HCC-Check: A Novel Diagnostic Tool for Early Detection of Hepatocellular Carcinoma Based on Cytokeratin-1 and Epithelial Membrane Antigen: A Cross-Sectional Study.

Background: Hepatocellular carcinoma is frequently diagnosed in advanced stages, leading to a poorer prognosis. Therefore, early diagnosis and identification of biomarkers may significantly improve outcomes. Methods: This cross-sectional study enrolled 486 participants distributed among 3 groups: F1 to F3 = 184, F4 = 183, and hepatocellular carcinoma = 119. Liver fibrosis staging was performed using FibroScan, while imaging features were used for hepatocellular carcinoma detection. Epithelial membrane antigen and cytokeratin-1 levels in serum were quantified through Western blot and ELISA, respectively. Results: Patients diagnosed with hepatocellular carcinoma exhibited significantly elevated levels of epithelial membrane antigen and cytokeratin-1 compared to non-hepatocellular carcinoma patients, with a highly significant statistical difference (P < .0001). Epithelial membrane antigen demonstrated diagnostic performance with an area under the curve of 0.75, a sensitivity of 69.0%, and a specificity of 68.5%. Cytokeratin-1 for the identification of hepatocellular carcinoma showed a sensitivity of 79.0% and a specificity of 81.4%, resulting in an area under the curve of 0.87. The developed HCC-Check, which incorporates epithelial membrane antigen, cytokeratin-1, albumin, and alpha-fetoprotein, displayed a higher area under the curve of 0.95 to identify hepatocellular carcinoma, with a sensitivity of 89.8% and a specificity of 83.9%. Notably, HCC-Check values exceeding 2.57 substantially increased the likelihood of hepatocellular carcinoma, with an estimated odds ratio of 50.65, indicating a higher susceptibility to hepatocellular carcinoma development than those with lower values. The HCC-Check diagnostic test exhibited high precision in identifying patients with hepatocellular carcinoma, particularly those with small tumor sizes (<5 cm) and a single nodule, as reflected in area under the curve values of 0.92 and 0.85, respectively. HCC-Check was then applied to the validation study to test its accuracy and reproducibility, showing superior area under the curves for identifying different stages of hepatocellular carcinoma. These outcomes underscore the effectiveness of the test in the early detection of hepatocellular carcinoma. Conclusion: The HCC-Check test presents a highly accurate diagnostic method for detecting hepatocellular carcinoma in its early stages.

Immunogenicity of a newly developed vaccine against Clostridium perfringens alpha-toxin in rabbits and cattle.

Background and Aim: Clostridium perfringens type A is an anaerobic bacterium that produces four major toxins (alpha, beta, epsilon, and iota) that cause various diseases. Most of the important C. perfringens-associated diseases of farm animals are caused by alpha-toxin. This study aimed to produce a vaccine against alpha-toxin using C. perfringens type A (ATCC 13124) and investigate its potency, stability, and safety. Materials and Methods: The vaccine was formulated of its constituents for 1 h. Each milliliter of the final vaccine product contained alpha toxoid 15 lecithovitellinase activity (Lv) by adding (0.375 mL containing 40 Lv) and approximately 0.2 mL from 3% concentrated aluminum hydroxide gel, <0.001% W/V thiomersal, <0.05% W/V formaldehyde, and nearly 0.425 mL phosphate-buffered saline (pH 7.2). The vaccine efficacy was evaluated in rabbits and cattle by performing potency, stability, and safety tests. Results: The vaccine produced approximately 8.8 and 4.9 IU/mL neutralizing antibodies in rabbits and cattle, respectively. These concentrations were higher than the lowest concentration recommended by various international protocols and the United States Department of Agriculture by 2.20-fold in rabbits and 1.23-fold in cattle. Interestingly, the formulated vaccine enhanced immune responses by 1.80-fold in rabbits compared with that in cattle; the difference was statistically significant (p < 0.0001). The vaccine was stable for 30 months. In vaccinated rabbits, the body temperature slightly increased temporarily during the first 10 h of vaccination; however, the temperature difference was not statistically significant (p > 0.05). Conclusion: This study describes a manufacturing process to obtain sufficient amounts of a vaccine against C. perfringens alpha-toxin. The formulated vaccine effectively elicited a higher level of neutralizing antibody response than the international standards. Furthermore, the vaccine was found to be stable, safe, and effective in preventing C. perfringens-related diseases in rabbits and cattle. Further studies are necessary to evaluate the efficacy of this vaccine in other farm animals.

Prevalence of Helicobacter pylori infection in patients with chronic hepatitis C.

BACKGROUND: Association between Helicobacter pylori (H. pylori) and chronic hepatitis C (CHC) still remains controversial. This work is concerned with assessing the potential role of H. pylori in the progression of hepatitis C virus (HCV)-related chronic liver disease. RESULTS: A total of 449 individuals constituted this study (200 individuals were used to validate the assay while 249 individuals were used to assess the correlation between H. pylori infection and CHC). H. pylori antigen was quantified in serum samples using ELISA. As a consequence, our findings showed that H. pylori positivity was increased significantly (P = 0.021) with liver fibrosis progression as it was found in 44.45% of fibrotic patients and 71.88% of cirrhotic patients. We demonstrated that patients with F4 were accompanied by a significant (P < 0.05) increase in the concentration of H. pylori antigen displaying 16.52-fold and 1.34-fold increase in its level over F0 and F1-F3, respectively. Patients co-infected with H. pylori and HCV are 3.19 times (219%) more likely to experience cirrhosis than those who are mono-infected with HCV. This suggests that the risk for developing F4 was found to increase upon H. pylori co-infection when compared to CHC mono-infected patients. CONCLUSION: The elevated levels of H. pylori-antigen in HCV/H. pylori co-infection suggest increased susceptibility of co-infected patients for promoting hepatic fibrosis progression.

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.

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.

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.

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.

Noninvasive diagnosis of liver fibrosis and cirrhosis in chronic hepatitis C patients.

BACKGROUND: We aimed to derive a simple noninvasive test for liver-fibrosis staging and then estimate its performance against four simple noninvasive tests in chronic hepatitis C (CHC) patients. METHODS: CHC patients were divided into two cohorts: an estimation set (n = 324) and a validation set (n = 524). Liver fibrosis was staged according to the METAVIR scoring system. Statistical analysis was done using stepwise linear discriminant analysis and area under receiver-operating characteristic curves (AUCs). RESULTS: Biotechnology Research Center (BRC) score was constructed combining several blood markers that proved useful to stage liver fibrosis. Aspartate aminotransferase /alanine aminotransferase ratio (AAR), aspartate to platelet ratio index (APRI), Fibro-α, King, and BRC scores correlated with the histological fibrosis stages with correlation coefficient 0.26, 0.36, 0.58, 0.45, and 0.73, respectively. BRC score produced AUCs 0.87, 0.83, and 0.89 for significant (F2-F4), advanced fibrosis (F3-F4), and cirrhosis (F4), respectively. These results were reproduced in the validation study with no significant difference yielding AUCs 0.85 for F2-F4, 0.82 for F3-F4, and 0.88 for F4. CONCLUSION: BRC score, a novel noninvasive test, is a useful and easy tool to evaluate liver fibrosis in CHC patients and seems more efficient than AAR, APRI, Fibro-α score, and King's score in this group of Egyptian patients.

Development of a novel score for liver fibrosis staging and comparison with eight simple laboratory scores in large numbers of HCV-monoinfected patients.

BACKGROUND: This study aimed to develop and evaluate a predictive score named Fibrosis Routine Test (FRT) for liver fibrosis staging and to compare FRT with APRI, Lok, GUCI, FI, FibroQ, FCI, FIB-4 and 4RLB scores in large numbers of untreated HCV-monoinfected patients. METHODS: Large numbers of estimation (N=2045) and validation patients (N=3212) were included in this study. Stepwise linear discriminant analysis and area under receiver-operating characteristic curves (AUCs) were used to create a predictive score comprising age, AFP, APRI and albumin. RESULTS: In the estimation study, FRT produced AUCs 0.84, 0.85 and 0.86 for significant (F2-F4), advanced fibrosis (F3-F4) and cirrhosis (F4), respectively. FRT > 4 was 83% specific and 73% sensitive for F2-F4, FRT > 5 was 83% specific and 71% sensitive for F3-F4 and FRT > 5.5 was 81% specific and 73% sensitive for F4. In the validation study, FRT produced AUCs 0.81, 0.89 and 0.95 for F2-F4, F3-F4 and F4, respectively. The above eight scores demonstrated lower AUCs than FRT. CONCLUSION: While liver biopsy is invasive, costly and associated with complications, Fibrosis Routine Test (FRT) is a non-invasive, inexpensive, simple and may reduce the need of liver biopsy.