Hepatitis C Assays: The Pitfalls of Polymerase Chain Reaction and Genotyping

Abstract

Hepatitis C virus (HCV) continues to represent a worldwide threat to the lives of millions of people chronically infected with the virus. Accurately identifying the virus in terms of genetic characteristics, as well as quantifying its replicative equilibrium both during steady state and in the presence of antiviral therapy, is critically important for providing optimal care and prognoses. Genotype testing continues to evolve; however, it is important to recognize its limitations, particularly as we enter the world of direct-acting antivirals, in which the precise subtyping of HCV may play an even more important role in guiding therapy. Polymerase chain reaction (PCR) has revolutionized the management of chronic viral illnesses, and the advent of real-time PCR has broadened the awareness of the hepatology community to the tenacity of HCV in the face of potent antiviral therapy. With new technology, however, comes the responsibility of learning how to harness the techniques and to avoid common pitfalls of improper use or interpretation.

Hepatitis C virus long‐term persistence in peripheral blood mononuclear cells in patients with haemophilia. Detection of occult genotype 1

“…One recognized deficiency of most genotyping assays is their limited sensitivity for detecting mixed‐genotype infections, and the reported prevalence is variable depending on the genotyping methodology used . Most of the available assays share the requirement of an initial target amplification step to generate appropriate amounts of template for genotypic analysis.…”

Mixed Genotypes in Hepatitis C Virus Infection

“…As a result, genotypes present at lower proportional levels in a mix or with lower affinity could be missed or mistyped (Hnatyszyn, 2005). Both direct sequencing and restriction fragment length polymorphism assays seem to require 10% to 30% prevalence of minor strains before they can detect them (Qian et al, 2000;Frederick, 2010). Hybridization based genotyping methods represent an attractive genotyping option compared to sequencing methods.…”

“…The potential advantage of hybridization assays is their ability to detect mixed populations of virus with a prevalence as low as 2% (Qian et al, 2000). However similar to most genotyping methods, it involves PCR amplification of target regions of the viral genome and is confined by the advantages and disadvantages associated with PCR (Hnatyszyn, 2006;Frederick, 2010). It was reported that the latest version of the LiPA (Versant LiPA v2.0, Bayer HealthCare-Diagnostics), which targets both the 5′ UTR and the core protein, seems to have improved on the problem of subtyping and has performed better than a direct sequencing assay (Trugene) as well as an earlier version of the hybridization assay (Versant LiPA v1.0).…”

“…Population-based DNA sequencing (i.e., cloning and sequencing of HCV cDNA) is so far, an accurate method for detection of mixed-genotype infections; however, this is not routinely feasible because it is time consuming and expensive. In addition, several clones would need to be completely sequenced in the presence of multiple coexisting genotypes (Frederick, 2010).…”

Quantitation assay of hepatitis C virus RNA using real-time PCR technique

“…Thus, many samples can be analysed simultaneously. In addition, real-time PCR makes quantitative assay of RNA much more precise and reproducible because it relies on threshold cycle values determined during the exponential phase of PCR rather than endpoint [14,15].…”