Also See : 2010 Hepatitis C:After 30yrs 15%–35% will develop cirrhosis
The aim of this study is to assess the role of HCV genotypes in severity of liver disease in Indian patients
Year : 2010 Volume : 53 Issue : 2 Page : 238-243
Date of Web Publication
Predictors of histological activity and fibrosis in chronic Hepatitis C infection: A study from North India
Sompal Singh1, Ruchika Gupta1, Veena Malhotra1, Shiv K Sarin21 Department of Pathology, G.B. Pant Hospital, New Delhi-110 002, India2 Department of Gastroenterology, G.B. Pant Hospital, New Delhi-110 002, India Abstract
Background and Aims:
The role of hepatitis C virus (HCV) genotypes in the severity of liver disease is still debatable and there is an occasional published report from India. . The aim of this study is to assess the role of HCV genotypes in severity of liver disease in Indian patients. An attempt has also been made to perform a multivariate analysis to identify the predictors of severity of liver disease in chronic HCV infection. .
Materials and Methods:
In this study, 31 newly diagnosed cases of chronic HCV infection over a period of two years were included. Age, sex and serum alanine transaminase (ALT) levels were recorded for each patient. HCV genotypes were identified using Line Probe assay (INNO-LiPA HCV II kit, Innogenetics, Belgium). Histological activity was graded and fibrosis was staged. Univariate and multivariate analysis was done to identify predictors of histological severity and fibrosis. Results: By univariate analysis, age of the patient, serum ALT levels and absence of genotype 3 (i.e., presence of HCV genotype other than genotype 3) showed association with histological activity score; whereas age and histological activity score showed association with fibrosis. However, on multivariate analysis, only serum ALT levels and absence of genotype 3 correlated well with activity score; while only activity score remained a significant predictor of stage of fibrosis. .
This study emphasizes the significant correlation of HCV genotype with severity of liver disease in chronic HCV infection. The stage of fibrosis showed correlation only with activity score as an independent factor. These results would further help in outlining algorithms for therapeutic stratification of patients with HCV infection. .
Keywords: Fibrosis stage, genotypes, hepatitis c virus, histological activity
How to cite this article:Singh S, Gupta R, Malhotra V, Sarin SK. Predictors of histological activity and fibrosis in chronic Hepatitis C infection: A study from North India. Indian J Pathol Microbiol 2010;53:238-43 .
How to cite this URL:Singh S, Gupta R, Malhotra V, Sarin SK. Predictors of histological activity and fibrosis in chronic Hepatitis C infection: A study from North India. Indian J Pathol Microbiol [serial online] 2010 [cited 2010 Nov 22];53:238-43. Available from: http://www.ijpmonline.org/text.asp?2010/53/2/238/64332
After discovery of Non-A, Non-B hepatitis (NANB) in 1975, it took another 14 years to identify hepatitis C virus (HCV) as its predominant causative agent.  Throughout the world, at least six main groups (genotypes) of sequence variants of HCV have been described, along with their more closely related sub-groups (subtypes).  Recent reports in literature suggest that genomic heterogeneity of HCV have a significant influence on disease severity and response to interferon (IFN) treatment.  In some studies, Type 1b, has been shown to be associated with more severe hepatic disease than other genotypes, however other studies have refuted this. , Rare studies have evaluated other predictive factors, including age at diagnosis, moderate/ severe steatosis and alcohol intake for severity of fibrosis. , Although there are a few studies on the distribution of various HCV genotypes in India, ,, there is a lack of studies exploring the relationship of HCV genotypes, if any, with the disease severity in Indian patients. A recent study by Hissar et al. in 2006 showed that there was no significant association between HCV genotype and degree of fibrosis or histological activity. However, they failed to analyze their data by multivariate analysis to evaluate other factors, which may act as predictors of histological activity and fibrosis in patients with chronic hepatitis C infection. The influence of other risk factors may be the cause of the existing discrepancy in literature. The available data in literature shows that the proportion of various HCV genotypes is different in India compared to the western countries. Hence, a study of the influence of HCV genotypes on the severity of chronic HCV infection in Indian patients is essential.The present study aims at identifying the role of HCV genotypes and other predictors of disease severity in chronic HCV infection in Indian patients.
. Materials and Methods
Thirty-one (31) consecutive new cases of chronic hepatitis C infection (anti-HCV positive using third generation enzyme-linked immunosorbent assay (ELISA)) diagnosed over a period of two years (Jan 1999 to Dec 2000) have been included in this study.
Patients with co-existent human immunodeficiency virus or hepatitis B virus infection as well as those with a history of consumption of alcohol or any drug known to cause liver cell injury were excluded from the study. Demographic data (age and sex) and serum alanine transaminase (ALT) levels were recorded in all the 31 patients included. Serum ALT levels were measured as per recommendations of international federation of clinical chemistry (IFCC). HCV ribonucleic acid (RNA) was isolated from serum samples using guanidinium iso-thiocyanate followed by phenol-chloroform extraction.  From the RNA thus isolated, complementary deoxyribonucleic acid (DNA (cDNA)) was synthesized at 42 O C for 60 minutes using avian myeloblastosis virus-reverse transcriptase (AMV-RT, Genei, Bangalore, India) followed by nested polymerase chain reaction (PCR) using two sets of primers (Innogenetics, Belgium) from highly conserved 5' non-coding region, as recommended in line probe assay.  In brief, initial denaturation of the cDNA was done at 94 O C for four minutes. This was followed by 35 cycles of 94 O C (30 sec), 58 O C (20 sec) and 72 O C (20 sec). Final extension was done at 72 O C for five minutes. A part of the first PCR product (5΅l) was re-amplified with internal set of primers for another 35 cycles using the same PCR conditions. Both the synthesis of cDNA and the nested PCR were programmed on a thermocycler (Perkin Elmer Gene Amp PCR System 2400, USA) Genotype of the amplified cDNA was determined using reverse hybridization in a line probe assay (INNO-LiPA HCV II kit, Innogenetics, Belgium). All cases underwent liver biopsy with informed written consent. The liver biopsies obtained from all the cases were adequate for morphologic examination. Sections were stained with hematoxylin and eosin (H and E), reticulin and Masson's trichrome stain for histological assessment, and for grading of fibrosis. The histological grading and staging was done independently by two pathologists. The severity of liver disease was graded on the basis of histological activity score and degree of fibrosis staged using Ishak modification of Knodell's scoring.  .
For meaningful statistical analysis, we divided our cases into those infected by HCV genotype 1, with genotype 3, with "other HCV genotypes" and those with mixed genotypes. The "other HCV genotype" group contained cases infected with a single genotype other than genotype 1 and genotype 3. Pair wise comparison of age and serum ALT levels among various genotypes was done either by student's t test or Kruskal Wallis test, depending on whether the data was normally distributed or not. ANOVA was done on serum ALT levels after logarithmic transformation of data. To identify the predictors of histological severity univariate logistic regression was done. The pair wise comparison was preceded by test (ANOVA or Kruskal Wallis) for overall difference in groups. A P-value of less than 0.05 was taken to be statistically significant. Although our sample size was less, an attempt for multivariate logistic regression analysis was made to find out the independent and significant predictors for severity of hepatitis and degree of fibrosis.
We found genotype 3 infection in 19 patients (61.3%), genotype 1 in five patients (16.1%), "other genotype" (genotype 2 in two cases and genotype 4 in one case) in three patient (9.6%) and mixed genotype infection in four patients (12.9%; with genotypes 4a+5; 4+5; 1+4; and 3+2). The HCV genotype distribution of part of these cases has been previously published.
Age and Genotypes .
The mean age of patients infected with HCV genotype 1 was 50.80 years (SD=5.07), which was higher than those with genotype 3 infection (38.79 years, SD=10.01). This difference was statistically significant (P=0.017). The mean age of cases infected with "other" genotypes was 38.67 years (SD=10.81), where as that of mixed genotype infection was 53.75 years (SD=22.03). The difference in age of patients with genotype 1 or genotype 3 and "other" genotypes was not statistically significant (P=0.068 and 0.984 respectively). However, patients with genotype 3 infection and mixed genotype infection had a significantly different mean age group (P=0.040). The pair wise tests for significance of difference in age were preceded by ANOVA to see for the overall difference in groups (P=0.038). .
Sex and Genotypes .
The percentage of males infected by genotype 1, genotype 3, "other" genotype and mixed genotypes were 40%, 63.2%, 100% and 25% respectively. Whereas the percentages of females were 60%, 36.8%, 0% and 75% in the corresponding groups respectively. . Serum ALT Levels and Genotypes The distribution of serum ALT levels was not statistically a normal distribution. The median serum ALT level in-patients infected with genotype 1 was 109 IU/L (quartile deviation=113IU/L). The median ALT level of patients with genotype 3 was 111IU/L (quartile deviation=37.5IU/L). The median and quartile deviation of serum ALT levels of patients with mixed genotype infection was 130.5 and 114.6IU/L, whereas that of patients infected with "other" genotypes was 56 and 223.5IU/L. Since the data was not normally distributed, a non-parametric test (Kruskal Wallis) was used to find the difference of serum ALT levels between various genotypes, which was found to be statistically insignificant (P=0.980). Analysis of variance (ANOVA) after logarithmic transformation of data yielded similar p value of 0.973.th .
Histology and Genotypes
Histological activity score was calculated for all biopsies using Ishak's modification of Knodell's scoring system (15). The total score (maximum 18) was divided into following groups: minimal hepatitis (score 1-3), mild hepatitis (score 4-8), moderate hepatitis (score 9-12) and severe hepatitis (score 13-18). Some of the histological features noted in our cases are illustrated in [Figure 1] and [Figure 2].
For statistical calculations, the cases were further grouped as minimal to mild hepatitis (score 1-8) and moderate to severe hepatitis (score 9-18). All cases (100%) with genotype 1 infection were in moderate to severe hepatitis group (score 9-18). Of patients infected with genotype 3, 68.4% were in minimal to mild hepatitis (score 1-8), where as 31.6% were in moderate to severe hepatitis group.
Seventy five percent (75%) of cases with mixed genotype infection were in moderate to severe hepatitis group. Of patients infected with "other" genotypes, 66.7% demonstrated moderate to severe hepatitis. The differences in histological activity score between genotype 1 and genotype 3 infections as well as between genotype 3 infection and non-3 infection were statistically significant (P=0.037 and 0.019 respectively, ANOVA).
Fibrosis was also staged using Ishak's modification of Knodell's scoring system (maximum score 6). For statistical analysis, the stages were divided as none to mild fibrosis (score 0-3) and extensive fibrosis to cirrhosis (score 4-6). All cases (100%) with genotype 1 infection showed extensive fibrosis to cirrhosis (score 4-6). Of patients infected with genotype 3, 52.6% were in none to mild fibrosis, where as 47.4% were in extensive fibrosis to cirrhosis group. Seventy five percent (75%) of cases with mixed genotype infection were in extensive fibrosis to cirrhosis group. Of patients infected with "other" genotypes, 66.7% showed extensive fibrosis to cirrhosis. The difference in fibrosis score between genotype 1 and genotype 3 infection was statistically significant (P=0.016, ANOVA), where difference between genotype 3 infection and non-3 infection was borderline (P=0.049, ANOVA). .
Univariate Logistic Regression for Predictors of Severity .
All the cases were divided into two categories, those having minimal to mild chronic hepatitis (n=15) and moderate to severe chronic hepatitis (n=16). Similarly groups having no to moderate fibrosis (n=12) and extensive fibrosis to cirrhosis (n=19) were made. The values of various possible predictors of liver disease are tabulated in [Table 1] and [Table 2]. To find the clinico-pathological predictors of moderate to severe hepatitis (activity score 9-18) and those for extensive fibrosis to cirrhosis (fibrosis stage 4 -6), univariate logistic regression analysis was done for each variable. The results of univariate analysis are shown in [Table 3]. Variables which showed significant correlation with occurrence of moderate to severe hepatitis included absence of genotype 3 infection, serum ALT levels and age of the patient. However, only age and histological activity score correlated with stage of fibrosis. The absence of genotype 3 had only marginal significance for extensive fibrosis (P=0.057) in univariate analysis. In our study, absence of genotype 3 means presence of genotype other than type 3; or presence of mixed genotype infection. Major genotypes (i.e. with more number of cases) included in "absence of genotype 3" were type 1 and mixed genotype infection. .
Multi-variate Logistic Regression for Predictors of Liver Histology
On multi-variate logistic regression, the effect of age on histological activity disappeared and the only independent factors correlating with severity of histological activity were absence of genotype 3, and serum ALT levels.The association of absence of genotype 3, and age, with extensive fibrosis disappeared on multi variate analysis. However, histological activity score remained an important predictor of stage of fibrosis.
Hepatitis C virus (HCV), discovered in 1989 as a causative agent of parenterally transmitted hepatitis, belongs to the family of Flaviviridae and genus Hepacivirus.  The genome of HCV consists of a single stranded RNA of positive polarity. HCV isolates from all over the world shows substantial genetic heterogeneity and currently, these isolates are divided into various genotypes and subtypes based on the phylogenetic analysis of nucleotide sequences of the 5' noncoding region (NS5 region). This division correlates well with the sub-classification based on other sub-genomic regions.  It is well known that the distribution of various genotypes differs according to the geographic region and this may provide clue about the origin of various HCV genotypes. The antigenic differences of genotypes and subtypes have implications for the development of an effective vaccine for HCV and for the optimal design of serological and confirmatory assays for HCV. For example it has been shown that RIBA-2 has diminished sensitivity to genotype 3. It has been suggested in various reports that the genomic heterogeneity among the various genotypes of HCV has important clinical and therapeutic implications. 
A multivariate study of 220 patients showed that HCV type 1 infection was highly prevalent among patients with severe liver disease, including cirrhosis and hepatocellular carcinoma. However, this study also concludes that age and duration of infection are major variables to be considered. 
Other studies have also shown that genotype 1b is associated with advanced liver damage and concluded that the pathogenecity of genotype 1b may be higher than other types. , Similarly, some studies demonstrated that patients with milder forms of chronic hepatitis C are infected by genotypes other than 1b.  However, a few studies have shown higher necro-inflammatory activity associated with HCV genotype 2 along with higher serum ALT levels. One report also suggested that there is no association between HCV genotype and progression of liver disease to cirrhosis.  Similar lack of correlation between HCV genotypes and progression of liver disease has been borne out in other reports as well. , Hence, there is still a prevailing confusion regarding the importance of HCV genotypes in severity of liver disease in chronic HCV infection.
In the present study, both univariate and multivariate analysis was performed to find out independent predictors of disease severity in HCV infection. In univariate analysis, absence of genotype 3 infection, age of the patient and serum ALT levels correlated significantly with histological activity of hepatitis. However, only absence of genotype 3 infection and serum ALT levels correlated well with severity during multivariate analysis. For the stage of fibrosis, univariate analysis showed its association with age of patient and histological activity while absence of genotype 3 was marginally significant; however, this association was lost on multivariate analysis, where histological activity score remained the only significant predictor of fibrosis. On the therapeutic front, studies have shown poor response to interferon-a in HCV type 1b infection while genotypes 2, 3 and 1a are associated with higher response to therapy. , Though there are few available studies evaluating the distribution of various genotypes of HCV in India, ,, only a single report in literature was found, which attempted to correlate HCV genotype with severity of liver disease in Indian patients.  Hissar et al. in their study, found genotype 3 to be the most prevalent genotype (as also reported by us in 2004). There was no significant difference in the activity score and fibrosis scores among the various genotypes in their study.
Although their study included more patients, no attempt was made to analyze the data with multivariate analysis to evaluate other clinical, biochemical or histological parameters for significant association with disease severity or fibrosis.  In contrast to their study, our results indicate, on univariate and multivariate logistic regression analysis, that absence of genotype 3 and serum ALT levels are associated with histological activity scores while activity score itself is an important predictor of stage of fibrosis.
Absence of genotype 3 in our study was the group with predominantly type 1 infection. This is consistent with reports form other part of the world that severity of liver disease in patients infected with genotype 1 infection was more than those infected with other genotypes. Duration of infection may also influence the severity of liver disease, but since the number of cases with known duration of infection (19 cases, 61.3%; data not shown) was less in this study, we could not analyze its role.
Multivariate analysis in our study has significantly improved the knowledge of various factors affecting the severity of liver disease in chronic HCV infection. Although age was significantly associated with both histological activity and fibrosis stage, its effect was lost on multivariate analysis. This can be explained by our finding of association between age and HCV genotype, in that most patients of genotype other than genotype 3 were of the older age group. As far as fibrosis stage is concerned, it is noteworthy that absence of genotype 3 is related to activity score, which in turn has an independent association with stage.
Hence, HCV genotypes may play a key role in predicting severity of liver disease in chronic HCV infection. However, larger studies are required to further validate the results of multivariate analysis of our study.In conclusion, our study emphasizes the significance of HCV genotype in the causation of severe liver damage in chronic hepatitis C infection. In our study, absence of genotype 3 correlated significantly with severe hepatitis. Also, histological activity score, as assessed using modified Knodell's score, is an important predictor of the stage of fibrosis. Further studies with adequate statistical analysis are needed to confirm our results, which may help the clinician in management of patients with chronic hepatitis C infection.
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