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C difficile is not another GI pathogen

3m Read

April 27, 2026

ANTIMICROBIAL STEWARDSHIP

Expert Perspective

C. difficile is not another GI pathogen: Why Testing Strategy matters

Editor’s note

This article summarizes content presented during Cepheid’s EU Excellence Champions Club, a scientific and educational forum that brings together leading laboratory and clinical experts from across Europe to exchange perspectives, share best practices, and explore the future of diagnostics.

The views expressed are those of the presenter(s) and may not represent the views of Cepheid.

Why we're sharing these insights

Our goal is to make the scientific discussions and real-world experiences shared during the Champions Club accessible to a broader audience, fostering continued learning and dialogue across the diagnostics community

In hospitals across Europe, Clostridioides difficile infection (CDI) remains a persistent and costly challenge. Despite decades of clinical experience and increasingly sophisticated diagnostics, accurately identifying those who truly have CDI and who does not remain a challenge for clinicians and microbiology laboratories alike. At the heart of the issue lies a fundamental truth: diagnosing CDI is not a single test decision, but a clinical process that demands nuance, context, and stewardship 1,2.

Recent discussions show that even well-intentioned diagnostic strategies can lead to both underdiagnosis and overdiagnosis, directly impacting patient outcomes, infection control, and antibiotic use1.

When sensitivity is not the whole story

Molecular tests, particularly NAATs, have transformed CDI diagnostics. NAATs offer greater sensitivity  than toxin enzyme immunoassays or GDH screening, making them central to many European testing algorithms3. However, factors beyond sensitivity also require attention.

Sensitivity alone does not guarantee clinical certainty. A positive molecular result confirms toxigenic C. difficile but does not always indicate active infection. Asymptomatic colonization is common in healthcare settings. Without careful interpretation, sensitive tests may identify the organism even when it is not causing disease4,5.

The hidden pitfalls of multistep algorithms

To balance accuracy and practicality, many laboratories rely on multistep diagnostic pathways combining GDH, toxin testing, and NAATs. While these algorithms aim to improve predictive value, they introduce their own vulnerabilities 6,7.

GDH screening, for example, can produce both false negatives and false positives. Some Clostridium species and even unrelated organisms may cross‑react, while certain toxigenic strains may be missed entirely. Toxin assays, meanwhile, are prone to variability and can generate misleading results when used in isolation6,8.

Certain strains challenge traditional assumptions. Some C. difficile isolates lack toxins A or B but carry binary toxin genes. Others, including epidemic ribotypes linked to severe disease, have genetic deletions that affect toxin regulation. Without accounting for these nuances, diagnostic tools may miss or delayed clinically significant cases 6,9,10.

What real-world evidence is telling us

Emerging clinical data are reshaping how laboratories think about CDI testing strategies. Studies evaluating reverse two-step algorithms and standalone NAAT approaches show tangible benefits: fewer hospital onset CDI cases, reduced antibiotic exposure, and more consistent infection control practices5,11.

Building on these insights, a notable finding is that some NAAT-positive patients may test negative by GDH screening. Clinically, using GDH alone as a gatekeeper may result in missed diagnoses, allowing unidentified cases to contribute to hospital transmission5.

Conversely, treating every molecular positive without considering symptoms or risk factors risks unnecessary antibiotic use. This reinforces a central message: laboratory results must be interpreted through a clinical lens, not in isolation4,12.

The stewardship imperative

As diagnostics become more powerful, the responsibility to use them wisely grows. Diagnostic stewardship, the principle of ordering the right test for the right patient at the right time, is now recognized as an essential partner to antimicrobial stewardship1,2.

In CDI, stewardship means resisting reflex testing in low probability situations, avoiding repeat testing without clinical justification, and embedding decision support into ordering workflows. Evidence from hospitals implementing stewardship have been associated with reductions in unnecessary testing and false‑positive results without clear evidence of increased missed diagnoses.13,14.

This is particularly relevant with the growing use of multiplex gastrointestinal panels. While these tools can rapidly identify a wide range of pathogens, they also increase the likelihood of detecting C. difficile colonization rather than infection. Studies indicate that only a minority of panel positive results ultimately represent true CDI, yet many patients still receive treatment5,15.

Moving forward: integration, not isolation

The future of CDI diagnosis does not lie in a single “perfect” test. Instead, it depends on integrating advanced molecular diagnostics with clear institutional policies, clinical judgment, and stewardship frameworks 1,16.

Laboratories and clinicians must work together to define when testing is appropriate, how results should be interpreted, and how diagnostic pathways align with patient management goals. This integrated approach helps avoid harm, reduces unnecessary antibiotic exposure, and supports better infection control decisions2.

Ultimately, improving CDI diagnosis is not just about detecting bacteria. It is about understanding disease. And in an era of powerful diagnostics, that understanding has never mattered more1.

CE-IVD. In vitro Diagnostic Medical Device. May not be available in all countries.

The content presented on this page is intended for informational and educational purposes. While it is available globally, it may reflect clinical practices or healthcare system considerations specific to a particular region.

References:

1.     Messacar K, Parker SK, Todd JK, Dominguez SR. Implementation of rapid molecular infectious disease diagnostics: the role of diagnostic and antimicrobial stewardship. J Clin Microbiol. 2017;55(3):715-723. doi:10.1128/JCM.02264-16. Available from: ASM Journal Article

2.     Dumm RE, Marlowe EM, Patterson L, Larkin PMK, She RC, Filkins LM. The foundation for the microbiology laboratory’s essential role in diagnostic stewardship: an ASM Laboratory Practices Subcommittee report. J Clin Microbiol. 2024 Oct 16;62(10):e00960-24. doi:10.1128/JCM.00960-24. Available from: https://journals.asm.org/doi/10.1128/jcm.00960-24

3.     Crobach MJT, Planche T, Eckert C, Barbut F, Terveer EM, Dekkers OM, et al. European Society of Clinical Microbiology and Infectious Diseases: update of the diagnostic guidance document for Clostridium difficile infection. Clin Microbiol Infect. 2016;22 Suppl 4:S63-81. doi:10.1016/j.cmi.2016.03.010. Available from: Clinical Microbiology and Infection article

4.     Prosty C, Hanula R, Katergi K, Longtin Y, McDonald EG, Lee TC. Clinical outcomes and management of NAAT-positive/toxin-negative Clostridioides difficile infection: a systematic review and meta-analysis. Clin Infect Dis. 2024;78(2):430-438. doi:10.1093/cid/ciad523. Available from: Clinical Infectious Diseases article

5.     Pender M, Throneberry SK, Grisel N, Leung DT, Lopansri BK. Syndromic panel testing among patients with infectious diarrhea: the challenge of interpreting Clostridioides difficile positivity on a multiplex molecular panel. Open Forum Infect Dis. 2023;10(5):ofad184. doi:10.1093/ofid/ofad184. Available from: Open Forum Infectious Diseases article

6.     Krutova M, Wilcox MH, Kuijper EJ. The pitfalls of laboratory diagnostics of Clostridium difficile infection. Clin Microbiol Infect. 2018;24(7):682-683. doi:10.1016/j.cmi.2018.02.026. Available from: Clinical Microbiology and Infection article

7.     Gateau C, Couturier J, Coia J, Barbut F. How to: diagnose infection caused by Clostridium difficile. Clin Microbiol Infect. 2018;24(5):463-468. doi:10.1016/j.cmi.2017.12.005. Available from: Clinical Microbiology and Infection article

8.     Kalacheva A, Popov M, Velev V, Stoyanova R, Mitova-Mineva Y, Velikova T, et al. Potential for misinterpretation in the laboratory diagnosis of Clostridioides difficile infections. Diagnostics (Basel). 2025;15(9):1166. doi:10.3390/diagnostics15091166. Available from: Diagnostics article

9.     Androga GO, Hart J, Foster NF, Charles A, Forbes D, Riley TV. Infection with toxin A-negative, toxin B-negative, binary toxin-positive Clostridium difficile in a young patient with ulcerative colitis. J Clin Microbiol. 2015;53(11):3702-3704. doi:10.1128/JCM.01810-15. Available from: Journal of Clinical Microbiology article

10.  Eckert C, Emirian A, Le Monnier A, Cathala L, De Montclos H, Goret J, et al. Prevalence and pathogenicity of binary toxin-positive Clostridium difficile strains that do not produce toxins A and B. New Microbes New Infect. 2015;3:12-17. doi:10.1016/j.nmni.2014.10.003. Available from: PMC Full Text Article

11.  Casari E, De Luca C, Calabrò M, Scuderi C, Daleno C, Ferrario A. Reducing rates of Clostridium difficile infection by switching to a stand-alone NAAT with clear sampling criteria. Antimicrob Resist Infect Control. 2018;7(1):1-4. doi:10.1186/s13756-018-0332-2. Available from: Antimicrobial Resistance & Infection Control article

12.  Tansarli GS, Falagas ME, Fang FC. Clinical significance of toxin EIA positivity in patients with suspected Clostridioides difficile infection: systematic review and meta-analysis. J Clin Microbiol. 2025;63(1):e00977-24. doi:10.1128/JCM.00977-24. Available from: Journal of Clinical Microbiology article

 

13.  Hitchcock MM, Gomez CA, Pozdol J, Banaei N. Effective approaches to diagnostic stewardship of syndromic molecular panels. J Appl Lab Med. 2024;9(1):104-115. doi:10.1093/jalm/jfad063. Available from: Journal of Applied Laboratory Medicine article

14.  Christensen AB, Barr VO, Martin DW, Anderson MM, Gibson AK, Hoff BM, et al. Diagnostic stewardship of C. difficile testing: a quasi-experimental antimicrobial stewardship study. Infect Control Hosp Epidemiol. 2019;40(3):269-275. doi:10.1017/ICE.2018.336. Available from: Infection Control & Hospital Epidemiology article

15.   lges D, Graf EH, Grant L, Long A, Siebeneck E, Seville MT, et al. Positive impact of a diagnostic stewardship intervention on syndromic panel ordering practices and inappropriate C. difficile treatment. Infect Control Hosp Epidemiol. 2025;46(1):71-76. doi:10.1017/ICE.2024.180. Available from: Infection Control & Hospital Epidemiology article

16.  Skinner AM, Guh AY, Petrella LA, Sambol S, Cheknis A, Johnson S, et al. Clostridioides difficile clinical diagnostic test methods and results are associated with recovery of C. difficile by stool culture. Microbiol Spectr. 2026;14(2):e03408-25. doi:10.1128/spectrum.03408-25. Available from: Microbiology Spectrum article

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