A case of community-acquired Clostridioides difficile infection causing intussusception, severe pneumonia, and severe hypokalemia.

BACKGROUND: Clostridioides difficile infection is associated with antibiotic use and manifests as diarrhea; however, emerging cases of fulminant diarrhea caused by binary toxin-producing C. difficile unrelated to prior antibiotic exposure have been reported. Although fulminant colitis caused by C. difficile has been documented, instances of intussusception remain scarce. Here, we present a case of adult intussusception with severe hypokalemia and pneumonia resulting from a community-acquired C. difficile infection in Japan. CASE PRESENTATION: An 82-year-old male presented with dizziness, progressive weakness, and diarrhea. Initial vital signs indicated severe respiratory and circulatory distress, and laboratory findings revealed hypokalemia, pneumonia, and septic shock. Imaging confirmed intussusception of the ascending colon. Although colonoscopy suggested a potential tumor, no malignancy was found. The C. difficile rapid test result was positive, indicating community-acquired C. difficile infection. Treatment with vancomycin was initiated; however, intussusception relapsed. Surgical intervention was successful and led to clinical improvement. The patient's complex pathophysiology involved community-acquired C. difficile-induced severe diarrhea, hypokalemia, hypermetabolic alkalosis, and subsequent intussusception. Although adult intussusception is uncommon, this case was uniquely linked to binary toxin-producing C. difficile. The identified strain, SUH1, belonged to a novel sequence type (ST1105) and clade 3, suggesting a highly virulent clone. Resistome analysis aligned with phenotypic susceptibility to metronidazole and vancomycin, confirming their treatment efficacy. CONCLUSION: This case report highlights a binary toxin-producing C. difficile that caused intussusception. The consideration of community-acquired C. difficile in the differential diagnosis of severe enteritis is necessary, even in Japan.

Ribotypes and New Virulent Strains Across Europe.

Clostridioides (formerly Clostridium) difficile is a major bacterial cause of post-antibiotic diarrhoea. The epidemiology of C. difficile infections (CDIs) has dramatically changed since the early 2000s, with an increasing incidence and severity across Europe. This trend is partly due to the emergence and rapid worldwide spread of the hypervirulent and epidemic PCR ribotype 027. Profiles of patients with CDI have also evolved, with description of community-acquired (CA) infections in patients with no traditional risk factors for CDI. However, epidemiological studies indicated that some European countries have successfully controlled the dissemination of the 027 clone whereas other countries reported the emergence of other virulent or unusual strains. The aims of this review are to summarize the current European CDI epidemiology and to describe the new virulent C. difficile strains circulating in Europe, as well as other potential emerging strains described elsewhere. Standardized typing methods and surveillance programmes are mandatory for a better understanding and monitoring of CDI in Europe.

Structural elucidation of the Clostridioides difficile transferase toxin reveals a single-site binding mode for the enzyme.

Clostridioides difficile is a Gram-positive, pathogenic bacterium and a prominent cause of hospital-acquired diarrhea in the United States. The symptoms of C. difficile infection are caused by the activity of three large toxins known as toxin A (TcdA), toxin B (TcdB), and the C. difficile transferase toxin (CDT). Reported here is a 3.8-Å cryo-electron microscopy (cryo-EM) structure of CDT, a bipartite toxin comprised of the proteins CDTa and CDTb. We observe a single molecule of CDTa bound to a CDTb heptamer. The formation of the CDT complex relies on the interaction of an N-terminal adaptor and pseudoenzyme domain of CDTa with six subunits of the CDTb heptamer. CDTb is observed in a preinsertion state, a conformation observed in the transition of prepore to ß-barrel pore, although we also observe a single bound CDTa in the prepore and ß-barrel conformations of CDTb. The binding interaction appears to prime CDTa for translocation as the adaptor subdomain enters the lumen of the preinsertion state channel. These structural observations advance the understanding of how a single protein, CDTb, can mediate the delivery of a large enzyme, CDTa, into the cytosol of mammalian cells.

Clostridioides difficile Binary Toxin Is Recognized by the Toll-Like Receptor 2/6 Heterodimer to Induce a Nuclear Factor-κB Response.

Clostridioides difficile infection (CDI) represents a significant burden on the health care system, one that is exacerbated by the emergence of binary toxin (CDT)-producing hypervirulent C. difficile strains. Previous work from our laboratory has shown that Toll-like receptor 2 (TLR2) recognizes CDT to induce inflammation. Here we explore the interactions of CDT with TLR2 and the impact on host immunity during CDI. We found that the TLR2/6 heterodimer, not TLR2/1, is responsible for CDT recognition, and that gene pathways including nuclear factor-κB and MAPK downstream of TLR2/6 are upregulated in mice with intact TLR2/6 signaling during CDI.

Influence of Binary Toxin Gene Detection and Decreased Susceptibility to Antibiotics among Clostridioides difficile Strains on Disease Severity: a Single-Center Study.

Clostridioides difficile infection (CDI) is the fifth leading cause of death from nonmalignant gastrointestinal disease in the United States. The contribution of resistance to C. difficile-active antibiotics to the outcomes of CDI is unclear. We evaluated the antimicrobial susceptibility of C. difficile isolates in a U.S. hospital and determined associations of clinical variables and binary toxin positivity with antibiotic resistance. C. difficile spores were cultured from fecal specimens of adult patients with CDI for genotyping and antimicrobial susceptibility assay (for clindamycin [CLI], fidaxomicin [FDX], metronidazole [MTZ], moxifloxacin [MXF], tigecycline [TGC], and vancomycin [VAN]). Electronic medical records were reviewed for clinical data extraction. Ninety-seven of 130 (75%) fecal samples grew toxigenic C. difficile in culture. Most of the isolates were tcdA+ tcdB+ cdtB- (80.4%), and 18.6% and 1% were tcdA+ tcdB+ cdtB+ and tcdA-tcdB+ cdtB+, respectively. Susceptibility to VAN, MTZ, FDX, TGC, MXF, and CLI was 96%, 94%, 100%, 100%, 8%, and 79%, respectively. Six isolates, all cdtB positive and belonging to the 027 ribotype, were resistant to VAN and/or MTZ. Higher MICs were found in isolates with a mutation in the VAN-related resistance gene vanR, but not vanS. In addition, cdtB+ isolates exhibited higher MICs of VAN, MTZ, TGC, CLI, and MXF compared to cdtB- strains. Patients with greater intestinal inflammation or severe disease were more likely to be infected with cdtB+ strains. Decreased susceptibility to antibiotics is not directly associated with either severe or recurrent CDI. However, antimicrobial susceptibility of C. difficile is decreased in strains positive for the binary toxin gene.

Purification, characterization and toxicity assessment of PirAB toxins from Photorhabdus akhurstii subsp. akhurstii K-1.

Photorhabdus insect related proteins A & B (PirA, PirB) from Photorhabdus and Xenorhabdus bacteria exhibit both oral and injectable toxicity against lepidopteran and dipteran insect pest. The pirA, pirAt (encoding 6 N-terminal truncated PirA), pirB genes, pirA-pirB (with ERIC sequences), pirA-pirB-mERIC (modified pirA-pirB with mutated ERIC sequences) and polycistronic-pirAB were cloned and expressed in Escherichia coli. However, PirA protein was expressed in insoluble form and therefore the pirA gene was modified to produce PirAt. Moreover, pirA-pirB-mERIC, polycistronic-pirAB and co-transformed pirA/pirB genes were not expressed in the studied prokaryotic expression systems. None of the single purified proteins or mixtures of the individually expressed and purified proteins were toxic to mosquito larvae of Aedes aegypti and Culex quinquefasciatus. However, PirA-PirB protein mixtures purified from pirA-pirB operon plasmid were toxic to A. aegypti and C. quinquefasciatus larvae with LC50 values of 991 and 614 ng/ml, respectively. The presence of ERIC sequences between the two orfs of the pirA-pirB operon could help to obtain the proteins in biologically active form. Further, results confirm that PirA-PirB proteins of P. akhurstii subsp. akhurstii K-1 are binary insecticidal toxins and ERIC sequences could play an important role in expression of Pir proteins. Reports of biophysical characterization of individually purified PirAt, PirB and expressed PirA-PirB toxin mixture could provide the structural insight into these proteins.

Three-year epidemiological analysis of Clostridioides difficile infection and the value of hospital-infection control measures in a tertiary-care teaching hospital in Japan using PCR-based open-reading frame typing.

INTRODUCTION: Clostridioides difficile is an important causative pathogen in antibiotic-associated colitis and nosocomial infections. This study aimed to assess immunochromatographic test results for C. difficile infection and the utility of PCR-based open-reading frame typing (POT) for potentially controlling the intra-ward transmission of C. difficile. METHODS: We conducted a molecular epidemiological analysis using POT to investigate 102 inpatients who tested positive for the C. difficile toxin using immunochromatography in a tertiary-care teaching hospital in Japan between 2016 and 2018; isolates from the patients were obtained and cultured. RESULTS: The number of POT numbers detected in 2016, 2017, and 2018 were 27 (among 34 patients), 20 (among 31 patients), and 28 (among 37 patients), respectively. During this three-year period, there were seven cases whose bacterial strains with the same POT number was identified in the same ward within 6 months. The intra-ward transmission rate was the highest in 2017 (16.1%). Intra-ward transmission was identified at a higher rate in patients whose sample cultures tested toxin-positive than in patients whose sample cultures tested toxin- and glutamate-dehydrogenase-positive via immunochromatography (16% vs. 3%, p < 0.05). CONCLUSIONS: We conclude that the use of immunochromatographic tests for C. difficile diagnosis and epidemiological analyses via POT may be helpful for evaluating intra-ward transmission of C. difficile.

The Importance of Therapeutically Targeting the Binary Toxin from Clostridioides difficile.

Novel therapeutics are needed to treat pathologies associated with the Clostridioides difficile binary toxin (CDT), particularly when C. difficile infection (CDI) occurs in the elderly or in hospitalized patients having illnesses, in addition to CDI, such as cancer. While therapies are available to block toxicities associated with the large clostridial toxins (TcdA and TcdB) in this nosocomial disease, nothing is available yet to treat toxicities arising from strains of CDI having the binary toxin. Like other binary toxins, the active CDTa catalytic subunit of CDT is delivered into host cells together with an oligomeric assembly of CDTb subunits via host cell receptor-mediated endocytosis. Once CDT arrives in the host cell's cytoplasm, CDTa catalyzes the ADP-ribosylation of G-actin leading to degradation of the cytoskeleton and rapid cell death. Although a detailed molecular mechanism for CDT entry and host cell toxicity is not yet fully established, structural and functional resemblances to other binary toxins are described. Additionally, unique conformational assemblies of individual CDT components are highlighted herein to refine our mechanistic understanding of this deadly toxin as is needed to develop effective new therapeutic strategies for treating some of the most hypervirulent and lethal strains of CDT-containing strains of CDI.

Genomic investigation of a sequence type 67 Clostridium difficile causing community-acquired fulminant colitis in Hong Kong.

In 2017, we identified a Clostridium difficile strain HKCD4 that caused community-acquired fulminant colitis in a previously healthy child. Phylogenetically, it belonged to clade 2, sequence type 67 and was resistant to fluoroquinolone and tetracycline. The strain was pathogenicity locus and binary toxin positive. It has a mutation in the trehalose repressor treR leading to the L172I substitution that was previously reported in the epidemic ribotype 027 lineage. HKCD4 has a tcdB sequence that shared very high identities with 3 highly virulent reference strains. It has a CpG depleted genome that is characteristic of hypervirulent C. difficile. The emergence of ST67 lineage with molecular feature of hypervirulence in the community is concerning and emphasizes the need for full characterization of strains causing severe disease in patients without classical risk factors.

In silico, in vitro and in vivo analysis of putative virulence factors identified in large clostridial toxin-negative, binary toxin- producing C. difficile strains.

The relevance of large clostridial toxin-negative, binary toxin-producing (A-B-CDT+) Clostridium difficile strains in human infection is still controversial. In this study, we investigated putative virulence traits that may contribute to the role of A-B-CDT+C. difficile strains in idiopathic diarrhea. Phenotypic assays were conducted on 148 strains of C. difficile comprising 10 different A-B-CDT+C. difficile ribotypes (RTs): 033, 238, 239, 288, 585, 586, QX143, QX444, QX521 and QX629. A subset of these isolates (n = 53) was whole-genome sequenced to identify genetic loci associated with virulence and survival. Motility studies showed that with the exception of RT 239 all RTs tested were non-motile. C. difficile RTs 033 and 288 had deletions in the F2 and F3 regions of their flagella operon while the F2 region was absent from strains of RTs 238, 585, 586, QX143, QX444, QX521 and QX629. The flagellin and flagella cap genes, fliC and fliD, respectively, involved in adherence and host colonization, were conserved in all strains, including reference strains. All A-B-CDT+C. difficile strains produced at least three extracellular enzymes (deoxyribonuclease, esterase and mucinase) indicating that these are important extracellular proteins. The toxicity of A-B-CDT+C. difficile strains in Vero cells was confirmed, however, pathogenicity was not demonstrated in a mouse model of disease. Despite successful colonization by most strains, there was no evidence of disease in mice. This study provides the first in-depth analysis of A-B-CDT+C. difficile strains and contributes to the current limited knowledge of these strains as a cause of C. difficile infection.

Phosphorylation and functionality of CdtR in Clostridium difficile.

The production of TcdA, TcdB and CDT in Clostridium difficile PCR ribotype 027, is regulated by the two-component system response regulator CdtR. Despite this, little is known about the signal transduction pathway leading to the activation of CdtR. In this study, we generated R20291ΔPalocΔcdtR model strains expressing CdtR phospho-variants in which our predicted phospho-accepting Asp, Asp61 was mutated for Ala or Glu. The constructs were assessed for their ability to restore CDT production. Dephospho-CdtR-Asp61Ala was completely non-functional and mirrored the cdtR-deletion mutant, whilst phospho-CdtR-Asp61Glu was functional, possessing 38-52% of wild-type activity. Taken together, these data suggest that CdtR is activated by phosphorylation of Asp61. The same principles were applied to assess the function of PCR ribotype 078-derived CdtR, which was shown to be non-functional owing to polymorphisms present within its coding gene. Conversely, polymorphisms present within its promoter region, provide significantly enhanced promoter activity compared with its PCR ribotype 027 counterpart. To ensure our data were representative for each ribotype, we determined that the cdtR nucleotide sequence was conserved in a small library of eight PCR ribotype 027 clinical isolates and nineteen PCR ribotype 078 isolates from clinical and animal origin.

An aromatic cluster in Lysinibacillus sphaericus BinB involved in toxicity and proper in-membrane folding.

The binary toxin from Lysinibacillus sphaericus has been successfully used for controlling mosquito-transmitted diseases. Based on structural alignments with other toxins, an aromatic cluster in the C-terminal domain of BinB (termed here BC) has been proposed to be important for toxicity. We tested this experimentally using BinB mutants bearing single mutations in this aromatic cluster. Consistent with the hypothesis, two of these mutations, F311A and F315A, were not toxic to Culex quinquefasciatus larvae and were unable to permeabilize liposomes or elicit ion channel activity, in contrast to wild-type BinB. Despite these effects, none of these mutations altered significantly the interaction between the activated forms of the two subunits in solution. These results indicate that these aromatic residues on the C-terminal domain of BinB are critical for toxin insertion in membranes. The latter can be by direct contact of these residues with the membrane surface, or by facilitating the formation a membrane-inserting oligomer.

Ribotypes and New Virulent Strains Across Europe.

Clostridium difficile is a major bacterial cause of post-antibiotic diarrhoea. The epidemiology of C. difficile infections (CDI) has dramatically changed since the early 2000s, with an increasing incidence and severity across Europe. This trend is partly due to the emergence and rapid worldwide spread of the hypervirulent and epidemic PCR ribotype 027. Profiles of patients with CDI have also evolved, with description of community-acquired (CA) infections in patients with no traditional risk factors for CDI. However, recent epidemiological studies indicated that some European countries have successfully controlled the dissemination of the 027 clone whereas other countries recently reported the emergence of other virulent or unusual strains. The aims of this review are to summarize the current European CDI epidemiology and to describe the new virulent C. difficile strains circulating in Europe, as well as other potential emerging strains described elsewhere. Standardized typing methods and surveillance programmes are mandatory for a better understanding and monitoring of CDI in Europe.

Evaluation of the Cepheid® Xpert®C. difficile binary toxin (BT) diagnostic assay.

Strains of Clostridium difficile producing only binary toxin (CDT) are found commonly in animals but not humans. However, human diagnostic tests rarely look for CDT. The Cepheid Xpert C. difficile BT assay detects CDT with equal sensitivity (≥92%) in human and animal faecal samples.

Binary Clostridium difficile toxin (CDT) - A virulence factor disturbing the cytoskeleton.

Clostridium difficile infection causes antibiotics-associated diarrhea and pseudomembranous colitis. Major virulence factors of C. difficile are the Rho-glucosylating toxins TcdA and TcdB. In addition, many, so-called hypervirulent C. difficile strains produce the binary actin-ADP-ribosylating toxin CDT. CDT causes depolymerization of F-actin and rearrangement of the actin cytoskeleton. Thereby, many cellular functions, which depend on actin, are altered. CDT disturbs the dynamic balance between actin and microtubules in target cells. The toxin increases microtubule polymerization and induces the formation of microtubule-based protrusions at the plasma membrane of target cells. Moreover, CDT causes a redistribution of vesicles from the basolateral side to the apical side, where extracellular matrix proteins are released. These processes may increase the adherence of clostridia to target cells. Here, we review the effects of the action of CDT on the actin cytoskeleton and on the microtubule system.

Antimicrobial resistance in large clostridial toxin-negative, binary toxin-positive Clostridium difficile ribotypes.

Antimicrobial resistance (AMR) is commonly found in Clostridium difficile strains and plays a major role in strain evolution. We have previously reported the isolation of large clostridial toxin-negative, binary toxin-producing (A-B-CDT+) C. difficile strains from colonised (and in some instances diarrhoeic) food animals, as well as from patients with diarrhoea. To further characterise these strains, we investigated the phenotypic and genotypic AMR profiles of a diverse collection of A-B-CDT+C. difficile strains. The in vitro activities of 10 antimicrobial agents were determined for 148 A-B-CDT+C. difficile strains using an agar dilution methodology. Whole-genome sequencing and in silico genotyping was performed on 53 isolates to identify AMR genes. All strains were susceptible to vancomycin, metronidazole and fidaxomicin, antimicrobials currently considered first-line treatments for C. difficile infection (CDI). Differences in antimicrobial phenotypes between PCR ribotypes (RTs) were observed but were minimal. Phenotypic resistance was observed in 13 isolates to tetracycline (TetR, MIC = 16 mg/L), moxifloxacin (MxfR, MIC = 16 mg/L), erythromycin (EryR, MIC ≥128 mg/L) and clindamycin (CliR, MIC = 8 mg/L). The MxfR strain (RT033) possessed mutations in gyrA/B, while the TetR (RT033) strain contained a tetM gene carried on the conjugative transposon Tn6190. All EryR and CliR strains (RT033, QX521) were negative for the erythromycin ribosomal methylase gene ermB, suggesting a possible alternative mechanism of resistance. This work describes the presence of multiple AMR genes in A-B-CDT+C. difficile strains and provides the first comprehensive analysis of the AMR repertoire in these lineages isolated from human, animal, food and environmental sources.

Pore-forming activity of clostridial binary toxins.

Clostridial binary toxins (Clostridium perfringens Iota toxin, Clostridium difficile transferase, Clostridium spiroforme toxin, Clostridium botulinum C2 toxin) as Bacillus binary toxins, including Bacillus anthracis toxins consist of two independent proteins, one being the binding component which mediates the internalization into cell of the intracellularly active component. Clostridial binary toxins induce actin cytoskeleton disorganization through mono-ADP-ribosylation of globular actin and are responsible for enteric diseases. Clostridial and Bacillus binary toxins share structurally and functionally related binding components which recognize specific cell receptors, oligomerize, form pores in endocytic vesicle membrane, and mediate the transport of the enzymatic component into the cytosol. Binding components retain the global structure of pore-forming toxins (PFTs) from the cholesterol-dependent cytotoxin family such as perfringolysin. However, their pore-forming activity notably that of clostridial binding components is more related to that of heptameric PFT family including aerolysin and C. perfringens epsilon toxin. This review focuses upon pore-forming activity of clostridial binary toxins compared to other related PFTs. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale.

Molecular Evolutionary Constraints that Determine the Avirulence State of Clostridium botulinum C2 Toxin.

Clostridium botulinum (group-III) is an anaerobic bacterium producing C2 toxin along with botulinum neurotoxins. C2 toxin is belonged to binary toxin A family in bacterial ADP-ribosylation superfamily. A structural and functional diversity of binary toxin A family was inferred from different evolutionary constraints to determine the avirulence state of C2 toxin. Evolutionary genetic analyses revealed evidence of C2 toxin cluster evolution through horizontal gene transfer from the phage or plasmid origins, site-specific insertion by gene divergence, and homologous recombination event. It has also described that residue in conserved NAD-binding core, family-specific domain structure, and functional motifs found to predetermine its virulence state. Any mutational changes in these residues destabilized its structure-function relationship. Avirulent mutants of C2 toxin were screened and selected from a crucial site required for catalytic function of C2I and pore-forming function of C2II. We found coevolved amino acid pairs contributing an essential role in stabilization of its local structural environment. Avirulent toxins selected in this study were evaluated by detecting evolutionary constraints in stability of protein backbone structure, folding and conformational dynamic space, and antigenic peptides. We found 4 avirulent mutants of C2I and 5 mutants of C2II showing more stability in their local structural environment and backbone structure with rapid fold rate, and low conformational flexibility at mutated sites. Since, evolutionary constraints-free mutants with lack of catalytic and pore-forming function suggested as potential immunogenic candidates for treating C. botulinum infected poultry and veterinary animals. Single amino acid substitution in C2 toxin thus provides a major importance to understand its structure-function link, not only of a molecule but also of the pathogenesis.

An Enterotoxin-Like Binary Protein from Pseudomonas protegens with Potent Nematicidal Activity.

Soil microbes are a major food source for free-living soil nematodes. It is known that certain soil bacteria have evolved systems to combat predation. We identified the nematode-antagonistic Pseudomonas protegens strain 15G2 from screening of microbes. Through protein purification we identified a binary protein, designated Pp-ANP, which is responsible for the nematicidal activity. This binary protein inhibits Caenorhabditis elegans growth and development by arresting larvae at the L1 stage and killing older-staged worms. The two subunits, Pp-ANP1a and Pp-ANP2a, are active when reconstituted from separate expression in Escherichia coli The binary toxin also shows strong nematicidal activity against three other free-living nematodes (Pristionchus pacificus, Panagrellus redivivus, and Acrobeloides sp.), but we did not find any activity against insects and fungi under test conditions, indicating specificity for nematodes. Pp-ANP1a has no significant identity to any known proteins, while Pp-ANP2a shows ∼30% identity to E. coli heat-labile enterotoxin (LT) subunit A and cholera toxin (CT) subunit A. Protein modeling indicates that Pp-ANP2a is structurally similar to CT/LT and likely acts as an ADP-ribosyltransferase. Despite the similarity, Pp-ANP shows several characteristics distinct from CT/LT toxins. Our results indicate that Pp-ANP is a new enterotoxin-like binary toxin with potent and specific activity to nematodes. The potency and specificity of Pp-ANP suggest applications in controlling parasitic nematodes and open an avenue for further research on its mechanism of action and role in bacterium-nematode interaction.IMPORTANCE This study reports the discovery of a new enterotoxin-like binary protein, Pp-ANP, from a Pseudomonas protegens strain. Pp-ANP shows strong nematicidal activity against Caenorhabditis elegans larvae and older-staged worms. It also shows strong activity on other free-living nematodes (Pristionchus pacificus, Panagrellus redivivus, and Acrobeloides sp.). The two subunits, Pp-ANP1a and Pp-ANP2a, can be expressed separately and reconstituted to form the active complex. Pp-ANP shows some distinct characteristics compared with other toxins, including Escherichia coli enterotoxin and cholera toxin. The present study indicates that Pp-ANP is a novel binary toxin and that it has potential applications in controlling parasitic nematodes and in studying toxin-host interaction.

Clostridium Difficile, Colitis, and Colonoscopy: Pediatric Perspective.

PURPOSE OF THE REVIEW: Review tests available for detection of Clostridium difficile (C. Diff) induced disease, including when such tests should be done in children and how they should be interpreted. RECENT FINDINGS: Multiple tests are available for detecting disease due to C. diff. These include colonoscopy and stool analysis. Colonoscopy with biopsy is the most sensitive test for detecting the presence of colitis. The toxins produced by the C. diff. (toxin A, toxin B, and binary toxin) are the agents that cause injury and disease. Only toxin producing C. diff. Strains will cause disease. Binary toxin by itself is not thought to produce disease. Binary toxin causes disease in humans when present with toxin A and B producing bacteria, and has been implicated with fulminant life threatening disease. Stool analyses vary in sensitivity and specificity depending on the assay used. The presence of toxin producing strains of C diff. in the stool does not equate with disease. The presence of a toxin-producing bacteria or toxins (A or B) only equates with disease if diarrhea or a diseased colon (toxic megacolon, ileus, and sepsis) is present. Nucleic acid amplification testing (NAAT), when used in the stool from patients with diarrhea, appears to be the most efficient study to detect the gene that encodes for toxin A and B and thus to diagnose C. diff.-induced disease. Infants have a high carriage rate of C. diff. and are believed not to develop disease from it or its toxins. Infants should not be tested for C. difficile. The NAAT is most specific when done on patients with diarrhea with liquid stools. Testing for C. difficile should only be done on patients with diarrhea. One can assume that a patient who has no diarrhea and is not ill does not have C. diff.-induced disease. Treatment should be limited to patients with diarrhea who test positive for C. diff. toxin (A or B) or toxin-producing bacteria. Direct testing for binary toxin is not commercially available. Binary toxin is only thought to cause disease in humans when C. diff. toxin (A and B)-producing bacteria are present.