ABSTRACT
BACKGROUND: Current diagnosis of Helicobacter pylori infection by biopsy-based tests requires invasive sampling. Non-invasive methods such as the H. pylori stool-antigen (HpSA) test may be the best alternative for diagnosis of active infection. However, due to the presence of antigenic-diversity among the strains, various commercial tests have shown some discrepancies in different geographical-areas. OBJECTIVES: This study evaluates a homemade HpSA kit developed by using the H. pylori antigens from Iranian-isolates for detection of H. pylori in the stool of infected patients. PATIENTS AND METHODS: Based on the endoscopic features and/or a rapid-urease test (RUT), 30 child and 50 adult patients, were recruited. From these candidates, three biopsies for RUT, culture and histology, and a stool-sample, were obtained. Patients were considered as H. pylori-positive if culture alone or RUT plus histology were found to be positive. Presence of H. pylori antigens in their stools was detected by the homemade HpSA test and an imported HpSA kit (Immundiagnostik, Germany). RESULTS: Using the biopsy-based tests with RUT, histology and culture, 53% (16/30) of children were diagnosed as H. pylori-positive while using the imported kit 57% and the homemade kit 50% of the candidates showed positive results. Also by the biopsy-based tests, 54% of the adults were diagnosed as H. pylori-positive while by the homemade kit 56% showed positive results. Considering the biopsy-based tests as the gold standard, sensitivity and specificity for the imported kit was 94% and 86%, respectively, while the mean sensitivity and specificity for the homemade kit was 96% and 98%, respectively. CONCLUSIONS: The homemade kit, compared with the imported kit and biopsy-proven tests may be a valid and reliable method for determining the presence of H. pylori infection in Iran.
ABSTRACT
BACKGROUND AND OBJECTIVE: An outer membrane protein (OMP) of Helicobacter pylori namely OipA, is an important virulence factor associated with peptic ulcer and gastric cancer risks. The purpose of this study was to isolate the 34 KDa OMP of H. pylori and evaluate its immunogenicity in experimental animals for rapid detection of more virulent H. pylori isolates. MATERIAL AND METHODS: Sarcosine insoluble fraction of membrane proteins (OMPs) were prepared from 15 clinical isolates of H. pylori and their profiles were analyzed by SDS-PAGE. Two out of 15 isolates which demonstrated higher expression for apparent 34 KDa proteins were selected. Under optimal conditions, 34 KDa protein was recovered from 5% SDS-Agarose gel, purified and injected into the New Zealand white rabbits with Fruend's adjuvant in multiple stages with two weeks intervals. Collected antiserum was purified through affinity chromatography with Sepharose column and its titer was determined by ELISA. Specific immune response was demonstrated by Dot blot and western blotting methods. RESULTS: The titer of antibody was determined about 1/3000 and western blotting demonstrated a 34 KD-protein. Screening of various strains by Dot blot method for its presence showed that its expression was more frequent in strains isolated from the patients with more severe pathology. CONCLUSION: High titer obtained for pAbs antibody, suggested the high immunogenicity of this protein in experimental animals. Detection of 34 KDa OMP in strains isolated from the patients with more severe pathology proposes the possible application of this pAbs in detecting more virulent strains of H. pylori.
ABSTRACT
We evaluated two protocols for isolation of Helicobacter pylori in stool from biopsied and nonbiopsied children. Twenty-three child patients whose presumptive positivity or negativity was diagnosed by endoscopy and a rapid urease test at site were used to compare biopsy-based tests with stool-based tests (H. pylori stool antigen test and stool culture). Their gastric activity and bacterial density were graded by the updated Sydney system. Biopsy and stool specimens were cultured on Campy-blood and Belo horizonte agar plates after enrichment in selective Campy-Thio medium. To compare two stool culture protocols, stools from 20 nonbiopsied children were tested by the HpSA test and cultured either as above or after treatment with cholestyramine. Grown colonies were screened by Gram staining, slide agglutination using anti-H. pylori monoclonal IgG; positive isolates were tested by biochemical tests and polymerase chain reaction for H. pylori-specific ureA gene. Coccoid H. pylori was isolated in stool samples from the biopsied patients whose bacterial density was two to four in histology. Their oxidase was slightly positive but became positive after two subcultures, while additional biochemical tests confirmed the isolation of H. pylori. Similar coccoid but oxidase positive H. pylori was isolated from three nonbiopsied children with the protocol of cholestyramine treatment only. The density of bacteria in the stomach may influence the recovery of H. pylori from stool; inactivation of bile with cholestyramine improves the yield in culture and favors isolation of an enhanced metabolic form of bacteria.
