Pre-existing mutations related to tenofovir in chronic hepatitis B patients with long-term nucleos(t)ide analogue drugs treatment by ultra-deep pyrosequencing

// Xiaxia Zhang 1 , Minran Li 2 , Hongli Xi 1 , Renwen Zhang 1 , Jianhong Chen 1 , Yu Zhang 1 , Xiaoyuan Xu 1 1 Department of Infectious Disease, Peking University First Hospital, Xicheng District, Beijing 100034, China 2 Division of Liver Disease, The Fifth Hospital of Shijiazhuang, Hebei Medical University, Shijiazhuang 050023, China Correspondence to: Xiaoyuan Xu, email: xiaoyuanxu6@163.com Keywords: hepatitis B virus, resistance, multi-drugs therapy, tenofovir, ultra-deep pyrosequencing Received: June 29, 2016     Accepted: August 26, 2016     Published: September 02, 2016 ABSTRACT Aims: The dynamics of resistance-associated mutations under combination therapy were explored. Methods: A total of 46 patients were classified into adefovir (n=14) and entecavir (n=32) groups. In the adefovir (ADV) group, six patients receiving combined therapy were DNA-positive after more than 3 years of therapy. Ultra-deep pyrosequencing was used to analyze the dynamics of multi-drugs resistance mutations. Results: At baseline, all 46 treatment-naive patients harbored rtA181V/T substitutions (1.2%-4.6%) and rtN236T substitutions (1.6%-6.1%). In the ADV group, eight patients with long-term treatment were consecutively HBV DNA-positive for more than 3 years. During treatment, the rtA181T resistance-associated site appeared with increasing frequency in six of eight patients (NOs. 1-6), and two patients (NOs.4 and 8) carrying the rtA181T resistance mutations increasingly showed high levels of rtN236T. One patient (NO. 8) experienced virological breakthrough. Other known pre-existing mutations showed no dynamic fluctuations, including in rtA194T, rtP177G, rtF249A, and rtD263E. In addition to the common substitutions, some previously unknown amino acid substitutions, such as rtD134N, rtL145M/S, rtF151Y/L, rtR153Q, and rtS223A, should be further studied. Conclusions: HBV-resistance substitutions conferring to nucleoside analogs are present at baseline. The dynamics of the HBV RT-region quasispecies variation are heterogeneous and complex.