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

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.

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Nucleic Acid Amplification Test Quantitation as Predictor of Toxin Presence in Clostridium difficile Infection

Journal of Clinical Microbiology ◽

10.1128/jcm.01316-17 ◽

2017 ◽

Vol 56 (3) ◽

Cited By ~ 13

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.

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Detection of Clostridium difficile in Feces of Asymptomatic Patients Admitted to the Hospital

Journal of Clinical Microbiology ◽

10.1128/jcm.01858-16 ◽

2016 ◽

Vol 55 (2) ◽

pp. 403-411 ◽

Cited By ~ 23

Author(s):

Elisabeth M. Terveer ◽

Monique J. T. Crobach ◽

Ingrid M. J. G. Sanders ◽

Margreet C. Vos ◽

Cees M. Verduin ◽

...

Keyword(s):

Clostridium Difficile ◽

Predictive Value ◽

Health Care Facilities ◽

Nucleic Acid Amplification ◽

Content Type ◽

Asymptomatic Patients ◽

Toxigenic Culture ◽

Stool Samples ◽

Sensitivity Specificity ◽

Low Prevalence

ABSTRACTRecent evidence shows that patients asymptomatically colonized withClostridium difficilemay contribute to the transmission ofC. difficilein health care facilities. Additionally, these patients may have a higher risk of developingC. difficileinfection. The aim of this study was to compare a commercially available PCR directed to both toxin A and B (artusC. difficileQS-RGQ kit CE; Qiagen), an enzyme-linked fluorescent assay to glutamate dehydrogenase (GDH ELFA) (Vidas, bioMérieux), and an in-house-developed PCR totcdB, with (toxigenic) culture ofC. difficileas the gold standard to detect asymptomatic colonization. Test performances were evaluated in a collection of 765 stool samples obtained from asymptomatic patients at admission to the hospital. TheC. difficileprevalence in this collection was 5.1%, and 3.1% contained toxigenicC. difficile. Compared toC. difficileculture, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of theC. difficileGDH ELFA were 87.2%, 91.2%, 34.7%, and 99.3%, respectively. Compared with results of toxigenic culture, the sensitivity, specificity, PPV, and NPV of the commercially available PCR and the in-house PCR were 95.8%, 93.4%, 31.9%, 99.9%, and 87.5%, 98.8%, 70%, and 99.6%, respectively. We conclude that in a low-prevalence setting of asymptomatically colonized patients, both GDH ELFA and a nucleic acid amplification test can be applied as a first screening test, as they both display a high NPV. However, the low PPV of the tests hinders the use of these assays as stand-alone tests.

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Evaluation of the cobas Cdiff Test for Detection of Toxigenic Clostridium difficile in Stool Samples

Journal of Clinical Microbiology ◽

10.1128/jcm.01135-17 ◽

2017 ◽

Vol 55 (12) ◽

pp. 3426-3436 ◽

Cited By ~ 6

Author(s):

Lance R. Peterson ◽

Stephen A. Young ◽

Thomas E. Davis ◽

Zi-Xuam Wang ◽

John Duncan ◽

...

Keyword(s):

Clostridium Difficile ◽

False Negative ◽

Reference Method ◽

Culture Method ◽

Nucleic Acid Amplification ◽

Culture Methods ◽

Content Type ◽

Toxigenic Culture ◽

Discrepancy Analysis ◽

Stool Samples

ABSTRACTNucleic acid amplification tests (NAATs) are reliable tools for the detection of toxigenicClostridium difficilefrom unformed (liquid or soft) stool samples. The objective of this study was to evaluate performance of the cobas Cdiff test on the cobas 4800 system using prospectively collected stool specimens from patients suspected of havingC. difficileinfection (CDI). The performance of the cobas Cdiff test was compared to the results of combined direct and broth-enriched toxigenic culture methods in a large, multicenter clinical trial. Additional discrepancy analysis was performed by using the XpertC. difficileEpi test. Sample storage was evaluated by using contrived and fresh samples before and after storage at −20°C. Testing was performed on samples from 683 subjects (306 males and 377 females); 113 (16.5%) of 683 subjects were positive for toxigenicC. difficileby direct toxigenic culture, and 141 of 682 subjects were positive by using the combined direct and enriched toxigenic culture method (reference method), for a prevalence rate of 20.7%. The sensitivity and specificity of the cobas Cdiff test compared to the combined direct and enriched culture method were 92.9% (131/141; 95% confidence interval [CI], 87.4% to 96.1%) and 98.7% (534/541; 95% CI, 97.4% to 99.4%), respectively. Discrepancy analysis using results for retested samples from a second NAAT (Xpert C. difficile/Epi test; Cepheid, Sunnyvale, CA) found no false-negative and 4 false-positive cobas Cdiff test results. There was no difference in positive and negative results in comparisons of fresh and stored samples. These results support the use of the cobas Cdiff test as a robust aid in the diagnosis of CDI.

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Validation of Active Surveillance Testing for Clostridium difficile Colonization Using the cobas Cdiff Test

Journal of Clinical Microbiology ◽

10.1128/jcm.01553-17 ◽

2018 ◽

Vol 56 (4) ◽

Cited By ~ 1

Author(s):

Parul A. Patel ◽

Donna M. Schora ◽

Kamaljit Singh ◽

Lance R. Peterson

Keyword(s):

Clostridium Difficile ◽

Active Surveillance ◽

Positive Culture ◽

The United States ◽

Nucleic Acid Amplification ◽

Hospital System ◽

Content Type ◽

Asymptomatic Patients ◽

Toxigenic Culture ◽

Active Surveillance Testing

ABSTRACT Clostridium difficile infection (CDI) is not declining in the United States. Nucleic acid amplification tests (NAAT) are used as part of active surveillance testing programs to prevent health care-associated infection. The objective of this study was to validate the cobas Cdiff Test on the cobas 4800 System (cobas) within a four-hospital system using prospectively collected perirectal swabs from asymptomatic patients at admission and during monthly intensive care unit (ICU) screening in an infection control CDI reduction program. Performance of the cobas was compared to that of toxigenic culture. Each positive cobas sample and the next following negative patient swab were cultured. The study design gave 273 samples processed by both cobas (137 positive and 136 negative) and culture (one negative swab was not cultured). Discrepant analysis was performed using a second NAAT, the Xpert C. difficile Epi test (Xpert). This strategy was compared to a medical record review for antibiotic receipt that would inhibit growth of C. difficile in colonic stool. None of the cobas-negative samples were culture positive. The cobas positive predictive value was 75.2% (95% confidence interval [CI], 66.9% to 82%) and positive percent agreement was 100% (95% CI, 96.0% to 100%). Overall agreement between cobas and direct toxigenic culture was 87.6% (95% CI, 83.1% to 91%). For the cobas-positive/culture-negative (discrepant) samples, 7 Xpert-positive samples were from patients receiving inhibitory antimicrobials; only 4 of 23 Xpert-negative samples received these agents ( P = 0.00006). Our results support use of the cobas as a reliable assay for an active surveillance testing program to detect asymptomatic carriers of toxigenic C. difficile .

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Infection with Toxin A-Negative, Toxin B-Negative, Binary Toxin-Positive Clostridium difficile in a Young Patient with Ulcerative Colitis

Journal of Clinical Microbiology ◽

10.1128/jcm.01810-15 ◽

2015 ◽

Vol 53 (11) ◽

pp. 3702-3704 ◽

Cited By ~ 25

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.

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Comparative Genome Analysis and Global Phylogeny of the Toxin Variant Clostridium difficile PCR Ribotype 017 Reveals the Evolution of Two Independent Sublineages

Journal of Clinical Microbiology ◽

10.1128/jcm.01296-16 ◽

2016 ◽

Vol 55 (3) ◽

pp. 865-876 ◽

Cited By ~ 29

Author(s):

M. D. Cairns ◽

M. D. Preston ◽

C. L. Hall ◽

D. N. Gerding ◽

P. M. Hawkey ◽

...

Keyword(s):

Clostridium Difficile ◽

Antibiotic Resistance Genes ◽

Binary Toxin ◽

Human Origins ◽

Comparative Genome Analysis ◽

Toxin B ◽

Content Type ◽

Global Spread ◽

Animal Isolates ◽

Evolutionary Lineages

ABSTRACT The diarrheal pathogen Clostridium difficile consists of at least six distinct evolutionary lineages. The RT017 lineage is anomalous, as strains only express toxin B, compared to strains from other lineages that produce toxins A and B and, occasionally, binary toxin. Historically, RT017 initially was reported in Asia but now has been reported worldwide. We used whole-genome sequencing and phylogenetic analysis to investigate the patterns of global spread and population structure of 277 RT017 isolates from animal and human origins from six continents, isolated between 1990 and 2013. We reveal two distinct evenly split sublineages (SL1 and SL2) of C. difficile RT017 that contain multiple independent clonal expansions. All 24 animal isolates were contained within SL1 along with human isolates, suggesting potential transmission between animals and humans. Genetic analyses revealed an overrepresentation of antibiotic resistance genes. Phylogeographic analyses show a North American origin for RT017, as has been found for the recently emerged epidemic RT027 lineage. Despite having only one toxin, RT017 strains have evolved in parallel from at least two independent sources and can readily transmit between continents.

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Sensitivity of Single-Molecule Array Assays for Detection of Clostridium difficile Toxins in Comparison to Conventional Laboratory Testing Algorithms

Journal of Clinical Microbiology ◽

10.1128/jcm.00452-18 ◽

2018 ◽

Vol 56 (8) ◽

Cited By ~ 11

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.

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Design and Validation of Transcription-Mediated-Amplification Nucleic Acid Amplification Tests forMycoplasma genitalium

Journal of Clinical Microbiology ◽

10.1128/jcm.00264-19 ◽

2019 ◽

Vol 57 (8) ◽

Cited By ~ 9

Author(s):

Brett Kirkconnell ◽

Barbara Weinbaum ◽

Katherine Santos ◽

Trisha Le Nguyen ◽

Bryan Vinluan ◽

...

Keyword(s):

Clinical Performance ◽

Mycoplasma Genitalium ◽

Vaginal Swab ◽

Nucleic Acid Amplification ◽

Clinical Validation ◽

Analytical Sensitivity ◽

Reference Standard ◽

Content Type ◽

Sensitivity Specificity

ABSTRACTMycoplasma genitaliumis a sexually transmitted bacterium linked to adverse sexual and reproductive health outcomes in women and men.M. genitaliumis difficult to culture, and in the absence of validated amplified molecular methods for diagnosis of infection, there is no reference standard available for use as a comparator for the validation of newM. genitaliumdiagnostic tests. We evaluated the analytical and clinical performance of three transcription-mediated amplification (TMA) tests forM. genitalium, each targeting unique rRNA sequences, for use as a composite comparator for clinical validation of the AptimaMycoplasma genitalium(AMG) assay, anin vitrodiagnostic (IVD) TMA test that targets 16 s rRNA ofM. genitalium. Analytical sensitivity, specificity, and strain inclusivity of all four TMA tests were determined using nine laboratory strains ofM. genitaliumand 56 nontarget bacteria, protozoa, and viruses. Analytical sensitivity of the tests forM. genitaliumranged from 0.017 to 0.040 genome equivalents/ml. None of the nontarget organisms evaluated cross-reacted with any test. A composite comparator reference standard consisting of the 3 alternate (Alt) TMA tests was used to evaluate the clinical performance of the AMG assay by testing residual vaginal swab, female urine, and male urine specimens obtained from 1,400 adult subjects from three U.S. clinical sites. Using this reference standard to establish infected specimen status, the sensitivity, specificity, and overall agreement of the AMG IVD assay were 100%, 99.9%, and 99.9%, respectively. These results demonstrate the utility of molecular composite reference standard methodology for the clinical validation of future IVD tests for this organism.

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