Biology of Clostridium Difficile: Implications For Epidemiology and Diagnosis

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Biology of Clostridium difcile: Implications for Epidemiology and Diagnosis

Karen C. Carroll1 and John G. Bartlett2
1
Annu. Rev. Microbiol. 2011.65:501-521. Downloaded from www.annualreviews.org by University of Texas - Arlington on 07/19/12. For personal use only.
Division of Medical Microbiology and 2 Division of Infectious Diseases, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21205; email: Kcarrol7@jhmi.edu
Annu. Rev. Microbiol. 2011. 65:50121 First published online as a Review in Advance on June 16, 2011 The Annual Review of Microbiology is online at micro.annualreviews.org This articles doi: 10.1146/annurev-micro-090110-102824 Copyright c 2011 by Annual Reviews. All rights reserved 0066-4227/11/1013-0501$20.00
Keywords
toxins, toxinotypes, hypervirulent, microbiota, risk factors
Abstract
Clostridium difcile is an anaerobic, spore-forming, gram-positive rod that causes a spectrum of antibiotic-associated colitis through the elaboration of two large clostridial toxins and other virulence factors. Since its discovery in 1978 as the agent responsible for pseudomembranous colitis, the organism has continued to evolve into an adaptable, aggressive, hypervirulent strain. Advances in molecular methods and improved animal models have facilitated an understanding of how this organism survives in the environment, adapts to the gastrointestinal tract of animals and humans, and accomplishes its unique pathogenesis. The advances in microbiology have been accompanied by some important clinical observations including increased rates of C. difcile infection, increased virulence, and multiple outbreaks. The major new risk is uoroquinolone use; there is also an association with proton pump inhibitors and increased recognition of cases in outpatients, pediatric patients, and patients without recent antibiotic use. The combination of more aggressive strains with mobile genomes in a setting of an expanded pool of individuals at risk has refocused attention on and challenged assumptions regarding diagnostic gold standards. Future research is likely to build upon the advancements in phylogenetics to create novel strategies for diagnosis, treatment, and prevention.
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Contents
INTRODUCTION . . . . . . . . . . . . . . . . . . THE BIOLOGY OF CLOSTRIDIUM DIFFICILE . . . . . . . Microbiology . . . . . . . . . . . . . . . . . . . . . . Summary of the Clostridium difcile Strain 630 Genome Sequence . . . The Pathogenicity Locus . . . . . . . . . . . Toxins A and B . . . . . . . . . . . . . . . . . . . . C. difcile Transferase . . . . . . . . . . . . . . Other Virulence Factors. . . . . . . . . . . . Toxin Variant Strains . . . . . . . . . . . . . . Pathophysiology . . . . . . . . . . . . . . . . . . . EPIDEMIOLOGY . . . . . . . . . . . . . . . . . . . People and Patients as Reservoirs . . . Risks for C. difcile Infection . . . . . . . . Changing Epidemiology . . . . . . . . . . . Emergence of Ribotype 027 . . . . . . . . DIAGNOSIS . . . . . . . . . . . . . . . . . . . . . . . . . Nucleic Acid Amplication Methods. . . . . . . . . . . . . . . . . . . . . . . . 502 502 502 503 504 504 506 507 507 508 508 508 509 510 510 510 513
Understanding the biology of C. difcile colonization as it relates to the human intestinal microbiota has been facilitated by the development of numerous mouse models such as gnotoxenic (germ-free animals) and monoxenic (single-species inoculation) mice (32). Human fecal ora remains stable and retains bacterial and enzymatic properties when inoculated into the gnotobiotic mice (32). Such human microbiota-associated (HMA) animals facilitate research on the effects of antimicrobial treatment on the human microbiota and on the protective effects of probiotics (32). Data from the human microbiome project are likely to extend the observations provided by the HMA mouse model. In addition to better animal models, molecular advances including whole-genome sequencing have enabled sophisticated phylogenetics studies (5, 66, 121, 127). This review summarizes to date our knowledge of the biology of C. difcile and the application of that knowledge to the understanding of important facets of C. difcile epidemiology and diagnosis.
INTRODUCTION
Major contributions to the current knowledge on Clostridium difcile and the disease it causes are the discovery of C. difcile in 1935 (63, 125), studies of antibiotic-associated enterocolitis in guinea pigs as reported by Hambra et al. (64) in 1943 and later by De Somer and colleagues (37), the recognition of a cytopathic toxin in the guinea pig model by Green in 1974 (62), the highly successful clinical experience with oral vancomycin for Staphylococcus aureus enterocolitis as reported by Kahn & Hall in 1966 (71), and the prospective study of clindamycin colitis by Tedesco et al. reported in 1974 (132). These observations set the stage to challenge S. aureus as the major pathogen of antibioticassociated colitis using rodent models and clinical observations (18, 19). These studies converged with reports published in the late 1970s dening the role of C. difcile as the major agent of antibiotic-associated colitis in people and animals (1417, 19, 54).
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THE BIOLOGY OF CLOSTRIDIUM DIFFICILE Microbiology

C. difcile is an obligate anaerobic, grampositive, spore-forming rod that is both saccharolytic and proteolytic. Its name is derived from observations by Hall & OToole (63) on the difculty in isolating the organism because of its slow growth (doubling time 40 to 70 min) compared with that of other Clostridium spp. (35, 63). During logarithmic growth, when vegetative cells predominate, the organism is very aerointolerant. C. difcile spores are the transmissible form, contribute to survival of the organism in the host, and are responsible for recurrence of disease when therapy is stopped. Like other bacterial spores, they are metabolically dormant, survive for long periods, and are resistant to harsh physical or chemical treatments. Lawley et al. (80) developed protocols to effectively purify spores of C. difcile strain
Phylogenetics: whole-genome comparisons of bacteria using a variety of molecular techniques to develop a model of bacterial evolution
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630. Transmission electron microscopy of these highly puried spores showed that they are composed of an electron-dense outer surface, with the spore coat composed of concentric inner rings (80). The outer surface of the spore coat contains an exosporium that is smooth in the dormant state but develops numerous lamentous projections that attach to the colonic microvilli during germination (101). Beyond the exosporium are the spore coat, a clearer cortex region, and then an inner membrane adjacent to the core (80). Densely packed ribosomes and nucleoproteins are seen in the core (80). Highly puried spores permitted the study of their biology and infectivity (80). Cholate, taurocholate, or glycocholate supplementation of brain heart infusion (BHI) increased germination rates 100- to 1000-fold more than BHI agar plates alone, an observation that has implications for recovery of C. difcile from clinical and environmental samples (80). Puried spores demonstrated resistance to high temperatures and 70% ethanol but were inactivated by sporicidal agents. Environmental spores infect mice in a dose-dependent manner; the dose required to infect 50% of the mice (ICD50) is 7 spores per cm2 . Genes encoding the spore proteins are dispersed throughout the bacterial genome. Proteases and stress response proteins that putatively protect the spore from oxidative stress during germination are abundant, as are metabolic proteins (80). Surface-exposed proteins may interact with the extracellular environment and may be important in cellular adhesion (e.g., S-layer protein, SlpA) and germination (80). These surface-exposed proteins are potential candidates for vaccine targets and novel diagnostic tests. Regulation of sporulation is intimately associated with toxin regulation (137).
Summary of the Clostridium difcile Strain 630 Genome Sequence

The complete genome sequence of C. difcile strain 630, an epidemic, toxinotype X clinical strain that is virulent and multi-drug resistant,
was determined by Sebaihia et al. (121). This work has provided insight into the genetic factors responsible for organism survival, resistance, and virulence. Some of the important observations are highlighted here. The genome of this organism has a circular chromosome (4,290,252 bp) and a plasmid (7,881 bp). When compared with the genomes of four other pathogenic Clostridium spp. including C. botulinum and C. tetani, C. difcile shares only 15% of its coding sequences with these species and 50% of the coding sequences are unique to C. difcile (121). Moreover, 11% of the genome consists of mobile genetic elements such as conjugative transposons capable of integrating into and excising from the host genome (121). One of the most important regions of the genome is the pathogenicity locus (PaLoc), which contains ve genes, tcdA, tcdB, tcdC, tcdR, and tcdE, responsible for the synthesis and regulation of toxins A (TcdA) and B (TcdB). The genes (cdtA and cdtB) encoding the binary toxin are not found on the PaLoc. Other important genetic loci important in virulence include slpA, a single gene that encodes the S-layer; genes encoding extracellular matrix-binding domains; a collagen protease gene, suggesting that digestion of collagen may occur in vivo; surfaceanchored proteins important for covalent attachment to peptidoglycan; a putative Type IV pilus biosynthesis locus involved in mbrial biosynthesis; and a cluster of genes involved in extracellular polysaccharide synthesis (121). C. difcile has a large number of genes important for survival in the gastrointestinal tract. Several of these coding sequences are dedicated to carbohydrate transport and metabolism. One distinguishing feature is the presence of genes that encode an enzyme p-hydroxyphenylacetate decarboxylase that allows C. difcile to produce and tolerate high concentrations of p-cresol, a bacteriostatic compound. The ability to tolerate this compound may provide a competitive advantage over other intestinal microbes and is responsible for the horse manure odor characteristic of this organism in culture. A 19-gene cluster exists that is involved in ethanolamine degradation, an important constituent of
www.annualreviews.org Clostridium difcile
Toxinotype: refers to a particular strain of C. difcile based on PCR-restriction fragment analysis of the PaLoc Pathogenicity locus (PaLoc): the section of the C. difcile genome that encodes the genes that regulate toxin production
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Hypervirulent: refers to toxin variant strains of C. difcile that are associated with increased toxin production and severe clinical disease Clade: a group of genetically related organisms
phospholipids and a source of carbon and nitrogen for C. difcile found in the hosts diet. Finally, C. difcile encodes an enzyme (missing in other sequenced clostridia) important for secondary bile acid synthesis, as well as other genes that allow it to tolerate bile acids. Stabler et al. (127) carried out wholegenome microarray analysis based on the sequenced genome of C. difcile strain 630. They applied Bayesian phylogeny after array hybridization. Their observations conrm those of Sebaihia et al. and provide some insight into the phylogenetic evolution of C. difcile. Fifty-ve human isolates, representing several hypervirulent strains, toxin variant isolates, and toxin-negative isolates, and 20 animal isolates were analyzed. This detailed analysis revealed four major clades: a hypervirulent clade that contained 20 of 21 hypervirulent isolates; a defective toxin clade with all 14 TcdA, TcdB+ isolates; and two clades, HA1 and HA2, in which animal isolates were intermixed with human isolates (127). The genomes of the hypervirulent strains had a number of deletions compared with strain 630. Some fragments of conjugative transposons (CTn2 and Ctn5) present in the epidemic and hypervirulent strains are not seen in nonepidemic older isolates. The function of the loci is not clear, but they may participate in drug transport (127). The toxin-defective clade consisted of TcdA, TcdB+ isolates from the United States, the United Kingdom, and Ireland, conrming widespread geographical distribution of this clone. HA1 had mostly human strains including toxinotype 0 and reference strain VPI 10463, a toxinotype 0 isolate that hyperproduces toxins A and B. The HA2 clade contains mostly animal isolates. Stabler et al. (127) also made several important observations, not detailed here, regarding deleted loci and genetic diversity that may be important for understanding host adaptation and virulence.
toxigenic strains (30, 138). Nontoxigenic strains lack the PaLoc; however, isolates with a defective PaLoc can still cause disease (30, 34). Five genes are present on the PaLoc, tcdA, tcdB, tcdC tcdE, and tcdR (Figure 1). The two toxin genes, tcdA and tcdB, are closely aligned, separated by an intervening sequence (tcdE), and both are transcribed in the same direction (138). tcdE encodes a holin, a protein whose pore-forming activity allows the release of TcdA and TcdB from the cell (40, 138). tcdR, found upstream of tcdB, is a major positive regulator of tcdA and tcdB expression (40, 138). It is responsive to environmental conditions and is increased during stationary phase (40, 138). tcdC is found downstream of tcdA (Figure 1) and is a negative regulator of toxin production, an antisigma factor that destabilizes the TcdR holoenzyme to prevent transcription of the PaLoc (30, 36, 40, 42, 90, 138). tcdC is expressed during the exponential phase of growth; all other genes are expressed during stationary phase (30, 36, 42, 90, 138). Dineen et al. (42) have also shown that toxin gene expression is repressed by another global gene regulator called CodY. This regulator acts by monitoring environmental nutrient factors. The authors demonstrated that in the presence of sufcient nutrients, such as certain amino acids and GTP, CodY binds to the promoter region of tcdR and represses toxin gene expression (42). When nutrients in the environment are lacking, toxin gene expression is derepressed (42). Identication of CodY as a toxin gene regulator may provide a potential target for the development of compounds that are useful in the treatment of C. difcile.
Toxins A and B
TcdA and TcdB are part of the large clostridial toxin family along with Clostridium sordellii lethal toxin and hemorrhagic toxin and Clostridium novyi alpha toxin (36, 138). Large clostridial toxins are so named because of their large size (205 to 308 kDa), cytotoxicity, and mechanism of action. Both toxins are glucosyltransferases. They transfer a UDP-glucose to small
The Pathogenicity Locus

The PaLoc of C. difcile is approximately 19.6 kb in size and is stable and conserved in
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a
tcdR tcdB
PaLoc (19 kb) tcdE tcdA tcdC
Catalytic domain 102 286 288 365 N Cysteine Trp DXD motif protease enzymatic activity Substrate specificity 516 544 767
Translocation domain 956 1,128 1,652 1,678
Binding domain
C Hydrophobic region Aspartate protease
b
cdtR 18 Catalytic domain N 50 kDa cdtA
CDT locus (4.3 kb) cdtB 1,383 1 2,631 Translocation and binding domain C N 100 kDa C
Figure 1 (a) Two large toxins, toxin A and toxin B (TcdA and TcdB), are encoded on the pathogenicity locus (PaLoc), which comprises ve genes. Both toxins are single-chain proteins, and several functional domains and motifs have been identied (see text for an explanation). TcdB is shown in detail below the PaLoc. (b) A third toxin, the binary toxin or CDT (C. difcile transferase), is encoded on a separate region of the chromosome (CdtLoc) and comprises three genes. The binary toxin is composed of two unlinked proteins, CdtB and CdtA. CdtB has a binding function and CdtA is the enzymatic component. Adapted by permission from Macmillan Publishers Ltd: Nature Reviews Microbiology, Rupnik M, et al. 7:532 copyright 2009 (116).
GTPases, such as Rho, Rac, and Cdc42, in the cell. These small proteins are important in regulating signaling pathways. Glycosylation disrupts these pathways, which results in morphological changes, inhibition of cell division and membrane trafcking, and eventual cell death (36, 138). TcdA (308 kDa), an enterotoxin, was originally believed to be the toxin associated with disease, and therefore necessary for virulence, until TcdA, TcdB+ strains were associated with outbreaks of severe C. difcile infection (52). TcdB (270 kDa), a cytotoxin, is 100- to 1000-fold more toxic to culture cells than TcdA is. Lyras et al. (88) reported that TcdB, not TcdA, is essential for virulence. Hamsters infected with toxin B mutants (active toxin A, no toxin B) were less likely to die, and the degree of in vitro cytotoxicity was less than that of wild-type animals (88). TcdA mutant
hamsters died as rapidly as wild-type animals. However, a recent paper by Kuehne et al. (74) refutes the assertion that only toxin B is essential for virulence and re-establishes the observation that both toxins can cause signicant disease. In that study, TcdA+, TcdB isolates were as likely to cause disease as the wild-type strains (74). The authors hypothesize that differences in the hamster models or in the C. difcile isolates themselves used in the respective studies may account for the differences in their observations (74, 88). Both toxins are large single-stranded proteins, and recent X-ray crystallography and small angle X-ray scattering models (SAXS) of TcdB suggest four structural domains (5). These domains include (a) a biologically active N-terminal glucosyltransferase protruding from the core of the protein; (b) a cysteine protease domain; (c) a middle translocation section
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that contains a hydrophobic region implicated in toxin delivery; and (d ) a C-terminal receptorbinding domain (5). Toxin activity is located in the N-terminal domain, and the rst 543 amino acids (aa) are sufcient to elicit full glucosyltransferase activity. This portion is delivered into the cytosol of host cells (68, 119). Cleavage of the biologically active 543-aa segment occurs between Leu543 and Gly544 in a highly conserved region (115). Separation occurs by autoproteolysis via the cysteine protease domain (5, 58, 68, 111, 115) and is dependent on host cell inositolphosphate (111). The C-terminal domain has short combined repetitive oligopeptides (CROPs) for receptor binding. In animal models of TcdA, carbohydrate structures play a role in toxin binding (5, 116, 138). These carbohydrates are not present in humans and the glycoprotein gp96 present in the human colon is the receptor for TcdA (116). The central translocation domain is large, taking up approximately 50% of the total size of the holotoxin. There is a hydrophobic stretch on the protein that likely facilitates membrane penetration during translocation (58). An understanding of the genetics, structure, and function of each of these domains has led to the following model of toxinogenesis. Toxins enter the host cells by receptor-mediated endocytosis, and they require an acidied endosome for translocation (36, 58, 111, 119, 138). Acidication induces a conformational change in both toxins, exposing the hydrophobic regions of the translocation domain and leading to subsequent pore formation through which the uncleaved catalytic domain, followed by the cysteine protease domain, translocates through the endosomal membrane into the cytosol (12, 58, 108, 111, 115, 119). Host cell inositol hexaphosphate induces autocleavage of the toxin by a C. difcile aspartate protease, resulting in biologically active toxin (3, 58, 111, 116). Once inside the cell, the toxins target the Ras superfamily of small GTPases (Rho, Rac, and Cdc) (42), modifying them through glycosylation (138). Glycosylation prevents the structural changes required for active
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conformation of Rho GTPases, signicantly altering the function of these proteins. Cells shrink and become rounded owing to disaggregation of the actin cytoskeleton, eventually dying (119, 138). Tight junctions between epithelial cells are disrupted. This allows neutrophils to migrate to the intestines, contributing to the inammatory response, typical of colitis (138). Biological inactivation of the GTPases results in serious physiological consequences such as inhibition of secretion, transcriptional regulation, and eventual apoptosis (119, 138). In addition to direct cytotoxic effects, TcdA stimulates release of tumor necrosis factor from activated macrophages as well as cytokine production (138). These activities cause uid accumulation and further the inammatory responses (138).
C. difcile Transferase
C. difcile transferase (CDTa), also known as binary toxin (120, 130), is encoded by the Cdt locus (CdtLoc). It is found in approximately 6% 12.5% of strains overall. Strains without binary toxin CDT have a conserved 68-bp sequence in place of the CdtLoc (36). CDT is an ADPribosylating toxin that disrupts the cytoskeleton of the cell, leading to excessive uid loss, rounding of the cell, and eventual cell death (130). CDT is composed of two subunits, CDTa and CDTb. Each component alone is not cytotoxic, together they cause cytotoxicity in vitro (57, 130). C. difcile CDT is similar to the binary toxins of other clostridia. The binding site for CDTa in the cell is unknown and its role in pathogenesis is unclear. The fact that the incidence of CDT is higher in some of the epidemic strains suggests that it contributes to the severity of disease. This is evidenced in the work by Geric et al. (57). The investigators challenged hamsters and inoculated rabbit ileal loops with a TcdA, TcdB, CDT+ strain. Marked nonhemorrhagic uid responses were seen in the rabbit ileal loops. Of the hamsters, 70%80% became colonized, but showed no signs of disease and no histological changes, and they did not die, compared with animals
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inoculated with TcdA+, TcdB+, CDT strains that did die (57). New ndings by Schwan et al. (120) reveal that CDT induces the formation of novel thin, dynamic, microtubules on the surface of epithelial cells, leading to increased adherence of Clostridia in vitro and in vivo. Electron microscopy showed that these protrusions increased the adherence of Clostridia to the epithelial cell surface by approximately vefold in vitro and fourfold in the mouse large intestine, thereby possibly playing an important role in intestinal colonization (120).
Other Virulence Factors

Although TcdA and TcdB are the major virulence mechanisms in C. difcile disease, other factors are likely important to disease. C. difcile has a crystalline or paracrystalline S-layer consisting of two proteins on the outer cell surface that coats the entire surface of the vegetative organism (26, 45, 50). These proteins are integral to adherence and stimulate inammation and induce antibody responses in the host (26, 45, 50, 116). Several other C. difcile proteins play a role in attachment to intestinal epithelial cells. Ampicillin and clindamycin stimulate the increased expression of these surface adhesins (36).
Toxin Variant Strains

Many strains of C. difcile have alterations in the sequences of one or more genes in the PaLoc compared with the sequences of the reference strain VPI 10463. Rupnik et al. (114) developed a polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) method for toxinotyping C. difcile based on mutations in these genes (114). Currently there are approximately 28 known toxinotypes (46). Despite the known role of TcdA in disease, the number of strains lacking TcdA recovered from patients with clinical disease has increased. These strains have TcdB but contain deletions in tcdA, resulting in a truncated form of the toxin. The rst A, B+ strain to be characterized was strain 8864 (toxinotype X) (46, 138).
It contained a 5.9-kb deletion in the 3 end of tcdA as well as an insertion in tcdE (138). This strain, which caused tissue damage in rabbit ileal loops, is uncommon in humans (46). Most TcdA strains have a 1.8-kb deletion in the 3 CROP region of tcdA, the region responsible for attachment to host cell receptors (see The Pathogenicity Locus, above). The absence of this region also prevents detection by diagnostic assays that use antibodies directed against this area of the toxin. These isolates have been rare causes of disease in the United States but constitute anywhere from 3% of all isolates in the United Kingdom and France to as high as 39% in Japan (46). The A, B+ strains described to date belong to toxinotypes VIII, X, XVI, and XVII. The last two also have CDT. Of these, toxinotype VIII seems to be most clinically signicant (46). Severe disease has been reported with these strains (ribotype 017) as has emergence of resistance to both the macrolidelincosamide-streptogramin antibiotics and uoroquinolones (36, 46). In addition to changes in tcdA and tcdB, changes in the other genes of the PaLoc may also alter virulence. In 2003, outbreaks of severe disease in the United States and Canada were caused by a clone of C. difcile designated North American pulsed-eld type 1 (NAP1), toxinotype III, ribotype 027, and restriction endonuclease analysis (REA) type B1 (84, 91). These isolates possessed binary toxin CDT and had an 18-bp deletion in the PaLoc tcdC (91). Quinolone resistance was also an important feature of the clone. Since the original description of the increased and widespread dissemination of this clone, several investigations into the genotypic and phenotypic characteristics of these isolates have been published (118, 126, 127, 139). In brief, Warny et al. (139) showed that 027 strains produced toxins A and B faster and in greater quantities than control (non-027) strains. In addition, others have shown that the duration of toxin production by 027 strains markedly exceeds that of other clones (118) and that these effects may be related partly to stimulation of germination and toxin production by quinolones (118). Toxin
B from both historical and modern 027 strains is more potent in vitro against multiple cell lines than toxinotype 0 is (127). Spigaglia et al. (126) studied modications to the accessory genes of the PaLoc. They detected 39- and 18-bp deletions in tcdC and they showed that nonsense mutations in tcdC reduced the TcdC protein from 232 to 61 aa (126). The 18-bp deletion has no effect on toxin production (48, 90). Rather, there is a correlation between truncation of TcdC, due to the single base-pair deletion at position 117 that results in the formation of a stop codon, and increased toxin production due to derepression of the PaLoc (34, 90, 126, 127). At least one study failed to demonstrate a consistent correlation between a truncated tcdC alone and increased toxin activity, and the authors caution against using mutations to diagnose patients as having more virulent strains and using this information to determine treatment (95). Aside from having altered TcdC, 027 strains have ve unique genetic regions not present in historical 027 strains (127). These genes include mutations that explain not only enhanced toxicity as described above, but motility, survival, increased sporulation (2), and antibiotic resistance (127). These factors combined may explain the increased severity of disease and mortality associated with these hypervirulent clones. In addition to emergence of 027 and the A, B+ 017 clone, strains of two other hypervirulent clones have recently been recognized. PCR ribotypes 053 and 078 (toxinotypes V) cause severe disease in humans. Strain 078 is interesting for a number of reasons. It causes disease in both animals, particularly calves and pigs, and humans. Studies to date have shown a high degree of genetic relatedness in the animal and human strains (38, 60). Both 027 and 078 strains have increased in frequency in the Netherlands, and clinical illness caused by both strains has similar presentations.
that ensure its survival in the gastrointestinal tract of humans and animals once introduced. Physiological factors that allow colonization include a disturbed or absent microbiota, an important barrier. The HMA model showed that amoxicillinclavulanic acid treatment of mice did not modify the total numbers of microbes but rather modied the type of microbiota. The authors showed that the BacteroidesPorphyromonas-Prevotella group increased and the Clostridium coccoidesEubacterium rectale group decreased dramatically (11). Others have assessed the impact of uoroquinolone treatment on gut microbiota, which decreases the quantities of enterococci and lactobacilli, Bacteroides spp., and bidobacteria (118). In this setting C. difcile endogenous or exogenous spores germinate and vegetative cells multiply. The organism adheres to the mucus layer by means of its multiple adhesins and penetrates the mucus with aid of agella and proteases. Once it penetrates mucus, the organism adheres to enterocytes and the rst phase of pathogenesis, namely colonization, begins. Putative virulence factors that promote colonization include the proteolytic enzyme cysteine protease Cwp84, adhesins such as S-layer proteins, a 66kDa cell wall protein Cwp66, the GroEL heat shock protein, a 68-kDa bronectin-binding protein, and the agella components FliC (agellin) and FliD (agellar cap protein) (40). Genes encoding these proteins are in close proximity on the genome. The second phase of the pathogenic process is toxin production (see The Pathogenicity Locus, above). As described above, alterations in genes that encode all these virulence factors, and not just those encoded in the PaLoc, are important for and explain strain variation in C. difcile disease.
EPIDEMIOLOGY People and Patients as Reservoirs

C. difcile is widely distributed in the environment and is often detected in the colon without pathologic consequences. C. difcile infection (CDI) is restricted to patients with C. difcile
Pathophysiology
As described above, the C. difcile genome enables the organism to express a variety of factors
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Table 1 Rates of recovery of C. difcile and frequency of positive cytotoxicity tests in culture positive specimensa Population tested Patients with pseudomembranous colitis Patients with antibiotic-associated diarrhea Antibiotic treatment without diarrhea Hospitalized patients Healthy adults Healthy neonates
a
Positive stool cultures 99%100% 15%25% 10%20% 10%25% 2%3% 5%70%
Positive toxin test 96% 10%20% <0%5% 2%8% <0.1% 5%63%
Adapted from Reference 13.
replication and toxin production. Rates of colonization and positive stool toxin tests based on multiple reports are summarized in Table 1 (13, 39, 53, 136). C. difcile has been detected in stool from 10% to 40% of most animal species, including cattle, horses, camels, donkeys, snakes, seals, dogs, and cats (82, 141). It is not clear that it causes disease in most of these animals except in antibiotic-treated horses (21) and rodent models. The organism has also been reported from diverse environmental sources; one report based on 2,580 samples showed positive cultures in 184 samples (7%) including 47% of samples from river water, 21% of soil samples, and 2% of home environment samples (4). Foodborne C. difcile infection. Foodborne CDI is an unproven but biologically plausible mechanism of CDI based on detection of C. difcile in an aggregate total of 90 of 592 (15%) retail meat supplies from diverse animal sources and geographic locations in the United States and Canada (61, 117, 140); the dominant strains in these reports are ribotypes 027 and 078.
Risks for C. difcile Infection

Major risks for CDI are antibiotic exposure, advanced age, and exposure to acute or chronic care facilities. Antibiotic treatment is always the leading risk for CDI in people and animals. The initial studies focused on clindamycin (132), which was highest in incidence, and cephalosporins, which were highest in
prevalence. In a review of 503 cases in Sweden from 1980 to 1982, the relative risk for these agents compared with other antibiotics was 10 to 70 times higher (6). The uoroquinolones emerged as major inducing agents and were implicated in several outbreaks that could be controlled only by restraining or prohibiting use of the entire class (55, 69). The prominent role of this class since 2000 presumably reects high use rates combined with the emergence of resistance by ribotype 027 (29, 55, 91). Advanced age is a risk, with most reports showing sharp increases in incidence in persons over 65 years and a direct correlation with age above that threshold (6, 102). A possible contributing factor is immunosenescence, based on reports showing humoral response to toxins A and/or B determines clinical expression (77) and protection afforded by monoclonal antibodies to toxins A and B (86). It is unclear that defective cell-mediated immunity is an important risk factor. Another important risk is contact with the healthcare system, which is heavily contaminated by C. difcile, especially case-associated areas (72). The potential sources of C. difcile acquisition include surface contamination, hospital personnel, patient cases and asymptomatic carriers, and hospital air (22, 31, 44, 52, 72). This risk is well veried by reports of C. difcile acquisition and CDI in hospitals and chronic care facilities. A prospective study of 428 admissions to Harborview Hospital in Seattle showed 6 (1.5%) patients were colonized on admission from home and 83 (21%) patients became colonized during their hospital stay (92).
Risks associated with C. difcile acquisition and infection include age over 65 years, ICU (intensive care unit) admission, antibiotic treatment, length of hospital stay, gastrointestinal procedures, and hospitalization in case-associated areas (10). There is increasing evidence that proton pump inhibitors also promote CDI (41). A systematic review of the literature from 1988 to 2005, based on 12 relevant publications and 2,948 patients with CDI, showed a relative risk of 1.94 (95% CI 1.42.8) for proton pump inhibitors (81, 83).
Changing Epidemiology
Since 2000, there has been a dramatic increase in rates and severity of CDI noted in North America and much of Europe. The rst reports were from Sherbrooke in Quebec, Canada, in 2003 showing a fourfold increase in CDI rates, from 35.6 to 156.3/100,000. Subsequent reports indicated this experience affected multiple hospitals in Quebec and included reviews showing the following: an attributable mortality of 6.9%, uoroquinolones as the dominant inducing agents, 10-fold-higher rates in persons over 65 years, and the hypervirulent ribotype 027 as the cause of the epidemic (84, 85, 103). In the United States an analysis by ICD-9 discharge data showed that the CDI rate doubled, the case mortality increased 80%, and the rate of colectomies for CDI tripled in 2003 compared with 1993 (47, 110, 113, 143). In addition, the U.S. experience suggested increases in selected patient populations including pediatric patients, persons without recent antibiotic exposure, community-acquired cases, and severe cases associated with pregnancy. European data also showed increased CDI rates and multiple hospital-associated outbreaks (52).
(<0.02%) typed strains collected from U.S. cases from 1984 to 1993, but was implicated in 96 of 187 (51%) of strains tested in eight U.S. outbreaks from 2000 to 2003 (91, 100). This strain has been associated with large outbreaks of severe CDI in hospitals in the United States, Canada, and Europe (75, 84, 96, 100). The factor thought to contribute to incidence is the strains resistance to uoroquinolones, and enhanced virulence may reect increased toxin production in vitro (139). In 2008, the European Center for Disease Control established a surveillance system to dene CDI incidence and implicated strains in 34 European countries. This showed a mean incidence of 5.5 cases per 10,000 patient days, and ribotype 027 accounted for just 5% of all cases (52). Unfortunately, there is no natural surveillance system in the United States, but a large clinical trial of a new therapeutic agent in 20052007 showed that ribotype 027 accounted for 36% of U.S. strains and 8% of European strains (56). The data reviewed provide strong support for the role of ribotype 027 in some devastating outbreaks, the important new role of uoroquinolones for driving incidence, and the critical role of uoroquinolone restriction in controlling some outbreaks (69, 75). However, this conclusion may be overly simplied because ribotype 027 accounted for only 5% of 89 typed strains in the 2008 European surveillance study (20); a case control study found no evidence to support assertions that ribotype 027 is more virulent than other PCR ribotypes (93). Some institutions report that beta-lactams are still much more important inducing agents than uoroquinolones (20) and that ribotype 078 has played an important part in some outbreaks (75).
DIAGNOSIS Emergence of Ribotype 027

The increasing incidence and severity of CDI in North America and much of Europe has been attributed to the emergence of ribotype 027. This strain accounted for only 14 of over 6,000
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Guidelines for diagnosis and treatment of C. difcile disease have been developed by the Society for Healthcare Epidemiology of America and the Infectious Diseases Society of America (SHEA/IDSA) in the United States
Bartlett
Table 2 Performance characteristics of various test methods Performance characteristics Methods/assays Toxigenic anaerobic culture Enzyme immunoassaysa Cell culture neutralization assaysb Glutamate dehydrogenasec Nucleic acid amplication testsd BD-GeneOhmTM Prodesse ProGastroTM GeneXpertTM illumigeneTM 8496 7792 94100 99 94100 9599 9399 98 Sensitivity (%, range) N/A 3199 6786 71100 Specicity (%, range) N/A 84100 97100 7698
Derived from References 33, 49, and 107 and includes both solid-phase and membrane assays combined. Data are not stratied by comparative method. b Compiled from References 9, 49, 105, and 129. c Compiled from References 33, 51, 124, 135, and 142. d Compiled from References 8, 9, 49, 59, 67, 70, 73, 76, 98, 99, 128, 129, 131, and 134.
and the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) (31, 33). Both groups recommend that testing be performed only on diarrheal (unformed) stool except in rare instances (31, 33). Asymptomatic patients should not be tested even for test of cure. Table 2 lists the variety of test methods available for the diagnosis of CDI and a summary of their performances based on published studies. Cell culture cytotoxicity neutralization assays (CCCNAs) are performed by inoculating a stool ltrate onto a monolayer of a particular cell line (e.g., human foreskin broblasts) and then observing for toxin-induced cytopathic effect (CPE). Once CPE is observed, neutralization with an antiserum, either C. sordellii antitoxin or C. difcile antitoxin, is performed. For many years, CCCNAs were considered the diagnostic gold standard. However, their performance has recently been re-evaluated in comparisons with toxigenic culture and nucleic acid amplication tests (NAATs) (9, 49, 105, 128) (Table 2). In general, CCCNAs are too insensitive (<90%) to be considered acceptable reference methods (31). Enzyme immunoassays (EIAs), the method most frequently used in U.S. laboratories, use
polyclonal or monoclonal antibodies targeting TcdA alone or both TcdA and TcdB. Because of the emergence of virulent TcdA, TcdB+ isolates, clinical laboratories should use a test that detects both toxins. About two dozen solid-phase, well-type, and rapid membrane assays are available. Several recent comprehensive reviews evaluating the performance of these assays have been published (33, 49, 107). Planche et al. (107) performed a comprehensive review of 28 studies assessing six Toxin A/B assays commonly used in the United Kingdom. The authors noted signicant heterogeneity among the assays, but the diagnostic odds ratio implied no difference in performance among them. Most of the assays had higher specicities than sensitivities, but none of the assays met the criteria for an acceptable test, dened as having a sensitivity of 90% and a false-positive rate of 3% (107). Eastwood et al. (49) evaluated six EIAs and three lateral ow assays on the same set of 600 diarrheal samples. Sensitivities ranged from 60% to 81% and specicities from 91% to 99.4% compared with toxigenic culture (49). None of the assays met the criteria as dened by Planche et al. (49). Because EIAs lack sensitivity, it has become common practice to perform stools for
C. difcile x 3. However, recent reports demonstrate the lack of utility of repeat testing regardless of the method used (1, 24, 27, 87, 97, 106). Given the poor sensitivity and positive predictive value in low-prevalence populations, both practice guidelines consider EIAs suboptimal for the diagnosis of C. difcile infections (31, 33). Glutamate dehydrogenase (GDH) is a metabolic enzyme expressed at high levels by all strains of C. difcile. GDH is present in both toxigenic and nontoxigenic strains, so a positive GDH test must be combined with an assay that also detects toxin. The C. DIFF CHEKTM -60 solid-phase microtiter plate GDH assay (TechLab, Blacksburg, Virginia) demonstrated high negative predictive values (98.5% to 99.7%) in several studies (51, 124, 135, 142). Therefore, many laboratories have adopted a two-step algorithm in which the GDH is performed rst, and if negative, no further testing is required. If the GDH test is positive, a toxin test should follow. If the GDH is positive but the toxin test is negative, either the patient is colonized with nontoxigenic C. difcile or (depending upon the assay) the toxin test is falsely negative. If both the GDH and the toxin test are positive, then the symptomatic patient likely has CDI. Although some investigators have recently noted suboptimal performance of the GDH assay compared with culture and/or molecular methods (76, 99, 133), a recent meta-analysis reports that GDH has a greater than 90% sensitivity with a negligible false-positive rate when compared with culture (123). This two-step method is the approach that is currently recommended by both ESCMID and SHEA/IDSA (31, 33). A newer immunochromatogenic membrane version of the test combines both GDH and toxin testing into a single device (C. DIFF Quik Chek CompleteTM , TechLab) and takes about 30 min to perform. In two published studies when this device was compared to toxigenic culture, the sensitivity of the GDH component was 100% (109, 131). However, the toxin component performed poorly; its sensitivity ranging from 61% to 78% (109, 131). Given the insensitivity of the toxin component of the
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assay, some laboratories are using the device in an expanded three-step approach, testing the GDH-positive, toxin-negative samples with another method such as a NAAT. The increase in the incidence of C. difcile infections, the emergence of hypervirulent strains causing more severe disease, and more recently, the need for a better method against which to assess evolving technologies, such as NAATs, have compelled laboratories to revisit anaerobic toxigenic culture. Toxigenic culture requires inoculating stool specimens to selective anaerobic media that contain substances inhibitory to normal stool microbiota while promoting the germination and growth of C. difcile. Both toxigenic and nontoxigenic isolates are recovered, so isolates must be tested for toxin production by EIA, CCCNA, or NAAT. Toxigenic culture performed after negative direct toxin testing by EIA or CCNA increases the yield of positives by 15% to 23% (25, 112). Although culture methods have not been standardized, there are some important caveats with respect to its performance. Spore enrichment signicantly enhances recovery and is useful when culturing is done for epidemiological purposes. There are two ways to enrich for spores: heat shock and treatment of the specimen with ethanol. The methods are detailed in References 28, 65, 78, and 89. A variety of media exist for culturing C. difcile. The original cycloserine, cefoxitin, fructose agar (CCFA), as described by George et al. (53) contained an egg yolk fructose agar base with 500 g ml1 of cycloserine and 16 g ml1 of cefoxitin. CCFA takes advantage of the ability of C. difcile to ferment fructose. CCFA variants with reduced concentrations of the antimicrobial agents are less sensitive than the formulation by George et al. (94, 104). Additives such as horse blood, taurocholate, and lysozyme improve recovery by enhancing vegetation (23; K. Carroll, personal observations). A recent study demonstrated improved recovery with CCF broth supplemented with 0.1% taurocholate compared with plating on solid agar (7). Whichever method is chosen, it is important to use prereduced media, as the failure to
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do so can affect the sensitivity of the culture method (104). Despite its sensitivity, toxigenic culture is not practical for use in most clinical laboratories. However, it is particularly useful in the setting of an outbreak, when evaluating a new test method, for surveillance of antimicrobial resistance, and for management of patients with recurrent/refractory disease.
Diarrheal stool specimen (N = 150)
C. DIFF Quik Chek CompleteTM
GDH positive (N = 22)
GDH negative (N = 114) C. difficile negative
Nucleic Acid Amplication Methods

In the United States there are currently four FDA-cleared NAATs available for diagnosis of CDI (Table 2): (a) the BD-GeneOhmTM Cdiff assay (BD, Franklin Lakes, New Jersey), (b) the Prodesse ProGastroTM Cd assay (Gen-Probe, Inc. San Diego, California), (c) the Cepheid GeneXpert R C difcile (Cepheid, Sunnyvale, California), and (d ) the illumigeneTM C. difcile assay (Meridian Diagnostics, Inc., Cincinnati, Ohio). The rst three assays target conserved regions of tcdB, and the last uses loop-mediated isothermal amplication to detect the PaLoc at a conserved region of tcdA that is present even in TcdA, TcdB+ strains. The assays vary considerably in terms of ease of use, instrumentation, and cost. The performance characteristics of these assays are well published in the literature and are highlighted in Table 2. In general, they are superior to all methods except toxigenic culture. To date, there is no published study that compares them all. An important observation came from the analysis of the data from the GeneXpert clinical trials. The authors noted that the performance of all the assays evaluated appeared to be affected by the ribotype of the strain causing the infection. Compared with toxigenic culture, the GDH assay and qPCR assay had comparable sensitivity for detection of ribotype 027 (91%) (133). However, for non-027 infections, the overall sensitivity for the GDH assay was 70% compared with 92% for the GeneXpert assay (133). These results were statistically significant (133). Likewise, the performance of two Toxin A/B EIAs revealed extensive variability, with sensitivities as low as 16% for some ribotypes (133). Although these observations can
Toxin positive (N = 11) Presumed CDI
Toxin negative (N = 11)
XpertTM C. difficile PCR
PCR positive (N = 8) CDI (N = 4) or toxigenic carriage (N = 4) Clinical assessment essential
PCR negative (N = 3) Nontoxigenic carriage (N = 2) or false-positive GDH (N = 1) No CDI
Figure 2 Proposed testing algorithm for detection of C. difcile in stool samples using the C. DIFF Quick Chek CompleteTM test followed by testing GDH-positive, EIA-negative specimens with a commercial real-time PCR method. The numbers in parentheses reect the numbers of specimens that required testing by each method in the authors study. Modied and reprinted with permission from Swindells J, et al. 2010. J. Clin. Microbiol. 48:6068 (131).
certainly explain the broad variability in test performance, especially for the EIA methods, it would be useful to see additional data from other sites and other platforms with respect to such observations. NAAT testing is rapidly replacing other methods in clinical microbiology laboratories, particularly in laboratories that already have the required instrumentation for testing. These new technologies have yet to be endorsed by professional society guidelines (31, 33). There are practical and theoretical concerns that, if claried, could reassure healthcare practitioners regarding their use. One of these concerns is whether genetic drift of tcdB or other gene targets in regions of primer/probe binding is likely to suddenly affect assay performance. As discussed above, there is a great deal of heterogeneity in the genome of various C. difcile
strains. However, the full and partial genome sequencing of several strains of C. difcile has elucidated regions that are conserved, including regions of genes on the PaLoc that have been targeted for these assays. At least one study showed performance variability based on ribotype (133). Information about the impact of NAATs on C. difcile transmission and their cost-effectiveness is also needed.
Given the costs of NAATs, some laboratories have explored using them on a limited basis in three-step algorithms as outlined in Figure 2. The algorithm begins with the C. DIFF Quik CHEK CompleteTM test, and NAAT testing is performed only on GDH-positive, EIA-negative specimens. Several studies have demonstrated the cost-effectiveness of such an approach (43, 79, 122).
SUMMARY POINTS
1. The complete genome of a clinical strain of C. difcile and partial genomes of toxin variant strains have been sequenced. 2. C. difcile causes disease by elaborating one or both large clostridial toxins encoded on a PaLoc with three additional genes that regulate toxin production. 3. Both toxins are large single-stranded proteins, and recent X-ray crystallography and small angle X-ray scattering models of TcdB suggest four structural domains are necessary for toxin entry and cytotoxicity. 4. Binary toxin, which is not encoded on the PaLoc, may contribute to disease severity by its cytotoxic activity and by induction of novel thin, dynamic microtubules on the surface of epithelial cells, leading to increased adherence of Clostridia. 5. Mutations in the PaLoc have resulted in the evolution of four clades of C. difcile inclusive of hypervirulent toxinotypes responsible for aggressive disease. 6. Risk factors such as quinolone antibiotics, proton pump inhibitors, and advanced age, in combination with more pathogenic strains, have created the perfect storm for recent epidemics. 7. The recognition of toxin variant strains in domestic animals and food sources may explain the onset of community disease. This needs further study. 8. Increased incidence and severity of disease has refocused attention on the inadequacy of EIAs and CCCNAs and has sparked development of diagnostic molecular assays.
DISCLOSURE STATEMENT
Dr. Karen C. Carroll has received research funding from BD Diagnostics (Sparks, MD) and Prodesse, now Gen-Probe, Inc.(San Diego, CA). Dr. John G. Bartlett has no relevant disclosures.
ACKNOWLEDGMENTS
The authors wish to thank Ms. Janet Landay for her secretarial assistance. LITERATURE CITED
1. Aichinger E, Schleck CD, Harmsen WS, Nyre LM, Patel R. 2008. Nonutility of repeat laboratory testing for detection of Clostridium difcile by use of PCR or enzyme immunoassay. J. Clin. Microbiol. 46:379597
514 Carroll
Bartlett
2. Akerlund T, Persson I, Unemo M, Noren T, Svenungsson B, et al. 2008. Increased sporulation rate of epidemic Clostridium difcile type 027/NAP1. J. Clin. Microbiol. 46:153033 3. Aktories K. 2007. Self-cutting to kill: new insights into the processing of Clostridium difcile toxins. ACS Chem. Biol. 2:22830 4. al Saif N, Brazier JS. 1996. The distribution of Clostridium difcile in the environment of South Wales. J. Med. Microbiol. 45:13337 5. Albesa-Jove D, Bertrand T, Carpenter EP, Swain GV, Lim J, et al. 2010. Four distinct structural domains in Clostridium difcile toxin B visualized using SAXS. J. Mol. Biol. 396:126070 6. Aronsson B, Mollby R, Nord CE. 1985. Antimicrobial agents and Clostridium difcile in acute enteric disease: epidemiological data from Sweden. J. Infect. Dis. 151:47681 7. Arroyo LG, Rousseau J, Willey BM, Low DE, Staempi H, et al. 2005. Use of a selective enrichment broth to recover Clostridium difcile from stool swabs stored under different conditions. J. Clin. Microbiol. 43: 534143 8. Babady NE, Stiles J, Ruggiero P, Khosa P, Huang D, et al. 2010. Evaluation of the Cepheid Xpert Clostridium difcile EPI assay for diagnosis of Clostridium difcile infection and typing of the NAP1 strain at a cancer hospital. J. Clin. Microbiol. 48:451924 9. Barbut F, Braun M, Burghoffer B, Lalande V, Eckert C. 2009. Rapid detection of toxigenic strains of Clostridium difcile in diarrheal stools by real-time PCR. J. Clin. Microbiol. 47:127677 10. Barbut F, Petit JC. 2001. Epidemiology of Clostridium difcileassociated infections. Clin. Microbiol. Infect. 7:40510 11. Barc MC, Bourlioux F, Janoir C, Charrin-Sarnel C, Janoir C, et al. 2004. Effect of amoxicillin-clavulanate on human fecal ora in a gnotobiotic mouse model measured with group specic 16SrRNA targeted oligonucleotide probes in combination with ow cytometry. Antimicrob. Agents Chemother. 48:136568 12. Barth H, Pfeifer G, Hofmann F, Maier E, Benz R, Aktories K. 2001. Low pH-induced formation of ion channels by Clostridium difcile toxin B in target cells. J. Biol. Chem. 276:1067076 13. Bartlett JG. 1994. Clostridium difcile: history of its role as an enteric pathogen and the current state of knowledge about the organism. Clin. Infect. Dis. 18(Suppl. 4):S26572 14. Bartlett JG, Chang TW, Gurwith M, Gorbach SL, Onderdonk AB. 1978. Antibiotic-associated pseudomembranous colitis due to toxin-producing clostridia. N. Engl. J. Med. 298:53134 15. Bartlett JG. 1980. Experimental studies of antibiotic associated colitis. Scand. J. Infect. Dis. (Suppl. 22):1115 16. Bartlett JG, Chang TW, Onderdonk AB. 1978. Will the real Clostridium species responsible for antibioticassociated colitis please step forward? Lancet 1:338 17. Bartlett JG, Moon N, Chang TW, Taylor N, Onderdonk AB. 1978. Role of Clostridium difcile in antibiotic-associated pseudomembranous colitis. Gastroenterology 75:77882 18. Bartlett JG, Onderdonk AB, Cisneros RL. 1977. Clindamycin-associated colitis in hamsters: protection with vancomycin. Gastroenterology 73:77276 19. Bartlett JG, Onderdonk AB, Cisneros RL, Kasper DL. 1977. Clindamycin-associated colitis due to a toxin-producing species of Clostridium in hamsters. J. Infect. Dis. 136:7015 20. Bauer MP, Notermans DW, Van Benthem BH, Brazier JS, Wilcox MH, et al. 2011. Clostridium difcile infection in Europe: a hospital-based survey. Lancet 377:6373 21. B verud V. 2002. Clostridium difcile infections in animals with special reference to the horse. A review. a Vet. Q. 24:20319 22. Best EL, Fawley WN, Parnell P, Wilcox MH. 2010. The potential for airborne dispersal of Clostridium difcile from symptomatic patients. Clin. Infect. Dis. 50:145057 23. Bliss DZ, Johnson S, Clabots CR, Savik K, Gerding DN. 1997. Comparison of cycloserine-cefoxitinfructose agar (CCFA) and taurocholate-CCFA for recovery of Clostridium difcile during surveillance of hospitalized patients. Diagn. Microbiol. Infect. Dis. 29:14 24. Borek AP, Aird DZ, Carroll KC. 2005. Frequency of sample submission for optimal utilization of the cell culture cytotoxicity assay for detection of Clostridium difcile toxin. J. Clin. Microbiol. 43:299495 25. Bouza E, Pelaez, T, Alonso R, Catalan P, Munoz P, Creixems MR. 2001. Second-look cytotoxicity: an evaluation of culture plus cytotoxin assay of Clostridium difcile isolates in the laboratory diagnosis of CDAD. J. Hosp. Infect. 48:23337
31. An authoritative guideline on current concepts in testing, treatment, and infection control.
26. Calabi E, Ward S, Wren B, Paxton T, Panico M, et al. Molecular characterization of the surface layer proteins from Clostridium difcile. Mol. Microbiol. 40:118799 27. Cardona DM, Rand KH. 2008. Evaluation of repeat Clostridium difcile enzyme immunoassay testing. J. Clin. Microbiol. 46:368689 28. Clabots CR, Gerding SJ, Olson MM, Peterson LR, Gerding DN. 1989. Detection of asymptomatic Clostridium difcile carriage by an alcohol shock procedure. J. Clin. Microbiol. 27:238687 29. Clements AC, Magalh es RJ, Tatem AJ, Paterson DL, Riley TV. 2010. Clostridium difcile PCR ribotype a 027: assessing the risks of further worldwide spread. Lancet Infect. Dis. 10:395404 30. Cohen SH, Tang YJ, Silva J Jr. 2000. Analysis of the pathogenicity locus in Clostridium difcile strains. J. Infect. Dis. 181:65963 31. Cohen SH, Gerding DN, Johnson S, Kelly CP, Loo VG, et al. 2010. Clinical practice guidelines for Clostridium difcile infection in adults: 2010 update by the Society for Healthcare Epidemiology of America (SHEA) and the infectious diseases society of America. Infect. Control Hosp. Epidemiol. 31:43155 32. Collignon A. 2010. Methods for working with the mouse model. In Clostridium difcile: Methods in Molecular Biology, ed. P Mullany, AP Roberts, pp. 22937. New York: Springer. 646 pp. 33. Crobach MJT, Dekkers OM, Wilcox MH, Kuijper EJ. 2009. European Society of Clinical Microbiology and Infectious Diseases (ESCMID): data review and recommendations for diagnosing Clostridium difcileinfection (CDI). Clin. Microbiol. Infect. 15:105366 34. Curry SR, Marsh JW, Muto CA, OLeary MM, Pasculle AW, Harrison LH. 2007. tcdC genotypes associated with severe TcdC truncation in an epidemic clone and other strains of Clostridium difcile. J. Clin. Microbiol. 45:21521 35. Curry S. 2010. Clostridium difcile. Clin. Lab. Med. 30:32942 36. Dawson LF, Valiente E, Wren BW. 2009. Clostridium difcilea continually evolving and problematic pathogen. Infect. Genet. Evol. 9:141017 37. De Somer P, Van de Voorde H, Eyssen H, Van Dijck P. 1955. A study on penicillin toxicity in guinea pigs. Antibiot. Chemother. 5:46369 38. Debast SB, Vaessen N, Choudry A, Wiegeers-Ligtvoet EA, van den Berg RJ, et al. 2009. Successful combat of an outbreak due to Clostridium difcile PCR ribotype 027 and recognition of specic risk factors. Clin. Microbiol. Infect. 15:42734 39. Delm e M, Verellen G, Avesani V, Francois G. 1988. Clostridium difcile in neonates: serogrouping and e epidemiology. Eur. J. Pediatr. 147:3640 40. Deneve C, Janoir C, Poilane I, Fantinato C, Collignon A. 2009. New trends in Clostridium difcile virulence and pathogenesis. Intern. J. Antimicrob. Agents 33:S2428 41. Dial S, Delaney JA, Barkun AN, Suissa S. 2005. Use of gastric acid-suppressive agents and risk of community-acquired Clostridium difcileassociated disease. JAMA 294:298995 42. Dineen SS, Villapakkam AC, Nordman JT, Sonenshein AL. 2007. Repression of Clostridium difcile toxin gene expression by CodY. Mol. Microbiol. 66:20619 43. Doing KM, Hintz MS, Keefe C, Horne S, LeVasseur S, Kulikowski ML. 2010. Reevaluation of the Premier Clostridium difcile toxin A and B immunoassay with comparison to glutamate dehydrogenase common antigen testing evaluating Bartels cytotoxin and Prodesse ProGastro Cd polymerase chain reaction as conrmatory procedures. Diagn. Microbiol. Infect. Dis. 66:12934 44. Donskey CJ. 2010. Preventing transmission of Clostridium difcile: Is the answer blowing in the wind? Clin. Infect. Dis. 50:145861 45. Drudy D, Calabi E, Kyne L, Sougioultzis S, Kelly E, et al. 2004. Human antibody response to surface layer proteins in Clostridium difcile infection. FEMS Immunol. Med. Microbiol. 41:23742 46. Drudy D, Fanning S, Kyne L. 2007. Toxin A-negative, toxin B-positive Clostridium difcile. Int. J. Infect. Dis. 11:510 47. Dubberke ER, Butler AM, Yokoe DS, Mayer J, Hota B, et al. 2010. Multicenter study of Clostridium difcile infection rates from 2000 to 2006. Infect. Control Hosp. Epidemiol. 31:103037 48. Dupuy B, Govind R, Antunes A, Matamouros S. 2008. Clostridium difcile toxin synthesis is negatively regulated by TcdC. J. Med. Microbiol. 57:68589
Carroll
516
Bartlett
49. Eastwood K, Else P, Charlett A, Wilcox M. 2009. Comparison of nine commercially available Clostridium difcile toxin detection assays, a real-time PCR assay for C. difcile tcdB, and a glutamate dehydrogenase detection assay to cytotoxin testing and cytotoxigenic culture methods. J. Clin. Microbiol. 47:321117 50. Fagan RP, Albesa-Jov D, Qazi O, Svergun DI, Brown KA, Fairweather NF. 2009. Structural insights e into the molecular organization of the S-layer from Clostridium difcile. Mol. Microbiol. 71:130822 51. Fenner L, Widmer AF, Goy G, Rudin S, Frei R. 2008. Rapid and reliable diagnostic algorithm for detection of Clostridium difcile. J. Clin. Microbiol. 46:32830 52. Freeman J, Bauer MP, Baines SD, Corver J, Fawley WN, et al. 2010. The changing epidemiology of Clostridium difcile infections. Clin. Microbiol. Rev. 23:52949 53. George WL, Sutter VL, Citron D, Finegold SM. 1979. Selective and differential medium for isolation of Clostridium difcile. J. Clin. Microbiol. 9:21419 54. George RH, Symonds JM, Dimock F, Brown JD, Arabi Y, et al. 1978. Identication of Clostridium difcile as a cause of pseudomembranous colitis. Br. Med J. 1:695 55. Gerding DN. 2004. Clindamycin, cephalosporins, uoroquinolones, and Clostridium difcileassociated diarrhea: This is an antimicrobial resistance problem. Clin. Infect. Dis. 38:64648 56. Gerding DN. 2010. Global epidemiology of Clostridium difcile infection in 2010. Infect. Control Hosp. Epidemiol. 31(Suppl. 1):S3234 57. Geric B, Carman RJ, Rupnik M, Genheimer CW, Sambol SP, et al. 2006. Binary toxin-producing large clostridial toxin-negative Clostridium difcile strains are enterotoxic but do not cause disease in hamsters. J. Infect. Dis. 193:114350 58. Giesemann T, Egerer M, Jank T, Aktories K. 2008. Processing of Clostridium difcile toxins. J. Med. Microbiol. 57:69096 59. Goldenberg SD, Dieringer T, French GL. 2010. Detection of toxigenic Clostridium difcile in diarrheal stools by real-time polymerase chain reaction. Diagn. Microbiol. Infect. Dis. 67:3047 60. Goorhuis A, Bakker D, Corver J, Debast, Harmanus C, et al. 2008. Emergence of Clostridium difcile infection due to a new hypervirulent strain, polymerase chain reaction ribotype 078. Clin. Infect. Dis. 47:116270 61. Gould LH, Limbago B. 2010. Clostridium difcile in food and domestic animals: a new foodborne pathogen? Clin. Infect. Dis. 51:57782 62. Green RH. 1974. The association of viral activation with penicillin toxicity in guinea pigs and hamsters. Yale J. Biol. Med. 47:16681 63. Hall IC, OToole E. 1935. Intestinal ora in newborn infants: with a description of a new pathogenic anaerobe, Bacillus difcilis. Am. J. Dis. Child. 49:390 64. Hambre DM, Rake G, McKee CM, MacPhillamy HB. 1943. The toxicity of penicillin as prepared for clinical use. Am. J. Med. Sci. 206:642 65. Hanff PA, Zaleznik DF, Kent KC, Rubin MS, Kelly E, et al. 1993. Use of heat shock for culturing Clostridium difcile from rectal swabs. Clin. Infect. Dis. 16(Suppl. 14):S24547 66. Heap JT, Pennington OJ, Cartman ST, Carter GP, Minton NP. 2007. The ClosTron: a universal gene knock-out system for the genus Clostridium. J. Microbiol. Methods 70:45264 67. Huang H, Weintraub A, Fang H, Nord CE. 2009. Comparison of a commercial multiplex real-time PCR to the cell cytotoxicity neutralization assay for diagnosis of Clostridium difcile infections. J. Clin. Microbiol. 47:372931 68. Jank T, Aktories K. 2008. Structure and mode of action of clostridial glucosylating toxins: the ABCD model. Trends Microbiol. 16:22229 69. Kallen AJ, Thompson A, Ristaino P, Chapman L, Nicholson A, et al. 2009. Complete restriction of uoroquinolone use to control an outbreak of Clostridium difcile infection at a community hospital. Infect. Control Hosp. Epidemiol. 30:26472 70. Karre T, Sloan L, Patel R, Mandrekar J, Rosenblatt J. 2011. Comparison of two commercial molecular assays to a laboratory-developed molecular assay for diagnosis of Clostridium difcile infection. J. Clin. Microbiol. 49:72527 71. Khan MY, Hall WH. 1966. Staphylococcal enterocolitis: treatment with oral vancomycin. Ann. Intern. Med. 65:18
52. Provides a comprehensive review of the epidemiology of C. difcile infections including devastating outbreaks in hospitals, regional trends in rates, and distribution of ribotypes.
517
72. Kim KH, Fekety R, Batts DH, Brown D, Cudmore M, et al. 1981. Isolation of Clostridium difcile from the environment and contacts of patients with antibiotic-associated colitis. J. Infect. Dis. 143:4250 73. Knetsch CW, Bakker D, de Boer RF, Sanders I, Hofs S, et al. 2011. Comparison of real-time PCR techniques to cytogenic culture methods for diagnosing Clostridium difcile infection. J. Clin. Microbiol. 49:22731 74. Kuehne SA, Cartman ST, Heap JT, Kelly ML, Cockayne A, Minton NP. 2009. The role of toxin A and toxin B in Clostridium difcile infection. Nature 467:71114 75. Kuijper EJ, van den Berg RJ, Debast S, Visser CE, Veenendaal P, et al. 2006. Clostridium difcile ribotype 027, toxinotype III, The Netherlands. Emerg. Infect. Dis. 12:82730 76. Kvach EJ, Ferguson D, Riska PF, Landry ML. 2010. Comparison of BD GeneOhm Cdiff real-time PCR assay with a two-step algorithm and a toxin A/B enzyme-linked immunosorbent assay for diagnosis of toxigenic Clostridium difcile infection. J. Clin. Microbiol. 48:10914 77. Kyne L. 2010. Clostridium difcilebeyond antibiotics. N. Engl. J. Med. 362:26465 78. Lahn M, Tyler G, Daubener W, Hadding U. 1993. Improvement of Clostridium difcile isolation by heat-shock and typing of the isolated strains by SDS-PAGE. Eur. J. Epidemiol. 9:32734 79. Larson AM, Fung AM, Fang FC. 2010. Evaluation of tcdB real-time PCR in a three-step diagnostic algorithm for detection of toxigenic Clostridium difcile. J. Clin. Microbiol. 48:12430 80. Lawley TD, Croucher NJ, Yu L, Clare S, Sebaihia M, et al. 2009. Proteomic and genomic characterization of highly infectious Clostridium difcile 630 spores. J. Bacteriol. 191:537786 81. Leonard J, Marshall JK, Moayyedi P. 2007. Systematic review of the risk of enteric infection in patients taking acid suppression. Am. J. Gastroenterol. 102:204756 82. Levett PN. 1986. Clostridium difcile in habitats other than the human gastrointestinal tract. J. Infect. 12:25363 83. Linsky A, Gupta K, Lawler EV, Fonda JR, Hermos JA. 2010. Proton pump inhibitors and risk for recurrent Clostridium difcile infection. Arch. Intern. Med. 170:77278 84. Loo VG, Poirier L, Miller MA, Oughton M, Libman MD, et al. 2005. A predominantly clonal multiinstitutional outbreak of Clostridium difcileassociated diarrhea with high morbidity and mortality. N. Engl. J. Med. 353:244249 85. Loo VG, Libman MD, Miller MA, Bourgault AM, Frenette CH, et al. 2004. Clostridium difcile: a formidable foe. Can. Med. Assoc. J. 171:4748 86. Lowy I, Molrine DC, Leav BA, Blair BM, Baxter R, et al. 2010. Treatment with monoclonal antibodies against Clostridium difcile toxins. N. Engl. J. Med. 362:197205 87. Luo RF, Banaei N. 2010. Is repeat PCR needed for diagnosis of Clostridium difcile infection? J. Clin. Microbiol. 48:373841 88. Lyras D, OConnor JR, Howarth PM, Sambol SP, Carter GP, et al. 2009. Toxin B is essential for virulence of Clostridium difcile. Nature 458:117679 89. Marler LM, Siders JA, Wolters LC, Pettigrew Y, Skitt BL, Allen SD. 1992. Comparison of ve cultural procedures for isolation of Clostridium difcile from stools. J. Clin. Microbiol. 30:51416 90. Matamouros S, England P, Dupuy B. 2007. Clostridium difcile toxin expression is inhibited by the novel regulator TcdC. Mol. Microbiol. 64:127488 91. McDonald LC, Kilgore GE, Thompson A, Owens RC Jr, Kazakova SV, et al. 2005. An epidemic, toxin gene-variant strain of Clostridium difcile. N. Engl. J. Med. 353:243341 92. McFarland LV, Mulligan ME, Kwok RY, Stamm WE. 1989. Nosocomial acquisition of Clostridium difcile infection. N. Engl. J. Med. 320:20410 93. Morgan OW, Rodrigues B, Elston T, Verlander NQ, Brown DF, et al. 2008. Clinical severity of Clostridium difcile PCR ribotype 027: a case-case study. PLoS One 3:e1812 94. Mundy LS, Shanholtzer CJ, Willard KE, Gerding DN, Peterson LR. 1995. Laboratory detection of Clostridium difcile. A comparison of media and incubation systems. Am. J. Clin. Pathol. 103:5256 95. Murray R, Boyd D, Levett PN, Mulvey MR, Alfa MJ. 2009. Truncation in the tcdC region of Clostridium difcile PathLoc of clinical isolates does not predict increased biological activity of Toxin B or Toxin A. BMC Infect. Dis. 9:103
518 Carroll
Bartlett
96. Muto CA, Pokrywka M, Shutt K, Mendelsohn AB, Nouri K, et al. 2005. A large outbreak of Clostridium difcileassociated disease with an unexpected proportion of deaths and colectomies at a teaching hospital following increased uoroquinolone use. Infect. Control Hosp. Epidemiol. 26:27380 97. Nemat H, Khan R, Ashraf MS, Matta M, Ahmed S, et al. 2009. Diagnostic value of repeated enzyme immunoassays in Clostridium difcile infection. Am. J. Gastroenterol. 104:203541 98. Nor n T, Alriksson I, Andersson J, Akerlund T, Unemo M. 2011. Rapid and sensitive loop-mediated e isothermal amplication test for Clostridium difcile detection challenges cytotoxin B cell test and culture as gold standard. J. Clin. Microbiol. 49:71011 99. Novak-Weekley SM, Marlowe EM, Miller JM, Cumpio J, Nomura JH, et al. 2010. Clostridium difcile testing in the clinical laboratory by use of multiple testing algorithms. J. Clin. Microbiol. 48:88993 100. OConnor JR, Johnson S, Gerding DN. 2009. Clostridium difcile infection caused by the epidemic BI/BNAP1/027 strain. Gastroenterology 136:191324 101. Panessa-Warren BJ, Tortora GT, Warren JB. 2007. High resolution FESEM and TEM reveal bacterial spore attachment. Microsc. Microanal. 13:25166 102. Pepin J, Valiquette L, Alary ME, Villemure P, Pelletier A, et al. 2004. Clostridium difcile associated diarrhea in a region of Quebec from 1991 to 2003: a changing pattern of disease severity. Can. Med. Assoc. J. 171:46672 103. P pin J, Valiquette L, Cossette B. 2005. Mortality attributable to nosocomial Clostridium difcile e associated disease during an epidemic caused by a hypervirulent strain in Quebec. Can. Med. Assoc. J. 173:103742 104. Peterson LR, Kelly PJ, Nordbrock HA. 1996. Role of culture and toxin detection in laboratory testing for diagnosis of Clostridium difcileassociated diarrhea. Eur. J. Clin. Microbiol. Infect. Dis. 15:330 36 105. Peterson LR, Manson RU, Paule SM, Hacek DM, Robicsek A, et al. 2007. Detection of toxigenic Clostridium difcile in stool samples by real-time polymerase chain reaction for the diagnosis of C. difcile associated diarrhea. Clin. Infect. Dis. 45:115260 106. Peterson LR, Robicsek A. 2009. Does my patient have Clostridium difcile infection? Ann. Int. Med. 151:17679 107. Planche T, Aghaizu A, Holliman R, Riley P, Poloniecki J, et al. 2008. Diagnosis of Clostridium difcile infection by toxin detection kits: a systematic review. Lancet Infect. Dis. 8:77784 108. QaDan M, Spyres LM, Ballard JD. 2000. pH-enhanced cytopathic conformational changes in Clostridium difcile toxin B. Infect. Immun. 68:247074 109. Quinn CD, Sefers SE, Babiker W, He Y, Alcabasa R, et al. 2010. C. Diff Quik Chek complete enzyme immunoassay provides a reliable rst-line method for detection of Clostridium difcile in stool specimens. J. Clin. Microbiol. 48:6035 110. Redelings MD, Sorvillo F, Mascola L. 2007. Increase in Clostridium difcilerelated mortality rates, United States, 19992004. Emerg. Infect. Dis. 13:141719 111. Reineke J, Tenzer S, Rupnik M, Koschinski A, Hasselmayer O, et al. 2007. Autocatalytic cleavage of Clostridium difcile toxin B. Nature 446:41519 112. Reller ME, Lema CA, Perl TM, Cai M, Ross TL, et al. 2007. Yield of stool culture with isolate toxin testing versus a two-step algorithm including stool toxin testing for detection of toxigenic Clostridium difcile. J. Clin. Microbiol. 45:36015 113. Ricciardi R, Rothenberger DA, Madoff RD, Baxter NN. 2007. Increasing prevalence and severity of Clostridium difcile colitis in hospitalized patients in the United States. Arch. Surg. 142:62431 114. Rupnik M, Avesani V, Janc M, von Eichel-Streiber C, Delmee M. 1998. A novel toxinotyping scheme and correlation of toxinotypes with serogroups of Clostridium difcile isolates. J. Clin. Microbiol. 36:2240 47 115. Rupnik M, Pabst S, Rupnik M, von Eichel-Streiber C, Urlaub H, Soling HD. 2005. Characterization of the cleavage site and function of resulting cleavage fragments after limited proteolysis of Clostridium difcile toxin B (TcdB) by host cells. Microbiology 151:199208 116. Rupnik M, Wilcox MH, Gerding DN. 2009. Clostridium difcile infection: new developments in epidemiology and pathogenesis. Nat. Rev. Microbiol. 7:52636
102. Represents the rst major report in North America on the regional outbreak of cases due to ribotype 027.
107. A useful review of 28 papers on EIAs for C difcile diagnosis that supports the view that these tests are suboptimal.
519
120. Explains how C. difcile binary toxin may contribute to the severity of disease.
117. Rupnik M. 2010. Clostridium difcile: (re)emergence of zoonotic potential. Clin. Infect. Dis. 51:58384 118. Saxton K, Baines SD, Freeman J, OConnor R, Wilcox MH. 2009. Effects of exposure of Clostridium difcile PCR ribotypes 027 and 001 to uoroquinolones in a human gut model. Antimicrob. Agents Chemother. 53:41220 119. Schirmer J, Aktories K. 2004. Large clostridial cytotoxins: cellular biology of Rho/Ras-glucosylating toxins. Biochim. Biophys. Acta 1673:6674 120. Schwan C, Stecher B, Tzivelekidis T, van Hamm M, Rohde M, et al. 2009. Clostridium difcile toxin CDT induces formation of microtubule-based protrusions and increases adherence of bacteria. PLoS Pathog. 5:114 121. Sebaihia M, Wren BW, Mullany P, Fairweather NF, Minton N, et al. 2006. The multi-drug resistant human pathogen Clostridium difcile has a highly mobile, mosaic genome. Nat. Genet. 38:77986 122. Sharp SE, Ruden LO, Pohl JC, Hatcher PA, Jayne LM, Ivie WM. 2010. Evaluation of the C. Diff Quik Chek Complete Assay, a new glutamate dehydrogenase and A/B toxin combination lateral ow assay for use in rapid, simple diagnosis of Clostridium difcile disease. J. Clin. Microbiol. 48:208286 123. Shetty N, Wren MWD, Coen PG. 2011. The role of glutamate dehydrogenase for the detection of Clostridium difcile in faecal samples: a meta-analysis. J. Hosp. Infect. 77:16 124. Snell H, Ramos M, Longo S, John M, Hussain Z. 2004. Performance of the TechLab C DIFF CHEK-60 enzyme immunoassay (EIA) in combination with the C. difcile Tox A/B II EIA kit, the Triage C. difcile panel immunoassay, and a cytotoxin assay for diagnosis of Clostridium difcileassociated diarrhea. J. Clin. Microbiol. 42:486365 125. Snyder MD. 1937. Further studies on Bacillus difcilis. J. Infect. Dis. 60:223 126. Spigaglia P, Mastrantonio P. 2002. Molecular analysis of the pathogenicity locus and polymorphism in the putative negative regulator of toxin production (TcdC) among Clostridium difcile clinical isolates. J. Clin. Microbiol. 40:347075 127. Stabler RA, Gerding DN, Songer JG, Drudy D, Brazier JS, et al. 2006. Comparative phylogenomics of Clostridium difcile reveals clade specicity and microevolution of hypervirulent strains. J. Bacteriol. 188:7297305 128. Stamper PD, Alcabasa A, Aird D, Babiker W, Wehrlin J, et al. 2009. Comparison of a commercial realtime PCR assay for tcdB detection to a cell culture cytotoxicity assay and toxigenic culture for direct detection of toxin-producing Clostridium difcile in clinical samples. J. Clin. Microbiol. 47:37378 129. Stamper PD, Babiker W, Alcabasa R, Aird D, Wehrlin J, et al. 2009. Evaluation of a new commercial TaqMan PCR assay for direct detection of the Clostridium difcile toxin B gene in clinical stool specimens. J. Clin. Microbiol. 47:384650 130. Sundriyal A, Roberts AK, Ling R, McGlashan J, Shone CC, Acharya KR. 2010. Expression, purication and cell cytotoxicity of actin-modifying binary toxin from Clostridium difcile. Protein Exp. Purif. 74:4248 131. Swindells J, Brenwald N, Reading N, Oppenheim B. 2010. Evaluation of diagnostic tests for Clostridium difcile infection. J. Clin. Microbiol. 48:6068 132. Tedesco FJ, Barton RW, Alpers DH. 1974. Clindamycin-associated colitis: a prospective study. Ann. Intern. Med. 81:42933 133. Tenover FC, Novak-Weekley S, Woods CW, Peterson LR, Davis T, et al. 2010. Impact of strain type on detection of toxigenic Clostridium difcile: comparison of molecular diagnostic and enzyme immunoassay approaches. J. Clin. Microbiol. 48:371924 134. Terhes G, Urb n E, Soki J, Nacsa E, Nagy E. 2009. Comparison of a rapid molecular method, the BD a GeneOhm Cdiff assay, to the most frequently used laboratory tests for detection of toxin-producing Clostridium difcile in diarrheal feces. J. Clin. Microbiol. 47:347881 135. Ticehurst JR, Aird DZ, Dam LM, Borek AP, Hargrove JT, Carroll KC. 2006. Effective detection of toxigenic Clostridium difcile by a two-step algorithm including tests for antigen and cytotoxin. J. Clin. Microbiol. 44:114549 136. Torres JF, Cedillo R, S nchez J, Dillman C, Giono S, Munoz O. 1984. Prevalence of Clostridium difcile a and its cytotoxin in infants in Mexico. J. Clin. Microbiol. 20:27475
Carroll
121. Summarizes the complete genome sequence of a clinical isolate of C. difcile.
127. Discusses and classies toxin variant strains and their clinical and epidemiological associations.
520
Bartlett
137. Underwood S, Guan S, Vijayasubhash V, Baines SD, Graham L, et al. 2009. Characterization of the sporulation initiation pathway of Clostridium difcile and its role in toxin production. J. Bacteriol. 191:7296305 138. Voth DE, Ballard JD. 2005. Clostridium difcile toxins: mechanism of action and role in disease. Clin. Microbiol. Rev. 18:24763 139. Warny M, Pepin J, Fang A, Killgore G, Thompson A, et al. 2005. Toxin production by an emerging strain of Clostridium difcile associated with outbreaks of severe disease in North America and Europe. Lancet 366:107984 140. Weese JS, Avery BP, Rousseau J, Reid-Smith RJ. 2009. Detection and enumeration of Clostridium difcile spores in retail beef and pork. Appl. Environ. Microbiol. 75:500911 141. Weese JS, Finley R, Reid-Smith RR, Janecko W, Rousseau J. 2010. Evaluation of Clostridium difcile in dogs and the household environment. Epidemiol. Infect. 138:11004 142. Zheng L, Keller SF, Lyerly DM, Carman RJ, Genheimer CW, et al. 2004. Multicenter evaluation of a new screening test that detects Clostridium difcile in fecal specimens. J. Clin. Microbiol. 42:383740 143. Zilberberg MD. 2009. Clostridium difcile-related hospitalization among US adults, 2006. Emerg. Infect. Dis. 15:12224
138. Discusses the pathogenesis of C. difcile toxins.
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ANNUAL REVIEWS

Biology of Clostridium difcile: Implications for Epidemiology and Diagnosis

THE BIOLOGY OF CLOSTRIDIUM DIFFICILE Microbiology

Summary of the Clostridium difcile Strain 630 Genome Sequence

The Pathogenicity Locus

PaLoc (19 kb) tcdE tcdA tcdC

Translocation domain 956 1,128 1,652 1,678

C Hydrophobic region Aspartate protease

Other Virulence Factors

Toxin Variant Strains

EPIDEMIOLOGY People and Patients as Reservoirs

Positive stool cultures 99%100% 15%25% 10%20% 10%25% 2%3% 5%70%

Positive toxin test 96% 10%20% <0%5% 2%8% <0.1% 5%63%

Adapted from Reference 13.

Risks for C. difcile Infection

DIAGNOSIS Emergence of Ribotype 027

Diarrheal stool specimen (N = 150)

C. DIFF Quik Chek CompleteTM

GDH positive (N = 22)

GDH negative (N = 114) C. difficile negative

Nucleic Acid Amplication Methods

Toxin positive (N = 11) Presumed CDI

Toxin negative (N = 11)

XpertTM C. difficile PCR

PCR positive (N = 8) CDI (N = 4) or toxigenic carriage (N = 4) Clinical assessment essential

PCR negative (N = 3) Nontoxigenic carriage (N = 2) or false-positive GDH (N = 1) No CDI

121. Summarizes the complete genome sequence of a clinical isolate of C. difcile.

138. Discusses the pathogenesis of C. difcile toxins.

www.annualreviews.org Clostridium difcile

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