Clinical Efficacy and Safety in Telbivudine- or Tenofovir-Treated Hepatitis B e Antigen-Positive Pregnant Women

Abstract

Background

Telbivudine (LdT) and tenofovir (TDF) are widely used in pregnant women to prevent vertical transmission; however, limited data are available on the differences in clinical efficacy and safety between the two drugs.

Methods

A total of 307 hepatitis B e antigen (HBeAg)-positive pregnant women with complete follow-up data were enrolled, the patients with alanine aminotransferase (ALT) levels <1xULN at baseline were enrolled to cohort 1 for treatment from 28 ±4 weeks gestation to delivery, while ALT levels >1xULN at baseline were enrolled to cohort 2 for treatment from 28 ±4 weeks gestation and continued after delivery. The clinical efficacy and safety was compared in LdT- and TDF-treated patients. In addition, 32 patients in cohort 1 were analysed for nucleoside analogue (NA)-related resistance mutations at baseline and after delivery.

Results

The results showed that HBV DNA levels were significantly lower at delivery than at baseline (P<0.001), but the decreases in HBV DNA, ALT, total bilirubin and total bile acid levels did not differ between the LdT- and TDF-treated patients at different time points (P>0.05) in the two cohorts. However, gastrointestinal adverse effects (vomiting) occurred more frequently in TDF-treated than LdT-treated patients (6.6% versus 0.0%; P=0.001). The results of NA-related resistance mutations analysis in cohort 1 revealed that short-term LdT or TDF treatment did not significantly change the NA-related resistance mutations (P>0.05).

Conclusions

This study revealed that the clinical efficacy in LdT- or TDF-treated HBeAg-positive Chinese pregnant women is similar, and gastrointestinal adverse effects occurred more frequently in TDF-treated patients.

Introduction

HBV infection is a serious global public health problem, and it is strongly related to hepatic failure, liver cirrhosis and hepatocellular carcinoma [1]. Although antiviral therapy can reduce the risk of severe liver diseases, achieving the goal of HBV eradication is difficult at present [2]. Therefore, the prevention of HBV transmission remains an important method of decreasing the disease burden of HBV infection. Mother to child transmission (MTCT) is the most common route for HBV infection in Asia, especially in China [3], and more than 90% of infants would be infected with HBV if immunoprophylaxis (immunoglobulin and hepatitis B vaccine) was not available [4]. Even if the infants receive effective immunoprophylaxis, the failure rate is still 10–15% according to the literature [5].

Previous studies revealed that a high level of HBV DNA in mothers is the most important risk factor to MTCT [6,7], especially the HBV DNA threshold higher than 2x6 log₁₀ IU/ml, the MTCT rate is significantly higher [8]. The object of antiviral therapy administered from the third trimester of pregnancy to delivery is to decrease the serum HBV DNA level and decrease perinatal transmission. Currently, nucleoside analogues (NAs) are widely used as therapeutic methods for HBV-positive pregnant women, and they are recommended by the European Association for the Study of the Liver (EASL), Asian Pacific Association for the Study of the Liver (APASL) and American Association for the Study of Liver Disease (AASLD) guidelines [9]. LdT and TDF are the most commonly used antiviral drugs in pregnant women and are approved as pregnancy category B drugs. Both LdT and TDF are effective at reducing the HBV DNA viral load in HBV-positive pregnant women and significantly decreasing the risk of MTCT [10]. In addition, LdT and TDF are safe for pregnant women and infants in the third trimester, even when used throughout the pregnancy period [11,12]. However, limited data are currently available comparing the clinical efficacy and safety of LdT and TDF in patients treated from the third trimester in pregnant women.

The genome of HBV is approximately 3.2 kb nucleotides long with four open reading frames (ORFs), namely, the pre-core/core, polymerase, surface and x open reading frames. The replication of HBV relies on RNA-dependent DNA polymerase, which lacks proofreading capability, so the rate of nucleotide change is much higher than in other DNA viruses. Therefore, large numbers of mutations are produced naturally or occur in a given replicative environment [13]. HBV mutations are associated with the efficacy of antiviral therapy and HBV-related liver disease, most importantly, viral mutations, especially drug resistance mutations, may yield important information for clinicians when they are choosing antiviral drugs after delivery, and the effect of short-term antiviral treatment on the occurrence of antiviral drug resistance mutations is unknown.

The aim of this study was to investigate the differences in clinical efficacy and safety in LdT- and TDF-treated hepatitis B e antigen (HBeAg)-positive pregnant women in China.

Methods

Study Subjects

The current study was retrospective. Among the 1,170 HBV-infected pregnant women treated from January 2012 to August 2019, 307 patients who received antiviral therapy from 28 ±4 weeks (w) gestation with complete follow-up records were enrolled. All of these patients were positive for HBeAg and received treatment at the Guangzhou Eighth People's Hospital of Guangzhou Medical University. The inclusion criteria included the following: patients never exposed to any antiviral drugs for the treatment of HBV; a history of hepatitis B surface antigen (HBsAg)-positivity longer than 6 months, HBeAg-positive and a serum HBV DNA level ≥2x6 log₁₀ IU/ml; a gestational age between 28 ±4 w and good compliance and a complete record of the follow-up data. The exclusion criteria were as follows: previous antiviral therapy for HBV, coinfected with HCV, HDV or HIV, and co-existance with autoimmune hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease, drug-induced liver disease, hepatic failure, liver cirrhosis or hepatocellular carcinoma.

The patients were allocated to two groups depending on different antiviral drugs: the LdT group comprised 185 HBV-infected pregnant women receiving LdT (Sebivo, 600 mg/day; Novartis, Beijing, China) and the TDF group comprised 122 HBV-infected pregnant women receiving TDF (Viread, 300 mg/day; GSK, Tianjin, China) from 28 ±4 w of gestation, all subjects underwent routine pregnancy tests every 4 w before delivery and follow-up at 4 w, 12 w and 24 w postpartum. Among these patients, 3 in the LdT group and 13 in the TDF group switched antiviral drugs due to drug-related adverse effects and were excluded for further analysis. According to the HBV treatment guidelines, the treatments were different based on different alanine aminotransferase (ALT) levels at baseline in HBV-infected pregnant women, so in this study, we classified two cohorts by ALT level to present the data, 147 LdT- and 76 TDF-treated patients with normal serum ALT levels (<1x upper limit of normal [ULN], 40.0 U/l, indicated the patients were at the immune tolerance phase) at baseline were enrolled to cohort 1, and initiated the antiviral therapy from 28 ±4 w of gestation to delivery. Meanwhile, 35 LdT- and 33 TDF-treated patients with abnormal ALT levels (>1xULN, indicated the patients were at the immune clearance phase) at baseline were enrolled to cohort 2, which also initiated the antiviral therapy at 28 ±4 w of gestation but continued the antiviral drugs after delivery. In addition, three LdT- and two TDF-treated patients in cohort 2 were excluded for further analysis because they requested to stop the antiviral drugs after delivery. Finally, 147 LdT- and 76 TDF-treated patients in cohort 1 and 32 LdT- and 31 TDF-treated patients in cohort 2 were enrolled for further analysis. Moreover, maternal serum samples were collected at 28 ±4 w of gestation (baseline, defined as T1) and 1 to 3 months postpartum (defined as T2) for NA-related resistance mutations analysis in cohort 1. All the infants received standard immunoprophylaxis including hepatitis B immunoglobulin and the first dose of the HBV vaccine within 12 h after birth and the second and third doses of the HBV vaccine at ages 1 and 6 months, respectively. The study design flow chart is shown in Figure 1.

Figure 1

The study design flow chart of patients enrolled in this study

The study protocol was approved by the Ethics Committee of Guangzhou Eighth People's Hospital, and it was compliant with the Helsinki Declaration of 1975, as revised in 2008, informed consent was obtained from each subject at recruitment.

Laboratory examination

For all study subjects, at least 5 ml of venous blood was collected at every visit. HBV serological markers were tested by chemiluminescence immunoassays (Abbott Laboratories, Chicago, IL, USA) or enzyme-linked immunosorbent assays (Wantai Biological, Beijing, China). ALT, total bilirubin (TBIL), total bile acid (TBA) and albumin (ALB) levels were determined with commercial kits using an AU2700 automatic biochemical analyzer (Olympus, Tokyo, Japan). Serum HBV DNA levels were measured by the TaqMan PCR assay (DaAn Gene, Guangzhou City, China; the detection limit is 100 IU/ml).

Viral nucleic acid extraction and PCR amplification

Serum samples from T1 and T2 were collected and stored at -80°C until analysis. HBV DNA was extracted from 200 ml of serum using Qiagen DNA blood mini kits (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Regions containing parts of the HBV RT gene (nt 368-827, based on D00330, defined as the HBV RT-1 fragment and nt 726–908, defined as the HBV RT-2 fragment) were amplified from the samples. Nested PCR was performed for all the samples with specific HBV primers designed for the HBV RT-1 and RT-2 genes [14], and the primer sequences are shown in Additional file 1. A pair of barcodes (6 nucleotides) for each inner primer was added to identify each sample. PrimeStar MAX DNA polymerase (Takara Biotechnology Co. Ltd, Dalian City, China) was used for the PCR as per the following procedure. The first round of PCR included predenaturation at 98°C for 3 min, 30 cycles of denaturation at 98°C for 10 s, annealing at 55°C for 5 s, extension at 72°C for 40 s–1 min, and a final extension at 72°C for 5 min. The second round of PCR included predenaturation at 98°C for 3 min, 30 cycles of denaturation at 98°C for 10 s, annealing at 55°C for 5 s, extension at 72°C for 30 s, and a final extension at 72°C for 5 min. Products from the second round of PCR were analysed on a 1% agarose gel. The amplicons of the HBV RT-1 and RT-2 genes were 472 bp and 195 bp in length, respectively. The PCR products were purified using TaKaRa MiniBEST Agarose Gel DNA Extraction Kit (Takara Biotechnology Co. Ltd).

Library construction and sequencing

HBV RT-1 fragment sequencing was performed on the Illumina platform with HiSeq PE250, while sequencing of the HBV RT-2 gene fragments was performed with HiSeq PE150. To evaluate the PCR and NGS error rates for HBV sequencing, the plasmid carrying the same genome of the HBV RT-1, RT-2 regions was included under the same conditions. The NEBNext Ultra DNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA) was used to construct the library.

Data analysis of NGS

The NGS data of the HBV RT-1 and RT-2 genes were first filtered with the FASTQ Quality Filter in the FASTX-Toolkit, and Q30 reads less than 90% were excluded. The paired-end reads of each sample were merged by Flash V1.2.9 with the relevant parameters (minimum overlap =10, maximum mismatch density =0.05), followed by confirmation of the mutations with Geneious R10.0.5 software. Drug resistance-related mutations in the RT gene region were analysed, included V173L, L180M, A181V/T, T184A, A194T, S202G/I, M204V/I/S, V207I, S213T, V214A, Q215S, N236T, P237H, K241E, M250V, respectively. Based on the HBV plasmid error rates in this study (less than 1%) and previous research [15], a positive cutoff value of 1% was defined. Reference sequences for HBV B (D00330) and HBV C (KM999990) were obtained from GenBank (NCBI). The HBV genotypes were determined by mapping the NGS data (HBV RT-1 gene) to the reference sequences and confirmed by MEGA 7.0 with the neighbour-joining method (shown in Additional file 2).

Statistical analysis

Demographic and clinical data were analysed for all study subjects. Continuous variables are represented as the mean ± standard deviation or median ± interquartile range (IQR); categorical variables are represented as percentages. The χ² test and Fisher's exact test were used for categorical variables, Student's t-test was used for normally distributed continuous variables, Mann-Whitney U test was used for nonparametric variables, the repeated measure analysis of covariance was used to compare the clinical data in different groups, and SPSS 13.0 was used for all analyses. A P-value less than 0.05 was considered statistically significant.

Results

Study population characteristics and the safety in the patients enrolled in this study

A total of 307 patients were enrolled at recruitment, including 185 LdT-treated patients and 122 TDF-treated patients. All subjects’ baseline characteristics in cohort 1 and 2 are shown in Table 1, among these patients, no significant differences were observed for age, serum ALT level, HBV DNA level, ALB level, TBIL level, TBA level and HBV genotype between LdT-treated and TDF-treated patients (P>0.05). Furthermore, paired serum samples were collected from a total of 32 patients in the LdT (n=16) and TDF (n=16) groups in cohort 1 for drug resistance mutations analysis, these patients’ clinical characteristics were also not significantly different at baseline (P>0.05), as detailed in Additional file 3. Among the 307 patients, gastrointestinal adverse effects, especially vomiting symptoms, occurred more frequently in TDF-treated patients (0.0% versus 6.6%; P=0.001; Table 2), 3 LdT- and 13 TDF-treated patients switched antiviral drugs to TDF and LdT, respectively, for the adverse effects, including nausea (1, 33.3%), dizziness (1, 33.3%), itchy skin (1, 33.3%) in 3 LdT-treated patients, and vomiting (7, 53.8%), vomiting and dizziness (1, 7.7%), dizziness (1, 7.7%), sour regurgitation and nausea (1, 7.7%), decreased appetite (1, 7.7%), skin itchy (1, 7.7%), dizziness, nausea and abdominal pain (1, 7.7%) in 13 TDF-treated patients respectively, these 16 patients were excluded for further analysis to more accurately compare the clinical efficacy between the two antiviral drugs.

Table 1

The clinical characteristics of LdT- and TDF-treated patients in cohort 1 and cohort 2

Clinical characteristics	LdT (cohort 1), n=147	TDF (cohort 1), n=76	P-value	LdT (cohort 2), n=32	TDF (cohort 2), n=31	P-value
Median age, years (IQR)	28.0 (26.0–31.0)	27.0 (25.0–30.0)	0.172	27.0 (25.0–29.0)	28.0 (26.0–30.0)	0.236
Median ALT, U/l (IQR)	18.0 (14.0–25.0)	17.0 (14.0–24.0)	0.873	78.0 (58.0–150.0)	117.0 (73.0–169.0)	0.118
ALT >1–2xULN, n (%)	0 (0.0)	0 (0.0)	–	15 (46.8)	9 (29.0)	0.145
ALT >2xULN, n (%)	0 (0.0)	0 (0.0)		17 (53.2)	22 (71.0)
Median TBIL, mmol/l (IQR)	6.2 (4.3–8.1)	6.9 (4.6–7.8)	0.284	9 (7–12)	9 (5–10)	0.270
Median TBA, mmol/l (IQR)	2.1 (1.1–3.5)	2.1 (1.2–3.2)	0.935	4.6 (3.3–8.7)	4.7 (2.1–8.9)	0.388
Median ALB, g/l (IQR)	38.0 (36.0–42.0)	38.0 (36.0–43.0)	0.826	39.0 (37.0–41.0)	38.0 (37.0–42.0)	0.738
HBeAg status
Positive, n (%)	162 (100.0)	86 (100.0)	1.000	32	31	1.000
Negative, n (%)	0 (0.0)	0 (0.0)		0	0
Mean HBV DNA, log₁₀ IU/l ±sd	7.8 ±0.7	7.9 ±0.6	0.250	7.4 ±0.9	7.3 ±0.9	0.695
HBV genotypes
B, n (%)	90 (61.2)	42 (55.3)	0.391	20	18	0.719
C, n (%)	57 (38.8)	34 (44.7)		12	13
Initial antiviral therapy time
24w, n (%)	22 (15.0)	11 (14.5)	0.922	4 (12.5)	3 (9.7)	0.518
26w, n (%)	25 (17.0)	9 (11.8)	0.309	5 (15.6)	4 (12.9)	0.521
28w, n (%)	78 (53.1)	37 (48.7)	0.535	15 (46.9)	16 (51.6)	0.707
29w, n (%)	5 (3.4)	3 (3.9)	0.835	0 (0.0)	0 (0.0)	1.000
30w, n (%)	7 (4.8)	9 (11.8)	0.052	5 (15.6)	4 (12.9)	0.521
32w, n (%)	10 (6.8)	7 (9.2)	0.521	3 (9.4)	4 (12.9)	0.482

ALB, albumin; ALT, alanine aminotransferase; HBeAg, hepatitis B e antigen; LdT, telbivudine; TBA, total bile acid; TBIL, total bilirubin; TDF, tenofovir; w, week.

Table 2

The adverse effects that occurred in LdT- and TDF-treated patients initiated from 28 ±4 weeks gestation

Adverse events	LdT (n=185)	TDF (n=122)	P-value
Nausea	1 (0.5)	3 (2.5)	0.305
Vomiting	0 (0.0)	8 (6.6)	0.001
Palpitation	0 (0.0)	1 (0.8)	0.397
Dizziness	1 (0.5)	3 (2.5)	0.305
Decreased appetite	0 (0.0)	1 (0.8)	0.397
Fidgety	0 (0.0)	2 (1.6)	0.157
Skin itchy	1 (0.5)	1 (0.8)	1.000
Abdominal pain	0 (0.0)	2 (1.6)	0.157
Numbness of the hands	0 (0.0)	1 (0.8)	0.397
Sour regurgitation	0 (0.0)	1 (0.8)	0.397
Elevation of creatine kinase^a	4 (2.2)	6 (4.9)	0.204

Data are n (%).

The patients experienced mild creatine kinase elevation (1-1.5xULN) and this returned to normal without treatment. LdT, telbivudine; TDF, tenofovir; ULN, upper limit of normal.

The clinical efficacy between short-term LdT and TDF treatment in cohort 1

In this cohort, the serum HBV DNA levels did not differ between LdT- and TDF-treated patients at different time points (P>0.05). The viral load levels at baseline and at delivery were 7.8 ±0.7 versus 7.9 ±0.6 log₁₀ IU/ml and 3.2 ±1.4 versus 3.5 ±1.1 log₁₀ IU/ml in LdT- and TDF-treated patients, respectively (P>0.05); however, at 4 w postpartum, the HBV DNA rose to 6.8 ±1.5 versus 7.0 ±1.8 log₁₀ IU/ml in the LdT- and TDF-treated groups (P>0.05) and remained at a high viraemia level at 12 w and 24 w postpartum. Serum HBV DNA levels were significantly decreased at delivery compared with baseline in LdT- and TDF-treated patients (4.6 ±1.2 versus 4.5 ±0.9 log₁₀ IU/ml; P<0.001). At delivery, HBV DNA was negative in 12.2% (18/147) of LdT-treated patients and 11.8% (9/76) of TDF-treated patients (P>0.05). Serum ALT levels at different time points also did not differ between the LdT- and TDF-treated groups (P>0.05), although the ALT levels were elevated after delivery. Similarly, TBIL and TBA levels were not different between the two groups (P>0.05). The results of multivariable analysis and repeated measure analysis also showed that HBV DNA, ALT, TBIL, TBA did not differ between the LdT- and TDF-treated groups (F=0.004, 0.028, 0.633 and 0.556; P=0.950, 0.869, 0.572 and 0.652 respectively), as detailed in Table 3 and Additional file 4.

The clinical efficacy between LdT and TDF treatment in cohort 2

In cohort 2, the serum HBV DNA levels also did not differ between LdT- and TDF-treated patients at different time points in this cohort (P>0.05). The viral load levels at baseline and at delivery were 7.4 ±0.9 versus 7.3 ±0.9 log₁₀ IU/ml and 3.0 ±0.8 versus 2.9 ±0.9 log₁₀ IU/ml in LdT- and TDF-treated patients, respectively (P>0.05). Serum HBV DNA levels were significantly decreased at delivery compared with baseline in LdT- and TDF-treated patients (4.4 ±0.8 versus 4.3 ±0.9 log₁₀ IU/ml; P<0.001). In addition, 18.5% (6/32), 37.5% (12/32), 87.5% (28/32), 100.0% (32/32) of patients in LdT-treated patients and 25.8% (8/31), 48.4% (15/31), 93.5% (29/31), 100.0% (31/31) of patients in TDF-treated patients serum HBV DNA was negative at delivery, 4 w, 12w, 24 w postpartum, respectively (P>0.05). Serum ALT levels were elevated in some patients in LdT- and TDF-treated patients after delivery although the HBV DNA remains suppressed, and TBIL and TBA levels were not different between the two groups (P>0.05). The results of multivariable analysis and repeated measure analysis also showed that HBV DNA, ALT, TBIL, TBA did not differ between the LdT-and TDF-treated groups (F=0.950, 0.037, 0.223 and 0.435; P=0.375, 0.851, 0.640 and 0.531, respectively), as detailed in Table 3 and Additional file 4.

Table 3

The clinical data (HBV DNA and ALT level) of LdT- and TDF-treated patients in cohort 1 and cohort 2

Clinical data	LdT (cohort 1), n=147	TDF (cohort 1), n=76	P-value	LdT (cohort 2), n=32	TDF (cohort 2), n=31	P-value
HBV DNA
0 w (at baseline)	7.8 ±0.7	7.9 ±0.6	0.250	7.4 ±0.9	7.3 ±0.9	0.695
4 w	4.7 ±1.0	4.8 ±1.0	0.877	4.0 ±1.4	3.6 ±1.8	0.376
8 w	3.9 ±1.3	4.0 ±1.1	0.774	3.4 ±1.1	3.3 ±1.0	0.984
Delivery	3.2 ±1.4	3.5 ±1.1	0.338	3.0 ±0.8	2.9 ±0.9	0.756
+4 w (4 w postpartum)^a	6.8 ±1.5	7.0 ±1.8	0.942	2.2 ±0.3	2.3 ±0.3	0.965
+12 w (12 w postpartum)^a	7.5 ±0.7	7.6 ±0.8	0.777	2.1 ±0.2	2.1 ±0.3	0.984
+24 w (24 w postpartum)^a	7.5 ±0.8	7.5 ±1.2	0.578	<2	<2	1.000
ALT level
0 w (at baseline)	18.0 (14.0–25.0)	17.0 (14.0–24.0)	0.873	78.0 (58.0–150.0)	117.0 (73.0–169.0)	0.118
4 w	17.0 (13.0–23.0)	18.0 (15.0–24.0)	0.754	72.0 (37.0–117.0)	60.0 (33.0–115.0)	0.626
8 w	17.0 (13.0–23.0)	19.0 (12.0–30.0)	0.187	49.0 (22.0–106.0)	43.0 (33.0–71.0)	0.705
Delivery	18.0 (13.0–28.0)	18.0 (14.0–28.0)	0.798	41.0 (25.0–47.0)	39.0 (30.0–49.0)	0.687
+4 w (4 w postpartum)^a	38.0 (26.0–54.0)	40.0 (25.0–55.0)	0.568	44.0 (21.0–121.0)	56.0 (33.0–247.0)	0.521
ALT >1–2xULN, n (%)	33 (22.4)	24 (31.2)	0.138	7 (21.9)	7 (22.6)	0.946
ALT >2xULN, n (%)	21 (14.3)	13 (17.1)	0.579	5 (15.6)	6 (19.4)	0.697
+12 w (12 w postpartum)^a	47.0 (28.0-77.0)	45.0 (30.0-80.0)	0.689	25 (20.0-29.0)	28.0 (24.0-39.0)	0.345
ALT >1–2xULN, n (%)	50 (34.0)	28 (36.8)	0.575	5 (15.6)	4 (12.9)	0.521
ALT >2xULN, n (%)	20 (13.6)	15 (19.7)	0.233	2 (6.3)	2 (6.5)	0.681
+24 w (24 w postpartum)^a	37.0 (19.0–50.0)	38.0 (20.0–51.0)	0.966	13.0 (11.0–15.0)	20.0 (15.0–25.0)	0.164
ALT >1–2xULN, n (%)	38 (25.9)	20 (26.3)	0.491	4 (12.5)	3 (9.7)	0.694
ALT >2xULN, n (%)	12 (8.2)	7 (9.2)	0.621	2 (6.3)	1 (3.2)	0.512

Data are means ±sd for HBV and median (IQR) for alanine aminotransferase (ALT).

Data from 4 weeks (w) to 24 w postpartum. LdT, telbivudine; TDF, tenofovir; ULN, upper limit of normal.

HBV infection rate of the infants in the two cohorts

The HBV infection rate in the infants at 28 w postpartum was also evaluated in the LdT- and TDF-treated groups in our cohorts. The results showed that no infants were positive for HBsAg and HBV DNA at week 28 postpartum in the LdT- or TDF-treated patients (P>0.05).

Analysis of the NA-related resistance mutations at T1 and T2 in cohort 1

The average number of reads generated for the HBV RT-1 and RT-2 genes at T1 and T2 were 10,249 ±4,165 versus 9,393 ±3,541, 12,741 ±2,697 versus 12,900 ±2,618 reads, respectively (P>0.05). The drug resistance mutations in the HBV RT region were no different at T1 and T2 in LdT- and TDF-treated patients (P>0.05). Among the LdT-treated patients, the pre-existing minor drug resistance mutations S213T were found in 1 patient (6.3%) at T1, and this mutation was also found at T2, and no other drug resistance mutations emerged at T2. Among the TDF-treated patients, no drug resistance mutation was found at T1 and T2.

Discussion

In China, approximately 7.18% of people are infected with HBV and approximately 5.5% to 7.5% of pregnant women test positive for HBsAg according to the literature [16–18]. Among HBsAg-positive women aged 15–39 years, 30% are positive for HBeAg [19]. MTCT is the most common route of HBV infection in China; therefore, management of HBV-positive pregnant women is critical to decrease the risk of HBV transmission. Routine immunoprophylaxis is performed, including administration of HBV immu-noglobulin and the hepatitis B vaccine, however, the failure rate of routine methods reaches 10–15%. LdT and TDF are widely used in the third trimester of pregnancy for high viraemia patients and significantly decrease the risk of MTCT. They have been approved as pregnancy category B drugs, and the safety and efficacy of these drugs for mothers and fetuses have been reported in some studies [20,21]. However, limited data are available on the difference between LdT and TDF treatments started from the third trimester of pregnancy.

Previous studies have analysed the efficacy and safety of LdT or TDF treatment; however, most of these studies investigated each antiviral drug alone. To the best of our knowledge, only one study focused on comparing the safety and efficacy of LdT and TDF in pregnant women and the difference in the prevention of HBV vertical transmission. That study enrolled 58 LdT-treated and 51 TDF-treated HBV-positive pregnant women and 36 patients who received no antiviral treatment as a control. The results showed that there were no differences in the clinical characteristics (HBV DNA and ALT level) of the mothers in the LdT- and TDF-treated groups, but both groups experienced significant decreases in HBV DNA levels compared with the no antiviral treatment group [22]. However, this study did not compare the differences of clinical efficacy based on different ALT levels in the LdT- and TDF-treated patients at baseline, and the sample size was relatively small, in addition, it did not compare the adverse effects that occurred in the patients.

The results of the present study revealed that antiviral therapy started from the third trimester can significantly decrease the HBV DNA level in HBeAg-positive pregnant women at delivery; however, after stopping the viral drugs in cohort 1, the HBV DNA level rose to nearly the same level as at the baseline. The serum ALT level was stable before delivery, but some of the patients experienced ALT level increases at 4 w, 12 w and 24 w postpartum in the two cohorts, which is consistent with the findings of previous studies. Serum TBIL and TBA levels were stable in the normal range throughout the period. In addition, the results showed that the kinetics of clinical data did not differ between LdT- and TDF-treated in the two cohorts, suggested no matter whether the patients were in immune tolerance or immune active phase, the clinical efficacy was nearly the same treated with the two drugs. Moreover, the present study analysed the differences of NA-related resistance mutations in short-term LdT- and TDF-treated patients in cohort 1 and the results showed that the no NA-related resistance mutations occurred after antiviral treatment, especially in LdT-treated patients. NAs are important drugs for treating HBV-infected patients and the major challenge for NA treatment are the occurrence of drug resistance mutations. LdT has a lower genetic barrier to resistance compared with TDF [23]; however, the drug resistance data of LdT usually originate from long-term treatment, and the occurrence of drug resistance mutations in patients treated with short courses (12w) is unknown. In this study, NGS was used to detect the mutations, and the results revealed that the short-term usage of LdT in the third trimester did not induce the emergence of new drug resistance mutations. Previous studies showed that approximately 11.3% of HBeAg-positive and 6.5% of HBeAg-negative patients developed drug resistance mutations in the third year of treatment [24], suggesting that long-term LdT treatment does induce drug resistance mutations. In addition, a study found two pregnant patients who received LdT in the third trimester and did not stop treatment in the postpartum period (n=38) had M204I mutations at 22 w and 71 w, respectively [25]. This is consistent with our research, which showed that short-term usage of LdT from the third trimester to delivery did not increase the risk of drug resistance mutations.

In this study, gastrointestinal side effects (vomiting, 6.6%) occurred more frequently in TDF-treated patients, whereas limited adverse effects were observed in LdT-treated patients, which is consistent with previous studies. A study from China found that 3.5% (2/59) TDF-treated pregnant women experienced vomiting while no patients experienced this adverse effects in the control group (none received antiviral treatment) [26], another study from Taiwan showed that TDF-treated pregnant women more frequently experienced gastrointestinal adverse effects, this study enrolled 62 TDF-treated patients from third trimester and 59 pregnant women as control, the results showed that 5 cases experienced nausea (8.4%) and 3 cases (5.1%) experienced vomiting symptoms in TDF-treated patients and no gastrointestinal adverse effects in control group [27]. However, approximately 0–2% of non-pregnant patients experienced nausea and no vomiting symptoms reported according to the literature [28,29]. The reason for the difference in gastrointestinal adverse effects in pregnant and non-pregnant patients using TDF treatment is unknown, and further research is needed. In this study, the adverse effects of patients receiving LdT treatment were less frequent, and the NA-related resistance mutations were not increased in short-term treatment, indicating that LdT remains a good choice in the third trimester in pregnant women to prevent vertical transmission.

This study had some limitations. This study was a retrospective study and thus may have some bias. Only 32 patients were included in the NA-related resistance mutations analysis because we only collected paired samples, so the enrolment of larger sample sizes in further studies is needed. In this study, no patients experienced HBsAg and HBeAg loss or seroconversion; however, in our cohort, some HBsAg and HBeAg results were qualitative data, so we cannot present and compare the change in HBsAg and HBeAg titres in the two groups. The clinical data was only presented from third trimester to 24 w postpartum because some patients were lost follow-up since 24 w postpartum. In this study, we did not focus on the safety for infants because previous studies have shown that LdT and TDF do not increase the incidence of congenital malfunctions.

In conclusion, this study investigated the clinical efficacy and viral characteristics of short-term TDF- or LdT-treated immune tolerance HBeAg-positive pregnant women in China. This information is crucial for clinicians when choosing appropriate antiviral drugs in HBeAg-positive pregnant women to prevent vertical transmission.

Footnotes

Acknowledgements

We sincerely thank Hongkai Wu (State Key Laboratory of Respiratory Disease, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou City, China) for assisting in the NGS data analysis. This work was supported by grants from the Medical and Health Technology Program of Guangzhou City (20191A011037), Science and Technology Project of Guangzhou City (2010GN-E00221) and Science and Technology Project of Guangzhou Eighth People's Hospital (2014by002).

The authors declare no competing interests.

Additional file 1: A table showing HBV RT-1 and RT-2 genes primers for PCR amplification can be found at

Additional file 2: A figure showing the phylogenetic tree used to determine the HBV genotypes can be found at

Additional file 3: A table showing the clinical characteristics of 32 patients enrolled for NAs-related resistance mutations analysis can be found at

Additional file 4: A table showing the clinical data (TBIL and TBA level) of LdT- and TDF-treated patients in cohort 1 and cohort 2 can be found at

References

Dienstag

J.L.

Hepatitis B virus infection. N Engl J Med 2008; 359: 1486–1500.

European Association for the Study of the Liver. EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection. J Hepatol 2017; 67: 370–398.

Peng

T.T.

, Cai

Q.E.

, Yang

Epidemiological trends and virological traits of hepatitis B virus infection in pregnant women and neonates. Arch Virol 2019; 164: 1335–1341.

Celen

M.K.

, Mert

, Ay

Efficacy and safety of tenofovir disoproxil fumarate in pregnancy for the prevention of vertical transmission of HBV infection. World J Gastroenterol 2013; 19: 9377–9382.

Buchanan

, Tran

T.T.

Management of chronic hepatitis B in pregnancy. Clin Liver Dis 2010; 14: 495–504.

Singh

A.E.

, Plitt

S.S.

, Osiowy

Factors associated with vaccine failure and vertical transmission of hepatitis B among a cohort of Canadian mothers and infants. J Viral Hepat 2011; 18: 468–473.

Burk

R.D.

, Hwang

L.Y.

, Ho

G.Y.

Outcome of perinatal hepatitis B virus exposure is dependent on maternal virus load. J Infect Dis 1994; 170: 1418–1423.

Wen

W.H.

, Chang

M.H.

, Zhao

L.L.

Mother-to-infant transmission of hepatitis B virus infection: significance of maternal viral load and strategies for intervention. J Hepatol 2013; 59: 24–30.

Chen

H.L.

, Wen

W.H.

, Chang

M.H.

Management of pregnant women and children: focusing on preventing mother-to-infant transmission. J Infect Dis 2017; 216: S785–S791.

10.

Jaffe

, Brown

R.S.

Jr. A review of antiviral use for the treatment of chronic hepatitis B virus infection in pregnant women. Gastroenterol Hepatol (N Y) 2017; 13: 154–163.

11.

, Li

M.H.

, Xie

Prospective cohort study on the efficacy and safety of telbivudine used throughout pregnancy in blocking mother-to-child transmission of hepatitis B virus. J Viral Hepat 2017; 24 Suppl 2: 49–56.

12.

Chen

J.Z.

, Liao

Z.W.

, Huang

F.L.

Efficacy and safety of tenofovir disoproxil fumarate in preventing vertical transmission of hepatitis B in pregnancies with high viral load. Sci Rep 2017; 7: 4132.

13.

Kay

, Zoulim

Hepatitis B virus genetic variability and evolution. Virus Res 2007; 127: 164–176.

14.

Deng

, Deng

, Liu

Naturally occurring antiviral drug resistance in HIV patients who are mono-infected or co-infected with HBV or HCV in China. J Med Virol 2018; 90: 1246–1256.

15.

Wang

, Deng

, Qian

Quasispecies characters of hepatitis B virus in immunoprophylaxis failure infants. Eur J Clin Microbiol Infect Dis 2018; 37: 1153–1162.

16.

Cui

, Jia

Update on epidemiology of hepatitis B and C in China. J Gastroenterol Hepatol 2013; 28 Suppl 2: 7–10.

17.

Lao

T.T.

, Sahota

D.S.

, Law

L.W.

Age-specific prevalence of hepatitis B virus infection in young pregnant women, Hong Kong Special Administrative Region of China. Bull World Health Organ 2014; 92: 782–789.

18.

Ding

, Sheng

, Ma

Chronic HBV infection among pregnant women and their infants in Shenyang, China. Virol J 2013; 10: 17.

19.

Liang

, Bi

, Yang

Evaluation of the impact of hepatitis B vaccination among children born during 1992– 2005 in China. J Infect Dis 2009; 200: 39–47.

20.

Shang

, Wen

, Wang

C.C.

Safety and efficacy of telbivudine for chronic hepatitis B during the entire pregnancy: long-term follow-up. J Viral Hepat 2017; 24 Suppl 2: 43–48.

21.

Pan

C.Q.

, Duan

, Dai

Tenofovir to prevent hepatitis B transmission in mothers with high viral load. N Engl J Med 2016; 374: 2324–2334.

22.

Zeng

, Zheng

, Li

Effectiveness of tenofovir or telbivudine in preventing HBV vertical transmission for pregnancy. Medicine (Baltimore) 2019; 98: e15092.

23.

Seifer

, Patty

, Serra

, Li

, Standring

Telbivudine, a nucleoside analog inhibitor of HBV polymerase, has a different in vitro cross-resistance profile than the nucleotide analog inhibitors adefovir and tenofovir. Antiviral Res 2009; 81: 147–155.

24.

Gane

E.J.

, Yuming

, Yun-Fan

Efficacy and safety of prolonged 3-year telbivudine treatment in patients with chronic hepatitis B. Liver Int 2011; 31: 676–684.

25.

Han

G.R.

, Cao

M.K.

, Zhao

A prospective and open-label study for the efficacy and safety of telbivudine in pregnancy for the prevention of perinatal transmission of hepatitis B virus infection. J Hepatol 2011; 55: 1215–1221.

26.

Lin

, Liu

, Ding

Efficacy of tenofovir in preventing perinatal transmission of HBV infection in pregnant women with high viral loads. Sci Rep 2018; 8: 15514.

27.

Chen

H.L.

, Lee

C.N.

, Chang

C.H.

Efficacy of maternal tenofovir disoproxil fumarate in interrupting mother-to-infant transmission of hepatitis B virus. Hepatology 2015; 62: 375–386.

28.

van Bömmel

, de Man

R.A.

, Wedemeyer

Long-term efficacy of tenofovir monotherapy for hepatitis B virus-monoinfected patients after failure of nucleoside/nucleotide analogues. Hepatology 2010; 51: 73–80.

29.

Heathcote

E.J.

, Marcellin

, Buti

Three-year efficacy and safety of tenofovir disoproxil fumarate treatment for chronic hepatitis B. Gastroenterology 2011; 140: 132–143.