Laboratory comparison between cell cytotoxicity neutralization assay and ultrasensitive single molecule counting technology for detection of Clostridioides difficile toxins A and B, PCR, enzyme immunoassays, and multistep algorithms

Sandlund, Johanna; Mills, Ray; Griego-Fullbright, Christen; Wagner, Aaron; Estis, Joel; Bartolome, Amelita; Almazan, Anna; Tam, Stanley; Biscocho, Sheryl; Abusali, Salina; Nolan, Niamh; Bishop, Jeffrey J.; Todd, John; Young, Stephen

doi:10.1016/j.diagmicrobio.2019.04.002

Cited by 13 publications

(10 citation statements)

References 25 publications

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“…This is in line with previous findings showing that the presence of toxin better correlates with severity and outcome than presence of toxigenic organism only (7,8). CDI is a toxin-mediated disease, and only 50% of NAAT ϩ patients in this study had detectable toxins, which is in line with other studies using an ultrasensitive toxin assay (11,19,20). Toxin gene expression is regulated by multiple different factors, including quorum signaling, temperature, and metabolism changes (21,22), and the presence of toxin gene(s) does not equal presence of toxins (23).…”

Section: Discussionsupporting

confidence: 93%

Increased Clinical Specificity with Ultrasensitive Detection of Clostridioides difficile Toxins: Reduction of Overdiagnosis Compared to Nucleic Acid Amplification Tests

Sandlund¹,

Estis²,

Katzenbach³

et al. 2019

J Clin Microbiol

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Clostridioides difficile infection (CDI) is one of the most common health care-associated infections, resulting in significant morbidity, mortality, and economic burden. Diagnosis of CDI relies on the assessment of clinical presentation and laboratory tests. We evaluated the clinical performance of ultrasensitive single-molecule counting technology for detection of C. difficile toxins A and B. Stool specimens from 298 patients with suspected CDI were tested with the nucleic acid amplification test (NAAT; BD MAX Cdiff assay or Xpert C. difficile assay) and Singulex Clarity C. diff toxins A/B assay. Specimens with discordant results were tested with the cell cytotoxicity neutralization assay (CCNA), and the results were correlated with disease severity and outcome. There were 64 NAAT-positive and 234 NAAT-negative samples. Of the 32 NAAT+/Clarity− and 4 NAAT−/Clarity+ samples, there were 26 CCNA− and 4 CCNA− samples, respectively. CDI relapse was more common in NAAT+/toxin+ patients than in NAAT+/toxin− and NAAT−/toxin− patients. The clinical specificity of Clarity and NAAT was 97.4% and 89.0%, respectively, and overdiagnosis was more than three times more common in NAAT+/toxin− than in NAAT+/toxin+ patients. The Clarity assay was superior to NAATs for the diagnosis of CDI, by reducing overdiagnosis and thereby increasing clinical specificity, and the presence of toxins was associated with negative patient outcomes.

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Section: Discussionsupporting

confidence: 93%

Increased Clinical Specificity with Ultrasensitive Detection of Clostridioides difficile Toxins: Reduction of Overdiagnosis Compared to Nucleic Acid Amplification Tests

Sandlund¹,

Estis²,

Katzenbach³

et al. 2019

J Clin Microbiol

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“…It could have been impacted by the differences in limits of detection between CCNA and Clarity, as CCNA has an estimated limit of detection of 50 to 100 pg/ml and Clarity has a cutoff of 12 pg/ml (21). The Clarity assay showed higher specificity than PCR while maintaining acceptable sensitivity (78 to 96%), which has been supported by other studies (11,(22)(23)(24). Predictably, specificity was reduced while sensitivity was improved for tests that detect the presence of either toxin genes or C. difficile antigens ( Table 1).…”

Section: Discussionsupporting

confidence: 61%

Evaluation of Cycle Threshold, Toxin Concentration, and Clinical Characteristics of Clostridioides difficile Infection in Patients with Discordant Diagnostic Test Results

et al. 2020

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Clostridioides difficile infection (CDI) is one of the most common health care-associated infections that can cause significant morbidity and mortality. CDI diagnosis involves laboratory testing in conjunction with clinical assessment. The objective of this study was to assess the performance of various C. difficile tests and to compare clinical characteristics, Xpert C. difficile/Epi (PCR) cycle threshold (CT), and Singulex Clarity C. diff toxins A/B (Clarity) concentrations between groups with discordant test results. Unformed stool specimens from 200 hospitalized adults (100 PCR positive and 100 negative) were tested by cell cytotoxicity neutralization assay (CCNA), C. diff Quik Chek Complete (Quik Chek), Premier Toxins A and B, and Clarity. Clinical data, including CDI severity and CDI risk factors, were compared between discordant test results. Compared to CCNA, PCR had the highest sensitivity at 100% and Quik Chek had the highest specificity at 100%. Among clinical and laboratory data studied, prevalences of leukocytosis, prior antibiotic use, and hospitalizations were consistently higher across all subgroups in comparisons of toxin-positive to toxin-negative patients. Among PCR-positive samples, the median CT was lower in toxin-positive samples than in toxin-negative samples; however, CT ranges overlapped. Among Clarity-positive samples, the quantitative toxin concentration was significantly higher in toxin-positive samples than in toxin-negative samples as determined by CCNA and Quik Chek Toxin A and B. Laboratory tests for CDI vary in sensitivity and specificity. The quantitative toxin concentration may offer value in guiding CDI diagnosis and treatment. The presence of leukocytosis, prior antibiotic use, and previous hospitalizations may assist with CDI diagnosis, while other clinical parameters may not be consistently reliable.

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“…The Singulex Clarity C. diff toxins A/B assay (herein, Clarity), for use on the Singulex Clarity system (Singulex, Inc., Alameda, CA, USA), was developed to meet this need. The automated platform utilizes singlemolecule counting technology for a rapid, ultrasensitive detection of analytes down to femtogram-per-milliliter concentrations (13,14). Regulatory studies require comparison of free-toxin tests with CCCNA, a reference method that is known to have issues related to toxin stability and subjectivity (4).…”

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confidence: 99%

Ultrasensitive Detection of Clostridioides difficile Toxins in Stool by Use of Single-Molecule Counting Technology: Comparison with Detection of Free Toxin by Cell Culture Cytotoxicity Neutralization Assay

Hansen

Young

et al. 2019

J Clin Microbiol

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Laboratory tests for Clostridioides difficile infection (CDI) rely on the detection of free toxin or molecular detection of toxin genes. The Singulex Clarity C. diff toxins A/B assay is a rapid, automated, and ultrasensitive assay that detects C. difficile toxins A and B in stool. We compared CDI assays across two prospective multicenter studies to set a cutoff for the Clarity assay and to independently validate the performance compared with that of a cell culture cytotoxicity neutralization assay (CCCNA). The cutoff was set by two sites testing fresh samples from 897 subjects with suspected CDI and then validated at four sites testing fresh samples from 1,005 subjects with suspected CDI. CCCNA testing was performed at a centralized laboratory. Samples with discrepant results between the Clarity assay and CCCNA were retested with CCCNA when the Clarity result agreed with that of at least one comparator method; toxin enzyme immunoassays (EIA), glutamate dehydrogenase (GDH) detection, and PCR were performed on all samples. The cutoff for the Clarity assay was set at 12.0 pg/ml. Compared to results with CCCNA, the Clarity assay initially had 85.2% positive agreement and 92.4% negative agreement. However, when samples with discrepant results between the Clarity assay and CCCNA in the validation study were retested by CCCNA, 13/17 (76.5%) Clarity-negative but CCCNA-positive samples (Clarity+/CCCNA−) became CCCNA−, and 5/26 (19.2%) Clarity+/CCCNA− samples became CCCNA+, resulting in a 96.3% positive agreement and 93.0% negative agreement between Clarity and CCCNA results. The toxin EIA had 59.8% positive agreement with CCCNA. The Clarity assay was the most sensitive free-toxin immunoassay, capable of providing CDI diagnosis in a single-step solution. A different CCCNA result was reported for 42% of retested samples, increasing the positive agreement between Clarity and CCCNA from 85.2% to 96.3% and indicating the challenges of comparing free-toxin results to CCCNA results as a reference standard.

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Laboratory comparison between cell cytotoxicity neutralization assay and ultrasensitive single molecule counting technology for detection of Clostridioides difficile toxins A and B, PCR, enzyme immunoassays, and multistep algorithms

Cited by 13 publications

References 25 publications

Increased Clinical Specificity with Ultrasensitive Detection of Clostridioides difficile Toxins: Reduction of Overdiagnosis Compared to Nucleic Acid Amplification Tests

Increased Clinical Specificity with Ultrasensitive Detection of Clostridioides difficile Toxins: Reduction of Overdiagnosis Compared to Nucleic Acid Amplification Tests

Evaluation of Cycle Threshold, Toxin Concentration, and Clinical Characteristics of Clostridioides difficile Infection in Patients with Discordant Diagnostic Test Results

Ultrasensitive Detection of Clostridioides difficile Toxins in Stool by Use of Single-Molecule Counting Technology: Comparison with Detection of Free Toxin by Cell Culture Cytotoxicity Neutralization Assay

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