| Congresso Brasileiro de Microbiologia 2023 | Resumo: 945-1 | ||||
Resumo:It is estimated that 58 million people are chronically infected with hepatitis C virus (HCV). The most prevalent subtypes are 1a and 1b, followed by 3a. The high rate of viral replication and the absence of HCV polymerase proofreading activity led to a heterogeneous classification and the detection of genetically distinct subpopulations with a dynamic process of mutation emergence. In Brazil, hepatitis C treatment has been evolving significantly with the licensing of direct-acting antivirals (DAAs). The selection of viral strains with resistance-associated substitutions (RAS) at HCV NS5A protein can be considered one of the limiting factors for achieving sustained virologic response (SVR) to DAAs. SVR values higher than 95% were observed in practical routine and controlled clinical trials however there are still isolated cases of treatment failure associated to viral resistance. Considering NS5A protein, RASs at amino acid positions 28, 30, 31 and 93 can reduce susceptibility to Brazilian available NS5A DAAs. Also, assessing viral genetic diversity from high-throughput sequencing (HTS) might be important for HCV genomic surveillance, especially for DAA-failure patients. This study aimed to evaluate genetic diversity in NS5A protein specific amino acid positions for HCV subtype 1a non-responder patients. A total of 7 serum samples were collected from non-responder patients after treatment with NS5A DAA daclatasvir. Initially, viral RNA extraction and RT-PCR with specific primers for NS5A HCV subtype 1a were done. Posteriorly, PCR amplicons (~1500 bp) were purified and submitted to end-repair. For barcode ligation, end prep products were added to a mixture with native barcodes. Following this, pooled libraries were used for adaptor insertion. A primed flow cell was applied to load the library and sequenced on MinION device. The rates of synonymous substitution per synonymous site (dS) and non-synonymous substitution per non-synonymous site (dN), as well as the dN/dS ratio, were obtained at SNAP Program. RASs at positions 28, 31 and 93 which are potentially associated with reduced response rates to NS5A DAAs were identified. NS5A mutation M28T (ATG>ACG) was observed in HCV sequence from two non-responder patients with a subpopulation distribution by 93.9% and 96.5%, respectively. Also, substitution L31M (CTG>ATG) was identified in 97.7% of HCV subpopulations for one patient indicating a possible NS5A antiviral resistance associated with this mutation. RASs at position 93, Y93N (TAC>AAC) and Y93H (TAC>CAC), were identified in HCV sequences from two and one patient, respectively, with a percentage of 97.8% and 89.5%. RAS Q30Y (CAA>TAT), which was not demonstrated to be clinically relevant due to low evidence in vivo, was observed for one patient. The dN/dS ratio was 6.3707 suggesting that there were more non-synonymous substitutions than synonyms and HCV subpopulations has been under evolutionary pressure to escape ancestral status (positive selection pressure). Viral factors, such as the infecting genotype, alongside drug resistance mutations, represent negative predictive factors to achieve SVR for Brazilian patients. This study highlighted the importance of identifying post-treatment NS5A RASs for HCV-1a sequences, indicating that there are still cases of treatment failure that reinforce the necessity of viral resistance evaluation to support physicians’ conduct in a rescue therapy with a different DAA. Palavras-chave: hepatitis C virus, molecular diagnosis, resistance, sequencing Agência de fomento:CAPES; FAPERJ | |||||