Detection of Mixed Populations ofClostridium difficilefrom Symptomatic Patients Using Capillary-Based Polymerase Chain Reaction Ribotyping

Objective.To investigate the simultaneous occurrence of more than 1Clostridium difficileribotype in patients' stool samples at the time of diagnostic testing.Methods.Stool samples submitted for diagnostic testing for the presence of toxigenicC. difficilewere obtained for 102 unique patients. A total of 95 single colonies ofC. difficileper stool sample were isolated on selective media, subcultured alongside negative (uninoculated) controls, and polymerase chain reaction (PCR) ribotyped using capillary gel electrophoresis.Results.Capillary-based PCR ribotyping was successful for 9,335C. difficileisolates, yielding a median of 93 characterized isolates per stool sample (range, 69-95). More than 1 C.difficileribotype was present in 16 of 102 (16%)C. difficileinfection (CDI) cases; 2 of the 16 mixtures were composed of at least 3 ribotypes, while the remaining 14 were composed of at least 2.Conclusions.Deep sampling of patient stool samples coupled with capillary-based PCR ribotyping identified a high rate of mixed CDI cases compared with previous estimates. Studies seeking to quantify the clinical significance of particular C.difficileribotypes should account for mixed cases of disease.