Purpose: Chronic hepatitis B virus infection affects more than 3 million people worldwide. The present study aimed to evaluate the role of pretreatment factor IV collagen as predictor of post treatment virological response with hepatic fibrosis regression. Materials and Methods: This prospective cohort study has been conducted on 74 naieve patients with chronic HBV infection with variable degree of hepatic fibrosis (F2, F3, ≥F4), viral load, and variable degree of abnormality in laboratory parameters of liver functions. All patients treated with Entecavir 0.5 mg/day or 1 mg/day according to severity of hepatic condition for 1 year. Liver fibrosis assessed using fibroscan, factor IV collagen, (APRI) and (FIB-4) scores evaluation. Results: All included patients in our study achieve post treatment Virological response with undetectable HBV DNA PCR (<16 IU/ml). With significant post treatment reduction in mean fibroscan value 10.70 ± 5.80 (p < 0.001). There were also significant end treatment improvements in mean FIB score and APRI score 1.56 ± 1.02 (p < 0.0001) and 0.50 ± 0.28 (p < 0.0001) respectively. There is significant end treatment improvement of mean factor IV collagen. In our study 49 (66.22%) of included patients showed post treatment reduction in their level of hepatic fibrosis (Responder) opposite to 25 (33.78%) had no post treatment improvement in degree of hepatic fibrosis (Non-responder). Conclusion: Low pretreatment factor IV collagen is significantly correlated with virologial response and hepatic fibrosis regression post Entecavir therapy in patients with chronic HBV infection.
Hepatitis B virus (HBV) infection is one of the major public health problems in Egypt; its prevalence was (1.4%) with higher prevalence in urban than rural area [
All patients with untreated chronic hepatitis B virus infection have a high risk of development of cirrhosis and hepatocellular carcinoma [
The aim of treatment is decreasing HBV DNA levels, decreasing hepatic inflammation and improving laboratory parameters of disease activity [
Entecavir is a potent oral guanosine nucleoside analogue for treatment of HBeAg-positive or negative chronic HBV infection; it’s safe and effective in treatment of naive and lamivudine resistant patients [
Chronic liver injury causes necrosis and apoptosis of parenchymal cells and replacement by extracellular matrix, with disease progression the liver parenchyma replaced by scar tissues with abnormal vascular architecture and finally organ dysfunction [
Coagulation cascade is one of main factors in development of hepatic fibrosis, in which Thrombin is cause change of fibrinogen to fibrin which appear in the hepatic tissue during acute and chronic liver injury [
Thrombotic risk factors play an important role in hepatic fibrosis in patients with chronic HBV or chronic HCV [
Type IV collagen is a direct marker of hepatic fibrosis which indicate hepatic extracellular matrix transformation, and its concentration affected by degree of liver cell loss and disturbed hepatic function [
Hepatic fibrosis is the main complication of chronic hepatic disease that eventually leads to cirrhosis with subsequent complications [
So, in this study we aimed to evaluate the role of pretreatment serum level of factor IV collagen as a predictor of post antiviral therapy virological response with hepatic fibrosis regression.
To evaluate the role of serum factor IV collagen level as a predictor of post antiviral therapy virological response with hepatic fibrosis regression.
This prospective cohort study has been conducted on 74 naieve patients with chronic HBV infection with variable degree of hepatic fibrosis (F2, F3, ≥F4), viral load (HBV DNA) from 16 up to >1,000,000 copies/ml, and variable degree of abnormality in laboratory parameters of liver functions. All patients attend out patient’s clinic of Tropical Medicine and Gastroenterology Departments during the period from April 2017 till May 2018.
Exclusion criteria included: 1) co-infection with HCV, HDV, or HIV; 2) excessive alcohol consumption (>21 drinks per week in men and >14 drinks per week in women); 3) dynamic CT imaging of HCC or history of HCC.
All patients treated with Entecavir 0.5 mg or 1 mg according to severity of hepatic condition, once daily oral on empty stomach for 1 year. Patients were treated according to EASL 2017 guidelines for HBV infection treatment [
At base line and one year post antiviral treatment all included patients subjected to complete history taking , full clinical examination, liver function tests, liver fibrosis assessment (F2- ≥ F4) using fibroscan, factor IV collagen measurement as a serum direct fibrosis markers, aspartate aminotransferase-to-platelet ratio index (APRI) score [
FIB- 4 = ( age × AST ) / ( platelet count × ( ALT ) 1 / 2 )
APRI = ( [ AST / UNL ] / platelet count ) × 100
8 ml of venous blood were collected from each patients under aseptic precautions and divided in 4 tubes: 1) 2 tubes containing EDTA (sterile EDTA containing Vacutainer) one for platelet count used directly and the other for HBV-DNA centrifuged, separated in sterile tube and frozen at −20˚C till the time of the assay. 2) Plain glass tube for liver function tests, then after clotting, the tube was centrifuged at 3000 rpm for 5 min at room temperature and then the serum was separated to be used for estimation of liver function (including liver enzymes, albumin, bilirubin-total and direct) and type IV collagen. 3) Sodium citrate containing tube (Vacutainer) for prothrombin time, prothrombin concentration and INR.
1) The platelet count was done by Celtak haematology analyzer (Nihon Kohden, Japan).
2) The liver function tests (total and direct bilirubin, serum albumin, AST and ALT) were done by BT 1500 full automated chemistry analyzer (Bioticnica, Italy).
3) The prothrombin time, concentration and INR was done by done using Thromborel S kits (Siemens, Germany) using BE coagulator (Germany).
4) HBV-DNA was done by StepOnePlus™ Real-Time PCR System (ThermoFisher Scientific, USA) using Taqman PCR mastermix (ThermoFisher Scientific, USA) after DNA extraction by Qiagen kit (Qiagen, inc, USA) and according the manufacture’s pamphlet.
5) Type IV collagen was assayed using quantitative sandwich ELISA technique with Human col IV ELISA kit (Mybiosource, inc, USA) and according the manufacture’s pamphlet.
Liver fibrosis assessment was done for all patients with Transient Elastography (FS-502 touch device, France) using both the M and XL probes. Transient elastography provides a continuous measure of liver stiffness measured in kilopascals (kPa). Transient elastography was performed after overnight fasting. Mild amplitude and low-frequency waves were transmitted through the liver and liver stiffness directly correlate with wave velocity [
10 valid measurements were done for every patient, which considered reliable when success rate ≥60% and an interquartile range (IQR)/median liver stiffness measures ≤30%. 10 All patients did ultrasound transient elastography examination at the baseline and one year after end of treatment. Results of transient elastography were correlated to METAVIR histological staging system. The cut-off values in our study were [
F0: till 5.4 kPa F0-F1: 5.5 - 5.9 kPa F1: 6 - 6.9 kPa F1-F2: 7 - 8.7 kPa
F2: 8.8 - 9.4 kPa F3: 9.5 - 12.4 kPa F3-F4: 12.5 - 14.4 kPa F4: ≥14.5 kPa
Responder patients to treatment: include any patients who show any decrease in their degree of fibrosis after one year of regular treatment in comparison to pretreatment one.
Non-Responder patients to treatment: include any patients with no change in their level of hepatic fibrosis or those who show any progression in hepatic fibrosis after one year of regular treatment compared to their pretreatment level.
Virological response during NA therapy: is defined as undetectable HBV DNA by a sensitive polymerase chain reaction (PCR) assay with a limit of detection of 10 IU/ml [
The study protocol was approved by the ethical committee of our institution and all our patients provided informed consent before inclusion in the study
The data were coded and verified prior to data entry. Computer program Statistical Package for Social Sciences (SPSS) (ver.21) Chicago. USA was used for analyzing the collected data and for drawing figures. Data expressed as mean ± standard deviation and number, percentage. Student-t-test used to determine significant for numeric variable. P value is considered significant if P value < 0.05 and not significant if P value > 0.05.
To calculate sample size in this study by “EBI” program at power 80%, with confidence 95.0%, Alpha 0.5 equal 74 patients.
The present study included 74 patients with naive chronic HBV-infection with their mean ages were 43.85 ± 10.12; 21 females (28.77%) and 53 males (72.6%) and their mean FIB-4 score and APRI score were 2.80 ± 1.61 and 1.24 ± 0.67 respectively.
The mean value for pretreatment fibroscan was 11.99 ± 4.96 kPa; twenty nine of them (39.19%) were F2, 30 (40.54%) were F3 and 15 (20.27%) were ≥F4 (
Regarding the post treatment fibroscan assessments of the included patients, the mean value was 10.70 ± 5.80 (p < 0.001**), 4 (5.41%) of them were F0, 17 (22.97%) were F1, 24 (32.43) were F2, 15 (20.27%) were F3 and 14 (18.92%) were F4 with evidence of cirrhosis.
The laboratory measures among chronic HBV infected patients including factor IV collagen, mean platelet count, and liver function tests together with mean FIB score and APRI score before and one year of regular therapy are presented in
All included patients achieve Virological response with undetectable HBV DNA PCR (<16 IU/ml) during regular treatment with Entecavir 0.5 mg/d or 1mg/day for one year (100%).
There were also significant end treatment improvements in mean FIB score and APRI score 1.56 ± 1.02 (p < 0.0001***) and 0.50 ± 0.28 (p < 0.0001***) respectively (
In our study 49 (66.22%) of included patients showed post treatment reduction in their level of hepatic fibrosis (Responder) opposite to 25 (33.78%) had no post treatment improvement in degree of hepatic fibrosis (Non-responder).
As regard hepatic fibroscan of the responder group:
・ 26 (53.06%) patients were F2 and after one year of regular treatment 4 (15.40%) of them became F0, 17 (65.40%) became F1and 5 (19.20%) didn’t show any change in their degree of hepatic fibrosis.
・ 20 (40.82%) patients were F3 and post treatment 17 (85.00%) of them became F2 and 3 (15%) of them showed no post treatment improvement in their hepatic fibrosis.
・ 3 (6.12%) patients were F4 and post treatment 2 (66.70%) of them became F2 and only one patients (33.30%) become F3.
With significant p value (<0.0001***). This is illustrated in
By comparing baseline laboratory parameters between responder and non-responder patients, the responder group had mild pretreatment impairment in their laboratory parameters.
This is illustrated in
ROC curve analysis of different pretreatment variables that show statistically significant difference between responder and non-responder group, revealed that pretreatment low level of serum bilirubin, mild impairment of serum INR, mild degree of liver stiffness as detected by fibroscan and low level of serum factor IV collagen were statistically significant factors associated with post treatment improvement in the level of hepatic fibrosis in naive chronic hepatitis B virus infected patients (
Variables | |
---|---|
Number | 74 |
Age (y) (Mean ± SD) | 43.85 ± 10.12 |
Male (N.%) | 53 (72.6%) |
Female (N.%) | 21 (28.77%) |
Platelet count (Mean ± SD) PLT (103/µl) | 147.03 ± 70.04 |
Liver function tests (Mean ± SD) | |
Bilirubin (mg/dl) | 1.55 ± 0.58 |
Albumin (gm/dl) | 3.52 ± 0.67 |
ALT (U/L) | 66.70 ± 32.76 |
AST (U/L) | 60.35 ± 23.67 |
INR | 1.31 ± 0.29 |
HBV DNA PCR level (IU/ml) (N. %) Mild (16 - 100,000 IU/ml) Moderate (100,000 - 1,000,000 IU/ml) High (>1,000,000 IU/ml) | 25 (33.80%) 25 (33.80%) 24 (32.40%) |
Factor IV collagen (mg/dl) | 219.04 ± 108.76 |
Liver Fibroscan (Kilo Pascal'' kPa'') (Mean ± SD) | 11.99 ± 4.96 |
Fibrosis stage (N.%) (Mean ± SD) | |
F2 | 29 (39.19%) 8.12 ± 0.59 |
F3 | 30 (40.54%) 11.49 ± 1.43 |
---|---|
F4 | 15 (20.27%) 20.46 ± 3.85 |
FIB-4 score (Mean ± SD) | 2.80 ± 1.61 |
APRI score (Mean ± SD) | 1.24 ± 0.67 |
Abbreviations: ALT: Alanine transaminase, AST: Aspartate transaminase, INR: International normalization ratio, HBV: Hepatitis B virus, FIB-4: Fibrosis index based on four factors APRI: Aspartate aminotransferase-to-platelet ratio index.
Variables (n = 74) | Before therapy | After one year of entecavir therapy | P. value |
---|---|---|---|
Platelet count (Mean ± SD) PLT (103/µl) | 147.03 ± 70.04 | 182.97 ± 76.22 | 0.002** |
Liver function tests (Mean ± SD) | |||
Bilirubin (mg/dl) | 1.55 ± 0.58 | 1.31 ± 0.42 | 0.224 |
Albumin (gm/dl) | 3.52 ± 0.67 | 3.62 ± 0.55 | 0.0001*** |
ALT (U/L) | 66.70 ± 32.76 | 37.38 ± 20.46 | 0.0001*** |
AST (U/L) | 60.35 ± 23.67 | 31.89 ± 14.87 | 0.0001*** |
INR | 1.31 ± 0.29 | 1.23 ± 0.24 | 0.111 |
HBV DNA PCR level (IU/ml) (N%) Mild viral load Moderate viral load High viral load | 25 (33.80%) 25 (33.80%) 24 (32.40%) | Undetectable | ------ |
Factor IV collagen (mg/dl) | 219.04 ± 108.76 | 171.34 ± 78.25 | 0.012* |
Liver Fibroscan (Kilo Pascal'' kPa'') (Mean ± SD) | 11.99 ± 4.96 | 10.70 ± 5.80 | 0.001 ** |
Fibrosis stage (N %) (Mean ± SD) F0 F1 F2 F3 F4 | ------- ------- 29 (39.19%) 8.12 ± 0.59 30 (40.54%) 11.49 ± 1.43 15 (20.27%) 20.46 ± 3.85 | 4 (5.41%) 5.17 ± 0.20 17 (22.97%) 6.11 ± 0.19 24 (32.43) 7.76 ± 0.80 15 (20.27%) 12.45 ± 1.03 14 (18.92%) 21 ± 4.01 | ---------- ---------- 0.018* 0.023* 0.972 |
FIB-4 score (Mean ± SD) | 2.80 ± 1.61 | 1.56 ± 1.02 | 0.0001*** |
APRI score (Mean ± SD) | 1.24 ± 0.67 | 0.50 ± 0.28 | 0.0001*** |
p < 0.05 indicate significant difference. Student-t-test was used, *P < 0.05 significant, **P < 0.001 moderate significance ***p < 0.000 highly significance.
Degree of liver fibrosis after treatment in responder | P value | ||||||
---|---|---|---|---|---|---|---|
F0 | F1 | F2 | F3 | F4 | |||
Degree of liver fibrosis before treatment in responder | F2 | 4 | 17 | 5 | 0 | 0 | <0.0001*** |
15.40% | 65.40% (17/26) | 19.20% (5/26) | 0.00% | 0.00% | |||
100.00% | 100.00% (17/17) | 22.70% (5/22) | 0.00% | 0.00% | |||
F3 | 0 | 0 | 17 | 3 | 0 | ||
0.00% | 0.00% | 85.00% (17/20) | 15% (3/20) | 0.00% | |||
0.00% | 0.00% | 77.30% (17/22) | 60.00% (3/5) | 0.00% | |||
F4 | 0 | 0 | 0 | 2 | 1 | ||
0.00% | 0.00% | 0.00% | 66.70% (2/3) | 33.30% (1/3) | |||
0.00% | 0.00% | 0.00% | 40.00% (2/5) | 100.00% (1/1) |
***p < 0.000 highly significance.
Variables (n = 74) | Pretreatment Responder (n = 49) | pretreatment parameters of Non-responder (n = 25) | P value |
---|---|---|---|
Platelet count (Mean ± SD) PLT (103/µl) | 168.57 ± 64.49 | 104.80 ± 61.65 | <0.0001*** |
Liver function tests (Mean ± SD) | |||
Bilirubin (mg/dl) | 1.33 ± 0.37 | 1.96 ± 0.68 | <0.0001*** |
Albumin (gm/dl) | 3.77 ± 0.61 | 3.03 ± 0.49 | <0.0001*** |
ALT (U/L) | 65.35 ± 25.39 | 69.36 ± 44.33 | 0.622 |
AST (U/L) | 62.22 ± 19.40 | 56.68 ± 29.00 | 0.331 |
INR | 1.19 ± 0.22 | 1.54 ± 0.26 | <0.0001*** |
Factor IV collagen (mg/dl) | 179.37 ± 76.04 | 296.80 ± 122.06 | <0.0001*** |
HBV DNA PCR level (IU/ml) (N.%) | |||
Mild viral load | 17 (34.69%) | 8 (32%) | 0.118 |
Moderate viral load | 16 (32.65%) | 9 (36%) | |
High viral load | 16 (32.65%) | 8 (32%) | |
Liver Fibroscan (kPa'') (Mean ± SD) | 9.89 ± 2.92 | 16.10 ± 5.59 | <0.0001*** |
FIB-4 score (Mean ± SD) | 2.09 ± 1.10 | 4.19 ± 1.56 | <0.0001*** |
APRI score (Mean ± SD) | 1.09 ± 0.61 | 1.53 ± 0.70 | 0.007** |
**p < 0.001 moderate significance ***p < 0.000 highly significance.
Area Under the Curve | |||||
---|---|---|---|---|---|
Test Result Variable(s) | Area | Std. Error | Asymptotic Sig. | Asymptotic 95% Confidence Interval | |
Lower Bound | Upper Bound | ||||
Bilirubin (mg/dl) | 0.872 | 0.043 | 0.000 | 0.788 | 0.956 |
Albumin (gm/dl) | 0.186 | 0.056 | 0.000 | 0.077 | 0.295 |
Platelet count (103/µl) | 0.460 | 0.067 | 0.579 | 0.329 | 0.591 |
INR | 0.838 | 0.053 | 0.000 | 0.734 | 0.943 |
Liver Fibroscan (kPa'') | 0.957 | 0.022 | 0.000 | 0.913 | 1.000 |
Factor Iv collagen (mg/dl) | 0.743 | 0.067 | 0.001 | 0.612 | 0.874 |
Progression of hepatic fibrosis is associated with increased risk of liver decompensation, and development of hepatocellular carcinoma (HCC) [
Chronic hepatitis B virus treatment with nucleotide analogs (NUCs) leads to clinical improvement and reduces risk of HCC development [
Entecavir is potent new NAs generation with low risk of resistance, however NAs don’t have immunomodulatory effects so, prolonged treatment is usually required to prevent viral relapse [
In this prospective study we try to assess degree of improvement in hepatic fibrosis after one year of regular treatment with entecavir 0.5 mg/day in naive chronic hepatitis B virus infection patients with different base line degree of fibrosis evaluated using hepatic fibroscan.
We have significant post treatment improvement in the level of hepatic fibrosis in 66.22% of our patients which assessed by evaluated by the decrease parameters of hepaticfibroscan.
This was in accordance with Chon et al. [
Also Kose et al. [
Both APRI and FIB-4 scores are suitable non-invasive markers for detecting marked hepatic fibrosis and liver cirrhosis in patients with chronic hepatitis B virus infection [
In our study we have significant improvement in both FIB-4 and APRI scores after one year or regular antiviral treatment compared to baselines values (P < 0.0001***).
With our study, Koksal et al. [
In contrary, Chon et al. [
On the other hand Kim et al. [
In our study we have significant post treatment increase in platelet count.
This come in agreement with Wang et al. [
In our study, we have significant improvement of bilirubin, albumin and liver enzymes after one year of regular antiviral therapy.
This come in agreement with Chang et al. [
Also Kim et al. [
In contrary, Wong et al. [
In our patients pretreatment ALT and bilirubin don’t exceed 4 times and 3 times upper limit of normal respectively.
The results of the current study as regard the therapeutic efficacy of entecavir as oral antiviral therapy revealed 100% virological response with undetectable HBV DNA after one year of regular therapy.
In agreement with this findings, Myung et al. [
On the opposite side, Chon et al. [
Factor IV collagen is non-invasive serological marker to identify significant fibrosis in patients with chronic hepatitis B [
In our study we have significant decrease in factor IV collagen one year post entecavir antiviral therapy (p < 0.012).
This come in agreement with Liang and Li [
Our study reveals that pretreatment low level of serum bilirubin, mild impairment of serum INR, mild degree of liver stiffness as detected by fibroscan and low level of serum factor IV collagen are associated with post treatment improvement in the level of hepatic fibrosis in naive chronic hepatitis B virus infected patients.
In conclusion, in chronic HBV infected patients, low pretreatment serum level of factor IV collagen are good predictor of post treatment virological response and hepatic fibrosis regression.
Lack of a long-term follow-up period after the treatment completion is the major limit of this study especially for non-responder patients.
The authors declare no conflicts of interest regarding the publication of this paper.
Ghweil, A.A., Osman, H.A. and Khodeary, A. (2018) Diagnostic Efficacy of Serum Factor IV Collagen of Hepatic Fibrosis Regression in Chronic Hepatitis B Virus Infected Patients. Advances in Infectious Diseases, 8, 241-254. https://doi.org/10.4236/aid.2018.84020