Clostridium difficile PCR Cycle Threshold Predicts Free Toxin

ABSTRACTThere is no stand-aloneClostridium difficilediagnostic that can sensitively and rapidly detect fecal free toxins. We investigated the performance of theC. difficilePCR cycle threshold (CT) for predicting free toxin status. Consecutive stool samples (n= 312) positive for toxigenicC. difficileby the GeneXpertC. difficile/EpitcdBPCR assay were tested with the rapid membrane C. Diff Quik Chek Complete immunoassay (RMEIA). RMEIA toxin-negative samples were tested with the cell cytotoxicity neutralization assay (CCNA) and tgcBIOMICS enzyme-linked immunosorbent assay (ELISA). Using RMEIA alone or in combination with CCNA and/or ELISA as the reference method, the accuracy ofCTwas measured at differentCTcutoffs. Using RMEIA as the reference method, aCTcutoff of 26.35 detected toxin-positive samples with a sensitivity, specificity, positive predictive value, and negative predictive value of 96.0% (95% confidence interval [CI], 90.2% to 98.9%), 65.9% (95% CI, 59.0% to 72.2%), 57.4% (95% CI, 52.7% to 62%), and 97.1% (95% CI, 92.8% to 98.9), respectively. Inclusion of CCNA in the reference method improvedCTspecificity to 78.0% (95% CI, 70.7% to 84.2%). Intercartridge lotCTvariability measured as the average coefficient of variation was 2.8% (95% CI, 1.2% to 3.2%). Standardizing the input stool volume did not improveCTtoxin specificity. The medianCTvalues were not significantly different between stool samples with Bristol scores of 5, 6, and 7, between pediatric and adult samples, or between presumptive 027 and non-027 strains. In addition to sensitively detecting toxigenicC. difficilein stool, on-demand PCR may also be used to accurately predict toxin-negative stool samples, thus providing additional results in PCR-positive stool samples to guide therapy.

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

Journal of Clinical Microbiology ◽

10.1128/jcm.00613-15 ◽

2015 ◽

Vol 53 (6) ◽

pp. 1942-1944 ◽

Cited By ~ 8

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.

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Comparison of GenomEra C. difficile and Xpert C. difficile as Confirmatory Tests in a Multistep Algorithm for Diagnosis of Clostridium difficile Infection

Journal of Clinical Microbiology ◽

10.1128/jcm.03093-14 ◽

2014 ◽

Vol 53 (1) ◽

pp. 332-335 ◽

Cited By ~ 11

Author(s):

Luis Alcalá ◽

Elena Reigadas ◽

Mercedes Marín ◽

Antonia Fernández-Chico ◽

Pilar Catalán ◽

...

Keyword(s):

Clostridium Difficile ◽

Positive Predictive Value ◽

Negative Predictive Value ◽

Clostridium Difficile Infection ◽

Predictive Value ◽

Confirmatory Test ◽

Content Type ◽

Diagnostic Algorithms ◽

Confirmatory Tests ◽

Sensitivity Specificity

We compared two multistep diagnostic algorithms based on C. Diff Quik Chek Complete and, as confirmatory tests, GenomEraC. difficileand XpertC. difficile. The sensitivity, specificity, positive predictive value, and negative predictive value were 87.2%, 99.7%, 97.1%, and 98.3%, respectively, for the GenomEra-based algorithm and 89.7%, 99.4%, 95.5%, and 98.6%, respectively, for the Xpert-based algorithm. GenomEra represents an alternative to Xpert as a confirmatory test of a multistep algorithm forClostridium difficileinfection (CDI) diagnosis.

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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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Diagnostic comparison of stool exam and point-of-care circulating cathodic antigen (POC-CCA) test for schistosomiasis mansoni diagnosis in a high endemicity area in northeastern Brazil

Parasitology ◽

10.1017/s0031182020002164 ◽

2020 ◽

pp. 1-7

Author(s):

Danielle de Freitas Bezerra ◽

Marta Cristhiany Cunha Pinheiro ◽

Luciene Barbosa ◽

Agostinho Gonçalves Viana ◽

Ricardo Toshio Fujiwara ◽

...

Keyword(s):

Predictive Value ◽

Point Of Care ◽

Northeastern Brazil ◽

Enzyme Linked Immunosorbent Assay ◽

False Negatives ◽

Parasitic Load ◽

Stool Samples ◽

Filter Test ◽

Sensitivity Specificity ◽

Spearman’S Correlation

Abstract This study aimed to evaluate the performance of the point-of-care circulating cathodic antigen (POC-CCA) test in a highly endemic area in Brazil, comparing it to the Kato-Katz (KK) technique for sensitivity, specificity and the intensity of the reaction of the test in relation to the parasitic load. The community in Sergipe, Brazil, participated in the study, providing three stool samples, one of urine (POC-CCA) and fingers tick blood sample was tested by enzyme-linked immunosorbent assay (ELISA). Sensitivity, specificity, positive predictive value, negative predictive value, accuracy, kappa coefficient and Spearman's correlation were calculated for the POC-CCA test using the KK as the reference. The prevalence of schistosomiasis by KK testing was 48.82%; POC-CCA (t+) 66.14%; POC-CCA (t−) 45.24%. ELISA results showed 100% agreement in individuals with high and moderate eggs per gram (EPG). POC-CCA presented good diagnostic performance in individuals with medium and high EPG, but there were a high number of false negatives in individuals with low intensity infections. As observed, POC-CCA-filter test improves accuracy and sensitivity compared to a conventional test.

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Use of a Toxin Neutralization Assay To Characterize the Serologic Response to Adenylate Cyclase Toxin after Infection with Bordetella pertussis

Clinical and Vaccine Immunology ◽

10.1128/cvi.00370-16 ◽

2016 ◽

Vol 24 (1) ◽

Cited By ~ 4

Author(s):

Joshua C. Eby ◽

Mary C. Gray ◽

Jason M. Warfel ◽

Tod J. Merkel ◽

Erik L. Hewlett

Keyword(s):

Adenylate Cyclase ◽

Bordetella Pertussis ◽

Neutralization Assay ◽

Enzyme Linked Immunosorbent Assay ◽

Immunologic Response ◽

Serum Samples ◽

Content Type ◽

Adenylate Cyclase Toxin ◽

Strong Candidate ◽

Candidate Antigen

ABSTRACT Adenylate cyclase toxin (ACT) is an essential virulence factor of Bordetella pertussis, and antibodies to ACT protect against B. pertussis infection in mice. The toxin is therefore a strong candidate antigen for addition to future acellular pertussis vaccines. In order to characterize the functionality of the immunologic response to ACT after infection, we developed an assay for testing the ability of serum samples from subjects infected with B. pertussis to neutralize ACT-induced cytotoxicity in J774 macrophage cells. Baboons develop neutralizing anti-ACT antibodies following infection with B. pertussis, and all sera from baboons with positive anti-ACT IgG enzyme-linked immunosorbent assay (ELISA) results neutralized ACT cytotoxicity. The toxin neutralization assay (TNA) was positive in some baboon sera in which ELISA remained negative. Of serum samples obtained from humans diagnosed with pertussis by PCR, anti-ACT IgG ELISA was positive in 72%, and TNA was positive in 83%. All samples positive for anti-ACT IgG ELISA were positive by TNA, and none of the samples from humans without pertussis neutralized toxin activity. These findings indicate that antibodies to ACT generated following infection with B. pertussis consistently neutralize toxin-induced cytotoxicity and that TNA can be used to improve understanding of the immunologic response to ACT after infection or vaccination.

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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.

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1089. Analytical Performance of an Ultrasensitive Immunoassay for Detection of Clostridium difficile Toxins in Stool

Open Forum Infectious Diseases ◽

10.1093/ofid/ofy210.924 ◽

2018 ◽

Vol 5 (suppl_1) ◽

pp. S326-S326

Author(s):

Amelita Bartolome ◽

Anna Almazan ◽

Salina Abusali ◽

Stanley Tam ◽

Eric Lee ◽

...

Keyword(s):

Clostridium Difficile ◽

Room Temperature ◽

High Sensitivity ◽

Turnaround Time ◽

Neutralization Assay ◽

Cross Reactivity ◽

Analytical Performance ◽

Freeze Thaw ◽

Gastrointestinal Pathogens ◽

Sensitivity Specificity

Abstract Background Clostridium difficile infection (CDI) is the main cause for nosocomial diarrhea. Currently available assays for the diagnosis of CDI show deficits in sensitivity, specificity, and/or turnaround time. The Singulex Clarity® C. diff toxins A/B assay, in development for the Singulex Clarity® system, was designed to provide an accurate and automated detection of C. difficile toxins A (TcdA) and B (TcdB) in stool. Here, the analytical performance of the assay is reported. Methods Limits of detection (LoD) for TcdA and TcdB in stool and buffer was determined, and a preliminary cutoff, as compared with cell cytotoxicity neutralization assay (CCNA), was established. Analytical reactivity against 38 toxigenic and nontoxigenic C. difficile strains of eight different toxinotypes was determined. Cross-reactivity against 53 other gastrointestinal pathogens and potential interference by 11 endogenous and exogenous substances were determined. Reproducibility was tested with triplicate samples (n = 85), and stability was evaluated in samples stored at room temperature, refrigerated, and frozen conditions, and subjected to three freeze-thaw cycles. Results The LoDs 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. Using a preliminary cutoff, the assay demonstrated 96.3% sensitivity and 96.1% specificity compared with CCNA. The Singulex Clarity® C. diff toxins A/B assay detected toxins from all tested strains and toxinotypes. No cross-reactivity or interference were detected. The repeatability was 99%, and samples for C. difficile toxin testing were stable up to 8 hours in room temperature, 1 week in 2–8°C, 6 months in −70°C, and up to three freeze–thaw cycles. Conclusion The Singulex Clarity C. diff toxins A/B assay (in development) can detect TcdA and TcdB at very low concentrations and it has high sensitivity and specificity compared with CCNA. The assay demonstrates reactivity to common C. difficile strains, does not show cross-reactivity to common gastrointestinal pathogens, is robust against common interferents, allows for toxin detection in both fresh and frozen stool samples and up to three freeze–thaw cycles, and provides results with high reproducibility. Disclosures A. Bartolome, Singulex, Inc.: Employee, Salary. A. Almazan, Singulex, Inc.: Employee, Salary. S. Abusali, Singulex, Inc.: Employee, Salary. S. Tam, Singulex, Inc.: Employee, Salary. E. Lee, Singulex, Inc.: Employee, Salary. A. Changavi, Singulex, Inc.: Employee, Salary. W. Trinh, Singulex, Inc.: Employee, Salary. K. Chau, Singulex, Inc.: Employee, Salary. J. Estis, Singulex, Inc.: Employee, Salary. B. Noland, Singulex, Inc.: Employee, Salary. J. Bishop, Singulex, Inc.: Employee, Salary.

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Comparative Evaluation of Four Phenotypic Methods for Detection of Class A and B Carbapenemase-Producing Enterobacteriaceae in China

Journal of Clinical Microbiology ◽

10.1128/jcm.00395-18 ◽

2018 ◽

Vol 56 (8) ◽

Cited By ~ 8

Author(s):

Menglan Zhou ◽

Danchen Wang ◽

Timothy Kudinha ◽

Qiwen Yang ◽

Shuying Yu ◽

...

Keyword(s):

Predictive Value ◽

Comparative Evaluation ◽

Detection Sensitivity ◽

Detection Methods ◽

Content Type ◽

Class A ◽

Carbapenem Resistant ◽

Class B ◽

Phenotypic Methods ◽

Sensitivity Specificity

ABSTRACT The objective of this study was to evaluate the performance of four phenotypic methods in the detection of carbapenemase-producing Enterobacteriaceae (CPE) in China. We evaluated the performance of four carbapenemase detection methods, the modified Hodge test (MHT), the Carba NP test, the meropenem hydrolysis assay (MHA) with 1- and 2-h incubation, and the modified carbapenem inactivation method (mCIM) with meropenem, imipenem, and ertapenem, on 342 carbapenem-resistant Enterobacteriaceae isolates (CRE) in China. PCR was used as the gold standard. The 2-h-incubation MHA performed the best in carbapenemase detection (overall sensitivity, specificity, positive predictive value, and negative predictive value all 100%). Second was the Carba NP test, with a sensitivity of 99.6%. The 1-h-incubation MHA performed poorly in Klebsiella pneumoniae carbapenemase (KPC) detection (sensitivity, 71.3%). For mCIM, the best performance was observed with the meropenem disk. The MHT exhibited the worst performance, with a specificity of 88.8%. All assays except 1-h-incubation MHA, which failed to identify 68 KPC-2s, had a sensitivity of >98% in the detection of 172 KPCs. Likewise, all assays had a sensitivity of >95% in the detection of 70 class B carbapenemases, except for MHT (82.9%). The 2-h-incubation MHA significantly improved the accuracy in CPE detection compared with that for 1-h incubation and performed the best in the detection of class A and B carbapenemases. Our findings suggest that the MHA is the most practical assay for carbapenemase detection. For those who cannot afford the associated equipment, both the Carba NP test and mCIM are good alternatives with regard to the practical requirements of time and cost.

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1094. Performance of Toxin Enzyme Immunoassays and PCR Cycle Threshold for Differentiating Clostridium difficile Infection From Colonization in Children With Diarrhea

Open Forum Infectious Diseases ◽

10.1093/ofid/ofy210.929 ◽

2018 ◽

Vol 5 (suppl_1) ◽

pp. S327-S328

Author(s):

Aakash Balaji ◽

Robyn Espinosa ◽

Kathleen Todd ◽

Kerry Steed ◽

Larry Kociolek

Keyword(s):

Clostridium Difficile ◽

Chart Review ◽

Operating Characteristic ◽

Neutralization Assay ◽

Stool Culture ◽

Research Support ◽

Parent Communication ◽

Cycle Threshold ◽

Assay Performance ◽

The Difference

Abstract Background Clostridium difficile colonization is common in children. PCR does not distinguish infection (CDI) from colonization. Toxin enzyme immunoassay (EIA) and PCR cycle threshold (Ct) may predict CDI in PCR+ adults, but assay performance in children is poorly understood. Methods Stools from children aged 2–21 years with laboratory-identified (labID) CDI (tcdB PCR+; GeneXpert) underwent: toxin EIA (QUIK CHEK Complete [QCC] and Immunocard [IC]); cell culture cytotoxicity neutralization assay (CCCNA); and C. difficile stool culture (Cx). Children were determined to have clinical CDI (cCDI) by chart review and/or parent communication if all were noted: at least three unformed stools (Bristol type 5–7) in 24 hours; response to CDI treatment within 5 days; and no other likely diarrheal etiology. EIA and PCR Ct performance were measured for various reference standards (RefStd) based on stool assay results and/or cCDI classification. Results A total of 253 PCR+ stools were included. All stools underwent QCC; 218 (86%) were quantity sufficient for IC. Discordant EIA results occurred in 19/218 (8.7%) stools. Table 1 lists EIA sensitivity (Sn), EIA specificity (Sp), and median PCR Ct for each RefStd. Figure 1 shows the receiver operating characteristic (ROC) curve for PCR Ct to identify PCR+/CCCNA+/cCDI+ children (area under curve = 0.76). The difference between sensitivity (71%) and specificity (72%) was minimized at Ct < 23.5. Conclusion Only a minority of PCR+ children meets strict clinical and laboratory CDI criteria. More stringent CDI definitions are associated with increasing toxin EIA Sn and lower PCR Ct (i.e., greater stool C. difficile inoculum). However, both toxin EIA and PCR Ct perform suboptimally as stand-alone tests to distinguish CDI from colonization in PCR+ children. Disclosures L. Kociolek, Alere/Techlab: Investigator, Research support.

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