Investigation of Helicobacter pylori antigenic variations by ELISA and Western blot methods in patients with dyspepsia

Helicobacter pylori (H. pylori) is a spiral-shaped, microaerophilic, Gram-negative bacteria which colonize gastric mucosal. H. pylori infection is one of the most common chronic bacterial infections occurring 20-50 % in developed countries and 80-90 % in developing countries. H. pylori is related to a number of upper gastrointestinal diseases, such as chronic gastritis, duodenal and gastric ulcer, gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma and has been classified as a class 1 carcinogen. Anti-H. pylori antibodies (IgG, IgA/IgG, CagA IgG) were determined in the sera and urine specimens of patients with dyspepsia by ELISA, Western blot, URINELISA and RAPIRUN methods. Also, the frequency of the antibodies againts virulence factors CagA, VacA and other H. pylori antigens were detected by Western blot method and their relationship were assessed with histopathology results in anti-H. pylori positive patient sera. Moreover, with the aim of making further researches to determine H. pylori antigenic variations in molecular level, as gold standard method H. pylori culture optimizations were performed.The study population consisted of 136 adult patients with dyspepsia (102 females, 34 males; mean age, 46.57±12.63 SD years; age range 21 to 78 years) from Outpatient Gastroenterology Service at Dokuz Eylül University Hospital between April 2006 and July 2008. All patients had dyspeptic symptoms and were referred to a diagnostic upper endoscopy. Four biopsy specimens (two antrum, two corpus) were taken from each patient; rapid urease test (RUT) and histopathological examination were applied.Infection by H. pylori was defined as positivity and negativity of histopathology and/or RUT. Morphologic examination of Helicobacter-like organisms (HLO), gastritis activity and to grade bacterial density of H. pylori were evaluated according to Updated Sydney System.Sera and urine specimens of these patients were examined by anti-H. pylori IgG ELISA, anti-H. pylori CagA IgG ELISA (EUROIMMUN Medizinische Labordiagnostika, Lübeck), H. pylori IgA/IgG ELISA (BIOHIT, Finland), anti-H. pylori IgG Western blot (EUROIMMUN Medizinische Labordiagnostika, Lübeck), anti-H. pylori IgG URINELISA and RAPIRUN (Otsuka Pharmacautical, Tokyo, Japan) methods.A hundred three of 136 patients (75.7 %) were diagnosed as positive and 33 (24.3 %) were negative for H. pylori infection by the gold standard methods.115 (84.5 %) were diagnosed positive and 21 (15.5 %) were diagnosed negative by anti- H. pylori IgG ELISA method. The sensitivity, specificity, positive and negative predictive values (PPV, NPV) and likelihood ratio (LR) were 91.1 %, 34.3 %, 80%, 57.2 % and 0.77, respectively. Statistical difference was found between the results of gold standard methods and anti- H. pylori IgG ELISA (p=0.02). 47 (34.6 %) were diagnosed positive and 89 (65.4 %) were diagnosed negative by anti- H. pylori CagA IgG ELISA method. The sensitivity, specificity, PPV, NPV and LR were 35.6 %, 65.7 % , 76.6 %, 25.9 % and 0.44, respectively. Statistical difference was found between the results of gold standard methods and anti- H. pylori CagA IgG ELISA (p=0.00). 100 (73.5 %) were diagnosed positive and 36 (26.5 %) were diagnosed negative by H. pylori IgA/IgG ELISA method. The sensitivity, specificity, PPV, NPV and LR were 85.2 %, 60 % , 86 %, 58.3 % and 0.89, respectively. No statistical difference was found between the results of gold standard methods and H. pylori IgA/IgG ELISA (p=1). 72 (52.9 %) were diagnosed positive and 64 (47.1 %) were diagnosed negative by anti-H. pylori IgG URINELISA method. The sensitivity, specificity, PPV, NPV and LR were 66.3 %, 85.7 % , 93.1 %, 46.9 % and 0.71, respectively. Statistical difference was found between the results of gold standard methods and anti-H. pylori IgG URINELISA (p=0.00). 70 (51.4 %) were diagnosed positive and 66 (48.6 %) were diagnosed negative by RAPIRUN method. The sensitivity, specificity, PPV, NPV and LR were 65.4 %, 82.9 % , 91.6 %, 45.3 % and 0.69, respectively. Statistical difference was found between the results of gold standard methods and RAPIRUN (p=0.00).Ninety-five (69.8 %) patients were positive, 25 (%18.4) were negative, and 16 were borderline (11.8 %) by anti- H. pylori IgG Western blot test. When the borderline results were not included, the sensitivity, specificity, PPV, NPV and LR were 85.7 %, 44.8 %, 82.9 %, 50 % and 0.76, respectively.The culture is the gold standard method in the diagnosis of H. pylori infection. H. pylori NCTC 11637 standard strain was used in the optimization of H. pylori culture using different culture media (10% human sera Columbia agar, 7% horse blood Columbia agar and 7% sheep blood Columbia agar with different supplements) and the optimization of bacterial DNA extraction was also performed for further molecular analysis of isolated H. pylori strains.In this study, the sensitivity of the anti- H. pylori IgG ELISA was in accordance but the the specificity was lower with the other studies on H. pylori. We suggest that H. pylori strains have a diverse genetic heterogeneity in the world, the serological test kits should be adapted to the most common H. pylori strain in that region.Key words: Helicobacter pylori, ELISA, Western blot, culture