Comparison of antigen and two molecular methods for the detection of Clostridium difficile toxins.

BACKGROUND: Clostridium difficile (CD) is considered an important cause of diarrhoea associated with the antimicrobial treatment of infections. The pathogenicity of CD is due to toxins A and B, produced by toxigenic CD strains. METHODS: We evaluated 3 methods for detecting CD toxins: the RIDASCREEN® enzyme immunoassay (EIA) (R-Biopharm)--one detecting toxins directly in the stool specimens and another detecting toxins from isolated CD strains--and 2 molecular methods, the illumigene™ loop-mediated isothermal amplification (LAMP) assay (Meridian) and RIDA®GENE polymerase chain reaction (PCR) assay (R-Biopharm), as direct identification methods from stool specimens. Toxigenic culture (TC) was used as the reference method. RESULTS: Altogether 884 stool samples were analyzed, of which 253 (29%) were positive by TC. Six hundred and seventy-two specimens were tested by RIDASCREEN EIA, 430 were tested with the illumigene LAMP assay, and 212 were tested with the RIDA GENE PCR assay. CD toxin A and B antigen tests by EIA were very insensitive, both directly from stool specimens (2 series; 57-61%) and in isolated CD strains (53%); consequently the negative predictive value remained low (84-93% and 91%, respectively). Specificity, however, was very good at 98-100%. The 2 molecular methods detected CD toxin genes excellently and equally, resulting in sensitivities, specificities, and positive and negative predictive values of 98%, 100%, 100%, and 98%, respectively. CONCLUSIONS: Both molecular assays were easy to use, rapid, sensitive, and specific for the detection of toxigenic CD strains.

Proteome and cytoskeleton responses in osteosarcoma cells with reduced OXPHOS activity.

We have recently shown disorganization of the vimentin network in cultured cells deficient in oxidative phosphorylation (OXPHOS). We describe here the cellular responses to OXPHOS deficiency in osteosarcoma cells upon complex I (CI) and complex IV (CIV) inhibition, and upon the lack of mitochondrial DNA (rho0 cells). We examined the cytoskeletal organization and the distribution of mitochondria and analysed total proteome by 2-DE and vimentin expression by ELISA. Upon CIV inhibition and in rho0 cells, the vimentin network had collapsed around the nucleus and formed thick bundles. The mitochondria formed a perinuclear crescent upon CIV inhibition, whereas they accumulated around the nucleus in the rho0 cells, where the amount of vimentin was increased. Analysis of the total proteome revealed that a lack of mitochondrial DNA or inhibition of CI or CIV led to changes in the expression of cytoskeletal and cytoskeleton-associated proteins and proteins involved in apoptosis, OXPHOS, glycolysis, the tricarboxylic acid cycle, and oxidative stress responses. Our findings suggest that a deficiency in the energy converting system and oxidative stress can lead to cytoskeletal changes.