Line Probe Assay for Monitoring Drug Resistance in Hepatitis B Virus-Infected Patients during Antiviral Therapy

Abstract

Since the introduction of antiviral compounds such as lamivudine and famciclovir in the treatment schedules of patients with chronic hepatitis B virus (HBV) infection, the accumulation of a variety of mutations in the HBV polymerase gene has been observed. The selection of these mutations is generally considered the cause of viral nonresponsiveness and treatment failure. Therefore, the detection of these mutations is of clinical importance. Previously genotyped HBV strains isolated from treated and untreated patients were amplified with primers specific for the HBV polymerase region from amino acids 465 to 562. Amplified products were cloned into plasmid vectors. The clones were used as reference strains. A set of 38 highly specific oligonucleotide probes covering three different codon positions, L528M, M552V/I, and V/L/M555I, were selected. These probes were applied as 19 different lines on a membrane strip. The strips were then hybridized with PCR fragments from the reference panel, revealing the amino acids at the three codon positions simultaneously for each clone. PCR products generated from two patients infected with HBV genotypes A and C, respectively, and treated with nucleoside analogs were analyzed on these strips. A gradual increase in genetic HBV polymerase complexity was observed in follow-up samples compared to that in pretreatment samples. Additional analysis of HBV polymerase DNA fragments in recombinant plasmid clones demonstrated the existence of (i) clones with double mutations, (ii) clones with single mutations at either codon 528, 552, or 555, and (iii) the simultaneous occurrence of two or more viral populations within one sample. This line probe assay detected the complex quasispecies nature of HBV and provided some insight into the dynamics of resistance mutations.

Early Detection of Viral Resistance by Determination of Hepatitis B Virus Polymerase Mutations in Patients Treated by Lamivudine for Chronic Hepatitis B

“…As already described in previous studies, the M552V mutant was found more frequently than the M552I mutant. [11][12][13][31][32][33] Interestingly, we found that in HBeAg-positive patients the M552I mutant was found either transiently or as a mixture with the M552V mutant suggesting that the M552V mutant had a replication advantage in this viral quasi-species, 13,21 while it was the predominant viral species in anti-HBe-positive patients infected with HBV genotype D. It will be interesting to see whether the pattern of mutation in the YMDD motif of the HBV polymerase is restricted by viral genotype on a larger number of patients. Moreover, we found that the C domain mutations were detected earlier than the B domain mutation in most of the patients, suggesting that the latter may represent a compensatory mutation.…”

“…[27][28][29] To determine genotypic resistance and detect polymerase gene variability at codon 528 in the B domain of the reverse transcriptase, and at codons 552 and 555 of the C domain, a research LiPA was used as described previously in detail. 21 As the HBV polymerase gene has a different nucleotide length depending on genotypes, genotype A corresponding to the longest polymerase gene sequence was used as a reference for amino acid numbering.…”

“…The recent development of a new genotypic assay for polymerase mutant detection based on the reverse hybridization technology provides a new tool to evaluate the clinical significance of the detection of HBV polymerase mutants on a large number of samples. 21 In the present study, we have investigated the molecular events associated with the emergence of drug-resistant strains in patients treated with lamivudine. Sequential serum samples from 20 consecutive patients with lamivudine resistance were analyzed to determine HBV-DNA levels and viral polymerase mutations.…”

Significance of hepatitis B genotype in acute exacerbation, HBeAg seroconversion, cirrhosis-related complications, and hepatocellular carcinoma

“…Serum on presentation was stored at Ϫ20°C then thawed to determine HBV genotype and precore and core promoter mutations. For determination of HBV genotype, HBV DNA was extracted as described by Stuyver et al 13 The HBsAg region spanning aa 107-205 was amplified and analyzed by the INNO-LiPA HBV Genotyping (Innogenetics NV, Gent, Belgium). The INNO-LiPA HBV Genotyping assay contained oligonucleotide probes specific for HBV genotypes A-G applied as 14 different lines on a membrane strip.…”

Nomenclature for antiviral-resistant human hepatitis B virus mutations in the polymerase region

“…70 rtM204V can cause sI195W196 to change into either sM195W196 or sK195W196; and the rtM204I might result in sI195X196, sI195L196, sI195S196, or sT195L196. 70 It is clear that the different nucleotide changes selected during lamivudine therapy might affect HBsAg differently. The importance of these HBsAg amino acid changes needs further investigation, because mutations in HBsAg could have a significant impact on immune recognition, replication capacity, and virulence.…”