Real-Time Cellular Analysis Coupled with a Specimen Enrichment Accurately Detects and Quantifies Clostridium difficile Toxins in Stool

Abstract

We describe here the use of an immunomagnetic separation enrichment process coupled with a modified real-time cellular analysis (RTCA) system (RTCA version 2) for the detection of C. difficile toxin (CDT) in stool. The limit of CDT detection by RTCA version 2 was 0.12 ng/ml. Among the consecutively collected 401 diarrheal stool specimens, 53 (13.2%) were toxin-producing C. difficile strains by quantitative toxigenic culture (qTC); bacterial loads ranged from 3.00 ؋ 10 1 to 3.69 ؋ 10 6 CFU/ml. The RTCA version 2 method detected CDT in 51 samples, resulting in a sensitivity of 96.2%, a specificity of 99. C lostridium difficile infection (CDI) is the leading bacterial cause of nosocomial diarrhea in hospitalized patients (1-4). The incidence of CDI has increased in the last decade and community onset disease has been encountered in patients without previous health care exposure or antibiotic use (5-7).A definitive laboratory diagnosis of CDI depends on a stool test result positive for the presence of toxigenic C. difficile or its toxins (CDT) or colonoscopic or histopathologic findings demonstrating pseudomembranous colitis (8). Currently, laboratory methods used for the diagnosis of CDI are based on detection of C. difficile-specific antigen (e.g., glutamate dehydrogenase enzyme immunoassay [EIA]), toxin proteins (toxin A/B EIA, toxigenic culture [TC], cell cytotoxicity neutralization assay [CCNA]), or toxin genes (real-time PCR [RT-PCR] and loop-mediated isothermal amplification assay) (9-14). Molecular diagnostic techniques, including RT-PCR and other molecular assays, have the advantages of higher sensitivities and rapid turnaround times and are increasingly being implemented for routine diagnostic use (15-17).While molecular-based tests have high sensitivity and are faster, these assays detect the presence of toxin-encoding genes, and they may not be able to distinguish between infection and colonization as well as live and dead C. difficile organisms in certain circumstances (18,19). Recent studies highlighted the importance of detecting and quantifying CDT (20,21). Planche et al. recently noted no increase in mortality when toxigenic C. difficile alone was present, while toxin determined by cytotoxin assay positivity correlated with clinical outcome. CDT correlated with the clinical severity of CDI (21). Leslie et al. found that toxin-negative patients had a lower level of C. difficile than toxin-positive patients (22). These results suggested that toxin quantification could be used clinically to predict toxin status and help distinguish patients with CDI from carriers with diarrhea due to other causes (21).We previously reported on the use of a real-time cellular analysis (RTCA) assay (ACEA Biosciences, San Diego, CA) for quantitative detection of CDT directly from stool (23). This assay is based on microelectronic sensor-based cellular analysis technology (23). Cell index (CI) is a dimensionless parameter to represent cell status based on the change of the electrode impedance. The impedance readout harness...

Rapid Tests for Detection of Main Clostridial Toxins

“…In the last two years, some immunoassays have been developed [116][117][118][119] that can overcome the stagnation of C. difficile toxin detection. These methods, are in many cases simples, fast and ultrasensitive, and have a large potential for clinical applications in the future.…”

“…This technique had a specificity of 99.6% and a sensitivity of 87.5% (28 of 32), which is higher than the EIA result. When a pre-step of immunomagnetic separation enrichment process is added, in which toxin B is first captured from supernatant via magnetic beads [116], the specificity raises to 99.7%.…”

Ultrasensitive Detection and Quantification of Toxins for Optimized Diagnosis of Clostridium difficile Infection

“…Arguing for the higher utility of toxin detection, multiple studies comparing the clinical features of patients with different test outcomes have demonstrated that NAAT-positive, toxin-negative patients have milder symptoms than NAAT-positive, toxin-positive patients (see, e.g., references 22, 23, and 24), and others have shown that toxin-positive patients have higher mortality than toxin-negative patients (see, e.g., references 19, 22, 25, 26, and 27). Further arguing for the clinical utility of toxin detection, disease severity has been correlated to stool toxin levels in some preliminary studies (17,19,25,28,29), suggesting that the ability to quantify toxin levels in stool could potentially be clinically valuable to predict disease and treatment outcomes and in identifying those who need aggressive therapy. Recent data (30) indicate that toxins also may be detectable in blood in some individuals with CDI, providing another potential use for an ultrasensitive toxin detection tool.…”

“…Their data indicated that almost half of the toxin-positive specimens in their study would not be detected by EIAs with LODs of ϳ1 ng/ml. Conventional cytotoxicity assays have demonstrated analytical LODs far below those of EIA for detection of toxin B in buffer (e.g., 1.5 pg/ml [38]), but achievable LODs for detection of toxins in stool samples appear to be higher (29,39). Older literature (40) states that "1 pg of toxin B is sufficient to cause rounding of the cells" in this assay format, but how this corresponds to an actual concentration of toxin in stool is unclear.…”

Real-time replication of swine vesicular disease virus (SVDV) in cell culture systems in vitro

“…CPE is a basis for studies using cell cultures. A novel method for the real-time assay of in vitro cells is real-time cell analysis (RTCA) (4,5,9,10,11,14), also applied to diagnosis of viral diseases (2,3,12,13), and it creates a possibility for improvement in that area. The aim of this experiment was to evaluate the applicability of RTCA in CPE detection in SVDV-infected cell cultures.…”