Nucleic Acid Amplification Test Quantitation as Predictor of Toxin Presence in Clostridium difficile Infection

Abstract

Multistep algorithmic testing in which a sensitive nucleic acid amplification test (NAAT) is followed by a specific toxin A and toxin B enzyme immunoassay (EIA) is among the most accurate methods for Clostridium difficile infection (CDI) diagnosis. The obvious shortcoming of this approach is that multiple tests must be performed to establish a CDI diagnosis, which may delay treatment. Therefore, we sought to determine whether a preliminary diagnosis could be made on the basis of the quantitative results of the first test in algorithmic testing, which provide a measure of organism burden. To do so, we retrospectively analyzed two large collections of samples (n = 2,669 and n = 1,718) that were submitted to the laboratories of two Dutch hospitals for CDI testing. Both hospitals apply a two-step testing algorithm in which a NAAT is followed by a toxin A/B EIA. Of all samples, 208 and 113 samples, respectively, tested positive by NAAT. Among these NAAT-positive samples, significantly lower mean quantification cycle (C_q ) values were found for patients whose stool eventually tested positive for toxin, compared with patients who tested negative for toxin (mean C_q values of 24.4 versus 30.4 and 26.8 versus 32.2; P < 0.001 for both cohorts). Receiver operating characteristic curve analysis was performed to investigate the ability of C_q values to predict toxin status and yielded areas under the curve of 0.826 and 0.854. Using the optimal C_q cutoff values, prediction of the eventual toxin A/B EIA results was accurate for 78.9% and 80.5% of samples, respectively. In conclusion, C_q values can serve as predictors of toxin status but, due to the suboptimal correlation between the two tests, additional toxin testing is still needed.

Keywords: Clostridium difficile; NAAT quantitation; diagnostic.

FIG 1

Flowchart of included samples. (Top) LUMC cohort. (Bottom) Amphia Hospital cohort. *, No culture was performed because a culture with positive results had been performed within the previous week. **, No culture was performed because a culture with positive results had been performed within the previous week (n = 1) or because positive TcdB results were obtained retrospectively during the implementation phase of the tcdB NAAT, when samples were routinely tested by toxin (Tox) A/B EIA only.

FIG 2

Dot plots of observed C_q values. (Top) LUMC cohort and asymptomatic carriers. (Bottom) Amphia Hospital cohort.

FIG 3

ROC curves assessing the ability of C_q values to predict the presence of toxin. Optimal cutoff values are shown in red. (Top) LUMC cohort. (Bottom) Amphia Hospital cohort.