Changes in indicators of fibrosis in patients with alcoholic liver cirrhosis associated with non-alcoholic fatty liver disease depending on the stage of cirrhosis

Material and methods. The study included 204 patients. Among them, 78 patients (Group I) had alcoholic liver cirrhosis (ALC) and 126 patients (Group II) had a combination of ALC with non-alcoholic fatty liver disease (NAFLD). Patients were subgrouped according to compensation classes by the Child-Pugh score (A, B, C). The degree of liver parenchyma fibrosis was assessed by calculating the FIB-4 (FibroIndex-4) and the liver parenchyma elasticity (kPa) determined by the shear wave elastography method.

According to the World Health Organization, alcohol abuse causes about 3.3 million deaths each year, which is 5.9 % of the total mortality rate. Almost 5 % of overall morbidity and disability are alcohol related. Alcoholic liver cirrhosis (ALC) is the most common disorder of all gastrointestinal tract diseases. In developed countries, alcohol, along with hepatitis C virus and metabolic syndrome are among the leading causes of hepatocellular carcinoma (HCC) [4].
Recently, it has been revealed that non-alcoholic fatty liver disease (NAFLD) is the most prevalent liver disease in Europe, the USA and Asia and is associated with increased mortality. According to Charlton et all, NAFLD is the third most popular indication for liver transplantation in the United States and the only indication that is steadily increasing in frequency [3,7]. The prevalence of NAFLD reaches more than 90 % among people suffering from obesity and diabetes [1,2,9,11].
Liver cirrhosis (LC) is considered to be a precursor to the HCC, and its frequency has been constantly increasing [4].
At present, it is important to develop and implement methods for the assessment of fibrosis, and these methods should be convenient-to-use and economically feasible to prevent the progression of prognostically adverse events.

Aim
Objective was to study changes in liver parenchyma fibrosis in patients with alcoholic liver cirrhosis associated with non-alcoholic fatty liver disease depending on decompensation using shear wave elastography and FIB-4 index.

Material and methods of research
The study involved 204 patients with diagnosed LC who were hospitalized in the gastroenterology department of the Ivano-Frankivsk Regional Clinical Hospital. Among them, 78 people were diagnosed with ALD at the stage of LC (group I) and 126 people had a combination of alcoholic liver cirrhosis (ALC) with NAFLD (group II). Among the patients of group I there were 24 women and 54 men (mean age 53.2 ± 11.4 years) and average duration of the disease 5.9 ± 2.1 years; among patients of group II there were 22 women and 104 men aged 47.8 ± 9.4 years and average duration of the disease 4.2 ± 2.7 years. Patients of groups I and II were subdivided depending on the Child-Pugh classes of LC compensation: IA (n = 17), ІВ (n = 38), ІС (n = 23); IIA (n = 44), IIB (n = 48), IIC (n = 34).
ALC was diagnosed according to the Adapted Clinical Guideline "Alcoholic Liver Disease" (the Ministry of Health of Ukraine, 2014). NAFLD was diagnosed according to the Adapted Clinical Guideline "Non-Alcoholic Fatty Liver Disease" (the Ministry of Health of Ukraine, 2014). The severity of LC was evaluated by Child-Pugh score. MELD index (Mayo Endstage Liver Disease, 2001) was calculated with electronic calculator: MELD index = 3.8 × log e serum bilirubin level (mg/dL) + 11.2 × log e serum creatinine level (mg/dL).
General clinical, instrumental and laboratory examinations. The degree of liver parenchyma fibrosis was assessed by calculating the FIB-4 (FibroIndex-4) and the liver parenchyma elasticity (kPa) determined by the shear wave elastography method on the GE Logiq E8 with assessment of the degree of fibrosis on METAVIR scale. The confirmation of the degree of F4 fibrosis by the method of elastography was the value of the elasticity of the liver parenchyma more than 11.9 kPa. The To determine the aetiology of the disease, more than 2 doses of alcohol (1 standard dose = 10 g of ethyl alcohol) per day for women and more than 4 doses for men were considered according to the recommendations of the World Health Organization, CAGE (Cut, Annoyed, Guilty, Eye-opener), AUDIT (Alcohol Use Disorders Identification Test,  Statistical processing of the obtained results was carried out using the software package Statistica v. 12.0 (StatSoft, USA, trial) and Microsoft Exсel. The data were checked using the Shapiro-Wilk test. Since the data correspond to the normal type of distribution (P > 0.05), the interval (M ± m) was chosen as a measure of central tendency. The Student's t-test was used to test the null hypothesis of establishing the difference between the comparison groups; the Newman-Keuls test was used to test the statistical significance of the difference in indicators. For quantitative data, the correlation was estimated using the Pearson parametric method of paired correlation. Statistically significant differences were considered at P < 0.05. The difference was statistically significant.

Results
Analyzing the clinical examination data it was found that the signs of astheno-vegetative, pain, dyspeptic, hepatorenal, hepatopulmonary syndromes, jaundice, medicamentally uncontrolled ascites, and manifestations of liver encephalopathy were more frequent in group II patients of the corresponding classes, which was accompanied by a more severe ALC by Child-Pugh severity score and MELD index, that is prognostic index for liver diseases taking into account bilirubin, international normalized ratio (INR) and serum creatinine.
In patients of both groups, they increased with ALC decompensation increasing. However, in patients of group II they were higher than those of group I by 22.74 % and 31.18 %, 21.06 % and 17.78 %, 13.72 % and 15.98 % of classes A, B, C, respectively (P < 0.05). Such results indicate a more severe clinical course and more pronounced rates of progression of hepatic failure in individuals with combination of ALC and NAFLD, which is caused by a more pronounced increase of inflammatory-necrotic process and the process of fibrosis in the liver and is accompanied by pronounced systemic changes in the blood flow, resulting in eventually to the development of multiple organ failure with adverse lethal consequences.
According to the results of the shear wave elastography, the elasticity of the liver parenchyma in all patients corresponded to stage F4 according to the METAVIR classification ( Table 1). In both groups, this indicator increased with increasing decompensation of the disease. In particular, in group I elasticity of liver parenchyma in class B exceeded that in class A patients by 1.34 times, and in class C exceeded that in class B persons by 1.24 times (P < 0.05). In group II persons, the class B liver parenchyma elasticity index exceeded that in class A by 1.37 times and in class C exceeded that in class B by 1.29 times (P < 0.05). Values of liver parenchyma elasticity were higher in group II patients compared to group I patients. In particular, in IIA, IIB and IIC persons, they were higher than the indicators of IA, IB and IC patients by 1.25, 1.27 and 1.32 times, respectively (P < 0.05).
The FIB-4 index in patients in both groups increased depending on the stage of decompensation. This indicator was higher in patients of group IВ compared to group IA indicator by 14.69 %, in the group IC patients compared with group IВ -by 7.14 %, in group IIB patients compared to group IIA indicator -by 15.36 %, in group IIC patients compared to group IIВ indicator -by 8.13 % (P < 0.05). FIB-4 indicators in patients of group II were higher than classes A, B, and C compared to group I by 7.26 %, 9.11 %, and 10.11 %, respectively (P < 0.05).
Higher values of liver parenchyma elasticity index and FIB-4 index in group II patients compared with group I patients show more pronounced fibrogenesis processes in patients with combination of ALC and NAFLD. These results indicate the effect of the combination of ALC and NAFLD on the increase in the intensity of fibrogenesis in the liver.
In patients with ALC in combination with NAFLD, direct correlations were established between the value of Child-Pugh score and the elasticity of the liver parenchyma (r = +0.69; P = 0.0006), the value of the MELD index and the elasticity of the liver parenchyma (r = +0.61; P = 0.0003), the value of Child-Pugh score and FIB-4 index (r = +0.67; P = 0.0005), the value of MELD index and FIB-4 index (r = +0.58; P = 0.0008), FIB-4 index and liver parenchyma elasticity (r = +0.72; P = 0.0005). Such correlations indicate a direct relationship of fibrosis (according to elastography and FIB-4 index) and Child-Pugh severity score and MELD prognostic index in patients with combination of ALC and NAFLD.

Discussion
Liver biopsy is considered to be the "gold standard" for the diagnosis of diffuse liver diseases. However, this method is invasive and carries considerable risks and costs [14]. Non-invasive techniques are alternative solution, which will allow evaluating the condition of the liver as a whole but not the particular samples of liver tissue [15].
Non-invasive methods for assessing the degree of liver fibrosis include ultrasound liver elastography and laboratory panels that are widely used in practical medicine. FIB-4 is a laboratory panel comprising four indicators (age, platelet count, alanine aminotransferase and aspartate aminotransferase levels); they are available at primary and secondary medical care and not expensive [5,6]. According to many studies, the use of FIB-4 index is recommended for assessment of the degree of liver fibrosis. Given its noninvasiveness and simplicity, the FIB-4 index has the advantage.
However, its use as a single method does not provide sufficient reason to replace the need for a liver biopsy [10,12,14]. Ultrasound elastography shows a fairly accurate assessment of the degree of fibrosis and is recommended as an alternative to liver biopsy in patients who cannot undergo invasive procedures [8,13].
In our work, to assess the degree of fibrosis in patients with combination of ALC and NAFLD, we used indicators of liver parenchyma elasticity according to the results of shear wave elastography and FIB-4 index. The fibrosis indicators in patients with combination of ALC and NAFLD were higher in A, B, and C classes in comparison with those in ALC patients without NAFLD.
According to our results, in patients with ALC the combination with NAFLD accompanied by a more severe clinical course according to the Child-Pugh score and the MELD index. In patients with combination of ALC and NAFLD we have found a direct correlation of fibrosis (according to elastography indicators and FIB-4 index) with Child-Pugh severity score and MELD index.
As the shear wave elastography and laboratory panel FIB-4, according to the literature [12], show fairly accurate results in the study of liver fibrosis and correlate with the indicators of Child-Pugh and MELD scales, a combination of these methods may, in our opinion, be an alternative to liver biopsy for the assessment of the degree of liver fibrosis and the prediction of the subsequent course of the disease in patients with combination of ALC and NAFLD. The perspective for the further scientific research is to study the treatment of alcoholic liver disease at the stage of cirrhosis associated with non-alcoholic fatty liver disease, depending on the compensation of the disease.

Funding
The study is carried out according to the plan of scientific works of SHEE "Ivano-Frankivsk National Medical University" and is a fragment of research work: "Diseases of internal organs in modern conditions, with combined pathology and lesions of target organs: features of the course, diagnosis and treatment", number of state registration: 0115U000995.