What C. difficile Toxin Amplified Probe Tests Detect
Nucleic acid amplification tests (NAATs) for C. difficile detect the genes that encode toxin production—specifically the tcdB gene (toxin B) or tcdC gene (toxin regulator)—not the actual toxins themselves. 1
Target of Detection
NAATs identify the presence of toxin-encoding genes in the bacterial DNA, confirming the presence of toxigenic (toxin-producing) C. difficile strains in stool specimens. 1
The most commonly targeted gene is tcdB, which regulates toxin B production, though some assays target the tcdC gene, a putative negative regulator of toxin A and B production. 1
NAATs do NOT detect the actual toxin proteins (toxin A or toxin B) present in the stool—they only detect the genetic capacity to produce toxins. 1, 2
Critical Distinction: Gene Detection vs. Toxin Detection
The fundamental limitation of NAATs is their inability to distinguish between active infection and asymptomatic colonization, since they detect toxin genes regardless of whether toxins are actually being produced. 1, 3
Approximately 7% of asymptomatic hospitalized patients are colonized with toxigenic C. difficile, meaning they carry the organism with toxin genes but are not actively infected. 1
This explains why NAAT-positive results can lead to overdiagnosis and overtreatment when used as a standalone test, with overdiagnosis being more than three times more common in NAAT+/toxin- patients compared to NAAT+/toxin+ patients. 2
Performance Characteristics
NAATs demonstrate excellent analytical sensitivity (80-100%) and specificity (87-99%) when compared to toxigenic culture as a reference standard. 1, 4
The positive likelihood ratio for NAAT-based testing is 46, and the negative likelihood ratio is 0.05, meeting "high information value" thresholds for diagnostic confidence. 3
However, clinical specificity is lower than analytical specificity due to detection of colonization—NAAT clinical specificity is approximately 89% compared to 97.4% for ultrasensitive toxin detection methods. 2
Clinical Implications
Testing should only be performed in patients with diarrhea (≥3 unformed stools in 24 hours), as testing asymptomatic patients or those with formed stool results in false positives and unnecessary treatment. 3
A two-step algorithm is recommended by most guidelines: GDH screening followed by confirmatory testing (either toxin EIA or NAAT), rather than NAAT alone, to balance sensitivity with clinical specificity. 3, 4
When GDH is positive but NAAT is negative, this represents a true negative for toxigenic C. difficile (likely non-toxigenic colonization), and treatment is not indicated, as approximately 20% of C. difficile strains are non-toxigenic. 4
Common Pitfall to Avoid
Do not repeat NAAT testing after an initial negative result unless there is a significant change in clinical presentation, as this leads to false positives and diagnostic confusion. 1, 3
Recognize that a positive NAAT does not automatically confirm CDI—clinical correlation is essential, and consideration of alternative diagnoses is warranted in patients without typical CDI symptoms despite positive molecular testing. 1, 2