Cytotoxigenic Culture Research Articles

Is there a relationship between the presence of the binary toxin genes in Clostridium difficile strains and the severity of C. difficile infection (CDI)?

Some strains of Clostridium difficile produce a binary toxin, in addition to the main C. difficile virulence factors (toxins A and B). There have been conflicting reports regarding the role of binary toxin and its relationship to the severity of C. difficile infection (CDI). Samples, isolates and clinical data were collected as part of a prospective multicentre diagnostic study. Clostridium difficile isolates (n=1259) were tested by polymerase chain reaction (PCR) assay to detect binary toxin genes cdtA and cdtB. The PCR binary toxin gene results were compared with clinical severity and outcome data, including 30-day all-cause mortality. The 1259 isolates corresponded to 1083 different patients (October 2010 to September 2011). The prevalence of binary toxin positive strains was significantly higher in faecal samples with detectable toxin A/B than in those without toxin but that were positive by cytotoxigenic culture (26.3% vs. 10.3%, p<0.001). The presence of binary toxin correlated moderately with markers of CDI severity (white cell count, serum albumin concentration and serum creatinine concentration). However, the risk ratio for all-cause mortality was 1.68 for binary toxin positive patients and patients were significantly less likely to survive if they had CDI caused by a binary toxin gene positive strain, even after adjusting for age (p<0.001). The presence of binary toxin genes does not predict the clinical severity of CDI, but it is significantly associated with the risk of all-cause mortality.

A Low Complexity Rapid Molecular Method for Detection of Clostridium difficile in Stool

Here we describe a method for the detection of Clostridium difficile from stool using a novel low-complexity and rapid extraction process called Heat Elution (HE). The HE method is two-step and takes just 10 minutes, no specialist instruments are required and there is minimal hands-on time. A test method using HE was developed in conjunction with Loop-mediated Isothermal Amplification (LAMP) combined with the real-time bioluminescent reporter system known as BART targeting the toxin B gene (tcdB). The HE-LAMP-BART method was evaluated in a pilot study on clinical fecal samples (tcdB +, n = 111; tcdB −, n = 107). The HE-LAMP-BART method showed 95.5% sensitivity and 100% specificity against a gold standard reference method using cytotoxigenic culture and also a silica-based robotic extraction followed by tcdB PCR to control for storage. From sample to result, the HE-LAMP-BART method typically took 50 minutes, whereas the PCR method took >2.5 hours.In a further study (tcdB +, n = 47; tcdB −, n = 28) HE-LAMP-BART was compared to an alternative commercially available LAMP-based method, Illumigene (Meridian Bioscience, OH), and yielded 87.2% sensitivity and 100% specificity for the HE-LAMP-BART method compared to 76.6% and 100%, respectively, for Illumigene against the reference method. A subset of 27 samples (tcdB +, n = 25; tcdB −, n = 2) were further compared between HE-LAMP-BART, Illumigene, GeneXpert (Cepheid, Sunnyvale, CA) and RIDA®QUICK C. difficile Toxin A/B lateral flow rapid test (R-Biopharm, Darmstadt, Germany) resulting in sensitivities of HE-LAMP-BART 92%, Illumigene 72% GeneXpert 96% and RIDAQuick 76% against the reference method. The HE-LAMP-BART method offers the advantages of molecular based approaches without the cost and complexity usually associated with molecular tests. Further, the rapid time-to-result and simple protocol means the method can be applied away from the centralized laboratory settings.

Differences in outcome according to Clostridium difficile testing method: a prospective multicentre diagnostic validation study of C difficile infection

SummaryBackgroundDiagnosis of Clostridium difficile infection is controversial because of many laboratory methods, compounded by two reference methods. Cytotoxigenic culture detects toxigenic C difficile and gives a positive result more frequently (eg, because of colonisation, which means that individuals can have the bacterium but no free toxin) than does the cytotoxin assay, which detects preformed toxin in faeces. We aimed to validate the reference methods according to clinical outcomes and to derive an optimum laboratory diagnostic algorithm for C difficile infection.MethodsIn this prospective, multicentre study, we did cytotoxigenic culture and cytotoxin assays on 12 420 faecal samples in four UK laboratories. We also performed tests that represent the three main targets for C difficile detection: bacterium (glutamate dehydrogenase), toxins, or toxin genes. We used routine blood test results, length of hospital stay, and 30-day mortality to clinically validate the reference methods. Data were categorised by reference method result: group 1, cytotoxin assay positive; group 2, cytotoxigenic culture positive and cytotoxin assay negative; and group 3, both reference methods negative.FindingsClinical and reference assay data were available for 6522 inpatient episodes. On univariate analysis, mortality was significantly higher in group 1 than in group 2 (72/435 [16·6%] vs 20/207 [9·7%], p=0·044) and in group 3 (503/5880 [8·6%], p<0·001), but not in group 2 compared with group 3 (p=0·4). A multivariate analysis accounting for potential confounders confirmed the mortality differences between groups 1 and 3 (OR 1·61, 95% CI 1·12–2·31). Multistage algorithms performed better than did standalone assays.InterpretationWe noted no increase in mortality when toxigenic C difficile alone was present. Toxin (cytotoxin assay) positivity correlated with clinical outcome, and so this reference method best defines true cases of C difficile infection. A new diagnostic category of potential C difficile excretor (cytotoxigenic culture positive but cytotoxin assay negative) could be used to characterise patients with diarrhoea that is probably not due to C difficile infection, but who can cause cross-infection.FundingDepartment of Health and Health Protection Agency, UK.

Evaluation of an Algorithmic Approach in Comparison with the Illumigene Assay for Laboratory Diagnosis of Clostridium difficile Infection

The following three diagnostic algorithms were evaluated in comparison with the Illumigene assay as a stand-alone test for Clostridium difficile detection: glutamate dehydrogenase antigen screen (GDH) followed by toxin A/B antigen testing (Tox A/B) with the cell cytotoxicity assay for discordant specimens (algorithm 1), GDH followed by the Illumigene (algorithm 2), and GDH followed by Tox A/B with the Illumigene for discordant specimens (algorithm 3). A total of 428 stool specimens submitted to three clinical microbiology laboratories in Manitoba, Canada, for C. difficile detection between June 2011 and April 2012 were included in the study. The prevalence of C. difficile in the stool specimens was 14.7% (63/428) based on toxigenic culture (microbiologic reference standard). The sensitivity and specificity of the Illumigene for C. difficile detection were 73.0% and 99.7%, respectively. The corresponding sensitivities and specificities were 65.1% and 100.0% for algorithm 1, 68.3% and 100.0% for algorithm 2, and 69.8% and 100.0% for algorithm 3. Using algorithm 1, a cell cytotoxicity assay was required for toxin detection in 37% of positive tests, prolonging turnaround time. However, the predictive value of a positive test based on a clinical reference standard (all tests positive or cytotoxigenic culture positive and clinical disease on chart review) was slightly higher with algorithm 1 than with the Illumigene assay as a stand-alone test or as part of an algorithm (algorithms 2 and 3). Based on a reduction in turnaround time, simplicity, and acceptable sensitivity and specificity, we recommend algorithm 2 (screening with the GDH antigen test and confirmatory testing with the Illumigene).

Comparison of Real-Time PCR Techniques to Cytotoxigenic Culture Methods for Diagnosing Clostridium difficile Infection

In the past decade, the incidence of Clostridium difficile infections (CDI) with a more severe course has increased in Europe and North America. Assays that are capable of rapidly diagnosing CDI are essential. Two real-time PCRs (LUMC and LvI) targeting C. difficile toxin genes (tcdB, and tcdA and tcdB, respectively) were compared with the BD GeneOhm PCR (targeting the tcdB gene), using cytotoxigenic culture as a gold standard. In addition, a real-time PCR targeting the tcdC frameshift mutation at position 117 (Δ117 PCR) was evaluated for detecting toxigenic C. difficile and the presence of PCR ribotype 027 in stool samples. In total, 526 diarrheal samples were prospectively collected and included in the study. Compared with those for cytotoxigenic culture, sensitivity, specificity, positive predicted value (PPV), and negative predicted value (NPV) were for PCR LUMC 96.0%, 88.0%, 66.0%, and 98.9%, for PCR LvI 100.0%, 89.4%, 69.7%, and 100.0%, for PCR Δ117 98.0%, 90.7%, 71.9%, and 99.5%, and for PCR BD GeneOhm 88.3%, 96.9%, 86.5%, and 97.4%. Compared to those with feces samples cultured positive for C. difficile type 027, the sensitivity, specificity, PPV, and NPV of the Δ117 PCR were 95.2%, 96.2%, 87.0%, and 98.7%. We conclude that all real-time PCRs can be applied as a first screening test in an algorithm for diagnosing CDI. However, the low PPVs hinder the use of the assays as stand-alone tests. Furthermore, the Δ117 PCR may provide valuable information for minimizing the spread of the epidemic C. difficile PCR ribotype 027.

C. Diff Quik Chek Complete Enzyme Immunoassay Provides a Reliable First-Line Method for Detection of Clostridium difficile in Stool Specimens

We evaluated a single membrane device assay for simultaneously detecting both Clostridium difficile glutamate dehydrogenase (GDH) and toxin A/B antigens against a standard that combines two PCR assays and cytotoxigenic culture. Results showing dual GDH and toxin A/B antigen positives and negatives can be reported immediately as true positives and negatives, respectively. Specimens with discrepant results for GDH and toxins A/B, which comprised 13.2% of the specimens, need to be retested.

Comparison of Nine Commercially Available Clostridium difficile Toxin Detection Assays, a Real-Time PCR Assay for C . difficile tcdB , and a Glutamate Dehydrogenase Detection Assay to Cytotoxin Testing and Cytotoxigenic Culture Methods

The continuing rise in the incidence of Clostridium difficile infection is a cause for concern, with implications for patients and health care systems. Laboratory diagnosis largely relies on rapid toxin detection kits, although assays detecting alternative targets, including glutamate dehydrogenase (GDH) and toxin genes, are now available. Six hundred routine diagnostic diarrheal samples were tested prospectively using nine commercial toxin detection assays, cytotoxin assay (CYT), and cytotoxigenic culture (CYTGC) and retrospectively using a GDH detection assay and PCR for the toxin B gene. The mean sensitivity and specificity for toxin detection assays were 82.8% (range, 66.7 to 91.7%) and 95.4% (range, 90.9 to 98.8%), respectively, in comparison with CYT and 75.0% (range, 60.0 to 86.4%) and 96.1% (91.4 to 99.4%), respectively, in comparison with CYTGC. The sensitivity and specificity of the GDH assay were 90.1% and 92.9%, respectively, compared to CYT and 87.6% and 94.3%, respectively, compared to CYTGC. The PCR assay had the highest sensitivity of all the tests in comparison with CYT (92.2%) and CYTGC (88.5%), and the specificities of the PCR assay were 94.0% and 95.4% compared to CYT and CYTGC, respectively. All kits had low positive predictive values (range, 48.6 to 86.8%) compared with CYT, assuming a positive sample prevalence of 10% (representing the hospital setting), which compromises the clinical utility of single tests for the laboratory diagnosis of C. difficile infection. The optimum rapid single test was PCR for toxin B gene, as this had the highest negative predictive value. Diagnostic algorithms that optimize test combinations for the laboratory diagnosis of C. difficile infection need to be defined.

Toxin A detection on Clostridium difficile colonies from 24-h cultures

OBJECTIVE: Performance of a combined approach for the detection of toxigenic strains in patients suspected of having Clostridium difficile-associated disease was evaluated. METHODS: In this approach, stools were cultured for 24 h on a selective medium supplemented with sodium taurocholate (TCCFA), in anaerobic conditions created with the Martreg Anoxomat system, and toxin A detection was performed directly on C. difficile colonies, by enzyme immunoassay (EIA). This method was compared with three others: cytotoxigenic culture consisting of a 48-h culture on selective medium followed by detection of in vitro cytotoxin production on cell monolayers, fecal cytotoxin detection and fecal toxin A detection by EIA. RESULTS: From 548 stools, 108 yielded a positive culture by at least one of the methods, and 81 isolates were cytotoxin producers. Cultures for 24 h on TCCFA were positive in 106 cases and EIA performed on colonies gave 73 positive results, giving a sensitivity of 90.1% and a specificity of 100%. By comparison, the sensitivity and specificity of cytotoxigenic culture, stool cytotoxin and stool toxin A were respectively 96.2% and 100%, 61.7% and 100%, and 66.7% and 95.9%. CONCLUSIONS: Performing EIA on colonies recovered after 24 h culture allows us to improve the detection of toxigenic strains in patients suspected of having C. difficile-associated disease.