The basic aim of this study was to find out the association of genotypes and viral load with host age, gender and biochemical outcome and, their association with liver fibrosis progression. Our patient's data showed no significant differences in genotype distribution in relation to gender. Various genotypes, particularly 1, 3 and 4 were equally distributed in gender. However, we observed more HCV dominance in patients with age ≤ 40 years. May be this was due to early assessment of disease. Prevalence of genotypes in our study was: genotype 3 (n = 4287, 70.9%), followed by genotype 1 (n = 802, 13.3%) and genotype 4 (n = 446, 7.4%). Subtypes 3a, 1a and 4a were predominant, whereas mix subtype 4a/5a was also found in some patients (n = 26, 0.4%). Among patients, 2.4% (n = 146) showed untypable genotype. The high prevalence of genotype 3 followed by 1a is according to the previous studies conducted in Pakistan [1, 8, 19]. Idress et al. 2008 reported increased prevalence of HCV infection due to genotype 4 and 1 without increase in frequency of genotype 3 in various areas of Pakistan mainly Khyber Pakhtoon Khawa (1a, 6.56% and 4, 2.30%) and Balochistan (1a, 25.80% and 4, 4.03%). Our results indicated a 4-5 fold increased prevalence of genotype 4 in Pakistan (1.49% to 7.4%). Moreover, genotype 4 is reported to be associated with liver cirrhosis ; there may be risk of increased liver cirrhosis in Pakistan as the percentage of genotype 4 is increasing.
Evaluating the correlation between different clinical markers with genotypes, we observed that four clinical markers AST, ALP, bilirubin level and AAR were significant in all three cohorts' one initial and two validation cohort. This may lead to conclude that these four biochemical markers may be used to differentiate genotypes. However, all genotypes except genotype 4 showed approximately same levels of serum markers bilirubin, ALP, ALT and AAR in patients as shown in Figure 2. We observed significantly high bilirubin, ALP and AST levels in patients with genotype 4 while, the AAR ratio for genotype 4 was considerably lower than other genotypes.
Concerning correlation of viral load with biochemical markers, we found a positive but very weak correlation of ALT with viral load, while a strong negative correlation with bilirubin and serum ALP levels. Weak correlation of serum viremia levels with ALT in our study was in agreement with outcome of Azzari et al. 2000 and Murakami et al. 2004 that the viral load was independent of ALT activity in HCV [13, 21]. Zechini et al. 2004 found a relation between HCV viral load and AST. However, we could not observe any correlation between viral load and AST that may be due to poor immune response resulting in uneven AST level and viral load and lead to liver damage .
High bilirubin level is usually associated with liver metastases and liver tumor involvement leading to hepatocellular carcinoma and liver cirrhosis by active or non-active HCV or HBV . Bilirubin has been reported as marker of liver injury and to determine the proper dose of interferon in patients with different genotypes . As different genotypes lead to diverse severity of liver disease so the treatment plan of chronic HCV infection with interferon varies with the genotype being treated .
Elevated aminotransferases levels act as indicators of liver cell injury [21, 25] and are usually predominant in liver cirrhosis with increased ALT levels . We observed mild increase in AST and elevated ALT level in genotype 4 as compared to other genotypes. These results could lead to the confirmation of association of genotype 4 with increased risk of cirrhosis .
In our study, patients with genotype 4 reflected high ALP levels as compared to others. Recent studies revealed that the higher levels of ALP are usually associated with liver metastasis, extra-hepatic bile obstruction, primary biliary cirrhosis, intrahepatic cholestasis, infiltrative liver disease, hepatitis, cirrhosis, primary sclerosing cholangitis, hepatic lymphoma, liver abscess, sarcoidosis and congestive cardiac failure. A change in ALP levels greater than 120 U/L can be indicative of advanced disease progression [27–29].
Although high AAR value indicates more chances of cirrhosis, we found low AAR levels for genotype 4 as compared to others leading less chances of cirrhosis; but according to Reedy et al. (1998), this test cannot predict significant cirrhosis in patients with chronic hepatitis C . We also observed slightly low Hb levels in HCV patients irrespective of genotype. As HCV is associated with many extra hepatic complications, decline of Hb level with the increase of viral load in HCV may lead to autoimmune haemolytic anemia (AIHA) that can contribute to enhance the liver cirrhosis especially in patients with genotypes 1-4 as the patients with HCV related AIHA have higher prevalence of cirrhosis . Higher ALP and bilirubin levels and mild increase in AST levels in patients with genotype 4 may also lead to cholestatic hepatitis that is a severe form of HCV recurrence after treatment and organ transplantation like liver, kidney and heart [24, 32].
We got consistent correlation of ALP, AST, bilirubin, with viral load and genotypes in all three cohorts that is initial cohort and two validation cohorts (Figure 3). We further evaluated the relation of these serum markers with fibrosis stages. As host factors reflect the disease progression, we found that serum bilirubin, ALP and AST gradually increased as fibrosis progressed (Table 4). Fibrosis progression was found to be independent of genotypes, while we found a significant correlation between serum HCV RNA levels and fibrosis stages as shown in Figure 4 (Table 4). As genotype 4 showed different results compare to other genotypes with serum markers we were unable to confirm these results in biopsy samples as we only get biopsy samples of genotype 3 and 1. Our previous pilot study also showed same trend . Kato et al. monitored significantly higher HCV RNA level in patients with chronic active hepatitis and cirrhosis compared to chronic persistent hepatitis . Serum HCV RNA level has linear relationship with amount of virus in the liver that is in accordance to De Moliner et al. 1998 results . In our study, we observed relatively high viral load in initial fibrosis stages (F0-F1) compared to advance fibrosis stages (F2-F3). An interesting finding was significantly lower viral RNA levels and high bilirubin, AST and ALP in cirrhosis (F4). The increased AST level had been attributed to mitochondrial injury associated with HCV infection and progression of liver fibrosis . In cirrhotic stage (F4) decline in serum HCV RNA levels could be due to reduce number of hepatocytes and advance fibrosis which results in shrinking of liver mass . We also observed a steady increase in serum ALP and bilirubin levels in advanced stage F3 as compared to initial fibrosis (F0-F2) stages that usually observed in significant liver disease or in hepatocellular carcinoma [23, 27, 28, 35, 36].
In conclusion, host serum biochemical factors were not found to be dependent on genotype except for genotype 4, where we observed high level of bilirubin, ALP, ALT and lower AAR. In general, viral load showed significant correlation with bilirubin, ALP and ALT. To confirm these results and finding; viral-host association with disease progression was evaluated in liver biopsy samples. We observed that lower viral load and elevated bilirubin; ALP and AST levels are associated with more advanced fibrosis leading to cirrhosis. It is conceivable that serum viral load, AST, ALP and bilirubin levels are suitable factors that can determine liver damage. Although, genotype 4 showed significant variable response to the serum markers, we recommend genotyping assay to find possible association with disease severity and guide about treatment duration and outcomes. Future studies are required to see the relation of the serum markers with genotype 4a infected biopsy samples to find any relation with disease progression.