Ultrasensitive Amplification Refractory Mutation System Real-Time PCR (ARMS RT-PCR) Assay for Detection of Minority Hepatitis B Virus-Resistant Strains in the Era of Personalized Medicine

Abstract

Resistance to antiviral treatment for chronic hepatitis B virus (HBV) has been associated with mutations in the HBV polymerase region. This study aimed at developing an ultrasensitive method for quantifying viral populations with all major HBV resistance-associated mutations, combining the amplification refractory mutation system real-time PCR (ARMS RT-PCR) with a molecular beacon using a LightCycler. The discriminatory ability of this method, the ARMS RT-PCR with molecular beacon assay, was 0.01 to 0.25% for the different HBV resistance-associated mutations, as determined by laboratory-synthesized wild-type (WT) and mutant (Mut) target sequences. The assay showed 100% sensitivity for the detection of mutant variants A181V, T184A, and N236T in samples from 41 chronically HBV-infected patients under antiviral therapy who had developed resistance-associated mutations detected by direct PCR Sanger sequencing. The ratio of mutant to wild-type viral populations (the Mut/WT ratio) was >1% in 38 (63.3%) of 60 samples from chronically HBV-infected nucleos(t)ide analogue-naive patients; combinations of mutations were also detected in half of these samples. The ARMS RT-PCR with molecular beacon assay achieved high sensitivity and discriminatory ability compared to the gold standard of direct PCR Sanger sequencing in identifying resistant viral populations in chronically HBV-infected patients receiving antiviral therapy. Apart from the dominant clones, other quasispecies were also quantified. In samples from chronically HBV-infected nucleos(t)ide analogue-naive patients, the assay proved to be a useful tool in detecting minor variant populations before the initiation of the treatment. These observations need further evaluation with prospective studies before they can be implemented in daily practice. N ucleos(t)ide analogues are a fundamental tool for the treatment of hepatitis B virus (HBV)-associated liver disease because of their potent antiviral activity in the absence of remarkable side effects and major contraindications (1). Longterm nucleos(t)ide analog therapy among patients significantly improves survival and reduces the risk of liver-related major complications, such as death, hepatocellular carcinoma (HCC), and liver decompensation (2). A major concern with nucleos(t)ide analogue therapy is the risk for the development of viral populations with resistance-associated mutations (3, 4). Standard direct PCR Sanger sequencing and point mutation assays are able to detect viral populations with resistance-associated mutations only when they become dominant, leaving a gap in the understanding of the dynamics in the development of resistance. Direct PCR Sanger sequencing detects, on average, mutations present at ratios of Ͼ20% of the circulating virus population (5). Clonal sequencing has a higher sensitivity for detecting low-prevalence HBV mutations, but it is costly and labor-intensive (6). Point mutation assays can detect specific variants at as low as 5% of the virus population, but only if well-established mutatio...

Progressive accumulation of mutations in the hepatitis B virus genome and its impact on time to diagnosis of hepatocellular carcinoma

“…Beyond providing information on overall risk for HCC, these findings suggest that the mutation score would help to predict short‐term risk, particularly in those who have preclinical HCC or are at high risk and will be diagnosed with HCC in the next 5 years. So far, different real‐time PCR platforms and methods for rapid quantification of HBV mutants associated with antiviral resistance have been published . Such assay systems are capable of detecting mutations at low amount of DNA (e.g., HBV‐DNA <100 copies/mL) and as few as <5% of mutant among WT virus.…”

Evaluation of the G145R Mutant of the Hepatitis B Virus as a Minor Strain in Mother-to-Child Transmission

“…Therefore, immunological pressure to induce amino acid/nucleotide mutations seems not to occur. Although previous studies have analysed viral sequences using Sanger sequencing, this method cannot detect minor populations that are less than 20% of the total population [52, 53]. Further studies are needed to confirm the association between HBeAg-positive status and the G145R mutant by sensitive detection systems or deep sequencing.…”

New real-time-PCR method to identify single point mutations in hepatitis C virus

“…In contrast to other methods used to detect resistance-associated substitutions, which require separate specific primers for G1a and G1b[14-16] our technique can identify a single point mutation using one set of primers for all G1 samples. Simple hybridization is then carried out, in this case with the specific probe that differentiated Q from K at a position 80.…”