Frequency of binary toxin genes among Clostridium difficile strains that do not produce large clostridial toxins

Pathogenic strains of Clostridium difficile commonly produce two large clostridial toxins (LCTs), A and B, virulence factors responsible for C. difficile disease. Some strains have been reported to produce an additional toxin, a binary toxin designated CDT. Binary toxin has cytotoxic effects on cells in culture, but its role in human disease is not yet defined. In this study we examined the frequency of binary toxin genes (cdtB and cdtA) among C. difficile isolates that do not produce LCTs (A(-) B(-)) from a large United States-based collection organized by restriction endonuclease analysis (REA) typing. Of 58 strains tested, 9 (15.5%) were cdtB and cdtA positive, including 4 of 46 (8.7%) non-LCT-producing REA groups, with an estimated prevalence of at least 2% of all non-LCT-producing isolates within the collection. Five of the binary toxin-positive strains belonged to toxinotype XI, which does not produce LCTs but has minor parts of the LCT coding region or pathogenicity locus (PaLoc). We describe two new binary toxin-positive variants, one without any remnant of the LCT genes. This previously unknown variation was found in three isolates that were unrelated by REA typing. LCT-negative, binary toxin-positive strains were isolated from symptomatic and asymptomatic patients and from the hospital environment.     

Intrarectal Instillation of Clostridium difficile Toxin A Triggers Colonic Inflammation and Tissue Damage: Development of a Novel and Efficient Mouse Model of Clostridium difficile Toxin Exposure

Clostridium difficile, a major cause of hospital-acquired diarrhea, triggers disease through the release of two toxins, toxin A (TcdA) and toxin B (TcdB). These toxins disrupt the cytoskeleton of the intestinal epithelial cell, increasing intestinal permeability and triggering the release of inflammatory mediators resulting in intestinal injury and inflammation. The most prevalent animal model to study TcdA/TcdB-induced intestinal injury involves injecting toxin into the lumen of a surgically generated “ileal loop.” This model is time-consuming and exhibits variability depending on the expertise of the surgeon. Furthermore, the target organ of C. difficile infection (CDI) in humans is the colon, not the ileum. In the current study, we describe a new model of CDI that involves intrarectal instillation of TcdA/TcdB into the mouse colon. The administration of TcdA/TcdB triggered colonic inflammation and neutrophil and macrophage infiltration as well as increased epithelial barrier permeability and intestinal epithelial cell death. The damage and inflammation triggered by TcdA/TcdB isolates from the VPI and 630 strains correlated with the concentration of TcdA and TcdB produced. TcdA/TcdB exposure increased the expression of a number of inflammatory mediators associated with human CDI, including interleukin-6 (IL-6), gamma interferon (IFN-γ), and IL-1β. Finally, we were able to demonstrate that TcdA was much more potent at inducing colonic injury than was TcdB but TcdB could act synergistically with TcdA to exacerbate injury. Taken together, our data indicate that the intrarectal murine model provides a robust and efficient system to examine the effects of TcdA/TcdB on the induction of inflammation and colonic tissue damage in the context of human CDI.     

Protective Efficacy Induced by Recombinant Clostridium difficile Toxin Fragments

Clostridium difficile is a spore-forming bacterium that can reside in animals and humans. C. difficile infection causes a variety of clinical symptoms, ranging from diarrhea to fulminant colitis. Disease is mediated by TcdA and TcdB, two large enterotoxins released by C. difficile during colonization of the gut. In this study, we evaluated the ability of recombinant toxin fragments to induce neutralizing antibodies in mice. The protective efficacies of the most promising candidates were then evaluated in a hamster model of disease. While limited protection was observed with some combinations, coadministration of a cell binding domain fragment of TcdA (TcdA-B1) and the glucosyltransferase moiety of TcdB (TcdB-GT) induced systemic IgGs which neutralized both toxins and protected vaccinated animals from death following challenge with two strains of C. difficile. Further characterization revealed that despite high concentrations of toxin in the gut lumens of vaccinated animals during the acute phase of the disease, pathological damage was minimized. Assessment of gut contents revealed the presence of TcdA and TcdB antibodies, suggesting that systemic vaccination with this pair of recombinant polypeptides can limit the disease caused by toxin production during C. difficile infection.     

Binary toxin and its clinical importance in <Emphasis Type="Italic">Clostridium difficile</Emphasis> infection,Belgium

Binary toxin-producing Clostridium difficile strains such as ribotypes 027 and 078 have been associated with increased Clostridium difficile infection (CDI) severity. Our objective was to investigate the association between presence of the binary toxin gene and CDI severity and recurrence. We performed a laboratory-based retrospective study including patients between January 2013 and March 2015 whose fecal samples were analyzed by polymerase chain reaction (PCR) for the presence of the genes for toxin B and binary toxin and a deletion in the tcdC gene, specific for ribotype 027. Clinical and epidemiological characteristics were compared between 33 binary toxin-positive CDI patients and 33 binary toxin-negative CDI patients. Subsequently, the characteristics of 66 CDI patients were compared to those of 66 diarrhea patients who were carriers of non-toxigenic C. difficile strains. Fifty-nine of 1034 (5.7 %) fecal samples analyzed by PCR were binary toxin-positive, belonging to 33 different patients. No samples were positive for ribotype 027. Binary toxin-positive CDI patients did not differ from binary toxin-negative CDI patients in terms of disease recurrence, morbidity, or mortality, except for a higher peripheral leukocytosis in the binary toxin-positive group (16.30?×?10⁹/L vs. 11.65?×?10⁹/L; p?=?0.02). The second part of our study showed that CDI patients had more severe disease, but not a higher 30-day mortality rate than diarrhea patients with a non-toxicogenic C. difficile strain. In our setting with a low prevalence of ribotype 027, the presence of the binary toxin gene is not associated with poor outcome.     

An Optimized,Synthetic DNA Vaccine Encoding the Toxin A and Toxin B Receptor Binding Domains of Clostridium difficile Induces Protective Antibody Responses In Vivo

Clostridium difficile-associated disease (CDAD) constitutes a large majority of nosocomial diarrhea cases in industrialized nations and is mediated by the effects of two secreted toxins, toxin A (TcdA) and toxin B (TcdB). Patients who develop strong antitoxin antibody responses can clear C. difficile infection and remain disease free. Key toxin-neutralizing epitopes have been found within the carboxy-terminal receptor binding domains (RBDs) of TcdA and TcdB, which has generated interest in developing the RBD as a viable vaccine target. While numerous platforms have been studied, very little data describes the potential of DNA vaccination against CDAD. Therefore, we created highly optimized plasmids encoding the RBDs from TcdA and TcdB in which any putative N-linked glycosylation sites were altered. Mice and nonhuman primates were immunized intramuscularly, followed by in vivo electroporation, and in these animal models, vaccination induced significant levels of both anti-RBD antibodies (blood and stool) and RBD-specific antibody-secreting cells. Further characterization revealed that sera from immunized mice and nonhuman primates could detect RBD protein from transfected cells, as well as neutralize purified toxins in an in vitro cytotoxicity assay. Mice that were immunized with plasmids or given nonhuman-primate sera were protected from a lethal challenge with purified TcdA and/or TcdB. Moreover, immunized mice were significantly protected when challenged with C. difficile spores from homologous (VPI 10463) and heterologous, epidemic (UK1) strains. These data demonstrate the robust immunogenicity and efficacy of a TcdA/B RBD-based DNA vaccine in preclinical models of acute toxin-associated and intragastric, spore-induced colonic disease.     

Evidence for holin function of tcdE gene in the pathogenicity of Clostridium difficile.

Toxigenic strains of Clostridium difficile produce two large bacterial toxins called toxins A (TcdA) and B (TcdB). tcdA and tcdB genes are located on the pathogenicity locus of C. difficile, a unique characteristic of toxigenic strains of this species. Intergenic to the two toxin genes is tcdE, a small 501-bp open reading frame of unknown function. Expression of the tcdE gene in Escherichia coli caused bacterial cell death. Computational analysis of the amino acid sequence of TcdE revealed structural features that are strikingly similar to a class of bacteriophage proteins called holins. Holins are cytolytic proteins that cause lysis of bacterial hosts to effect the release of progeny phages. Further analysis of the recombinant clone expressing TcdE by transmission electron microscopy confirmed that the site of action of TcdE is on the bacterial cell membrane. The results provide evidence that TcdE is structurally and functionally similar to holin proteins. TcdE may function as a lytic protein to facilitate the release of TcdA and TcdB to the extracellular environment, as these toxins lack signal peptide.     

Epidemiology of Clostridium difficile infection in a large teaching hospital in Singapore

AIMS: We undertook this study to define the incidence of toxigenic Clostridium difficile in our hospital and to characterise the isolates. METHODS: All unformed stool was tested for the presence of Toxin A (TcdA) and Toxin B (TcdB), and cultured for C. difficile. Culture filtrates were also tested for TcdA and TcdB. Detection of tcdA and tcdB genes was carried out for A(-)B(+) strains by polymerase chain reaction (PCR).The minimum inhibitory concentrations (MICs) of metronidazole, vancomycin and clindamycin for all isolates were tested using the Etest. PCR ribotyping was carried out on all isolates. RESULTS: The incidence of Clostridium difficile associated disease (CDAD) was 3.2 cases per 1000 admissions or discharges and 53.8 cases per 100 000 patient days. Most cases occurred in renal and haematology patients. CDAD was more common in patients aged over 50 years and of male gender. The Indian population was under-represented. Fourteen (11.8%) isolates were A(-)B(+). All strains were susceptible to metronidazole but one strain showed intermediate resistance to vancomycin. Only 12.8% of the isolates were susceptible to clindamycin. Thirty-five isolates had PCR ribotype A, of which 29 (83%) had a clindamycin MIC >256 mg/L. Thirty-three had PCR ribotype B, of which only one (3%) had a clindamycin MIC >256 mg/L. The 14 A(-)B(+) strains were all PCR ribotype C, and had a range of MICs for clindamycin from 2 to >256 mg/L. CONCLUSIONS: The incidence of CDAD in our hospital is relatively low. Isolates remain susceptible to metronidazole and vancomycin.     

A chimeric toxin vaccine protects against primary and recurrent Clostridium difficile infection

The global emergence of Clostridium difficile infection (CDI) has contributed to the recent surge in severe antibiotic-associated diarrhea and colonic inflammation. C. difficile produces two homologous glucosylating exotoxins, TcdA and TcdB, both of which are pathogenic and require neutralization to prevent disease occurrence. However, because of their large size and complex multifunctional domain structures, it has been a challenge to produce native recombinant toxins that may serve as vaccine candidates. Here, we describe a novel chimeric toxin vaccine that retains major neutralizing epitopes from both toxins and confers complete protection against primary and recurrent CDI in mice. Using a nonpathogenic Bacillus megaterium expression system, we generated glucosyltransferase-deficient holotoxins and demonstrated their loss of toxicity. The atoxic holotoxins induced potent antitoxin neutralizing antibodies showing little cross-immunogenicity or protection between TcdA and TcdB. To facilitate simultaneous protection against both toxins, we generated an active clostridial toxin chimera by switching the receptor binding domain of TcdB with that of TcdA. The toxin chimera was fully cytotoxic and showed potent proinflammatory activities. This toxicity was essentially abolished in a glucosyltransferase-deficient toxin chimera, cTxAB. Parenteral immunization of mice or hamsters with cTxAB induced rapid and potent neutralizing antibodies against both toxins. Complete and long-lasting disease protection was conferred by cTxAB vaccinations against both laboratory and hypervirulent C. difficile strains. Finally, prophylactic cTxAB vaccination prevented spore-induced disease relapse, which constitutes one of the most significant clinical issues in CDI. Thus, the rational design of recombinant chimeric toxins provides a novel approach for protecting individuals at high risk of developing CDI.     

New types of toxin A-negative,toxin B-positive strains among Clostridium difficile isolates from Asia

A total of 56 C. difficile strains were selected from 310 isolates obtained from different hospitals in Japan and Korea and from healthy infants from Indonesia. Strains that had been previously typed by pulsed-field gel electrophoresis and PCR ribotyping, were characterized by toxinotyping and binary toxin gene detection. When toxinotyped, 35 strains were determined to be toxinotype 0, whereas 21 strains showed variations in toxin genes and could be grouped into 11 variant toxinotypes. Six of the toxinotypes had been described before (I, III, IV, VIII, IX, and XII). In addition, five new toxinotypes were defined (XVI to XX). Three of the new toxinotypes (XVIII, XIX, and XX) vary only in repetitive regions of tcdA and produce both toxins. In two strains from toxinotypes XVI and XVII, the production of TcdA could not be detected with commercial immunological kits. Strain J9965 (toxinotype XVII) was in PaLoc similar but not identical to another known A(-)B(+) strain, C. difficile 8864. Strain SUC 36 (toxinotype XVI), on the other hand, was similar to well-defined group consisting of toxinotypes V, VI, and VII, which thus far includes only A(+)B(+) strains. Toxinotypes XVI and XVII represent two new groups of A(-)B(+) strains. Strains of the well-known A(-)B(+) group from toxinotype VIII have a nonsense mutation at the beginning of tcdA gene, and the introduction of a stop codon at amino acid position 47 results in nonproduction of TcdA. The 5'-end sequence of tcdA in two newly described A(-)B(+) strains does not contain an identical mutation. The prevalence of variant C. difficile strains varied greatly among nine hospitals. Only five strains from four different hospitals were positive in PCR for amplification of the binary toxin gene.     

A Mixture of Functionally Oligoclonal Humanized Monoclonal Antibodies That Neutralize Clostridium difficile TcdA and TcdB with High Levels of In Vitro Potency Shows In Vivo Protection in a Hamster Infection Model

Clostridium difficile infections are a major cause of antibiotic-associated diarrhea in hospital and care facility patients. In spite of the availability of effective antibiotic treatments, C. difficile infection (CDI) is still a major cause of patient suffering, death, and substantial health care costs. Clostridium difficile exerts its major pathological effects through the actions of two protein exotoxins, TcdA and TcdB, which bind to and disrupt gut tissue. Antibiotics target the infecting bacteria but not the exotoxins. Administering neutralizing antibodies against TcdA and TcdB to patients receiving antibiotic treatment might modulate the effects of the exotoxins directly. We have developed a mixture of three humanized IgG1 monoclonal antibodies (MAbs) which neutralize TcdA and TcdB to address three clinical needs: reduction of the severity and duration of diarrhea, reduction of death rates, and reduction of the rate of recurrence. The UCB MAb mixture showed higher potency in a variety of in vitro binding and neutralization assays (∼10-fold improvements), higher levels of protection in a hamster model of CDI (82% versus 18% at 28 days), and higher valencies of toxin binding (12 versus 2 for TcdA and 3 versus 2 for TcdB) than other agents in clinical development. Comparisons of the MAb properties also offered some insight into the potential relative importance of TcdA and TcdB in the disease process.     

Recent emergence of an epidemic clindamycin-resistant clone of Clostridium difficile among Polish patients with C. difficile-associated diarrhea

Analysis of both the antibiotic resistance and the virulence characteristics of anaerobic human microbial pathogens is important in order to improve our understanding of a number of clinically significant infectious diseases, including Clostridium difficile-associated diarrhea (CDAD). We determined the presence of the clindamycin resistance-associated gene ermB and the ribotype of 33 C. difficile strains isolated from Polish patients suffering from CDAD. While all strains produced cytotoxin B (TcdB), enterotoxin A (TcdA) was produced by a subset of 15 strains only. The results showed that a single ermB-positive, TcdA(-)B(+) C. difficile strain with ribotype A has disseminated widely in the two Warsaw hospitals under investigation. Although different strains with the same phenotype were detected, the genotype A strain appeared to be the only one with a clear epidemic character. Apparently, enhanced local spread of CDAD-causing C. difficile may be restricted to a limited number of bacterial genotypes only.     

Development and Optimization of a High-Throughput Assay To Measure Neutralizing Antibodies against Clostridium difficile Binary Toxin

Clostridium difficile strains producing binary toxin, in addition to toxin A (TcdA) and toxin B (TcdB), have been associated with more severe disease and increased recurrence of C. difficile infection in recent outbreaks. Binary toxin comprises two subunits (CDTa and CDTb) and catalyzes the ADP-ribosylation of globular actin (G-actin), which leads to the depolymerization of filamentous actin (F-actin) filaments. A robust assay is highly desirable for detecting the cytotoxic effect of the toxin and the presence of neutralizing antibodies in animal and human sera to evaluate vaccine efficacy. We describe here the optimization, using design-of-experiment (DOE) methodology, of a high-throughput assay to measure the toxin potency and neutralizing antibodies (NAb) against binary toxin. Vero cells were chosen from a panel of cells screened for sensitivity and specificity. We have successfully optimized the CDTa-to-CDTb molar ratio, toxin concentration, cell-seeding density, and sera-toxin preincubation time in the NAb assay using DOE methodology. This assay is robust, produces linear results across serial dilutions of hyperimmune serum, and can be used to quantify neutralizing antibodies in sera from hamsters and monkeys immunized with C. difficile binary toxin-containing vaccines. The assay will be useful for C. difficile diagnosis, for epidemiology studies, and for selecting and optimizing vaccine candidates.     

Caspase Activation as a Versatile Assay Platform for Detection of Cytotoxic Bacterial Toxins

Pathogenic bacteria produce several virulence factors that help them establish infection in permissive hosts. Bacterial toxins are a major class of virulence factors and hence are attractive therapeutic targets for vaccine development. Here, we describe the development of a rapid, sensitive, and high-throughput assay that can be used as a versatile platform to measure the activities of bacterial toxins. We have exploited the ability of these toxins to cause cell death via apoptosis of sensitive cultured cell lines as a readout for measuring toxin activity. Caspases (cysteine-aspartic proteases) are induced early in the apoptotic pathway, and so we used their induction to measure the activities of Clostridium difficile toxins A (TcdA) and B (TcdB) and binary toxin (CDTa-CDTb), Corynebacterium diphtheriae toxin (DT), and Pseudomonas aeruginosa exotoxin A (PEA). Caspase induction in the cell lines, upon exposure to toxins, was optimized by toxin concentration and intoxication time, and the specificity of caspase activity was established using a genetically mutated toxin and a pan-caspase inhibitor. In addition, we demonstrate the utility of the caspase assay for measuring toxin potency, as well as neutralizing antibody (NAb) activity against C. difficile toxins. Furthermore, the caspase assay showed excellent correlation with the filamentous actin (F-actin) polymerization assay for measuring TcdA and TcdB neutralization titers upon vaccination of hamsters. These results demonstrate that the detection of caspase induction due to toxin exposure using a chemiluminescence readout can support potency and clinical immunogenicity testing for bacterial toxin vaccine candidates in development.     

Release of TcdA and TcdB from Clostridium difficile cdi 630 is not affected by functional inactivation of the tcdE gene

The small open reading frame tcdE is located between the genes tcdA and tcdB which encode toxin A (TcdA) and B (TcdB), respectively, within the pathogenicity locus of Clostridium difficile. Sequence and structure similarities to bacteriophage-encoded holins have led to the assumption that TcdE mediates the release of the toxins from C. difficile into the extracellular environment. A TcdE-deficient C. difficile 630 strain was generated by insertional inactivation of the tcdE gene. Data revealed that TcdE does not regulate or affect growth or sporogenesis. TcdE-deficiency was accompanied by a moderately increased accumulation of TcdA and TcdB prior to sporulation in this microorganism. Interestingly, this observation did not correlate with a delayed or inhibited toxin release: inactivation of TcdE neither significantly altered kinetics of release nor the absolute level of secreted TcdA and TcdB, indicating that TcdE does not account for the pathogenicity of C. difficile strain 630. Furthermore, mass spectrometry analysis could not reveal differences in the secretome of wild type and TcdE-deficient C. difficile, indicating that TcdE did not function as a secretion system for protein release. TcdE was expressed as a 19 kDa protein in C. difficile, whereas TcdE expressed in Escherichia coli appeared as a 19 and 16 kDa protein. Expression of the short 16 kDa TcdE correlated with bacterial cell death. We conclude that TcdE does not exhibit pore-forming function in C. difficile since in these cells only the non-lytic full length 19 kDa protein is expressed.     

Toll-Like Receptor 5-Dependent Immunogenicity and Protective Efficacy of a Recombinant Fusion Protein Vaccine Containing the Nontoxic Domains of Clostridium difficile Toxins A and B and Salmonella enterica Serovar Typhimurium Flagellin in a Mouse Model of Clostridium difficile Disease

Clostridium difficile is a spore-forming bacillus that produces toxin-mediated enteric disease. C. difficile expresses two major virulence factors, toxin A (TcdA) and toxin B (TcdB). Human and animal studies demonstrate a clear association between humoral immunity to these toxins and protection against C. difficile infection (CDI). The receptor binding-domains (RBDs) of TcdA and TcdB are known to be immunogenic. Here, we tested the immunoadjuvant properties of Salmonella enterica serovar Typhimurium flagellin (FliC) subunit D1 as an innate immune agonist expressed as a recombinant fusion vaccine targeting the RBDs of TcdA and TcdB in mice. Intraperitoneally immunized mice developed prominent anti-TcdA and anti-TcdB immunoglobulin G in serum. The protective efficacy of the recombinant vaccines, with or without an adjuvant, was tested in a mouse model of CDI that closely represents the human disease. Following intraperitoneal immunization equivalent to two doses of toxoid A and toxoid B vaccine adjuvanted with alum and oral challenge with C. difficile VPI 10463, C57BL/6 mice were able to mount a protective immune response that prevented diarrhea and death compared to mice immunzed with alum alone. These results are significantly different from those for control mice (P < 0.001). These results provide evidence that a recombinant protein-based vaccine targeting the RBDs of the C. difficile toxins adjuvanted with S. Typhimurium flagellin can induce rapid, high-level protection in a mouse model of CDI when challenged with the homologous strain from which the vaccine antigens were derived and warrant further preclinical testing against clinically relevant C. difficile strains in the mouse and hamster models of CDI.     

Lack of association between clinical outcome of Clostridium difficile infections, strain type, and virulence-associated phenotypes

Clostridium difficile strain NAP1/027 (North American pulsed-field gel electrophoresis [PFGE] type 1 and PCR ribotype 027 [R027]) has been associated with recent outbreaks in North America and Europe. It has been associated with more severe disease symptoms, higher mortality rates, and greater risk of relapse. This strain is thought to produce more toxins and sporulate to higher levels. However, recent studies suggest that this may not always be the case. The objective of our study was to assess, in a nonoutbreak situation, whether specific strains, such as NAP1/027, were associated with more severe disease symptoms, higher toxin production, and/or greater sporulation in vitro. We isolated and characterized C. difficile strains from 21 patients with mild to moderate, severe, or complicated symptoms of C. difficile infection (CDI). The isolates were characterized by different molecular typing methods, including PCR ribotyping, tandem repeat sequence typing (TRST), and sequencing of the tcdC gene. Fourteen isolates were of PCR ribotype 027 with deletions in tcdC, but no association with severity or clinical outcome was found. We show by immunodot blot detection of toxins with monoclonal antibodies that all R027 isolates produced more TcdA and TcdB than other strains. On the other hand, they consistently produced fewer spores than non-R027 isolates. Taken together, our data suggest that NAP1/027 isolates are not always associated with more severe disease, even though they may produce larger amounts of toxins. Our study also suggests that current assertions regarding the NAP1/027 may not apply to all isolates and that other factors may come into play.     

Laboratory Detection of Clostridium difficile in Piglets in Australia

Clostridium difficile is a well-known enteric pathogen of humans and the causative agent of high-morbidity enteritis in piglets aged 1 to 7 days. C. difficile prevalence in Australian piglets is as high as 70%. The current diagnostic assays have been validated only for human infections, and there are no published studies assessing their performance in Australian piglets. We evaluated the suitability of five assays for detecting C. difficile in 157 specimens of piglet feces. The assays included a loop-mediated isothermal amplification (LMIA)-PCR for tcdA (illumigene C. difficile; Meridian), a real-time PCR for tcdB (GeneOhm Cdiff; Becton Dickinson), two-component enzyme immunoassays (EIA) for C. difficile glutamate dehydrogenase (GDH) (EIA-GDH) and TcdA/TcdB (EIA-TcdA/TcdB) (C. diff Quik Chek; Alere), and direct culture (DC) (C. difficile chromID agar; bioMérieux). The assays for detection of the organism were compared against enrichment culture (EC), and assays for detection of toxins/toxin genes were compared against EC followed by PCR for toxin genes (toxigenic EC [TEC]). The recovery of C. difficile by EC was 39.5% (n = 62/157), and TEC revealed that 58.1% (n = 36/62) of isolates were positive for at least one toxin gene (tcdA/tcdB). Compared with those for EC/TEC, the sensitivities, specificities, positive predictive values, and negative predictive values were, respectively, as follows: DC, 91.9, 100.0, 100.0, and 95.0%; EIA-GDH, 41.9, 92.6, 78.8, and 71.0%; EIA-TcdA/TcdB, 5.6, 99.2, 66.7, and 77.9%; real-time PCR, 42.9, 96.7, 78.9, and 85.4% and LMIA-PCR, 25.0, 95.9, 64.3, and 81.1%. The performance of the molecular methods was poor, suggesting that the current commercially available assays for diagnosis of C. difficile in humans are not suitable for use in piglets. C. difficile recovery by the DC provides a cost-effective alternative.     

Rapid and simple method for detecting the toxin B gene of Clostridium difficile in stool specimens by loop-mediated isothermal amplification

We applied the loop-mediated isothermal amplification (LAMP) assay to the detection of the toxin B gene (tcdB) of Clostridium difficile for identification of toxin B (TcdB)-positive C. difficile strains and detection of tcdB in stool specimens. tcdB was detected in all toxin A (TcdA)-positive, TcdB-positive (A(+)B(+)) and TcdA-negative, TcdB-positive (A(-)B(+)) C. difficile strains but not from TcdA-negative, TcdB-negative strains. Of the 74 stool specimens examined, A(+)B(+) or A(-)B(+) C. difficile was recovered from 39 specimens, of which 38 specimens were LAMP positive and one was negative. Amplification was obtained in 10 specimens that were culture negative, indicating that LAMP is highly sensitive. The LAMP assay was applied to detection of tcdB in DNA extracted by a simple boiling method from 47 of those 74 specimens, which were cultured overnight in cooked-meat medium (CMM). Twenty-two of 24 culture-positive specimens were positive for LAMP on DNA from the culture in CMM. Four specimens were culture negative but positive by LAMP on DNA from CMM cultures. The LAMP assay is a reliable tool for identification of TcdB-positive C. difficile as well as for direct detection of tcdB in stool specimens with high sensitivity. Detection of tcdB by LAMP from overnight cultures in CMM could be an alternative method of diagnostic testing at clinical laboratories without special apparatus.     

Algorithm Combining Toxin Immunoassay and Stool Culture for Diagnosis of Clostridium difficile Infection

Enzyme immunoassays (EIA) to detect glutamate dehydrogenase or toxins A (TcdA) and B (TcdB), a cytotoxicity assay, and bacteriologic culture have disadvantages when applied individually to diagnosis of Clostridium difficile infections. Stool specimens (n = 1,596) were subjected to toxin detection via an enzyme-linked fluorescent immunoassay (ELFA; Vidas CDAB assay) and bacteriologic culture for toxigenic C. difficile in a three-step algorithm with additional toxigenic culture. Isolates (n = 163) from ELFA-negative stool specimens were examined via ELFA for toxin production. We amplified tcdA and tcdB from C. difficile isolates and tcdB from stool specimens that were ELFA positive or equivocal and culture negative, and we compared the results to those obtained with the three-step algorithm. More than 26% of stool specimens (419/1,596) were culture positive, yielding 248 isolates (59.2%) with both toxin genes (tcdA- and tcdB-positive isolates), 88 isolates (21.0%) with either tcdA or tcdB, and 83 (19.8%) that had no toxin genes (tcdA- and tcdB-negative isolates). Among 49 (culture-negative/ELFA-positive or -equivocal) stool specimens, 53.1% (26/49) represented tcdB-positive isolates. Therefore, the total number of PCR-positive cases was 362, and 27.1% (98/362) of these were detected through toxigenic culture. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 63.3%, 96.7%, 90.5%, and 92.4% (ELFA alone); 92.8%, 93.3%, 80.2%, and 97.8% (culture); and 70.7%, 91.4%, 95.5%, and 100% (three-step algorithm ELFA and bacterial culture with toxigenic culture), respectively, with culture and PCR for tcdA and tcdB as the standards. Thus, sensitivity and specificity were highest using culture and ELFA, respectively, but we recommend the three-step algorithm comprising EIA to detect both toxins and toxigenic culture for C. difficile as a practical method for achieving better PPV and NPV.Clostridium difficile is an important nosocomial pathogen, causing antimicrobial-associated diarrhea and pseudomembranous colitis. Toxins A (TcdA) and B (TcdB) mediate the pathogenesis of C. difficile infection (CDI), and toxin detection is an important part of diagnosis. A cytotoxicity neutralization assay (CNA) is the reference method for toxin detection, but it is expensive and time-consuming and requires tissue culture facilities (34, 35). Most laboratories now use a commercial enzyme immunoassay (EIA) to detect TcdA and/or TcdB, with the benefits of rapid turnaround time and ease of use (3, 21, 22, 23, 26, 27, 33, 35). The putative >90% sensitivity of toxin EIAs is not often realized in practice, but EIA is the only toxin detection method available to many routine medical laboratories. The demand for EIA kits detecting both TcdA and TcdB has increased due to increased worldwide prevalence of TcdA-negative, TcdB-positive (TcdA− TcdB+) strains (1, 12, 24, 29, 32).A two-step algorithm, based upon EIA-based detection of species-specific antigen glutamate dehydrogenase (GDH-Ag) and toxin detection via CNA, was suggested to have improved sensitivity and specificity in the detection of toxigenic C. difficile (34). However, the GDH-Ag assay detects both nontoxigenic and toxigenic strains, and the aforementioned shortcomings of the CNA assay make it unavailable to many routine laboratories.Bacteriologic culture can be time-consuming, but it is more straightforward and sensitive than CNA for the detection of toxigenic C. difficile. Furthermore, it provides isolates for characterization, yielding information about CDI epidemiology and antimicrobial susceptibility (11, 28, 36). We evaluated the combination of bacteriologic culture and EIA-based detection of TcdA and TcdB as a new strategy for diagnosis of CDI, especially in areas where TcdA− TcdB+ strains are prevalent.