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Detection of Clostridium difficile in Fecal Specimens: a Comparative Evaluation of Nucleic Acid Amplification Test and Toxigenic Culture by Shima Lotfian, Masoumeh Douraghi, Amir Aliramezani, Sedighe Ghourchian, Abdolfatah Sarrafnejad, Hojjat Zeraati

Background: The available data regarding Clostridium difficile infections (CDIs) in developing countries are scarce. This may be related in part to the complexity of anaerobic bacterial culture and/or cytotoxicity assays of C. difficile. Here, we evaluated the diagnostic efficacy of PCR in comparison with toxigenic culture for direct detection of conserved genes as well as toxin genes of C. difficile in fecal specimens of patients with clinical symptoms of CDI.
Methods: Loose or soft feces from 171 patients suspected of having C. difficile associated diarrhea (CDAD) were subjected to DNA extraction, PCR of cdu-2, cdd-3, gdh, tpi, tcdA/B, and toxigenic culture (TC). Limit of detection (LoD) was defined as the lowest concentration of DNA at which the target gene was amplified via PCR. The Kappa agreement between two diagnostic tests was calculated.
Results: The in-house extraction method extracted DNA successfully as confirmed by amplification of conserved genes of C. difficile. LoD of PCR for total DNA was 0.064 ng/μL. Only 10 specimens were positive for C. difficile via both PCR and TC. Among 10 identified C. difficile strains, 8 were tcdA+B+, but 2 were tcdA-B+. A very good agreement was observed between TC as reference method and PCR (κ = 1).
Conclusions: Despite the high concordance between PCR and TC, this in-house nucleic acid amplification test can be used to identify symptomatic patients who harbor high amounts of bacteria. This procedure allows primary and same day diagnosis of C. difficile, and clinical laboratories in low-income countries may adopt the method for sample extraction and PCR assay at least for symptomatic patients.

DOI: 10.7754/Clin.Lab.2016.160112