Best Laboratory Practices for Clostridioides difficile Diagnosis in Adults

clostridium difficile
clostridium difficile
Repeat testing of nucleic acid amplification tests is not recommended for detection of the presence of Clostridioides (formerly Clostridium) difficile in adults.

Repeat testing of nucleic acid amplification tests (NAAT) is not recommended for detection of Clostridioides (formerly Clostridium) difficile presence in adults, according to review results published in Clinical Microbiology Reviews.

In the United States, C difficile infection is a leading cause of healthcare-associated infections with 453,000 documented cases and 29,000 deaths in 2015. Further, in all healthcare settings C difficile infection accounts for 15% to 25% of cases of healthcare-associated diarrhea. In combination with clinical presentation, several laboratory assays are available to support C difficile infection diagnoses including toxigenic cultures, cell cytotoxicity neutralization assays, enzyme immunoassays and immunochromatographic assays for the detection of glutamate dehydrogenase; toxin A, B, or both; and, in the last 10 years, NAATs.

The toxigenic culture and the cell cytotoxicity neutralization assay tests currently serve as reference methods for diagnosis of C difficile infection, but are both slow and labor-intensive. Although there is a wide variation of testing methods and diagnostic algorithms, it remains controversial which of these methods may be the gold standard for diagnosing C difficile infection in the laboratory. Further, it is unclear whether the potential differences in accuracy of NAAT only or an algorithmic strategy would affect patient management or outcomes. Therefore, this systematic review evaluated the diagnostic accuracy of laboratory testing algorithms that include NAATs to detect the presence of C difficile.

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In total, 238 studies were eligible and included population, intervention, comparison, and outcome factors and assessed the diagnostic accuracy of NAAT alone or following glutamate dehydrogenase enzyme immunoassays or glutamate dehydrogenase enzyme immunoassays plus C difficile toxin enzyme immunoassays. Of the eligible studies, 72 had sufficient data for meta-analysis. To perform the systematic review, 2 analytic approaches were used: qualitative determinations of overall strength of evidence and quantitative meta-analysis.

The meta-analysis results supported the use of testing algorithms that use NAAT alone or in combination with glutamate dehydrogenase or glutamate dehydrogenase plus toxin enzyme immunoassays to detect the presence of C difficile in adults. When comparing the sensitivities and specificities based on whether stool met the criteria reported in the studies, the NAAT-only studies had a sensitivity of P <.001 and a specificity of P <.001. The glutamate dehydrogenase/NAAT had a sensitivity of P=.02 and a specificity of P =.16 and the glutamate dehydrogenase/toxin/NAAT had a sensitivity of P <.001 and a specificity of P =.58.

In patients suspected of having C difficile infection, NAAT-only testing, glutamate dehydrogenase/NAAT algorithm, and glutamate dehydrogenase/toxin/NAAT are all recommended practice for detecting the C difficile toxin gene, organism/toxin gene, and organism/toxin/toxin gene, respectively. However, there is no recommendation for or against repeated testing by NAAT only within 7 days when the result is negative due to insufficient evidence. These results aligned with the recommendations of the American Society for Microbiology.

Overall, the study investigators concluded that, “Findings from this system[at]ic review provide clarity to diagnostic testing strategies and highlight gaps, such as low numbers of [glutamate dehydrogenase]/toxin/PCR studies, in existing evidence on diagnostic performance, which can be used to guide future clinical research studies.”


Kraft CS, Parrott JS, Cornish NE, et al. A laboratory medicine best practices systematic review and meta-analysis of nucleic acid amplification tests (NAATs) and algorithms including NAATs for the diagnosis of Clostridioides (Clostridium) difficile in adults [published online May 29, 2019]. Clin Microbiol Rev. doi:10.1128/CMR.00032-18