Multicenter Evaluation of the Verigene Clostridium difficile Nucleic Acid Assay

Abstract

hThe Verigene Clostridium difficile Nucleic Acid test (Verigene CDF test) (Nanosphere, Northbrook, IL) is a multiplex qualitative PCR assay that utilizes a nanoparticle-based array hybridization method to detect C. difficile tcdA and tcdB in fecal specimens. In addition, the assay detects binary toxin gene sequences and the single base pair deletion at nucleotide 117 (⌬ 117) in tcdC to provide a presumptive identification of the epidemic strain 027/NAP1/BI (referred to here as ribotype 027). This study compared the Verigene CDF test with anaerobic direct and enriched toxigenic culture on stool specimens from symptomatic patients among five geographically diverse laboratories within the United States. The Verigene CDF test was performed according to the manufacturer's instructions, and the reference methods performed by a central laboratory included direct culture onto cycloserine cefoxitin fructose agar (CCFA) and enriched culture using cycloserine cefoxitin mannitol broth with taurocholate and lysozyme. Recovered isolates were identified as C. difficile using gas liquid chromatography and were tested for toxin using a cell culture cytotoxicity neutralization assay. Strains belonging to ribotype 027 were determined by PCR ribotyping and bidirectional sequencing for ⌬ 117 in tcdC. A total of 1,875 specimens were evaluable. Of these, 275 specimens (14.7%) were culture positive by either direct or enriched culture methods. Compared to direct culture alone, the overall sensitivity, specificity, positive predictive value, and negative predictive value for the Verigene CDF test were 98.7%, 87.5%, 42%, and 99.9%, respectively. Compared to combined direct and enriched culture results, the sensitivity, specificity, positive predictive value, and negative predictive values of the Verigene CDF test were 90.9%, 92.5%, 67.6%, and 98.3%, respectively. Of the 250 concordantly culture-positive specimens, 59 (23.6%) were flagged as "hypervirulent"; 53 were confirmed as ribotype 027, and all 59 possessed ⌬ 117 in tcdC. Time to results was approximately 2.5 h per specimen. The Verigene CDF test is a novel nucleic acid microarray that reliably detects both C. difficile toxins A and B in unformed stool specimens and appears to adequately identify ribotype 027 isolates. The optimum testing method for the diagnosis of Clostridium difficile is still a matter of debate. A variety of testing methods are available and include enzyme immunoassays (EIAs) for detection of toxins A and B, cell culture cytotoxicity neutralization assays (CCNAs), anaerobic toxigenic culture, detection of glutamate dehydrogenase, and nucleic acid amplification tests (NAATs). Although EIAs provide same-day results and are relatively inexpensive and easy to perform, there is general agreement in the United States and in some European countries that EIAs should not be performed as stand-alone tests due to their lack of sensitivity (1, 2). CCNAs and anaerobic toxigenic culture do not provide timely results and have never been standardized. Laboratories are beginning...

Clinical Utility of Laboratory Detection of Clostridium difficile Strain BI/NAP1/027

“…Carroll and colleagues reported data from their investigation of the Verigene assay using 1,875 clinical stool specimens collected from patients with diarrhea (25). Of the 58 specimens that were presumptively identified as BI/NAP1/027 by the Verigene assay and also underwent PCR ribotyping, 53 (91%) were confirmed to be BI/NAP1/027.…”

“…Cepheid (Sunnyvale, CA) and Nanosphere (Northbrook, IL) both offer commercially available nucleic acid amplification tests (NAATs) that can presumptively identify BI/NAP1/027 directly from clinical stool specimens with very short turnaround times (i.e., several hours) (25)(26)(27). Like most C. difficile NAATs, both the Cepheid Xpert C. difficile/Epi assay and the Nanosphere Verigene assay detect tcdB (and the Verigene assay additionally detects tcdA).…”

Emerging Technologies for the Clinical Microbiology Laboratory

“…A clinical evaluation of the CDF assay demonstrated 98.7% sensitivity and 87.5% specificity for detection of toxigenic C. difficile based on the presence of tcdA and/or tcdB, the primary toxinencoding genes present in toxigenic strains of C. difficile (94). In addition, the CDF assay contains capture probes for detection of the ⌬117 deletion in tcdC, which encodes the repressor of tcdA and -B expression, and genes encoding binary toxin (cdtA and cdtB) (94).…”

“…In addition, the CDF assay contains capture probes for detection of the ⌬117 deletion in tcdC, which encodes the repressor of tcdA and -B expression, and genes encoding binary toxin (cdtA and cdtB) (94). Strains with the ⌬117 deletion produce up to 23-fold more toxin than wild-type strains (105).…”

“…4) has offers microarraybased tests for identification of respiratory viruses (RVϩ), C. difficile (CDF), blood cultures containing Gram-positive bacteria (BC-GP) or Gram-negative bacteria (BC-GN), and identification of genetic variants, including Factor V Leiden and CYP450 2C19 *2 and *3, which impact patients with coagulation disorders (94)(95)(96)(97)(98)(99)(100)(101)(102).…”