Abbreviations

ALT – alanine aminotransferase

AST – aspartate aminotransferase

BMI – body mass index

CHC – chronic hepatitis C

EBR – early biochemical response

EHR – early histological response

EIA – enzyme immunoassay

EVR – early virological response

HAI – hepatitis activity index

HCV – hepatitis C virus

HCV RNA - hepatitis C virus ribonucleic acid

Helicobacter spp. – Helicobacter species

H. pylory – Helicobacter pylori

IFN – interferon α-2b

PCR – polymerase chain reaction

PEG IFN – peginterferon

RBV – ribavirin

SVR – sustained virological response

SBR – Sustained biochemical response

INTRODUCTION

Chronic hepatitis due to hepatitis C virus (HCV) infection is a worldwide disease representing a serious public health problem. Chronic hepatitis C (CHC) infection affects nearly 170-200 million people worldwide. The prevalence of anti-HCV at this time in the general adult population of Lithuania is nearly 50 thousand (0.9%).

Without effective treatment strategies, hepatitis C - related morbidity and mortality is expected to increase nearly 3-fold by the year 2015. Current hepatitis C therapies are aimed at achieving eradication of HCV infection as a means of delaying progression to end-stage liver disease and preventing the development of hepatocellular carcinoma. The treatment options include interferon (IFN), ribavirin (RBV) and peg interferon’s a-2a and a-2b (PEG IFN). IFN-based regiments for the treatment of CHC have become increasingly effective and are to eradicate virus in more than one half of cases. In CHC, combination therapy with IFN and RBV represents an important advance both for naive patients, and for those who did not respond to or went into relapse after IFN monotherapy. The efficacy of IFN plus RBV combination therapy is enhanced significantly over that of IFN alone and is reported to be around 20–40%. In the economically developed Western countries the new therapy of CHC with PEG IFN’s has been shown to achieve 60–80% HCV eradication rates. Unfortunately, because of high cost or delays in approval/distribution, the PEG IFN’s are not universally available. In the majority of Eastern European, Asian, South American countries, as well as in Lithuania the combination therapy with IFN and RBV is still the standard treatment for patients with CHC.

The impact of wide range of viral patient factors on response has been evaluated to determine whether a successful outcome could be predicted before initiating IFN-based therapy. Overall, the most consistent pre-treatment predictors of sustained virological response rate to IFN-based therapy are genotype non-1, low viral load, lighter body weight, younger age and absence of bridging fibrosis/cirrhosis. But as treatment of HCV infection has evolved, additional outcomes have gained importance, such as histological response (improvement of inflammation and fibrosis in liver biopsy). The value of liver biopsy in predicting treatment response is incompletely defined and the relation of pretreatment liver biopsy findings to standard IFN and RBV treatment outcomes is heterogeneous.

Chronic inflammation per se is known to be a factor of progression towards cancer, though HCV in itself does not appear to foster a strong inflammatory mechanism. Recently, some Helicobacter species (Helicobacter spp.) associated with the pathogenesis of gastric and extra-digestive manifestations have been detected in the liver of persons suffering from cholestatic diseases and hepatocellular carcinoma arising from non-cirrhotic liver. Helicobacter hepaticus is currently the best studied of the enterohepatic Helicobacter spp. and has many features common with Helicobacter pylori: both persistently infect their hosts and cause chronic inflammation which can progress to carcinoma. Whether Helicobacter spp. could act as a cofactor in the progression towards cirrhosis and carcinogenesis in humans with viral hepatitis, is still questionable. The discovery of this Helicobacter spp., in conjunction with a number of clinical observations, has raised the possibility of relationship between Helicobacter infection and liver diseases.

Therapy has improved in recent years and eradication of HCV by treatment is a reality in many chronically infected patients, but the issue of a substantial number of non-responders is still unsolved. Currently, other approaches are under evaluation ‘in vitro’ studies. Four broad groups of new therapeutic agents appear to be promising: modified IFN’s and RBV, viral lifecycle targets, immunomodulators, and antifibrotic agents.

THE AIM AND OBJECTIVES OF THE STUDY

Aim of the study – to assess peculiarities of patients with chronic hepatitis C, and to define the predicting factors of interferon a-2b and ribavirin treatment outcomes.

Objectives

1. To evaluate clinical, biochemical, virological and morphological peculiarities of patients with chronic hepatitis C.

2. To define whether Helicobacter species DNA could be found in Helicobacter pylori-seropositive patients with chronic hepatitis C.

3. To analyze changes of biochemical parameters and changes of liver lesions (grade of necroinflammation, stage of fibrosis and grade of steatosis) after 24-week interferon a-2b and ribavirin combination therapy.

4. To assess early virological, biochemical and histological treatment responses.

5. To assess sustained virological and biochemical treatment responses.

6. To define the predicting factors of interferon a-2b and ribavirin treatment outcomes.

MATERIAL AND METHODS

Characteristics of studies, carried out in 1997-2003 in Kaunas University of Medicine Clinic of Gastroenterology have been included in the doctoral dissertation, and they are presented in Table 1.

Assessment of peculiarities of patients with chronic hepatitis C

From January, 1997 to December, 2003, 141 adult patients with established CHC referred to the Kaunas Medical University Clinic of Gastroenterology were included into the study. The study was approved by the Kaunas regional ethics committee of biomedical trials (protocol Nr 163).

Patients were eligible for the study if they had biochemical, virological and histopathological signs of CHC. Patients with serological evidence of active B or D virus infections, decompensate cirrhosis, alcohol or intravenous drug abuse, pregnancy, malignancy, autoimmune-type disease, changes in hematological values, pre-existing psychiatric disease, and seizure disorders, were excluded.

Patients were defined as treatment-naive patients, non-responders to previous IFN monotherapy (continued presence of detectable hepatitis C virus ribonucleic acid (HCV RNA) by polymerase chain reaction (PCR) at the end of treatment), and relapses after IFN monotherapy (the absence of detectable HCV RNA in the serum at the end of treatment followed by subsequent detectability of HCV RNA in the 24 weeks after the end of treatment).

On the day of inclusion in the study clinical characteristics of the disease for patients with CHC were estimated during interview and blood sample was obtained for laboratory analysis. A general questionnaire form was applied for collecting the clinical and life style information from such patients. The questionnaire included information about a source of transmission of HCV, data and age at the time of acquiring HCV, previous CHC treatment methods, information on the duration, amount, and type of alcohol consumed.

Testing for qualitative HCV RNA by PCR and HCV genotyping (virological characteristics) performed by a line probe assay was carried out in a single laboratory for all patients according to manufacturer’s instructions. Sera for alanine aminotransferase (ALT) and aspartate aminotransferase (AST) analysis (biochemical characteristics) were drawn from all patients. ALT and AST were evaluated by an automatic analyzer without pyridoxal-5-phosphate according to the method of International Federation Clinical Chemistry (IFCC).

Percutaneous liver biopsy with standard Menghini (1.4–1.6 mm diameter, 70–88 mm length) needle or transjugular liver biopsy with Tru-Cut (2.2 mm diameter, 15 cm length) needle was performed, and morphological characteristics of the disease for patients with CHC were assessed. A single pathologist who was unaware of the patient’s diagnosis and clinical data scored liver biopsies for steatosis, hepatitis activity index (HAI) and fibrosis stage (morphological characteristics).

Detection of Helicobacter species DNA in hepatic tissue of patients with chronic hepatitis C

All in all, 20 patients with CHC and 34 patients with various liver diseases (control group) without contraindications for liver biopsy were included into the study. Percutaneous liver biopsy was performed by standard Menghini (1.4–1.6 mm diameter, 70–88 mm length) needle and liver specimens were collected from patients with CHC, primary billiary cirrhosis, primary sclerosing cholangitis; extra – hepatic cholestasis due to choledocholithiasis and pancreatitis; fatty liver and steatohepatitis, and some other chronic liver diseases (hemochromatosis, drug related hepatitis, chronic hepatitis B). Paraffin embedded liver biopsy samples were tested by PCR for presence of genomic 16S ribosomal RNA (16S rRNA) of Helicobacter spp. using specific primers and a gene encoding a 26 kDa surface protein. DNA extraction was performed using QIA amp DNA mini kits (Germany) and purified using QIA amp kits up to 50 kb in size with fragments of approximately 20–30 kb predominating. 2.5 U/ml of rTth (MBI Fermentas, Vilnius, Lithuania), 25 mM MgCl2 were used for PCR amplification.

Immunoglobulin G antibodies against Helicobacter pylori (H. pylori) in sera were detected by enzyme immunoassay (EIA) (human gamma globulin, Pharmacia & Upjohn, Stockholm, Sweden, Exel Gel, Pharmacia Biotech AB, Uppsala, Sweden, Millipore, Intertech, Bedeford, Massachusetts, USA).

Assessment of interferon a-2b and ribavirin treatment efficacy for patients with chronic hepatitis C and evaluation of factors which may influence treatment outcomes

One hundred and forty one patients with inclusion and exclusion criteria mentioned in previous section was eligible for the present study. Patients were assigned to treatment course of intramuscular IFN (RealdironÒ, Sicor Baltic, Lithuania) at a dose of 3 MU three time a week in combination with oral RBV (RebetolÒ, Schering-Plough), administered twice daily according to body weight (<65kg: 800 mg daily; 65–85 kg: 1000 mg daily; >85 kg: 1200 mg daily). According with guidelines of scientific societies in Lithuania, IFN plus RBV combination therapy was stopped, if HCV RNA by PCR were detectable at the end of 24-week IFN and RBV treatment period.

Patients were assigned to treatment and assessed as outpatients for tolerance and efficacy every 24-week. The early treatment results: early virological, biochemical, histological, and complete responses were evaluated according to the level of ALT and AST, HCV RNA and changes in liver lesions (grade of necroinflammation and stage of fibrosis) at the end of 24-week treatment period. Early virological response (EVR) was defined as undetectable HCV RNA in serum, early biochemical response (EBR) – as normal ALT and AST level, early histological response (EHR) – as a 2-point or greater decrease in inflammatory score comparing the final with pre-treatment biopsy. Improvement was assessed for inflammation using HAI according to Ishak method (range 0–18) and fibrosis according to Metavir (range 0–4). Early complete response to treatment was defined in the case of association of virological, biochemical and histological response rates.

Patients with EVR were assigned for further 24–weeks combination therapy with IFN and RBV. At the end of 48–week treatment period, therapy with IFN and RBV was stopped. After 24–weeks of further follow-up without therapy, late treatment results, i.e. sustained virological response (SVR) and sustained biochemical response (SBR) were assessed. SVR was defined as a negative HCV RNA, SBR – as normal ALT level 24–weeks after the completion of therapy. When adverse events others than anemia (thrombopenia, leukopenia) were reported as intolerable by the patient, IFN dose was reduced. The dose of RBV was adjusted relying on hemoglobin levels.

Clinical, biochemical and morphological factors that may have contributed to the response to treatment were evaluated (gender, age, BMI, route of HCV transmission, HCV genotypes, consumption of alcohol, ALT, AST level, HAI, fibrosis and steatosis).

Statistical analysis

SPSS for Windows 10.0 statistical package was used for research analysis of study data. Statistical significance between parameters was evaluated according to the probability criteria included in the statistical program Statistica for Windows 5.0. Student criteria (t) was applied for evaluation hypothesis about probability equation. ANOVA statistical analysis was applied for calculation of mean and variance statistics. The obtained data were analyzed and compared using χ2 or Student's t-test. To test the association Pearson (r) and Spearman correlation coefficient was used. For evaluation of continuous variables the statistical mean and standard deviation (SD) were used. Values of p<0.05 were considered significant.

RESULTS

Peculiarities of patients with chronic hepatitis C

One hundred and forty one patients with a mean age of 48.1±14.1 year, 73 (51.8%) men and 68 (48.2%) women, had a mean duration of the disease – 18.6±10.1 year, mean age at the time of acquiring HCV was 28.5±13.4 year. Patients were divided into subgroups according to the previous treatment method: 82 (58.2%) – treatment naive, 40 (28.4%) – non-responders and 19 (13.5%) – relapses after IFN monotherapy. No statistically significant difference between the subgroups of patients was noted for age, gender, body mass index (BMI), ALT, AST activity (Table 2).

The distribution of HCV genotypes was as follows: genotype 1 – 82.0%, (1a – 9%, 1b – 73%); genotype 2 – 6.0%, (2a – 3%, 2a/2c – 3%); genotype 3 – 12.0%.

HCV transmission route: transfusion of blood products – 39 (27.7%), blood donation – 25 (17.7%), tattooing – 9 (6.4%), intravenous drug abuse – 3 (2.1%), exposure to healthcare – 3 (2.1%), haemodialysis – 1 (0.7%). For 61 (43.3%) patients HCV transmission route was not identified.

Rarely used alcohol (<3 standard alcohol units) – 58 (41.1%), never use alcohol – 76 (53.9%) patients, there were 40 (28.4%) smokers and 101 (71.6%) – non smokers. There was statistical significant association between the amount of alcohol intake and ALT, AST activity. For patients who consume alcohol, ALT and AST was higher (186.8±104.3 U/l, 124.9±65.3 U/l, respectively) to compare with non drinkers (125.3±92.3 UI/l, 92.3±78.6 U/l, respectively) (p<0.05).

Liver histology results are shown in Table 3. More severe HAI, focal necrosis and portal inflammation was assessed in naive patients. These differences may be due to previous treatment with IFN monotherapy.

In our sample, 25 (17.7%) patients had cirrhosis (fibrosis stage ≥4).

Association of clinical, biochemical and morphological characteristics of patients is presented in Tables 4 and 5. There was low correlation between age and duration of the disease, age and fibrosis stage. No statistically significant association between ALT, AST activity, AST/ALT ratio and liver lesions (grade of necroinflammation and fibrosis stage) were noted (Table 4). There was a low association between AST/ALT ratio and the stage of fibrosis (Table 5).

Liver steatosis is a frequent finding in the biopsy of patients with CHC. In our own study 67% of patients had liver steatosis. Steatosis was graded as mild in 60.3%, moderate in 5.7%, severe in 1.4%. Compared with patients without steatosis, patients with steatosis had a higher BMI and reported a higher alcohol consumption (Table 6).

Whatever the cause, liver steatosis may contribute to progression of fibrosis in patients with HCV. Therefore, we evaluated the association of grade of liver steatosis with liver lesions (both necroinflammation and fibrosis) (Table 7). Grade of necroinflammation was more severe in patients without steatosis. There were no association between presence of steatosis and fibrosis stage.

Detection of Helicobacter species DNA in hepatic tissue of patients with chronic hepatitis C

Investigation for Helicobacter spp. DNA was performed of liver specimens of 54 patients: 20 (37.0%) patients with CHC and 34 (63.0%) with other liver diseases (control group). The study group comprised 29 (53.7%) men and 25 (46.3%) women, mean age 47.0±12.3 years.

Forty seven (87%) of investigated patients: 16 (80%) patients with CHC and 31 (91.2%) with other liver diseases were H. pylori-seropositive by EIA. In 13 (24.1%) H. pylori-seropositive patients, Helicobacter spp. DNA in liver samples were detected: 3 (18.8%) – with CHC (1 of them with cirrhosis) and 10 (32.3%) – with other liver diseases. Helicobacter spp. DNA in liver samples was not detected in H. pylori-seronegative patients. Helicobacter spp. DNA in liver specimens was detected statistically significantly more frequently in subgroup of patients with extra hepatic cholestasis (60 %) than in patients with CHC (15%), p=0.046 (fig. 1).

Assessment of interferon a-2b and ribavirin treatment efficacy for patients with chronic hepatitis C and evaluation of factors which may influence treatment outcomes

One hundred and forty one patients with the prevalence (82.0%) of HCV genotype 1 were assigned to a 24-week IFN and RBV combination therapy. The baseline characteristics of patients are given in part previous section. Flowchart of treated patients is presented in fig. 2.

Twelve (8.5%) patients discontinued treatment earlier than at 24 weeks due to side effects of IFN and RBV therapy. In 6 (4.2%) patients after 24-week treatment period liver biopsy was not performed. Therefore, 123 patients, mean age 47.1±14.6 year, 68 (55.3%) men and 55 (44.7%) women, completed 24-week treatment period. 68 (55.3%) naive patients, 37 (30.1%) – non-responders and 18 (14.6%) – relapses after IFN monotherapy were included for per protocol analysis. The clinical, biochemical and morphological characteristics of patients are presented in Table 8. No statistically significant differences among the subgroups were noted for age, gender, BMI, ALT, HAI and fibrosis.

Early treatment results of interferon α-2b and ribavirin combination therapy

EVR was achieved in 38 (30.9%), EBR – in 80 (65.0%), EHR – in 97 (78.9%), early complete response – in 38 (30.9%) patients. No statistically significant difference between subgroups of patients was noted for EVR, EBR and complete response rates (Table 9). Only EHR rate was more frequent in naive patients (89.7%) to compare with relapse patients subgroup (44.4%).

For assessment of early treatment response rates between treatment-naive patients and patients treated with IFN, we have joined non-responders and relapses after IFN monotherapy into one group and called them as non-naive patients. Fig. 3 presents data on the prevalence of early treatment response rates in subgroups of treatment-naive and non-naive patients. Treatment-naive patients had better early treatment response rates than non-naive patients in all dimensions, but statistical significance is reached only on evaluating EHR (89.7%; 65.5%, respectively).

Long term treatment results of interferon α-2b and ribavirin combination therapy

Thirty eight patients with EVR (out of 123) have completed a follow-up visit at the end of 72-weeks. In 25 (20.3%) patients, SVR was achieved: in 17 (25%) naive patients, in 5 (13.5%) non-responders and in 3 (16.7%) relapses after IFN monotherapy. Of 38 patients, who had EVR, 65.7% achieved SVR.

For assessment of SVR rates between treatment-naive patients and patients treated with IFN, we have joined non-responders and relapses after IFN monotherapy into one group and called them as non-naive patients. Fig. 4 presents results of SVR rates in patient’s subgroups. Naive patients (25%) have a better chance to achieve SVR than non-naive patients (14.5%).

Assessment of clinical, biochemical and morphological factors which may influence treatment outcomes

Extensive studies have investigated the underlying reasons for the insufficient treatment response rate in patients with chronic HCV infection. In our own study, some pre-treatment clinical, biochemical and morphological factors and their association with long term treatment results were assessed. There is no correlation between the age of patients, duration of the disease, BMI, intake of alcohol, smoking habits, pre-treatment ALT, AST activity, liver lesions (grade of necroinflammation, stage of fibrosis, and the grade of steatosis) and SVR rates (Table 10). We have noted that there is a trend for patients, who achieved SVR, to have lower BMI but no level of significance was achieved.

In the group of 123 patients, association of liver lesions (grade of necroinflammation and fibrosis stage) with EVR was analyzed. In all treated patients (with and without EVR) there was an obvious drop in total HAI and in scores of histological activity features at the end of 24-week treatment period. According to the baseline findings, only confluent necrosis was found to be significantly lower in patients, who achieved EVR, p<0.05 (Fig. 5). Fibrosis stage was not influenced by the treatment success, p>0.05 (Fig. 6).

Association of liver lesions (grade of necroinflammation and fibrosis stage) with SVR is presented in the Table 11. In those with SVR, there was statistically significant lower fibrosis at baseline and after 24-week of combination therapy than in the patients without SVR (p<0.001). No correlation between total HAI grade, grade of histological activity features and SVR was noted (p>0.05).

Changes in liver steatosis after 24-week of IFN and RBV combination therapy with regard to the EVR is presented in the Fig. 7. Although there is a trend in the improvement of steatosis, but it is rather insignificant in both patients groups: with and without EVR.

CONCLUSIONS

1. In patients with chronic hepatitis C referred to the Kaunas University of Medicine Clinic of Gastroenterology, clinical, biochemical and morphological characteristics were established:

1.1. The vast majority (82.0%) of patients were infected with hepatitis C virus genotype 1.

1.2. Activities of alaninine aminotransferase and aspartate aminotransfe-rase were not associated with the grade of necroinflammation activity. The ratio of aspartate aminotransferase and alanine aminotransferase weakly correlated with liver fibrosis (r=0.396, p<0.001).

1.3. Higher grade of steatosis was more common among those who reported a higher alcohol consumption, and who had a higher body mass index (p<0.05). The higher grade of steatosis was significantly associated with the lower grade of necroinflammation activity (p<0.05). There was no significant association between the higher grade of steatosis and liver fibrosis (p>0.05).

1.4. Age of patients weakly correlated with fibrosis stage (r=0.391, p<0.01)

2. Helicobacter species DNA in the liver samples of H. pylori-seropositive patients with chronic hepatitis C could be found. This non- specific phenomenon was found in 18.8% of patients with chronic hepatitis C and in 32.3% of patients with other liver diseases.

3. In 97.4% of patients, who achieved early virological response and in 50.6% who did not achieved early virological response after 24-week interferon a-2b and ribavirin combination therapy normal alanine aminotransferase levels were assessed (p<0.01). In 94.7% and in 72.9% of patients, respectively, the grade of necroinflammation activity improved (p<0.05). Fibrosis has not been influenced by the treatment success.

4. In 30.9% of patient’s early virological response, in 65.0% – early biochemical response and in 78.9% – early histological response was achieved. In 65.7% of patients with early virological response, sustained virological response was achieved.

5. In 20.3% of patients sustained virological response and in 22% of patients sustained biochemical response were achieved.

6. Less pre-treatment confluent necrosis was significantly associated with early virological response (p<0.05). Sustained virological response was associated with lower grade of liver fibrosis at baseline and after 24-week of interferon a-2b and ribavirin combination therapy (p<0.001).

CLINICAL RECOMMENDATIONS

Liver biopsy is probably not indicated after a course of treatment in the majority of patients but a repeated liver biopsy for patients with CHC has a real value in the evaluation of treatment efficacy and can provide information on changes of liver inflammation and especially liver fibrosis after antiviral therapy. For evaluation of liver histology in patients affected by bleeding disorders transjugular liver biopsy are recommended. Our study showed that some liver lesions, i.e. confluent necrosis and stage of fibrosis are associated with treatment results and could be predictors of antiviral therapy outcomes. For the most difficult to treat patients, i. e. infected with HCV genotype 1 who remain stable in evaluating liver fibrosis after interferon a-2b and ribavirin combination therapy, antiviral therapy with peginterferon plus ribavirin should be recommended. It is reasonable because achievement of liver fibrosis reduction after antiviral therapy is very important as a means of delaying progression of chronic hepatitis C to end-stage liver disease and for preventing the development of hepatocellular carcinoma.

Raktažodžiai: priešvirusinis gydymas, helicobacter, hepatitis C, ribavirin, histology, helicobacter, ribavirinas, interferon a, interferonas alfa, histologija, hepatitas C, antiviral treatment