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

ABSTRACT 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.

Download Full-text

Ultrasensitive Detection of Clostridioides difficile Toxins A and B by Use of Automated Single-Molecule Counting Technology

Journal of Clinical Microbiology ◽

10.1128/jcm.00908-18 ◽

2018 ◽

Vol 56 (11) ◽

Cited By ~ 19

Author(s):

Johanna Sandlund ◽

Amelita Bartolome ◽

Anna Almazan ◽

Stanley Tam ◽

Sheryl Biscocho ◽

...

Keyword(s):

Single Molecule ◽

Neutralization Assay ◽

Cross Reactivity ◽

Nucleic Acid Amplification ◽

Analytical Sensitivity ◽

Content Type ◽

Testing Procedures ◽

Clostridioides Difficile ◽

A Cell ◽

Stool Samples

ABSTRACT Current tests for the detection of Clostridioides (formerly Clostridium) difficile free toxins in feces lack sensitivity, while nucleic acid amplification tests lack clinical specificity. We have evaluated the Singulex Clarity C. diff toxins A/B assay (currently in development), an automated and rapid ultrasensitive immunoassay powered by single-molecule counting technology, for detection of C. difficile toxin A (TcdA) and toxin B (TcdB) in stool. The analytical sensitivity, analytical specificity, repeatability, and stability of the assay were determined. In a clinical evaluation, frozen stool samples from 311 patients with suspected C. difficile infection were tested with the Clarity C. diff toxins A/B assay, using an established cutoff value. Samples were tested with the Xpert C. difficile/Epi assay, and PCR-positive samples were tested with an enzyme immunoassay (EIA) (C. Diff Quik Chek Complete). EIA-negative samples were further tested with a cell cytotoxicity neutralization assay. The limits of detection for TcdA and TcdB were 0.8 and 0.3 pg/ml in buffer and 2.0 and 0.7 pg/ml in stool, respectively. The assay demonstrated reactivity to common C. difficile strains, did not show cross-reactivity to common gastrointestinal pathogens, was robust against common interferents, allowed detection in fresh and frozen stool samples and in samples after three freeze-thaw cycles, and provided results with high reproducibility. Compared to multistep PCR and toxin-testing procedures, the Singulex Clarity C. diff toxins A/B assay yielded 97.7% sensitivity and 100% specificity. The Singulex Clarity C. diff toxins A/B assay is ultrasensitive and highly specific and may offer a standalone solution for rapid detection and quantitation of free toxins in stool.

Download Full-text

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

Journal of Clinical Microbiology ◽

10.1128/jcm.01629-19 ◽

2019 ◽

Vol 58 (2) ◽

Cited By ~ 1

Author(s):

Marie L. Landry ◽

Jeffrey E. Topal ◽

Joel Estis ◽

Phoebe Katzenbach ◽

Niamh Nolan ◽

...

Keyword(s):

Standard Of Care ◽

Neutralization Assay ◽

High Agreement ◽

Content Type ◽

Clostridioides Difficile ◽

Automated Assay ◽

Toxin Assay ◽

Stool Samples ◽

Testing Algorithm ◽

Positive Agreement

ABSTRACT 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.

Download Full-text

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

Journal of Clinical Microbiology ◽

10.1128/jcm.00945-19 ◽

2019 ◽

Vol 57 (11) ◽

Cited By ~ 5

Author(s):

Johanna Sandlund ◽

Joel Estis ◽

Phoebe Katzenbach ◽

Niamh Nolan ◽

Kirstie Hinson ◽

...

Keyword(s):

Nucleic Acid ◽

Patient Outcomes ◽

Single Molecule ◽

Clinical Performance ◽

Neutralization Assay ◽

Nucleic Acid Amplification ◽

Content Type ◽

Clostridioides Difficile ◽

Health Care Associated Infections ◽

Stool Specimens

ABSTRACT 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.

Download Full-text

Evaluation of six commercial SARS-CoV-2 serology assays detecting different antibodies for clinical testing and serosurveillance

Open Forum Infectious Diseases ◽

10.1093/ofid/ofab239 ◽

2021 ◽

Author(s):

Suellen Nicholson ◽

Theo Karapanagiotidis ◽

Arseniy Khvorov ◽

Celia Douros ◽

Francesca Mordant ◽

...

Keyword(s):

Cell Culture ◽

Neutralization Test ◽

Serological Test ◽

Neutralizing Antibody ◽

Neutralization Assay ◽

Virus Neutralization Test ◽

Virus Neutralization ◽

Binding Assays ◽

Humoral Immune ◽

A Cell

Abstract Background Serological testing for SARS-CoV-2 complements nucleic acid tests for patient diagnosis and enables monitoring of population susceptibility to inform the COVID-19 pandemic response. It is important to understand the reliability of assays with different antigen or antibody targets to detect humoral immunity after SARS-CoV-2 infection and to understand how antibody (Ab) binding assays compare to those detecting neutralizing antibody (nAb), particularly as we move into the era of vaccines. Methods We evaluated the performance of six commercially available Enzyme-linked Immunosorbent Assays (ELISAs), including a surrogate virus neutralization test (sVNT), for detection of SARS-CoV-2 immunoglobulins (IgA, IgM, IgG), total or nAb. A result subset was compared to a cell culture-based microneutralisation (MN) assay. We tested sera from patients with prior RT-PCR confirmed SARS-CoV-2 infection, pre-pandemic sera and potential cross-reactive sera from patients with other non-COVID-19 acute infections. Results For sera collected > 14 days post-symptom onset, the assay achieving the highest sensitivity was the Wantai total Ab at 100% (95% confidence interval: 94.6-100) followed by 93.1% for Euroimmun NCP-IgG, 93.1% for GenScript sVNT, 90.3% for Euroimmun S1-IgG, 88.9% for Euroimmun S1-IgA and 83.3% for Wantai IgM. Specificity for the best performing assay was 99.5% for the Wantai total Ab and for the lowest performing assay was 97.1% for sVNT (as per IFU). The Wantai Total Ab had the best agreement with MN at 98% followed by Euroimmun S1-IgA, Euro NCP-IgG and sVNT (as per IFU) with (97%, 97% and 95% respectively) and Wantai IgM having the poorest agreement at 93%. Conclusion Performance characteristics of the SARS-CoV-2 serology assays detecting different antibody types are consistent with those found in previously published reports. Evaluation of the surrogate virus neutralization test in comparison to the Ab binding assays and a cell culture-based neutralization assay showed good result correlation between all assays. However correlation between the cell-based neutralization test and some assays detecting Ab’s not specifically involved in neutralization was higher than with the sVNT. This study demonstrates the reliability of different assays to detect the humoral immune response following SARS-CoV-2 infection, which can be used to optimise serological test algorithms for assessing antibody responses post SARS-CoV-2 infection or vaccination.

Download Full-text

1088. Ultrasensitive Detection of C. difficile Toxins in Stool Using Single Molecule Counting Technology: A Multicenter Study for Evaluation of Clinical Performance

Open Forum Infectious Diseases ◽

10.1093/ofid/ofy210.923 ◽

2018 ◽

Vol 5 (suppl_1) ◽

pp. S325-S326 ◽

Cited By ~ 1

Author(s):

Stephen Young ◽

Ray Mills ◽

Christen Griego-Fullbright ◽

Aaron Wagner ◽

Emily Herding ◽

...

Keyword(s):

Single Molecule ◽

Multicenter Study ◽

Clinical Performance ◽

Turnaround Time ◽

Neutralization Assay ◽

Single Step ◽

Reporting Structure ◽

One Step ◽

Automated Immunoassay ◽

Higher Sensitivity

Abstract Background Commercially available tests for Clostridium difficile infection (CDI) make test selection by the laboratory difficult due to the following unsatisfactory characteristics: long turnaround time, poor sensitivity, and/or poor specificity. The Singulex Clarity® C. diff toxins A/B assay (in development) is a rapid and automated immunoassay for the detection of C. difficile toxins A and B in stool, with analytical limits of detection for toxins A and B at 2.0 and 0.7 pg/mL, respectively. In this multicenter study, the clinical performance of the Singulex Clarity C. diff toxins A/B assay was compared with standalone PCR, a multistep algorithm with enzyme immunoassay (EIA) and PCR, and cell cytotoxicity neutralization assay (CCNA). Methods Fresh samples from 267 subjects with suspected CDI were tested at two sites (Minneapolis Medical Research Foundation and TriCore Reference Laboratories) with the Singulex Clarity assay, PCR (Xpert®C. difficile), and EIA (C. Diff Quik Chek Complete®) for GDH and toxin testing. The performance of the assays and multistep algorithms were evaluated against CCNA (Microbiology Specialists, Inc.). Results The overall CDI prevalence was 15.7%. The Singulex Clarity C. diff toxins A/B assay had 90.5% sensitivity and 96.0% specificity, with a 98.2% negative predictive value when compared with CCNA, and the Clarity assay’s AuROC was 0.9534. PCR had 90.5% sensitivity and 91.1% specificity. Compared with CCNA, the toxin EIA had 47.6% sensitivity and 100% specificity. Testing with a multistep algorithm using EIA with discordant results reflexed to PCR resulted in 85.7% sensitivity and 94.7% specificity. Conclusion The ultrasensitive Singulex Clarity C. diff toxins A/B assay is equivalent to the sensitivity of PCR while providing higher specificity. Compared with a multistep algorithm, the Clarity assay provides higher sensitivity and specificity while providing faster time-to-result in a simpler-to-understand, one-step reporting structure, allowing for a standalone, single-step solution for detection of C. difficile toxins in patients with suspected CDI. Disclosures E. Friedland, Singulex, Inc.: Employee, Salary. A. Bartolome, Singulex, Inc.: Employee, Salary. A. Almazan, Singulex, Inc.: Employee, Salary. S. Tam, Singulex, Inc.: Employee, Salary. S. Biscocho, Singulex, Inc.: Employee, Salary. S. Abusali, Singulex, Inc.: Employee, Salary. J. Sandlund, Singulex, Inc.: Employee, Salary. J. Estis, Singulex, Inc.: Employee, Salary. J. Bishop, Singulex, Inc.: Employee, Salary.

Download Full-text

2356. Increased Clinical Specificity with Ultrasensitive Detection of Clostridioides difficile Toxins: Reduction of Overdiagnosis Compared with Nucleic Acid Amplification Tests

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz360.2034 ◽

2019 ◽

Vol 6 (Supplement_2) ◽

pp. S811-S812 ◽

Cited By ~ 1

Author(s):

Johanna Sandlund ◽

Joel Estis ◽

Phoebe Katzenbach ◽

Niamh Nolan ◽

Kirstie Hinson ◽

...

Keyword(s):

Nucleic Acid ◽

Disease Severity ◽

Single Molecule ◽

Clinical Performance ◽

Neutralization Assay ◽

Nucleic Acid Amplification ◽

Clostridioides Difficile ◽

Percent Agreement ◽

Clostridioides Difficile Infection ◽

Healthcare Associated

Abstract Background Clostridioides difficile infection (CDI) is one of the most common healthcare-associated infections, resulting in significant morbidity, mortality, and economic burden. Diagnosis of CDI relies on the assessment of clinical presentation and laboratory tests. We have evaluated the clinical performance of ultrasensitive Single Molecule Counting technology for detection of C. difficile toxins A and B. Methods Stool specimens from 298 patients with suspected CDI were tested with nucleic acid amplification test (NAAT; BD MAX™ Cdiff assay or Xpert® C. difficile assay) and Singulex Clarity® C. difficile toxins A/B assay. Specimens with discordant results were tested with cell cytotoxicity neutralization assay (CCNA), and results were correlated with disease severity and outcome. Results 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 or overall death was more common in NAAT+/toxin+ patients than in NAAT+/toxin− and NAAT−/toxin− patients, and NAAT+/toxin+ patients were 3.7 times more likely to experience relapse or death (Figure 1). The clinical specificity of Clarity and NAAT was 97.4% and 89.0%, respectively, and overdiagnosis was over three times more common in NAAT+/toxin− than in NAAT+/toxin+ patients (Figure 2). Negative percent agreement between NAAT and Clarity was 98.3%, and positive percent agreement increased from 50.0% to effective 84.2% and 94.1% after CCNA testing and clinical assessment. Conclusion The Clarity assay was superior to NAATs in diagnosis of CDI, by reducing overdiagnosis and thereby increasing clinical specificity, and presence of toxins was associated with disease severity and outcome. Disclosures All authors: No reported disclosures.

Download Full-text

Antibody-Mediated Neutralization of Authentic SARS-CoV-2 B.1.617 Variants Harboring L452R and T478K/E484Q

Viruses ◽

10.3390/v13091693 ◽

2021 ◽

Vol 13 (9) ◽

pp. 1693

Author(s):

Alexander Wilhelm ◽

Tuna Toptan ◽

Christiane Pallas ◽

Timo Wolf ◽

Udo Goetsch ◽

...

Keyword(s):

Cell Culture ◽

Monoclonal Antibodies ◽

Combination Therapy ◽

Neutralization Assay ◽

A Cell

The capacity of convalescent and vaccine-elicited sera and monoclonal antibodies (mAb) to neutralize SARS-CoV-2 variants is currently of high relevance to assess the protection against infections. We performed a cell culture-based neutralization assay focusing on authentic SARS-CoV-2 variants B.1.617.1 (Kappa), B.1.617.2 (Delta), B.1.427/B.1.429 (Epsilon), all harboring the spike substitution L452R. We found that authentic SARS-CoV-2 variants harboring L452R had reduced susceptibility to convalescent and vaccine-elicited sera and mAbs. Compared to B.1, Kappa and Delta showed a reduced neutralization by convalescent sera by a factor of 8.00 and 5.33, respectively, which constitutes a 2-fold greater reduction when compared to Epsilon. BNT2b2 and mRNA1273 vaccine-elicited sera were less effective against Kappa, Delta, and Epsilon compared to B.1. No difference was observed between Kappa and Delta towards vaccine-elicited sera, whereas convalescent sera were 1.51-fold less effective against Delta, respectively. Both B.1.617 variants Kappa (+E484Q) and Delta (+T478K) were less susceptible to either casirivimab or imdevimab. In conclusion, in contrast to the parallel circulating Kappa variant, the neutralization efficiency of convalescent and vaccine-elicited sera against Delta was moderately reduced. Delta was resistant to imdevimab, which, however, might be circumvented by combination therapy with casirivimab together.

Download Full-text

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

Journal of Clinical Microbiology ◽

10.1128/jcm.01681-19 ◽

2020 ◽

Vol 58 (5) ◽

Author(s):

Megan D. Shah ◽

Joan-Miquel Balada-Llasat ◽

Kelci Coe ◽

Erica Reed ◽

Johanna Sandlund ◽

...

Keyword(s):

Clinical Characteristics ◽

Laboratory Data ◽

Antibiotic Use ◽

Neutralization Assay ◽

Test Results ◽

Toxin Concentration ◽

Content Type ◽

Cycle Threshold ◽

Clostridioides Difficile ◽

Clostridioides Difficile Infection

ABSTRACT 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.

Download Full-text

Tulane Virus Recognizes the A Type 3 and B Histo-Blood Group Antigens

Journal of Virology ◽

10.1128/jvi.02595-14 ◽

2014 ◽

Vol 89 (2) ◽

pp. 1419-1427 ◽

Cited By ~ 28

Author(s):

Dongsheng Zhang ◽

Pengwei Huang ◽

Lu Zou ◽

Todd L. Lowary ◽

Ming Tan ◽

...

Keyword(s):

Cell Culture ◽

Blood Group ◽

Culture Method ◽

Blood Group Antigens ◽

Content Type ◽

Tulane Virus ◽

A Cell ◽

Type 3

ABSTRACTTulane virus (TV), the prototype of theRecovirusgenus in the calicivirus family, was isolated from the stools of rhesus monkeys and can be cultivatedin vitroin monkey kidney cells. TV is genetically closely related to the genusNorovirusand recognizes the histo-blood group antigens (HBGAs), similarly to human noroviruses (NoVs), making it a valuable surrogate for human NoVs. However, the precise structures of HBGAs recognized by TV remain elusive. In this study, we performed binding and blocking experiments on TV with extended HBGA types and showed that, while TV binds all four types (types 1 to 4) of the B antigens, it recognizes only the A type 3 antigen among four types of A antigens tested. The requirements for HBGAs in TV replication were demonstrated by blocking of TV replication in cell culture using the A type 3/4 and B saliva samples. Similar results were also observed in oligosaccharide-based blocking assays. Importantly, the previously reported, unexplained increase in TV replication by oligosaccharide in cell-based blocking assays has been clarified, which will facilitate the application of TV as a surrogate for human NoVs.IMPORTANCEOur understanding of the role of HBGAs in NoV infection has been significantly advanced in the past decade, but direct evidence for HBGAs as receptors for human NoVs remains lacking due to a lack of a cell culture method. TV recognizes HBGAs and can replicatein vitro, providing a valuable surrogate for human NoVs. However, TV binds to some but not all saliva samples from A-positive individuals, and an unexplained observation of synthetic oligosaccharide blocking of TV binding has been reported. These issues have been resolved in this study.

Download Full-text