Molecular diagnostics of HBV resistance and HDV viremia

HBV mutations frequently involved in resistance to antivirals

Doerig C et al. Rev Med Suisse 2009; 5:203 - 208    LAM: lamivudine; ADV: adefovir; ETV: entecavir; LdT: telbivudine; TDF: tenofovir. Note that the mutations indicated in grey reduce the susceptibility of HBV to TDF in vitro but that virological breakthrough has not been documented yet in patients.

Well known mutations associated with drug resistance are reported above. HBV resistance to antivirals is due to mutations concentrated in the rt domain of the polymerase, near the YMDD peptide motif found at the active site of rt. Mutations are numbered according to the amino acid position within the rt domain. The codon for Methione (M) at position 204 is a key target for mutations changing M to Isoleucine (I) or to Valine (V). These mutations are reported as M204I or M204V respectively. While M204I can be found isolated in patients infected with LAM resistant viruses, M204V is always accompanied by another mutation like L180M. Such a mutation is necessary to compensate the replication defect induced by M204V, thus improving the fitness of the variant virus in the presence of the antiviral drug.

Molecular tools to identify mutations

The identification of mutations can only be done after polymerase chain reaction (PCR) amplification of rt with DNA extracted from the plasma of infected patients. Primers used in our laboratory are depicted in green on the HBV genome displayed in the background section.

Precise identification of mutations can be done by reverse blotting hybridization against a panel of probes (for known mutations, high sensitivity of detection), or by DNA sequencing (for known as well as for unknown mutations) provided they represent at least 20% of the whole population. This level of detection is sufficient for routine diagnostics. Once the DNA sequence is determined, it is translated into its corresponding amino acid stretch. Mutations can then be identified after alignment of the amino acid sequence against reference HBV protein databases. This method, used in our laboratory, is illustrated below by sequencing chromatograms of two samples from the same patient, either before (upper strand) or after failure treatment with telbivudine (lower strand). The G/T transversion indicated by the blue shade leads to the M204I mutation known to confer resistance to this drug.

HDV RNA testing

HDV is a subviral agent that requires the envelope proteins of HBV for its maturation. As a consequence, HDV can only persist in HBV-infected patients. The genetic information of HDV is carried by a circular RNA molecule, with extensive base pairing, posing a challenge for PCR techniques aimed at the quantitative assessment of HDV viremia. We have developed a two-step reverse transcription-PCR directed against all known HDV genotypes referenced in Genbank (status April 2009). This assay has been evaluated retrospectively with known samples and is currently made available, with the understanding that it is in a prospective, final evaluation phase.