. 3794 patients were excluded because EBVDNA was not assessed, and 10 patients were exclued because of various reasons (Fig. 1). A total of 97 patients with EBVDNA assessed were enrolled (Fig. 1), of which 36 (37.1%) were EBV DNA-positive, with a mean of 9573.4 copies/mL. The patients consisted of 52 males and 45 females who had a mean age of 55.9 years; 43.3% of the patients were at least 60 years of age. Hepatitis B infection caused cirrhosis in 51.6% of the patients, 68% of patients had ascites, and 26.8% of patients had bacterial infections. 13.4% patients had ACLF, of which 3 patients (3.1%) were in grade 1, 9 patients (9.3%) were in grade 2, 1 patients (1.0%) were in grade 3. The livers were reduced and the spleens were enlarged in all patients in this study. The mean size of liver was 8.52 ± 0.791 cm, and the mean size of spleen was 4.87 ± 0.815; There was no significant difference in liver and spleen size between EBV infected and uninfected patients. The demographic and clinical characteristics of the participants are presented in Table 1.

All indices of liver function, including ALT, AST, TB, DB, CHE, ALP, and GGT, did not differ significantly (P > 0.05) between EBV-infected and -non-infected patients; however, the albumin level was lower in EBV-infected patients (30.92 ± weve1 g/L) than non-infected patients (33.65 ± 5.199 g/L); this difference was statistically significant (P = 0.019). Also, the difference in A/G was statistically significant (P = 0.013); specifically, EBV-infected patients had a lower A/G (1.05 ± 0.366) than non-infected patients (1.27 ± 0.432). For the EBV-infected and -non-infected patients, the Child-Pugh scores were 9.83 ± 2.864 and 8.64 ± 2.470, respectively; EBV-infected patients had higher Child-Pugh scores (P = 0.033). Nevertheless, the MELD scores of the two groups were also not significantly different (P > 0.05). (Table 2).

There were 8 patients had ACLF in patients with EBV infected, including 2 patients with grade 1 and 6 patients with grade 2. There were 5 patients had ACLF in patients with EBV un-infected, including 1 patients with grade 1, 3 patients with grade 2 and 1 patients with grade 3. patients with EBV infected had higher rate of ACLF. Compared to patients with EBV un-infected, patients with EBV infected had more higher ACLF rate (P = 0.050). However, there were no significant difference in ACLF grade between these two groups. (Table 2).

In this study, we found that 20 (20.6%), 24 (24.7%), and 29 (29.9%) patients died within 28, 90, and 180 days, respectively.

Univariate and multivariate logistic regression were used to assess the factors studied and the possible relationships to mortality. For 28- and 90-day transplant-free mortality in univariate analysis, age, ascites, gastrointestinal bleeding, hepatocellular carcinoma, hepatorenal syndrome, bacterial infection, EBV infection, and serum creatinine (Scr) were shown not to be risk factors; however, the Child-Pugh score, MELD score, ACLF, hepatic encephalopathy, hypoalbuminemia, and INR were shown to be risk factors. Of note, we found that the Child-Pugh score and ACLF were the risk factors by multivariate logistic regression. Based on univariate analysis, the Child-Pugh score, MELD score, ACLF, hepatic encephalopathy, hypoalbuminemia, INR, ascites, and hepatocellular carcinoma were shown to be risk factors for 180-day transplant-free mortality; however, we also found that the Child-Pugh score and ACLF were the risk factors by multivariate logistic regression. (Tables 3, 4, 5).