In our study, we found that the overall viral quasispecies complexity was 0.40 ± 0.09 and 30 of 73 patients (41.1%) had rtM204I/V positive, while 71.2% of pregnant women had serum HBV DNA load more than 103 IU/mL at delivery. It was reported that the pre-existing primary resistance mutations could reduce the susceptibility of anti-HBV monotherapy or even combined-therapy, such as rtM204I was refractory to LAM and TBV, the efficacy of the corresponding NAs could be affected. We tested the association between the mutation frequency of rtM204I and the HBV DNA load decrease, no significant association was found in either high mutation frequency group or low mutation frequency group (Fig. 3), which indicated that the pre-existing primary resistance might have no influence on the short-term TBV treatment. Besides, rtN236T/A mutation that was related to decreasing sensitivity of tenofovir disoproxil fumarate (TDF) presented in 53.4% of the pregnant women, which had no difference with the proportion of the patients with rtM204I/V mutation. This indicated that TDF with high genetic barrier to HBV resistance might not be superior to TBV for the pregnant women to prevent MTIT from the view of pre-existing primary resistance mutations.
We used UDPS to detect the drug resistance mutations, which is much more sensitive than the methods of many studies used before (5–20% variants detected in NAs-naïve patients)[10–12]. The UDPS can detect minor HBV variants and reveal the massive genetic heterogeneity by parallel amplification and detection of abundant small size sequences; moreover, it can provide longer reads than other techniques and is suitable for viral resistance studies. As far as we know, this is the first work to evaluate the pre-existing NA resistance mutations by UDPS in a moderate sample of pregnant women with chronic HBV infection. In the present study, 41.1% of patients were rtM204I/V positive, while only 9.6% (7/73) patients had rtM204I/V frequency 20% or more. In addition, the average frequency of the mutation was 0.13 ± 0.11, both of which were consistent with the previous study findings [10–12]. Two previous studies conducted rtM204I/V mutation testing with sensitive methods. Kirishima et al. reported 22.2% (4/18) NA-naïve patients had rtM204I/V mutation by peptide nucleic acid mediated polymerase chain reaction clamping which could detect mutation rate as low as 0.01 − 0.001% , and Ayres et al. detected 12.5% (3/24) pregnant women had the mutation by UDPS , which were lower than the rate in our study, the difference may be associated with the very limited sample sizes in the above two studies.
Drug-resistant HBV variants were reported to emerge in the mothers accepted short-term LAM treatment during late pregnancy . Han et al. reported rtM204 mutation arose in two mothers at 22 weeks and 71 weeks of TBV treatment, respectively . In our study, approximately 7 months TBV treatment was administrated in the pregnant women. No increases of the viral quasispecies complexity and the frequency of rtM204I mutation were observed, which supplemented the safety of TBV treatment in late pregnancy. Furthermore, some pregnant women had multi-base mutations combined with rtM204I/V at baseline, including rtM204I + rtA181T/V, rtM204I/V + rtL80I/V, rtM204I/V + rtN236T, and rtM204I/V + rtI233V, which may affect their sensitivity to LAM, TBV, ADV and TDF. The complexity of viral quasispecies and the frequency of rtM204I mutation had no significant increase after TBV treatment in those pregnant women; however, caution has to be taken for them to choose NAs in subsequent long-term therapy due to the drug resistance mutations.
In this study, the prevalence of HBV pre-existing resistant mutants in pregnant women, the influence of the efficacy of short-term TBV treatment and the drug-resistant mutations after TBV therapy were assessed retrospectively. Although the number of subjects was moderate, a prospective cohort study with larger sample size is necessary to evaluate the relationship between the HBV mutations and the antiviral treatment effect.
In conclusion, the prevalence of pre-existing HBV mutation among the pregnant women was as high as 41.1%. However, the pre-existing HBV mutation had limited influence on the efficacy of short-term TBV treatment, and TBV treatment during late pregnancy seemed not to increase the risk of emerging HBV resistant mutants.