scholarly journals Detoxification of toxin A and toxin B by copper ion-catalyzed oxidation in production of a toxoid-based vaccine against Clostridioides difficile

2020 ◽  
Vol 160 ◽  
pp. 433-446
Author(s):  
Aria Aminzadeh ◽  
Manish Kumar Tiwari ◽  
Srwa Satar Mamah Mustapha ◽  
Sandra Junquera Navarrete ◽  
Anna Bielecka Henriksen ◽  
...  
Keyword(s):  
Copper Ion ◽  
Toxin A ◽  
Toxin B ◽  
Clostridioides Difficile ◽  
Catalyzed Oxidation

scholarly journals Host Immune Markers Distinguish Clostridioides difficile Infection From Asymptomatic Carriage and Non–C. difficile Diarrhea

2019 ◽  
Author(s):  
Ciaran P Kelly ◽  
Xinhua Chen ◽  
David Williams ◽  
Hua Xu ◽  
Christine A Cuddemi ◽  
...  
Keyword(s):  
Adaptive Immunity ◽  
Symptomatic Patient ◽  
Nucleic Acid Amplification ◽  
Toxin A ◽  
Toxin B ◽  
Innate And Adaptive Immunity ◽  
Clostridioides Difficile ◽  
Asymptomatic Carriage ◽  
Positive Stool ◽  
Tnf Α

Abstract 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.

scholarly journals Toxin A–Predominant Pathogenic Clostridioides difficile: A Novel Clinical Phenotype

2019 ◽  
Vol 70 (12) ◽  
pp. 2628-2633 ◽  
Author(s):  
Qianyun Lin ◽  
Nira R Pollock ◽  
Alice Banz ◽  
Aude Lantz ◽  
Hua Xu ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Toxin Production ◽  
Toxin A ◽  
Toxin B ◽  
Clostridioides Difficile ◽  
Quantitative Immunoassay ◽  
Clinical Detection ◽  
Stool Samples

Abstract 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 (&lt;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&gt;&gt;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 (&gt;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.

scholarly journals Systematic Evaluation of Parameters Important for Production of Native Toxin A and Toxin B from Clostridioides difficile

Toxins ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 240
Author(s):  
Aria Aminzadeh ◽  
René Jørgensen
Keyword(s):  
Sandwich Elisa ◽  
Toxin Production ◽  
Culture Conditions ◽  
Systematic Evaluation ◽  
Toxin A ◽  
Toxin B ◽  
Melting Temperatures ◽  
Clostridioides Difficile ◽  
Differential Scanning Fluorimetry ◽  
Yeast Extract Medium

In the attempt to improve the purification yield of native toxin A (TcdA) and toxin B (TcdB) from Clostridioides difficile (C. difficile), we systematically evaluated culture parameters for their influence on toxin production. In this study, we showed that culturing C. difficile in a tryptone-yeast extract medium buffered in PBS (pH 7.5) that contained 5 mM ZnCl2 and 10 mM glucose supported the highest TcdB production, measured by the sandwich ELISA. These culture conditions were scalable into 5 L and 15 L dialysis tube cultures, and we were able to reach a TcdB concentration of 29.5 µg/mL of culture. Furthermore, we established a purification protocol for TcdA and TcdB using FPLC column chromatography, reaching purities of >99% for both toxins with a yield around 25% relative to the starting material. Finally, by screening the melting temperatures of TcdA and TcdB in various buffer conditions using differential scanning fluorimetry, we found optimal conditions for improving the protein stability during storage. The results of this study present a complete protocol for obtaining high amounts of highly purified native TcdA and TcdB from C. difficile.

scholarly journals Infection with Toxin A-Negative, Toxin B-Negative, Binary Toxin-Positive Clostridium difficile in a Young Patient with Ulcerative Colitis

2015 ◽  
Vol 53 (11) ◽  
pp. 3702-3704 ◽  
Author(s):  
Grace O. Androga ◽  
Julie Hart ◽  
Niki F. Foster ◽  
Adrian Charles ◽  
David Forbes ◽  
...  
Keyword(s):  
Ulcerative Colitis ◽  
Clostridium Difficile ◽  
Young Patient ◽  
Diagnostic Methods ◽  
Binary Toxin ◽  
Toxin A ◽  
Toxin B ◽  
Content Type ◽  
Severe Diarrhea ◽  
Clinically Significant

Large clostridial toxin-negative, binary toxin-positive (A−B−CDT+) strains ofClostridium difficileare almost never associated with clinically significantC. difficileinfection (CDI), possibly because such strains are not detected by most diagnostic methods. We report the isolation of an A−B−CDT+ribotype 033 (RT033) strain ofC. difficilefrom a young patient with ulcerative colitis and severe diarrhea.

scholarly journals Nucleic Acid Amplification Test Quantitation as Predictor of Toxin Presence in Clostridium difficile Infection

2017 ◽  
Vol 56 (3) ◽  
Author(s):  
M. J. T. Crobach ◽  
N. Duszenko ◽  
E. M. Terveer ◽  
C. M. Verduin ◽  
E. J. Kuijper
Keyword(s):  
Nucleic Acid ◽  
Clostridium Difficile ◽  
Characteristic Curve ◽  
Nucleic Acid Amplification Test ◽  
Nucleic Acid Amplification ◽  
Toxin A ◽  
Toxin B ◽  
Content Type ◽  
Quantitative Results ◽  
Testing Algorithm

ABSTRACT Multistep algorithmic testing in which a sensitive nucleic acid amplification test (NAAT) is followed by a specific toxin A and toxin B enzyme immunoassay (EIA) is among the most accurate methods for Clostridium difficile infection (CDI) diagnosis. The obvious shortcoming of this approach is that multiple tests must be performed to establish a CDI diagnosis, which may delay treatment. Therefore, we sought to determine whether a preliminary diagnosis could be made on the basis of the quantitative results of the first test in algorithmic testing, which provide a measure of organism burden. To do so, we retrospectively analyzed two large collections of samples ( n = 2,669 and n = 1,718) that were submitted to the laboratories of two Dutch hospitals for CDI testing. Both hospitals apply a two-step testing algorithm in which a NAAT is followed by a toxin A/B EIA. Of all samples, 208 and 113 samples, respectively, tested positive by NAAT. Among these NAAT-positive samples, significantly lower mean quantification cycle ( C q ) values were found for patients whose stool eventually tested positive for toxin, compared with patients who tested negative for toxin (mean C q values of 24.4 versus 30.4 and 26.8 versus 32.2; P < 0.001 for both cohorts). Receiver operating characteristic curve analysis was performed to investigate the ability of C q values to predict toxin status and yielded areas under the curve of 0.826 and 0.854. Using the optimal C q cutoff values, prediction of the eventual toxin A/B EIA results was accurate for 78.9% and 80.5% of samples, respectively. In conclusion, C q values can serve as predictors of toxin status but, due to the suboptimal correlation between the two tests, additional toxin testing is still needed.

scholarly journals Impact of Two-Step Testing Algorithm on Reducing Hospital-Onset Clostridioides difficile Infections

2021 ◽  
Vol 1 (S1) ◽  
pp. s42-s43
Author(s):  
Bhagyashri Navalkele ◽  
Wendy Winn ◽  
Sheila Fletcher ◽  
Regina Galloway ◽  
Jason Parham ◽  
...  
Keyword(s):  
Medical Center ◽  
Molecular Testing ◽  
Toxin A ◽  
Clostridioides Difficile ◽  
Contact Isolation ◽  
Academic Center ◽  
Mississippi Medical ◽  
True Infection ◽  
Testing Algorithm ◽  
The University

Clostridioides difficile infection (CDI) is one of the leading causes of hospital–onset infections. Clinically distinguishing true CDI versus colonization with C. difficile is challenging and often requires reliable and rapid molecular testing methods. At our academic center, we implemented a 2-step testing algorithm to help identify true CDI cases. The University of Mississippi Medical Center is a 700+ bed academic facility located in Jackson, Mississippi. Hospital-onset (HO) CDI was defined based on NHSN Laboratory Identified (LabID) event as the last positive C. difficile test result performed on a specimen using a multistep testing algorithm collected >3 calendar days after admission to the facility. HO-CDI data were collected from all inpatient units except the NICU and newborn nursery. HO-CDI outcomes were assessed based on standardized infection ratio (SIR) data. In May 2020, we implemented a 2-step testing algorithm (Figure 1). All patients with diarrhea underwent C. difficile PCR testing. Those with positive C. difficile PCR test were reflexed to undergo enzyme immunoassay (EIA) glutamate dehydrogenase antigen (Ag) testing and toxin A and B testing. The final results were reported as colonization (C. difficile PCR+/EIA Ag+/Toxin A/B−) or true CDI case (C. difficile PCR+/EIA +/Toxin A/B +) or negative (C. difficile PCR−). All patients with colonization or true infection were placed under contact isolation precautions until diarrhea resolution for 48 hours. During the preintervention period (October 2019–April 2020), 25 HO-CDI cases were reported compared to 8 cases in the postintervention period (June 2020–December 2020). A reduction in CDI SIR occurred in the postintervention period (Q3 2020–Q4 2020, SIR 0.265) compared to preintervention period (Q4 2019–Q1 2020, SIR 0.338) (Figure 2). We successfully reduced our NHSN HO-CDI SIR below the national average after implementing a 2-step testing algorithm for CDI. The 2-step testing algorithm was useful for antimicrobial stewardship to guide appropriate CDI treatment for true cases and for infection prevention to continue isolation of infected and colonized cases to reduce the spread of C. difficile spores.Funding: NoDisclosures: None

scholarly journals Identification of Toxin A-Negative, Toxin B-Positive Clostridium difficile by PCR

1998 ◽  
Vol 36 (8) ◽  
pp. 2178-2182 ◽  
Author(s):  
Haru Kato ◽  
Naoki Kato ◽  
Kunitomo Watanabe ◽  
Naoichi Iwai ◽  
Haruhi Nakamura ◽  
...  
Keyword(s):  
Cell Culture ◽  
Clostridium Difficile ◽  
Pcr Amplification ◽  
Enzyme Linked Immunosorbent Assay ◽  
Pcr Assay ◽  
Toxin A ◽  
Toxin B ◽  
Cell Monolayers ◽  
Toxigenic Strains ◽  
Cell Culture Assay

Toxigenic strains of Clostridium difficile have been reported to produce both toxins A and B nearly always, and nontoxigenic strains have been reported to produce neither of these toxins. Recent studies indicate that it is not always true. We established a PCR assay to differentiate toxin A-negative, toxin B-positive (toxin A−, toxin B+) strains from both toxin-positive (toxin A+, toxin B+) strains and both toxin-negative (toxin A−, toxin B−) strains as an alternative to cell culture assay and enzyme-linked immunosorbent assay (ELISA). By using the PCR primer set NK11 and NK9 derived from the repeating sequences of the toxin A gene, a shorter segment (ca. 700 bp) was amplified from toxin A−, toxin B+ strains compared to the size of the segment amplified from toxin A+, toxin B+ strains (ca. 1,200 bp), and no product was amplified from toxin A−, toxin B− strains. We examined a total of 421 C. difficile isolates by PCR. Of these, 48 strains showed a shorter segment by the PCR, were negative by ELISAs for the detection of toxin A, and were positive by cell culture assay. Although the cytotoxin produced by the toxin A−, toxin B+ strains was neutralized by anti-toxin B serum, the appearance of the cytotoxic effects on Vero cell monolayers was distinguishable from that of toxin A+, toxin B+ strains. By immunoblotting, the 44 toxin A−, toxin B+ strains were typed to serogroup F and the remaining four strains were serogroup X. Pulsed-field gel electrophoresis separated the 48 strains into 19 types. The PCR assay for the detection of the repeating sequences combined with PCR amplification of the nonrepeating sequences of either the toxin A or the toxin B gene is indicated to be useful for differentiating toxin A−, toxin B+ strains from toxin A+, toxin B+ and toxin A−, toxin B− strains and will contribute to elucidation of the precise role of toxin A−, toxin B+ strains in intestinal diseases.

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