Specific detection of toxigenic strains of Clostridium difficile in stool specimens

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Abstract
Clostridium difficile is the infectious agent responsible for antibiotic-associated colitis. We report the use of the polymerase chain reaction technique to identify toxigenic strains of C. difficile in human stool specimens. A set of primers based on the nucleotide sequence of the toxin B gene, which amplified a 399-bp fragment from isolates producing toxin B, was designed. We examined 28 known toxigenic strains, which were all positive by this assay. DNAs from the nontoxigenic strains examined and from strains of Clostridium sordellii and C. bifermentans were not amplified with these primers. The sensitivity of this assay allowed us to identify as little as 10% toxigenic C. difficile cells in the presence of 90% nontoxigenic cells and to detect the toxin B gene in 1 pg of DNA from a toxigenic strain. DNAs extracted from 18 clinical stool specimens that were positive for toxin B by the tissue culture cytotoxicity assay were also positive by this assay. In addition, we detected toxin B sequences in DNA from 2 of 18 stool specimens that were negative for toxin B by the cytotoxicity assay. These two stool specimens were from patients who had a clinical pattern of colitis that was compatible with C. difficile causation. This rapid, sensitive assay will be useful for specific identification of toxigenic C. difficile and for revealing cases that are undetected by analysis of fecal samples for toxin B alone.