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General Demographic Data A total of 290 pairs of newborns and their HBsAg-positive mothers were included and their demographic data are shown in Table 1 (Total column). The rate of HBV intrauterine transmission was 11.72% (34/290).
Table 1. Summary of the Demographic Feature of the Mothers and Newborns
Group Total n (%) (N = 290) HBV Intrauterine Transmission, n (%) χ2 P Value Yes (n = 34) No (n = 256) Mothers Age (y) 8.56 0.014 18-23 77 (26.55) 16 (20.78) 61 (79.22) 24-29 153 (52.76) 14 (9.15) 139 (90.85) 30-48 60 (20.69) 4 (6.67) 56 (93.33) Education level 3.50 0.174 Junior secondary school and below 115 (39.66) 14 (12.17) 101 (87.83) High school and junior college 116 (38.80) 17 (14.66) 99 (85.34) Undergraduate and above 59 (20.34) 3 (5.08) 56 (94.92) Family history of HBV infection 0.02 0.089 Yes 108 (37.24) 13 (12.04) 95 (87.96) No 182 (62.76) 21 (11.54) 161 (88.46) Hepatitis B Vaccine injection 1.000a Yes 2 (0.69) 0 (0.00) 2 (100.00) No 288 (99.31) 34 (11.81) 254 (88.19) HBIG injectionb 0.530a Yes 6 (2.11) 1 (16.67) 5 (83.33) No 284 (97.93) 33 (11.62) 251 (88.38) PBMC HBV cccDNA 9.28 0.002 Positive 49 (16.90) 12 (24.49) 37 (75.51) Negative 241 (83.10) 22 (9.13) 219 (90.87) Serum HBV DNA 0.63 0.430 Positive 167 (57.59) 22 (13.17) 145 (86.83) Negative 123(42.41) 12 (9.76) 111 (90.24) Serum HBeAg 10.10 0.001 Positive 115 (39.66) 22 (19.13) 93 (80.87) Negative 175 (60.34) 12 (6.86) 163 (93.14) Serological patternsc 12.38 0.002 A 99 (34.14) 21 (21.21) 78 (78.79) B 173 (59.66) 12 (6.94) 161 (93.06) Others 18 (6.20) 1 (5.56) 17 (94.44) Mode of delivery 12.88 < 0.001 Vaginal delivery 138 (47.59) 26 (18.84) 112 (81.16) Cesarean section 152 (52.41) 8 (5.26) 144 (94.74) Newborn Characteristics Gestational weeks 0.715a 35-36 20 (6.90) 3 (15.00) 17 (85.00) 37-41 270 (93.10) 31 (11.48) 239 (88.52) Gender 0.15 0.700 Boy 154 (53.10) 17 (11.04) 137 (88.96) Girl 136 (46.90) 17 (12.50) 119 (87.50) Birth weight (g) 0.709a 2, 500-3, 999 271 (93.45) 33 (12.18) 238 (87.82) 4, 000-5, 000 19 (6.55) 1 (5.26) 18 (94.74) Birth length (cm) 45-53 288 (99.31) 34 (11.81) 254 (88.19) 1.000a 54-55 2 (0.69) 0 (0.00) 2 (100.00) Note. aresults from Fisher's Exact Test; bHBIG, hepatitis B immunoglobulin; cPattern A is 'HBsAg (+), HBeAg (+), anti-HBc (+)' and pattern B is 'HBsAg (+), anti-HBe (+), anti-HBc (+)'. The details of serological patterns are presented in Table 3. The Association of PBMC HBV cccDNA with Serum HBV DNA and Serum HBeAg As shown in Table 2, the PBMC HBV cccDNA positive group had significantly more HBV DNA positive serum than in the negative group (χ2 = 13.96, P < 0.001), and serum HBeAg positivity in the PBMC HBV cccDNA positive group was significantly higher than in the negative group (χ2 = 47.74, P < 0.001).
Table 2. The Relationship between PBMC HBV cccDNA and Serological Markers in HBsAg-positive Mothers
HBV Replication Markers PBMC HBV cccDNA, n (%) Total χ2 P Value Positive Negative HBV DNA 13.96 < 0.001 Positive 40 (81.63) 127 (52.70) 167 Negative 9 (18.37) 114 (47.30) 123 Total 49 241 290 HBeAg Positive 41 (83.67) 74 (30.71) 115 47.74 < 0.001 Negative 8 (16.33) 167 (60.29) 175 Total 49 241 290 Serological patternsa 48.48 < 0.001 A 37 (75.51) 62 (25.73) 99 B 8 (16.33) 165 (68.46) 173 Others 4 (8.16) 14 (5.81) 18 Total 49 241 290 Note.aPattern A is 'HBsAg (+), HBeAg (+), anti-HBc (+)' and pattern B is 'HBsAg (+), anti-HBe (+), anti-HBc (+)'. The details of serological patterns are presented in Table 3. The values of PBMC HBV cccDNA, HBV DNA and HBeAg were transformed logarithmically, and Pearson linear correlation analysis was used to analyze the association of PBMC HBV cccDNA with serum HBV DNA and HBeAg. Our results revealed that the number of positive cases of PBMC HBV cccDNA was positively correlated with that of HBV DNA (r = 0.436, P < 0.001) and HBeAg (r = 0.403, P < 0.001).
The Relationship between HBV cccDNA of PBMC and HBV Serological Patterns Six HBV serological patterns are summarized (Table 3) from data on the 290 HBsAg-positive mothers. Pattern A 'HBsAg (+), HBeAg (+), anti-HBc (+)' accounted for 34.14% (99/290) of the cases, while 59.66% (173/290) were pattern B 'HBsAg (+), anti-HBe (+), anti-HBc (+)' with the remaining four patterns accounting for 6.21%.
Table 3. Serological Patterns of 290 HBsAg-positive Mothers
Serological Patterns HBV Serological Markers n (%) HBsAg Anti-HBs HBeAg Anti-HBe Anti-HBc A + - + - + 99 (34.14) B + - - + + 173 (59.66) C + + + - + 6 (2.07) D + + - + + 2 (0.69) E + - + + + 9 (3.10) F + - + - - 1 (0.34) Note. '+' positive; '-' negative. Of note the case distribution of HBV serological pattern A and B between PBMC HBV cccDNA positive and negative group was significantly different (χ2 = 48.48, P < 0.001), with the PBMC HBV cccDNA positive group accounting for the majority of pattern A and the negative group accounting for the majority of pattern B (Table 2).
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Factors Influencing HBV Intrauterine Transmission Of the maternal characteristics listed in Table 1, our analysis revealed that PBMC HBV cccDNA, serum HBV DNA, HBeAg, serological patterns, mode of delivery, and age were significantly associated with HBV intrauterine transmission.
The Effect of PBMC HBV cccDNA and HBV Infection Status on HBV Intrauterine Transmission We combined the data on maternal serum HBV DNA and HBeAg with the PBMC HBV cccDNA data to further analyze the relationship between PBMC HBV cccDNA and HBV intrauterine transmission. The results are shown in Table 4. Taking serum HBeAg and PBMC HBV cccDNA double negativity as a control, our analysis demonstrated that the risk of intrauterine transmission was 2.43 times higher (OR = 2.43, 95% CI: 1.02-5.78) when either HBeAg or HBV cccDNA was positive, and 5.20 times higher when they were both positive. The Mantel-Haenszel test also showed a greater risk of HBV intrauterine transmission when HBeAg and HBV cccDNA were both positive (χ2 = 13.71, P < 0.001). For the same reason, the risk of HBV intrauterine transmission was much higher when HBV DNA and HBV cccDNA were both positive (OR = 3.39, 95% CI: 1.24-9.27; χ2 = 6.01, P = 0.014). When the HBeAg and HBV DNA data were combined to analyze the relationship between PBMC HBV cccDNA and HBV intrauterine transmission, it was found that the risk of HBV intrauterine transmission was highest in the 'HBeAg (+) HBV DNA (+) cccDNA (+)' group (OR = 3.69, 95% CI: 1.30-10.42); χ2 = 8.08, P = 0.004).
Table 4. The Relationship between Maternal PBMC HBV cccDNA, HBV DNA, and HBeAg and HBV Intrauterine Transmission
HBV Replication Markers HBV Intrauterine Transmission χ2 P Value OR (95% CIa) Yes No HBeAg (-) HBV cccDNA (-) 11 (32.35) 156 (60.94) 13.71 < 0.001 HBeAg (+) / HBV cccDNA (+) 12 (35.30) 70 (27.34) 2.43 (1.02, 5.78) HBeAg (+) HBV cccDNA (+) 11 (32.35) 30 (11.72) 5.20 (2.07, 13.08) HBV DNA (-) HBV cccDNA (-) 9 (26.47) 105 (41.02) 6.01 0.014 HBV DNA (+) / HBV cccDNA (+) 16 (47.06) 120 (46.87) 1.56 (0.66, 3.67) HBV DNA (+) HBV cccDNA (+) 9 (26.47) 31 (12.11) 3.39 (1.24, 9.27) HBeAg (-) HBVDNA (-) cccDNA (-) 8 95 8.08 0.004 Other cccDNA (-)b 14 124 1.34 (0.54, 3.33) Other cccDNA (+)c 3 8 4.45 (0.98, 20.17) HBeAg (+) HBV DNA (+) cccDNA (+) 9 29 3.69 (1.30, 10.42) Note. aCI, confidence interval. bOther cccDNA (-) is HBeAg (+) HBVDNA (-) cccDNA (-), HBeAg (-) HBVDNA (+) cccDNA (-), HBeAg (+) HBVDNA (+) cccDNA (-). cHBeAg (+) HBVDNA (-) cccDNA (+), HBeAg (-) HBVDNA (+) cccDNA (+), HBeAg (-) HBVDNA (-) cccDNA (+). As shown in Table 5, the risk of HBV intrauterine transmission was highest (OR = 5.89, 95% CI: 2.36-14.72) when the subject was PBMC HBV cccDNA positive and exhibited serological pattern A, suggesting the critical role of PBMC HBV cccDNA in HBV intrauterine transmission.
Table 5. The Relationship between Maternal PBMC HBV cccDNA and Serological Pattern A on HBV Intrauterine Transmission
HBV cccDNA Pattern A HBV Intrauterine Transmission P Value OR (95% CI) Yes No - - 12 (35.30) 167 (65.23) 1.00 - + 10 (29.41) 52 (20.31) 0.026 2.67 (1.09, 6.34) + - 1 (2.94) 11 (4.30) 0.582a 1.27 (0.15, 10.64) + + 11 (32.35) 26 (10.16) < 0.001 5.89 (2.36, 14.72) Note.aIndicates the use of Fisher's Exact Test. The Bayesian Network of HBV Intrauterine Transmission Factors statistically significant in univariate analysis and HBV DNA except for serum HBeAg were put into a Bayesian model. It was excluded because it was already covered by serological pattern. As shown in Figure 1, the Bayesian network model revealed that maternal PBMC HBV cccDNA was directly related to HBV intrauterine transmission whereas maternal serum
Figure 1. The bayesian network of HBV intrauterine transmission. Age: Low means 18-23, Medium means 24-29, and High means 30-48. Mode_of_delivery: V means vaginal delivery, and C means cesarean section. Serological patterns: P1 means pattern A ['HBsAg (+), HBeAg (+), anti-HBc (+)'], and P2 means pattern B ['HBsAg (+), anti-HBe (+), anti-HBc (+)'].
HBV DNA was indirectly related to HBV intrauterine transmission via maternal PBMC HBV cccDNA. The possibility of HBV intrauterine transmission was much higher when PBMC HBV cccDNA was positive (25.49%) than when it was negative (9.47%), according to the conditional probability distribution.
doi: 10.3967/bes2019.043
Relationship between Maternal PBMC HBV cccDNA and HBV Serological Markers and its Effect on HBV Intrauterine Transmission
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Abstract:
Objective To investigate the relationship between maternal peripheral blood mononuclear cells (PBMC) hepatitis B virus (HBV) covalenty closed circular deoxyribonucleic acid (cccDNA) and other HBV serological markers and its effects on HBV intrauterine transmission. Methods We enrolled 290 newborns and their hepatitis B surface antigen (HBsAg) positive mothers. HBV cccDNA in PBMC and HBV DNA in serum were detected by a real-time PCR-TaqMan probe while HBV serological markers were detected with an electrochemiluminescence immunoassay. Results There was a positive correlation between the levels of PBMC HBV cccDNA and serum HBV DNA and HBeAg (r=0.436 and 0.403, P < 0.001). The detection rate of pattern A['HBsAg (+), HBeAg (+), and anti-HBc (+)'] was significantly higher in the PBMC HBV cccDNA positive group than in the control group (χ2=48.48, P < 0.001). There was a significant association between HBV intrauterine transmission and PBMC HBV cccDNA (χ2=9.28, P=0.002). In the presence of serum HBV DNA, HBeAg, and PBMC HBV cccDNA, the risk of HBV intrauterine transmission was three times higher (OR=3.69, 95% CI:1.30-10.42) than that observed in their absence. The risk of HBV intrauterine transmission was the greatest (OR=5.89, 95% CI:2.35-14.72) when both PBMC HBV cccDNA and pattern A were present. A Bayesian network model showed that maternal PBMC HBV cccDNA was directly related to HBV intrauterine transmission. Conclusion PBMC HBV cccDNA may be a direct risk factor for HBV intrauterine transmission. Our study suggests that serological markers could be combined with PBMC-related markers in prenatal testing. -
Figure 1. The bayesian network of HBV intrauterine transmission. Age: Low means 18-23, Medium means 24-29, and High means 30-48. Mode_of_delivery: V means vaginal delivery, and C means cesarean section. Serological patterns: P1 means pattern A ['HBsAg (+), HBeAg (+), anti-HBc (+)'], and P2 means pattern B ['HBsAg (+), anti-HBe (+), anti-HBc (+)'].
Table 1. Summary of the Demographic Feature of the Mothers and Newborns
Group Total n (%) (N = 290) HBV Intrauterine Transmission, n (%) χ2 P Value Yes (n = 34) No (n = 256) Mothers Age (y) 8.56 0.014 18-23 77 (26.55) 16 (20.78) 61 (79.22) 24-29 153 (52.76) 14 (9.15) 139 (90.85) 30-48 60 (20.69) 4 (6.67) 56 (93.33) Education level 3.50 0.174 Junior secondary school and below 115 (39.66) 14 (12.17) 101 (87.83) High school and junior college 116 (38.80) 17 (14.66) 99 (85.34) Undergraduate and above 59 (20.34) 3 (5.08) 56 (94.92) Family history of HBV infection 0.02 0.089 Yes 108 (37.24) 13 (12.04) 95 (87.96) No 182 (62.76) 21 (11.54) 161 (88.46) Hepatitis B Vaccine injection 1.000a Yes 2 (0.69) 0 (0.00) 2 (100.00) No 288 (99.31) 34 (11.81) 254 (88.19) HBIG injectionb 0.530a Yes 6 (2.11) 1 (16.67) 5 (83.33) No 284 (97.93) 33 (11.62) 251 (88.38) PBMC HBV cccDNA 9.28 0.002 Positive 49 (16.90) 12 (24.49) 37 (75.51) Negative 241 (83.10) 22 (9.13) 219 (90.87) Serum HBV DNA 0.63 0.430 Positive 167 (57.59) 22 (13.17) 145 (86.83) Negative 123(42.41) 12 (9.76) 111 (90.24) Serum HBeAg 10.10 0.001 Positive 115 (39.66) 22 (19.13) 93 (80.87) Negative 175 (60.34) 12 (6.86) 163 (93.14) Serological patternsc 12.38 0.002 A 99 (34.14) 21 (21.21) 78 (78.79) B 173 (59.66) 12 (6.94) 161 (93.06) Others 18 (6.20) 1 (5.56) 17 (94.44) Mode of delivery 12.88 < 0.001 Vaginal delivery 138 (47.59) 26 (18.84) 112 (81.16) Cesarean section 152 (52.41) 8 (5.26) 144 (94.74) Newborn Characteristics Gestational weeks 0.715a 35-36 20 (6.90) 3 (15.00) 17 (85.00) 37-41 270 (93.10) 31 (11.48) 239 (88.52) Gender 0.15 0.700 Boy 154 (53.10) 17 (11.04) 137 (88.96) Girl 136 (46.90) 17 (12.50) 119 (87.50) Birth weight (g) 0.709a 2, 500-3, 999 271 (93.45) 33 (12.18) 238 (87.82) 4, 000-5, 000 19 (6.55) 1 (5.26) 18 (94.74) Birth length (cm) 45-53 288 (99.31) 34 (11.81) 254 (88.19) 1.000a 54-55 2 (0.69) 0 (0.00) 2 (100.00) Note. aresults from Fisher's Exact Test; bHBIG, hepatitis B immunoglobulin; cPattern A is 'HBsAg (+), HBeAg (+), anti-HBc (+)' and pattern B is 'HBsAg (+), anti-HBe (+), anti-HBc (+)'. The details of serological patterns are presented in Table 3. Table 2. The Relationship between PBMC HBV cccDNA and Serological Markers in HBsAg-positive Mothers
HBV Replication Markers PBMC HBV cccDNA, n (%) Total χ2 P Value Positive Negative HBV DNA 13.96 < 0.001 Positive 40 (81.63) 127 (52.70) 167 Negative 9 (18.37) 114 (47.30) 123 Total 49 241 290 HBeAg Positive 41 (83.67) 74 (30.71) 115 47.74 < 0.001 Negative 8 (16.33) 167 (60.29) 175 Total 49 241 290 Serological patternsa 48.48 < 0.001 A 37 (75.51) 62 (25.73) 99 B 8 (16.33) 165 (68.46) 173 Others 4 (8.16) 14 (5.81) 18 Total 49 241 290 Note.aPattern A is 'HBsAg (+), HBeAg (+), anti-HBc (+)' and pattern B is 'HBsAg (+), anti-HBe (+), anti-HBc (+)'. The details of serological patterns are presented in Table 3. Table 3. Serological Patterns of 290 HBsAg-positive Mothers
Serological Patterns HBV Serological Markers n (%) HBsAg Anti-HBs HBeAg Anti-HBe Anti-HBc A + - + - + 99 (34.14) B + - - + + 173 (59.66) C + + + - + 6 (2.07) D + + - + + 2 (0.69) E + - + + + 9 (3.10) F + - + - - 1 (0.34) Note. '+' positive; '-' negative. Table 4. The Relationship between Maternal PBMC HBV cccDNA, HBV DNA, and HBeAg and HBV Intrauterine Transmission
HBV Replication Markers HBV Intrauterine Transmission χ2 P Value OR (95% CIa) Yes No HBeAg (-) HBV cccDNA (-) 11 (32.35) 156 (60.94) 13.71 < 0.001 HBeAg (+) / HBV cccDNA (+) 12 (35.30) 70 (27.34) 2.43 (1.02, 5.78) HBeAg (+) HBV cccDNA (+) 11 (32.35) 30 (11.72) 5.20 (2.07, 13.08) HBV DNA (-) HBV cccDNA (-) 9 (26.47) 105 (41.02) 6.01 0.014 HBV DNA (+) / HBV cccDNA (+) 16 (47.06) 120 (46.87) 1.56 (0.66, 3.67) HBV DNA (+) HBV cccDNA (+) 9 (26.47) 31 (12.11) 3.39 (1.24, 9.27) HBeAg (-) HBVDNA (-) cccDNA (-) 8 95 8.08 0.004 Other cccDNA (-)b 14 124 1.34 (0.54, 3.33) Other cccDNA (+)c 3 8 4.45 (0.98, 20.17) HBeAg (+) HBV DNA (+) cccDNA (+) 9 29 3.69 (1.30, 10.42) Note. aCI, confidence interval. bOther cccDNA (-) is HBeAg (+) HBVDNA (-) cccDNA (-), HBeAg (-) HBVDNA (+) cccDNA (-), HBeAg (+) HBVDNA (+) cccDNA (-). cHBeAg (+) HBVDNA (-) cccDNA (+), HBeAg (-) HBVDNA (+) cccDNA (+), HBeAg (-) HBVDNA (-) cccDNA (+). Table 5. The Relationship between Maternal PBMC HBV cccDNA and Serological Pattern A on HBV Intrauterine Transmission
HBV cccDNA Pattern A HBV Intrauterine Transmission P Value OR (95% CI) Yes No - - 12 (35.30) 167 (65.23) 1.00 - + 10 (29.41) 52 (20.31) 0.026 2.67 (1.09, 6.34) + - 1 (2.94) 11 (4.30) 0.582a 1.27 (0.15, 10.64) + + 11 (32.35) 26 (10.16) < 0.001 5.89 (2.36, 14.72) Note.aIndicates the use of Fisher's Exact Test. -
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