A biprobe real-time PCR protocol, followed by hybridization melting point analysis, to detect point mutations in the 23S rRNA gene of Helicobacter pylori associated with clarithromycin resistance was established and evaluated in a clinical study. Of 92 patients who underwent endoscopy, 45 were found to be H. pylori infected and invariably were also culture positive. Of the 45 isolates, 11 were shown to be resistant to clarithromycin by E-test. With respect to the detection of H. pylori infection, PCR showed sensitivities of 100% in biopsies and 98% in stool specimens and a specificity of 98% in both biopsy and stool samples. All clarithromycin-sensitive cases were identified as such by PCR in both biopsy and stool samples. Of the cases with a resistant strain, eight were identified as such in stool DNA and nine were identified in biopsy DNA. Failure of PCR to detect the resistant genotype in the biopsy DNA, stool DNA, or both (one case) was associated with mixed populations. In these cases, patients had not been treated for H. pylori infection before, and the sensitive population showed to be present in considerably higher numbers than the resistant population. In five of six cases in which infection with a resistant genotype only was identified by PCR, the patients had received clarithromycin-based eradication therapy in the past. Thus, the assay presented provides a highly accurate noninvasive method to detect H. pylori infection in stool and at the same time allows for culture-independent clarithromycin susceptibility testing.Helicobacter pylori is a gram-negative bacterium associated with different digestive diseases, such as gastritis, peptic ulcer, mucosa-associated lymphoid tissue lymphoma, and gastric cancer (3). At present, several diagnostic assays for H. pylori detection are available (23). Invasive methods requiring gastric endoscopy include rapid urease testing, culture, histology, and molecular diagnostics. Noninvasive approaches include fecal antigen detection, serologic testing, and urea breath testing. During recent years, noninvasive methods gained in importance; however, no information on resistance against antibiotics can yet be obtained with these tests. Clarithromycin is an integral part of first-line therapies to treat H. pylori infection. As demonstrated by a meta-analysis of published data, susceptibility or resistance to clarithromycin result in eradication rates of 81 to 95% and 0 to 48%, respectively (8). Since clarithromycin is a widely used antimicrobial drug, the prevalence of clarithromycin-resistant H. pylori strains is increasing continuously.Resistance of H. pylori to clarithromycin is mainly due to an adenine-to-guanine transition at positions 2142 and 2143 and to an adenine-to-cytosine transversion at position 2142, which are included in the peptidyltransferase loop of the 23S rRNA (24). Recently, several PCR based methods, such as PCR-restriction fragment length polymorphism (17), PCR-DNA-enzyme immunoassay (13), reverse hybridization line probe assay (22), and real-time PCR...

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Desmin ful¢ls important functions in maintenance of muscle cells and mutations in the desmin gene have been linked to a variety of myopathies. To ascertain the role of desmin's amino-terminal domain in muscle cells we generated embryonic stem cells constitutively expressing desmin v1À48 in a null background and investigated muscle cell development in vitro. Desmin v1À48 lacking the ¢rst 48 amino acid residues promotes fusion of myoblasts, rescues myogenesis and down-regulates vimentin expression in embryoid bodies, but hampers cardiomyogenesis and blocks smooth muscle development. These results demonstrate that desmin's amino-terminus has di¡erent roles in skeletal, cardiac, and smooth muscle cell development and function. ß