Background-The development of macrolide resistance in Helicobacter pylori is considered an essential reason for failure of antibiotic eradication therapies. The predominant mechanism of resistance to macrolides, particularly clarithromycin, is based on three defined mutations within 23S rRNA, resulting in decreased binding of the antibiotic to the bacterial ribosome. Aim-To develop an rRNA based whole cell hybridisation method to detect Helicobacter species in situ within gastric tissue, simultaneously with its clarithromycin resistance genotype. Methods-A set of fluorescent labelled oligonucleotide probes was developed, binding either to H pylori 16S rRNA or 23S rRNA sequences containing specific point mutations responsible for clarithromycin resistance. After hybridisation and stringent washing procedures, labelling of intact single bacteria was monitored by fluorescence microscopy. The new approach was compared with PCR based assays, histology, and microbiological culture. Results-In comparison with the phenotypic resistance measurement by E test, the genotypic clarithromycin resistance correlated perfectly (100%) for 35 H pylori isolates analysed. In a set of gastric biopsy specimens (27) H pylori infection was confirmed by histology (17/27) and correctly detected by whole cell hybridisation. Five clarithromycin resistant strains were identified in gastric tissue specimens directly. Furthermore, non-cultivable coccoid forms of H pylori were easily detectable by whole cell hybridisation. Conclusions-Whole cell hybridisation of rRNA holds great promise for cultivation independent, reliable, and rapid (three hours) genotypic determination of clarithromycin resistance in H pylori. Compared with PCR techniques it is independent of nucleic acid preparations, not prone to inhibition, and allows semiquantitative visualisation of the bacteria within intact tissue samples. (Gut 2000;46:608-614)

The Gram negative bacterium Helicobacter pylori is a human pathogen which infects the gastric mucosa and causes an inflammatory process leading to gastritis, ulceration and cancer. A systematic, proteome based approach was chosen to detect candidate antigens of H. pylori for diagnosis, therapy and vaccine development and to investigate potential associations between specific immune responses and manifestations of disease. Sera from patients with active H. pylori infection (n = 24), a control group with unrelated gastric disorders (n = 12) and from patients with gastric cancer (n = 6) were collected and analyzed for the reactivity against proteins of the strain HP 26695 separated by two-dimensional electrophoresis. Overall, 310 antigenic protein species were recognized by H. pylori positive sera representing about 17% of all spots separated. Out of the 32 antigens most frequently recognized by H. pylori positive sera, nine were newly identified and 23 were confirmed from other studies. Three newly identified antigens which belong to the 150 most abundant protein species of H. pylori, were specifically recognized by H. pylori positive sera: the predicted coding region HP0231, serine protease HtrA (HP1019) and Cag3 (HP0522). Other antigens were recognized differently by sera from gastritis and ulcer patients, which may identify them as candidate indicators for clinical manifestations. The data from these immunoproteomic analyses are added to our public database (http://www.mpiib-berlin.mpg.de/2D-PAGE). This platform enables one to compile many protein profiles and to integrate data from other studies, an approach which will greatly assist the search for more immunogenic proteins for diagnostic assays and vaccine design.

Gastric infection withHelicobacter pylori is one of the most common chronic infections in humans, causing substantial morbidity and mortality. The diagnosis of H. pylori infection usually involves upper endoscopy with biopsy since the only noninvasive method of comparable accuracy, the [ 13 C]urea breath test, requires technical equipment that is not available in most gastroenterological units. Serological methods for detection of H. pylori infection have reached sufficient accuracy to be used as screening tests before endoscopy or for seroepidemiological surveys. In the present study we evaluated different interpretation criteria for use with immunoglobulin G immunoblotting for the diagnosis of H. pylori infection. We applied five different sets of interpretation criteria, four of which had been published previously, to the Western blot results of 294 patients with different gastrointestinal symptoms. Since it is known that less than 2% of patients who are infected with H. pylori fail to seroconvert, an optimally sensitive Western blotting system should be able to detect approximately 98% of active infections. When the different criteria were applied to our patient population, it became apparent that the abilities of the systems to detect active H. pylori infection were quite varied. The results for the sensitivity and specificity, according to the different applied criteria, ranged from 62.8 to 95.9% and from 85.7 to 100.0%, respectively. Positive predictive values and negative predictive values, according to the published criteria, ranged from 97.2 to 100.0% and from 37.7 to 82.4%, respectively. Recommendations for the optimal use of the different interpretation criteria are discussed.