Ultrasensitive and Quantitative Toxin Measurement Correlates With Baseline Severity, Severe Outcomes, and Recurrence Among Hospitalized Patients With Clostridioides difficile Infection.

BACKGROUND: Stool toxin concentrations may impact Clostridioides difficile infection (CDI) severity and outcomes. We correlated fecal C difficile toxin concentrations, measured by an ultrasensitive and quantitative assay, with CDI baseline severity, attributable outcomes, and recurrence. METHODS: We enrolled 615 hospitalized adults (≥18 years) with CDI (acute diarrhea, positive stool nucleic acid amplification testing, and decision to treat). Baseline stool toxin A and B concentrations were measured by single molecule array. Subjects were classified by baseline CDI severity (4 scoring methods) and outcomes within 40 days (death, intensive care unit stay, colectomy, and recurrence). RESULTS: Among 615 patients (median, 68.0 years), in all scoring systems, subjects with severe baseline disease had higher stool toxin A+B concentrations than those without (P < .01). Nineteen subjects (3.1%) had a severe outcome primarily attributed to CDI (group 1). This group had higher median toxin A+B (14 303 pg/mL [interquartile range, 416.0, 141 967]) than subjects in whom CDI only contributed to the outcome (group 2, 163.2 pg/mL [0.0, 8423.3]), subjects with severe outcome unrelated to CDI (group 3, 158.6 pg/mL [0.0, 1795.2]), or no severe outcome (group 4, 209.5 pg/mL [0.0, 8566.3]) (P = .003). Group 1 was more likely to have detectable toxin (94.7%) than groups 2-4 (60.5%-66.1%) (P = .02). Individuals with recurrence had higher toxin A+B (2266.8 pg/mL [188.8, 29411]) than those without (154.0 pg/mL [0.0, 5864.3]) (P < .001) and higher rates of detectable toxin (85.7% versus 64.0%, P = .004). CONCLUSIONS: In CDI patients, ultrasensitive stool toxin detection and concentration correlated with severe baseline disease, severe CDI-attributable outcomes, and recurrence, confirming the contribution of toxin quantity to disease presentation and clinical course.

Higher In Vivo Fecal Concentrations of Clostridioides difficile Toxins A and B in Patients With North American Pulsed-Field Gel Electrophoresis Type 1/Ribotype 027 Strain Infection.

Ultrasensitive, quantitative Clostridioides difficile stool toxin measurement demonstrated significantly higher concentrations of toxins A and B in patients infected with the North American pulsed-field gel electrophoresis type 1/ribotype 027 (NAP-1/027) strain compared with other strains, providing in vivo confirmation of the in vitro association between NAP-1/027 and elevated toxin production.

Absence of Toxemia in Clostridioides difficile Infection: Results from Ultrasensitive Toxin Assay of Serum.

Clostridioides difficile infection (CDI) is caused by Toxins A and B, secreted from pathogenic strains of C. difficle. This infection can vary greatly in symptom severity and in clinical presentation. Current assays used to diagnose CDI may lack the required sensitivity to detect the exotoxins circulating in blood. The ultrasensitive single molecule array (Simoa) assay was modified to separately detect toxin A and toxin B in serum with a limit of detection at the low picogram level. When applied to a diverse cohort, Simoa was unable to detect toxins A or B in serum from patients with CDI, including many classified as having severe disease. The detection of toxin may be limited by the inference of antitoxin antibodies circulating in serum. This result does not support the hypothesis that toxemia occurs in C. difficile infection, conflicting with the findings of other published reports.

Host Immune Markers Distinguish Clostridioides difficile Infection From Asymptomatic Carriage and Non-C. difficile Diarrhea.

BACKGROUND: Recent data indicate that Clostridioides difficile toxin concentrations in stool do not differentiate between C. difficile infection (CDI) and asymptomatic carriage. Thus, we lack a method to distinguish a symptomatic patient with CDI from a colonized patient with diarrhea from another cause. To address this, we evaluated markers of innate and adaptive immunity in adult inpatients with CDI (diagnosed per US guidelines), asymptomatic carriage, or non-CDI diarrhea. METHODS: CDI-NAAT patients had clinically significant diarrhea and positive nucleic acid amplification testing (NAAT) and received CDI treatment. Carrier-NAAT patients had positive stool NAAT but no diarrhea. NAAT-negative patients (with and without diarrhea) were also enrolled. A panel of cytokines and anti-toxin A and B immunoglobulin (Ig) were measured in serum; calprotectin and anti-toxin B Ig A/G were measured in stool. NAAT-positive stool samples were tested by an ultrasensitive toxin assay (clinical cutoff, 20 pg/mL). RESULTS: Median values for interleukin (IL)-4, IL-6, IL-8, IL-10, IL-15, granulocyte colony-stimulating factor (GCSF), MCP-1, tumor necrosis factor α (TNF-α), and IgG anti-toxin A in blood and IgA/G anti-toxin B in stool were significantly higher in CDI patients compared with all other groups (P < .05). Concentration distributions for IL-6, GCSF, TNF-α, and IgG anti-toxin A in blood, as well as IgA and IgG anti-toxin B in stool, separated CDI patients from all other groups. CONCLUSIONS: Specific markers of innate and adaptive immunity distinguish CDI from all other groups, suggesting potential clinical utility for identifying which NAAT- and toxin-positive patients with diarrhea truly have CDI.

Toxin A-Predominant Pathogenic Clostridioides difficile: A Novel Clinical Phenotype.

BACKGROUND: Most Clostridioides difficile toxinogenic strains produce both toxins A and B (A+B+), but toxin A-negative, toxin B-positive (A-B+) variants also cause disease. We report the identification of a series of pathogenic clinical C. difficile isolates that produce high amounts of toxin A with low or nondetectable toxin B. METHODS: An ultrasensitive, quantitative immunoassay was used to measure toxins A and B in stool samples from 187 C. difficile infection (CDI) patients and 44 carriers. Isolates were cultured and assessed for in vitro toxin production and in vivo phenotypes (mouse CDI model). RESULTS: There were 7 CDI patients and 6 carriers who had stools with detectable toxin A (TcdA, range 23-17 422 pg/mL; 5.6% of samples overall) but toxin B (TcdB) below the clinical detection limit (<20 pg/mL; median TcdA:B ratio 17.93). Concentrations of toxin A far exceeded B in in vitro cultures of all 12 recovered isolates (median TcdA:B ratio 26). Of 8 toxin A>>B isolates tested in mice, 4 caused diarrhea, and 3 of those 4 caused lethal disease. Ribotyping demonstrated strain diversity. TcdA-predominant samples were also identified at 2 other centers, with similar frequencies (7.5% and 6.8%). CONCLUSIONS: We report the discovery of clinical pathogenic C. difficile strains that produce high levels of toxin A but minimal or no toxin B. This pattern of toxin production is not rare (>5% of isolates) and is consistently observed in vitro and in vivo in humans and mice. Our study highlights the significance of toxin A in human CDI pathogenesis and has important implications for CDI diagnosis, treatment, and vaccine development.

Comparison of Clostridioides difficile Stool Toxin Concentrations in Adults With Symptomatic Infection and Asymptomatic Carriage Using an Ultrasensitive Quantitative Immunoassay.

Background: We used an ultrasensitive, quantitative single molecule array (Simoa) immunoassay to test whether concentrations of Clostridioides (formerly Clostridium) difficile toxins A and/or B in the stool of adult inpatients with C. difficile infection (CDI) were higher than in asymptomatic carriers of toxinogenic C. difficile. Methods: Patients enrolled as CDI-NAAT had clinically significant diarrhea and a positive nucleic acid amplification test (NAAT), per US guidelines, and received CDI treatment. Potential carriers had recently received antibiotics and did not have diarrhea; positive NAAT confirmed carriage. Baseline stool samples were tested by Simoa for toxin A and B. Results: Stool toxin concentrations in both CDI-NAAT (n = 122) and carrier-NAAT (n = 44) cohorts spanned 5 logs (0 pg/mL to >100000 pg/mL). Seventy-nine of 122 (65%) CDI-NAAT and 34 of 44 (77%) carrier-NAAT had toxin A + B concentration ≥20 pg/mL (clinical cutoff). Median toxin A, toxin B, toxin A + B, and NAAT cycle threshold (Ct) values in CDI-NAAT and carrier-NAAT cohorts were similar (toxin A, 50.6 vs 60.0 pg/mL, P = .958; toxin B, 89.5 vs 42.3 pg/mL, P = .788; toxin A + B, 197.2 vs 137.3 pg/mL, P = .766; Ct, 28.1 vs 28.6, P = .354). However, when CDI/carrier cohorts were limited to those with detectable toxin, respective medians were significantly different (A: 874.0 vs 129.7, P = .021; B: 1317.0 vs 81.7, P = .003, A + B, 4180.7 vs 349.6, P = .004; Ct, 25.8 vs 27.7, P = .015). Conclusions: Toxin concentration did not differentiate an individual with CDI from one with asymptomatic carriage. Median stool toxin concentrations in groups with CDI vs carriage differed, but only when groups were defined by detectable stool toxin (vs positive NAAT).

Sensitivity of Single-Molecule Array Assays for Detection of Clostridium difficile Toxins in Comparison to Conventional Laboratory Testing Algorithms.

Guidelines recommend the use of an algorithm for the laboratory diagnosis of Clostridium difficile infection (CDI). Enzyme immunoassays (EIAs) detecting C. difficile toxins cannot be used as standalone tests due to suboptimal sensitivity, and molecular tests suffer from nonspecificity by detecting colonization. Sensitive immunoassays have recently been developed to improve and simplify CDI diagnosis. Assays detecting CD toxins have been developed using single-molecule array (SIMOA) technology. SIMOA performance was assessed relative to a laboratory case definition of CDI defined by positive glutamate dehydrogenase (GDH) screen and cell cytotoxicity neutralizing assay (CCNA). Samples were tested with SIMOA assays and a commercial toxin EIA to compare performance, with discrepancy resolution using a commercial nucleic acid-based test and a second cell cytotoxicity assay. The SIMOA toxin A and toxin B assays showed limits of detection of 0.6 and 2.9 pg/ml, respectively, and intra-assay coefficients of variation of less than 10%. The optimal clinical thresholds for the toxin A and toxin B assays were determined to be 22.1 and 18.8 pg/ml, respectively, with resultant sensitivities of 84.8 and 95.5%. In contrast, a high-performing EIA toxin test had a sensitivity of 71.2%. Thus, the SIMOA assays detected toxins in 24% more samples with laboratory-defined CDI than the high performing toxin EIA (95% [63/66] versus 71% [47/66]). This study shows that SIMOA C. difficile toxin assays have a higher sensitivity than currently available toxin EIA and have the potential to improve CDI diagnosis.