Evolution Of Multi-drug Resistant Hepatitis B Virus During Nucleos(t)ide Analog Therapy

ObjectiveThe aim of this study was to determine the evolution patterns and formation mechanisms of multi-drug resistant hepatitis B virus during nucleos(t)ide analog therapy in patients with chronic hepatitis B.Methods1)706 patients diagnosed as chronic hepatitis B and with unsatisfactory curative effect during nucleos(t)ide analog therapy were enrolled in this study. Amino acid sequence of HBV RT domain which amplified by PCR and then directly sequenced was analysized. 15 serum samples from five representative patients with complete clinical data were selected out for clonal analysis using'PCR-cloning-sequencing'method. Additionally, dynamic detection of consecutive serum samples was carried out in four patients to determine the evolution of HBV strains.Results1) The total multi-drug resistance detection rate of the patients who received nucleos(t)ide analogue monotherapy, sequential therapy or combination therapy was 3.5%. Among which, double resistance to LAM and ADV was 72%; triple resistance to LAM, ADV and ETV was 28%. 2) Multi-drug resistance can not only lead to viral rebound, biochemical breakthrough, hepatitis B recurrence but also liver failure or even death. 3) Clonal analysis revealed that the evolution of quasispecies can be divided into the following three models.â‘ During sequential therapy, progressive evolution was from single drug resistant HBV mutations only to mixtures of clones that have multi-drug resistant mutations and clones that have single drug resistant HBV mutations only.â‘¡Progressive evolution was from single drug resistant HBV mutations only to mixtures of clones that have multi-drug resistant mutations during sequential therapy.â‘¢Within drug selective pressure, progressive evolution of the variants was from mixtures of clones that have multi-drug resistant mutations and clones that have single drug resistant HBV mutations only, ultimately to the single drug resistant HBV mutations as dominant variant for its high replication ability. 4) Combination therapy with ETV and ADV reduced HBV DNA to the detection limit of 500copies/ml within 12 weeks in 3 patients.Conclusion1) Multi-drug resistance can be induced during nucleos(t)ide analogue monotherapy, sequential therapy or combination therapy. 2) Multi-drug resistance can not only lead to viral breakthrough, but also liver failure or even death. 3) Clonal analysis showed mutations conferring multi-drug resistance colocate on the same viral genome. 4) Dynamic changes of HBV quasispecies had relation to the selective pressure of NAs therapy, distinct replicative advantage of the variants and replication space. 5) Combination therapy with ETV and ADV may be effective for some multi-drug resistant patients.