A two-step algorithm for the detection of Clostridium difficile by the use of C. Diff Quik Chek (TechLab, Blacksburg, VA) and a tissue culture cytotoxicity neutralization assay was found to be more sensitive than the widely used solid-phase enzyme immunoassay (EIA), the Premier toxin A and B EIA (Meridian Bioscience, Cincinnati, OH), and a newly developed, rapid single-test EIA for C. difficile toxins A and B (Tox A/B Quik Chek; TechLab).The recent emergence of a highly virulent strain of Clostridium difficile, with its associated increased morbidity and increased mortality, has placed a renewed emphasis on the laboratory diagnosis of C. difficile disease (7, 10). For well over a decade, first the Premier toxin A enzyme immunoassay (EIA) and then the Premier toxin A and B EIA (A/B EIA; Meridian Bioscience, Inc., Cincinnati, OH) were used to detect this toxin (21). The latter is the most widely used test for the detection of C. difficile toxins in the United States (4). Recent studies (11,20) have shown that this assay has both excellent sensitivity (Ͼ90%) and excellent specificity (Ͼ95%) compared to the results of a tissue culture cytotoxicity neutralization (CTN) assay, an assay frequently considered the "gold standard" for the diagnosis of C. difficile disease (1,9,11,12,15,18,19,21). Other data suggest that the detection of toxigenic strains of C. difficile is actually a more sensitive approach to the diagnosis of C. difficile disease (5,6,13,14), but culture is infrequently done for diagnostic purposes in the United States because of its slow turnaround time and the need for special isolation media and technical expertise. In addition, the specificity of toxigenic culture for the diagnosis of C. difficile disease has also been questioned (1,14).Given the emergence of a highly virulent C. difficile strain in the United States, a recent report by Ticehurst and colleagues was a cause for concern (18). They showed that another widely used EIA, the C. difficile Tox A/B II assay (TechLab, Blacksburg, VA), which has performance characteristics similar to those of the A/B EIA (11,12,19), had a sensitivity of only 36% for C. difficile detection when the results were compared to those obtained by the use of a two-step algorithm in which specimens were first tested for the presence of the glutamate dehydrogenase (GDH) antigen by a solid-phase EIA (C. Diff Chek-60; TechLab) and the positive results were confirmed by the CTN assay. The test for the GDH antigen proved to be an excellent screening test, with a sensitivity of Ͼ95% and a negative predictive value of Ͼ99% compared with the results of the CTN assay (18). In practical terms, that meant that GDHnegative specimens could be reported to be negative without further testing. By using the CTN assay as a reference method, it was found that the assay for the GDH antigen had a positive predictive value of 53%, indicating that a confirmatory test was required to detect specimens containing C. difficile toxins.I applied the two-step algorithm approach using a modification ...