scholarly journals Clostridium difficile Infections in Children: Impact of the Diagnostic Method on Infection Rates

2016 ◽  
Vol 37 (9) ◽  
pp. 1087-1093 ◽  
Author(s):  
Mohammad AlGhounaim ◽  
Yves Longtin ◽  
Milagros Gonzales ◽  
Joanna Merckx ◽  
Nicholas Winters ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Pediatric Population ◽  
Toxin Production ◽  
Enzyme Linked Immunosorbent Assay ◽  
Medical Problems ◽  
Toxin A ◽  
Crude Odds Ratio ◽  
Entire Cohort ◽  
Elisa Testing ◽  
Standardize Laboratory

BACKGROUNDPolymerase chain reaction (PCR) assays based on the detection of the toxin B gene are replacing enzyme-linked immunosorbent assay (ELISA)–based toxin production detection or cell cytotoxicity assay in most laboratories.OBJECTIVETo determine the proportion of pediatric patients diagnosed withClostridium difficile infection by PCR who would have also been diagnosed by ELISA and to compare the clinical characteristics of PCR+/ELISA+ vs PCR+/ELISA− patients.METHODSUsing the microbiology laboratory information system, stool samples positive for C. difficile by PCR between October 2010 and July 2014 were identified. Using frozen stool specimens, an ELISA for toxin A and B was performed. A retrospective medical chart review was conducted to obtain demographic and clinical data. Duplicate samples were excluded.RESULTSA total of 136 PCR-positive samples underwent ELISA testing: 54 (40%) were positive for toxin A or B. The mean (SD) age of the entire cohort was 8.5 (6.2) years. There was no difference in age, gender, clinical manifestation, previous medical problems, and management between patients positive or negative by ELISA. However, patients positive by ELISA were more likely to have had a recent exposure to antibiotics (67.9% vs 50%; crude odds ratio, 2.1 [95% CI, 1.03–4.28]).CONCLUSIONIn our pediatric population, 60% of patients with C. difficile diagnosed by PCR had no toxin detectable by ELISA. ELISA-negative patients were less likely to have received an antibiotic recently compared with ELISA-positive patients. These results highlight the need to standardize laboratory criteria for the diagnosis of C. difficile infections in children.Infect Control Hosp Epidemiol 2016;37:1087–1093

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.

scholarly journals Evaluating the Effects of Surotomycin Treatment on Clostridium difficile Toxin A and B Production, Immune Response, and Morphological Changes

2016 ◽  
Vol 60 (6) ◽  
pp. 3519-3523 ◽  
Author(s):  
Bradley T. Endres ◽  
Eugénie Bassères ◽  
Mohammed Khaleduzzaman ◽  
M. Jahangir Alam ◽  
Laurent Chesnel ◽  
...  
Keyword(s):  
Immune Response ◽  
Clostridium Difficile ◽  
Morphological Changes ◽  
Toxin Production ◽  
Morphological Data ◽  
Growth Media ◽  
Toxin A ◽  
Content Type ◽  
Cyclic Lipopeptide ◽  
The Impact

Surotomycin is a cyclic lipopeptide in development forClostridium difficile-associated diarrhea. This study aimed to assess the impact of surotomycin exposure onC. difficiletoxin A and B concentrations and the associated changes in immune response in comparison to vancomycin and metronidazole. Time-kill curve assays were performed using strain R20291 (BI/NAP1/027) at supra-MICs (4× and 40×) and sub-MICs (0.5×) of surotomycin and comparators. Following treatment, CFU counts, toxin A and B concentrations, and cellular morphological changes using scanning electron microscopy were examined. Inflammatory response was determined by measuring interleukin-8 (IL-8) concentrations from polarized Caco-2 cells exposed to antibiotic-treatedC. difficilegrowth media. Supra-MICs (4× and 40×) of surotomycin resulted in a reduction of vegetative cells over 72 h (4-log difference,P< 0.01) compared to controls. These results correlated with decreases of 77% and 68% in toxin A and B production at 48 h, respectively (P< 0.005, each), which resulted in a significant reduction in IL-8 concentration compared to controls. Similar results were observed with comparator antibiotics. Bacterial cell morphology showed that the cell wall was broken apart by surotomycin treatment at supra-MICs while sub-MIC studies showed a “deflated” phenotype plus a rippling effect. These results suggest that surotomycin has potent killing effects onC. difficilethat results in reduced toxin production and attenuates the immune response similar to comparator antibiotics. The morphological data also confirm observations that surotomycin is a membrane-active antibiotic.

scholarly journals Intestinal Secretory Factor Released by Macrophages Stimulated with Clostridium difficile Toxin A: Role of Interleukin 1β

1998 ◽  
Vol 66 (10) ◽  
pp. 4910-4916 ◽  
Author(s):  
M. F. G. Rocha ◽  
A. M. Soares ◽  
C. A. Flores ◽  
T. S. Steiner ◽  
D. M. Lyerly ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Receptor Antagonist ◽  
Short Circuit ◽  
Intestinal Secretion ◽  
Interleukin 1Β ◽  
Enzyme Linked Immunosorbent Assay ◽  
Protein Synthesis Inhibitor ◽  
Lipoxygenase Inhibitor ◽  
Toxin A ◽  
Tnf Α

ABSTRACT Clostridium difficile toxin A is associated with enterocolitis in animals and humans. However, the mechanisms of its secretory and damaging effects are not totally understood. In this work, we examined the intestinal secretion of electrolytes and water caused by supernatants from macrophages stimulated with toxin A in rabbit ileal mucosa mounted in Üssing chambers. We also investigated the mechanism by which the intestinal secretory factor (ISF) is released from stimulated macrophages. Supernatants from macrophages stimulated with toxin A caused potent intestinal secretion (change in short-circuit current [ΔIsc], 76 μA · cm−2; P < 0.01). The release of the ISF was pertussis toxin sensitive (reduction, 61%; P < 0.01) and was also reduced (P < 0.05) by a protein synthesis inhibitor (67%), protease inhibitors (57%), a phospholipase A2 inhibitor (54%), a cyclo-oxygenase inhibitor (62%), a dual cyclo- and lipoxygenase inhibitor (48%), a platelet-activating factor (PAF) receptor antagonist (55%), and tumor necrosis factor alpha (TNF-α) synthesis inhibitors (48%). However, this release was not inhibited by a lipo-oxygenase inhibitor. Monoclonal anti-interleukin 1β (IL-1β) but not anti-IL-1α antibody blocked (72%; P < 0.01) the secretory action of the ISF, as did recombinant human IL-1 receptor antagonist (80%;P < 0.01). High levels of IL-1β (3,476 pg/ml) were detected by an enzyme-linked immunosorbent assay in the above supernatants. Furthermore, the addition of IL-1β to the serosal side caused a potent secretory effect (ΔIsc, 80 μA · cm−2; P < 0.01). These results show that macrophages stimulated with toxin A release an ISF capable of provoking intestinal secretion. The regulation of this factor is dependent upon the activation of the G protein. In addition, prostaglandins, PAF, and TNF-α are involved in the release of the ISF. We conclude that IL-1β is probably the ISF released by macrophages in response to toxin A.

scholarly journals Anti-Clostridium difficile bovine immunoglobulin concentrate inhibits cytotoxicity and enterotoxicity of C. difficile toxins.

1996 ◽  
Vol 40 (2) ◽  
pp. 373-379 ◽  
Author(s):  
C P Kelly ◽  
C Pothoulakis ◽  
F Vavva ◽  
I Castagliuolo ◽  
E F Bostwick ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Biological Effects ◽  
Human Fibroblasts ◽  
Enzyme Linked Immunosorbent Assay ◽  
Toxin A ◽  
Ileal Loop ◽  
Cytotoxic Effects ◽  
Bovine Immunoglobulin

Clostridium difficile diarrhea and colitis result from the actions of bacterial exotoxins on the colonic mucosa. This study examined the ability of hyperimmune bovine colostral antibodies to neutralize the biological effects of these toxins. Anti-C. difficile bovine immunoglobulin concentrate was prepared from the colostral milk of Holstein cows previously immunized with C. difficile toxoids. The anti-C. difficile bovine immunoglobulin concentrate contained high levels of bovine immunoglobulin G specific for C. difficile toxins A and B, as evaluated by enzyme-linked immunosorbent assay. Anti-C. difficile bovine immunoglobulin concentrate neutralized the cytotoxic effects of purified toxin A and toxin B on cultured human fibroblasts, whereas control bovine immunoglobulin concentrate had little toxin-neutralizing activity. Anti-C. difficile bovine immunoglobulin concentrate also blocked the binding of toxin A to its enterocyte receptor and inhibited the enterotoxic effects of C. difficile toxins on the rat ileum, as measured by an increased rat ileal loop weight/length ratio (63% inhibition; P < 0.01), increased mannitol permeability (92% inhibition; P < 0.01), and histologic grading of enteritis (P < 0.01 versus nonimmune bovine immunoglobulin concentrate). Thus, anti-C. difficile bovine immunoglobulin concentrate neutralizes the cytotoxic effects of C. difficile toxins in vitro and inhibits their enterotoxic effects in vivo. This agent may be clinically useful in the prevention and treatment of C. difficile diarrhea and colitis.

scholarly journals Effect of sub-MIC concentrations of metronidazole, vancomycin, clindamycin and linezolid on toxin gene transcription and production in Clostridium difficile

2008 ◽  
Vol 57 (6) ◽  
pp. 776-783 ◽  
Author(s):  
Michael Gerber ◽  
Christiane Walch ◽  
Birgit Löffler ◽  
Kristin Tischendorf ◽  
Udo Reischl ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Gene Transcription ◽  
Toxin Production ◽  
Transcriptomic Analysis ◽  
Toxin Gene ◽  
Toxin A ◽  
Toxin Genes ◽  
Cell Culture Assay ◽  
Hospital Acquired ◽  
The Impact

Clostridium difficile is the major cause of hospital-acquired infectious diarrhoea. Several antimicrobials are known to induce and promote C. difficile-associated diarrhoea (CDAD). The impact of metronidazole (MTR), vancomycin (VAN), clindamycin (CLI) and linezolid (LZD) on growth, toxin gene transcription and toxin production in C. difficile was investigated. Four C. difficile strains were grown with and without sub-MIC concentrations of MTR, VAN, CLI and LZD (0.5× MIC) and growth was measured by colony counts. Toxin production was detected using ELISA (for toxin A) and a cytotoxicity assay (for toxin B) in culture supernatants and also in sonicated cells. Real-time PCR was used to measure transcription of the toxin A and B genes. The aim of this work was to combine analysis of toxin A and B production by ELISA or cell culture assay with transcriptomic analysis. The four strains showed similar growth and different levels of toxin production in the absence of antibiotics. An antibiotic-free control showed toxin production at a late stage when the plateau phase of bacterial growth was reached, whereas antibiotic-exposed strains showed earlier toxin production. All of the antibiotics used except CLI increased the transcription rate of toxin genes. The findings of this study show that sub-MIC concentrations of antibiotics can cause changes in gene transcription of the major virulence factors of C. difficile. This study describes a new method for transcriptomic analysis of toxin genes in C. difficile.

scholarly journals Effects of ciprofloxacin on the expression and production of exotoxins by Clostridium difficile

2013 ◽  
Vol 62 (5) ◽  
pp. 741-747 ◽  
Author(s):  
Michael John Aldape ◽  
Aaron Eugene Packham ◽  
Drew William Nute ◽  
Amy Evelyn Bryant ◽  
Dennis Leroy Stevens
Keyword(s):  
Gene Expression ◽  
Clostridium Difficile ◽  
Untreated Control ◽  
Protein Production ◽  
Temporal Dynamics ◽  
Toxin Production ◽  
Resistant Isolate ◽  
Toxin Gene ◽  
New Paradigm ◽  
Toxin A

Hypervirulent BI/NAP1/027 strains of Clostridium difficile have been associated with increased mortality of C. difficile infection (CDI). The emergence of highly fluoroquinolone (FLQ)-resistant BI/NAP1/027 strains suggests that FLQ exposure may be a risk factor for CDI development. However, the mechanism for this is not clear. We compared the effects of subinhibitory concentrations of ciprofloxacin on Toxin A and B gene expression and protein production in recent (strain 039) and historical (strain 5325) BI/NAP1/027 clinical isolates with high- and low-level ciprofloxacin resistance, respectively. In the highly ciprofloxacin-resistant isolate (strain 039), ciprofloxacin significantly and dose-dependently increased Toxin A gene expression and shifted its expression to earlier in its growth cycle; TcdB gene expression also increased but was less sensitive to low-dose ciprofloxacin. Maximal Toxin A/B production (4 ng ml−1) was increased twofold and occurred significantly earlier than in the untreated control. In strain 5325, ciprofloxacin at 0.25×MIC markedly increased both tcdA and tcdB expression but their temporal dynamics were unchanged. Maximal toxin production (250 ng ml−1) was reduced approximately threefold compared with that of the untreated control. These results demonstrate significant differences in ciprofloxacin-induced toxin gene expression and protein production among BI/NAP1/027 isolates, and offer a new paradigm for FLQ-associated CDI caused by recent, highly antibiotic-resistant strains.

scholarly journals Synergistic Effects of Antimicrobial Peptides and Antibiotics against Clostridium difficile

2014 ◽  
Vol 58 (10) ◽  
pp. 5719-5725 ◽  
Author(s):  
Sabine Nuding ◽  
Tina Frasch ◽  
Martin Schaller ◽  
Eduard F. Stange ◽  
Lutz T. Zabel
Keyword(s):  
Clostridium Difficile ◽  
Antimicrobial Peptides ◽  
Bacterial Cell ◽  
Synergistic Effects ◽  
Toxin Production ◽  
Enzyme Linked Immunosorbent Assay ◽  
Bacterial Killing ◽  
Content Type ◽  
Membrane Perturbation

ABSTRACTAccelerating rates of health care-associated infections caused byClostridium difficile, with increasing recurrence and rising antibiotic resistance rates, have become a serious problem in recent years. This study was conducted to explore whether a combination of antibiotics with human antimicrobial peptides may lead to an increase in antibacterial activity. Thein vitroactivities of the antimicrobial peptides HBD1 to HBD3, HNP1, HD5, and LL-37 and the antibiotics tigecycline, moxifloxacin, piperacillin-tazobactam, and meropenem alone or in combination against 10 toxinogenic and 10 nontoxinogenicC. difficilestrains were investigated. Bacterial viability was determined by flow cytometry and toxin production by enzyme-linked immunosorbent assay (ELISA). When combined at subinhibitory concentrations, antimicrobial peptides and antibiotics generally led to an additive killing effect against toxinogenic and nontoxinogenicC. difficilestrains. However, LL-37 and HBD3 acted in synergism with all the antibiotics that were tested. Electron microscopy revealed membrane perturbation in bacterial cell walls by HBD3. In 3 out of 10 toxinogenic strains, HBD3, LL-37, piperacillin-tazobactam, and meropenem administration led to an increased toxin release which was not neutralized by the addition of HNP1. Antimicrobial peptides increase the bacterial killing of antibiotics againstC. difficileregardless of the antibiotics' mode of action. Membrane perturbation in or pore formation on the bacterial cell wall may enhance the uptake of antibiotics and increase their antibacterial effect. Therefore, a combination of antibiotics with antimicrobial peptides may represent a promising novel approach to the treatment ofC. difficileinfections.

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