To investigate correlations between serum HBV RNA and HBV DNA level, ALT activity in naive patients with chronic hepatitis B and HBV-related cirrhosis. Subjects and methods: This cross-sectional study involved 39 chronic hepatitis B and 19 cirrhosis patients, aged 19 - 78 years. Serum samples at admission were used to quantify HBV RNA, HBV DNA level and ALT activity. Spearman’s correlation analysis was used for correlations of HBV RNA and HBV DNA level, ALT activity. For HBV RNA quantification, a specific real-time PCR technique was used with a lower limit of detection of 100 copies/mL. Results: The HBV RNA and HBV DNA detection rate was 61.54% and 100% in chronic hepatitis B patients and 42.11% and 89.47% in cirrhosis cases, respectively. HBV RNA level and ALT activity in chronic hepatitis B patients were significantly higher than in cirrhosis cases (6.29 ± 1.42 log10 copies/mL vs 5.1 ± 1.58 log10 copies/mL, p < 0.05 and 571.04 ± 574.94 U/l vs. 88.92 ± 155.06 U/l, p < 0.001, respectively). No significant HBV DNA level difference was found between those.
Journal of military pharmaco-medicine n02-2019 CORRELATION BETWEEN SERUM HBV RNA AND HBV DNA LEVEL, ALT ACTIVITY IN PATIENTS WITH CHRONIC HEPATITIS B AND HBV-RELATED CIRRHOSIS Nguyen Van Dien1; Ho Huu Tho2; Hoang Vu Hung1 SUMMARY Objectives: To investigate correlations between serum HBV RNA and HBV DNA level, ALT activity in naive patients with chronic hepatitis B and HBV-related cirrhosis Subjects and methods: This cross-sectional study involved 39 chronic hepatitis B and 19 cirrhosis patients, aged 19 - 78 years Serum samples at admission were used to quantify HBV RNA, HBV DNA level and ALT activity Spearman’s correlation analysis was used for correlations of HBV RNA and HBV DNA level, ALT activity For HBV RNA quantification, a specific real-time PCR technique was used with a lower limit of detection of 100 copies/mL Results: The HBV RNA and HBV DNA detection rate was 61.54% and 100% in chronic hepatitis B patients and 42.11% and 89.47% in cirrhosis cases, respectively HBV RNA level and ALT activity in chronic hepatitis B patients were significantly higher than in cirrhosis cases (6.29 ± 1.42 log10 copies/mL vs 5.1 ± 1.58 log10 copies/mL, p < 0.05 and 571.04 ± 574.94 U/l vs 88.92 ± 155.06 U/l, p < 0.001, respectively) No significant HBV DNA level difference was found between those There was a significant positive correlation between HBV RNA and HBV DNA level in chronic hepatitis B (r = 0.78; p < 0.001) and HBeAg (+), chronic hepatitis B (r = 0.81; p < 0.01) groups but not in cirrhosis patients HBV RNA level significantly correlated with ALT activity in cirrhosis (r = 0.91; p < 0.005) and HBeAg (+) CHB (r = -0.76; p < 0.001) patients Conclusion: HBV RNA level correlated with HBV DNA level in chronic hepatitis B and HBeAg (+), chronic hepatitis B patients, with ALT activity in cirrhosis and HBeAg (+), chronic hepatitis B patients * Keywords: Chronic hepatitis B; Cirrhosis; Serum HBV RNA; HBV DNA; ALT INTRODUCTION Hepatitis B virus (HBV) infection remains a global public health problem Worldwide, an estimated billion persons have been infected with HBV, more than 240 million persons have chronic infections, and 500,000 - 700,000 persons died as a result of HBV infection [10] In Vietnam, the prevalence of HBsAg (hepatitis B surface antigen) in healthy subjects ranges from 5.7 - 24.7% depending on study setting [6] Treatment of patients with HBV infection has remarkably improved with the advent of oral nucleos(t)ide analogues (NA) It has been proposed that covalently closed circular (ccc) DNA level in the liver is much more valuable in monitoring therapy compared to serum HBV DNA - one of most widely used marker in clinical practice 103 Military Hospital Vietnam Military Medical University Corresponding author: Nguyen Van Dien (nguyenvandien8290@gmail.com) Date received: 20/12/2018 Date accepted: 18/01/2019 185 Journal of military pharmaco-medicine n02-2019 However, intra-hepatic cccDNA measurement seems ill-suited for clinical use because it requires a liver biopsy Recently, it is confirmed that HBV pgRNA, an intermediate of HBV replication cycle, is present in hepatitis B patients‟ sera It gradually decreases during treatment but is still detected over an extended period, even undetectable serum HBV DNA level goal is achieved, especially with NA drugs [9] Recent studies have shown clinical significance of serum HBV RNA that includes: Estimate levels of cccDNA in the liver, reflect the antiviral potency of NA drugs; independently predict response to treatment; guide NA discontinuation; and monitor the emergence of viral mutation during antiviral treatment Although the clinical significance and nature of serum HBV RNA have been intensively investigated, little is known about the factors influencing its level Additionally, in Vietnam, quantification of serum HBV RNA still has not been widely performed In this study, we measured serum HBV RNA and analyzed its characteristics in CHB and HBV-related cirrhosis patients Furthermore, serum HBV RNA level was compared to serum HBV DNA and ALT levels to clarify whether there exist any clinical significance correlations between them in these patients SUBJECTS AND METHODS Patients and samples In this study, 58 adult treatment - naive patients (39 chronic hepatitis B [CHB] and 19 HBV-related cirrhosis), who admitted to Department of Infectious Diseases and Department of Gastrointestinal Diseases, 186 103 Military Hospital, from - 2016 to 11 - 2017, were recruited The diagnosis criteria were according to the guideline of American Association for the Study of Liver Diseases (AASLD - 2009) [2] for CHB patients and Bacon BR.„s criteria (Harrison's, 2008) [3] for cirrhosis patients This study was approved by the Ethics Committee of Vietnam Military Medical University Written informed consent was obtained from each patient Serum samples were collected at the admission, preserved in heparin-coated tubes and EDTA coated tubes depending on types of assays Samples for HBV RNA quantification were stored at -800C for further examination Methods * Quantification of HBV RNA: This assay was performed at Department of Genomics, Institute of Biomedicine & Pharmacy, Vietnam Military Medical University using a protocol optimized from that of Jing Wang et al (2016) [9] Extracted RNA (using E.Z.N.A.® Blood RNA Mini Kit, Omega Bio-tek, Inc., USA) was used to synthesize cDNA, then quantified by real-time PCR (Rotor Gene™ Q,QIAGEN, Germany) in 20 µL reactions, including µL of cDNA solution The experiments were performed using the protocol of cycle at 95°C for minutes, 45 cycles at 95°C for 30 seconds, 60°C for 15 seconds, and 72°C for 30 seconds The lower detection limit for the HBV RNA assay was determined as 100 copies/mL Journal of military pharmaco-medicine n02-2019 * Other laboratory tests: HBsAg, HBV-e antigen, HBV-e antibody, anti-HBc antibody, and hepatitis C virus antibody were measured by Elecsys HBsAg, Elecsys HBeAg, Elecsys anti-HBe, Elecsys anti-HBs, Elecsys anti-HBc - Roche kit, respectively that were based on electrochemiluminescence immunoassay principle Serum HBV DNA level was extracted from samples using E.Z.N.A.® Blood DNA Mini Kit (Omega Bio-tek, Inc., USA) and quantified by using real-time polymerase chain reaction (PCR) technique with a 20 μL volume on the Rotor Gene™ Q (QIAGEN, Germany) The limit of detection was 100 copies/mL * Statistical analysis: HBV nucleic acids were logarithmically transformed (log10) for analysis, and data are expressed as mean or median and range and percentage SPSS 22.0 software for Window with student‟s t-test, Chi-square tests or Fisher‟s exact tests, and Spearman‟s correlation test were used for statistical analysis as appropriate A p-value < 0.05 was considered to be statistically significant RESULTS Overall characteristics of study population Table 1: (A) CHB patients p value p value (B) HBeAg (+), (C) HBeAg (-), (D) Cirrhosis CHB patients CHB patients patients (A) vs (D) (B) vs (C) Case number 39 18 21 19 Age (year), mean (range) 37.53 ± 12.47 (19 - 71) 31.94 ± 8.94 (20 - 58) 42.14 ± 13.51 (19 - 71) 56.90 ± 11.63 (33 - 78) < 0.001 < 0.01 Gender, male/female 36/3 18/0 18/3 16/3 > 0.05 > 0.05 < 0.001 > 0.05 ALT (U/l) 571.04 ± 574.94 627.11 ± 695.97 522.98 ± 459.45 88.92 ± 155.06 (55.4 - 2547) (104.8 - 2574) (55.4 - 1851.15) (19.24 - 723.2) HBV DNA detection rate (%) 100 100 100 89.47 > 0.05 > 0.05 HBV DNA (log10 copies/mL) 6.66 ± 1.76 (2.15 - 9.18) 7.44 ± 1.29 (5.09 - 9.18) 5.99 ± 1.86 (2.15 -8.08) 6.37 ± 2.35 (2.10 - 9.45) > 0.05 < 0.01 Cirrhosis patients were generally older than CHB patients Among CHB group , the mean age of HBeAg (-) patients were lower than HBeAg (+) ones There was no statistically significant diff erence in sex and HBV DNA detectability between the two study group and HBV DNA level in CHB patients was non-significantly higher than cirrhosis ones Further analysis in CHB patients showed a higher HBV DNA level in HBeAg (+) patients in comparison to HBeAg (-) subjects ALT activity was shown to be significantly higher in CHB patients compared to cirrhosis ones but not different among CHB patients with different HBeAg status 187 Journal of military pharmaco-medicine n02-2019 Serum HBV RNA level in participants A B Log 10 copies/mL p < 0.05 log10copies/ml Log 10 copies/mL 6.29 5.10 6.95 6.95 L Cirrhosis p < 0.05 5.62 5.62 HBeAg HBeAg(-)(-) HBeAg (+)(+) HbeAg n(n=12) = HBV 22 RNAn(n=12) = 12 CHB Serum HBV RNA level (n = 8) (n = 24) Figure 1: Comparison of HBV RNA level among groups of patients Comparison of HBV RNA level between CHB and cirrhosis patients (A); comparison of HBV RNA level between HBeAg (+), CHB patients and HBeAg (-), CHB patients (B) HBV RNA was detected in 24/39 CHB (61.54%) and 08/19 cirrhosis patients (42.11%) As shown in fig 1A, level of HBV RNA in CHB patients was significantly higher than in cirrhosis ones (p < 0.05) Among CHB patients, in relation to HBeAg status, HBV RNA was detected in the sera of CHB patients at the rate of 12/21 and 12/18 in HBeAg (-) and HBeAg (+) subjects, respectively (p > 0.05) The average level of HBV RNA of CHB patients with HBeAg (+) was observed to be significantly higher than of CHB patients with HBeAg (-) (p < 0.05) (fig 1B) Correlations between HBV RNA and HBV DNA level A: r = 0.78; p < 0.001; n = 24 7.50 5.50 3.50 2.50 4.50 6.50 8.50 10.00 9.00 8.00 7.00 6.00 5.00 2.50 HBV DNA HBV DNA 9.50 B: r = 0.64; p > 0.05; n = HBV RNA 10.00 9.00 8.00 7.00 6.00 5.00 4.00 5.00 6.00 HBV RNA 7.00 8.50 D: r = 0.60; p > 0.05; n = 12 HBV DNA HBV DNA C: r = 0.81; p < 0.01; n = 12 4.50 6.50 HBV RNA 8.00 9.00 8.00 7.00 6.00 5.00 3.00 5.00 7.00 9.00 HBV RNA Figure 2: Correlations between HBV RNA and HBV DNA level (A: CHB patients; B: Cirrhosis patients; C: HBeAg (+), CHB patients; D: HBeAg (-), CHB patients HBV DNA (log10 copies/mL); HBV RNA (log10 copies/mL) 188 Journal of military pharmaco-medicine n02-2019 In correlations analysis, we excluded patients with HBV RNA level under the limit of detection In analyzing the entire study cohort, while the level of HBV RNA was highlighted to be strongly correlated with that of HBV DNA in CHB patients (r = 0.78; p < 0.001), there was no statistically significant relationship between them in cirrhosis patients (r = 0.64; p > 0.05) (fig 2A & 2B) The HBV RNA - HBV DNA correlation in CHB patients was then further analyzed and interestingly found that the positive correlation between them was confirmed in the group of HbeAg (+) patients (r = 0.81; p < 0.01) whereas no significant correlations was found in the others (r = 0.06; p > 0.05) (fig 2C & 2D) Correlations between HBV RNA level and ALT activity A: r = -0.17; p > 0.05; n = 24 B: r = 0.91; p < 0.05; n = 100 80 ALT ALT 3000 2500 2000 1500 1000 500 2.50 40 4.50 6.50 HBV RNA 20 2.00 8.50 C: r = -0.76; p < 0.01; n = 12 2500 ALT ALT 2000 1500 1000 500 5.00 6.00 7.00 HBV RNA 4.00 6.00 HBV RNA 8.00 D: r = 0.15; p > 0.05; n = 12 3000 4.00 60 8.00 1400 1200 1000 800 600 400 200 2.50 4.50HBV RNA 6.50 8.50 Figure 3: Correlations between HBV-RNA level and ALT activity (A: CHB patients; B: Cirrhosis patients; C: HBeAg (+), CHB patients; D: HBeAg (-), CHB patients HBV RNA (log10 copies/mL); ALT (U/l) In order to determine whether HBV RNA level is linked to liver, we also tested for associations between serum HBV RNA level and liver injury in study population In CHB patients, there was no statistically significant relationship between ALT activity and HBV RNA level (r = -0.17; p > 0.05) (fig 3A) However, when stratifying the patients according to HBeAg status, a strongly negative correlation in the HBeAg (+), CHB subjects between them (r = -0.75; p < 0.01) (Spearman‟s correlation analysis) were observed (fig 3C) We did not find any significant 189 Journal of military pharmaco-medicine n02-2019 correlations between HBV RNA levels and ALT activity (r = 0.152; p > 0.05) in HBeAg (-), CHB patients (fig 3D) significantly higher ALT activity than cirrhosis ones Similar observations were also highlighted in previous study [1] Our analysis also highlighted a strong and positive correlation between HBV RNA level and ALT activity in the group of patients with cirrhosis (r = 0.91; p < 0.05) (8 patients) (fig 3D) Interestingly, HBV RNA level strongly, positively correlated with HBV DNA level in CHB patients and HbeAg (+), CHB patients Among CHB patients, while no significant correlations was revealed between HBV RNA level and ALT activity in CHB patients and HBeAg (-), CHB patients, HBV RNA level was identified to strongly, negatively correlated with ALT activity in HBeAg (+) patients A nonsignificant correlation between HBV RNA and HBV DNA level and a strongly significant, positive correlation between HBV RNA and ALT activity were highlighted in patients with cirrhosis DISCUSSION In this study, 61.54% (24/39) and 42.11% (08/19) detection rate for HBV RNA were found in CHB and cirrhosis patients, respectively HBV RNA detectability in our study was higher than that in the report by Huang et al (2015) (21/52 = 40.38% of the untreated CHB patients with 46.5% of patients with HBeAg (+)) [4], but lower than in Yutang Li‟s study (412/483 = 85.3% patients [5] This difference may be attributed to the differences between study populations The patients in the current study had a significantly lower HBeAg positive ratio (18/39 = 46.15%) compared with their patients (426/483 = 88.2%) Our findings clearly showed that HBV RNA level was significantly higher in CHB compared to cirrhosis patients as well as in CHB subjects with HBeAg (+) compared to HBeAg (-) CHB ones This is in good agreement with findings demonstrated in other studies in CHB patients [8] There was no significant difference between CHB and cirrhosis patients in both HBV DNA detectability and level However, among CHB patients, we found a significantly higher HBV DNA level in subjects with HBeAg (+) compared to others In addition, CHB patients had a 190 These were in line with previous studies conducted in CHB patients A strongly, positive correlation between HBV RNA and HBV DNA level were revealed by Akinori Rokuhara (2006) (r = 0.801; p < 0.001 [7] and van Bommel F (2015) (r = 0.59; p < 0.001) [8] Among HBeAg (+) CHB patients, the relationship between HBV RNA and HBV DNA was also indicated by van Bommel F (2015) [8] The above results suggested that as an intermediate in HBV replication cycle, HBV pgRNA-containing virion is also produced and released into sera along with HBV DNA-containing virion (Dane particles) at a certain rate compared to Dane particles In term of HBV RNA level - ALT activity correlation, in the same line, van Bommel F (2015) revealed that HBV RNA level did not correlate with serum ALT activity Journal of military pharmaco-medicine n02-2019 (r = 0.01) [8] This apparent lack of correlation between HBV RNA level and ALT activity in CHB patients could be accounted for the pathophysiology of chronic HBV infection In immune clearance phase (the predominance of our participants is patients with exacerbation of CHB), while HBV replication is partially suppressed, infected hepatocytes is thought to be damaged by the immune-mediated lysis mechanism resulting into ALT elevation However, that phenomenon differs among patients depending on various factors including HBeAg status In HBeAg positive CHB patients, infected hepatocytes lysis caused by sufficient immune clearance results into ALT elevation and a decrease in the level of viral markers, including HBV DNA, HBV RNA, even leads to seroconversion of some of them Therefore, HBV RNA has a tendency to negatively correlate with ALT activity Further analysis in patients with HBeAg negative CHB, it is thought that there is an insufficient immune against HBV that is characterized by ALT elevation without decreasing in serum viral markers Regarding cirrhosis ones, little is known about serum HBV RNA to date Our findings provide clues for further investigation into the roles of serum HBV RNA in natural history of HBV infection and HBV infection management The difference in serum HBV RNA level between cirrhosis and CHB patients suggests that it could help to have a more precise view in the nature of viral replication and pathogenesis of disease among different phases of chronic HBV infection Additionally, significant correlations found between serum HBV RNA and HBV DNA level, ALT activity indicate that serum HBV RNA could be a promising biomarker in HBV infection evaluation and management This is the first publication on serum HBV RNA in Vietnam, however, the findings must be interpreted in the context of a number of potential limitations Firstly, this is a crosssectional study assessed data at one-time point of admission only Thus, it is difficult to establish a causal relationship between serum HBV RNA and liver histological changes, HBV DNA dynamics because it requires longitudinal observations Secondly, this study investigated a quite small sized sample which may have reduced the statistical power CONCLUSION This study demonstrates significant differences in baseline serum HBV RNA level between CHB and cirrhosis patients Serum HBV RNA level correlated with HBV DNA level in CHB and HBeAg (+) CHB patients, and ALT activity in patients with cirrhosis and HBeAg (+) CHB Serum HBV-RNA may help clinicians in managing persistent HBV infection Acknowledgments We thank all study participants for their donation of blood samples This work was supported by Department of Infectious diseases, 103 Military Hospital and Department of Genomics, Institute of Biomedicine and Pharmacy of Vietnam Military Medical University 191 Journal of military pharmaco-medicine n02-2019 REFERENCES Nguyen Huu Quyen Serum HBsAg level, viral load and ALT activity in forms of patients with chronic HBV infection Master Thesis in Medicine Vietnam Military Medical University Hanoi 2015 Anna S.F Lok, Brian J McMahon Chronic hepatitis B AASLD practice guidelines Hepatology 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envelope antigen seroconversion during treatment with polymerase inhibitors Hepatology 2015, 61 (1), pp.66-76 Wang J, Shen T, Huang X et al Serum hepatitis B virus RNA is encapsidated pregenome RNA that may be associated with persistence of viral infection and rebound J Hepatol 2016, 65 (4), pp.700-710 10 World Health Organization (WHO) Prevention and control of viral hepatitis infection: Framework for global action WHO, Geneva 2012 ... differences in baseline serum HBV RNA level between CHB and cirrhosis patients Serum HBV RNA level correlated with HBV DNA level in CHB and HBeAg (+) CHB patients, and ALT activity in patients with cirrhosis. .. nonsignificant correlation between HBV RNA and HBV DNA level and a strongly significant, positive correlation between HBV RNA and ALT activity were highlighted in patients with cirrhosis DISCUSSION In this... between HBV- RNA level and ALT activity (A: CHB patients; B: Cirrhosis patients; C: HBeAg (+), CHB patients; D: HBeAg (-), CHB patients HBV RNA (log10 copies/mL); ALT (U/l) In order to determine