Hepatitis C Genotyping Tests
Hepatitis C genotyping can be performed using direct sequence analysis, reverse hybridization, or restriction fragment mass polymorphism (RFMP), with most assays analyzing the 5'-untranslated region (UTR) and HCV core regions where nucleotide sequences are highly conserved. 1
Primary Genotyping Methods
HCV genotyping is essential before treatment to determine the optimal therapeutic duration and dose of ribavirin. The main methods for determining HCV genotypes include:
Recommended Methods
- Direct sequence analysis (Sanger sequencing) of NS5B or core regions 1
- Next Generation Sequencing (NGS) of NS5B or another coding region 1
- Reverse hybridization using line probe assays 1
- Restriction Fragment Mass Polymorphism (RFMP) 1
Most clinical laboratories analyze the 5'-untranslated region (UTR) and HCV core regions where nucleotide sequences are highly conserved. With analysis of the 5'-UTR region, HCV genotyping errors occur at a rate of <3%, but subtyping errors may occur in 10-25% of cases 1.
Accuracy and Limitations
- Population sequencing (Sanger) or deep sequencing with phylogenetic analysis provides the most accurate determination of HCV genotype and subtype 1
- Line probe assays accurately identify genotypes 1-6 and subtypes 1a and 1b, but misclassify many less common subtypes 1
- Genotyping is not possible in <5% of patients due to low HCV levels, problems with PCR amplification, or high nucleotide variability 1
- Real-time RT-PCR assays have shown good correlation with restriction fragment length polymorphism (RFLP) methods, particularly for genotypes 1,2, and 3 2
Clinical Importance of Accurate Subtyping
While subtyping was not necessary with older interferon-based therapies, it has become more important with direct-acting antivirals (DAAs):
- Subtypes may need to be confirmed since DAAs act differently according to genotype 1a and 1b 1
- Some subtypes (e.g., 1l, 4r, 3b, 3g, 6u, 6v) harbor natural polymorphisms that confer inherent resistance to NS5A inhibitors 1
- For migrants from regions where these resistant subtypes are prevalent, determination of genotype and subtype by population or deep sequencing of NS5B is recommended 1
Emerging Technologies
Whole genome sequencing (WGS) using next-generation sequencing technology offers advantages over traditional methods:
- Higher accuracy than legacy AVR and genotyping assays 3
- Ability to distinguish between mixed infections, cross-contaminants, and recombinant viruses 3
- Simultaneous determination of genotype and antiviral resistance profile 3
Practical Considerations
- HCV genotype should be assessed before treatment for determining the optimal therapeutic duration and dose of ribavirin 1
- With newer pangenotypic regimens (sofosbuvir/velpatasvir or glecaprevir/pibrentasvir), treatment can be initiated without knowledge of genotype with high probability of success 1
- However, genotype determination remains useful where drug procurement or pricing dictates genotype-specific treatment 1
Common Pitfalls
- Using assays based only on 5'-UTR region may not accurately discriminate between subtypes 1a and 1b 1
- Relying on reverse hybridization with line probe assays for patients from regions with uncommon subtypes may lead to misclassification 1
- Not considering the possibility of mixed infection (1-30% prevalence) which may affect treatment outcomes 1
- Using non-recommended methods like 5'UTR PCR amplification alone for genotyping 1
For optimal patient outcomes, laboratories should use sequence analysis of NS5B or core regions, or whole genome sequencing with NGS when possible, especially for patients from regions where uncommon subtypes are prevalent.