CLINICAL MANAGEMENT OF RESISTANCE

The efficacy of antiviral therapy is influenced by a number of factors:

• Adverse short-term side effects
• Long-term toxicity
• Sub optimal therapy due to:
• inadequate drug dose
• drug interactions and/or pharmacological properties
• cross resistance leading to reduced susceptibility
• patient compliance
• infection with drug resistant virus

Measuring the HBV DNA titre from patient serum is the best indicator of HBV replication and therefore drug efficacy. Reductions in serum HBV DNA always precede biochemical and histological responses. A minimum reduction in serum HBV DNA of >1log10 IU/ml within 3 months from the pretreatment baseline is defined as an “antiviral effect”. Failure to achieve such a reduction in HBV DNA constitutes "primary treatment failure”. “Secondary treatment failure” is defined by an increase in serum HBV DNA of >log10 IU/ml from baseline following an initial antiviral response.

Currently the approved therapeutic options for the HBV infection include two immunomodulators: interferon alpha and pegylated interferon and five nucleos(t)ide analogs (NA): Lamivudine (LMV), Adefovir (ADV), Entecavir (ETV), Telbivudine (L-dT) and Tenofovir (TDF). Antiviral therapy with NA while effective does not completely inhibit replication of the virus. As a consequence almost inevitably drug resistant HBV emerges. To date the primary resistance mutations have been identified for four out of the five currently approved NA. The emergence of drug resistance presents a significant challenge to clinicians:

• Limited treatment options
• One class of agents with one viral target (the polymerase)
• Reported clinical outcomes of combination therapy limited
• High level of cross resistance within structural groups of compounds and reduced sensitivity across the different structural groups of compounds

Until antiviral agents which target different stages of the viral life cycle become available, understanding the molecular mechanisms of NA drug resistance is paramount when deciding on the best rescue option for patients who have failed antiviral therapy. Developing methods for defining, detecting and quantifying drug resistance plays a vital role in patient management.

Why Detect Drug Resistance?

A virological flare due to drug resistance often can result in serious and sometimes-fatal clinical outcomes especially in patients with cirrhosis. Patients can experience increases in serum ALT levels, hepatic flares, hepatic decompensation and although rare, can result in death due to liver failure.

Early detection of drug resistance means early therapeutic intervention, this practice can avoid the clinical complications associated with virological breakthrough. In addition early intervention minimizes the further evolution of resistance through the selection of compensatory mutations.

Measuring viral load is the best indicator for monitoring patients on antiviral therapy.  Factors, which can affect the viral load, include:

• Suboptimal response
• Poor patient compliance
• Virological breakthrough due to genotypic resistance

In each case, a positive HBV viral load test (HBV DNA > 200IU/ml) should always be investigated for the possibility of the emergence of genotypic drug resistance. Instituting a new regimen, early during the development of drug resistance, offers the patient the best chance of therapeutic success.

Resistance testing methods:

To date there are currently only two commercially available resistance testing assays of HBV isolates:

1. Direct population based Sequencing

• Allows for bi-directional sequencing of the amplified product.
• Will only detect viral variation if present >20% of the total viral population.
• Will detect all mutations across the area sequenced, this in turn allows for the identification of all substitutions including primary, compensatory and potentially new resistance mutations for existing as well as new antiviral agents.
• Can identify HBsAg amino acid changes (in the overlapping reading frame) which can occur as a consequence of the emergence of drug resistance.
• Allows for the direct comparison of all HBV sequence variation from HBV isolates obtained before treatment commences and during virological breakthrough.

2. INNO LiPA Hybridisation (INNOGENETICS N.V, INNOLiPA HBV DR v2)

• PCR amplified products are hybridized with specific oligonucleotide probes immobilized on membrane based strips.
• Assay is more sensitive, will detect viral resistance variants when they constitute 5% or more of the total viral population.
• Can only detect pre specified mutations within the HBV polymerase known to be associated with drug resistance to LMV and ADV.

Several other techniques have been developed as an alternative to direct sequencing to perform genotypic resistance testing for HBV. Most are sequence-specific (like the LiPA assay) and are regarded as “in-house” methods as they are not available commercially.

These include:

• Restriction length polymorphism (Allen et al J Clin Micro 1999: 37:3338-3347)
• Restriction fragment mass polymorphism – (Hong et al J Hepatol 2004: 40:837-844)
• Oligonucleotide microarrays – (tran Eta l 2006 JCM 44:2792, Li et al Viral Hep 2005, 12: 168-75)
• Real-time PCR using mutation specific primers – (Wang et al World J Gastro 2006 12, 1308-1311)
• MALDI-TOF mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry – (Hong et al J Hepatol 2004: 40:837-844, Kim et al Ant Vir Ther, 2005:10:441-449)

Next Generation Sequencing Platforms such as:

• Ultra Deep Pyrosequencing – GS FLX Roche Platform (Solmone et al J Virol. 2009 Feb;83(4):1718-26. Epub 2008 Dec 10).