The role of hepatitis C virus genotyping in evaluating the efficacy of INF-based therapy used in treating hepatitis C infected patients in Libya

Background: Hepatitis C virus (HCV) therapy has been evolved over years and many parameters were used to evaluate such therapy particularly genotyping. In North Africa and Eastern Mediterranean the genotypes vary from most of world and rarely studies were conducted to assess the influence of such genotypes on the HCV therapy. Aims: This study was designed to determine; the role of HCV genotyping in assessing the efficacy of interferon therapy and to analyze the rates of sustained virological response (SVR) in INF and PegIFN-based regimens according on HCV genotype infecting Libyan patients with chronic hepatitis C infection. Methods: A total of 479 patients with chronic HCV registered at Tripoli Medical Centre were treated with ‘INF alfa or PEGINFPegylated INF alfa 2a in combination with ribavirin’ for a five year period. These patients were registered and followed up from January 2007 to October 2012. The information were reviewed and data were collected from each patient regarding age, gender, ALT level, and viral load, viral genotype using qualitative PCR. The statistical analysis were carried using SPSS version 11.5. Results: Of the patients studied 86 patients were treated with INF based regimen, only 54% of them had end treatment response (ETR) and 28% had SVR. Off 143 patients treated with peg-INF alfa 2a based therapy; 69% had ETR and 36% had SVR. The SVR of Peg-INF based regimen was higher than INF based regimen in all genotypes except for genotype 4. The relationship between SVR and gender was significant in patients who were given INF based therapy comparable to PEG INF based regimen, though the relationship between SVR and age, basal viral load and basal ALT were also reported in both regimens. Conclusions: HCV genotyping has been found to play an important valuable role in determining the efficacy of Hepatitis C therapy. SVR vary according to the HCV genotype involved. HCV genotype 1 and 4 were found to be the prevalent resistant genotypes infecting Libyan patients. Such findings are particularly important in guiding the clinical therapy of patients infected with hepatitis C virus.


Introduction
Hepatitis C virus (HCV) has been considered to be one of the most ongoing causes of viral hepatitis. Deaths due this virus are expected to be tripled in next 20 years, particularly among developing countries [1]. This imposes a major personal and social burdens on infected individual further to a heavy economic hurdles on health and insurance sectors [2]. Hence then having proper valuable therapy, easily monitored will be priority for clinicians and researchers involved in the management of HCV infection.
Treatment of Hepatitis of C infection has been evolved over years and many regimens has been introduced [3,4]. The early used drug was interferon (INF) alpha; the addition of a polyethylene glycol (Peg) moiety to INF (termed, Peg-INF) has dramatically enhances its span life in blood and thus it reduces the needed dose [5,6]. This efficiency was significantly improved with the addition of guanosine analogue ribavirin (RBV). Combination therapy particularly (Peg-INF) with RBV has been considered to be effective therapies for chronic HCV [7,8]. Such therapy was not well tolerated with all patients. Hence then more effective and tolerable treatments were introduced among them, direct-acting antiviral (DAA) drugs such as telaprevir, and boceprevir, which were approved for clinical use recently [9,10]. These could be used in combination with pegylated interferon and ribavirin particularly in treatment-na¨ıve and problematic patients [11]. Despite the increasing advancement in such therapy and the ultimate increase in the clearance rate which reached up to 80% in different clinical trials, emergence of resistant viral variants has been reported [12]. Hence then, therapy of HC Infection should be effectively monitored and different parameters were introduced to assess its efficacy. Both host and viral factors were found to be associated with such treatment particularly with non-response to PEG-INF. Viral factors include viral load, genotype and quasi-species of Virology Discovery ISSN 2052-6202 HCV. Each patient should be anticipated to follow a specific criterion that might include, progress of disease, predisposing factors, symptomology and viral status [13,14].
Hepatitis C Genotyping plays an important role in the clinico-epidemiological manifestations of HC infection and the existence of six major types virus genotypes and about 100 subtypes, which, have been identified with distinct geographic distributions. Genotypes 1, 2 & 3 accounted for the majority of HCV infections worldwide [15]. Genotype-1, the most common genotype, it is dominant in USA, Europe and most Asia-Pacific [16]. Genotype-2 in Japan, South Korea and southern Taiwan [17,18]. Genotype-3 is prevalent on the Indian subcontinent and Australia. Genotype-4 is predominantly found in the Middle East and North Africa [2] though genotype -5 was limited to South Africa [19], and HCV-6-11 was found in South-East Asia [20]. Such variation starts to change due worldwide massive social surge and cultural diversity.
HCV genotype is the strongest baseline predictor of IFN response and the efficacy of HC therapy has been influenced by HCV genotypes and different genotypes showed different clinical responses to HCV therapy. Hence then, the use of HCV genotyping in assessing the therapy of Hepatitis C infection is becoming valuable important aspect [9,21]. Monitoring of HCV drug resistance before and during treatment is likely to provide important information for management of patients undergoing anti-HCV therapy. The objectives of this study were to evaluate the efficacy of IFN and peg-INF-based regimens and determine factors that influence the response and resistance of the regimens used and the role of HCV genotyping in predicting on monitoring the response of such therapy.

Patients population
A total of 479 patients with hepatitis C virus were studied. The patients were recruited from the Department of Infectious Diseases at Tripoli Medical Centre, Tripoli. All were registered and followed up at Out Patient Department from January 2007 to October 2012. Three hundred and three patients were male and 176 patients were female. (Male; Female ratio 1.7:1). The age was ranged from16 to 84 years with an average age of 40 years at the entry of the study.
The data collected was designed to extract information from patients that may influence the outcome of viral therapy such as age, gender and year of diagnosis of HCV. The participation was voluntary in accordance of with the guidelines for observational and interventional studies of national ethical committees as the research was conducted according to Helsinki Declaration [22] and Libyan National committee for ethical approval number LETC/THCV-T77/2006.

Patient exclusion criterion
Each patient has to fulfil the specific criteria that include; no co infection with human immune-deficiency syndrome (AIDS) virus or with hepatitis B virus and HDV, and none of them had liver cirrhosis, or undergo haemodialysis; and no concomitant metabolic or autoimmune disorder or underlying systemic diseases.

Study design
This randomised clinical study was conducted at Tripoli Medical Centre as patients were randomly assigned. Antiviral therapy was administrated to 229 patients, 86 patients were given INF-alfa plus Ribavirin combination therapy and 143 were given pegylated interferon-alfa 2a plus Ribavirin combination therapy. The INF alfa dose were 3 million IU subcutaneously three times a week for 24 weeks for genotypes (2 and 3) and for 48 weeks for genotypes (1 and 4

Laboratory and Clinical Evaluation of HCV Infection Virological Studies
A serum specimen was collected from each patient and was tested positive for HCV antibody (Anti-HCV) using and 3 rd generation commercial Enzyme Linked Immunosorbant Assays (ELISA) The INNO-LIA TM HCV Ab III update is 3 rd generation line immunoassay which incorporates HCV antigens derived from the core region, the E2 hyper-variable region, the NS3 helicase region, the NS4A, and NS5A regions. The antigens were coated on a nylon strip with plastic backing as 6 discrete lines. Then, in each strip every four control lines were coated: strepavidin control, 3+Positive Control (antihuman Ig), 1+Positive Control (human IgG) and+cut-off line (human IgG). Incubated with test sample then purified alkaline phosphatase was added-labelled goat anti-human IgG and lastly we added conjugate [1].

Determination of HCV genotypes
The genotyping in this study was carried out by gene amplification using COBAS-Amblicor HCV test as early described [1], detected by reverse-transcribing HCV RNA into cDNA by PC, hybridizing amplified cDNA with an oligonucleotid probe that binds enzyme, and catalyzing conversion of substrate to a colored product that is recognized by COBAS AMBLICOR Analyzer (Roche, Diagnostic, Basal, Switzerland).

Determination of viral load
HCV load was performed by COBAS R TaqMan R HCV Test, v2.0 which based on three major processes [1] manual specimen preparation to extract HCV RNA, [2] automated reverse transcription of the target RNA to generation complementary DNA, [3] PCR amplification of target cDNA using HCV specific complementary primers and simultaneous detection of cleaved dual fluorescent dye-labelled oligonucleotide probes. The Master Mix reagent contains primer pairs and probes specific for both HCV RNA and HCV Quantitation Standard RNA as previously published [1]. The detection of amplified using target specific and Quantitation Standard specific dual labelled oligonucliotide probes that permit independent identification of HCV amplicon and HCV Quantitation Standard amplicon, and also quantitation of HCV viral RNA is performed using the HCV Quantitation Standard. The test has limits of detection approximately 50 IU per ml, (Roche, Diagnostic, Basal, Switzerland). Biochemical parameters such as ALT (alanine aminotransferase) (GPT) and AST (aspartate aminotransferase) (GOT) were also analysed and evaluated for each patients.

Statistical analysis
Quantitative variables were expressed as mean ± standard deviation and were compared by Student's test (t-test). Differences in proportion of qualitative variables were tested with non-parametric tests (X2) Yates correlation. Fisher exact test and a p value < 0.05 were considered significant. A multivariate analysis was conducted using logistic regression in order to verify which variables statistically had an influence on HCV infection. such as gender (male vs. female), IV drug abuser (yes or no), blood transfusion (yes or no) surgical Intervention (yes or no), dental care (yes or no); Heterosexuality and homosexuality (yes or no). The calculation of 95% Confidence Interval (CI) to compare groups was determined and the data were analyzed using SPSS version 11.5 to evaluate the efficacy of therapy and its association with genotypes, gender, ALT, viral load, and also to assess the sustained virological response (SVR) in different genotype, and finally the different regimens of therapy efficacy were compared.

Efficacy of HCV therapeutic regimens
Initially 479 patients with chronic HCV were recruited to be included in this study their clinical characteristics were shown in

Monitoring of therapeutic regimens
Both liver function tests and viral load were monitored in this study, off 26 patients on INF based therapy who had ETR had normal ALT and 20 of them had raised abnormal ALT at diagnosis. Though,14 patients who had SVR had normal ALT level at diagnosis where as 10 patient who had SVR had abnormal ALT level at diagnosis. In PEG INF based group Where 57 patients who had ETR had normal ALT and 41 of them had elevated ALT. Furthermore, 25 patients of them who had SVR had normal ALT level at diagnosis where as 26 patients who had SVR had abnormal ALT level at diagnosis. The analysis of viral load showed that, in the first group based regimen 24 of patients who had ETR had low viremia at diagnosis (viral load less than 2 million and 22 of them had abnormal high viremia(viral load higher than2 million) at diagnosis. While 12 patients who had SVR had low viremia at diagnosis at diagnosis where as 12 patient who had SVR had high viremia at diagnosis. In the second group 61of patients who had (PEG INF based therapy) ETR had low viremia and 37of them had high viremia, While 33 patients who had SVR had low viremia at diagnosis where as 18 patients who had SVR had high viremia at diagnosis.

Role of HCV genotyping in monitoring therapy
The ETR and SVR of both regimens were correlated with the different HCV genotypes of the studied patients as shown in Table 3 Figure 1 Illustrates the correlation between Viral response rates of different HCV genotypes and both End of Treatment Response (ETR) and sustained Virologic Response (SVR), according to intention -to -treat analysis.

Discussion
Hepatitis C virus infection results in chronic active hepatitis in more than 80% of infected patients; 20 to 30% of these patients develop progressive fibrosis and cirrhosis, whereas only approximately 10 to 20% of the infected people spontaneously eliminate the virus [23]. Treatment regimens for chronic hepatitis C have significantly improved during the last decade, resulting in higher sustained virological response (SVR) rates [24]. The dual anti-HCV therapy is based on administration  of long-acting pegylated alpha interferon (IFN) and ribavirin (RBV). Different factors have been evaluated as predictors of the sustained response to treatment, with controversial results [25]. In the present study we analyzed the efficiency of two regimens INF or PEG-INF alpha 2; either one was given in combination with RBV in treatment HCV infected patients.    [27]. This is consistent with our result, though we could not analyze the effect on SVR of discontinuing in certain patients. Despite no specific correlation could be made between these studies, it's obvious that the tolerability of antiviral regimens used was satisfactory. Different factors have been known to predict clinical response of antiviral-therapy among CHV patients these may include patient, viral and biochemical parameters [28,29]. Patient associated factors such as gender, age and the ethnic clan, have been studied by many investigators [2,30]. In this study we found a significant relationship between gender and both ETR and SVR in INF based therapy. Female patients had a higher SVR than male patients and the relationship was insignificant in PEG INF based therapy. Such results are compatible with other studies that revealed male sex had worse prognosis to therapy than female, that is may be because male patients have more ability to have liver cirrhosis [25].
The relationship between age groups and SVR were assessed in this study. A younger patients less than 40 years of age, were found to have a higher SVR rate compared with the older ones. This however, is an agreement with other studies, who reported that SVR rate in older patients was remarkably low at 17.4% compared to SVR rate in all patients included in study which was 36.0% these results were in disagreement with other studies carried in Pakistan where a significant relationship between age group and SVR as they found younger patients had higher SVR [31,32].
In this study we evaluated the biochemical parameters that have their influence on the SVR of both regimens used. Its well documented that most patients with an SVR normalize their serum ALT, AST shortly after discontinuing treatment unless other liver disease is present. The relationship between ALT and viral load level at diagnosis were insignificant in both regimens, some studies found inverse relationship between basal viral load and response to therapy [29]. Others did not found such relationship [33]. High ALT levels are correlates with liver cirrhosis but some studies revealed no relationship between response to therapy and ALT level before treatment [5,32]. This however is not the case of our study as those with live cirrhosis did not fit with patient selection criterion we applied.
Different viral related factors have been found to be involved in response of interferon treatment. Viral genotype, genetic diversity, viral load and kinetics where found to be among the most important ones involved. Recently, our group [34] and others [35,36] reported different rates of infection with different virus genotypes which was reflected on the viral response to the therapy used in treating HC infection. Interestingly, several studies have demonstrated that the chance to respond to IFN treatment is related to the baseline viral load. In this study strong association was found between viral load and ETR of patients treated INF based compared with those treated with Peg INF alfa2a therapy (P=0.976). Patients with a high viral load are less sensitive to the treatment than patients with a low viral load. Thus, patients with genotypes, low baseline viral load and RVR may be treated for a short period (eg., 24 weeks), while patients with genotypes, high baseline viral load and without RVR may require longer period (eg., 48 weeks) of treatment.
In this study according to genotypes and on intention to treat, ETR of patients treated PEG INF alfa 2a based regimen were superior to INF alfa based regimen in all genotypes though it was particularly significant in HCV genotype 1 ( 'P value=0.025'). These results were in agreement with the studies carried at University of Southampton [37]. Further more in both regimens HCV genotype 2, had a higher ETR than genotype 1 and 4 which same as previous study was carried in Cameroon [38]. However, both therapeutic regimens were less effective in patients infected with HCV genotype 1 and 4 although much higher ETR rates are reached in individuals infected with genotype 2.
Other studies have shown no differences in the clinical response of patients infected with HCV genotype 2 and 3, when they were treated with PEG INF alfa2a or INF alfa combination therapies [38,39]. The SVR of Peg INF based regimen was higher than INF based Regimen in all genotypes except for genotype 4 because as 11 patient infected with this genotype had ETR of Peg INF miss the follow up and thus excluded for the data analysis. Though, for those patients treated with INF based regimen, SVR was higher among patients infected with genotype 4 comparable to genotype 2 and 3 this may be related to discontinuation of therapy among the patients. The SVR in genotype 1 infected patients was the least in both regimens comparable to other genotypes. These results are similar to other studies carried in France on selected multiethnic patients from France, Egypt and other African countries who were treated with Peg INF alfa 2a based regimen [26,39]. In their study the selected patients were French and Egyptian and other African who had HCV genotype 4 infection on Peg INF alfa 2a they found. SVR was variable according to the ethnicity as it was 54% among Egyptian, 40.3% in French patient and among Africans it was 32.4% [40]. The relationship between HCV subtypes and ETR and SVR were also studied. No significant relationship was found, particularly with patients with HCV genotype 1a and 1b.
Our studies are in accordance with those carried by Zeuzem; who did find heterogeneous virological response rates to interferon-based therapy in patients with chronic hepatitis C based on genotypes and subtypes of HCV [37,41]. Hence then virus clearance rates are variable different depending on viral genotypes and that could be used to monitor HCV therapy.
Despite the limitations that this study facing due to the inability of some patients to finish the due course of the therapy and the discontinuation of the regimens among others. Our results highlights the importance of monitoring antiviral therapy and the factors that influence such therapy among treated patients.
In conclusion, we have found that Responsiveness to hepatitis C virus therapy is variable according to the different regimens used. Certain factors such as gender, age, viral load and genotypes influence viral response rate. Furthermore, genotype 1 and 4 were more resistant comparable with other genotypes. Therefore, New drug therapies such as antiviral protease and antipolymerase should be combined with the ongoing therapy both to increase SVR among non responsive patients and reduce the duration of treatment particularly of those who were infected with resistant genotypes.