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 Evaluation of the QiagenartusC. difficile QS-RGQ Kit for Detection of Clostridium difficile Toxins A and B in Clinical Stool Specimens

2015 ◽  
Vol 53 (6) ◽  
pp. 1942-1944 ◽  
Author(s):  
Nathalie Jazmati ◽  
Pia Wiegel ◽  
Božica Ličanin ◽  
Georg Plum
Keyword(s):  
Nucleic Acid ◽  
Clostridium Difficile ◽  
Positive Predictive Value ◽  
Negative Predictive Value ◽  
Predictive Value ◽  
Nucleic Acid Amplification Test ◽  
Nucleic Acid Amplification ◽  
Content Type ◽  
Stool Specimens ◽  
Sensitivity Specificity

We compared the QiagenartusC. difficile QS-RGQ kit, a new nucleic acid amplification test for the detection ofClostridium difficiletoxins in stool specimens, with the Cepheid XpertC. difficiletest. The sensitivity, specificity, positive predictive value, and negative predictive value for the QiagenartusC. difficile QS-RGQ test were 100%, 89.5%, 60.9%, and 100%, and those for the Cepheid XpertC. difficiletest were 100%, 90%, 62.2%, and 100%, respectively.

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 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 Development and Validation of Digital Enzyme-Linked Immunosorbent Assays for Ultrasensitive Detection and Quantification of Clostridium difficile Toxins in Stool

2015 ◽  
Vol 53 (10) ◽  
pp. 3204-3212 ◽  
Author(s):  
Linan Song ◽  
Mingwei Zhao ◽  
David C. Duffy ◽  
Joshua Hansen ◽  
Kelsey Shields ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Cytotoxicity Assay ◽  
Nucleic Acid Amplification ◽  
Analytical Sensitivity ◽  
Toxin B ◽  
Content Type ◽  
Toxigenic Culture ◽  
Immunosorbent Assays ◽  
Digital Elisa ◽  
Detection And Quantification

The currently available diagnostics forClostridium difficileinfection (CDI) have major limitations. Despite mounting evidence that toxin detection is paramount for diagnosis, conventional toxin immunoassays are insufficiently sensitive and cytotoxicity assays too complex; assays that detect toxigenic organisms (toxigenic culture [TC] and nucleic acid amplification testing [NAAT]) are confounded by asymptomatic colonization by toxigenicC. difficile. We developed ultrasensitive digital enzyme-linked immunosorbent assays (ELISAs) for toxins A and B using single-molecule array technology and validated the assays using (i) culture filtrates from a panel of clinicalC. difficileisolates and (ii) 149 adult stool specimens already tested routinely by NAAT. The digital ELISAs detected toxins A and B in stool with limits of detection of 0.45 and 1.5 pg/ml, respectively, quantified toxins across a 4-log range, and detected toxins from all clinical strains studied. Using specimens that were negative by cytotoxicity assay/TC/NAAT, clinical cutoffs were set at 29.4 pg/ml (toxin A) and 23.3 pg/ml (toxin B); the resulting clinical specificities were 96% and 98%, respectively. The toxin B digital ELISA was 100% sensitive versus cytotoxicity assay. Twenty-five percent and 22% of the samples positive by NAAT and TC, respectively, were negative by the toxin B digital ELISA, consistent with the presence of organism but minimal or no toxin. The mean toxin levels by digital ELISA were 1.5- to 1.7-fold higher in five patients with CDI-attributable severe outcomes, versus 68 patients without, but this difference was not statistically significant. Ultrasensitive digital ELISAs for the detection and quantification of toxins A and B in stool can provide a rapid and simple tool for the diagnosis of CDI with both high analytical sensitivity and high clinical specificity.

Detection of Clostridium difficile in Fecal Specimens: a Comparative Evaluation of Nucleic Acid Amplification Test and Toxigenic Culture

2016 ◽  
Vol 62 (10/2016) ◽  
Author(s):  
Shima Lotfian ◽  
Masoumeh Douraghi ◽  
Amir Aliramezani ◽  
Sedighe Ghourchian ◽  
Abdolfatah Sarrafnejad ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Clostridium Difficile ◽  
Comparative Evaluation ◽  
Nucleic Acid Amplification Test ◽  
Nucleic Acid Amplification ◽  
Toxigenic Culture

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 Supplementary Testing Is Not Required in the cobas 4800 CT/NG Test for Neisseria gonorrhoeae Weak-Positive Urogenital Samples

2014 ◽  
Vol 53 (1) ◽  
pp. 327-328 ◽  
Author(s):  
Collette Bromhead ◽  
Nadika Liyanarachchy ◽  
Julia Mayes ◽  
Arlo Upton ◽  
Michelle Balm
Keyword(s):  
Nucleic Acid ◽  
Neisseria Gonorrhoeae ◽  
Nucleic Acid Amplification Test ◽  
Pretest Probability ◽  
Nucleic Acid Amplification ◽  
Test Results ◽  
Content Type

Weak-positiveNeisseria gonorrhoeaenucleic acid amplification test results are difficult to interpret. We show that the frequency of unconfirmedN. gonorrhoeaeresults from the cobas 4800 test rises exponentially after 38.0 cycles, where the likelihood of an unconfirmed result exceeds 29%. Supplementary testing of such samples should be avoided; instead, treatment should be based on clinical pretest probability.

scholarly journals Pooled Nucleic Acid Amplification Test for Screening of Stool Specimens for Shiga Toxin-Producing Escherichia coli

2016 ◽  
Vol 54 (11) ◽  
pp. 2711-2715
Author(s):  
Agatha N. Jassem ◽  
Frank Y. Chou ◽  
Cathevine Yang ◽  
Matthew A. Croxen ◽  
Katarina D. M. Pintar ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nucleic Acid ◽  
Shiga Toxin ◽  
Large Scale ◽  
Nucleic Acid Amplification Test ◽  
Nucleic Acid Amplification ◽  
Accurate Estimation ◽  
Content Type ◽  
Pooling Strategy ◽  
Stool Specimens

Shiga toxin-producingEscherichia coli(STEC)-associated enteric illness is attributed to O157 and non-O157 serotypes; however, traditional culture-based methods underdetect non-O157 STEC. Labor and cost of consumables are major barriers to implementation of the CDC recommendation to test all stools for both O157 and non-O157 serotypes. We evaluated the feasibility of a pooled nucleic acid amplification test (NAAT) as an approach for screening stool specimens for STEC. For retrospective evaluation, 300 stool specimens were used to create pools of 10 samples each. The sensitivity was 83% for the preenrichment pooling strategy and 100% for the postenrichment pooling strategy compared with those for individual NAAT results. The difference in cycle threshold (CT) between individual and pooled NAAT results for specimens was significantly lower and more consistent for postenrichment pooling (stx1mean = 3.90,stx2mean = 4.28) than those for preenrichment pooling (excluding undetected specimens;stx1mean = 9.34,stx2mean = 8.96) (P≤ 0.0013). Cost of consumables and labor time savings of 48 to 81% and 6 to 66%, respectively, were estimated for the testing of 90 specimens by the postenrichment pooled NAAT strategy on the basis of an expected 1 to 2% positivity rate. A 30-day prospective head-to-head clinical trial involving 512 specimens confirmed the sensitivity and labor savings associated with the postenrichment pooled NAAT strategy. The postenrichment pooled NAAT strategy described here is suitable for efficient large-scale surveillance of all STEC serotypes. Comprehensive detection of STEC will result in accurate estimation of STEC burden and, consequently, appropriate public health interventions.

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.

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