Tenofovir is a more suitable treatment than entecavir for chronic hepatitis B patients carrying naturally occurring rtM204I mutations

doi:10.3748/wjg.v25.i33.4985

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World Journal of Gastroenterology

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World J Gastroenterol. Sep 7, 2019; 25(33): 4985-4998
Published online Sep 7, 2019. doi: 10.3748/wjg.v25.i33.4985

Table 1 Primers and LNA probes developed for identification of hepatitis B virus wild type (YMDD) and rtM204I variants (YIDD) by real-time PCR

Primer/probe	Sequence (5'-3') ¹	T_m (°C)²	Target	Channel
Primers (product: 127 bp)
Forward	TGGGCCTCAGTCCGTTTCT	65.4	HBV RT gene
Reverse	TGTACAGACTTGGCCCCCAAWAC	65.2-66.1	HBV RT gene
LNA Probes
YMDD	5' HEX-TAT+A+T+G+G+AT+GAT- 3' IB®FQ	58	YMDD (wild type)	HEX
YIDD	5' 6-FAM-TAT+A+T+T+G+AT+GAT- 3' IB®FQ	53	YIDD	FAM

¹LNA nucleotides are written +A, +C, +T or +G;

²Primer melting temperature was calculated by using LC PDS software V 2.0 and probe melting temperature by https://www.exiqon.com/ls/pages /exiqontmpredictiontool.aspx. T_m: Melting temperature; RT: Reverse transcriptase; HBV: Hepatitis B virus.

Table 2 Measurement of melting temperatures of hepatitis B virus wild type (YMDD) and rtM204I variants (YIDD) by LNA real-time PCR

Sample (copies, number of samples n)	*Measured T_m* (°C) at channel**
	FAM			HEX
	Min	Max	Mean ± SD (number of positives)	Min	Max	Mean ± SD (number of positives)
YIDD positive control DNA (24-2400000, n = 48)	51.3	52.2	51.7 ± 0.2 (48, 100%)	(-)	(-)	(-)
WT positive control DNA (24-2400000, n = 48)	(-)	(-)	(-)	61.9	63.3	62.6 ± 0.4 (46, 95.8%)
YIDD and WT standard mixtures (24-2400000, n = 280)	50.1	52.6	51.5 ± 0.3 (280, 100%)	61.9	64.0	63.0 ± 0.4 (280, 100%)
YIDD and WT standard mixtures (24, n = 56)	49.9	51.9	51.2 ± 0.5 (45, 80.3%)	60.9	63.8	63.0 ± 0.5 (44, 78.6%)
YIDD of clinical samples in duplicate (82-180000, n = 17)	49.6	51.9	51.3 ± 0.5	(-)	(-)	(-)
YMDD of clinical samples in duplicate (65-13000000, n = 397)	(-)	(-)	(-)	61.1	64.0	62.8 ± 0.3

¹The variants were identified by direct sequencing of PCR products. (-) indicates no significant melting temperature; SD: standard deviation; T_m: Melting temperature.

Table 3 Rates of positive detection of hepatitis B virus wild type (YMDD) and rtM204I variant (YIDD) in 410 samples by LNA real-time PCR

Type of detection	No. of samples	Percentage
Clinical samples	410	100%
Identified	403	98.3%
YIDD	17	4.1%
YIDD exclusively	9	2.2%
YIDD + YMDD	3	0.7%
YIDD + YMDD + YIDD with ATA codon¹	1	0.2%
YIDD + YMDD	1	0.5%
YIDD + YMDD	3	0.7%
YMDD	394	96.3%
YMDD exclusively	385	93.9%
YMDD + unknown variant	1	0.2%
Unidentified	7	1.7%
Non-target T_m	2	0.5%
HEX 50.4 °C; YVDD¹	1	0.2%
HEX 57.9 °C; YMDD with Y of TAC codon¹	1	0.2%
No positive signal	5	1.2%

Bold type indicates dominant form.

¹The variants were identified by direct sequencing of PCR products. T_m: Melting temperature.

Table 4 Baseline characteristics of 403 treatment-naïve patients with chronic hepatitis B

Characteristics	Pre-existing rtM204I n = 17	Wild type rtM204 n = 386	P value
Gender (M/F)	14/3	230/156	0.060
Age (yr)	48.8 ± 9.3	43.7 ± 12.6	0.101
HBeAg (positive/negative)	10/7	228/158	0.984
HBV-DNA (log₁₀ IU/mL)	6.33 ± 0.66	6.06 ± 1.77	0.519
qHBsAg (log ₁₀ IU/mL)	3.43 ± 0.30	3.59 ± 0.70	0.334
AST (IU/L)	104.7 ± 40.9	78.7 ± 65.2	0.103
ALT (IU/L)	77.8 ± 29.4	85.4 ± 84.1	0.709
Total bilirubin (mg/dL)	0.93 ± 0.33	0.88 ± 0.60	0.743
Albumin (g/dL)	4.02 ± 0.43	4.14 ± 0.52	0.338
Prothrombin time (INR)	1.07 ± 0.10	1.02 ± 0.16	0.246
Platelet count (× 10³/mm³)	155.7 ± 55.6	181.4 ± 64.4	0.106
FIB-4	4.38 ± 2.33	2.91 ± 2.92	0.041
APRI	1.94 ± 0.98	1.34 ± 1.35	0.068
Significant fibrosis¹ (presence/absence)	12/5	145/241	0.006
Liver cirrhosis (presence/absence)	7/10	80 / 306	0.045
HCC (presence/absence)	5/12	46 / 340	0.034

¹Significant fibrosis was defined as aspartate aminotransferase to platelet ratio index > 1.5 or fibrosis-4 index > 3.25. ALT: Alanine aminotransferase; APRI: Aspartate aminotransferase to platelet ratio index; AST: Aspartate aminotransferase; FIB-4: Fibrosis-4 Index; HCC: Hepatocellular carcinoma; INR: International normalized ratio; qHBsAg: Quantitative hepatitis B surface antigen levels; HBV: Hepatitis B virus.

Table 5 Independent factors for pre-existing rtM204I variants in treatment-naïve chronic hepatitis B patients

	Pre-existing rtM204I (n = 17)	Wild type rtM204 (n = 386)	Univariate			Multivariate
	Pre-existing rtM204I (n = 17)	Wild type rtM204 (n = 386)	OR	95%CI	P value	OR	95%CI	P value
Gender (male)	14 (82.4%)	230 (59.6%)	3.165	(0.895-11.197)	0.074
Age, yr	48.8 ± 9.3	43.7 ± 12.6	1.033	(0.993-1.074)	0.105
HBeAg status (positive)	10 (58.8%)	228 (59.1%)	0.990	(0.369-2.656)	0.984
HBV-DNA, log₁₀ IU/mL	6.33 ± 0.66	6.06 ± 1.77	1.098	(0.827-1.458)	0.519
qHBsAg, log₁₀ IU/mL	3.43 ± 0.30	3.59 ± 0.70	0.555	(0.336-0.916)	0.021
AST, IU/L	104.7 ± 40.9	78.7 ± 65.2	1.005	(0.999-1.011)	0.109
ALT, IU/L	77.8 ± 29.4	85.4 ± 84.1	0.999	(0.992-1.005)	0.709
Total bilirubin, mg/dL	0.93 ± 0.33	0.88 ± 0.60	1.140	(0.522-2.487)	0.742
Albumin, g/dL	4.02 ± 0.43	4.14 ± 0.52	0.655	(0.275-1.559)	0.339
Prothrombin time, INR	1.07 ± 0.10	1.02 ± 0.16	4.625	(0.339-63.020)	0.250
Platelet count, × 10³/mm³	155.7 ± 55.6	181.4 ± 64.4	0.993	(0.985-1.001)	0.108
Significant fibrosis¹	12 (70.6%)	145 (37.6%)	3.989	(1.377-11.553)	0.011	3.397	(1.119-10.319)	0.031
Liver cirrhosis	7 (41.2%)	80 (20.7%)	2.677	(0.988-7.255)	0.053
HCC	5 (29.4%)	46 (11.9%)	3.080	(1.038-9.139)	0.043	1.961	(0.626-6.143)	0.248

Table 6 Independent factors for complete response at 12 mo of antiviral therapy in 232 patients who were treated with nucleos(t)ide analogues

	Complete response (n = 199)	Incomplete response (n = 33)	Univariate			Multivariate
	Complete response (n = 199)	Incomplete response (n = 33)	OR	95%CI	P value	OR	95%CI	P value
Gender (male)	130 (65.3%)	20 (60.6%)	1.225	(0.575-2.610)	0.600
Age, yr	47.3 ± 11.9	43.6 ± 10.9	1.028	(0.996-1.062)	0.087
HBeAg status (positive)	106 (53.3%)	30 (90.9%)	0.114	(0.034-0.386)	< 0.001	0.438	(0.086-2.226)	0.320
HBV-DNA, log₁₀ IU/mL	5.97 ± 1.40	7.69 ± 1.13	0.402	(0.290-0.559)	< 0.001	0.185	(0.083-0.412)	< 0.001
qHBsAg, log₁₀ IU/mL	3.46 ± 0.57	3.89 ± 0.74	0.270	(0.134-0.544)	< 0.001	1.492	(0.501-4.447)	0.473
AST, IU/L	94.6 ± 69.5	83.0 ± 47.2	1.003	(0.997-1.009)	0.361
ALT, IU/L	103.5 ± 92.7	91.6 ± 57.5	1.002	(0.997-1.006)	0.474
Total bilirubin, mg/dL	0.95 ± 0.57	0.76 ± 0.30	2.201	(0.964-5.024)	0.061
Albumin, g/dL	4.06 ± 0.53	4.14 ± 0.44	0.743	(0.351-1.573)	0.438
Prothrombin time, INR	1.05 ± 0.16	1.01 ± 0.10	8.323	(0.444-155.931)	0.156
Platelet count, × 10³/mm³	164.6 ± 63.8	182.6 ± 53.0	0.995	(0.990-1.001)	0.128
Significant fibrosis	105 (52.8%)	13 (39.4%)	1.718	(0.810-3.644)	0.158
Liver cirrhosis	58 (29.1%)	6 (18.2%)	1.851	(0.726-4.720)	0.197
HCC	33 (16.6%)	3 (9.1%)	1.988	(0.573-6.899)	0.279
High genetic barrier	180 (90.5%)	12 (36.4%)	16.579	(7.069-38.882)	< 0.001	82.076	(14.945-450.760)	< 0.001
Pre-existing rtM204I	9 (4.5%)	8 (24.2%)	0.148	(0.052-0.419)	< 0.001	0.014	(0.002-0.096)	< 0.001

ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; INR: International normalized ratio; qHBsAg: Quantitative hepatitis B surface antigen levels; HBV: Hepatitis B virus; HCC: Hepatocellular carcinoma.

Table 7 Treatment responses during nucleos(t)ide analogues in patients with pre-existing rtM204I variants

Outcome	Tenofovir (n = 7)	Entecavir (n = 6)	Low genetic barriers¹ (n = 4)	P value
Reduction of HBV-DNA (log₁₀ IU/mL), mean ± SD
Mo 3	-3.22 ± 0.74	-2.12 ± 0.53²	-2.22 ± 0.40²	0.011
Mo 6	-3.97 ± 0.75	-2.71 ± 0.43²	-2.92 ± 0.51²	0.005
Mo 9	-4.44 ± 0.70	-3.33 ± 0.48²	-3.28 ± 0.35²	0.004
Mo 12	-4.75 ± 0.59	-3.65 ± 0.43²	-3.52 ± 0.60²	0.003
Complete virologic response, cumulative incidence
Mo 12	100%	16.7%²	25%²	0.021

¹The low genetic barriers include lamivudine, adefovir, or combination of lamivudine and adefovir;

²The same number of superscripted indicates non-specific difference between groups. The continuous variables were tested by one-way ANOVA among groups, and categorical variables were tested by Fisher’s exact test. SD: standard deviation; HBV: Hepatitis B virus.

Citation: Choe WH, Kim K, Lee SY, Choi YM, Kwon SY, Kim JH, Kim BJ. Tenofovir is a more suitable treatment than entecavir for chronic hepatitis B patients carrying naturally occurring rtM204I mutations. World J Gastroenterol 2019; 25(33): 4985-4998
URL: https://www.wjgnet.com/1007-9327/full/v25/i33/4985.htm
DOI: https://dx.doi.org/10.3748/wjg.v25.i33.4985