Changes in clinical, biochemical, immunological and integrative parameters in patients with chronic hepatitis C virus infection according to the virus genotype and the grade of activity

The aim of this study is to determine the features of the course of chronic hepatitis C virus (CHCV) infection and the dependence of changes in integrative indicators of endogenous intoxication, nonspecific immunoreactivity and inflammation on the virus genotype and the grade of activity. Materials and methods. In total, 287 CHCV patients were examined and their inpatient and outpatient medical cards were analyzed. The study included 55 healthy individuals. In addition to the general group, which included all the patients, the examined people were divided into groups, depending on the grade of activity (minimal activity – 210 people, moderate – 68, expressed – 9) and the virus genotype (1b – 150 people, 2 – 19, 3a – 102). Clinical and laboratory examination was performed according to the protocol. The integrative indices of severity, nonspecific reactivity, indices of inflammation and intoxication activity were also calculated.The statistical processing of the obtained results was carried out by Microsoft Office Excel 2010 and IBM SPSS Statistic 23 computer software. Results. The groups were sex- and age-representative. The 1b genotype (52.30 %), moderate liver fibrosis (F2 – 31.25 %) and minimal activity (73.17 %) were the most frequently encountered in patients with CHCV. The most frequent clinical manifestations were asthenovegetative

More than 71 million people or about 1.0 % of the world's population [1], are infected with hepatitis C virus (HCV). A predominant liver lesion characterizes hepatitis C; it has a light yellowish form in the acute period, frequent chronicity with long-term asymptomatic course and complications including cirrhosis and hepatocellular carcinoma [2].
The fact is that only about 10 % of acute hepatitis C overlap with clear clinical signs, resulting in most of them not being timely detected. In the manifested disease, elimination of the virus is higher (up to 50 %) than among those who remain without marked symptoms of hepatitis [3]. Chronic hepatitis C virus (CHCV) is also usually clinically undetectable in the early years. Some patients complain of weakness, fatigue and malaise [4]. Compared to other viral hepatitis, arthralgia and myalgia are more commonly reported in CHCV [5]. The levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) usually range from the normal to two-to fourfold increase, rarely exceeding 200 IU / l in the absence of other concomitant liver diseases (alcohol-induced liver injury, etc.). An accurate determination of viral load is not very important in most clinical situations and on average, it is around 2 million IU/l. Low viral load is associated with faster recovery after the treatment, but is not associated with low levels of aminotransferases or less pronounced clinical symptoms. Thus, patients might not be aware of the disease for decades; diagnosis is made during screening and detection of abnormal levels of transferases, or as a result of risk group screening [4].
Besides, HCV can cause various extrahepatic lesions that should be considered in the diagnosis of CHCV. Possible co-morbidities, including alcoholism, heart disease, renal impairment, autoimmune, genetic or metabolic liver Original research diseases (e.g. genetic hemochromatosis, diabetes, obesity) and a possibility of drug-induced hepatotoxic effects should be evaluated [6].
The effect of the fibrosis degree on hematological and biochemical parameters has been sufficiently studied at this time [7]. But the data on the virus genotype impact on these parameters are controversial. In some previous studies, patients with CVHC did not show any association between clinical and biochemical parameters and the virus genotype [8,9], whereas other scientific papers reported on a correlation between the virus genotype and enzymatic activity of leukocytes and monocytes [10] as well as proliferative activity of lymphocytes in peripheral blood [11]. The relationship between the grade of the process activity and the hematological-biochemical data remains understudied.
In addition, it is proved that the level of ALT significantly increases with increasing degree of liver fibrosis [7], which provides a theoretical basis for studying the effect of the grade of activity on indicators that are changed in liver fibrosis progression.

Aim
The aim of this study is to determine the features of the course of CHCV infection and the dependence of changes in integrative indicators of endogenous intoxication, nonspecific immunoreactivity and inflammation on the virus genotype and the grade of activity.

Materials and methods
In the furtherance of the purpose, 287 patients with established diagnosis of CHCV who were undergoing treatment at Sumy Regional Infectious Diseases Clinical Hospital named after Z. Y. Krasovytsky from 2015 to 2019 were examined. Their inpatient and outpatient medical cards were also analyzed.
Inclusion criteria were as follows: the presence of typical clinical signs of CVHC, epidemiological data, no malignant diseases at the time of examination or in the anamnesis (including hepatocellular carcinoma), CVHС confirmed by ELISA and PCR (quality method for RNA detection).
Exclusion criteria were as follows: presence of acute conditions or major complications of decompensated pathology (decompensated cirrhosis; hepatic insufficiency III), concomitant pathology, which may affect the indicators of inflammation, endogenous intoxication and allergies (other infectious diseases, inflammatory diseases of different organs and systems, acute allergic reactions, malignancies, decompensated diabetes mellitus); not detected viral RNA by PCR, detection of hepatitis B virus DNA or antibodies to HBcor-Ag.
The comparison group included 55 apparently healthy individuals who underwent a preventive medical examination at Sumy State University Clinic in 2018-2019. The patients were divided into 3 groups depending on the virus genotype (1b -150 people, 2 -19, 3a -102) and the grade of activity (minimal activity -210 people, moderate -68, expressed -9). Patients with undetectable virus genotype and those who were not tested, as well as individuals with genotype 1b were included in the total sample only. General integrative indices (integral severity index -ISI, entropy of leukocyte formula), indices of nonspecific reactivity (resistance coefficient -RC, immunoreactivity index -IIR, neutrophil and monocyte ratio index -NMRI, lymphocytic index -Ilimph, eosinophils and lymphocytes ratio index ELRI, allergy index -IA, nuclear index -NI); indexes of inflammation activity (total index of inflammation -TII, Krebs index -KI, lymphocyte-granulocyte index -ILG, index of leukocytes and ESR ratio -IL ESR), indexes of intoxication (leukocyte index of intoxication -LII, aggression index -Iagr, hematological index of intoxication -HII, leukocyte shift index -ILS, indicator of intoxication -IIS, reactive neutrophil response -NRR) were calculated [12,13].
A clinical and laboratory examination was performed according to the protocol. Clinical blood test (CobasMicros), biochemical blood test were done at the hospital laboratory (SOBASEMira) and at the Sinevo Commercial Laboratory where serological studies -enzyme-linked immunosorbent assay for the determination of antinuclear antibody titer (ANA), antimitochondrial antibodies (AMA), thyroid peroxidase antibodies (ATPO), antibodies to thyroglobulin (ATTG), and polymerase chain reaction (PCR, detection of ribonucleic acid (RNA), virus genotype detection) were also carried out.
The grade of the process activity in the liver was determined by the generally accepted International Classification of Liver Diseases (Los Angeles, 1994), depending on the ALT level.
In terms of epidemiological and sex characteristics, age composition and degree of fibrosis, all groups were representative. Concomitant pathology in all the patients was compensated and in remission. All patients were examined prior to the start of etiotropic therapy.
The statistical processing was performed in Microsoft Office Excel 2010 and IBM SPSS Statistic 23 computer software. Data were checked for group distribution normality using the Shapiro-Wilk test. We used non-parametric methods because the data obtained did not follow a normal distribution. Mann-Whitney U-test was used to analyze the quantitative data. To determine the significance of the differences between the frequency indices in the different groups when comparing the qualitative characteristics, contingency tables using the Pearson χ 2 criterion were constructed. All the used tests were two-sided, P < 0.05 values were considered statistically significant. The results of the study in the text and tables are presented in the form of a median, interquartile range (25th to 75th percentiles).

Results
Among the CHCV patients examined, there were 1.96 times (66.20 %) more men than women (33.80 %). The average age of patients in the total group was 46 (36-55) years.
The vast majority of patients had a subclinical course of acute hepatitis that was detected during a preventive medical examination (97.21 %), and only 2.79 % of patients indicated previously experienced acute viral hepatitis C, while 9.04 % had acute viral hepatitis C in the past.
Comparing the total sample with the HCV genotype 1b patients, the latter were found to be 1.2 times more likely to have telangiectasia (8.67 % versus 6.97 %) (P < 0.05), but no significant difference was observed.
Patients with genotype 2 did not experience increased discomfort in the left hypochondrium, skin rash and jaundice, feeling of a bitter taste in the mouth was 2.9 times less than in the total group (5.26 %), skin itching was twice less frequently (5.26%), hepatomegaly -1.6 times (47.37 %), splenomegaly -1.5 times (15.79 %), heaviness in the right hypochondrium -1.4 times (47.37 %) less frequently, and subicterus or yellowing of the sclera was 1.4 times more frequently (21.05 %). However, there was no significant difference in the severity of these clinical features compared to the total group except for hepatomegaly (P < 0.05).
In patients with virus genotype 3a, itching (5.88 %), telangiectasia (3.92 %) were 1.8 times less common, arthralgia and myalgia -1.5 times less frequent (7.84 %), the presence of skin rashes -1.4 times less frequent (2.94 %), subicterus and yellowing of the sclera (12.45 %) and feeling of a bitter taste in the mouth -1.2 times less frequent (12.7 %), but none of the clinical signs was significantly different from the total group. Thus, the clinical features did not depend on hepatitis C virus genotype and the groups were homogeneous.
In the total group, leukopenia (15.68 %), erythrocytopenia (18.47 %), anemia (6.62 %) and thrombocytopenia (33.10 %) were more common compared with the healthy group (P < 0.05). People with different HCV genotypes also demonstrated these dynamics, except for the patients with 2 genotype, who did not have anemia.
In groups with different genotypes, the changes corresponded to the overall sample, except AI that was higher only in the total group and with 3a genotype; ESR in patients with 3a genotype was lower than in the total group but did not differ from the comparison group; IIS was higher in the group with 1b genotype of HCV than in the comparison group, and it was 1.7 times lower in 3a genotype compared

Оригинальные исследования
to all patients. In contrast to the total group, in patients with 2 genotype, the TII was decreased even more, but NMRI, IA, and ELRI were not changed compared with the healthy subjects ( Table 1). Among individuals with minimal activity, endogenous intoxication indices (Iagr, HII, IIS, ILS) were higher than in the comparison group. Differences from the comparison group were observed in the distribution of leukocyte formula entropy values, RC, Ilimph, ELRI, TII, KI, ILG, IL ESR, ILS, HII, IIS and NRR ( Table 2).
In patients with moderate activity, in addition to the previous group, IA was increased, NMRI, LII were decreased, and ELRI, HII, IIS -without changes. Compared to the total group, LII, Iagr, II had lower values.
The values of the integrative indicators of the sample with expressed activity differed from the total group Iagr, and from the comparison group -in the value of ISI, entropy, KI, ILG, ILS ( Table 2).   [15]. However, more than half of the examined patients with CHCV in Brazil (54.4 %) had F4 [16]. Among all the patients, the vast majority had a clear asthenovegetative syndrome (81.88 %) and a feeling of heaviness in the right hypochondrium (64.76 %). Other authors' publications also state that chronic HCV infection in the early years does not have a clear manifestation.
Only some patients complain of weakness, fatigue and malaise [4].
It is known that CHCV can cause various extrahepatic lesions that need to be considered for diagnosis. This implies the need for examinations on the presence of comorbidities (alcoholism, heart disease, impaired renal function, autoimmune, genetic or metabolic diseases of the liver) [6]. The examination of CHCV patients found an autoimmune component in the disease pathogenesis: cryoglobulinemia, psoriasis, autoimmune thyroiditis, glomerulonephritis, rheumatic heart disease, Recklinghausen disease, although they are rare. According to a systematic review and meta-analysis, the nine most common diseases associated with HCV infection are known. These include mixed cryoglobulinemia, chronic kidney or end-stage renal disease, type 2 diabetes mellitus, B-cell lymphoma, Sjogren's syndrome, late-stage porphyria, rheumatoid arthritis. The authors reported that type 2 diabetes mellitus (15 %) and depression (25 %) had the highest incidence among HCV-infected patients. In addition, 4.9 % of patients could develop symptomatic mixed cryoglobulinemia, and 30 % had true cryoglobulinemia. In fact, CHCV patients had a 12-fold higher risk of mixed cryoglobulinemia than healthy patients did. Other researchers also found that CHCV patients had an increased risk of developing kidney disease and / or endstage kidney disease by 23 %, an increased risk of type 2 diabetes mellitus, and a 60 % higher risk of developing lymphoma. In addition, HCV-infected patients were twice as likely to develop flat lichen, Sjogren's syndrome, rheumatoid arthritis, and depression, with an 8-fold increased risk of late-onset porphyria [17]. The mechanisms by which extrahepatic lesions develop include immunological disorders when chronic virus persistence results in the circulation of immune complexes and other autoimmune phenomena that are directly caused by the virus and associated with its tropism to other tissues [18]. Cardiovascular disorders (from 4.18 % to 37.63 %) had a significant role in patients. Other researchers also evaluated the impact of HCV on the incidence of cardioand cerebrovascular lesions and found that cardiovascular disease was more frequent in CHCV patients by 20 % and cerebrovascular -by 35 % compared to those without HCV [19]. However, some European studies showed very different features of concomitant pathology in CHCV, when the most common disease was diabetes mellitus (20.8 %), metabolic syndrome (15.5 %), and coronary heart disease in a small number of patients (6.2 %) [15], the majority of HCV-positive patients also had diabetes mellitus (18.7 %), chronic kidney disease (4.4 %), and end-stage renal insufficiency requiring hemodialysis (2.6 %) [20]. These differences are explained by the peculiarities of different nosologies incidence in the population of some countries. It is well-known fact that the highest level of metabolic disorders (including diabetes mellitus) among US residents is related to their lifestyle and diet. According to the WHO, the relative mortality rate from cardiovascular pathology in Ukraine is 68 %, and in the USA -31 %, while the death rate from diabetes mellitus in Ukraine is 1 %, and in the USA -3 % [21].
Typical signs were observed in patients with CVHC: thrombocytopenia, erythrocytopenia, leukocytopenia, which was confirmed by the literature data. However, according to the previous studies, the mean count of erythrocytes, leukocytes and platelets did not differ from the values of the control group [22,23], while in our study, platelet count