Automated Detection of Toxigenic Clostridium difficile in Clinical Samples: Isothermal tcdB Amplification Coupled to Array-Based Detection

Abstract

dClostridium difficile can carry a genetically variable pathogenicity locus (PaLoc), which encodes clostridial toxins A and B. In hospitals and in the community at large, this organism is increasingly identified as a pathogen. To develop a diagnostic test that combines the strengths of immunoassays (cost) and DNA amplification assays (sensitivity/specificity), we targeted a genetically stable PaLoc region, amplifying tcdB sequences and detecting them by hybridization capture. The assay employs a hot-start isothermal method coupled to a multiplexed chip-based readout, creating a manual assay that detects toxigenic C. difficile with high sensitivity and specificity within 1 h. Assay automation on an electromechanical instrument produced an analytical sensitivity of 10 CFU (95% probability of detection) of C. difficile in fecal samples, along with discrimination against other enteric bacteria. To verify automated assay function, 130 patient samples were tested: 31/32 positive samples (97% sensitive; 95% confidence interval [CI], 82 to 99%) and 98/98 negative samples (100% specific; 95% CI, 95 to 100%) were scored correctly. Largescale clinical studies are now planned to determine clinical sensitivity and specificity.

Multicenter Clinical Evaluation of the Portrait Toxigenic C. difficile Assay for Detection of Toxigenic Clostridium difficile Strains in Clinical Stool Specimens

“…Indeed, 87/90 (96.7%) "lowpositive" samples tested positive by the Portrait test, which is compatible with the expected results. The testing of the "highnegative"-density (C 5 [cell density at which 5% of tests are expected to give a positive result]) samples resulted in 4/90 (4.4%) positive tests, which is consistent with the expected 5% positive samples.…”

“…EIAs directed against C. difficile toxins have reported sensitivities of 25% to 86% (1,5,8,14). Similarly, the reported sensitivities of toxin-based lateral flow tests range from 43% to 73% (3,5,14). Given this low sensitivity, it was suggested that toxin EIAs and lateral flow tests lack utility as confirmatory methods for GDH-positive specimens in two-step testing algorithms (5).…”

“…Similarly, the reported sensitivities of toxin-based lateral flow tests range from 43% to 73% (3,5,14). Given this low sensitivity, it was suggested that toxin EIAs and lateral flow tests lack utility as confirmatory methods for GDH-positive specimens in two-step testing algorithms (5). The cell cytotoxin neutralization assay is another method used for the confirmation of toxigenic C. difficile in GDH-positive specimens; however, when conducted without bacterial enrichment (i.e., directly from stool), the CCNA may be only 54% sensitive (11).…”

“…This test utilizes the isothermal heli-case-dependent amplification of a 78-nucleotide 3= region of the tcdB gene followed by detection using an immobilized capture probe on a slide array (5). Results were compared to toxigenic bacterial culture (TBC)/CCNA as a gold standard.…”

“…The Society for Healthcare Epidemiology of America (SHEA) and the Infectious Diseases Society of America (IDSA) recommend a multistep approach that includes an initial screen for GDH followed by the detection of toxin using a cell cytotoxin neutralization assay (CCNA) or an enzyme immunoassay (EIA) (3). EIAs directed against C. difficile toxins have reported sensitivities of 25% to 86% (1,5,8,14). Similarly, the reported sensitivities of toxin-based lateral flow tests range from 43% to 73% (3,5,14).…”