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.
Trang 1CORRELATION BETWEEN SERUM HBV RNA AND HBV DNA LEVEL,
ALT ACTIVITY IN PATIENTS WITH CHRONIC HEPATITIS B AND
HBV-RELATED CIRRHOSIS
Nguyen Van Dien 1 ; Ho Huu Tho 2 ; Hoang Vu Hung 1
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 log 10 copies/mL vs 5.1 ± 1.58 log 10 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 2 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
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
1 103 Military Hospital
2 Vietnam Military Medical University
Corresponding author: Nguyen Van Dien (nguyenvandien8290@gmail.com)
Date received: 20/12/2018
Date accepted: 18/01/2019
Trang 2However, 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
1 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,
103 Military Hospital, from 9 - 2016 to
11 - 2017, were recruited The diagnosis criteria were according to the guideline of American Association for the Study of
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
2 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
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 3 µL of cDNA solution The experiments were performed using the protocol of 1 cycle at 95°C for
5 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
Trang 3* 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
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
1 Overall characteristics of study population
Table 1:
(A) CHB patients
(B) HBeAg (+), CHB patients
(C) HBeAg (-), CHB patients
(D) Cirrhosis patients
p value (A) vs (D)
p value (B) vs (C)
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,
ALT (U/l) 571.04 ± 574.94
(55.4 - 2547)
627.11 ± 695.97 (104.8 - 2574)
522.98 ± 459.45 (55.4 - 1851.15)
88.92 ± 155.06 (19.24 - 723.2) < 0.001 > 0.05 HBV DNA
HBV DNA (log 10
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
Trang 42 Serum HBV RNA level in participants
A B
6.29
5.10
0
2
4
6
8
Serum HBV RNA level
Cirrhosis CHB
(n = 8) (n = 24)
5.62
HBV RNA
log10copies/ml
HBeAg (+) HBeAg (-)
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)
3 Correlations between HBV RNA and HBV DNA level
3.50
5.50
7.50
9.50
HBV RNA
5.00 6.00 7.00 8.00 9.00 10.00
HBV RNA
5.00
6.00
7.00
8.00
9.00
10.00
4.00 5.00 6.00 7.00 8.00
HBV RNA
5.00 6.00 7.00 8.00 9.00
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 (log 10 copies/mL); HBV RNA (log 10 copies/mL)
p < 0.05 Log 10 copies/mL p < 0.05
HBeAg (+) HbeAg (-)
n = 22 n = 12 Log 10 copies/mL
6.95 5.62
Trang 5In 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)
4 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 = 8
0 500
1000
1500
2000
2500
3000
2.50 4.50 6.50 8.50
HBV RNA
20 40 60 80 100
2.00 4.00HBV RNA6.00 8.00
0 500
1000
1500
2000
2500
3000
4.00 5.00 6.00 7.00 8.00
HBV RNA
0 200 400 600 800 1000 1200 1400
2.50 4.50HBV RNA6.50 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 (log 10 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
Trang 6correlations between HBV RNA levels
and ALT activity (r = 0.152; p > 0.05)
in HBeAg (-), CHB patients (fig 3D)
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)
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
significantly higher ALT activity than cirrhosis ones Similar observations were also highlighted in previous study [1]
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 non-significant 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
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
Trang 7(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 cross-sectional 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
Trang 8REFERENCES
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