Dynamics of Genotypic Mutations of the Hepatitis B Virus Associated With Long-Term Entecavir Treatment Determined With Ultradeep Pyrosequencing: A Retrospective Observational Study

Abstract

The aim of the study is to explore the evolution of genotypic mutations within the reverse transcriptase region in partial virological responders (PVRs) receiving long-term entecavir (ETV) treatment. A total of 32 patients were classified as completely virological responders (CVRs) (n = 12) or PVRs (n = 20). Five partial responders were hepatitis B virus (HBV)-DNA positive after long-term therapy, which lasted for >3 years. A total of 71 serum samples from these 32 patients were assayed by ultra-deep pyrosequencing (UDPS): 32 samples were from all patients at baseline, and 39 were from PVRs with sequential inter-treatment. Approximately 84,708 sequences were generated per sample. At baseline, the quasispecies heterogeneity did not significantly differ between the 2 groups. The frequencies of substitutions indicating pre-existence of nucleos(t)ide analog resistant (NAr) mutants ranged from 0.10% to 6.70%, which did not statistically differ between groups either. However, the substitutions associated with the NAr mutants were significantly different from those associated with the non-NAr mutants in 13 patients; 6 of these patients were PVRs and the others were CVRs. Five patients were HBV DNA positive after regular ETV monotherapy for >3 years, and 4 of these patients underwent mild NAr substitution fluctuations (<20%). One patient developed virological breakthrough while bearing single, double, and triple (rtL180 M, rtM204 V, rtS202G) substitutions. In addition to the common substitutions, unknown amino acid substitutions, such as rtL145 M/S, rtF151Y/L, rtR153Q, rtI224 V, rtN248H, rtS223A, rtS256C, need to be further verified. NAr substitutions are observed at frequencies of 0.10% to 6.7% before therapy. Long-term ETV therapy generally results in virological responses, as long as the proportion of resistance mutations remains at a relatively low level. Genotypic resistance to ETV is detected in all PVRs receiving long-term ETV therapy.

FIGURE 1

The dynamic changes in HBV DNA levels and resistant variants of the RT domains during ETV treatment of patient 1. (A) The serum HBV DNA level declined gradually and was undetectable after treatment for 3 years and followed-up for 7 years. (B) The dynamic changes in the RT domains of HBV variants, as determined by UDPS. At baseline, this patient displayed rtM204I/V (3.5%), rtL180 M (2.1%), and rtS202G (3.4%) substitutions. A wave of resistant variants (rtM204 V [19.95%], rtL180 M [19.12%], and rtS202G [18.33%]) was detected at 1 year and then decreased to <1% by 2 years. The rtA181T substitution rose from 2.17% to 64.15% by 2 years and certain nonresistance mutations (rtN248A, rtI224 V, and rtS223A) increased significantly. ETV = entecavir, DNA = di-ribonucleic acid, HBV = hepatitis B virus, RT = reverse transcriptase, UDPS = ultra-deep pyrosequencing.

FIGURE 2

The dynamic changes in HBV DNA levels and resistant variants of the RT domains during ETV treatment of patient 2. (A) The serum HBV DNA level was undetectable after treatment with ETV for 3 years. (B) At baseline, the virus harbored an rtA181T substitution (1.4%), and other resistance mutations were present at low levels (<1%). These mutations did not obviously fluctuate during the follow-up period. Several other variants fluctuated at relatively high levels (ranging from 3.56% to 7.89%), including rtN248H, rtS223A, rtS256C, and rtI224 V mutations. ETV = entecavir, DNA = di-ribonucleic acid, HBV = hepatitis B virus, RT = reverse transcriptase.

FIGURE 3

The dynamic changes in HBV DNA levels and resistant variants of the RT domains during ETV treatment of patient 3. (A) The serum HBV DNA level declined gradually and was undetectable by the 5th year of ETV treatment. (B) At baseline, the virus only harbored an rtA181T substitution (1.2%) and had low fluctuations during the follow-up period. Resistant variants (rtM204I/rtV173 M/A) increased to 9.23%/1.87% in 24 weeks and declined to <1% at 1 year. Two variants (rtI187L/rtV191I) rose to a high level during the 4th year (84.43% /83.41%), and 2 other variants, namely, rtN248H and rtS256G, were maintained at a high level (>60%). ETV = entecavir, DNA = di-ribonucleic acid, HBV = hepatitis B virus, RT = reverse transcriptase.

FIGURE 4

The dynamic changes in HBV DNA levels and resistant variants of the RT domains during ETV treatment of patient 4. (A) The serum HBV DNA level was undetectable after treatment for 5 years. (B) Resistance mutations displayed low levels of fluctuation (<7%), and nonresistant variants (rtN248H/rtS223A) rose to 12.88%/13.1% by 2 years. Additionally, rtS256G substitutions were maintained at a high level (>55%).ETV = entecavir, DNA = di-ribonucleic acid, HBV = hepatitis B virus, RT = reverse transcriptase.

FIGURE 5

The dynamic changes in HBV DNA levels and resistant variants of the RT domains during ETV treatment of patient 5. (A) The HBV DNA declined to a low level (10² IU/mL) at 1 year, but viral replication increased (10⁷ IU/mL) by 2 years. During gradual ETV therapy, the DNA level began to decrease after a nadir at 6 years, and a virological breakthrough appeared in the 7th year (10³ IU/mL). (B) From 1 to 2 years, the resistant variants (rtL180 M, rtM204 V, rtS202G) began to rise to a high peak (84.60%, 79.56%, 76.88%), and double amino acid substitutions (rtL180M+rtM204 V) and triple substitutions (rtL180M+rtM204V+rtS202G) also began to rise. All of the single- and double-resistant variants maintained high values. ETV = entecavir, DNA = di-ribonucleic acid, HBV = hepatitis B virus, RT = reverse transcriptase.