Systemic antibody response to Clostridium difficile in colonized patients with and without symptoms and matched controls

1 Centre for Infectious Diseases, University of Edinburgh College of Medicine and Veterinary Medicine, The Chancellor's Building, 49 Little France Crescent, Edinburgh EH16 4SB, UK 2 Geriatric Medicine Unit, University of Edinburgh College of Medicine and Veterinary Medicine, Edinburgh EH16 4SB, UK Correspondence Ian R. Poxton i.r.poxton{at}ed.ac.uk Received 23 October 2007 Accepted 30 January 2008 It has been proposed that patients who develop Clostridium difficile -associated disease (CDAD) do so because they are unable to mount an adequate immune response. Serum was collected from three groups of elderly in-patients: (i) cases ( n =21) of CDAD, being toxin A/B-positive; (ii) carriers ( n =21) asymptomatic for CDAD (no diarrhoea) but at least toxin or culture positive; and (iii) controls ( n =26) asymptomatic for CDAD and negative for both C. difficile toxin and culture. The age and gender of each group were compared, and the colonizing strains were ribotyped and toxinotyped. Serum antibodies (IgG and IgM) were measured by ELISA using different antigen preparations: EDTA extract (containing cell-surface proteins and carbohydrates), guanidine hydrochloride extract (surface-layer proteins), aqueous phenol-extracted lipocarbohydrate (LC); crude toxin (dialysis culture supernatant) and purified toxin A. LPS from Escherichia coli was used as a control antigen. Antibodies were also tested for toxin neutralization on tissue monolayers and for binding to EDTA-extracted antigens by Western blotting. IgG antibody measurements to cytomegalovirus (CMV) were included as an indicator of potential immunosenescence. Results showed that the patient groups were well matched by age and gender, and the colonizing strains were similar in cases and carriers, being predominantly ribotype 001 and toxinotype 0. By ELISA, IgG levels to most of the antigens were highest in the cases and lowest in the controls, with the exception of antibodies to the LC, which were higher in the controls than the cases. Levels in the carriers tended to be of intermediate level or similar to the controls. For all antigens, the levels of IgM were not significantly different among cases, carriers and controls. Serum from all groups was able to neutralize the cytotoxic action of toxin on both Vero and Caco2 cells, and all to a similar extent. Western blots showed an overall higher level of IgG antibodies to the EDTA-extracted antigens in the cases. The results of the CMV ELISA showed that specific IgG w