Comparison of Enzyme Immunoassay (EIA), Glutamate Dehydrogenase (GDH), and Cytotoxin Assays to Toxigenic Culture for the Detection of Clostridium difficile in Stool Samples

Background: C. difficile (Cdif) is the most frequent pathogen of infectious diarrhea in hospitalized patients. Diagnosis currently depends upon EIA detection of toxins A or A and B in stool produced by pathogenic strains. GDH common antigen assays are often used as a screening assay for Cdif before toxin testing.

Objective:

This study compared the performance of 4 toxin EIAs, 2 GDH assays, and tissue culture cytotoxin versus toxigenic culture for the detection of toxigenic Cdif.

Methods:

A total of 350 liquid or semi-liquid stool samples were tested that were collected for routine Cdif testing. EIA tests included Wampole^TM C.difficile TOX A/B II (EIA1), Meridian Immunocard Toxins A&B (EIA2), Meridian Primer Toxins A&B (EIA3), and TechLab C. diff Quick Check Complete^TM (EIA4). GDH assays used were TechLab C. diff Quick Check Complete^TM (GDH1) and Wampole^TM C. diff Check^TM-60 (GDH2). EIA4 and GDH1 are available as a single assay that combines both tests for the detection of Cdif antigen and toxins. Testing and interpretation for all EIA and GDH assays were performed according to the package insert. The cytotoxin assay was performed with the Clostridium difficile Toxin/Antitoxin Kit (TechLab). Samples were processed according to the package insert, followed by inoculation onto MRC-5 cells (ViroMed). Cytopathic effects on MRC-5 were examined after 24 and 48 hours. Anaerobic culture was performed with pre-reduced cycloserine-cefoxitin-fructose agar (CCFA-VA formulation; Remel, Lenexa, KS) media. Each sample was inoculated into Cycloserine Cefoxitin Mannitol Broth with Tauocholate Lysozyme Cysteine (CCMB-TAL) for enriched culture. Plates and broths were incubated anaerobically at 35^◦C. After 48 hrs the plates were examined, suspicious colonies were confirmed by Gram stain, aerotolerance and a Pro-disk test (Key Scientific). CCMB-TAL broths were plated to CCFA-VA only if the original culture was negative. All Cdif isolates were tested for toxigenicity by growing in anaerobic chopped meat broth (Remel) and performing toxigenicity with the TechLab C. difficile Toxin/Antitoxin Kit. A second culture method (Stamper et al, J. Clin. Microbiol 47:373, 2009) was used to resolve discrepancies.

Results:

The sensitivity and specificity for all tests compared to toxigenic culture were: EIA1 52.5% and 99%; EIA2 57.5% and 99.7%; EIA3 42.5% and 99.4%; EIA4 52.5% and 99%; GDH1 92.5% and 95.5%; GDH2 75% and 95.5%; cytotoxin assay 55% and 99.7% respectively.

Conclusions:

Other than GDH1, the rapid detection assays tested remain insensitive. Both GDH assays had inferior specificity. It appears none of these tests should be considered as reliable assays in the diagnosis of Cdif infection (CDI).