Low-level persistence of drug resistance mutations in hepatitis B virus-infected subjects with a past history of Lamivudine treatment

Abstract

We sought to determine the prevalence of hepatitis B virus (HBV) lamivudine (LAM)-resistant minority variants in subjects who once received LAM but had discontinued it prior to virus sampling. We performed direct PCR Sanger sequencing and ultradeep pyrosequencing (UDPS) of HBV reverse transcriptase (RT) of plasma viruses from 45 LAM-naive subjects and 46 LAM-experienced subjects who had discontinued LAM a median of 24 months earlier. UDPS was performed to a depth of ∼3,000 reads per nucleotide. Minority variants were defined as differences from the Sanger sequence present in ≥0.5% of UDPS reads in a sample. Sanger sequencing identified ≥1 LAM resistance mutations (rtL80I/V, rtM204I, and rtA181T) in samples from 5 (11%) of 46 LAM-experienced and none of 45 LAM-naive subjects (0%; P = 0.06). UDPS detected ≥1 LAM resistance mutations (rtL80I/V, rtV173L, rtL180M, rtA181T, and rtM204I/V) in 10 (22%) of the 46 LAM-experienced subjects, including 5 in whom LAM resistance mutations were not identified by Sanger sequencing. Overall, LAM resistance mutations were more likely to be present in LAM-experienced (10/46, 22%) than LAM-naive subjects (0/45, 0%; P = 0.001). The median time since LAM discontinuation was 12.8 months in the 10 subjects with a LAM resistance mutation compared to 30.5 months in the 36 LAM-experienced subjects without a LAM resistance mutation (P < 0.001). The likelihood of detecting a LAM resistance mutation was significantly increased using UDPS compared to Sanger sequencing and was inversely associated with the time since LAM discontinuation.

Fig 1

Distribution of months of past lamivudine (LAM) treatment (A) and of months since LAM discontinuation (B) among the 46 LAM-experienced subjects.

Fig 2

Months since discontinuation of lamivudine (LAM) for the 10 subjects with and 36 subjects without viruses containing LAM resistance mutations (median: 11.2 versus 30.5 months; P = 0.0001, Mann-Whitney test).

Fig 3

Inverse relationship between the time since lamivudine (LAM) discontinuation and the log-transformed value of the most prevalent LAM resistance mutations within each of the 46 LAM-experienced subjects (r = 0.69, P < 0.001; Spearman's rank correlation coefficient). A single point (x = 30.5 months and y = 0.1%) is used to represent the 36 subjects who had discontinued LAM a median of 30.5 months prior to sampling and who did not have detectable LAM resistance mutations.