The Silent Face of Chronic Hepatitis B: Biopsy-Supported Fibrosis Detection and the Reliability of Non-Invasive Scores (FIB-4, APRI) in Inactive, Gray Zone, and Immune-Tolerant Cases

Introduction

Chronic hepatitis B (CHB) infection remains one of the leading causes of liver cirrhosis and hepatocellular carcinoma worldwide. Its natural course consists of 4 phases: immune-tolerant, immune-active, inactive carrier, and reactivation [1,2].

The immune-tolerant phase is marked by elevated hepatitis B virus (HBV) DNA and positive Hepatitis B Virus Early Antigen (HBeAg), while alanine aminotransferase (ALT) remains normal. In contrast, the inactive phase is defined by HBeAg negativity, low HBV DNA levels (<2000 IU/mL), and normal ALT values [2,3].

However, a subgroup of patients present with HBeAg negativity, HBV DNA ≥2000 IU/mL, and persistently normal ALT levels; test results that do not fit clearly into the standard classification system. This group is often referred to as the “gray zone.” Although these patients may be classified as inactive carriers, repeated liver biopsies and non-invasive assessments have revealed that some of them may experience subclinical but progressive hepatic injury [4,5].

These patient groups are generally followed without antiviral treatment, and liver biopsy is rarely performed. However, recent studies have demonstrated that these “silent” phases may not be as histologically benign as previously thought [6,7]. In particular, patients who have remained in the immune-tolerant phase for an extended period or those with potential for immune transition due to aging may develop silent fibrosis.

Although liver biopsy is considered the gold standard for assessing liver fibrosis, its invasive nature, risk of complications, and limited patient acceptance restrict its routine use. Consequently, non-invasive biochemical scoring systems such as Fibrosis-4 Index (FIB-4) and Aspartate Aminotransferase-to-Platelet Ratio Index (APRI) have emerged as alternative diagnostic tools, especially in cases where biopsy is not feasible [3,6,8].

The diagnostic value of non-invasive scores in silent phases of HBV infection remains unclear. This study aims to assess the association between histologically confirmed fibrosis stages (F<3 vs F ≥3) and FIB-4 and APRI scores in patients with immune-tolerant, inactive carrier, or gray zone status, and to evaluate their usefulness in detecting significant fibrosis.

Material and Methods

HISTOPATHOLOGICAL EVALUATION:

Liver biopsy samples from the study participants were assessed using the histological activity index (HAI). In this scoring system, liver fibrosis severity is rated on a scale ranging from 0 to 6. According to the fibrosis scores obtained, patients were categorized into 2 groups: those with mild or no significant fibrosis (score <3) and those with moderate to advanced fibrosis (score ≥3).

Since the primary objective of the study was to evaluate the diagnostic performance of FIB-4 and APRI scores in predicting histological fibrosis, patient subgroup analyses were classified directly according to liver fibrosis stages rather than clinical phases. This approach was adopted because non-invasive scoring systems such as FIB-4 and APRI primarily aim to estimate the degree of histological fibrosis. Clinical phase classifications are mainly based on virological and biochemical parameters, which may not always correlate directly with fibrosis severity. Therefore, to more accurately assess the true diagnostic performance of these scoring systems, grouping was performed according to fibrosis scores.

Biochemical data for the same patients were used to calculate APRI [9] and FIB-4 [10] scores. The upper reference limit, also known as upper limit of normal (ULN), for ALT and aspartate aminotransferase (AST) was accepted as 32 IU/L, based on the reference ranges used in our hospital laboratory.

The formulas used were as follows:

The obtained FIB-4 and APRI scores were compared with histological fibrosis stages determined according to the HAI system.

ETHICAL APPROVAL:

This retrospective study was approved by the Clinical Research Ethics Committee of Mersin University Faculty of Medicine (Approval Date: June 25, 2025; Approval No: 722).

STATISTICAL ANALYSIS:

Continuous variables were expressed as mean±standard deviation with minimum and maximum values, while categorical variables were reported as numbers (n) and percentages (%). Group comparisons for continuous variables were performed using t-test.

Receiver operating characteristic (ROC) curve analysis was used to determine optimal cut-off values for FIB-4 and APRI scores. Area under the curve (AUC) values with 95% confidence intervals were calculated. Sensitivity, specificity, and likelihood ratios were used to assess diagnostic performance. ROC curves were also compared using pairwise analysis.

A P value <0.05 was considered statistically significant. Analyses were conducted using SPSS v25.0 (IBM Corp., NY, USA) and MedCalc software.

Results

A total of 230 patients in the inactive, immune-tolerant, and gray zone phases were included in this retrospective cross-sectional study. The mean age of the patients was 44.4±12.2 years, with 46.1% (n=106) being male and 53.9% (n=124) female. The need for treatment was present in 37.4% (n=86) of the patients (Table 1).

The mean serum ALT level was 21.05±6.07 U/L, AST was 22.08±8.76 U/L, and PLT was 236.66±61.99 (×103/μL). The mean HAI score was 4.9±2.2, and the mean fibrosis score was 2.1±0.89. The average FIB-4 and APRI values were 1.04±0.80 and 0.32±0.18, respectively (Table 1).

When compared according to fibrosis stage, patients with a fibrosis score ≥3 had significantly higher FIB-4 (1.53±0.90) and APRI (0.44±0.23) values than those with a fibrosis score <3 (P=0.003 for FIB-4, P=0.001 for APRI) (Table 2).

In the ROC curve analysis, FIB-4 demonstrated an AUC of 0.70 (P=0.001) with an optimal cut-off value above 1.06. At this threshold, sensitivity was 70%, specificity 75%, positive predictive value (PPV) 66%, and negative predictive value (NPV) 87%. For APRI, the AUC was 0.68 (P=0.001), with a cut-off set at >0.38, yielding a sensitivity of 65%, specificity of 86%, PPV of 66%, and NPV of 87% (see Table 3 and Figure 1).

When the diagnostic performances of FIB-4 and APRI were compared, the difference between the ROC curves was not statistically significant (AUC difference: 0.01; 95% CI: −0.07 to 0.09; P=0.80) (Table 4).

Discussion

LIMITATIONS:

This study has several limitations. Its retrospective, cross-sectional design limits causal inference and may be affected by missing data. The relatively small sample size, especially in the advanced fibrosis subgroup, restricts the generalizability of the findings. Inclusion was limited to biopsy-confirmed cases, which may not fully represent real-world clinical use of noninvasive tests. Variability in laboratory test timing and standardization may have influenced results. Finally, as a single-center study from a specific geographic region, the applicability of findings to other populations is limited. Prospective multicenter studies with larger cohorts are needed.

Conclusions

FIB-4 and APRI scores demonstrated utility in predicting advanced fibrosis (F ≥3) among CHB patients in immune-tolerant, inactive, and gray zone phases. Both tests exhibited high negative predictive values and were effective in excluding advanced fibrosis. However, AUC values below 0.80 indicate limited accuracy in mild to moderate fibrosis. Therefore, FIB-4 and APRI should be used as complementary tools alongside clinical and laboratory data to guide biopsy decisions.