Molecular Profile of Hepatitis B Virus Resistance to Lamivudine, Entecavir, and Tenofovir in Brazil Over Time

Hepatitis B virus (HBV) resistance to nucleos(t)ide analogues is a major public health challenge. Continuous molecular surveillance is essential to monitor the emergence and spread of resistant viral variants. To evaluate the prevalence and temporal evolution of HBV resistance to lamivudine (LVD), entecavir (ETV), and tenofovir (TDF) in a cohort of chronic hepatitis B patients from different Brazilian regions over time, assessing associations with geographic distribution and viral genotypes. Descriptive study including 258 samples from patients with chronic HBV infection, collected between November 2007 and February 2024 in São Paulo, Rio Grande do Sul, Espírito Santo, and Amazonas. Genomic amplification used two approaches: full-genome amplification (∼3,200 bp) in a single PCR reaction, and nested PCR with overlapping fragments in low-viral load samples. Fragments were sequenced on the Illumina NextSeq2000 platform and analyzed with Geno2Pheno to identify resistance mutations. Resistance frequencies were evaluated over time and stratified by state of origin and genotype. The frequencies of mutations conferring resistance to LVD and entecavir were 8.91% (23/258) and 0.78% (2/258), respectively. Among analyzed samples, 8.14% (21/258) had mutations associated with partial resistance to ETV. Temporal analysis showed a significant increase in resistance starting in 2022. A heterogeneous geographic distribution was observed. Amazonas had the highest frequency of samples with mutations associated with full resistance to LVD (17.36%, 21/121) and to ETV (1.65%, 2/121). Among identified genotypes, genotype D had the highest frequency of LVD resistance (14.89%, 14/94) and ETV resistance (1.06%), and also partial ETV resistance (13.83%, 13/94). No mutations associated with tenofovir resistance were identified. The data indicate a recent increase in the frequency of resistance mutations to LVD and ETV among patients with chronic hepatitis B, especially in Amazonas and among genotype D carriers. Incorporating genomic surveillance can contribute to more effective monitoring of antiviral resistance and support individualized therapeutic choices.