Simultaneous Genotyping And Quantification Of Hepatitis B Virus For Genotypes B And C By Real-time Pcr Assay And Application For Drug Sensitivity To Genotypes B And C

Hepatitis B virus is one of the most serious and prevalent health problems, affecting more than 2 billion people worldwide. Chronic HBV infection greatly increases the risk for liver cirrhosis and hepatocellular carcinoma (HCC). HBV infection is associated with up to 80–90% of HCC patients in China, India, Korea, Singapore and Vietnam. Although highly effective vaccines against hepatitis B virus have been available since 1982, there are still more than 400 million chronic carriers, 75% of whom reside in Asia Pacific region. The evolutionary history of major hepatitis viruses in different human populations includes the origin of phylogenetic variants named genotypes. Several clinical and epidemiological observations suggest that genetic differences in viral genotypes may underlie differences in biological and clinical behaviours. Similarly to hepatitis C virus (HCV), HBV persistence and progression to chronic liver disease are thought to result from a combination of viral and host factors. More importantly, although for HCV infections genotyping has become an essential part of therapeutic algorithms, the evidence that HBV genotypes play any role in response to antiviral treatment is much less clear. The problem is further compounded by the fact that drugs which have exclusive antiviral activity against HBV, such as nucleos(t)ide analogues (NUC's), seem not to be influenced by genotypes, whereas interferon-a does.Several studies indicate that HBV genotypes may affect the disease profiles and clinical outcome in chronic HBV infection. Genotypes B and C are the two most common HBV genotypes in China accounting for approximately 95% of patients. Here we wanted to develop a rapid and sensitive method for concurrent quantification and identification of HBV genotypes B and C in a single-step reaction. And we used this novel method to evaluate the relationship with HBV genotype and drug sensitivity.Background/Aims. Hepatitis B virus (HBV) is an important cause of human chronic liver diseases and is a major public health problem. Viral load and HBV genotype play critical roles in determining clinical outcome and response to antiviral treatment in hepatitis B patients. Viral genotype detection and quantitation assays are currently in use with a different effectiveness. In this study, a real time genotyping and quantitative PCR (RT-GQ-PCR)-based method was wanted to develop and to provide simultaneous identification and quantification of genotypes B and C in a single reaction. And we investigated the genotype changes of hepatitis B virus (HBV) genotypes B and C in chronic hepatitis B (CHB) patients during adefovir dipivoxil treatment and try to evaluate the relationship with HBV genotype and drug sensitivity.Methods. The genotype-specific PCR primers and hybridization probes were selected to genotype and quantify viral loads simultaneously for HBV genotypes B and C from infected patients. Our RT-GQ-PCR correctly identified all predefined genotypes B and C, and no cross-reaction between genotypes was observed. The RT-GQ-PCR identified more cases of HBV infections with mixed genotypes B and C than direct sequencing did. Genotyping 127 samples from HBV infected Chinese patients with RT-GO-PCR revealed 56.7% HBV as genotype B, 13.4% as genotype C and 29.8% as mixed-genotypes B and C. Longitudinal study of HBV genotypes was performed in 98 patients with adefovir dipivoxil treatment. Serum samples from 53 CHB patients were included in seven time points (0, 4, 8, 12, 24, 36 and 48 week) and 45 in five time points (0, 8, 12, 24 and 48 week).Results. At the baseline of adefovir dipivoxil treatment, genotypes were B 68.37% (67/98) of cases, C 10.20% (10/98) and mixed B/C 21.43% (21/98). After 48 weeks'treatment, there are B 80.41% (78/97) of cases, mixed genotype B/C 15.59% (19/97) and genotype C 0% (0/97), with one negative patient. All genotype C (100%, 10/10) were changed, while only 10.61% (7/66) of genotype B changed. In the mixed genotype B/C, 14 samples had genotype changes, of which 10 samples (71.43% [10/14]) converted into a single genotype B at 48 weeks. Genotype C has the higher HBeAg seroconverison rate (70.00% [7/10]) and the higher rate (80.00% [8/10]) of HBV-DNA levels reduction compared to genotype B (42.84% [29/67]; 49.25% [33/67]) and mixed genotype B/C (57.14% [12/21]; 47.62% [10/21]). Genotype changes were observed in 32 patients (32.99%, 32/97). In the 48 weeks of ADV treatment, a total of 38 patients (38.78%, 38/98) there have been mixed genotypes.Conclusions: This assay provides a reliable, efficient, and cost-effective means for quantification of the B and C genotypes of HBV in single or mixed infections and is suitable for diagnosis of HBV, sequential monitoring of viral load levels in response to antiviral therapy, and determining the relationship between the genotype, viral load and stage of disease in Asians. We found that hepatitis genotype C is more sensitive and has better virologic response than genotype B to adefovir dipivoxil treatment. Our data suggest that the existence of genotype switch and mixed genotypes is ubiquitous in the antiviral therapy. Identification for HBV genotypes should be considered in future clinical trials of antiviral therapy of chronic hepatitis B.