Establishment of a new quantitative detection approach to adefovir-resistant HBV and its clinical application

Table 1 Sensitivity and specificity of the new method for the detection of ADV-associated HBV quasispecies

Plasmid	HBV DNA reagent	rtA181 reagent	rtN236 reagent
Wild strain plasmid of Type B	7.27E+06	-	-
Wild strain plasmid of Type C	7.23E+06	-	-
rtA181V variant plasmid	8.78E+06	1.88E+06	-
rtA181T variant plasmid	1.14E+07	2.26E+06	-
rtN236T variant plasmid of Type B	1.14E+07	-	1.25E+06
rtN236T variant plasmid of Type C	1.12E+07	-	1.31E+06
Mixed variants plasmid	5.92E+06	2.00E+06	1.18E+06

ADV: Adefovir; HBV: Hepatitis B virus.

Table 2 Sensitivity of the new method for the detection of mixed variants

Variant: wild strain	HBV DNA reagent	rtA181 reagent	rtN236 reagent	a/b	a/c
0:1	6.06E+06	-	-
1:1	2.94E+06	4.80E+06	5.10E+06	0.61	0.58
1:10	3.98E+06	486 276.3	337 566.8	8.18	11.79
1:50	4.28E+06	83 242.16	85 867.71	51.42	49.84
1:200	5.63E+06	23  130.8	22 000	243.4	255.91
1:1000	5.76E+06	7880	6223.36	730.96	925.55
1:10 000	6.62E+06	-	-

a/b: The total DNA to rtA181 mutation-associated DNA; a/c: The total DNA to rtN236 mutation-associated DNA.

Table 3 PCR of HBV DNA, mutations of rtA181 and rtN236, a/b and a/c in patients who did not receive ADV treatment

Serum samples	HBV DNA reagent	rtA181 reagent	rtN236 reagent	a/b	a/c
Sample 1	2.45E+07	541.19	-	45 270.61
Sample 2	6.79E+06	-	-
Sample 3	1.09E+07	935.07	-	11 656.88
Sample 4	7.48E+06	-	-
Sample 5	3.24E+07	4285.91	185.27	7559.65	174 879.91
Sample 6	3.02E+07	1652.39	-	18 276.56
Sample 7	4.22E+06	-	-
Sample 8	1.64E+07	2871.36	-	5711.58

PCR: Polymerase chain reaction.

Table 4 Quantification of rtA181 and rtN236 mutations

a/b	n	rtA181 mutation (log10 IU/mL)	P1	a/c	n	rtN236 mutation (log10 IU/mL)	P2
1-10	6	6.22 ± 0.55	< 0.0001	1-200	8	5.31 ± 0.25	< 0.0001
10-200	14	4.78 ± 0.21
≥ 200	12	3.14 ± 0.33		≥ 200	24	2.49 ± 0.19

¹Analysis of one way variance (ANOVA) result; ²t-test result. n: Number of samples.

Table 5 ADV-resistant mutations detected by sequence analysis and their quantification

Patients	rtA181	rtN236	a/b	a/c
#1	rtA181T		8.32
#2	rtA181V		1.66
#3	rtA181T		0.931
#4	rtA181T		1.76
#5	rtA181T	rtN236T	1.03	3.11
#6	rtA181T		7.13

¹The ratio below 1 due to PCR error.