Laboratory diagnosis of Clostridium difficile infection can molecular amplification methods move us out of uncertainty?

Abstract

The laboratory diagnosis of Clostridium difficile infection (CDI) continues to be challenging. Recent guidelines from professional societies in the United States note that enzyme immunoassays for toxins A and B do not have adequate sensitivity to be used alone for detecting CDI, yet the optimal method for diagnosing this infection remains unclear. Nucleic acid amplification tests (NAATs) that target chromosomal toxin genes (usually the toxin B gene, tcdB) show high sensitivity and specificity, provide rapid results, and are amenable to both batch and on-demand testing, but these tests were not universally recommended for routine use in the recent guidelines. Rather, two-step algorithms that use glutamate dehydrogenase (GDH) assays to screen for C. difficile in stool specimens, followed by either direct cytotoxin testing or culture to identify toxin-producing C. difficile isolates, were recommended in one guideline and either GDH algorithms or NAATs were recommended in another guideline. Unfortunately, neither culture nor direct cytotoxin testing is widely available. In addition, this two-step approach requires 48 to 92 hours to complete, which may delay the initiation of therapy and critical infection control measures. Recent studies also show the sensitivity of several GDH assays to be <90%. This review considers the role of NAATs for diagnosing CDI and explores their potential advantages over two-step algorithms, including shorter time to results, while providing comparable, if not superior, accuracy.

Figure 1

Amino acid heterogeneity (red) and predicted antigenicity of toxin B protein (black lines) based on an alignment of amino acid sequences of toxin B from 16 C. difficile isolates. The red-shaded plot is the variability of the amino acid sequences of toxin B at each amino acid position; great amino acid diversity is indicated by higher peaks. The black line indicates the predicted antigenicity of the protein based on the algorithm of Kolaskar and Tongaonkar (KT) (high peaks indicate high antigenicity). The blue bars indicate regions of DNA sequence conservation among the 16 isolates. The 16 isolates include strains of PCR ribotypes 001, 017, 027, and 078 and pulsed-field gel electrophoresis types NAP1, NAP7, and NAP8.

Figure 2

Steps involved in four FDA-cleared nucleic acid amplification assays for the detection of CDI. Data were extracted from package inserts and published literature. rxn, reaction.