Samples
A total of 449 consecutive Egyptian individuals composed of three different groups constituted the present study. Informed consents were obtained from all participants and they were fully informed concerning the diagnostic procedures involved and disease nature. The study protocol was approved by the Ethics Committee of the Mansoura University hospitals, Mansoura University, Egypt. The consent to participate was obtained verbally and approved by Ethics Committee. The first group included serum samples of 100 H. pylori-infected individuals collected from the Gastro-Enterology and Surgery Center, Mansoura University, Mansoura, Egypt. They were diagnosed based on culture as a gold standard.
The second group included serum samples of another 249 chronic hepatitis C patients [95 with F0, 90 with F1‑F3, and 64 with F4] collected from the Tropical Medicine Department, Mansoura University hospitals, Mansoura, Egypt. These patients were tested positive for the presence of HCV-ribonucleic acid (RNA) using quantitative polymerase chain reaction assay (COBAS Ampliprep/COBAS TaqMan, Roche Diagnostics, Pleasanton, USA). Histopathological classification for liver fibrosis and cirrhosis was performed according to the METAVIR score [12]. Liver fibrosis was defined as a METAVIR score of ≤ 3 (F1‑F3) whereas cirrhosis was defined as a METAVIR score of 4 (F4). The third group included serum samples of another 100 healthy volunteers used as a control group. Overall, the first and the third groups were used to validate the assay while the second group was dedicated to assessing the correlation between H. pylori infection and chronic hepatitis C.
Laboratory tests
All serum samples constituted this study were subjected to laboratory investigations including liver function tests [alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, albumin, and alkaline phosphatase (ALP)] measured on an automated biochemistry analyzer (A15, Biosystem, Spain). Alpha fetoprotein (AFP) level was performed by chemiluminescence, with Immulite AFP (1000) kit (Diagnostic Products Corporation; Los Angeles, CA, USA).
Detection of 58-kDa H. pylori antigen using ELISA
First of all, the target 58-kDa H. pylori antigen was previously identified at 58-kDa molecular weight based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blot as published by Attallah et al. [9]. In this work, the quantification of 58-kDa H. pylori antigen was performed based on ELISA technique using its mono-specific antibodies (ABC Diagnostics, New Damietta, Egypt) as according to Attallah et al. [9]. Color intensity was proportional to the amount of bound conjugate and therefore is a function of the concentration of H. pylori antigen present in the serum sample.
Statistical analysis
All statistical calculations were done by the SPSS software v.15.0 (SPSS Inc., Chicago, IL) and GraphPad Prism package v.5.0 (GraphPad Software, San Diego, CA). Continuous variables were expressed as mean ± standard error of mean. One-way analysis of variance (ANOVA) and Tukey’s post hoc test were used to compare continuous variables while chi-square test was used to compare proportions. All tests were two-tailed and statistical significance assessed at the 0.05 level. Area under the curve (AUC) was used to assess the diagnostic value of 58-kDa H. pylori antigen to distinguish between patients infected with H. pylori and non-infected individuals. An AUC equal to 1.0 is characteristic of an ideal test, whereas 0.5 indicates a test without diagnostic value. The nearer a curve shifts to the top left-hand corner of the graph, the more useful the marker is for the diagnosis. Results obtained by the H. pylori culture test as a gold standard; common indicators of 58-kDa H. pylori antigen accuracy (sensitivity, specificity, efficiency, and odds ratio) were derived from such a 2 × 2 contingency table. Odds ratio (with 95% confidence intervals) was calculated to estimate the risk of a target disorder from subjects without it.