Current molecular methods for the detection of hepatitis B virus quasispecies

Abstract

Chronic HBV infection affects more than 240 million people worldwide and is associated with a broad range of clinical manifestations including liver cirrhosis, liver failure and hepatocellular carcinoma. Because of the lack of an efficient cure for chronic hepatitis B, the main goal of antiviral therapy is the prevention of liver disease progression coupled with prolonged survival of patients. Because HBV viral load has been shown to be a crucial determinant of the progression of liver damage, these goals can be achieved as long as HBV replication can be suppressed. Unfortunately, long-term therapy with the low-to-moderate genetic barrier drugs, which are still recommended in a majority of developing countries, are strongly associated with HBV resistance development and treatment failure. In such cases, the precise and accurate determination of drug-resistant variants in an individual patient before treatment is important for a proper choice of first-line potent therapy. Nowadays, a number of techniques are available to study HBV quasispecies evolution. This review describes the advantages and limitations of various assays detecting drug-resistant HBV variants. Copyright © 2016 John Wiley & Sons, Ltd.

Ultraspecific and Amplification-Free Quantification of Mutant DNA by Single-Molecule Kinetic Fingerprinting

“…A common application of current PCR-based assays for mutant DNA detection is for quantification of MUT ctDNA extracted from human biofluids 1–2,29–30 . In order to examine matrix effects on our assay, we tested the robustness of our super-resolution kinetic fingerprinting assay in urine—a compositionally complex biological sample matrix in which trans-renal ctDNA can be detected 31 .…”

“…Detection and quantification of rare mutant DNA alleles is increasingly important for a broad set of applications in biological and clinical research, including tracking the emergence of rare alleles responsible for drug resistance in populations of microbes, viruses or cancer cells 1–2 . For example, rare fragments of circulating tumor DNA (ctDNA) are present in biofluids ( e.g.…”

How viral genetic variants and genotypes influence disease and treatment outcome of chronic hepatitis B. Time for an individualised approach?

“…158 To date, several tools relying on different technologies are available to evaluate HBV genome variability. 159 The recent advent of next-generation sequencing (NGS) technologies, [160][161][162] with ever decreasing costs, 163 paves the way for more rational treatment strategies from the perspective of precision medicine. 164 In the context of HBV infection, NGS are technologies of choice as they can feed temporal data into a multidimensional matrix for both the virus and the host, at the genome, epigenome and transcriptome levels, from acute HBV infection to development of cirrhosis and HCC.…”

Primary resistance of hepatitis B virus to nucleoside and nucleotide analogues

“…However, little information is available on the prevalence of resistant HBV variants in treatment‐naïve patients. Classical techniques, such as population sequencing, reverse DNA hybridization, restriction fragment length polymorphism or cloning of PCR products followed by sequencing of the clones, have been widely used for HBV resistance testing in both in vitro and in vivo research studies . These molecular methods have limitations relating to their specificity, sensitivity, ability to detect novel mutations and reporting of qualitative results.…”