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.

Laxative Use Does Not Preclude Diagnosis or Reduce Disease Severity in Clostridiodes difficile Infection.

BACKGROUND: To optimize utility of laboratory testing for Clostridiodes difficile infection (CDI), the 2017 Infectious Diseases Society of America-Society for Healthcare Epidemiology of America (IDSA-SHEA) clinical practice guidelines recommend excluding patients from stool testing for C. difficile if they have received laxatives within the preceding 48 hours. Sparse data support this recommendation. METHODS: Patients with new-onset diarrhea (≥3 bowel movements in any 24-hour period in the 48 hours before stool collection) and a positive stool C. difficile nucleic acid amplification test were enrolled. Laxative use within 48 hours before stool testing, severity of illness (defined by 4 distinct scoring methods), and clinical outcomes were recorded. RESULTS: 209 patients with CDI were studied, 65 of whom had received laxatives. There were no significant differences in the proportion of patients meeting severe CDI criteria by 4 severity scoring methods in patients receiving versus not receiving laxatives (66.2% vs 56.3%, respectively; P = .224) by IDSA-SHEA, the primary scoring system. Similar rates of serious outcomes attributable to CDI, including death, intensive care unit admission, and colectomy, were observed in the laxative and no laxative groups. CONCLUSIONS: Our study found similar rates of severe CDI and serious CDI-attributable clinical outcomes in CDI-diagnosed patients who did or did not receive laxatives. Precluding recent laxative users from CDI testing, as proposed by the IDSA-SHEA guideline, carries a potential for harm due to delayed diagnosis and treatment.

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).

Filter-based assay for Escherichia coli in aqueous samples using bacteriophage-based amplification.

This paper describes a method to detect the presence of bacteria in aqueous samples, based on the capture of bacteria on a syringe filter, and the infection of targeted bacterial species with a bacteriophage (phage). The use of phage as a reagent provides two opportunities for signal amplification: (i) the replication of phage inside a live bacterial host and (ii) the delivery and expression of the complementing gene that turns on enzymatic activity and produces a colored or fluorescent product. Here we demonstrate a phage-based amplification scheme with an M13KE phage that delivers a small peptide motif to an F(+), α-complementing strain of Escherichia coli K12, which expresses the ω-domain of ß-galactosidase (ß-gal). The result of this complementation-an active form of ß-gal-was detected colorimetrically, and the high level of expression of the ω-domain of ß-gal in the model K12 strains allowed us to detect, on average, five colony-forming units (CFUs) of this strain in 1 L of water with an overnight culture-based assay. We also detected 50 CFUs of the model K12 strain in 1 L of water (or 10 mL of orange juice, or 10 mL of skim milk) in less than 4 h with a solution-based assay with visual readout. The solution-based assay does not require specialized equipment or access to a laboratory, and is more rapid than existing tests that are suitable for use at the point of access. This method could potentially be extended to detect many different bacteria with bacteriophages that deliver genes encoding a full-length enzyme that is not natively expressed in the target bacteria.