scholarly journals In VivoPhysiological and Transcriptional Profiling Reveals Host Responses to Clostridium difficile Toxin A and Toxin B

2013 ◽  
Vol 81 (10) ◽  
pp. 3814-3824 ◽  
Author(s):  
Kevin M. D'Auria ◽  
Glynis L. Kolling ◽  
Gina M. Donato ◽  
Cirle A. Warren ◽  
Mary C. Gray ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Neutrophil Infiltration ◽  
Small Gtpase ◽  
Host Responses ◽  
Transcriptional Responses ◽  
Toxin A ◽  
Toxin B ◽  
Altered Expression ◽  
Content Type ◽  
Cell Counts

ABSTRACTToxin A (TcdA) and toxin B (TcdB) ofClostridium difficilecause gross pathological changes (e.g., inflammation, secretion, and diarrhea) in the infected host, yet the molecular and cellular pathways leading to observed host responses are poorly understood. To address this gap, we evaluated the effects of single doses of TcdA and/or TcdB injected into the ceca of mice, and several endpoints were analyzed, including tissue pathology, neutrophil infiltration, epithelial-layer gene expression, chemokine levels, and blood cell counts, 2, 6, and 16 h after injection. In addition to confirming TcdA's gross pathological effects, we found that both TcdA and TcdB resulted in neutrophil infiltration. Bioinformatics analyses identified altered expression of genes associated with the metabolism of lipids, fatty acids, and detoxification; small GTPase activity; and immune function and inflammation. Further analysis revealed transient expression of several chemokines (e.g.,Cxcl1andCxcl2). Antibody neutralization of CXCL1 and CXCL2 did not affect TcdA-induced local pathology or neutrophil infiltration, but it did decrease the peripheral blood neutrophil count. Additionally, low serum levels of CXCL1 and CXCL2 corresponded with greater survival. Although TcdA induced more pronounced transcriptional changes than TcdB and the upregulated chemokine expression was unique to TcdA, the overall transcriptional responses to TcdA and TcdB were strongly correlated, supporting differences primarily in timing and potency rather than differences in the type of intracellular host response. In addition, the transcriptional data revealed novel toxin effects (e.g., altered expression of GTPase-associated and metabolic genes) underlying observed physiological responses toC. difficiletoxins.

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

2018 ◽  
Vol 56 (8) ◽  
Author(s):  
Alice Banz ◽  
Aude Lantz ◽  
Brigitte Riou ◽  
Agnès Foussadier ◽  
Mark Miller ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Single Molecule ◽  
Laboratory Diagnosis ◽  
Case Definition ◽  
Cell Cytotoxicity ◽  
Toxin A ◽  
Toxin B ◽  
Content Type ◽  
High Performing ◽  
Molecular Tests

ABSTRACT 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.

scholarly journals Fidaxomicin and OP-1118 Inhibit Clostridium difficile Toxin A- and B-Mediated Inflammatory Responses via Inhibition of NF-κB Activity

2017 ◽  
Vol 62 (1) ◽  
Author(s):  
Hon Wai Koon ◽  
Jiani Wang ◽  
Caroline C. Mussatto ◽  
Christina Ortiz ◽  
Elaine C. Lee ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Intestinal Inflammation ◽  
Inflammatory Responses ◽  
Human Colon ◽  
Toxin A ◽  
Primary Metabolite ◽  
Necrosis Factor Alpha ◽  
Toxin B ◽  
Content Type ◽  
Tnf Α

ABSTRACTClostridium difficilecauses diarrhea and colitis by releasing toxin A and toxin B. In the human colon, both toxins cause intestinal inflammation and stimulate tumor necrosis factor alpha (TNF-α) expression via the activation of NF-κB. It is well established that the macrolide antibiotic fidaxomicin is associated with reduced relapses ofC. difficileinfection. We showed that fidaxomicin and its primary metabolite OP-1118 significantly inhibited toxin A-mediated intestinal inflammation in micein vivoand toxin A-induced cell roundingin vitro. We aim to determine whether fidaxomicin and OP-1118 possess anti-inflammatory effects against toxin A and toxin B in the human colon and examine the mechanism of this response. We used fresh human colonic explants, NCM460 human colonic epithelial cells, and RAW264.7 mouse macrophages to study the mechanism of the activity of fidaxomicin and OP-1118 against toxin A- and B-mediated cytokine expression and apoptosis. Fidaxomicin and OP-1118 dose-dependently inhibited toxin A- and B-induced TNF-α and interleukin-1β (IL-1β) mRNA expression and histological damage in human colonic explants. Fidaxomicin and OP-1118 inhibited toxin A-mediated NF-κB phosphorylation in human and mouse intestinal mucosae. Fidaxomicin and OP-1118 also inhibited toxin A-mediated NF-κB phosphorylation and TNF-α expression in macrophages, which was reversed by the NF-κB activator phorbol myristate acetate (PMA). Fidaxomicin and OP-1118 prevented toxin A- and B-mediated apoptosis in NCM460 cells, which was reversed by the addition of PMA. PMA reversed the cytoprotective effect of fidaxomicin and OP-1118 in toxin-exposed human colonic explants. Fidaxomicin and OP-1118 inhibitC. difficiletoxin A- and B-mediated inflammatory responses, NF-κB phosphorylation, and tissue damage in the human colon.

scholarly journals A Combination of Three Fully Human Toxin A- and Toxin B-Specific Monoclonal Antibodies Protects against Challenge with Highly Virulent Epidemic Strains of Clostridium difficile in the Hamster Model

2015 ◽  
Vol 22 (7) ◽  
pp. 711-725 ◽  
Author(s):  
Natalie G. Anosova ◽  
Leah E. Cole ◽  
Lu Li ◽  
Jinrong Zhang ◽  
Anna M. Brown ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Clostridium Difficile ◽  
Toxin A ◽  
Healthy Human ◽  
Hamster Model ◽  
Toxin B ◽  
Content Type ◽  
Highly Virulent

ABSTRACTClostridium difficileinfection (CDI) is the principal cause of nosocomial diarrhea and pseudomembranous colitis associated with antibiotic therapy. Recent increases in the number of outbreaks attributed to highly virulent antibiotic-resistant strains underscore the importance of identifying efficacious alternatives to antibiotics to control this infection. CDI is mediated by two large exotoxins, toxins A and B. Strong humoral toxin-specific immune responses are associated with recovery and a lack of disease recurrence, whereas insufficient humoral responses are associated with recurrent CDI. Multiple approaches targeting these toxins, including intravenous immunoglobulin, neutralizing polymers, active vaccines, and, most recently, monoclonal antibodies (MAbs), have been explored, with various degrees of success. In this study, we describe the characterization of the first MAbs isolated from healthy human donors using a high-throughput B-cell cloning strategy. The MAbs were selected based on their ability to inhibit the actions of toxins A and Bin vitroand because of theirin vivoefficacy in a hamster challenge model. A potent 2-MAb cocktail was identified and then further potentiated by the addition of a second anti-toxin B MAb. This 3-MAb combination protected animals against mortality and also reduced the severity and duration of diarrhea associated with challenge with highly virulent strains ofC. difficiletoxinotypes 0 and III. This highly efficacious cocktail consists of one MAb specific to the receptor binding domain of toxin A and two MAbs specific to nonoverlapping regions of the glucosyltransferase domain of toxin B. This MAb combination offers great potential as a nonantibiotic treatment for the prevention of recurrent CDI.

scholarly journals Human Monoclonal Antibodies against Clostridium difficile Toxins A and B Inhibit Inflammatory and Histologic Responses to the Toxins in Human Colon and Peripheral Blood Monocytes

2013 ◽  
Vol 57 (7) ◽  
pp. 3214-3223 ◽  
Author(s):  
Hon Wai Koon ◽  
David Q. Shih ◽  
Tressia C. Hing ◽  
Jun Hwan Yoo ◽  
Samantha Ho ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Clostridium Difficile ◽  
Peripheral Blood ◽  
Blood Monocytes ◽  
Human Monoclonal Antibodies ◽  
Toxin A ◽  
Toxin B ◽  
Peripheral Blood Monocytes ◽  
Content Type ◽  
Tnf Α

ABSTRACTClostridium difficileinfection (CDI) is a common and debilitating nosocomial infection with high morbidity and mortality.C. difficilemediates diarrhea and colitis by releasing two toxins, toxin A and toxin B. Since both toxins stimulate proinflammatory signaling pathways in human colonocytes and both are involved in the pathophysiology of CDI, neutralization of toxin A and B activities may represent an important therapeutic approach against CDI. Recent studies indicated that human monoclonal antibodies (MAbs) against toxins A and B reduce their cytotoxic and secretory activities and prevent CDI in hamsters. Moreover, anti-toxin A and anti-toxin B MAbs together with antibiotics also effectively reduced recurrent CDI in humans. However, whether these MAbs neutralize toxin A- and toxin B-associated immune responses in human colonic mucosa or human peripheral blood monocyte cells (PBMCs) has never been examined. We used fresh human colonic biopsy specimens and peripheral blood monocytes to evaluate the effects of these antibodies against toxin A- and B-associated cytokine release, proinflammatory signaling, and histologic damage. Incubation of anti-toxin A (MK3415) or anti-toxin B (MK6072) MAbs with human PBMCs significantly inhibited toxin A- and toxin B-mediated tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) expression. MK3415 and MK6072 also diminished toxin A- and toxin B-mediated NF-κB p65 phosphorylation in human monocytes, respectively, and significantly reduced toxin A- and B-induced TNF-α and IL-1β expression as well as histologic damage in human colonic explants. Our results underline the effectiveness of MK3415 and MK6072 in blockingC. difficiletoxin A- and toxin B-mediated inflammatory responses and histologic damage.

scholarly journals A New Type of Toxin A-Negative, Toxin B-Positive Clostridium difficile Strain Lacking a CompletetcdAGene

2014 ◽  
Vol 53 (2) ◽  
pp. 692-695 ◽  
Author(s):  
Sandra Janezic ◽  
Mercedes Marín ◽  
Adoración Martín ◽  
Maja Rupnik
Keyword(s):  
Clostridium Difficile ◽  
Virulence Factors ◽  
Diagnostic Tests ◽  
Integration Site ◽  
Molecular Diagnostic ◽  
Binary Toxin ◽  
Toxin A ◽  
Toxin B ◽  
Content Type ◽  
New Type

Toxins A and B are the main virulence factors ofClostridium difficileand are the targets for molecular diagnostic tests. Here, we describe a new toxin A-negative, toxin B-positive, binary toxin CDT (Clostridium difficiletransferase)-negative (A−B+CDT−) toxinotype (XXXII) characterized by a variant type of pathogenicity locus (PaLoc) withouttcdAand with atypical organization of the PaLoc integration site.

scholarly journals An Optimized, Synthetic DNA Vaccine Encoding the Toxin A and Toxin B Receptor Binding Domains of Clostridium difficile Induces Protective Antibody ResponsesIn Vivo

2014 ◽  
Vol 82 (10) ◽  
pp. 4080-4091 ◽  
Author(s):  
Scott M. Baliban ◽  
Amanda Michael ◽  
Berje Shammassian ◽  
Shikata Mudakha ◽  
Amir S. Khan ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Dna Vaccine ◽  
Receptor Binding ◽  
Nonhuman Primates ◽  
Colonic Disease ◽  
Toxin A ◽  
Lethal Challenge ◽  
Toxin B ◽  
Content Type ◽  
Binding Domains

ABSTRACTClostridium difficile-associated disease (CDAD) constitutes a large majority of nosocomial diarrhea cases in industrialized nations and is mediated by the effects of two secreted toxins, toxin A (TcdA) and toxin B (TcdB). Patients who develop strong antitoxin antibody responses can clearC. difficileinfection and remain disease free. Key toxin-neutralizing epitopes have been found within the carboxy-terminal receptor binding domains (RBDs) of TcdA and TcdB, which has generated interest in developing the RBD as a viable vaccine target. While numerous platforms have been studied, very little data describes the potential of DNA vaccination against CDAD. Therefore, we created highly optimized plasmids encoding the RBDs from TcdA and TcdB in which any putativeN-linked glycosylation sites were altered. Mice and nonhuman primates were immunized intramuscularly, followed byin vivoelectroporation, and in these animal models, vaccination induced significant levels of both anti-RBD antibodies (blood and stool) and RBD-specific antibody-secreting cells. Further characterization revealed that sera from immunized mice and nonhuman primates could detect RBD protein from transfected cells, as well as neutralize purified toxins in anin vitrocytotoxicity assay. Mice that were immunized with plasmids or given nonhuman-primate sera were protected from a lethal challenge with purified TcdA and/or TcdB. Moreover, immunized mice were significantly protected when challenged withC. difficilespores from homologous (VPI 10463) and heterologous, epidemic (UK1) strains. These data demonstrate the robust immunogenicity and efficacy of a TcdA/B RBD-based DNA vaccine in preclinical models of acute toxin-associated and intragastric, spore-induced colonic disease.

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.

Sign in / Sign up

Export Citation Format

Share Document