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 [2] .

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 [10] and fibrosis index based on four factors (FIB-4) score [11] where increase in FIB-4 and APRI scores correlate with increase in hepatic fibrosis [12] [13] , they calculated according to the following equation.

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 [14] .

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 [15] :

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 [2] .

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 (Table 1).

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 Table 2, with significant improvement of them except INR which show non-significant post treatment improvement (p < 0.111).

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 (Table 2).

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 Table 3.

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 Table 4.

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 (Table 5, Figure 1).

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.

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.

***p < 0.000 highly significance.

**p < 0.001 moderate significance ***p < 0.000 highly significance.