Effective Detection of Toxigenic Clostridium difficile by a Two-Step Algorithm Including Tests for Antigen and Cytotoxin

Abstract

We evaluated a two-step algorithm for detecting toxigenic Clostridium difficile: an enzyme immunoassay for glutamate dehydrogenase antigen (Ag-EIA) and then, for antigen-positive specimens, a concurrent cell culture cytotoxicity neutralization assay (CCNA). Antigen-negative results were >99% predictive of CCNA negativity. Because the Ag-EIA reduced cell culture workload by Ϸ75 to 80% and two-step testing was complete in <3 days, we decided that this algorithm would be effective. Over 6 months, our laboratories' expenses were US$143,000 less than if CCNA alone had been performed on all 5,887 specimens.Clostridium difficile-associated diarrhea is an important illness among patients who are extensively treated with antibacterial or other chemotherapeutic agents (4,7,19). While definitive evidence of toxigenic C. difficile comes from microbiologic testing, laboratories are challenged to provide accurate results rapidly and cost-effectively (23). Cell culture assays for cytotoxin (toxin B) are considered the gold standard but require up to 4 days for results, expensive cells and media, and labor-intensive expertise (4,17,19).Consequently, many laboratories use immunoassays for C. difficile toxins, or "common" glutamate dehydrogenase antigen (2,6,13,16,19,21,23,24). Toxin enzyme immunoassays (EIAs) are frequently used as stand-alone assays but clinical sensitivity may be suboptimal, particularly if only toxin A is detected (4,6,8,9,12,19). Current antigen EIAs (Ag-EIAs) accurately detect an essential and constitutively synthesized enzyme (23), thereby rapidly identifying C. difficile while overcoming the low sensitivity of toxin EIAs and suboptimal performance of older, latex-agglutination antigen assays. Because Ag-EIAs detect nontoxigenic as well as toxigenic C. difficile, however, they must be used in combination with a toxin-detecting assay to provide specific laboratory evidence of C. difficile-associated diarrhea (2,3,10,11,16,19,(21)(22)(23)(24).Both of our institutions' C. difficile-testing laboratories had adopted a stand-alone EIA approach for detecting toxins A and B, using C. DIFFICILE TOX A/B II (ToxAB-EIA; TechLab, Blacksburg, Va.; distributed by Wampole Laboratories, Princeton, N.J.). These laboratories are in acute-care hospitals: one in the 900-bed Johns Hopkins Hospital (JHH), which also serves the 190-bed acute-care Howard County General Hospital in Columbia, Md.; the other is at the 350-bed Johns Hopkins Bayview Medical Center (JHBMC) and serves the 220-bed Johns Hopkins Care Center, a colocated facility for subacute and long-term care. During late 2003, it became apparent that the sensitivity of the ToxAB-EIA was unacceptably low at JHH (see below for analogous JHBMC data from 2004). After determining that the performance of similar assays was inadequate (unpublished data; e.g., 71% sensitivity, 73% specificity, and 25% negative predictive value for Premier C. difficile Toxin AϩB [Meridian Diagnostics, Cincinnati, Ohio] versus cytotoxin testing for 63 specimens), we decided to develop an alternati...

Loop-Mediated Isothermal Amplification Compared to Real-Time PCR and Enzyme Immunoassay for Toxigenic Clostridium difficile Detection

“…They can detect glutamate dehydrogenase (GDH) (so-called common antigen) and/or major toxins A and B and are inexpensive, rapid, and easy to perform. A drawback of EIA toxin tests is a lack of sensitivity (8,9,13,15,16,20,22,27,30,32). Conversely, EIA GDH tests have good sensitivity but lack specificity, as they cannot distinguish toxigenic from nontoxigenic C. difficile (8, 13, 15, 23-25, 30, 32).…”

Laboratory Diagnosis of Clostridium difficile Infection

“…1,45 Because GDH assays are purported to be highly sensitive but not specific for toxin-producing C. difficile isolates, this assay is usually used to screen stool specimens as part of a two-or three-step algorithm. Data from recent studies 46,47 supported the use of a two-step approach, although another study 7 raised concerns about using EIA tests for toxins A and B as confirmatory tests for GDH-positive samples because of the low sensitivity of the toxin EIAs. The SHEA-IDSA guidelines recommend screening liquid stools using a GDH EIA test and confirming positive results with either CCCN or toxigenic culture.…”

Clinical update for the diagnosis and treatment ofClostridium difficileinfection

“…The EIA for toxin A/B has been adopted by most clinical laboratories because it is fast, convenient and inexpensive. Recent studies have shown however, that the sensitivity can be as low as 38% [55] . The EIA requires 100-1000 picograms of toxin as compared to the ability of the CCCNA to detect less than 10 picograms of toxin [53] .…”