Clinical and Phylogenetic Analysis of Hepatitis B Virus Infection and Associations of its Genotypes with Antiviral Therapy in Pakistani Patients.

Abstract

Genotypes of hepatitis B virus and their role in disease severity and treatment response has not been studied from all over the Pakistan. This research was aimed to study HBV genotype prevalence in all parts of Pakistan, relationship of HBV genotypes with viral and biochemical factors, influence of different treatments and pretreatment factors on patients' response, and the mutations responsible for resistance. A total of 840 samples was collected from all over the Pakistan and genotyped by type specific primer PCR method. Viral load, ALT and HBsAg tests were performed for 154 treatment naive patients and the values were compared by ANOVA. One hundred and sixty two chronic HBV patients were compared for three treatments viz. Tenofovir, Entecavir and Peg-Interferon. Patients were monitored for virological response, combined response, HBsAg clearance and HBeAg clearance. The data was analyzed by logistic regression and Chi square tests. Viral reverse transcriptase domain of 20 non-responder patients was sequenced and mutations were detected by aligning the sequences with wild type strains. Genotype D was found to be the most prevalent genotype from all parts of the country as it was present in 71.2% of the samples. The second major sample population (17.3%) was found to be infected with a mixture of different combinations of genotypes, with the dominant combination of A+D (13.7%) while genotype A was the third more prevalent genotype (7.7%). Genotypes B, C and E were found from less than 1% of the samples. Significantly higher values of viral load were found in patients with genotype A+D while no relation of genotype with ALT and HBsAg levels was found. Correlation, however was found between the levels of HBsAg and the HBV DNA quantities of the patients having HBeAg negative infection. Combine response of peginterferon treated patients was higher than entecavir and tenofovir treated patients which have higher rate of virological response than peginterferon treated patients. Genotype A and low baseline viral load were associated to better treatment response. HBV genotype, baseline viral load, HBeAg and ALT were found to be significant factors in at least two of the treatment arms. In tenofovir group, only HBeAg and ALT had a significant influence on response rate, where high base line ALT and negative HBeAg were related with better virologic response. In both entecavir and peginterferon arms, genotype A, low viral load, low ALT and negative HBeAg were associated with better virologic response of the patients. Universally accepted resistant mutations were identified from 8 samples while some new mutations were also found which are still not known for their role in resistance. Known mutations rtL180M/I and rtA181V were found from 6 (30%) samples while rtM204V and rtV173L were found from 5 (25%) samples each. New mutations, rtY135S and rtN248H were found from 13 (65%) and 12 (60%) samples respectively. The study concludes that HBV/D is the most prevalent HBV genotype in Pakistan followed by A+D and A genotypes. Mix infection with A+D is associated with high HBV DNA quantities and low rate of treatment response. Genotype A or D alone, low viral load and negative HBeAg, are significant predictors of high response rate. Treatment with tenofovir and entecavir have better virologic response rate as compared to peginterferon treatment.