Deep Sequencing Analysis of Hepatitis C Virus Subtypes and Resistance-Associated Substitutions in Genotype 4 Patients Resistant to Direct-Acting Antiviral (DAA) Treatment in Egypt

Egypt has the highest global prevalence of hepatitis C virus (HCV), with genotype 4 (G4) in over 94% of cases. Direct-acting antivirals (DAAs) yield sustained virologic response (SVR) rates above 95%. Second-generation DAAs are recommended for patients with virological failure, achieving over 90% eradication. This study aimed to classify and evaluate the pattern of HCV resistance-associated substitutions (RASs) in patients who failed DAA treatment in Egypt. A total of 1778 chronically infected HCV patients from Egypt's Nile Delta were enrolled (2016-2018). Among them, 37 relapsed, and high-quality serum samples from 22 patients were available, including 6 cases with pre- and post-treatment samples. Next-generation sequencing (NGS) was performed for HCV subtyping and RAS identification. Among the 22 analyzed cases, 21 (95.4%) were G4: 11 were classified as subtype G4a, seven G4o, and three G4m. One patient (4.5%) was identified as G1g. One case shifted from G4a pre- to G4o post-treatment, suggesting reinfection. The RAS pattern in rare G4 subtypes (G4m/G4o) differs from the G4a subtype. The combination of L28M/L30S mutations was detected in 8/11 G4a samples; in contrast, RASs in G4o were characterized by T30S or Y93C/H/N/S substitutions. Notably, some substitutions identified as RASs may represent fixed polymorphisms in regional viral populations, such as those in Egypt's Nile Delta. HCV subtypes significantly influence the RAS pattern, particularly within the NS5A region, after DAA-treatment failure. The RAS pattern differs among G4 subtypes, particularly in rare ones, predisposing patients to resistance and underscoring the importance of NGS in regional populations to optimize treatment strategies.