Identification of hepatitis C virus genotype 3 by a commercial assay challenged by natural polymorphisms detected in Spain from patients with diverse origins

• We report the largest series of hepatitis C virus (HCV) isolates genotyped by the Abbott assay.
• An indeterminate result was obtained in 7.95% of HCV-3 cases at a Spanish center.
• These indeterminate results were caused by one to three mismatches in the HCV-3 probe-binding region.
• These natural polymorphisms were detected in patients with diverse origins.

BackgroundHepatitis C virus (HCV) genotyping is crucial in clinical practise for determining the type and duration of antiviral therapy. Between 2009 and 2014, 24 (7.95%) of all HCV genotype 3 (HCV-3) cases obtained indeterminate results via the RealTime HCV Genotype II assay (Abbott) at a tertiary care center in Spain. HCV-3 is the second most common genotype worldwide. Moreover, it has been associated with a higher risk of liver disease progression and a lower response to the latest antivirals.ObjectiveGiven the clinical significance of accurately identifying HCV-3, we aimed to characterize the genetic diversity of the HCV 5′ untranslated region (5′ UTR), the target of genotyping assays, by ultradeep pyrosequencing (UDPS).Study designFor the 24 indeterminate samples, the 5′ UTR-core was amplified and subjected to UDPS with the 454/GS-Junior platform (Roche). The genotype/subtype of each identified haplotype was assigned by phylogenetic analysis. For comparison, three additional samples correctly identified as HCV-3 by the real-time assay were also analyzed.ResultsHCV genotyping based on 5′ UTR-core UDPS was in agreement with NS5B Sanger sequencing in all cases, confirming the absence of mixed infections and recombination events. The generated 5′ UTR sequences proved the presence of one to three polymorphisms at the probe-binding site of the Abbott assay, thereby differentiating indeterminate from correctly genotyped HCV-3 samples.ConclusionsThe observed naturally occurring polymorphisms provide insight into regional differences observed with genotype 3, their impact on genotyping assay performance, and potential improvement and designing options.