Comparison of real-time PCR, “in house” PCR, enzyme immunoassay and toxigenic culture in diagnosis of Clostridium difficile infection

Abstract

Background: The aim of the present study was to compare the performance of a commercial real-time PCR (RT-PCR) assay, a characterized in house approach and an enzyme immunoassay (EIA) on set of stool samples from patients with suspected Clostridium difficile infection (CDI) during the period January-May 2017. Methods & Materials: A total of 97 consecutive feces samples were obtained from hospitalized patients with suspected CDI. All specimens were processed as follows: 1) culture in anaerobic atmosphere on CHROMagar™C.difficille medium. Colonies suspected to be C. difficile were confirmed by MALDI-TOF (Bruker). Toxin production was tested by EIA and PCR 2) EIA using kit C.Diff-Quick-Check-Complete for the detection of glutamate dehydrogenase (GDH), toxin (Tx) A and B 3) RT-PCR using the LightCycler. This PCR detect the presence of the tcdC gene, as well as the 18-bp deletions which have been associated with the “epidemic strain” ribotype 027 4)“in-house” PCR targeting the C. difficile toxin gene tcdB. Results: Toxigenic C. difficile was isolated in 14 of the 97 specimens (14,4%). The RT-PCR and “in-house” PCR had a concordance of 92,9% (13/14) and 100% (14/14) with toxigenic culture, respectively. 18 bp deletions in the tcdC gene were detected in 2 of the 13 positive samples using RT-PCR by melting curve analysis. Six samples were positive for GDH and Tx (Ag + Tx+), “in-house” PCR and toxigenic culture. Of 15 positive GDH and negative Tx (Ag + Tx-), 7 were positive by both PCRs and toxigenic culture. Non-toxigenic C. difficile was isolated in 8 samples (Ag + Tx-) and negative PCR. All specimens which were negative by culture were also negative by PCR. Using toxigenic culture as the “gold standard”, the sensitivities, specificities, and positive and negative predictive values, respectively, of the assays were 42,9%, 90,4%, 42,9%, and 90,4% for the EIA; 92,9%, 100%, 100%, and 98,8% for the RT-PCR; and 100%, 100%, 100%, and 100% for the “in-house” PCR. Conclusion: According to results, the molecular methods have an sensitivity and specificity higher than the EAI. Nevertheless, EIAs are still very useful for screening due to the high negative predictive value. For inconclusive results (Ag + Tx-) we recommend performing a molecular method.