Assessment of Clostridium difficile Infections by Quantitative Detection of tcdB Toxin by Use of a Real-Time Cell Analysis System

We explored the use of a real-time cell analysis (RTCA) system for the assessment of Clostridium difficile toxins in human stool specimens by monitoring the dynamic responses of the HS27 cells to tcdB toxins. The C. difficile toxin caused cytotoxic effects on the cells, which resulted in a dose-dependent and time-dependent decrease in cell impedance. The RTCA assay possessed an analytical sensitivity of 0.2 ng/ml for C. difficile toxin B with no cross-reactions with other enterotoxins, nontoxigenic C. difficile, or other Clostridum species. Clinical validation was performed on 300 consecutively collected stool specimens from patients with suspected C. difficile infection (CDI). Each stool specimen was tested in parallel by a real-time PCR assay (PCR), a dual glutamate dehydrogenase and toxin A/B enzyme immunoassay (EIA), and the RTCA assay. In comparison to a reference standard in a combination of the three assays, the RTCA had a specificity of 99.6% and a sensitivity of 87.5% (28 of 32), which was higher than the EIA result (P = 0.005) but lower than the PCR result (P = 0.057). In addition, the RTCA assay allowed for quantification of toxin protein concentration in a given specimen. Among RTCA-positive specimens collected prior to treatment with metronidazole and/or vancomycin, a significant correlation between toxin protein concentrations and clinical CDI severities was observed (R² = 0.732, P = 0.0004). Toxin concentrations after treatment (0.89 ng/ml) were significantly lower than those prior to the treatment (15.68 ng/ml, Wilcoxon P = 0.01). The study demonstrates that the RTCA assay provides a functional tool for the potential assessment of C. difficile infections.Clostridium difficile is recognized as the leading cause of infectious diarrhea that develops in patients after hospitalization and/or in patients receiving antibiotic treatment (3, 12). Moreover, the recently emerged, highly virulent strain BI-NAP1-027 has been associated with increased morbidity and mortality (14, 16). A definitive diagnosis depends on the detection of C. difficile-specific toxin B production in the laboratory, which allows for prompt treatment and isolation procedures to prevent further nosocomial spread of infection (12, 19). There are a number of methods available that have been used for the laboratory diagnosis of C. difficile infection (CDI). The well-accepted standard is cytotoxigenic culture, which is conducted by culturing C. difficile from the stool and then performing a cytotoxin assay on the isolate (21). This standard is labor-intensive, subjective, and time-consuming and therefore is not widely used in the clinical setting. Several enzyme immunoassays (EIAs) detect C. difficile antigens in stool, including glutamate dehydrogenase (GDH), as well as toxins A and B (7, 19, 20, 22-24, 28, 29). PCR-based molecular assays that detect toxin A or B, or both, have shown promising performance (4, 10, 18, 24, 30).Currently, recommended therapies for CDI include oral administration of metronidazole and/or vancomycin for 10 to 14 days (6). However, increased percentages of patients experience infection relapse after completion of treatment (3, 12, 17). The emergence and spread of resistance in C. difficile are complicating treatment and prevention (9). While new antibiotics and therapeutic methods are available, providing a rapid and accurate laboratory tool for monitoring disease severity and therapy efficacy is clinically desirable. The C. difficile toxin assay, which detects cell toxicity caused by toxin B, is a direct determination of whether a functional C. difficile toxin exists in stool. However, this assay is labor- and time-intensive and provides only qualitative results.A real-time cell analysis (RTCA) assay (ACEA Biosciences, San Diego, CA) was developed for monitoring the cell status using electronic impedance technology. Utilizing a dimensionless parameter called the cell index (CI), the RTCA system detects the changes to the cell layers cultured on gold microelectrodes on the glass substrates integrated in the bottom of the microelectronic plates (25). This technology has been applied in a number of cell-based assays, including cytotoxicity, cell adhesion and spreading, functional monitoring of receptor-mediated signaling, and cell invasion and migration (2, 11, 31, 32). In this study, we adapt the system for assessment of C. difficile toxin directly from stool specimens. Analytical and diagnostic sensitivities and specificities of this system for the diagnosis and monitoring of CDI were determined. We also explored the assessment of clinical CDI severities and therapeutic efficacies by using toxin concentrations in stool specimens as determined by the RTCA assay.(This study was presented in part at the 110th American Society for Microbiology Annual Meeting, San Diego, CA, 23 to 27 May 2010.)