A variety of tests is available for detection of C. difficile
toxins, but a lack of guidelines for appropriate use can lead to difficulties in choice of test and to uncertain outcomes. CNA is generally considered the gold standard for diagnosis of CDI but is not performed by most laboratories; the assay is tedious, requires tissue culture facilities, may lack sensitivity under some conditions, and is difficult to standardize. An assay for GDH-Ag has a high false-positive rate, perhaps due to detection of nontoxigenic C. difficile
). Bacteriologic culture is strongly recommended by some authors, and its advantages include availability of isolates for determination of toxinogenesis (toxigenic culture), more effective study of epidemiology, and determination of antimicrobial susceptibility (11
). EIA for TcdA and/or TcdB is preferred in most laboratories because a positive test provides indirect evidence that toxigenic C. difficile
is in stool specimens; the EIA is readily available and less time-consuming than are CNA and C. difficile
One or two of these methods are usually selected as diagnostic tools for CDI, depending on the situation in each laboratory. Results of a European survey revealed that 93% of laboratories undertook direct detection of toxins in stool specimens and ~80% used a commercial EIA; 41.6% of the laboratories used both culture and toxin detection (7
). A two-step algorithm (GDH-Ag EIA and CNA) was suggested to enhance detection rates (34
). It has several advantages, but also limitations, in that routine medical laboratories may not be equipped for cell culture and the GDH-Ag test does not detect TcdA or TcdB.
Therefore, we established and evaluated a three-step algorithm based upon bacteriologic culture and detection of TcdA and TcdB (in stool specimens and by isolates) by EIA, which makes it more accessible to most laboratories. This allows direct detection of toxin in stool specimens and reliable ruling out of CDI.
In fact, variant strains of C. difficile
have been described with increasing prevalence worldwide, ranging from 0.2 to 56% in studies from the United States, Europe, and Asia (6
), and many recent studies have revealed that TcdA− TcdB+ strains are involved in a wide spectrum of CDI, ranging from colonization to uncomplicated diarrhea to pseudomembranous colitis (1
). TcdA+ TcdB− strains have been reported only rarely worldwide. However, one case was reported (10
), and in this work, we encountered two cases (0.5%) involving this strain type. Therefore, a diagnostic method capable of detecting both TcdA and TcdB is likely the best choice, owing to the high prevalence of TcdA− TcdB+ strains in many countries and the possible emergence of TcdB− strains.
In our study, 59 (67.0%) and eight (9.1%) strains among 88 cases yielding tcdAv/tcdB-positive, tcdA-negative/tcdB-positive, and tcdA-positive/tcdB-negative C. difficile strains yielded positive and equivocal ELFA results, respectively. These may have been missed had we used a method detecting TcdA or TcdB alone, although 23.9% (21/88) of these variant strains were not detected with ELFA.
The sensitivities of toxin EIAs are reportedly variable, ranging from 55% to >90% (2
). The limited sensitivity of both toxin EIA and CNA might be due to inhibitors in stools, by variable amounts of toxins in stools, and by instability of products and procedures (11
). In this work, sensitivity of the dual-toxin EIA was not so high (63.3%). However, the rate of case detection (positive predictive value) via our three-step algorithm (using culture and ELFA) was higher than with another such algorithm, which was based upon detection of GDH-Ag and CNA (95.5% versus 82.1%). There was no significant difference in specificity between the GDH-Ag/CNA algorithm and our ELFA/culture algorithm. Moreover, the processing time with our algorithm (2 to 3 days) was comparable to that of the earlier one.
Culture also allowed identification of additional ELFA-positive cases that would otherwise have been missed, through application of ELFA to toxigenic culture (5
). In our study, 98 (60.1%) of 163 toxigenic culture cases were tcdA
positive. Among them, 69 strains were ELFA positive (n
= 51) or equivocal (n
= 18), respectively. These may have been missed had we not used toxigenic culture, although 31.6% (31/98) of them were not detected with ELFA.
Interpretation of equivocal ELFA results is not part of the manufacturer's instructions, in contrast to the toxin A detection kit (CDA2, Vidas; bioMérieux, France), but isolates in 86.4% (51/59) of culture-positive/ELFA-equivocal cases (including ELFA-equivocal cases of toxigenic culture) were PCR positive for tcdA and tcdB. Thus, if C. difficile culture is positive, ELFA-equivocal results can likely be interpreted as toxin positive. Therefore, if we regarded equivocal ELFA results as positive, the sensitivity of the three-step algorithm would increase to 84.8% (307/362).
Positivity rates in the EIA were highly associated with the outcome of semiquantitative culture—the higher the grade, the higher the positivity rates in the ELFA (chi-square test for trend, P < 0.0001). This suggests that detection rates in the EIA depend on the quantity of TcdA or TcdB produced by C. difficile, and a false-negative EIA result is associated, at least in part, with lower numbers of C. difficile isolates (and, thus, its toxins) in stool. The manufacturer suggests that the limit of detection in the ELFA is 3 ng/ml of TcdA and 1 ng/ml of TcdB, and samples in which these limits were not reached would be reported as negative. However, culture positivity in EIA-negative stools, with subsequent demonstration of tcdA and/or tcdB by a PCR assay, may increase sensitivity. Culture was the most sensitive method for detection of toxigenic C. difficile, but it also missed 7.2% (26/362) of hypothetical positive cases. The prevalence of such cases was very low (3.0%), but it suggested that ELFA-positive or -equivocal/culture-negative cases may be associated with CDI.
In conclusion, we recommended a three-step algorithm comprising EIA (detecting TcdA and TcdB) and toxigenic bacteriologic culture. This approach, which is supported by good positive and negative predictive values, allows direct and rapid assay for toxins and toxigenic C. difficile in stool specimens to confirm or rule out CDI in a reasonable time period. The method is readily accessible for routine microbiology laboratories, and the availability of C. difficile isolates will facilitate epidemiologic study and determination of antimicrobial susceptibility.