Using 42 isolates contributed by laboratories in Canada, The Netherlands, the United Kingdom, and the United States, we compared the results of analyses done with seven Clostridium difficile typing techniques: multilocus variable-number tandem-repeat analysis (MLVA), amplified fragment length polymorphism (AFLP), surface layer protein A gene sequence typing (slpAST), PCR-ribotyping, restriction endonuclease analysis (REA), multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). We assessed the discriminating ability and typeability of each technique as well as the agreement among techniques in grouping isolates by allele profile A (AP-A) through AP-F, which are defined by toxinotype, the presence of the binary toxin gene, and deletion in the tcdC gene. We found that all isolates were typeable by all techniques and that discrimination index scores for the techniques tested ranged from 0.964 to 0.631 in the following order: MLVA, REA, PFGE, slpAST, PCR-ribotyping, MLST, and AFLP. All the techniques were able to distinguish the current epidemic strain of C. difficile (BI/027/NAP1) from other strains. All of the techniques showed multiple types for AP-A (toxinotype 0, binary toxin negative, and no tcdC gene deletion). REA, slpAST, MLST, and PCR-ribotyping all included AP-B (toxinotype III, binary toxin positive, and an 18-bp deletion in tcdC) in a single group that excluded other APs. PFGE, AFLP, and MLVA grouped two, one, and two different non-AP-B isolates, respectively, with their AP-B isolates. All techniques appear to be capable of detecting outbreak strains, but only REA and MLVA showed sufficient discrimination to distinguish strains from different outbreaks.

Acquired neuromyotonia is characterized by hyperexcitability of motor nerves leading to muscle twitching, cramps, and weakness. The symptoms may improve following plasma exchange, and injection of immunoglobulin G (IgG) from 1 neuromyotonia patient into mice increased the resistance of neuromuscular transmission to d-tubocurarine. Here we examine nerves and muscle in vitro from mice injected with plasma or purified IgG from 6 neuromyotonia patients or pooled control subjects, and cultured dorsal root ganglion cells after treatment with IgG. Three of the patients had antibodies against human voltage-gated potassium channels labeled with 125I-alpha-dendrotoxin. The quantal release of acetylcholine (quantal content) at end-plates in diaphragms from mice treated with neuromyotonia IgG preparations was increased by 21% relative to control values (p = 0.0053). With one IgG preparation, the duration of the superficial peroneal nerve compound action currents was increased by 93%. The dorsal root ganglion cells treated with this IgG showed a marked increase in repetitive firing of action potentials. All effects were similar to those obtained with aminopyridines. We conclude that at least some patients with acquired neuromyotonia have antibodies directed against aminopyridine- or alpha-dendrotoxin-sensitive K+ channels in motor and sensory neurons, and they are likely to be implicated in the disease process.

Using the genomic sequence of Clostridium difficile strain 630, we developed multiple-locus variable-number tandem-repeat analysis (MLVA) with automated fragment analysis and multicolored capillary electrophoresis as a typing method for C. difficile. All reference strains, representing 31 serogroups, 25 toxinotypes, and 7 known subtypes of PCR ribotype 001, could be discriminated from each other. Application of MLVA to 28 isolates from 7 outbreaks due to the emerging hypervirulent PCR ribotype 027-pulsed-field gel electrophoresis type NAP1 resulted in recognition of 13 clusters. Additionally, 29 toxin A-negative, toxin B-positive isolates belonging to PCR ribotype 017 from eight different countries revealed eight country-specific clusters. MLVA is a highly discriminatory genotyping method and a new tool for subtyping of newly emerging variants of C. difficile.

In a prospective multicenter study, 367 fecal samples from 300 patients with diarrhea were tested for Clostridium difficile-associated diarrhea (CDAD) with a new immunochromatography assay for toxins A and B (ICTAB), a real-time PCR on the toxin B gene, and the cell cytotoxicity assay. Twenty-three (6.2%) of the 367 fecal samples were positive by the cell cytotoxicity assay. With the cell cytotoxicity assay as the "gold standard," the sensitivity, specificity, positive predictive value, and negative predictive value for the ICTAB assay and real-time PCR were 91, 97, 70, and 99%, and 87, 96, 57 and 99%, respectively. In conclusion, both the ICTAB and the real-time PCR can be implemented as rapid screening methods for patients suspected of having CDAD.Clostridium difficile-associated diarrhea (CDAD) is the most important infectious cause of nosocomial diarrhea and pseudomembranous colitis. The enteropathogenicity depends on the production of enterotoxin A (308 kDa) and cytotoxin B (270 kDa). Several authors have suggested that all fecal samples for C. difficile from patients with diarrhea hospitalized for more than 72 h be investigated (3) irrespective of the physician's request, since length of hospitalization is simple to implement as an inclusion criterion. Conventional diagnostic methods for CDAD are the cell cytotoxicity assay and the enzyme immunoassays (EIA) to detect fecal toxins A (TcdA) and B (TcdB). The cell cytotoxicity assay is considered the "gold standard." However, with a turnaround time of more than 48 h, this method is laborious and time-consuming. Frequently, EIA are used because of their more rapid turnaround time. Rapid diagnosis of CDAD is important, since it may result in early treatment and prevention of nosocomial transmission.A new rapid immunochromatography test, the ImmunoCard Toxins A&B (ICTAB; Meridian), has recently been introduced. The ICTAB is a single-test enzyme immunoassay for the detection of TcdA and TcdB in fecal samples within 20 min. No sample pretreatment is required, and an internal procedure control is integrated in each card. The performance of this rapid assay was evaluated in comparison with an in-housedeveloped, real-time PCR using tcdB and the cell cytotoxicity assay. A positive PCR result for a fecal sample is indicative of the presence of a C. difficile strain capable of producing TcdB. Fecal samples from adult patients with diarrhea for whom there was a request for C. difficile diagnosis and samples from patients hospitalized for more than 72 h were included. All samples were stored within 6 hours after arrival at the laboratory at Ϫ20°C in two individual vials for subsequent testing by the cell cytotoxicity assay and real-time PCR at the LUMC. The ICTAB was performed in the Erasmus MC and the LUMC. All fecal samples were thawed only once for a specific test.The ICTAB was performed according to the respective manufacturer's instructions. Briefly, enzyme conjugate was added to specimen diluent before the addition of 25 l of the fecal sample or the control. After i...

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