The Molecular Edge
Viral hepatitis is a serious worldwide health problem, especially when caused by hepatitis B and C viruses. Both viruses are divided into different genotypes with diverse distribution around the world. Several approaches for genotypic determination are taken with commercially available kits using a variety of technologies, including Line Probe Assay (LiPA), direct Sanger sequencing and real-time PCR. Laboratory-developed tests (LDTs) also have been reported in the literature. Furthermore, besides determining the viral genotype, some methods also determine the presence of drug resistance-related mutations.1
Finally, the use of sequencing and real-time PCR methodologies for genetic characterization of the host is becoming more important to determine how the infected patient will respond to treatment. Several papers have been published describing molecular markers to predict treatment response for viral hepatitis, especially for HCV. A good example is IL28B genotyping and response to pegylated interferon/Ribavirin treatment.2
Hepatitis B Virus (HBV)
Despite significant progress in vaccine development, Hepatitis B virus (HBV) infection remains one of the major diseases affecting humankind. HBV is a small DNA virus with a partial double-stranded 3.2 kb genome that has been classified into nine genotypes (A-I) and further segregated into subgenotypes based on sequence divergence over the entire genome.
Effective treatment of chronic hepatitis B is necessary to prevent progression into end-stage liver disease and hepatocellular carcinoma. One of the issues drawing more attention recently is the classification of HBV at the genotype and subgenotype levels and their influence on disease progression and response to antiviral therapies. Substantial advances have been made for chronic hepatitis B treatment during the last decade, since approved treatments have expanded from just one agent to several, including new interferon formulations and nucleos(t)-ide analogues (NA). NAs are better tolerated than interferon and best suited for prolonged treatment, but such drugs are associated with the appearance of drug resistance mutations.
HBV employs its own polymerases for replication; one such enzyme, reverse transcriptase, demonstrates a high rate of nucleotide misincorporation due to the absence of proofreading activity. This feature leads to the appearance of mutations in the viral genome that can be selected due to treatment. Mutations leading to drug resistance have been described for all NAs used so far (lamivudine, tenofovir, entecavir, adefovir, emtricitabine, etc.). HBV genotypes C and D are considered to respond less well to antiviral treatment and some authors propose NAs as the best first option for treatment, while interferon treatment should be considered for treating infections due to HBV genotypes A and B.
Molecular characterization of HBV is a useful tool in the management of infected patients, and data concerning genotype frequency and drug resistance mutations present in different countries are important. We developed a sequencing method in our laboratory that allows the identification of HBV genotypes and subgenotypes, the detection of mutations eliciting resistance to major antiviral drugs, and mutations in the envelope gene (S) related to resistance to HBIg, anti-HBs and vaccine. The report generated by this method assigns the closest consensus sequence, allowing the determination of the viral genotype/subgenotype and a mutation table showing the published mutations from international guidelines. This workflow reduces overall costs associated with separate HBV genotyping and mutational analysis reactions.
In a four-year follow-up in our population, the number of patients with any known drug resistance mutations rose from 28.6% in the first year to 42% in the last year, suggesting that drug resistance is increasing. Our results also showed that there are an increasing number of patients resistant to multiple drugs, especially to lamivudine (LMV), which has been used since 1998. It is known that LMV has a low genetic barrier since one single mutation leads to complete resistance, and about 70% of patients are resistant to it after three to four years of treatment. Several consensus studies for hepatitis B treatment do not recommend LMV as the first drug of choice for HBV treatment, with only few exceptions. Entecavir (ETV) has a high genetic barrier, but mutations conferring resistance to this drug are already present in 2.1% of our patients, as the genetic barrier is lower when the virus is already resistant to LMV. Tenofovir (TDF) is currently chosen by the Brazilian Ministry of Health as the first option for HBV antiviral treatment. Only two mutations associated with partial resistance have been described and are found in only 3.4% of patients.
Hepatitis C Virus (HCV)
HCV is an important human pathogen that causes acute and chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. According to the World Health Organization, 180 million individuals in the world are infected with HCV. The number of different subtypes reported from different geographical locations exceeds 80. Recent studies indicate that diverse genotypes are beginning to emerge in the U.S. and Europe with greater frequency than before as a result of extensive immigration and travel, necessitating a more broad-based means of genotyping samples.
This virus, a family member of Flaviviridae, is a positive-stranded RNA virus with a genome size of 10 kilobases. The genome organization of several prototype isolates has been completely determined. Comparison of these isolates showed considerable variability in the envelope and non-structural regions, while the 5' untranslated region (UTR) and, to a lesser extent, the core region are more highly conserved.
Genotyping is clinically important because genotypes 1 and 4 are more resistant than genotypes 2 and 3 to the current standard of care, pegylated interferon and ribavirin combination therapy. Indeed, most treatment protocols require genotype information to tailor dose and duration of treatment. Typically, only 40-50% of genotype 1 chronically infected individuals exhibit complete and permanent clearance of virus infection after being placed on combination therapy. This long-term response rate is much lower than the 70-80% of treated patients infected with HCV genotypes 2 or 3. This difference has proven to be highly significant in patient management and has led to the use of higher doses and longer durations of treatment for genotypes 1 and 4 infections to achieve better efficacy. In numerous multivariate analyses, genotype-specific differences in treatment response have been shown to be independent of host variables, such as stage of disease progression, age, duration of infection, sex, and HIV and other viral co-infections. It is similarly independent of virus-specific factors, such as pre-treatment viral load, although this also correlates independently (inversely) with response.
As HCV treatment options are growing due to the pipeline of new drugs in clinical trials, drug resistance for these drugs will also become a concern for HCV management, similar to what has been observed with HBV.
Regarding host factors, several recently published studies have shown a correlation between specific polymorphisms located near the IL28b gene, located on chromosome 19, with a significant decrease of sustained virologic response (SVR). Characterization of these polymorphisms has proven to be a better SVR predictor than viral genotyping. The protective alleles of these polymorphisms have a higher frequency in European than in African populations and could explain part of the difference in response between these populations. A testing regimen combining IL28b genotyping, virus genotyping and viral load measurement will help in predicting response to treatment and optimizing the ideal treatment for each patient.
HBV and HCV genotyping are relevant tools for designing an individualized and effective therapy regimen for the treatment of patients with chronic hepatitis. The characterization of human genetic markers such as IL28B is also becoming more important for determining an appropriate course of therapy.
Roberta Sitnik, Cristóvão Luís Pitangueira and João Renato Rebello Pinho work at Albert Einstein Diagnostic Medicine, São Paulo, Brazil.
1. Zoulim F. New nucleic acid diagnostic tests in viral hepatitis. Semin Liver Dis 2006;26(4):309-17.
2. Imazeki F, Yokosuka O, Omata M. Impact of IL-28B SNPs on control of hepatitis C virus infection: A genome-wide association study. Expert Rev Anti Infect Ther 2010 May;8(5):497-9.