Next-Generation Sequencing of Hepatitis C Virus (HCV) Mixed-Genotype Infections in Anti-HCV-Negative Blood Donors

The infection with more than one hepatitis C virus (HCV) genotype especially in subjects with a high risk of multiple HCV exposures has been demonstrated. The role of HCV mixed-genotype infection in viral persistence and treatment effect is not fully understood. The prevalence of such infection varies greatly depending on the technique used for genotype determination and studied population. Next-generation sequencing (NGS) which is suitable for extensive analysis of complex viral populations is a method of choice for studying mixed infections. The aim of the present study was to determine the prevalence of mixed-genotype HCV infections in the Polish seronegative, HCV-RNA-positive blood donors (n = 76). Two-step PCR was used for amplification of 5′-UTR of HCV. Using pyrosequencing altogether, 381,063 reads were obtained. The raw reads were trimmed and subjected to similarity analysis against the entire unfiltered NCBI nt database. Results obtained from NGS were compared with the standard genotyping. One (1.3%) mixed-genotype [3a, 2989 reads (94.8%); 1b, 164 reads (5.2%)] infection was found in a sample diagnosed as genotype 3a only by routine testing. Two samples were identified with different genotypes, compared to routine testing. In conclusion, NGS is a sensitive method for HCV genotyping. The prevalence of mixed-genotype HCV infections in blood donors is low.