The association between serum HBsAg LEVEL, HBV viral load and transaminase enzymes in subjects with chronic hepatitis B virus infection

T¹p chÝ y - d−îc häc qu©n sù sè 8-2020 101 THE ASSOCIATION BETWEEN SERUM HBsAg LEVEL, HBV VIRAL LOAD AND TRANSAMINASE ENZYMES IN SUBJECTS WITH CHRONIC HEPATITIS B VIRUS INFECTION Nguyen Dinh Ung1,2, Do Thi Le Quyen2, Hoang Tien Tuyen2 Le Phuong Ha3, Nguyen Trong Chinh2 Ho Huu Tho1,4, Vu Thi Tuong Van5 SUMMARY Objectives: To assess the association between serum HBsAg level, hepatitis B virus (HBV) viral load and transaminase enzymes in subjects with chronic HBV infection. Subjects and methods: Serum samples were collected from 256 participants with chronic HBV infection. The samples were obtained at the Gastroenterology Consulting Room, Outpatient Department, Bach Mai Hospital. These serum samples were extracted, tested to detect HBeAg, quantified HbsAg, and measured HBV DNA level by Realtime PCR reaction. Results: The serum HBsAg level and HBV viral load showed moderate correlation (r = 0.38, p < 0.001). In HBeAg-positive patients, HBsAg levels correlated with HBV viral load (r = 0.41, p = 0.003), whereas in HBeAg-negative patients, there was no correlation between HBsAg levels and HBV viral load (r = 0.12, p = 0.274). In chronic hepatitis B patient group, the serum HBsAg level had a moderate linear correlation with the serum HBV viral load (r = 0.47, p < 0.001). However, the correlation between transaminase enzymes (AST, ALT, GGT) and HBsAg levels was not observed (p > 0.05). Conclusion: Serum HBsAg level may be an independent factor with HBV DNA to assess the efficacy of treatment and prognosis for chronic HBV infection. * Keywords: Chronic hepatitis B virus; HBV DNA; HbsAg; Transaminase enzyme. INTRODUCTION Hepatitis B virus infection is a global problem, with an estimated 2 billion people infected with HBV worldwide, of which nearly 292 million are chronically infected with HBV, accounting for 3.9% of the world population [5]. Although hepatitis B vaccine has been introduced in expanded immunization nationwide, the clinical symptoms of hepatitis B virus infection are diverse, ranging from mild constitutional symptoms of fatigue and nausea to more marked symptoms of hepatitis. Prolonged viral infections include inactive HBV carriers or progression to chronic hepatitis, cirrhosis, or primary liver cancer that can be life-threatening [1]. 1Genomics and Cytogenetic Department, Institute of Biomedicine & Pharmacy, Vietnam Military Medical University 2Infectious Diseases Department, Military Hospital 103, Vietnam Military Medical University 3University of Science and Technology of Hanoi 4Microbiology Department, Military Hospital 103, Vietnam Military Medical University 5Microbiology Department, Bach Mai Hospital Corresponding author: Nguyen Dinh Ung (dr.ungd4.vmmu@gmail.com) Date received: 14/8/2020 Date accepted: 09/10/2020 T¹p chÝ y - d−îc häc qu©n sù sè 8-2020 102 Currently, HBV load tests are used in predicting and monitoring treatment response for HBV patients. Besides, quantitative serum HBsAg assay index is also becoming the concern of clinicians in assessing patients' treatment results. Some recent studies show that the level of hepatitis B surface antigen (HBsAg) is correlated with HBV viral load, especially strongly correlated with the total amount of HBV DNA in the liver and cccDNA (covalently circular closed DNA). Low HBsAg and HBV DNA levels predict response to treatment by pegylated interferon to entecavir better than HBV- DNA [6]. HBsAg levels are associated with HBV viral load and play a role in identifying inactive virus carriers and reducing HBsAg levels during analog nucleoside (NA) treatment in HBeAg- positive patients to help identify cases of seroconversion or HBsAg loss after that [7]. The HBsAg quantitative index is also an important risk factor for increasing liver cancer in low HBV viral load patients [8]. Because of these reasons, we conducted this study: To assess the association between HBsAg level and HBV viral load, and transaminase enzymes, as a basis for further studies on the role of this marker in clinical practice and HBV infection management in the community. SUBJECTS AND METHODS 1. Subjects and materials 256 patients with chronic HBV infection who referred to the Gastroenterology Consulting Room, Department of Outpatient, Bach Mai Hospital for medical examination and treatment in a period of February 2013 to July 2013 were enrolled. - Sampling criteria: The group of chronic hepatitis B (CHB) and group of the inactive HBV carrier were selected according to the criteria of American Association for the Study of Liver Diseases (AASLD) 2009 [7]. The HBV related cirrhotic group was selected according to the Bacon BR‘s criteria (Harrison's - 2008) [9] and the HBV related hepatocellular carcinoma group was selected according to the Conclusion of the Barcelona 2000 EASL conference (clinical management of hepatocellular carcinoma) [3]. - Exclusion criteria: Potential subjects who met any of the following criteria will be excluded from the study enrollment such as HIV/HCV-coinfected; alcohol abuse; refusal to give informed consent. 2. Methods * Study design: Using convenience sampling with cross-sectional survey. * Procedures: - HBV DNA viral load test was performed on the COBAS AmpliPrep system, using the COBAS AmpliPrep/COBASkit TaqMan HBV test (Roche). - HBsAg quantitative assay was performed on COBAS 6000 system, using HBsAg II quantitative biological kit (Roche). - HBsAg quantitative according to the principle of "sandwich" through two incubation, with a quantitative threshold of 20 IU/mL, the total implementation time was 18 minutes. - Realtime reaction to quantify HBV DNA: Using the COBAS AmpliPrep/COBAS TaqMan HBV test kit with nucleic acid T¹p chÝ y - d−îc häc qu©n sù sè 8-2020 103 amplification principle to quantify the HBV viral load in the patient’s serum. The sample preparation process was done automatically by the COBAS AmpliPrep devices and automatic detection COBAS system with TaqMan48 Analyzer. The detection threshold for the system was 116 IU/mL. * Data processing: Statistical analysis was performed by SPSS 22.0 software. * Research ethics: All participants were fully explained about the purpose of the study, the implementation process, the risks involved, the rights, and the confirmation of participation in the study. Information about participants is confidential. RESULTS AND DISCUSSION 1. General characteristics of the study subjects Table 1: Age distribution. Gender Number of patients Percentage (%) Age (X̅ ± SD) p-value Male 140 54.69 39.78 + 13.56 Female 116 45.31 43. 42 + 14.48 < 0.05 Total 256 100.0 41.76 + 14.14 - The mean age was 41.76 ± 14.14 years (15 - 68 years). There was a significant difference between the mean age of male and female patients (p < 0.05); males were younger than females (p < 0.001). Similar to previous studies, the study by Ho Tuan Dat et al on 122 cases of CHB patients also found that the mean age was 33.9 ± 10.4 [2]. Azita Ganji et al studied 97 patients in Mashhad, Iran (2009) with the mean age of 39 ± 11 years [11]. In a total of 256 patients, 54.69% of patients were male. The results obtained by several authors suggested that the prevalence of chronic HBV infection in the male group was higher than in the female group. Ngo Quynh Trang et al’s study on 186 patients in Phu Cuong, Kim Dong, and Hung Yen communes showed that 54.6% of men had HBsAg (+), while women with HBsAg (+) only accounted for 45.4% [3]. Table 2: Clinical phenotypes of chronic HBV. Clinical phenotypes Number of patients Percentage (%) The inactive HBV carriers 6 2.34 Chronic hepatitis B 198 77.34 Cirrhosis 27 10.55 Liver cancer 25 9.77 Total 256 100.0 T¹p chÝ y - d−îc häc qu©n sù sè 8-2020 104 Table 3: HBeAg, anti-Hbe distribution according to gender. Male (n = 140) Female (n = 116) Total (n = 256) Group n % n % n % HBeAg positive 49 35.00 50 43.10 99 38.67 Anti-HBe positive 87 62.14 74 63.79 161 62.89 Overall, the proportion of patients with HBeAg-positive accounted for 38.67%, and with anti-HBe (+) accounted for 62.89%. * Transaminase enzymes and bilirubin characteristics of HBV:*-++ The mean value of AST, ALT, GGT was 49.43 ± 6.65 IU/L, 51.95 ± 6.58 IU/L, and 54.46 ± 11.11 IU/L, respectively. Total and direct bilirubin index was 16.54 ± 8.64 µmol/L and 7.01 ± 1.86 µmol/L, respectively. Table 4: HBsAg levels and HBV viral load characteristics of chronic HBV patients. Index HBeAg - positive (X̅ ± SD) HbeAg - negative (X̅ ± SD) p-value Total (X̅ ± SD) n 99 157 - 256 HbsAg (IU/mL) 9,856.44 ± 576.18 1,605.30 ± 308.93 < 0.001 4,793.22 ± 479.91 HBsAg log10 (IU/mL) 3.98 ± 0.81 2.77 ± 0.89 - 3.24 ± 1.04 HBV DNA (IU/mL) 2.06 x 10 8 ± 7.82 x 107 3.05 x 104 ± 1.82 x 104 < 0.001 9.82 x 107 ± 2.89 x 107 HBV DNA log10 (IU/mL) 8.31 ± 2.50 4.49 ± 1.12 - 7.99 ± 1.80 Serum HBsAg levels in HBeAg-positive patients (9,856.44 ± 576.18 IU/mL) were higher than in HBeAg-negative patients (1,605.30 ± 308.93 IU/mL). We observed a significant difference between two groups (p < 0.001). Our result was higher than A Ganji et al’sfindings [11] on 97 CHB patients: HBsAg levels reached 4,021 ± 2,305 IU/mL. This could be explained due to the different techniques and selected subjects. HBV viral load in HBeAg-positive patients (8.31 ± 2.50 Log10 IU/mL) was higher than in HBeAg-negative patients (4.49 ± 1.12 Log10IU/mL), this difference was statistically significant (p < 0.001). Tran Ngoc Anh and Chien-Hung Chen also reported similar results (7.30 ± 1.67 Log10 IU/mL [4] and 7.40 ± 0.93 Log10 IU/mL, respectively [12]). T¹p chÝ y - d−îc häc qu©n sù sè 8-2020 105 2. Correlation between serum HBsAg level and HBV viral load in chronic HBV infection Figure 1: Correlation between HBsAg level and HBV viral load (A: HbeAg-positve group; B: HbeAg-negative group). Table 5: Correlation between HBsAg level and HBV viral load in patients with chronic HBV infection. HBsAg (IU/mL) HbsAg (log10 IU/mL) HBV DNA (log10 IU/mL) n (X̅ ± SD) (X̅ ± SD) (X̅ ± SD) 9,856.44 ± 576.18 3.98 ± 0.81 8.31 ±1.65 HBeAg - positive 99 r = 0.41, p = 0.003 157 1,605.30 ± 308.93 2.77 ± 0.89 5.49 ± 1.21 HBeAg- negative r = 0.12, p = 0.274 256 4,793.22 ± 479.91 3.24 ± 1.04 7.99 ± 1.80 Total r = 0.38, p <0.001 It can be seen that HBsAg levels and HBV viral load between two groups had a moderate correlation (r = 0.38, p < 0.001). The similar results were found in study by E Gupta (India, 2012) on 198 patients. In E. Gupta's study, in HBeAg-positive patients, the correlation between HBsAg level and HBV viral load was better than in HBeAg- negative patients (r = 0.402, p < 0.01 vs. r = 0.193, p = 0.05, respectively) [12]. Chien-Hung Chen (2004) also reported a good correlation between HBsAg and HBV viral load (r = 0.709, p < 0.001) when studying 67 HBV carrier [13]. However, compared to the study by Tran Ngoc Anh on 278 CHB patients, there was no correlation in both HBeAg-positive patients (r = 0.049, p = 0.732) and HBeAg-negative patients (r = 0.223, p = 0.226). The difference could be due to randomized selection and a large number of patients who were not previously received antiretroviral treatment. T¹p chÝ y - d−îc häc qu©n sù sè 8-2020 106 Table 6: The correlation between HBsAg level and HBV viral load according to the patient's clinical phenotypes. HBsAg (IU/mL) HbsAg (Log10IU/mL) HBV DNA (Log10IU/mL) Clinical phenotypes n (X̅ ± SD) (X̅ ± SD) (X̅ ± SD) 325.14 ± 56.55 3.27 ± 0.34 2.04 ± 0.84 Inactive HBV carrier 6 r = 0.87, p = 0.332 5,790.44 ± 1,073.60 3.18 ± 0.94 8.05 ± 1.21 Chronic hepatitis B 198 r = 0.47, p < 0.001 1,362.70 ± 97.00 3.00 ± 0.37 7.99 ± 1.25 Cirrhosis 27 r = 0.47, p = 0.572 1,254.50 ± 1,147.27 2.67 ± 1.01 6.47 ± 1.12 Liver cancer 25 r = 0.16, p = 0.584 There was a moderate linear correlation between HBsAg level and HBV viral load in CHB patients (r = 0.47, p < 0.001). Whereas in the remaining patient groups, we did not find any correlation between HBsAg level and HBV viral load (r = 0.87; 0.47; 0.16, p > 0.05). Similar to our result, in the study by Jaroszewics et al (2010), 226 HBV-mono-infected patients, there was a good linear correlation between HBsAg levels and HBV viral load and the case of HBeAg-positive CHB (r = 0.60, p < 0.001) and HBeAg-negative CHB (r = 0.64, p < 0.001) [14]. However, our results were also different from some previous studies. Tran Ngoc Anh’s findings [4] on 278 CHB patients showed that there was no correlation between HBsAg level and HBV viral load (r = 0.1136, p = 0.223). Ganji carried out the study on 97 CHB patients treated in Iran, reported that there was no correlation between HBsAg and HBV DNA levels (r = 0.53, p = 0.606) [11]. Table 7: Correlation between HBsAg level and transaminase enzymes. HBsAg level (IU/mL) < 1,500 (n = 124) 1,500 - 20,000 (n = 116) > 20,000 (n = 16) Enzymes (IU/L) r p r p r p AST 0.00 0.91 0.22 0.09 0.00 0.98 ALT 0.05 0.68 0.21 0.10 0.89 0.63 GGT 0.05 0.74 0.15 0.26 0.26 0.53 T¹p chÝ y - d−îc häc qu©n sù sè 8-2020 107 There was no correlation between AST, ALT, GGT and HBsAg level in HBsAg < 1,500 IU/mL patients (r = 0.00; 0.05; 0.05, respectively; p > 0.05), and in HBsAg range of 1,500 - 20,000 IU/mL patients (r = 0.22; 0.21; 0.15, respectively; p > 0.05) and in HBsAg > 20,000 IU/mL patients (r = 0.00; 0.89; 0.26, respectively; p > 0.05). This can be explained by the pathogenesis of chronic HBV infection. During the immunological tolerance period, the virus had a strong replication due to the absence of the destruction of liver cells, almost all liver enzyme indices were within the normal range, although at this time, qualitative of HBsAg was very high. During the immune clearance phase, there was a partial inhibition of viral replication, but liver cells were destroyed due to the participation of immune mechanisms that increased liver enzymes in the blood. Jerzy Jaroszewicz et al’s findings (2010) were also similar. The author assessed 226 HBV-monoinfection patients who were untreated, with the subgroups including the normal liver patients, and the enzyme index increased more than 2 times. For both groups, there was no linear correlation between serum HBsAg level and AST, ALT values (r = 0.07; p = 0.69 and r = 0.10; p = 0.61) [14]. 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