Correlation between Clostridium difficile Bacterial Load, Commercial Real-time PCR Cycle Thresholds, and Results of Diagnostic Tests Based on Enzyme Immunoassay and Cell-Culture Cytotoxicity Assay

Background: The impact of C. difficile fecal load on diagnostic test results is not deeply understood but could have clinical importance. Methods: We investigated the relationship between C. difficile fecal bacterial load and four assays: glutamate deshydrogenase (GDH) enzyme immunoassay (EIA), toxin A/B antigen EIA (ToxAB), cell-culture cytotoxicity assay (CCA) and PCR detecting tcdB gene. We compared the median fecal load with the results from these assays. We also compared PCR cycle threshold (CT) with the result of quantitative culture using Spearman’s rank correlation coefficient. Finally, we sequenced the genome of 24 strains with different detection profiles. Results: 203 clinical samples harboring toxigenic C. difficile were analysed and sorted into four groups: 17 PCR+ (group 1), 37 PCR+GDH+ (group 2), 24 PCR+GDH+CCA+ (group 3) and 125 PCR+GDH+ToxAB+ (group 4). The overall median fecal load in log10 CFU/g was 6,67 (Interquartile range [IQR], 5,57 to 7,54). The median fecal bacterial load of groups 1, 2, 3 and 4 were as follows: 4,15 (IQR, 3,00 to 4,98), 5,74 (IQR, 4,75 to 6,16), 6,20 (IQR, 5,23-6,80) and 7,08 (IQR, 6,35-7,83). Group 1 samples had lower fecal loads than each of the other groups (p<0,001). Group 2 samples had lower fecal loads than group 3 and 4 (p<0,001). There was a significant correlation between PCR CT and bacterial loads (Spearman p= -0,697; p<0,001). NAP1 strains were associated to the detection of toxins.