Study of the effects of antivirus therapy in patients with chronic liver disease due to hepatitis C virus infection Abbreviated title : Efficacy of antiviral therapy in CLD

Background: Hepatitis C virus (HCV) infection is one of the leading causes of chronic liver disease (CLD). About 80% of those exposed to the virus develop a chronic inf ectio . Hyperhomocysteinemia may develop in these patients although alter ed alanine amino transferase (ALT) enzyme levels are generally associated wit h damage to liver cells. The gold standard therapy for chronic hepatitis C patients is pegylated i n rferon combined with an anti-viral drug (ribavirin). The aim of the present study was to investigate the effect of this combined therapy and the diagnostic significance of homocysteine (Hc y) levels in addition to other parameters in these patients. Methods: 532 CLD patients and 70 healthy controls were recruited for the stud y. All patients were subjected to laboratory investigations including HCV-RNA le vels, complete blood cell counts, serum levels of homocysteine, ALT, ALP, lipid profile and liver ultrasonographic examination. The outcome of treatment with pegylated interferon α plus ribavirin treatment and sustained virologic response (SVR) was determined 6-9 months post-therapy. Results: Hyperhomocysteinemia was found in 91.35 % of CLD patients. The differ enc in plasma Hcy concentrations reached statistical significance bet ween the patient and control groups. ALT, cholesterol and triglyceride levels were found higher t an normal in the patients group. After receiving a combined therapy for 24 weeks, 43.66 % pa tients showed an SVR; 30.98 % patients were non-responders while 25.35 % patients initia lly responded to therapy but again retrieved positive status of HCV infection six months post-therapy (relapsecirrhotic). The mean levels of plasma Hcy, ALT and ALP were significantly reduced in the responders within 10 weeks of therapy when compared with non-responders a el psecirrhotic. Conclusion: This study strengthens the evidence that supports the importance of standard interferon α plus ribavirin treatment in patients with chronic liver disease du to HCV infection and diagnostic significance of homocysteine levels in addi tion to other laboratory parameters. Introduction Chronic infection with hepatitis C virus (HCV) is one of the lea ding causes of chronic liver disease; about 170 million people worldwide are estimated to be infect ed. Hepatitis C infection causes acute symptoms in only 15% of patients exposed to HC V infection while about 80% patients develop chronic infection [1]. Chronic hepatitis C result s in formation of high levels of free radicals in the liver cells, which put serious xidative stress depleting protective antioxidants and eventually kill the liver cells. Chronic he patitis C infection progresses very slowly and is marked by episodes of acute hepatiti s characterized by liver inflammation and elevated ALT levels. A hepatitis screen i s recommended for patients whereby the disease can be diagnosed by the presence of antibodies f or h patitis C or by the direct presence of the virus or viral products in the blood [2]. Serum levels of liver enzymes such as alanine aminotransferase (ALT), aspartate aminotransfer ase (AST) and alkaline phosphatase (ALP) are analyzed to estimate damage caused to liver . Ele ated ALT, ALP & AST levels are associated with varying degrees of damage to the liver c ells [3]. Homocysteine levels are also altered in chronic liver disease. H omocysteine is a sulphurcontaining amino acid belonging to the group of intracellular thiols. Numerous clinical and epidemiological studies have reported that elevated plasma homocysteine concentrations reflect impaired cellular metabolism [4] and may be considered as an independent risk factor for atherosclerotic vascular disease and thromboembolism [5]. Expe rimental data in transgenic mice deficient in homocysteine metabolism enzymes ha ve shown the presence of severe liver steatosis with occasional steatohepatitis. In huma n beings, many studies have found a correlation between homocysteine and steatosis. Hyperhomocystei nema may result from defects in homocysteine-metabolizing genes; vitamin B 6, 12, or folate deficiencies resulting from nutritional conditions; or chronic alcohol c nsumption [6]. Homocysteine is mainly synthesized and metabolized in the liver, si nce metabolism of majority of dietary methionine occurs in this organ, where about 85% of the whole body capacity for transmethylation resides. Therefore, genes involved in methionine and homocysteine metabolism are expressed in a specific pattern in t he liver [7]. Homocysteine is formed as an intermediate in methionine metabolism; therefore, impa ired liver function leads to altered methionine and homocysteine metabolism [8]. The gold standard therapy for chronic hepatitis C patients is a combined therapy with interferon (pegylated interferon) and an anti-viral drug (ribaviri n). The first trial of interferon as therapy for chronic non-A, non-B hepatitis was reported in 1986. A sust ained virologic response results from combined therapy with both pegylated interfer on and ribavirin in 28% to 50% of patients with genotype 1, while in patients with genotype 2, sustained response rates are higher (76% to 82%) [9,10]. The recommended duration of treatme nt for HCV genotype 2 and 3 is 24 weeks and for genotype 1 is 48 weeks. Sustained vi rologic response (SVR) is usually accompanied by a return to normal serum ALT l evels and improvement in inflammation within the liver. Apparently, the usual effect of int erferon in patients with chronic hepatitis C who respond to this therapy is viral suppression but ot eradication or cure. The genotype and level of viremia are important factors that affect the initial and longterm response to this therapy. The optimal treatment for nonrespond ers and relapsers is not well established. However, it is expected that a minority of nonre sponders (6% to 12%) will respond to a second course of pegylated interferon and ribavirin [11]. The aim of the present study was to investigate the effects of combined antiviral therapy (interferon with an antiviral drug) in patients with CLD due to HCV infection and recovery of normal serum ALT, ALP and Hcy levels in such patients. Subjects and Methods Selection of Subjects Approval for the study was obtained from the Quaid-i-Azam university institutional review board. Informed consent was obtained from all subjects who participated in the study. All the patients were positive for hepatitis C infection while the control subjects were all healthy. The patients were recruited from the KRL hospital Islamabad. Pa tients with hepatitis B virus infection, schistosomiasis, chronic parenchymal and obstructive renal diseases and alcohol abuse were excluded from the study. 532 patients (20-68 years; 272 male s, 260 females) with chronic liver disease (CLD) (Group I) and 70 healthy control subjects (18-55 years; 42 males, 28 females) (Group II) were included in the study. All the patients and controls were subjected to the following investigations: thorough history taking and phys ical examination, laboratory tests including complete blood cell counts (CBC), liver enz ymes (ALT, ALP & AST), lipid profile (serum cholesterol, triglycerides, HDL, LDL) , viral markers (PCR for HCV-RNA for those who tested positive for HCV-Ab), and plasma homocys teine concentration. Abdominal ultrasound and liver biopsy was performed for all s ubjects to evaluate liver and exclude any renal parenchymal disease or obstructive ur opathy. Treatment with Interferon The patient group was given interferon therapy: as a first line of defence, all the patients received 3 million U of uniferon α-2B plus an antiviral drug Ribazole (1000 to1200 mg / day) for at least 24 weeks. A second line of defense therapy (Uniferon or Pegasys + Ribazole) was given after 6 months of completion of first dose only to those patients who were either nonresponders or relapsed back first line of defence. Serum levels of HCV RNA were determined during a follow-up peri od (6-9 months posttreatment) with use of a reverse-transcriptase-PCR assay. A ll the patients were again subjected to thorough physical examination, lab tests, liver biopsy and a bdominal ultrasound through and after the period of medication. Assessment of treatment efficacy A sustained virologic response, defined by the detectable levels of HCV RNA in serum 24 weeks after the end of treatment was taken as primary end point. The absence of the detectable levels of HCV RNA in serum at the end of therapy and the normalization of serum ALT levels were considered as secondary end points. Statistical analysis All values were expressed as arithmetic mean ± SD. The diffe renc of biochemical and other parameters between the control and patient group before treatment wa s tested for statistical significance using t-test. For overall comparisons, one-way ana lysis of variance (ANOVA) was used. The difference in serum homocysteine and liver enzyme le vels between the differently responding-patient groups after interferon treatment wa s investigated by Tukey’s honestly significant difference (HSD) test. A P-value < 0.0001 was considered extremely statistically significant, < 0.001 as very statistically significant and < 0.01 as statistically significant. Results Hyperhomocysteinemia and serum ALT levels before interferon trea tment 532 patients (272 males; 260 females) affected with chronic liver di sease (CLD) were studied. 70 healthy individuals (42 males; 28 females) were included as a control group. Hyperhomocysteinemia was defined as plasma Hcy level > 15μmol/L . The mean level of plasma Hcy was significantly higher in the patient group (25.66+ 8.89) when compared to control group (12.36+ 1.64). Hyperhomocysteinemia was observed in 486 patients (253 males; 233 females). The difference in plasma Hcy concentration reached statistical significance between the control and patient groups (table 1; fi gure 1). Overall ALT levels were found quite higher than the normal in the patient group (