Clinical outcomes and treatment necessity in patients with toxin-negative Clostridioides difficile stool samples.

PURPOSE: The clinical significance of negative toxin enzyme immunoassays (EIA) for Clostridioides difficile infections (CDIs) is unclear. Our study aimed to investigate the significance of toxin EIA-negative in the diagnosis and prognosis of CDI. METHODS: All stool specimens submitted for C. difficile toxin EIA testing were cultured to isolate C. difficile. In-house PCR for tcdA, tcdB, cdtA, and cdtB genes were performed using C. difficile isolates. Stool specimens were tested with C. difficile toxins A and B using EIA kit (RIDASCREEN Clostridium difficile toxin A/B, R-Biopharm AG, Darmstadt, Germany). Characteristics and subsequent CDI episodes of toxin EIA-negative and -positive patients were compared. RESULTS: Among 190 C. difficile PCR-positive patients, 83 (43.7%) were toxin EIA-negative. Multivariate analysis revealed independent associations toxin EIA-negative results and shorter hospital stays (OR = 0.98, 95% CI 0.96-0.99, p = 0.013) and less high-risk antibiotic exposure in the preceding month (OR = 0.38, 95% CI 0.16-0.94, p = 0.035). Toxin EIA-negative patients displayed a significantly lower white blood cell count rate (11.0 vs. 35.4%, p < 0.001). Among the 54 patients who were toxin EIA-negative and did not receive CDI treatment, three (5.6%) were diagnosed with CDI after 7-21 days without complication. CONCLUSION: Our study demonstrates that toxin EIA-negative patients had milder laboratory findings and no complications, despite not receiving treatment. Prolonged hospitalisation and exposure to high-risk antibiotics could potentially serve as markers for the development of toxin EIA-positive CDI.

Human Leukemia T-Cell Lines as Alternatives to Animal Use for Detecting Biologically Active Staphylococcal Enterotoxin Type B.

Staphylococcal enterotoxin type B (SEB) is associated with food poisoning. Current methods for the detection of biologically active SEB rely upon its ability to cause emesis when administered to live kittens or monkeys. This technique suffers from poor reproducibility and low sensitivity and is ethically disfavored over concerns for the welfare of laboratory animals. The data presented here show the first successful implementation of an alternative method to live animal testing that utilizes SEB super-antigenic activity to induce cytokine production for specific novel cell-based assays for quantifiable detection of active SEB. Rather than using or sacrificing live animals, we found that SEB can bind to the major histocompatibility complex (MHC) class II molecules on Raji B-cells. We presented this SEB-MHC class II complex to specific Vß5.3 regions of the human T-cell line HPB-ALL, which led to a dose-dependent secretion of IL-2 that is capable of being quantified and can further detect 10 pg/mL of SEB. This new assay is 100,000 times more sensitive than the ex vivo murine splenocyte method that achieved a detection limit of 1 µg/mL. The data presented here also demonstrate that SEB induced proliferation in a dose-dependent manner for cells obtained by three different selection methods: by splenocyte cells containing 22% of CD4+ T-cells, by CD4+ T-cells enriched to >90% purity by negative selection methods, and by CD4+ T-cells enriched to >95% purity by positive selection methods. The highly enriched and positively isolated CD4+ T-cells with the lowest concentration of antigen-presenting cells (APC) (below 5%) provided higher cell proliferation than the splenocyte cells containing the highest concentration of APC cells.

¿La detección de toxinas de Clostridioides difficile es necesaria cuando se detecta la enzima glutamato deshidrogenasa? / Is Clostridioides difficile toxins detection necessary when the glutamate dehydrogenase enzyme is detected?

Resumen Introducción: Clostridioides difficile causa diarrea y colitis pseudomembranosa. Su diagnóstico se realiza con la detección de glutamato-deshidrogenasa (GDH) o las toxinas A y B y se confirma con pruebas de amplificación de ácidos nucleicos. Objetivo: Definir si la determinación de GDH es redundante a la de las toxinas. Métodos: Estudio observacional retrospectivo de muestras fecales de pacientes con sospecha de infección por Clostridioides difficile. Las toxinas y GDH se determinaron mediante inmunocromatografía. Se realizó una simulación bayesiana con los cocientes de probabilidad; se consideró significativo un valor de p < 0.05. Resultados: Se analizaron 329 resultados de GDH y toxinas A y B. Se encontró una prevalencia de infección de Clostridioides difficile de 18.2 %. La sensibilidad y especificidad de la prueba de GDH fue de 0.90 y 0.89, respectivamente. El cociente de probabilidad positivo fue de 8.9 y el negativo, de 0.11. Conclusiones: Un resultado negativo de GDH disminuye considerablemente la probabilidad de infección, pero no la descarta. La detección de toxinas de Clostridioides difficile puede ser necesaria en instituciones donde la amplificación de ácidos nucleicos no es económica o accesible.

Abstract Introduction: Clostridioides difficile causes diarrhea and pseudomembranous colitis. Its diagnosis is made with glutamate dehydrogenase (GDH) or toxins A and B detection and is confirmed with nucleic acid amplification tests. Objective: To define if GDH determination is redundant to that of toxins. Methods: Retrospective, observational study in diarrheal stools of patients with suspected Clostridioides difficile infection. Toxins and GDH were determined by immunochromatography. Bayesian simulation was performed with likelihood ratios; a p-value < 0.05 was regarded as significant. Results: 329 GDH and toxin A and B results were analyzed. Clostridioides difficile infection prevalence was 18.2 %. Sensitivity and specificity of the GDH test were 0.90 and 0.89, respectively. Positive likelihood ratio was 8.9, and negative was 0.11. Conclusions: A negative GDH result considerably reduces the probability of infection but does not rule it out. Clostridioides difficile toxins detection may be necessary in institutions where nucleic acid amplification is not affordable or accessible.

Clostridioides difficile laboratory diagnostic techniques: a comparative approach of rapid and molecular methods.

Clostridioides difficile infection is a public health problem because of it is easily spread; with harmful consequences, it is essential to reduce hospital costs and prevent its dissemination by having a precise diagnosis. The gold standard for its diagnosis is polymerase chain reaction (PCR); however, the technique is not available for all laboratories due to the high cost. New approaches using non-molecular tests to detect C. difficile and toxin A/B production has been proposed to improve cost benefits. The objective of this study is to compare molecular methods (PCR) and rapid methods (immunochromatographic test and enzymatic immunoassay). A series of tests comprising these diagnostic techniques was performed with 50 patients with a clinical diagnosis for Clostridioides difficile on GeneXpert® devices test; a calculation of the sensitivity was executed, followed by a comparison of the efficiency of all techniques. Greater sensitivity was observed in the PCR-based methods (BD MAX™ and BioFire FilmArray®) and the GDH-based assays (RIDASCREEN® and Alere Techlab®). The proposed algorithm represents minor monetary disadvantages but a significant temporal optimization of 10%. Future studies concerning both positive and negative results could be advantageous because of the possibility of calculating more method concordance indexes, such as the specificity and Kappa index, in addition to being able to indicate a monetary profit if the proposed algorithm was applied due to the nonproceeding PCR cases.

The reference method influence on the sensitivity of the Clostridium difficile enzyme immunoassays: A meta analysis.

The use of enzyme immunoassays to screen for toxins A and B produced by Clostridium difficile is a common procedure in algorithms designed for its detection. Moreover, the absence of a unique test capable of providing reliable results at low cost motivates a great discussion about which algorithm is the best. Thus, several studies have evaluated the performance of these enzyme immunoassays. However, all fail to provide sufficient explanations for the different behaviours observed in different studies that evaluate the same index test against a common reference method. Our main goal was to find out which factors affect the sensitivity of these assays, since the specificity is very close to 1. In this research, we verified that sensitivity increases with the prevalence rate and with the proportion of reported cases of onset diarrhea. Therefore, its use is advisable for high prevalence rates (e.g. in an epidemic setting). As far as reference methods are concerned, nucleic acid amplification tests can be used as a reference method, with a performance similar to the well-accepted toxigenic culture. The method chosen for toxigenicity screening in a toxigenic culture also seems to affect the evaluation performance of tests and should be better studied in the future.

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

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.

Brote de intoxicación alimentaria en un jardín de infantes de la provincia de Buenos Aires / Food poisoning outbreak in a kindergarten in the Province of Buenos Aires, Argentina

Resumen El 27 de noviembre de 2008 ocurrió un brote de intoxicación alimentaria asociado al consumo de salpicón de ave en un jardín de infantes de Hurlingham, provincia de Buenos Aires. Treinta y siete niños y 10 adultos presentaron síntomas gastrointestinales. Cinco niños fueron internados con signos de deshidratación, y uno de ellos requirió cuidados intensivos. Se aisló Staphylococcus aureus subsp. aureus del alimento involucrado, de 4/5 muestras de materia fecal de pacientes y de 3/5 manipuladores (nariz del manipulador 1, manos de manipuladores 2 y 3). Las cepas aisladas portaban los genes que codifican las enterotoxinas SEA y SED. Por electroforesis de campo pulsado con la enzima SmaI, los patrones de macrorrestricción presentaron 100% de similitud. La investigación oportuna del brote permitió identificar al agente causal de la intoxicación, determinar las fallas en la elaboración del alimento e implementar las medidas correctivas correspondientes.

Abstract On November 27, 2008, a foodborne disease outbreak associated with the consumption of chicken salad occurred in a kindergarten in the District of Hurlingham, Province of Buenos Aires. Thirty-seven children and 10 adults with gastrointestinal symptoms were affected. Five children were hospitalized with signs of dehydration, one of them requiring intensive care. Staphylococcus aureus subsp. aureus was isolated from the mentioned food in 4 out of 5 stool specimens from the patients, and in 3 out of 5 food handlers (nose of food handler #1, hands of food handlers #2 and 3). The isolates carried the genes coding for enterotoxins SEA and SED. The macrorestriction patterns showed 100% similarity by pulsed-field gel electrophoresis using the SmaI enzyme. A timely outbreak investigation allowed us to identify the causative agent of the food poisoning as well as the failures in food processing and to implement corrective measures.

Study of the effectiveness of staphylococcins in biopreservation of Minas fresh (Frescal) cheese with a reduced sodium content.

Reducing salt content in foods such as cheeses, while limiting the growth of spoilage microorganisms and foodborne pathogens, is a difficult challenge. One method that may prove useful is use of staphylococcins, which are bacteriocins produced by staphylococci. Therefore, staphylococcin antimicrobial activity against six strains of S. aureus isolated from cheese was tested aiming at their industrial application in biopreservation of Minas fresh (Frescal) cheese with reduced sodium content. Three staphylococcins were selected for these tests: Pep 5, aureocin A53 and lysostaphin. All three staphylococcins proved to be bacteriolytic against all six strains of S. aureus. The antimicrobial activity of the partially purified staphylococcins was subsequently investigated against strains S. aureus Q1 and QJ3 in cheese matrices (6.0 log CFU/g) with different NaCl contents (control, a 25% reduction, and a 50% reduction), kept under refrigeration at 4 °C, for 21 days. Both strains were shown to be of concern for food industry as they carry the SEA, SEB and SEH enterotoxin genes, and are resistant to ß-lactam drugs and moderate biofilm formers when grown in TSB. When used singly, Pep5, aureocin A53 and lysostaphin reduced approximately 95%, 99% and 99.99% of the viable cell counts, respectively, irrespective of the sodium content of the cheese matrix. The combined action of aureocin A53 and Pep5 resulted in an additional and significant reduction (p < 0.05) of ~1.0 log CFU/g when compared with the reduction caused by the use of either one singly. The combined action of lysostaphin and aureocin A53 or lysostaphin and Pep5 resulted in a reduction similar to or slightly smaller (p > 0.05) than that observed when lysostaphin was employed singly. Lysostaphin also proved to reduce the number of the staphylococcal viable cells to a level (~ 2.0 log CFU/g) at which enterotoxin production should not reach a sufficient quantity to cause food poisoning. Therefore, lysostaphin may have a practical application in the food industry to control staphylococcal contamination of Minas fresh cheese with a sodium content reduced up to 50%, providing consumers with more safe options to reduce their intake of sodium.

Quantification of staphylococcal enterotoxin type A in cow milk by using a stable isotope-labelled peptide via liquid chromatography-tandem mass spectrometry.

In this study, the staphylococcal enterotoxin type A (SEA) contaminant was quantified in cow milk by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with the use of a stable isotope-labelled peptide of SEA as an internal standard. SEA was cleaned up in a two-step process that included pH control and trichloroacetic acid (TCA) precipitation. The pH control phase eliminated other proteins. TCA precipitation cleaned up SEA without special equipment. An appropriate enzyme-to-protein ratio maximised tryptic digestion. A desalting process guaranteed the stable retention of SEA-digested peptides. The coverage of amino-acid sequences (>10%) clearly identified the toxin's presence. SEA was accurately quantified using LC-MS/MS based on a multiple-reaction monitoring mode. The developed method was validated based on spiked recovery tests at 50 and 100 µg kg-1 conducted with two samples collected on a daily basis for five days based on Japanese validation guidelines. The new method exhibited good accuracy which ranged from 80% to 82%. The relative standard deviations of repeatability were 13-14% and the relative standard deviations of within-laboratory reproducibility were 13-18%. These standard deviations satisfied the criteria of the Japanese validation guidelines. The quantification limit was estimated to be 10 µg kg-1.

Magnetic nanocomposites: versatile tool for the combination of immunomagnetic separation with flow-based chemiluminescence immunochip for rapid biosensing of Staphylococcal enterotoxin B in milk.

Immunomagnetic separation (IMS) was combined with flow-based chemiluminescence sandwich immunoassays (CL-SIA) for the quantification of Staphylococcal enterotoxin B in milk. Therefore, iron oxide-shell silica-core magnetic nanocomposites were conjugated to biotinylated anti-SEB antibodies (MNC-IgGs). MNC-IgGs were applied successfully for (i) capturing SEB in milk samples by an affinity reaction, (ii) magnetophoretic collection on antibody spots in a channel of a flow-based immunochip, and (iii) sensitive enzymatic chemiluminescence detection of biotin labels by poly(horseradish peroxidase)-streptavidin. IMS was performed in 0.6 mL and 100 mL milk samples resulting in detection limits of 50 ng L-1 and 0.39 ng L-1, respectively, for the combined analytical method. It was shown that the assay sensitivity was dramatically improved by the combination of IMS with flow-based CL-SIA compared to CL-SIA directly applied with milk samples (detection limit 130 ng L-1). The IMS-CL-SIA has a time-to-result of 2-3 h. The reported combined analytical method can be used for a rapid control of SEB in complex food matrices such as milk. In future, even the monitoring of multiple contaminants in food or water may be performed by IMS-CL-SIA. Graphical abstract.

Laboratory Diagnosis of Clostridium difficile Infection in Korea: The First National Survey.

In May 2015, we conducted a voluntary online survey on laboratory diagnostic assays for Clostridium difficile infection (CDI) across clinical microbiology laboratories in Korea. Responses were obtained from 66 laboratories, including 61 hospitals and five commercial laboratories. Among them, nine laboratories reported having not conducted CDI assays. The toxin AB enzyme immunoassay (toxin AB EIA), nucleic acid amplification test (NAAT), and C. difficile culture, alone or in combination with other assays, were used in 51 (89.5%), 37 (64.9%), and 37 (64.9%) of the remaining 57 laboratories, respectively, and 23 (40.4%) of the laboratories performed all three assays. Only one laboratory used the glutamate dehydrogenase assay. Nine laboratories used the toxin AB EIA as a stand-alone assay. The median (range) of examined specimens in one month for the toxin AB EIA, NAAT, and C. difficile culture was 160 (50-2,060), 70 (7-720), and 130 (9-750), respectively. These findings serve as valuable basic data regarding the current status of laboratory diagnosis of CDI in Korea, offering guidance for improved implementation.

Rapid and Sensitive Detection of Staphylococcal Enterotoxin B by Recombinant Nanobody Using Phage Display Technology.

Staphylococcal enterotoxin B, from Staphylococcus aureus (S. aureus), is one of the most potent bacterial superantigens with profound toxic effects on the immune system. It is associated with food poisoning, toxic shock, atopic dermatitis, asthma, and nasal polyps in humans. The current diagnostic methods for staphylococcal enterotoxin are mainly based on traditional monoclonal antibodies which hardly meet the requirements for clinical applications, and hybridoma clones lose their ability to secrete antibodies during time. The present study investigates the development of a novel, highly specific, low-cost, and sensitive nanobody capable of being used in immunoassays for Staphylococcal enterotoxin B (SEB) detection in suspicious foods. For this purpose, Camelus dromedarius was immunized against SEB toxin. After obtaining acceptable titration, a high-quality phage display nanobody library (4 × 1010 PFU/ml) was constructed. High-affinity SEB-specific nanobodies were retrieved from constructed libraries. After phage rescue and five round of biopanning, clone screening was performed by phage ELISA. Recombinant nanobodies which were expressed from C7 and C21 clone showed the highest affinity for SEB. The presence of high quality and pure nanobody band at ~ 15 kDa was confirmed by SDS-PAGE and western blotting. The affinity constant which was measured by ELISA was calculated to be around 10-9 M. The results suggest that the proposed detection method by nanobodies is an alternative diagnostic tool enabling a rapid, inexpensive, and specific detection of the SEB.

Evaluation of the TRANSIA® PLATE Staphylococcal Enterotoxins Kit for the Detection of Staphylococcal Enterotoxins in Selected Foods.

The TRANSIA® PLATE Staphylococcal Enterotoxins enzyme immunoassay (EIA) was validated according to AOAC INTERNATIONAL guidelines for validating qualitative binary chemistry and microbiological methods. Five food matrixes were analyzed to determine the probability of detection (POD) for staphylococcal enterotoxins (SE), including SEA, SEB, SEC1, SEC2, SEC3, SED, and SEE, by the TRANSIA ^{PLATE Staphylococcal Enterotoxins EIA. The food matrixes tested were food types implicated in staphylococcal enterotoxin outbreaks and included raw milk cheese, liquid infant formula, eclairs, ready-to-eat ham, and canned mushrooms. Cheese and infant formula were tested with and without dialysis/concentration. The infant formula was also tested by an independent laboratory. Each food matrix was inoculated with specific toxins at low levels to yield fractional recoveries (0.015-0.20 ng/g of food) for POD analysis. One hundred percent recovery was achieved at concentrations ranging from <0.10 ng/g to 0.25 ng/g of toxin in the various food matrixes. At the same time, 50 Staphylococcus aureus strains known to produce toxins and 30 non-toxin producing bacteria (including 22 Staphylococcus strains) were grown and tested. All the SE toxin-producing strains yielded positive results and all of the exclusivity strains were negative. Robustness studies showed that changes in sample volume, sample pH, and EIA assay temperature had no significant effect on performance. Stability studies showed that kits stored for 12+ months performed as well as newly made kits. This assay has been approved as a Performance Tested MethodSM.}

Clostridium difficile colonization among patients with clinically significant diarrhea and no identifiable cause of diarrhea.

OBJECTIVE: To determine the prevalence of Clostridium difficile colonization among patients who meet the 2017 IDSA/SHEA C. difficile infection (CDI) Clinical Guideline Update criteria for the preferred patient population for C. difficile testing. DESIGN: Retrospective cohort. SETTING: Tertiary-care hospital in St. Louis, Missouri.PatientsPatients whose diarrheal stool samples were submitted to the hospital's clinical microbiology laboratory for C. difficile testing (toxin EIA) from August 2014 to September 2016.InterventionsElectronic and manual chart review were used to determine whether patients tested for C. difficile toxin had clinically significant diarrhea and/or any alternate cause for diarrhea. Toxigenic C. difficile culture was performed on all stool specimens from patients with clinically significant diarrhea and no known alternate cause for their diarrhea. RESULTS: A total of 8,931 patients with stool specimens submitted were evaluated: 570 stool specimens were EIA positive (+) and 8,361 stool specimens were EIA negative (-). Among the EIA+stool specimens, 107 (19% of total) were deemed eligible for culture. Among the EIA- stool specimens, 515 (6%) were eligible for culture. One EIA+stool specimen (1%) was toxigenic culture negative. Among the EIA- stool specimens that underwent culture, toxigenic C. difficile was isolated from 63 (12%). CONCLUSIONS: Most patients tested for C. difficile do not have clinically significant diarrhea and/or potential alternate causes for diarrhea. The prevalence of toxigenic C. difficile colonization among EIA- patients who met the IDSA/SHEA CDI guideline criteria for preferred patient population for C. difficile testing was 12%.

Observational study of chemotherapy-induced Clostridium difficile infection in patients with lung cancer.

BACKGROUND: Diarrhea post-antibiotic use is primarily attributed to Clostridium difficile infection (CDI)-induced mucosal lesions, and evidence of CDI in patients undergoing chemotherapy without prior antibiotic treatment is also increasing. However, few studies have investigated the relationship between chemotherapy use and diarrhea. This study aimed to determine whether the incidence of CDI increased in patients with lung cancer undergoing chemotherapy even without prior antibiotic treatment. METHODS: We conducted a retrospective study and investigated the presence of Clostridium difficile (C. difficile) and its toxins in patients who experience diarrhea during chemotherapy. If grade 2 or higher diarrhea was noted, a stool culture was performed to detect anaerobic organisms and C. difficile toxins A and B. RESULTS: A total of 345 consecutive patients (492 in terms of chemotherapy regimens) were enrolled in the study. Grade 2 or higher diarrhea was observed in patients using 36 (7.3%) of these regimens, among which CDI without prior antibiotic exposure was confirmed in patients using 8 regimens (22.2%). CONCLUSIONS: CDI may remain undetected in patients undergoing chemotherapy even in those who had not received antibiotic treatment previously, unless due attention is paid to its possibility. Testing for C. difficile toxins is highly recommended to expedite timely treatment for diarrhea in such patients. Further studies are needed to clarify the relationship between chemotherapy drug use and CDI to facilitate prevention.

Quantitative Thresholds Enable Accurate Identification of Clostridium difficile Infection by the Luminex xTAG Gastrointestinal Pathogen Panel.

Clostridium difficile colonizes the gastrointestinal (GI) tract, resulting in either asymptomatic carriage or a spectrum of diarrheal illness. If clinical suspicion for C. difficile is low, stool samples are often submitted for analysis by multiplex molecular assays capable of detecting multiple GI pathogens, and some institutions do not report this organism due to concerns for high false-positive rates. Since clinical disease correlates with organism burden and molecular assays yield quantitative data, we hypothesized that numerical cutoffs could be utilized to improve the specificity of the Luminex xTAG GI pathogen panel (GPP) for C. difficile infection. Analysis of cotested liquid stool samples (n = 1,105) identified a GPP median fluorescence intensity (MFI) value cutoff of ≥1,200 to be predictive of two-step algorithm (2-SA; 96.4% concordance) and toxin enzyme immunoassay (EIA) positivity. Application of this cutoff to a second cotested data set (n = 1,428) yielded 96.5% concordance. To determine test performance characteristics, concordant results were deemed positive or negative, and discordant results were adjudicated via chart review. Test performance characteristics for the MFI cutoff of ≥150 (standard), MFI cutoff of ≥1,200, and 2-SA were as follows (respectively): concordance, 95, 96, and 97%; sensitivity, 93, 78, and 90%; specificity, 95, 98, and 98%; positive predictive value, 67, 82, and 81%;, and negative predictive value, 99, 98, and 99%. To capture the high sensitivity for organism detection (MFI of ≥150) and high specificity for active infection (MFI of ≥1,200), we developed and applied a reporting algorithm to interpret GPP data from patients (n = 563) with clinician orders only for syndromic panel testing, thus enabling accurate reporting of C. difficile for 95% of samples (514 negative and 5 true positives) irrespective of initial clinical suspicion and without the need for additional testing.

Evaluation of antigen-positive toxin-negative enzyme immunoassay results for the diagnosis of toxigenic Clostridium difficile infection.

Clostridium difficile (C. difficile)-associated diarrhea (CDAD) is a challenging nosocomial infectious disease. C. DIFF Quik Chek Complete assay is widely used to detect glutamate dehydrogenase (GDH) antigen and toxin A/B of C. difficile simultaneously. However, the interpretation of GDH positive/toxin negative results is problematic. We performed a retrospective study of patients with GDH positive/toxin negative results to determine the probability of detecting toxigenic C. difficile and its risk factors. Between April 2012 and March 2017, we investigated cultures of fecal specimens followed by toxin detection tests. The clinical histories of patients with and without toxigenic C. difficile were compared using univariate- and multivariate-analyses. In total, 2675 patients were examined using C. Diff Quik Chek Complete assay. Among 356 GDH positive/toxin negative patients, cultures were performed in 220 cases and toxigenic C. difficile was recovered from 139 (63.2%) specimens. Patients with toxigenic C. difficile had significantly lower body mass index than those without. Over half the GDH positive/toxin negative patients were infected with toxigenic C. difficile. Lower BMI was a CDAD risk factor in this patient population. These data can be utilized to initiate isolation and clinical interventions before confirmatory test results are available. J. Med. Invest. 65:131-135, February, 2018.

Impact of simultaneous glutamate dehydrogenase and toxin A/B rapid immunoassay on Clostridium difficile diagnosis and treatment in hospitalized patients with antibiotic-associated diarrhea in a university hospital of Brazil.

BACKGROUND AND AIM: Clostridium difficile is a major cause of health care-associated infection, but disagreement between diagnostic tests is an ongoing barrier to clinical decision-making. Conventional enzyme immunoassay (EIA) for toxin detection is currently the most frequently used technique for C. difficile infection (CDI) diagnosis, but its low sensitivity makes the development of an alternative strategy necessary for improving the diagnosis in developing countries. METHODS: Between years 2011 and 2015, 154 stool samples from patients with antibiotic-associated diarrhea were examined by toxigenic culture and EIA for the diagnosis of CDI. In the year 2015, when glutamate dehydrogenase (GDH) test was first available in Brazil, 53 of those fecal specimens were also tested by the C. diff Quik Chek Complete rapid immunoassay. At this time, we prospectively assessed the impact of this test on CDI treatment rates before and after it was introduced in clinical practice. RESULTS: The GDH component of C. diff Quik Chek Complete test had a sensitivity of 100% and specificity of 95.1% compared with toxigenic culture, with 89.8% concordance. The Tox A/B II EIA and the toxin portion of C. diff Quik Chek Complete yielded sensitivities between values of 50-58.3%, with 100% specificities. The introduction of GDH test increased the number of treated patients with CDI from 57.7% to 100%. CONCLUSIONS: Glutamate dehydrogenase test is a reliable method for the diagnosis of CDI and greatly increases the number of properly treated patients with CDI. Therefore, this exam should be considered the mainstay for the laboratory diagnosis of CDI in developing countries.

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

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 (Cq ) values were found for patients whose stool eventually tested positive for toxin, compared with patients who tested negative for toxin (mean Cq 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 Cq values to predict toxin status and yielded areas under the curve of 0.826 and 0.854. Using the optimal Cq cutoff values, prediction of the eventual toxin A/B EIA results was accurate for 78.9% and 80.5% of samples, respectively. In conclusion, Cq values can serve as predictors of toxin status but, due to the suboptimal correlation between the two tests, additional toxin testing is still needed.

A two-step approach improves the diagnosis of Clostridium difficile infection.

Clostridium difficile infection (CDI) is a major cause of health care-associated diarrhea. The aim of the present study was to evaluate a two-step approach for the diagnosis of CDI. The two-step procedure consisted of GDH-toxin A/B EIA (Enzyme immunoassay targeting enterotoxin A and Cytotoxin B), followed by PCR detecting toxigenic C. difficile. Results indicate that EIAs provide a rapid screening assay for the laboratory diagnosis of CDI but, in GDH-positive and toxins-negative samples, EIA should be always followed by PCR to distinguish toxigenic vs nontoxigenic strains. GDH-toxin A/B EIA-rapid test has high specificity but low sensitivity to detect CDI. The implementation of a two-step procedure significantly increases the diagnostic accuracy to detect CDI and provides a toxigenic type characterization of C. difficile isolates.