Development and Validation of Digital Enzyme-Linked Immunosorbent Assays for Ultrasensitive Detection and Quantification of Clostridium difficile Toxins in Stool

Abstract

The currently available diagnostics for Clostridium difficile infection (CDI) have major limitations. Despite mounting evidence that toxin detection is paramount for diagnosis, conventional toxin immunoassays are insufficiently sensitive and cytotoxicity assays too complex; assays that detect toxigenic organisms (toxigenic culture [TC] and nucleic acid amplification testing [NAAT]) are confounded by asymptomatic colonization by toxigenic C. difficile. We developed ultrasensitive digital enzymelinked immunosorbent assays (ELISAs) for toxins A and B using single-molecule array technology and validated the assays using (i) culture filtrates from a panel of clinical C. difficile isolates and (ii) 149 adult stool specimens already tested routinely by NAAT. The digital ELISAs detected toxins A and B in stool with limits of detection of 0.45 and 1.5 pg/ml, respectively, quantified toxins across a 4-log range, and detected toxins from all clinical strains studied. Using specimens that were negative by cytotoxicity assay/TC/NAAT, clinical cutoffs were set at 29.4 pg/ml (toxin A) and 23.3 pg/ml (toxin B); the resulting clinical specificities were 96% and 98%, respectively. The toxin B digital ELISA was 100% sensitive versus cytotoxicity assay. Twenty-five percent and 22% of the samples positive by NAAT and TC, respectively, were negative by the toxin B digital ELISA, consistent with the presence of organism but minimal or no toxin. The mean toxin levels by digital ELISA were 1.5-to 1.7-fold higher in five patients with CDI-attributable severe outcomes, versus 68 patients without, but this difference was not statistically significant. Ultrasensitive digital ELISAs for the detection and quantification of toxins A and B in stool can provide a rapid and simple tool for the diagnosis of CDI with both high analytical sensitivity and high clinical specificity.T he recent increases in the global incidence and severity of Clostridium difficile infection (CDI) (1-3) are of major concern, and CDI now consumes substantial resources for diagnosis, treatment, and infection control (2, 4). A recent U.S. prevalence survey of health care-associated infections (HAI) (5) found that C. difficile was the most commonly reported pathogen, causing 12.1% of HAI. Despite available therapies, treatment failure and recurrence are common (1, 2, 6). The emergence of epidemic strains capable of toxin hyperproduction and increased disease severity/mortality (2, 7, 8) has further increased the urgency to improve methods for diagnosis and treatment. Accurate diagnosis remains the cornerstone of effective management.Disease caused by C. difficile infection is due to the effects of two large protein exotoxins, toxins A and B (9, 10). While the presence of toxin is necessary to cause disease, the optimal method for diagnosing CDI remains controversial. Appropriate patient selection can improve diagnostic accuracy for all assays (2, 11). The classic gold standard stool assay, toxigenic culture (TC), is complex, lengthy, and unstandardized; furthermore, it is...

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.…”

“…al. [117], an ultrasensitive toxin detection based on single molecule array technology. This assay was validated using culture filtrates prepared from a panel of clinical C. difficile strains and adult clinical stool specimens.…”

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

“…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.…”

“…An alternative immunoassay approach to ultrasensitive toxin detection has recently been developed based on single-molecule array (Simoa) technology (39). Simoa technology (Quanterix; Lexington, MA), also known as "digital enzyme-linked immunosorbent assay (ELISA)," is based on efficient capture, labeling, and detection of single protein molecules on paramagnetic beads in arrays of femtoliter-sized wells; in terms of achievable LODs, digital ELISA is typically 1,000-fold more sensitive than conventional ELISA (42).…”

Strategies for Optimizing the Diagnostic Predictive Value of Clostridium difficile Molecular Diagnostics

“…Ideally, assays that can more reliably differentiate patients with C. difficile carriage from those with CDI are necessary to completely overcome the discordant analytical and diagnostic specificities of NAATs. An ultrasensitive toxin enzyme-linked immunosorbent assay currently under clinical investigation may be one such assay that provides improved diagnostic specificity while overcoming the potentially poor sensitivity of existing toxin EIAs (24). However, even if this important diagnostic challenge for CDI is solved with improved diagnostics, highly sensitive molecular diagnostics will continue to emerge and be more broadly utilized for other infectious diseases.…”