Mutation analysis of lamivudine resistant hepatitis B virus strains by TaqMan PCR

Abstract

Hepatitis B virus infected patients on long-term lamivudine treatment are exposed to a 15–32% risk compounded annually of developing resistance mutations. Such resistance results in a progression of the liver damage caused by chronic hepatitis B, and may also impair the effect of other antivirals through cross-resistance. At present lamivudine is used frequently as monotherapy because of its relatively low price and negligible side effects. Thus, simple methods for identifying resistance mutations are required. A method based on real-time polymerase chain reaction with TaqMan chemistry is described. The method combines both primer specificity, in order to target wild type and mutant viral strains at codon 180, and a mixture of three minor groove binding probes distinguishing the YMDD wild type and the YVDD and YIDD variants at codon 204. The accuracy of the method was verified by concordance with results of direct sequencing and restriction fragment length polymorphism when examining 27 samples from five patients, in whom lamivudine resistance was known to have developed. This method is rapid, cost effective, and should prove useful for monitoring patients treated with lamivudine.

Introduction

Hepatitis B virus (HBV) infection is a major health problem with around 400 million carriers worldwide, according to the World Health Organization. Chronic hepatitis B may be complicated by liver cirrhosis or hepatocellular carcinoma (HCC), and causes one million deaths every year. Current treatment includes interferon-alpha and nucleoside analogues such as lamivudine (LAM) and adefovir (ADV).

Lamivudine is a well-documented, potent inhibitor of HBV replication. It is less expensive than newer drugs, safe and well-tolerated, and has been shown to reduce long-term progression of liver damage (Liaw et al., 2004). However, prolonged monotherapy with lamivudine is associated with a relatively rapid selection of resistant virus strains (Tipples et al., 1996), and a subsequent reduction of the virological response resulting in progression of liver damage (Liaw et al., 2004). The accumulating evidence of resistance when using long-term lamivudine monotherapy suggests that this treatment is no longer suitable (Hadziyannis et al., 2000, Osborn and Lok, 2006). Instead, newer drugs, less prone to induce resistance, or a combination of two drugs are preferred as first line therapy. However, it is likely that monotherapy with lamivudine will continue in many regions due to cost factors. There is also cross-resistance between lamivudine and new drugs such as emtricabin, clevudine and telbivudine (Lee et al., 2006, Zoulim, 2004). Therefore, it is of interest to introduce simplified methods for detection of resistance mutations related to lamivudine.

There are two main positions in the viral reverse transcriptase (RT) associated with lamivudine resistance. At codon 204 in the highly conserved tyrosine-methionine-aspartate-aspartate (YMDD) motif of the nucleotide-binding site, a methionine (M) is in general replaced by either valine (V) or isoleucine (I) while at codon 180, a leucine (L) is replaced by a methionine (Allen et al., 1998, Ling et al., 1996). Typically, the M204V and L180M mutations are linked, while the M204I mutation may occur alone or is associated with L180M (Li et al., 2005). The emergence of lamivudine resistant HBV strains may be identified by direct sequencing. However, this is a laborious and time-consuming method, and therefore simpler and more accurate molecular techniques have been developed. These include restriction fragment length polymorphism (RFLP) (Allen et al., 1999, Chayama et al., 1998, Jardi et al., 1999), 5′ nuclease assay (Allen et al., 1999), melting point analysis (Cane et al., 1999), and real-time PCR using mutation-specific primers (Wang et al., 2006). Most of them, however, focus only on position 204.

This study describes a rapid method, based on real-time polymerase chain reaction (PCR), for the detection of lamivudine resistant mutations in HBV. The assay identifies resistance mutations at codons 180 and 204 in the RT region of the HBV polymerase gene, and uses a combination of primer specificity and minor groove binding (MGB) TaqMan probes (de Kok et al., 2002).