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

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Abstract
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.

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