This study included 120 CLD children of different etiologies; they were 70 males and 50 females with a mean age of 11.1 ± 5.3 years ranged from 2 years to 17 years. While the 60 healthy control children were 29 males and 31 females with a mean age of 10.3 ± 4.4 years ranged from 2 years to 17 years. Both groups were sex and age-matched (P = 0.320, 0.212 respectively).
Etiology and clinical presentation of CLD in the patient group
The diagnosis of the studied CLD patients was achieved via comprehensive investigations and the underlying etiologies were verified with liver biopsies which also determined the exact fibrosis stage in each case (Table 1). The underlying etiologies of CLD in the studied patients were genetic-metabolic disorders [glycogen storage disease (21 case) (17.5%) [GSD-I (12 cases), GSD-II (4 cases), GSD-III (5 cases)], Niemen Pick disease (3 cases) (type A (2 cases) and type B (1case)) (2.5%), Wilson disease (5 cases) (4.1%), and tyrosinemia 2 cases (1.7%)], autoimmune hepatitis type-I (29 cases) (24.2%), congenital hepatic fibrosis (9 cases) (7.5%), chronic hepatitis for differential diagnosis (11 cases) (9.2%), infective hepatitis (HBV) (5 cases) (4.10%) and HCV (18 cases) (15%), and cholestatic liver disease (biliary atresia) (14 cases) (11.7%), familial intrahepatic cholestasis (PFIC-1) (3 cases) (2.5%).
Clinical presentation of CLD patients were jaundice (48.3%), abdominal distention (19.2%), clay color stool (11.7%), abdominal pain (5%), faltering of growth (5.7%), convulsion (4.2%), and pallor (4.2%). Abdominal ultrasonography revealed that 80% of patients had hepatomegaly and 33% had splenomegaly.
Laboratory characteristics and histopathological evaluation of the studied subjects
Baseline laboratory data of studied subjects showed that there was statistically significant difference between CLD patients and healthy controls regarding liver function tests (ALT, AST, PT, albumin, bilirubin) as they were higher in CLD patients (Table 2).
Histopathological evaluation of liver biopsy according to the METAVIR scoring  revealed that disease activity was mild (A1) in 51.7%, moderate (A2) in 25.8%, and severe (A3) in 22.5%. Meanwhile, degree of fibrosis (55.8%) had minimal fibrosis (F1), 22.5% had moderate fibrosis (F2), 8.3% had severe fibrosis (F3), and 13.3% had cirrhosis (F4).
Non-invasive biomarkers for assessment of hepatic fibrosis
Applying the non-invasive surrogate blood indices of liver fibrosis revealed that the mean ± SD of APRI and FIB-4 scores (1.1 ± 0.85 and 0.53 ± 0.54 respectively) in all studied cases and they showed a significant trend of increase with increasing the degree of fibrosis stage evident with histological METAVIR scoring system (P 0.001) (Table 3).
Serum matrix metalloproteinase-1 assessment
Serum matrix metalloproteinases-1 (MMP-1) level was significantly lower in CLD patients compared to healthy controls (P < 0.001) (Table 2).
Serum MMP-1 concentration was decreased significantly with increasing the degree of fibrosis in CLD patients (Table 3).
Serum MMP-1 level did not differ according to CLD etiologies in studied patients (P > 0.05) (Table 4).
Serum MMP-1 was found to be negatively correlated with AST (r = −0.448, P < 0.001), ALT (r = −0.389, P < 0.001), GGT (r = −0.544, P < 0.001), ALP (r = −0.675, P < 0.001), PT (r = −0.361, P <0.001), degree of fibrosis (r =−0.960, P < 0.001), (APRI) (r =−0.653, P < 0.001), and FIB-4 (r = −0.557, P < 0.009) and positively correlated with serum albumin (r = 0.312, P < 0.001), hemoglobin level (r = 0.327, P < 0.001), and platelets count (r = 0.446, P < 0.001).
Diagnostic performance and predictive value of serum MMP-1, APRI, and FIB-4
The diagnostic performances of studied biomarkers to detect the presence of fibrosis (> F0) in children with CLD indicated that serum MMP-1 at a cut-off value < 21.3 ng/ml had a 95% sensitivity, 95% specificity with a fair area under the ROC curve (AUC) of 0.972 (95% confidence interval, 0.909‑0.987), p < 0.001, while APRI at cut-off index ≥ 0.35 had a 95.8% sensitivity, 100% specificity with a fair AUC of 0.965 (95% confidence interval, 0.934‑0.995), p < 0.001. For FIB-4, at a cut-off index ≥ 0.11, it had a 90.1% sensitivity, 100% specificity with AUC of 0.978 (95% confidence interval, 0.940‑0.994), p < 0.001 (Fig. 1).
While at cut-off value < 10.5 ng/ml with 100% sensitivity, 100% specificity, with excellent AUC of 1 (95% confidence interval, 1‑1), p < 0.001 serum MMP-1 could detect severe fibrosis (≥ F3 METAVIR). While, APRI could detect severe fibrosis (≥ F3 METAVIR) at cut-off index ≥ 1.44, with sensitivity 100%, specificity 89.7%, with a good AUC of 0.975 (95% confidence interval, 0.952‑0.998), p < 0.001. Also, FIB-4 at cut-off value ≥ 0.44 with 100% sensitivity, 75.6% specificity, with a good AUC of 0.939 (95% confidence interval, 0.890‑0.981), p < 0.001 could detect severe fibrosis (≥ F3 METAVIR) in children with CLD (Fig. 2).
Multiple linear regression analysis revealed that platelet, AST, ALT, PT, APRI, FIB-4, and MMP-1 are potential predictors for detecting fibrosis in children with CLD (Table 5).