Ultrasensitive Detection of Clostridium difficile Toxins Reveals Suboptimal Accuracy of Toxin Gene Cycle Thresholds for Toxin Predictions

Sandlund, Johanna; Wilcox, Mark H.

doi:10.1128/jcm.01885-18

Cited by 10 publications

(5 citation statements)

References 24 publications

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“…In the current study, 22.8% of all PCR ϩ /toxin Ϫ samples had tcdB C T values of Ͻ27.0, and 14.3% of all PCR ϩ /toxin ϩ samples had C T values of Ͼ27.0. Similar results have also recently been reported (13,32). Although there is a correlation between tcdB C T values and toxin concentration, the accuracy appears to be suboptimal for use in clinical practice.…”

Section: Discussionsupporting

confidence: 84%

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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Section: Discussionsupporting

confidence: 84%

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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“…This testing algorithm has already shown similar efficacy in a pediatric context (31). However, some have argued that the CT-toxin lacks 100% analytical sensitivity and lacks specificity to differentiate between true CDI and colonization (32,33), and in a commentary published earlier this year, Sandlund and Wilcox specifically argue for use of alternative testing modalities such as ultrasensitive toxin immunoassays using single-molecule counting technology, which purports to offer a highly sensitive and more specific result than PCR while maintaining rapid turnaround time (34,35). While a potential option, the ultrasensitive toxin immunoassay currently is not available for use in routine clinical practice.…”

Section: Discussionmentioning

confidence: 99%

Dual Reporting of Clostridioides difficile PCR and Predicted Toxin Result Based on PCR Cycle Threshold Reduces Treatment of Toxin-Negative Patients without Increases in Adverse Outcomes

et al. 2019

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Nucleic acid amplification tests are commonly used to diagnose Clostridioides difficile infection (CDI). Two-step testing with a toxin enzyme immunoassay is recommended to discriminate between infection and colonization but requires additional resources. Prior studies showed that PCR cycle threshold (CT) can predict toxin positivity with high negative predictive value. Starting in October 2016, the predicted toxin result (CT-toxin) based on a validated cutoff was routinely reported at our facility. To evaluate the clinical efficacy of this reporting, all adult patients with positive GeneXpert PCR results from October 2016 through October 2017 underwent a chart review to measure the recurrence of or conversion to a CT-toxin+ result and 30-day all-cause mortality. There were 482 positive PCR tests in 430 unique patients, 282 CT-toxin+ and 200 CT-toxin−. Patient characteristics were similar at testing, though CT-toxin+ patients had higher white blood cell (WBC) counts (12.5 × 103 versus 9.3 × 103 cells/μl; P = 0.001). All cases (n = 21) of fulminant CDI had a CT-toxin+ result. Index CT-toxin+ patients were significantly more likely to have a CT-toxin+ result within 90 days than CT-toxin− patients (17.4% [n = 49] versus 8.0% [n = 16], respectively; P = 0.003). Thirty-day all-cause mortality was higher in CT-toxin− patients (11.1% versus 6.8%; P = 0.1), though no deaths in CT-toxin− patients were directly attributable to CDI. Of the 200 CT-toxin− patients, 51.5% (n = 103) were treated for CDI. The rates of conversion to a CT-toxin+ result (8.8% versus 7.2%; P = 0.8) and all-cause mortality (8.8% versus 13.4%; P = 0.3) were similar between treated and untreated CT-toxin− patients, respectively. CT-based toxin prediction may identify patients at higher risk for CDI-related complications and reduce treatment among CT-toxin− patients.

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“…Two patients with the highest CCNA titers, 1:1,000 and 1:10,000, appeared to have CDI and responded to therapy. Of note, their NAAT cycle threshold (C T ) values were higher than the cutoff of 27, which has been reported to correlate with the presence of free toxins (15)(16)(17). Of the other 12 patients with CCNA-positive results of 1:10 or 1:100, 3 had no diarrhea in the hospital.…”

Section: Resultsmentioning

confidence: 95%

High Agreement Between an Ultrasensitive Clostridioides difficile Toxin Assay and a C. difficile Laboratory Algorithm Utilizing GDH-and-Toxin Enzyme Immunoassays and Cytotoxin Testing

Landry

Topal

Estis³

et al. 2020

J Clin Microbiol

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The Singulex Clarity C. diff toxins A/B (Clarity) assay is an automated, ultrasensitive immunoassay for the detection of Clostridioides difficile toxins in stool. In this study, the performance of the Clarity assay was compared to that of a multistep algorithm using an enzyme immunoassay (EIA) for detection of glutamate dehydrogenase (GDH) and toxins A and B arbitrated by a semiquantitative cell cytotoxicity neutralization assay (CCNA). The performance of the assay was evaluated using 211 residual deidentified stool samples tested with a GDH-and-toxin EIA (C. Diff Quik Chek Complete; Techlab), with GDH-and-toxin discordant samples tested with CCNA. The stool samples were stored at –80°C before being tested with the Clarity assay. For samples discordant between Clarity and the standard-of-care algorithm, the samples were tested with PCR (Xpert C. difficile; Cepheid), and chart review was performed. The testing algorithm resulted in 34 GDH+/toxin+, 53 GDH−/toxin−, and 124 GDH+/toxin− samples, of which 39 were CCNA+ and 85 were CCNA−. Clarity had 96.2% negative agreement with GDH−/toxin− samples, 100% positive agreement with GDH+/toxin+ samples, and 95.3% agreement with GDH+/toxin−/CCNA− samples. The Clarity result was invalid for one sample. Clarity agreed with 61.5% of GDH+/toxin−/CCNA+ samples, 90.0% of GDH+/toxin−/CCNA+ (high-positive) samples, and 31.6% of GDH+/toxin−/CCNA+ (low-positive) samples. The Singulex Clarity C. diff toxins A/B assay demonstrated high agreement with a testing algorithm utilizing a GDH-and-toxin EIA and CCNA. This novel automated assay may offer an accurate, stand-alone solution for C. difficile infection (CDI) diagnostics, and further prospective clinical studies are merited.

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Ultrasensitive Detection of Clostridium difficile Toxins Reveals Suboptimal Accuracy of Toxin Gene Cycle Thresholds for Toxin Predictions

Cited by 10 publications

References 24 publications

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

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

Dual Reporting of Clostridioides difficile PCR and Predicted Toxin Result Based on PCR Cycle Threshold Reduces Treatment of Toxin-Negative Patients without Increases in Adverse Outcomes

High Agreement Between an Ultrasensitive Clostridioides difficile Toxin Assay and a C. difficile Laboratory Algorithm Utilizing GDH-and-Toxin Enzyme Immunoassays and Cytotoxin Testing

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