Cell Cytotoxicity Neutralization Assay Research Articles

Diagnostic Guidance for C. difficile Infections.

Diagnosis of Clostridioides difficile infection (CDI) can be challenging. First of all, there has been debate on which of the two reference assays, cell cytotoxicity neutralization assay (CCNA) or toxigenic culture (TC), should be considered the gold standard for CDI detection. Although the CCNA suffers most from suboptimal storage conditions and subsequent toxin degradation, TC is reported to falsely increase CDI detection rates as it cannot differentiate CDI patients from patients asymptomatically colonised by toxigenic C. difficile. Several rapid assays are available for CDI detection and fall into three broad categories: (1) enzyme immunoassays for glutamate dehydrogenase, (2) enzyme immunoassays or single-molecule array assays for toxins A/B and (3) nucleic acid amplification tests detecting toxin genes. All three categories have their own limitations, being suboptimal specificity and/or sensitivity or the inability to discern colonised patients from CDI patients. In light of these limitations, multi-step algorithmic testing has been advocated by international guidelines (IDSA/SHEA and ESCMID) in order to optimize diagnostic accuracy. As a result, a survey performed in 2018-2019 in Europe revealed that most of all hospital sites reported using more than one test to diagnose CDI. CDI incidence rates are also influenced by sample selection criteria, as several studies have shown that if not all unformed stool samples are tested for CDI, many cases may be missed due to an absence of clinical suspicion. Since methods for diagnosing CDI remain imperfect, there has been a growing interest in alternative testing strategies like faecal microbiota biomarkers, immune modulating interleukins, cytokines and imaging methods. At the moment, these alternative methods might play an adjunctive role, but they are not suitable to replace conventional CDI testing strategies.

711. High Prevalence of Positive Cell Cytotoxicity Neutralization Assay in Symptomatic and Asymptomatic Pediatric Oncologic Patients

Abstract Background Clostridium difficile infection (CDI) is the most common cause of nosocomial diarrhea in the United States. This burden is more prevalent and severe in the oncologic population and is associated with greater morbidity and mortality. Diagnostic modalities are limited, and misdiagnosis is high with resultant mismanaged care – inappropriate antibiotics, and potentially missed opportunities for appropriate targeted care towards graft-versus-host disease or other etiologies. To date, no large-scale evaluations have been performed to characterize the incidence of true disease in this population or to help delineate predictive markers for true infections (cell cytotoxicity neutralization assay (CCNA) positivity). Methods This is a multi-center, prospective stool banking study from January 2020 to date enrolling pediatric patients with an oncologic diagnosis admitted at Advocate Children’s Hospital and Comer Children’s Hospital. Stool was collected weekly along with relevant clinical histories and symptomology. Isolates of C. difficile were cultured and CCNA performed. Stool was also collected during episodes of PCR diagnosed CDI. Results 65 unique subjects were enrolled over 183 admissions. 11 samples from 10 patients were thought to represent CDI with PCR positivity noted. Five of these specimens were confirmed as CCNA positive while the remainder did not have active toxin presence. Bristol type 4 stools or > 3 stools in 24 hours were significant factors for modeling CCNA positivity, but not for modeling PCR positivity. Notably 26 stool specimens were CCNA positive, and 21 did not have PCR tests done. Among all CCNA positive specimens, no significant difference was noted in symptomatology or antibiotic use. Conclusion Differentiating true CDI can be challenging. However, even in patients with high risk of alternative GI pathology, the frequency of stooling and stool characteristics can be used. Although considered the gold standard for assessment of disease, CCNA positivity in this population was noted in patients who were not evaluated for disease by their primary providers. Underdiagnosis is possible, but potentially less likely given the absence of clinically significant differentiation in symptoms. Host factors predisposing to disease are likely present. Disclosures Larry K. Kociolek, MD, MSCI, Merck: Grant/Research Support

394. Impact of Misdiagnosis of Clostridioides difficile Infection (CDI) by Standard-of-care Specimen Collection and Testing on Estimates of Hospitalized CDI Incidence Among Adults in Louisville, Kentucky, 2019-2020

Abstract Background Public health surveillance indicates that there is a high population-based incidence of laboratory-confirmed hospitalized CDI cases in the United States. Although reported CDI cases are identified via standard-of-care (SOC) specimen collection and CDI testing practices, the impact of SOC misdiagnosis on the reported CDI incidence is uncertain. Methods Active surveillance from Oct 14, 2019, to Apr 11, 2020, identified inpatients aged ≥50 years with diarrhea (≥3 stools with Bristol score ≥5 in 24 hours) at all wards at 8 of the 9 adult hospitals in Louisville, Kentucky (population >50 years = 276 456). Study stool specimens from inpatients with diarrhea were screened by rapid GDH/toxin membrane enzyme immunoassay and the positive samples tested by PCR and cell cytotoxicity neutralization assay (CCNA). A study CDI case was a patient with PCR positive/CCNA positive stool or PCR positive stool with pseudomembranous colitis (PMC). Incidence (non-recurrent CDI cases/100 000 persons aged >50 years per year [PY]) was adjusted for the hospitalization share of participating hospitals and, in a sensitivity analysis, for patients with diarrhea without a CDI test result. SOC stool specimen CDI testing occurred independent of the study. Results Among 1541 inpatients with diarrhea, study testing identified 109 non-recurrent CDI cases; 18 (16.5%) had PMC, 36 (33.0%) were admitted to intensive care, and 21 (19.3%) died during the 90-day follow-up. Study hospitalized CDI incidence was 154/100 000 PY (202/100 000 PY in the sensitivity analysis). SOC hospitalized CDI incidence was 121/100 000 PY. Of the 109 study CDI cases, 44 (40%) were not SOC-diagnosed (SOC under-diagnosis). Of the 75 SOC CDI cases that also had study testing, 12 (16%) were not study CDI cases (SOC over-diagnosis). SOC-undiagnosed and SOC-diagnosed CDI cases had similar demographics, medical histories, and clinical outcomes. Study testing identified 24% more CDI cases than SOC testing. Conclusion There was a high incidence of hospitalized CDI in persons aged >50 years (154-202/100,000 PY). Of the hospitalized CDI cases, one-third were admitted to ICU and one-fifth died. Public health surveillance estimates of the incidence of laboratory-confirmed hospitalized CDI cases, which are based on SOC testing, may be under-estimated by 24%. Disclosures Frederick Angulo, DVM PhD, Pfizer: Employee|Pfizer: Stocks/Bonds Joann M. Zamparo, MPH, Pfizer: Employee|Pfizer: Stocks/Bonds Elisa Gonzalez, MPH, Pfizer: Employee|Pfizer: Stocks/Bonds Pingping Zhang, MS, Pfizer: Employee|Pfizer: Stocks/Bonds Michael W. Pride, PhD, Pfizer: Employee|Pfizer: Stocks/Bonds Sharon Gray, MPH, Pfizer: Employee|Pfizer: Stocks/Bonds Catia Matos Ferreira, PhD, Pfizer: Employee|Pfizer: Stocks/Bonds Forest W. Arnold, DO, MSc, Gilead Sciences, Inc.: Grant/Research Support Raul E. Isturiz, MD, Pfizer: Employee|Pfizer: Stocks/Bonds Nadia Minarovic, PhD, Pfizer: Employee|Pfizer: Stocks/Bonds Jennifer Moisi, PhD, Pfizer: Employee|Pfizer: Stocks/Bonds Luis Jodar, PhD, Pfizer: Employee|Pfizer: Stocks/Bonds.

658. Diagnostic Testing Among Patients with Suspected Recurrent Clostridioides difficile Infection (rCDI) in ECOSPOR III a Phase 3 Clinical Trial: Implications for Clinical Practice vs Clinical Trials

BackgroundAccurate diagnosis of rCDI is challenging because of limitations in test performance and alternative causes of recurrent diarrhea, such as post-infectious irritable bowel syndrome (IBS). Stool enzyme immunoassay (EIA) toxin testing (TOX) is the best predictor of active disease, but may miss cases of CDI when toxins are below the limit of detection. In contrast, glutamate dehydrogenase (GDH) or PCR have high sensitivity but cannot differentiate colonization from infection, leading to possible overdiagnosis due to low specificity. In ECOSPOR III, SER-109, an investigational purified microbiome therapeutic, was superior to placebo in reducing rCDI (12.4% vs 39.8%, respectively; p-value < 0.001). We examined diagnostic testing patterns among screened subjects. MethodsPatients with ≥2 prior episodes and ≥3 unformed bowel movements over 48 hours were screened. To ensure enrollment of patients with active CDI, toxin testing was required at entry via a local certified or central lab (Eurofins; Framingham, MA). Subjects with discordant GDH+/TOX- tests at the central lab had reflex confirmatory testing with a cell cytotoxicity neutralization assay (CCNA), considered the “gold standard” for toxin testing.ResultsThe leading reason for screen failure among 281 subjects screened was a negative toxin test (50/99; 50.5%). Of 182 patients enrolled, 59 (32.4%) qualified with EIA TOX+ at the local lab (33 TOX+; 25 GDH+/TOX+) and 122 (67.0%) qualified by the central lab (Table 1). Of these 122 subjects, 87 qualified by GDH+/TOX+ but 35 required additional reflex testing by CCNA due to discordant GDH+/TOX-results; all 35 were positive. Diagnostic Testing for Qualifying C. difficile Episode in ITT Population ConclusionThese diagnostic testing patterns suggest a subset of patients with suspected rCDI have toxin concentrations below the EIA threshold for detection or may have an alternative cause of diarrhea, such as post-infectious IBS. Thus, the limitations of EIA toxin testing need to be considered in clinical practice when evaluating patients with compatible symptoms of rCDI and a high prior probability of infection. In contrast, in trials of investigational agents, toxin testing assures enrollment of patients with active disease and accurate estimates of efficacy. Disclosures Matthew Sims, MD, PhD, FACP, FIDSA, Astra Zeneca (Independent Contractor)Diasorin Molecular (Independent Contractor)Epigenomics Inc (Independent Contractor)Finch (Independent Contractor)Genentech (Independent Contractor)Janssen Pharmaceuticals NV (Independent Contractor)Kinevant Sciences gmBH (Independent Contractor)Leonard-Meron Biosciences (Independent Contractor)Merck and Co (Independent Contractor)OpGen (Independent Contractor)Prenosis (Independent Contractor)Regeneron Pharmaceuticals Inc (Independent Contractor)Seres Therapeutics Inc (Independent Contractor)Shire (Independent Contractor)Summit Therapeutics (Independent Contractor) Sahil Khanna, MBBS, MS, Seres (Grant/Research Support) Darrell Pardi, MD, seres (Consultant)Vedanta (Consultant) Paul Feuerstadt, MD, FACG, Ferring/Rebiotix Pharmaceuticals (Consultant, Scientific Research Study Investigator, Speaker's Bureau)Finch Pharmaceuticals (Scientific Research Study Investigator)Merck and Co (Speaker's Bureau)SERES Therapeutics (Consultant, Scientific Research Study Investigator)Takeda Pharmaceuticals (Consultant) Elaine E. Wang, MD, Seres Therapeutics (Employee) Elaine E. Wang, MD, Seres Therapeutics (Employee, Shareholder) Barbara McGovern, MD, Seres Therapeutics (Employee, Shareholder) Lisa von Moltke, MD, Seres Therapeutics (Employee, Shareholder)

Development and clinical validation of an automated cell cytotoxicity neutralization assay for detecting Clostridioides difficile toxins in clinically relevant stools samples

ObjectivesTo improve the diagnostic accuracy of Clostridioides difficile infection, current U.S. and E.U. guidelines recommend multistep testing that detects the presence of C. difficile and toxin in clinically relevant stool samples to confirm active disease. An accepted gold standard to detect C. difficile toxins is the cell cytotoxicity neutralization assay (CCNA). Although highly sensitive, the traditional CCNA has limitations. One such limitation is the subjective interpretation of an analyst to recognize cytopathic effects in cultured cells exposed to a fecal sample containing toxin. To overcome this limitation, an automated CCNA was developed that replaces most human pipetting steps with robotics and incorporates CellTiterGlo® for a semi-quantitative, non-subjective measure of cell viability instead of microscopy. MethodsTo determine sample positivity and control for non-specific cytopathic effects, two thresholds were defined and validated by evaluating the sample with/without antitoxin antisera (sample-antitoxin/sample + antitoxin): 1) a >70% cell viability threshold was validated with samples containing anti-toxin, and 2) a >1.2-fold difference cut-off where sample results above the cut-off are considered positive. ResultsAssay validation demonstrated excellent accuracy, precision, and sample linearity with an LOD of 126.9 pg/mL toxin-B in stool. The positivity cut-offs were clinically validated by comparing 322 diarrheal stool sample results with those run in a predicate, microscopic readout-based CCNA. The automated CCNA demonstrated 96% sensitivity and 100% specificity compared with the predicate CCNA. Conclusions: Overall, the automated CCNA provides a specific, sensitive, and reproducible tool to support determination of CDI epidemiology or the efficacy of interventions such as vaccines.

Association Between Federal Value-Based Incentive Program Implementation and Hospital-Onset C. difficile Infection Rates

Background: The Hospital-Acquired Conditions Reduction Program (HACRP) and Hospital Value-Based Purchasing (HVBP) are federal value-based incentive programs that financially reward or penalize hospitals based on quality metrics. Hospital-onset C. difficile infection (HO-CDI) rates reported to the CDC NHSN became a target quality metric for both HACRP and HVBP in October 2016, but the impact of these programs on HO-CDI rates is unknown. Methods: We used an interrupted time-series design to examine the association between HACRP/HVBP implementation in October 2016 and quarterly rates of HO-CDI per 10,000 patient days among incentive-eligible acute-care hospitals conducting facility-wide HO-CDI NHSN surveillance between January 2013 and March 2019. Generalized estimating equations were used to fit negative binomial regression models to assess for immediate program impact (ie, level change) and changes in the slope of HO-CDI rates, controlling for each hospital’s predominant method for CDI testing (nucleic acid amplification including PCR (NAAT), enzyme immunoassay for toxin (EIA), or other testing method including cell cytotoxicity neutralization assay and toxigenic culture). Results: Of the 265 study hospitals studied, most were medium-sized (100–399 beds, 55%), not-for-profit (77%), teaching hospitals (70%), and were located in a metropolitan area (87%). Compared to EIA, rates of HO-CDI were higher when detected by NAAT (incidence rate ratio [IRR], 1.55; 95% CI, 1.41–1.70) or other testing methods (IRR, 1.47; 95% CI, 1.26–1.71). Controlling for CDI testing methods, HACRP/HVBP implementation was associated with an immediate 6% decline in HO-CDI rates (IRR, 0.94; 95% CI, 0.89–0.99) and a 4% decline in slope per year-quarter thereafter (IRR, 0.96; 95% CI, 0.95–0.97) (Fig. 1). Conclusions: HACRP/HVBP implementation was associated with both immediate and gradual improvements in HO-CDI rates, independent of CDI testing methods of differing sensitivity. Future research may evaluate the precise mechanisms underlying this improvement and if this impact is sustained in the long term.Funding: NoneDisclosures: None

Two-step Testing for Clostridioides Difficile is Inadequate in Differentiating Infection From Colonization in Children.

Recent Infectious Disease Society of America guidelines recommend multistep testing algorithms to diagnose Clostridioides difficile infection (CDI), including a combination of nucleic acid amplification-based testing (NAAT) and toxin enzyme immunoassay (EIA). The use of these algorithms in children, including the ability to differentiate between C. difficile colonization and CDI, however, has not been evaluated. We prospectively enrolled asymptomatic pediatric patients with cancer, cystic fibrosis (CF), or inflammatory bowel disease (IBD) and obtained a stool sample for NAAT testing. If positive by NAAT (colonized), EIA was performed. In addition, children with symptomatic CDI who tested positive by NAAT via the clinical laboratory were enrolled, and EIA was performed on residual stool. A functional cell cytotoxicity neutralization assay (CCNA) was also applied to stool samples from both the colonized and symptomatic cohorts. Of the 225 asymptomatic children enrolled in the study, 47 (21%) were colonized with C. difficile including 9/59 (15.5%) with cancer, 30/92 (32.6%) with CF, and 8/74 (10.8%) with IBD. An additional 41 children with symptomatic CDI were enrolled. When symptomatic and colonized children were compared, neither EIA positivity (44% vs 26%, P = 0.07) nor CCNA positivity (49% vs 45%, P = 0.70) differed significantly or were able to predict disease severity in the symptomatic cohort. Use of a multistep testing algorithm with NAAT followed by EIA failed to differentiate symptomatic CDI from asymptomatic colonization in our pediatric cohort. As multistep algorithms are moved into clinical care, the pediatric provider will need to be aware of their limitations.

4345 Two-step Algorithm for Clostridioides difficile is Inadequate for Differentiating Infection from Colonization in Children

OBJECTIVES/GOALS: In 2017, new guidelines recommended multi-step algorithms for CDI diagnosis, and clinical centers rapidly implemented changes despite limited pediatric data. We assessed a multi-step algorithm using NAAT followed by EIA for ability to differentiate symptomatic CDI from colonization in children. METHODS/STUDY POPULATION: We prospectively enrolled pediatric patients with cancer, cystic fibrosis, or inflammatory bowel disease who were not being tested or treated for CDI and obtained a stool sample for NAAT. If positive by NAAT (colonized), EIA was performed. Children with symptomatic CDI who tested positive by NAAT via the clinical laboratory were also enrolled and EIA performed on residual stool. A functional cell cytotoxicity neutralization assay (CCNA) was performed in addition. RESULTS/ANTICIPATED RESULTS: Of the 138 asymptomatic children enrolled, 24 (17%) were colonized. An additional 37 children with symptomatic CDI were enrolled. Neither EIA positivity (41% versus 21%, P = 0.11) or CCNA positivity (49% versus 46%, P = 0.84) were significantly different between symptomatic versus colonized children. When both EIA and CCNA were positive, children were more commonly symptomatic than colonized (33% versus 13%, P = 0.04). DISCUSSION/SIGNIFICANCE OF IMPACT: A multi-step testing algorithm with NAAT and EIA failed to differentiate symptomatic CDI from colonization in our pediatric cohort. As multi-step algorithms are moved into clinical care, pediatric providers will need to be aware of the continued limitations in diagnostic testing.

Impact of ribotype on Clostridioides difficile diagnostics

This study investigates the performance of diagnostic methods for detection of Clostridioides difficile infection in Sweden, including impact of PCR ribotype on diagnostic performance. Between 2011 and 2016, a total of 17,878 stool samples from 26 laboratories were tested by either well-type enzyme immunoassays (EIAs), membrane bound EIAs, cell cytotoxicity neutralization assay (CTA), or nucleic acid amplification tests (NAATs) and subsequently cultured for C. difficile. Roughly half of the samples (9454/17878) were subjected to diagnostic testing both on the fecal sample and on the 1323 isolated C. difficile strains. All C. difficile isolates were typed by PCR ribotyping, and the isolates were classified as toxigenic or non-toxigenic based on the empirical knowledge of the association between toxin-positivity and ribotype. The overall sensitivity, specificity, and positive and negative predictive values were highest for NAATs and membrane EIAs. Ribotype-specific sensitivity varied greatly between methods and ribotypes. All methods had 100% sensitivity against ribotype 027 and 013. For other types, the sensitivity ranged from 33 to 85% in fecal samples and from 78 to 100% on isolates. For the most prevalent ribotypes (014, 020, and 001), the sensitivity varied between 38 and 100% in the fecal samples, with the lowest sensitivity observed for well-type EIAs and CTA. The large variation in diagnostic sensitivity implies that type distribution significantly affects the outcome when evaluating diagnostic performance. Furthermore, performing comparative studies of diagnostic tests in settings with high prevalence of ribotype 027 will overestimate the general performance of diagnostic tests.

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

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.MethodsStool 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.ResultsThere 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.ConclusionThe 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.

2357. Toxin Detection Using Single Molecule Counting Technology: The Best of Both Worlds?

BackgroundAccurate diagnosis of CDI remains challenging as there is no standalone laboratory test with adequate clinical sensitivity and specificity. Thus, many clinical laboratories currently employ a multistep algorithm incorporating a sensitive screening test followed by a specific toxin test. An automated ultrasensitive toxin immunoassay (Singulex Clarity® C. difficile toxins A/B assay) has demonstrated excellent performance compared with cell cytotoxicity neutralization assay (CCNA). In this study, the Clarity assay was evaluated relative to glutamate dehydrogenase (GDH), toxin EIA, toxin B gene PCR, multistep algorithms, and C. difficile culture with ribotyping.MethodsResidual clinical stool samples (n = 293) were collected from patients with suspected CDI. The samples were tested on-site with GDH (C. DIFF CHEK™-60), PCR (EntericBio realtime® C. difficile assay), a membrane-type toxin EIA (Tox A/B Quik Chek®), and culture and ribotyping. In total, 188 samples were tested with GDH and 239 samples were tested by PCR. All PCR-positive samples (n = 148) and prospectively tested GDH samples (n = 97) were tested with the toxin EIA. Culture and ribotyping information were available for 205 samples.ResultsThree of the samples tested gave no result using the Clarity assay and were excluded from the analysis. The Singulex Clarity C. difficile toxins A/B assay had high positive percent agreement (PPA) and low negative percent agreement (NPA) compared with toxin EIA and multistep algorithms ending with toxin EIA. The Clarity assay had high NPA and low PPA compared with PCR, GDH, and the multistep algorithm ending with PCR (figure). Less than 70% of the detected C. difficile PCR positive samples had toxins present. There was no difference in toxin concentration between the ribotypes.ConclusionThe Clarity assay had strong PPA compared with toxin EIA and strong NPA compared with PCR. The low NPA and PPA compared with toxin EIA and PCR, respectively, may reflect the poor sensitivity of current toxin EIAs and low specificity of PCR. The Clarity assay detected 30 different ribotype strains, and less than 70% of samples (by PCR) or strains (by ribotyping) had toxins present. The Clarity assay may be considered for use as a standalone test for CDI diagnosis. Disclosures All authors: No reported disclosures.

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

Diagnostic tests for Clostridioides difficile infection (CDI) lack either specificity (nucleic acid amplification tests) or sensitivity (enzyme immunoassays; EIAs). The performance of the Singulex Clarity® C. diff toxins A/B assay was compared to cell cytotoxicity neutralization assay. Testing was also performed using an EIA for glutamate dehydrogenase (GDH) and C. difficile toxins A and B (C. Diff Quik Chek Complete®), polymerase chain reaction (PCR) (BD MAX™ Cdiff Assay), and 2 multistep algorithms: algorithm 1 (discordant GDH/toxin results arbitrated by PCR) and algorithm 2 (PCR-positive samples tested with toxin EIA). The Clarity assay and PCR both had 97% sensitivity, while specificity was 100% for Clarity and 79% for PCR. Algorithm 1 yielded 41% discordant results, and both toxin EIA and algorithm 2 had 58% sensitivity. Median toxin concentrations, as measured by the Clarity C. difficile toxin assay, were 3590, 11.5, 0.4, and 0 pg/mL for GDH+/toxin+, GDH+/toxin−/PCR+, GDH+/toxin−/PCR−, and GDH−/toxin− samples, respectively (P < 0.001). The Clarity assay may offer a single-test solution for CDI.

1093. Single Molecule Counting Technology for Ultrasensitive Quantification of Clostridium difficile Toxins A and B

Background Clostridium difficile, a spore-forming, anaerobic, Gram-positive bacterium, is the leading cause of nosocomial diarrhea. C. difficile infection (CDI) is mediated by two toxins, A (TcdA) and B (TcdB), and the role of each toxin in CDI pathogenesis remains unclear. Many assays used in CDI diagnostics, such as most NAATs and cell cytotoxicity neutralization assay (CCNA), detect presence of only tcdB or TcdB. In this study, an ultrasensitive immunoassay (UIA) powered by Single Molecule Counting technology was used for quantification of TcdA and TcdB, to assess toxin dynamics in CDI.MethodsBanked samples from 46 patients with suspected CDI were tested with PCR (BD MAX™ Cdiff Assay) and CCNA, and TcdA and TcdB were quantified using the UIA (tested in triplicate). The limits of detection (LoDs) for the TcdA and TcdB assays are 0.04 and 0.12 pg/mL, respectively.ResultsThere were 21 PCR+/CCNA+ and 25 PCR−/CCNA− samples. Both toxins were measured above LoD in all PCR+/CCNA+ samples, ranging up to 100,000 pg/mL. The average CV for the PCR+/CCNA+ samples was 9%. The median TcdA concentrations in PCR−/CCNA− and PCR+/CCNA+ samples were 0.19 pg/mL (IQR 0.12–0.67) and 3,301 pg/mL (125–8,737), respectively. The median TcdB concentrations in PCR−/CCNA− and PCR+/CCNA+ samples were 0.12 pg/mL (0.12–0.21) and 2,690 pg/mL (145–30,307), respectively. In the PCR+/CCNA+ samples, TcdA was one or more logs higher than TcdB in two samples, one or more logs lower than TcdB in six samples, and within one log of TcdB in 13 samples. In one sample (4.8% of PCR+/CCNA+ samples), TcdA was at moderately high concentration while TcdB was below a provisional cutoff, indicating that only TcdA was expressed. There was a significant correlation between TcdA and TcdB (Spearman r = 0.753).ConclusionThe UIA allows for toxin quantification over a concentration range of ≥5 logs, suggesting that the quantitative TcdA and TcdB assays could be of value in CDI characterization and clinical decision making. The TcdA/TcdB ratio varied, and toxin quantification could be a useful tool in further understanding their individual roles in CDI. The TcdA concentration was not lower than TcdB (trended higher), indicating that detection of tcdB or TcdB alone may not be sufficient for accurate CDI diagnostics. Disclosures P. Katzenbach, Singulex, Inc.: Employee, Salary. G. Dave, Singulex, Inc.: Employee, Salary. A. Mukherjee, Singulex, Inc.: Employee, Salary. J. Todd, Singulex, Inc.: Employee, Salary. J. Bishop, Singulex, Inc.: Employee, Salary. J. Sandlund, Singulex, Inc.: Employee, Salary. J. Estis, Singulex, Inc.: Employee, Salary.

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

BackgroundCommercially 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).MethodsFresh 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.).ResultsThe 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.ConclusionThe 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.

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

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.MethodsLimits 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.ResultsThe 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.ConclusionThe 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.

Advances in the diagnosis and treatment of Clostridium difficile infections

Clostridium difficile is a leading cause of antibiotic-associated diarrhea worldwide. The diagnosis of C. difficile infection (CDI) requires both clinical manifestations and a positive laboratory test for C. difficile and/or its toxins. While antibiotic therapy is the treatment of choice for CDI, there are relatively few classes of effective antibiotics currently available. Therefore, the development of novel antibiotics and/or alternative treatment strategies for CDI has received a great deal of attention in recent years. A number of emerging agents such as cadazolid, surotomycin, ridinilazole, and bezlotoxumab have demonstrated activity against C. difficile; some of these have been approved for limited clinical use and some are in clinical trials. In addition, other approaches such as early and accurate diagnosis of CDI as well as disease prevention are important for clinical management. While the toxigenic culture and the cell cytotoxicity neutralization assay are still recognized as the gold standard for the diagnosis of CDI, new diagnostic approaches such as nucleic acid amplification methods have become available. In this review, we will discuss both current and emerging diagnostic and therapeutic modalities for CDI.

Diagnostic Guidance for C. difficile Infections.

Diagnosis of Clostridium difficile infection (CDI) can be challenging. First of all, there has been debate on which of the two reference assays, cell cytotoxicity neutralization assay (CCNA) or toxigenic culture (TC) should be considered the gold standard for CDI detection. Although the CCNA suffers most from suboptimal storage conditions and subsequent toxin degradation, TC is reported to falsely increase CDI detection rates as it cannot differentiate CDI patients from patients asymptomatically colonised by toxigenic C. difficile. Several rapid assays are available for CDI detection and fall into three broad categories: (1) enzyme immunoassays for glutamate dehydrogenase, (2) enzyme immunoassays for toxins A/B and (3) nucleic acid amplification tests detecting toxin genes. All three categories have their own limitations, being suboptimal specificity and/or sensitivity or the inability to discern colonised patients from CDI patients. In light of these limitations, multi-step algorithmic testing has now been advocated by international guidelines in order to optimize diagnostic accuracy. Despite these recommendations, testing methods between hospitals vary widely, which impacts CDI incidence rates. CDI incidence rates are also influenced by sample selection criteria, as several studies have shown that if not all unformed stool samples are tested for CDI, many cases may be missed due to an absence of clinical suspicion. Since methods for diagnosing CDI remain imperfect, there has been a growing interest in alternative testing strategies like faecal biomarkers, immune modulating interleukins, cytokines and imaging methods. At the moment, these alternative methods might play an adjunctive role, but they are not suitable to replace conventional CDI testing strategies.

Clostridium difficile PCR Cycle Threshold Predicts Free Toxin.

There is no stand-alone Clostridium difficile diagnostic that can sensitively and rapidly detect fecal free toxins. We investigated the performance of the C. difficile PCR cycle threshold (CT ) for predicting free toxin status. Consecutive stool samples (n = 312) positive for toxigenic C. difficile by the GeneXpert C. difficile/Epi tcdB PCR 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 of CT was measured at different CT cutoffs. Using RMEIA as the reference method, a CT cutoff 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 improved CT specificity to 78.0% (95% CI, 70.7% to 84.2%). Intercartridge lot CT variability measured as the average coefficient of variation was 2.8% (95% CI, 1.2% to 3.2%). Standardizing the input stool volume did not improve CT toxin specificity. The median CT values 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 toxigenic C. difficile in 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.

Diagnostic test accuracy of glutamate dehydrogenase for Clostridium difficile: Systematic review and meta-analysis.

We performed this systematic review and meta-analysis to assess the diagnostic accuracy of detecting glutamate dehydrogenase (GDH) for Clostridium difficile infection (CDI) based on the hierarchical model. Two investigators electrically searched four databases. Reference tests were stool cell cytotoxicity neutralization assay (CCNA) and stool toxigenic culture (TC). To assess the overall accuracy, we calculated the diagnostic odds ratio (DOR) using a DerSimonian-Laird random-model and area the under hierarchical summary receiver operating characteristics (AUC) using Holling’s proportional hazard models. The summary estimate of the sensitivity and the specificity were obtained using the bivariate model. According to 42 reports consisting of 3055 reference positive comparisons, and 26188 reference negative comparisons, the DOR was 115 (95%CI: 77–172, I2 = 12.0%) and the AUC was 0.970 (95%CI: 0.958–0.982). The summary estimate of sensitivity and specificity were 0.911 (95%CI: 0.871–0.940) and 0.912 (95%CI: 0.892–0.928). The positive and negative likelihood ratios were 10.4 (95%CI 8.4–12.7) and 0.098 (95%CI 0.066–0.142), respectively. Detecting GDH for the diagnosis of CDI had both high sensitivity and specificity. Considering its low cost and prevalence, it is appropriate for a screening test for CDI.

Low sensitivity of fecal toxin A/B enzyme immunoassay for diagnosis of Clostridium difficile infection in immunocompromised patients

The optimal approach in laboratory diagnosis of Clostridium difficile infection (CDI) is still not well defined. Toxigenic culture (TC) or alternatively fecal toxin assay by cell cytotoxicity neutralization assay are considered to be the reference standard, but these methods are time-consuming and labor intensive. In many medical centers, diagnosis of CDI is therefore still based on fecal toxin A/B enzyme immunoassay (EIA) directly from stool alone, balancing cost and speed against limited diagnostic sensitivity. The aim of the study was to assess in which patient population the additional workload of TC is justified. All consecutive stool specimens submitted for diagnosis of suspected CDI between 2004 and 2011 at a tertiary-care center were examined by toxin EIA and TC. Clinical data of patients with established diagnosis of CDI were collected in a standardized case-report form. From 12 481 stool specimens submitted to the microbiologic laboratory, 480 (3.8%) fulfilled CDI criteria; 274 (57.1%) were diagnosed by toxin EIA; and an additional 206 (42.9%) were diagnosed by TC when toxin EIA was negative. Independent predictors for negative toxin EIA but positive TC were high-dose corticosteroids (odds ratio (OR) 2.97, 95% confidence interval (CI) 1.50–5.90, p 0.002), leukocytopenia <1000/μL (OR 2.52, 95% CI 1.22–5.23, p 0.013) and nonsevere CDI (OR 2.21, 95% CI 1.39–3.50, p 0.001). There was no difference in outcomes such as in-hospital mortality and recurrence between both groups. In conclusion, negative toxin EIA does not rule out CDI in immunocompromised patients in the setting of relevant clinical symptoms. Methods with improved sensitivity such as TC or PCR should be used, particularly in this patient population.