Krüppel-like factor 6-mediated loss of BCAA catabolism contributes to kidney injury in mice and humans.

Altered cellular metabolism in kidney proximal tubule (PT) cells plays a critical role in acute kidney injury (AKI). The transcription factor Krüppel-like factor 6 (KLF6) is rapidly and robustly induced early in the PT after AKI. We found that PT-specific Klf6 knockdown (Klf6PTKD) is protective against AKI and kidney fibrosis in mice. Combined RNA and chromatin immunoprecipitation sequencing analysis demonstrated that expression of genes encoding branched-chain amino acid (BCAA) catabolic enzymes was preserved in Klf6PTKD mice, with KLF6 occupying the promoter region of these genes. Conversely, inducible KLF6 overexpression suppressed expression of BCAA genes and exacerbated kidney injury and fibrosis in mice. In vitro, injured cells overexpressing KLF6 had similar decreases in BCAA catabolic gene expression and were less able to utilize BCAA. Furthermore, knockdown of BCKDHB, which encodes one subunit of the rate-limiting enzyme in BCAA catabolism, resulted in reduced ATP production, while treatment with BCAA catabolism enhancer BT2 increased metabolism. Analysis of kidney function, KLF6, and BCAA gene expression in human chronic kidney disease patients showed significant inverse correlations between KLF6 and both kidney function and BCAA expression. Thus, targeting KLF6-mediated suppression of BCAA catabolism may serve as a key therapeutic target in AKI and kidney fibrosis.

Loss of proximal tubular transcription factor Krüppel-like factor 15 exacerbates kidney injury through loss of fatty acid oxidation.

Loss of fatty acid ß-oxidation (FAO) in the proximal tubule is a critical mediator of acute kidney injury and eventual fibrosis. However, transcriptional mediators of FAO in proximal tubule injury remain understudied. Krüppel-like factor 15 (KLF15), a highly enriched zinc-finger transcription factor in the proximal tubule, was significantly reduced in proximal tubule cells after aristolochic acid I (AAI) treatment, a proximal tubule-specific injury model. Proximal tubule specific knockout of Klf15 exacerbated proximal tubule injury and kidney function decline compared to control mice during the active phase of AAI treatment, and after ischemia-reperfusion injury. Furthermore, along with worsening proximal tubule injury and kidney function decline, knockout mice exhibited increased kidney fibrosis as compared to control mice during the remodeling phase after AAI treatment. RNA-sequencing of kidney cortex demonstrated increased transcripts involved in immune system and integrin signaling pathways and decreased transcripts encompassing metabolic pathways, specifically FAO, and PPARα signaling, in knockout versus control mice after AAI treatment. In silico and experimental chromatin immunoprecipitation studies collectively demonstrated that KLF15 occupied the promoter region of key FAO genes, CPT1A and ACAA2, in close proximity to transcription factor PPARα binding sites. While the loss of Klf15 reduced the expression of Cpt1a and Acaa2 and led to compromised FAO, induction of KLF15 partially rescued loss of FAO in AAI-treated cells. Klf15, Ppara, Cpt1a, and Acaa2 expression was also decreased in other mouse kidney injury models. Tubulointerstitial KLF15 independently correlated with eGFR, PPARA and CPT1A appearance in expression arrays from human kidney biopsies. Thus, proximal tubule-specific loss of Klf15 exacerbates acute kidney injury and fibrosis, likely due to loss of interaction with PPARα leading to loss of FAO gene transcription.

Simplified HCC-ART score for highly sensitive detection of small-sized and early-stage hepatocellular carcinoma in the widely used Okuda, CLIP, and BCLC staging systems.

BACKGROUND: Small-sized HCC can be effectively cured by surgery with good clinical outcomes. A highly sensitive HCC α-fetoprotein routine test (HCC-ART) for HCC diagnosis as well as a simplied form of the HCC-ART were reported in the British Journal of Cancer. Here, we verified and studied the applicability of the HCC-ART to the detection of early-stage HCC. METHODS: 341 cirrhotic patients and 318 HCC patients were included in this study. For each, the HCC-ART score was calculated, and then the sensitivity, specificity, and results of an ROC curve analysis were compared between the HCC-ART and AFP when these biomarkers were used to detect small-sized HCC. RESULTS: Different HCC-ART cutoffs were set for the detection of different tumor sizes. The HCC-ART (AUC = 0.871, 70% sensitivity, 97% specificity) and the simplified HCC-ART (AUC = 0.934, 82% sensitivity, 100% specificity) were found to have high predictive power when attempting to separate cirrhotic patients from those with small-sized HCC. The simplified HCC-ART score was superior to AFP for determining stages according to the early Okuda (0.950 AUC, 84% sensitivity, 99% specificity), CLIP (0.945 AUC, 84% sensitivity, 99% specificity), and BCLC (1.000 AUC, 100% sensitivity, 99% specificity) staging systems. The simplified HCC-ART score was more strongly correlated than AFP and other staging systems with HCC tumor size (P < 0.0001; r = 0.8). CONCLUSION: The HCC-ART is superior to AFP for diagnosing early-stage HCC. Due to its advantages of minimal variability and a wide continuous scale for assessing HCC severity, the simplified HCC-ART has the potential to be more widely used than the original HCC-ART.

Circulating levels of collagen III and MMP-1 in patients with chronic hepatitis C co-infected with hepatitis B virus.

BACKGROUND: There is controversial data in the literature about the characteristics and features of dual hepatitis B and hepatitis C infection. This work is concerned with estimating the extent to which HBV could influence circulating levels of hepatitis C viral nonstructural-4 (HCV-NS4) in addition to some direct fibrosis markers in chronic hepatitis C. METHODS: Thirty-eight HCV mono-infected and 87 HCV/HBV co-infected patients constituted this study. Western-blot and ELISA were used for identifying HCV-NS4, hepatitis B surface antigen (HBsAg), collagen III and matrixmetalloproteinase-1 (MMP-1) in patients' sera. RESULTS: Hepatitis B surface antigen (HBsAg) provided area under curve (AUC) of 0.97 for identifying HBV-patients with 89% sensitivity and 94% specificity, while HCV-NS4 antigen provided an AUC of 0.95 for identifying HCV-patients with 89% sensitivity and absolute specificity (100%). In general, patients with significant fibrosis (F2-F4) showed significantly higher concentration of collagen III (P = 0.009) and lower concentrations of MMP-1 (P = 0.007) when compared to patients with minimal fibrosis (F1). However, HCV/HBV co-infected patients with F1 and F2-F4 did not show any significant difference (P > 0.05) from HCV mono-infected patients with respect to HCV-NS4, collagen III and MMP-1. These results indicate that HBV does not influence the rate of HCV-NS4 synthesis and the deposition of extracellular matrix in HCV/HBV co-infected patients and subsequently does not affect the progression rates of hepatic fibrosis. CONCLUSION: HCV/HBV co-infected and HCV- mono-infected patients had similar clinical characteristics and there is no effect of HBV co-infection on the progression rates of liver fibrosis in chronic hepatitis C patients.

GPC-HCC model: a combination of glybican-3 with other routine parameters improves the diagnostic efficacy in hepatocellular carcinoma.

Conflicting results for circulating glypican-3 (GPC3) were reported in hepatocellular carcinoma (HCC) diagnosis. We aimed to improve the diagnostic power of GPC3 by developing a GPC-HCC model for diagnosing HCC. GPC3 was tested for HCC (138), liver cirrhosis (56), and fibrosis (62) patients by ELISA. Data from patient groups were retrospectively analyzed. A novel score, GPC-HCC, based on combination of GPC3 and routine laboratory tests, was developed for HCC diagnosis. The GPC-HCC model values produced a significant 1.7-fold increase in liver cirrhosis and 3.2-fold increase in HCC, in comparison with liver fibrosis. In contrast to GPC3 and alpha fetoprotein (AFP), the GPC-HCC model showed high HCC diagnostic power with area under the curve (AUC) of 0.939, sensitivity 93 %, specificity 93 %, positive predictive value 89 %, negative predictive value 95 %, and efficiency 93 %. GPC-HCC AUC in HCC with single tumor, absent vascular invasion, and tumor size ≤3 cm were 0.93, 0.92, and 0.92, respectively, compared with 0.63, 0.63, and 0.64, respectively, for GPC3 and 0.69, 0.70, 0.55, respectively, for AFP. In conclusion, owing to these promising findings, the combination of GPC3 with other laboratory simple routine tests (GPC-HCC model) could improve the diagnostic power of GPC3 in HCC screening and follow up of cirrhotic patients.

Levels of Schistosoma mansoni Circulating Antigen in Chronic Hepatitis C Patients with Different Stages of Liver Fibrosis.

The goal of this study was to determine the levels of S. mansoni antigen in different liver fibrosis stages with chronic hepatitis C (CHC) Egyptian patients. A total of 174 CHC patients showing HCV-NS4 antigen and HCV- RNA in their sera were included. S. mansoni antigen was detected in serum using Western blot and ELISA. The levels of interferon-γ (IFN- γ) were determined using ELISA. The 50 kDa S. mansoni antigen discriminated patients infected with S. mansoni from healthy individuals with 0.93 area under curve (AUC), 92% sensitivity, and 97% specificity. The level of S. mansoni antigen (µg/ml) was significantly (P < 0.0001) increased with the progression of liver fibrosis stages (26.9 ± 17.5 in F1, 42.1 ± 25.2 in F2, 49.8 ± 30.3 in F3 and 62.2 ± 26.3 µg/mL in F4 liver cirrhosis), 26.9 ± 17.59 in significant fibrosis (F2-F4); 51.2 ± 27.9 in advanced fibrosis (F3-F4). A significant correlation (r = 0.506; P < 0.0001) was shown between the levels of the S. mansoni antigen and the HCV-NS4 antigen. In conclusion, the presence of S. mansoni antigen in different liver fibrosis stages of CHC patients confirming that concomitant schistosome infection aggravates liver disease.

Interferon-gamma is associated with hepatic dysfunction in fibrosis, cirrhosis, and hepatocellular carcinoma.

The relation between interferon-gamma (IFN-γ) levels and the severity of liver diseases through fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) has not been fully clarified. Thus, we aimed to characterize IFN-γ levels in liver-diseased patients. IFN-γ levels were determined by Western-blot and ELISA in sera from 30 healthy individuals, 53 patients with non-significant fibrosis (F0-F1), 47 with moderate/severe fibrosis (F2-F3), 44 cirrhotic patients (F4), and 50 with HCC. Enhanced levels of IFN-γ were associated with the progression of liver disease. The differences were statistically significant (P < 0.0001) when patients with F2-F3, F4, or HCC were compared with F0-F1 or healthy controls. The increase in IFN-γ was associated with HCC (OR = 0.98, 95% CI 0.97-0.99, P = 0.002). There was no statistically significant association between IFN-γ levels and HCV-RNA (IU/ml) (r = 0.1, P = 0.43) or HCV-NS4 (µg/mL) (r = 0.1, P = 0.17). There was significant (P < 0.0001) association between IFN-γ levels and the fibrosis stages and activity, albumin, platelet count, total bilirubin, and international normalized ratio (INR). In conclusion, elevated concentrations of IFN-γ represent a characteristic feature of liver disease severity regardless of underlying disease. Significant correlations with indices of hepatic dysfunction suggest that enhanced IFN-γ levels represent a consequence of liver dysfunction rather than of inflammatory disease.

HCC-DETECT: a combination of nuclear, cytoplasmic, and oncofetal proteins as biomarkers for hepatocellular carcinoma.

Currently, the search for suitable hepatocellular carcinoma (HCC) biomarkers is very intensive. Besides, efficacy and cost/effectiveness of screening and surveillance of cirrhotics for the diagnosis of HCC is still debated. So, the present study is concerned with the evaluation of cytokeratin-1 (CK-1) and nuclear matrix protein-52 (NMP-52) for identifying HCC. Two-hundred and eighty individuals categorized into three groups [liver fibrosis (F1-F3), cirrhosis (F4), and HCC] constituted this study. Western blot was used for identifying CK-1 and NMP-52 in serum samples. As a result, a single immunoreactive band was shown at 67 and 52 kDa corresponding to CK-1 and NMP-52, respectively. Both CK-1 and NMP-52 bands were cut and electroeluted separately. These markers were quantified in sera using ELISA. Patients with HCC were associated with higher concentrations of CK-1 and NMP-52 than those without HCC with a significant difference (P < 0.0001). CK-1 showed an area under receiver-operating characteristic curve (AUC) of 0.83 with 75 % sensitivity and 82 % specificity while NMP-52 yielded 0.72 AUC with 62 % sensitivity and 70 % specificity for identifying HCC. HCC-DETECT comprising CK-1 and NMP-52 together with AFP was then constructed yielding 0.90 AUC for identifying HCC with 80 % sensitivity and 92 % specificity. HCC-DETECT was then tested for separating HCC from F1-F3 showing 0.94 AUC with 80 % sensitivity and 93 % specificity. In conclusion, CK-1 in conjunction with NMP-52 and AFP could have a potential role for improving the detection of HCC with a high degree of accuracy.

Fibro-check: a combination of direct and indirect markers for liver fibrosis staging in chronic hepatitis C patients.

UNLABELLED: BACKGROUND AND RATIONALE FOR THE STUDY: The assessment of liver fibrosis provides useful information not only for diagnosis but also for therapeutic decision. This study was concerned with determining the levels of collagen III and its degrading enzyme matrix metalloproteinase-1 (MMP-1) as direct and complementary markers for liver fibrosis staging. RESULTS: A total of 269 chronic hepatitis C patients constituted this study. Western blotting was used for identifying collagen III and MMP-1 in serum samples. As a result, collagen III and MMP-1 were identified, respectively, at 70 and 245 kDa using their respective mono-specific antibodies. These two markers were quantified in sera of patients using ELISA. Next, Fibro-check was constructed combining collagen III and MMP-1 together with other indirect markers which reflect alteration in hepatic functions that proved useful to stage liver fibrosis. Fibro-check produced area under the receiver-operating characteristic curve (AUC) 0.91 and 0.83 for significant (F2-F4) and cirrhosis (F4), respectively. Additionally, we estimated the performance of Fibro-check in comparison with aspartate to platelet ratio index (APRI) and fibrosis index. Fibro-check seems to be more efficient than both of them. Fibro-check was then applied to the validation study to test its accuracy and reproducibility showing AUCs 0.90 for F2-F4 and 0.86 for F4. CONCLUSIONS: Fibro-check combining 'direct' and 'indirect' markers using a mathematical formula may improve the staging of liver fibrosis with a high degree of accuracy and seems more efficient than APRI and Fibrosis index in this group of Egyptian patients.

A simple diagnostic index comprising epithelial membrane antigen and fibronectin for hepatocellular carcinoma.

UNLABELLED: Background and rationale for the study. Continuing search for suitable tumor-markers is of clinical value in managing patients with various malignancies. These markers may be presented as intracellular substances in tissues or may be released into the circulation and appear in serum. Therefore, this work is concerned with identification and quantitative determination of epithelial membrane antigen (EMA) and fibronectin and estimating their performances as surrogate markers for identifying hepatocellular carcinoma (HCC). RESULTS: A total of 627 individuals constituted this study [fibrosis (F1-F3) = 217; cirrhosis = 191; HCC = 219]. Western-blot was used for identifying EMA and fibronectin in sera. As a result, a single immunoreactive band was shown at 130-kDa and 90-kDa corresponding to EMA and fibronectin, respectively. They were quantified using ELISA providing values in HCC higher than fibrosis or cirrhosis with a significant difference (P < 0.0001). For identifying HCC, EMA showed 0.82 area under receiver-operating characteristic curve (AUC) with sensitivity = 70% and specificity = 78% while fibronectin yielded AUC = 0.70 with sensitivity = 67% and specificity = 82%. FEBA-Test comprising fibronectin and EMA together with total-bilirubin and AFP was constructed yielding AUC = 0.92 for identifying HCC from cirrhosis with sensitivity = 89% and specificity = 85%. FEBA-Test was then tested for differentiating HCC from fibrosis showing AUC = 0.97 with sensitivity = 90% and specificity = 89%. FEBA-Test enabled the correct identification of HCC patients with CLIP 0-1 and size ≤ 3 cm with AUC = 0.80 and AUC = 0.84, respectively, indicating its ability in identifying early HCC. CONCLUSIONS: A four-marker index may improve the early detection of HCC with a high degree of accuracy.

Circulating levels and clinical implications of epithelial membrane antigen and cytokeratin-1 in women with breast cancer: can their ratio improve the results?

Immunohistochemical studies proved that the presence of breast cancer (BrCa) is accompanied by elevated levels of epithelial membrane antigen (EMA) and decreased levels of cytokeratin-1 (CK1). We, therefore, hypothesize that the serum EMA/CK1 ratio may serve as a promising biomarker for early diagnosis of breast cancer. The circulating levels of EMA and CK1 were determined by Western blot and enzyme-linked immunosorbent assay (ELISA) in sera from 102 women with BrCa and 90 women as controls (40 with benign breast disease and 50 healthy). EMA at 130 kDa and CK1 at 67 kDa were identified, purified, and quantified in sera of BrCa patients using ELISA. EMA/CK1 ratio values were found to discriminate BrCa patients from controls (P < 0.0001) with high diagnostic ability (area under the curve [AUC] = 0.901, sensitivity = 82, specificity = 76). The sensitivity and specificity for early-stage (≤ T2) BrCa were 72 and 76%, respectively. The ratio values of patients with late-stage (>T2) tumors were significantly higher than those of patients with early-stage (≤ T2) tumors. Moreover, higher grades (grades 2-3) were associated with higher values than grade 1 tumors. AUC values in different BrCa patients who had early stage, low grade, or size ≤ 2 cm were 0.855, 0.762, and 0.839, respectively. AUC values of patients with positive lymph node or positive distant metastasis were 0.907 and 0.913, respectively. We show for the first time the impact of serum EMA and CK1 ratio in BrCa detection. Differential EMA/CK1 values may serve as a diagnostic marker in early-stage breast cancer patients.

Diagnostic value of fibronectin discriminant score for predicting liver fibrosis stages in chronic hepatitis C virus patients.

BACKGROUND: Several noninvasive predictive models were developed to substitute liver biopsy for fibrosis assessment. AIM: To evaluate the diagnostic value of fibronectin which reflect extracellular matrix metabolism and standard liver functions tests which reflect alterations in hepatic functions. MATERIAL AND METHODS: Chronic hepatitis C (CHC) patients (n = 145) were evaluated using ROC curves and stepwise multivariate discriminant analysis (MDA) and was validated in 180 additional patients. Liver biochemical profile including transaminases, bilirubin, alkaline phosphatase, albumin, complete blood count were estimated. Fibronectin concentration was determined using monoclonal antibody and ELISA. RESULTS: A novel index named fibronectin discriminant score (FDS) based on fibronectin, APRI and albumin was developed. FDS produced areas under ROC curves (AUC) of 0.91 for significant fibrosis and 0.81 for advanced fibrosis. The FDS correctly classified 79% of the significant liver fibrosis patients (F2-F4) with 87% sensitivity and 75% specificity. The relative risk [odds ratio (OR)] of having significant liver fibrosis using the cut-off values determined by ROC curve analyses were 6.1 for fibronectin, 4.9 for APRI, and 4.2 for albumin. FDS predicted liver fibrosis with an OR of 16.8 for significant fibrosis and 8.6 for advanced fibrosis. The FDS had similar AUC and OR in the validation group to the estimation group without statistically significant difference. CONCLUSION: FDS predicted liver fibrosis with high degree of accuracy, potentially decreasing the number of liver biopsy required.

Impact of Hepatitis C Virus/Schistosoma mansoni Coinfection on the Circulating Levels of HCV-NS4 Protein and Extracellular-Matrix Deposition in Patients with Different Hepatic Fibrosis Stages.

Hepatitis C virus (HCV)/Schistosoma mansoni coinfection is common in Egypt and other developing countries. This study aimed to investigate the influence of HCV/S. mansoni coinfection on the concentration of HCV-nonstructural protein-4 (NS4) in addition to collagen III and matrix metalloproteinase-1 (MMP-1) in different hepatic fibrosis stages. We found that coinfected patients (N = 186) showed significantly (P < 0.05, Mann-Whitney U test) higher concentrations of HCV-NS4, collagen III, and collagen III/MMP-1 ratio (CMR) than those with HCV monoinfection (N = 104) in different fibrosis stages. Conversely, coinfected patients showed significantly lower concentrations of MMP-1 when compared with HCV monoinfection. The elevated levels of CMR in case of HCV monoinfection yielded an estimated odds ratio of 1.8 and 2.6 for developing significant fibrosis (F2-F4) and cirrhosis (F4), respectively. HCV/S. mansoni coinfection increased the risk for developing F2-F4 and F4 several fold yielding an estimated odds ratio of 11.1 and 5.2, respectively. This means that coinfected patients have a 6-fold and 2-fold increased risk of developing F2-F4 and F4, respectively, over HCV-monoinfected patients. Thus, elevated levels of HCV-NS4 and CMR in HCV/S. mansoni coinfection suggest increased susceptibility of coinfected patients, compared with those with HCV monoinfection, for accelerating hepatic fibrosis progression.

Combined use of epithelial membrane antigen and nuclear matrix protein 52 as sensitive biomarkers for detection of bladder cancer.

BACKGROUND: The advent of noninvasive urine-based markers as well as other novel modalities has yielded improved diagnostic accuracy. However, the new markers failed to reach higher sensitivity and specificity. We therefore evaluated the potential role of epithelial membrane antigen (EMA) and nuclear matrix protein 52 (NMP-52) singly and combined as noninvasive biomarkers for the detection of bladder cancer (BC). METHODS: A total of 160 individuals including 66 patients with BC, 54 patients with benign urologic disorders and 40 healthy volunteers were investigated. Urinary EMA at 130 kDa and NMP at 52 kDa were identified, purified and quantified by Western blot, electroelution and enzyme-linked immunosorbent assay (ELISA). The diagnostic performance of each biomarker and their combination were compared using area under receiver operating characteristic curves (AUC). RESULTS: Mean urinary EMA, 2.42 µg/mL, and NMP-52, 17.85 µg/mL, were significantly elevated in patients with BC compared to controls, 1.18 and 3.44 µg/mL, respectively (p<0.0001). The combined use of these markers yielded values which were increased 4.4- and 13.7-fold in the benign and malignant disease groups, respectively, with respect to the normal group. The values of EMA and NMP-52 were significantly higher in patients with higher-grade tumors than those with lower-grade tumors (p<0.0001). Moreover, this combination could predict all BC stages and grades with 0.91 AUC, 94% sensitivity and 80% specificity. CONCLUSIONS: EMA and NMP-52 in combination could be promising noninvasive biomarkers for BC detection.

Perinatal transmission of hepatitis C antigens: envelope 1, envelope 2 and non-structural 4.

BACKGROUND: Perinatal exposure to hepatitis C virus (HCV) antigens during pregnancy may affect the developing immune system in the fetus. We aimed to study the perinatal transmission of HCV structural and non-structural antigens. METHODS: Sera from 402 pregnant mothers were tested for anti-HCV antibody and HCV RNA. HCV antigens were determined in sera from 101 HCV-infected mothers and their cord blood. RESULTS: In both serum and cord blood samples, HCV NS4 (non-structural 4) at 27 kDa, E1 (envelope 1) at 38 kDa and E2 (envelope 2) at 40 kDa were identified, purified and quantified using western blotting, electroelution and ELISA. Maternal sera and neonate cord blood samples had similar detection rates for NS4 (94.1%), E1 (90.1%) and E2 (90.1%). The mean maternal serum levels (optical density, OD) of HCV NS4 (0.87 ± 0.01), E1 (0.86 ± 0.01) and E2 (0.85 ± 0.01) did not differ significantly (p > 0.05) from those of neonatal cord blood (0.83 ± 0.01, 0.87 ± 0.01 and 0.85 ± 0.01, respectively). Also, strong correlations (p < 0.0001) were shown between sera and cord blood sample levels of HCV NS4, r = 0.77; E1, r = 0.76 and E2, r = 0.80. The vertical transmission of these antigens in vaginal delivery did not differ significantly (p > 0.05) from those in caesarean section. CONCLUSIONS: These findings indicate that vertical transmission of HCV NS4, E1 and E2 antigens was very high. Thus, exposure to these antigens may influence the developing immune responses to natural infection or future vaccination.

Fibro-α score as a simple and useful non-invasive test for predicting significant liver fibrosis in chronic hepatitis C patients.

BACKGROUND AND STUDY AIMS: Non-invasive methods for the assessment of liver fibrosis are clinically important where hepatitis C virus (HCV) is common in Egypt. Our aim was to evaluate the diagnostic performance of a panel of simple blood markers of liver fibrosis in chronic hepatitis C (CHC) patients. PATIENTS AND METHODS: A total of 199 patients with CHC evaluated for eligibility for antiviral therapy were included. Liver biochemical profile including transaminases, bilirubin, alkaline phosphatase, serum albumin, complete blood count prothrombin time and AFP were estimated. Liver biopsy was done. Statistical analyses were performed by logistic regression and receiver operating characteristic (ROC) curves to assess and compare diagnostic accuracy of blood markers. A stepwise combination algorithm was developed and validated prospectively in 135 additional patients. RESULTS: α-Foetoprotein (AFP) was the most efficient marker among other markers tested. The areas under the curves (AUCs) of AFP were 0.77 for significant liver fibrosis (F2-F4), 0.75 for advanced liver fibrosis (F3-F4) and 0.76 for liver cirrhosis (F4). The stepwise multivariate discriminant analysis (MDA) selected a novel non-invasive index for discriminating patients with significant liver fibrosis, named Fibro-α score. Fibro-α score=(1.35 (numeric constant) +AFP (IUml(-1))×0.009584+aspartate aminotransferase (AST)/alanine aminotransferase (ALT)×0.243-platelet count (×10(9)l(-1))×0.001624). The Fibro-α score was used for patients with advanced liver fibrosis and liver cirrhosis. The AUCs of Fibro-α score were 0.82 for patients with advanced liver fibrosis and 0.80 for those with cirrhosis. These results were reproduced in a validation study with no significant difference. CONCLUSION: While liver biopsy is invasive, expensive and, in some settings, impossible to do, Fibro-α score is simple, cheap, non-invasive and may be useful for predicting significant liver fibrosis.