Molecular detection of toxigenic C difficile: Toxin A or B gene?

Clostridium difficile infections (CDIs) are the leading cause of hospital-acquired diarrhea, primarily affecting patients exposed to antibiotics. Infections have been associated with prolonged hospital stays and significant healthcare costs. Recent changes in the epidemiology of CDI include increased incidence and disease severity, partly due to the emergence of the (BI/NAP1/027) strain which is now endemic in many U.S. hospitals.¹ Clinicians have also observed increased infections in previously low-risk populations and cases of community-acquired CDI in the absence of traditional risk factors.

Only toxin-producing C diff strains cause disease and toxins A and B (encoded by the tcdA and tcdB genes) appear to play important roles. The toxins are pro-inflammatory enterotoxins, but toxin B is a more potent cytotoxin.² Direct stool cytotoxicity, which detects toxin B, was the first clinically useful diagnostic assay to be developed. In the early 1990s, rapid enzyme immunoassays (EIAs) were developed to detect toxin A which is more immunogenic than toxin B. Many labs adopted these assays because they were convenient and easier to use.

In 2000, outbreaks of severe cases of CDI due to strains that lack toxin A (A-B+ variants) were the first clue that toxin A was not essential for clinical disease.^3,4 CDI patients harboring these variant strains were misdiagnosed because their stool samples were negative by toxin A-specific EIAs.³ In the absence of appropriate treatment, some patients developed severe CDI complications with poor outcomes, including death.^3,4 In response to the emergence of toxin A-negative variant C diff strains, kit manufacturers developed new EIAs that also detected toxin B because it was no longer acceptable to detect toxin A alone.

The majority of disease-causing toxigenic C diff produce both toxins. Toxin A-B+ strains, however, account for 2% to 11% of CDI cases⁵ with higher estimates in Ireland⁶ and Asia.⁷ These strains, which are characterized as having large deletions in the tcdA gene cause the same disease spectrum as those producing both toxins, ranging from mild diarrhea to pseudomembranous colitis.⁴ Some reports suggest, however, that disease caused by toxin A-B+ strains is more likely to be severe.⁷

In contrast, reports of naturally occurring disease-causing strains that do not produce toxin B (toxin A+B- variants) are extremely rare. The only report in the literature of infection with an A+B- strain was a patient with recurrent CDI, from whom C diff isolates harboring both tcdA and tcdB genes were previously isolated.⁸ Stool samples from the patient during the prior diarrheal episodes were positive for toxin B by a cytotoxicity assay. An investigation of the A+B- strain from the third episode revealed a complete absence of the tcdB gene or other genes necessary for toxin production and a deletion in the tcdA gene.⁹ Futhermore, the A+B- variant failed to produce either toxin A or toxin B in vitro, bringing into question its relevance as the cause of the patient’s symptoms.

The ability to genetically engineer C diff that produces only toxin A (A+B- mutants) or toxin B (A-B+ mutants) and infects susceptible hamsters with the mutants has led to two investigations on the individual importance of these toxins in the disease process. In one study, the authors concluded that toxin B is essential for disease, while toxin A is not required.¹⁰ The second study showed that either toxin was capable of causing disease, but that the mutant strains producing toxin B alone caused more severe disease.¹¹ While the two studies seem to contradict one another, the findings of Lyras, et al, are more consistent with what is observed in clinical practice to date, in that all clinically relevant C diff strains (including A+B+ and A-B+ variants) produce toxin B and the absence of naturally occurring disease-causing strains that produce only toxin A.

Laboratory testing for detection of toxigenic C diff to confirm a CDI diagnosis in patients remains a challenge due to the performance of commonly used toxin A/B EIAs or a turnaround times for more sensitive methods like toxigenic culture.¹² Rapid molecular testing for toxigenic C diff has significantly improved the accuracy with which CDI patients can be diagnosed and managed. With reliable sensitivities and specificities for toxigenic culture, clinicians can trust laboratory results that confirm their clinical suspicion of CDI or rule out C diff as the source a patient’s symptoms. According to the new SHEA/IDSA (Society for Healthcare Epidemiology of America and Infectious Diseases Society of America) clinical practice guidelines for CDI in adults, PCR testing appears to be rapid, sensitive, and specific and, ultimately, may address testing concerns.¹²

All FDA-cleared real-time polymerase chain reaction, or PCR, assays target the toxin B gene (tcdB). The choice of tcdB as a molecular target is ideal for several reasons: the essential role of toxin B in the disease process^10,11; the presence of toxin B and tcdB in all disease-producing strains; and evidence that detection of tcdB in symptomatic patients correlates well with accurate diagnosis of CDI.¹³

The rationale for other targets such as the tcdA gene is less clear. Many A-B+ variant C diff strains have deletions in the toxin A gene¹⁴ and may not be reliable as a target. Unlike the tcdB gene, studies demonstrating that detection of tcdA correlates with clinical disease are lacking; and because the gene may also be found alone in non-disease-causing strains,¹⁵ detection of tcdA could potentially lead to misdiagnosis of CDI.

CDI diagnosis requires a combination of clinical symptoms and accurate detection of toxigenic C diff in a symptomatic patient’s stool.¹² When used appropriately and limited to patients with symptoms consistent with clinical disease, PCR assays that target the tcdB gene may facilitate accurate diagnosis of CDI which, in turn, may lead to better patient management with appropriate therapy and prompt implementation of infection-control measures.

Diane Kawa, PhD, SM(ASCP),

is the director, Scientific Affairs at BD in Franklin Lakes, NJ.

References

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