Clonal spread of a Clostridium difficile strain with a complete set of toxin A, toxin B, and binary toxin genes among Polish patients with Clostridium difficile-associated diarrhea

Clinically relevant Clostridium difficile strains usually produce toxins A and B. Some C. difficile strains can produce an additional binary toxin. We report clonality among five strains carrying all toxin genes from Polish patients with C. difficile-associated diarrhea. In another strain, possible recombination between binary toxin genes is documented.     

Epidemics of diarrhea caused by a clindamycin-resistant strain of Clostridium difficile in four hospitals

BACKGROUND: Large outbreaks of diarrhea caused by a newly recognized strain of Clostridium difficile occurred in four hospitals located in different parts of the United States between 1989 and 1992. Since frequent use of clindamycin was associated with the outbreak in one of the hospitals, we examined the resistance genes of the epidemic-strain isolates and studied the role of clindamycin use in these outbreaks. METHODS: Case-control studies were performed at three of the four hospitals to assess the relation of the use of clindamycin to C. difficile-associated diarrhea. All isolates of the epidemic strain and representative isolates of other strains identified during each outbreak were tested for susceptibility to clindamycin. Chromosomal DNA from these representative isolates was also analyzed by dot blot hybridization and amplification with the polymerase chain reaction (PCR) with the use of probes and primers from a previously described determinant of erythromycin resistance - the erythromycin ribosomal methylase B (ermB) gene - found in C. perfringens and C. difficile. RESULTS: In a stratified analysis of the case-control studies with pooling of the results according to the Mantel-Haenszel method, we found that the use of clindamycin was significantly increased among patients with diarrhea due to the epidemic strain of C. difficile, as compared with patients whose diarrhea was due to nonepidemic strains (pooled odds ratio, 4.35; 95 percent confidence interval, 2.02 to 9.38; P<0.001). Exposure to other types of antibiotics or hospitalization in a surgical ward was not significantly associated with the risk of C. difficile-associated diarrhea due to the epidemic strain. All epidemic-strain isolates were highly resistant to clindamycin (minimal inhibitory concentration, >256 microg per milliliter). DNA hybridization and PCR analysis showed that all these isolates had an ermB gene, which encodes a 23S ribosomal RNA methylase that mediates resistance to macrolide, lincosamide, and streptogramin antibiotics. Only 15 percent of the nonepidemic strains were resistant to clindamycin. CONCLUSIONS: A strain of C. difficile that is highly resistant to clindamycin was responsible for large outbreaks of diarrhea in four hospitals in different states. The use of clindamycin is a specific risk factor for diarrhea due to this strain. Resistance to clindamycin further increases the risk of C. difficile-associated diarrhea, an established complication of antimicrobial use.     

tcdC genotypes associated with severe TcdC truncation in an epidemic clone and other strains of Clostridium difficile

Severe Clostridium difficile associated disease is associated with outbreaks of the recently described BI/NAP1 epidemic clone. This clone is characterized by an 18-bp deletion in the tcdC gene and increased production of toxins A and B in vitro. TcdC is a putative negative regulator of toxin A&B production. We characterized tcdC genotypes from a collection of C. difficile isolates from a hospital that experienced an outbreak caused by the BI/NAP1 epidemic clone. Sequence analysis of tcdC was performed on DNA samples isolated from 199 toxigenic C. difficile isolates (31% BI/NAP1) from 2001 and 2005. Sequences obtained from 36 (18.6%) isolates predicted wild-type TcdC (232 amino acid residues), whereas 12 (6.1%) isolates had tcdC genotypes with previously described 18- or 39-bp deletions. The remaining isolates comprised 15 unique genotypes. Of these, 5 genotypes contain 18- or 36-bp deletions. Of these five genotypes, one is characterized by a single nucleotide deletion at position 117 resulting in a frameshift that introduces a stop codon at position 196, truncating the predicted TcdC to 65 amino acid residues. All 62 of the isolates in this collection comprising the epidemic clone are characterized by this genotype. This result suggests that severe truncation of TcdC is responsible for the increased toxin production observed in strains belonging to the BI/NAP1 clone and that the 18-bp deletion is probably irrelevant to TcdC function. Further investigations are required to determine the effect of this and other tcdC genotypes on toxin production and clinical disease.     

Molecular analysis of Clostridium difficile strains isolated from 18 cases of recurrent clostridium difficile-associated diarrhea

Recurrence of Clostridium difficile-associated diarrhea (CDAD) occurs in 15 to 20% of patients after discontinuation of treatment. Arbitrarily primed PCR was used to investigate the epidemiology of recurrent CDAD in 18 patients. Reinfection with a new strain occurred in 6 of 18 patients (33.3%), while 12 patients relapsed with the original strain shortly after discontinuation of treatment. These data suggest that reinfection with exogenous C. difficile is a common problem and that not all recurrences are due to relapse.     

Variability of Clostridium difficile surface proteins and specific serum antibody response in patients with Clostridium difficile-associated disease

Pathogen attachment is a crucial early step in mucosal infections. This step is mediated by important virulence factors, such as surface proteins. Clostridium difficile surface proteins have been identified as (i) adhesins (the flagellar cap protein FliD; the flagellin FliC; and the cell wall protein Cwp 66 with a two domain-structure [Cw 66 N-terminal and Cwp 66 C-terminal domains]) and (ii) protease (the Cwp 84 protein). To address the roles of these proteins in the pathogenesis of Clostridium difficile and to identify vaccine antigen candidates, we analyzed the variability of the proteins and their immunogenicities in 17 patients with C. difficile-associated disease. PCR-restriction fragment length polymorphism analysis of amplified gene products revealed interstrain homogeneity with fliC and fliD, in contrast to cwp 66 genes. Immunoblot analysis showed that FliC and FliD were detected in the majority of isolates. The N-terminal domain of Cwp 66 and Cwp 84 were present in all strains tested, in contrast to the Cwp 66 C-terminal domain, the expression of which was heterogeneous. The 17 sera from the corresponding patients were analyzed by enzyme-linked immunosorbent assay to detect antibodies directed against these proteins. Many patients developed antibodies to FliC, FliD, Cwp 84, and the Cwp 66 C-terminal domain, but not to the Cwp 66 N-terminal domain. In conclusion, this study confirms the expression of these surface proteins of C. difficile during the course of the disease. In addition, the FliC, FliD, and Cwp 84 proteins appeared to be good potential vaccine candidates.     

Unnecessary repeat Clostridium difficile PCR testing in hospitalized adults with C. difficile-negative diarrhea

The aim of this study was to determine the extent and associated costs of repeat Clostridium difficile stool polymerase chain reaction (PCR) assays in patients with initially negative PCRs. C. difficile stool PCRs were done on adult hospitalized patients with diarrhea. The number/time course of repeat PCRs on initially negative PCR patients was determined. Of 5,027 C. difficile stool PCRs, 814 (16.2 %) were positive and 4,213 (83.8 %) were negative. Ninety-seven of the initially PCR-negative patients had >2 repeat tests 1–59 days after the initial negative stool PCR. Repeat negative PCR testing rarely resulted in a subsequent positive result (0.05 %). The unnecessary costs of 97 repeat PCRs was $32,658.00. Many of these patients were originally given empiric oral anti-C. difficile therapy, in spite of repeatedly negative PCRs.     

Characterization of a toxin A-negative, toxin B-positive strain of Clostridium difficile responsible for a nosocomial outbreak of Clostridium difficile-associated diarrhea

Clostridium difficile-associated diarrhea (CAD) is a very common nosocomial infection that contributes significantly to patient morbidity and mortality as well as to the cost of hospitalization. Previously, strains of toxin A-negative, toxin B-positive C. difficile were not thought to be associated with clinically significant disease. This study reports the characterization of a toxin A-negative, toxin B-positive strain of C. difficile that was responsible for a recently described nosocomial outbreak of CAD. Analysis of the seven patient isolates from the outbreak by pulsed-field gel electrophoresis indicated that this outbreak was due to transmission of a single strain of C. difficile. Our characterization of this strain (HSC98) has demonstrated that the toxin A gene lacks 1.8 kb from the carboxy repetitive oligopeptide (CROP) region but apparently has no other major deletions from other regions of the toxin A or toxin B gene. The remaining 1.3-kb fragment of the toxin A CROP region from strain HSC98 showed 98% sequence homology with strain 1470, previously reported by M. Weidmann in 1997 (GenBank accession number Y12616), suggesting that HSC98 is toxinotype VIII. The HSC98 strain infecting patients involved in this outbreak produced the full spectrum of clinical illness usually associated with C. difficile-associated disease. This pathogenic spectrum was manifest despite the inability of this strain to alter tight junctions as determined by using in vitro tissue culture testing, which suggested that no functional toxin A was produced by this strain.     

Commercial latex agglutination test for detection of Clostridium difficile-associated diarrhea.

A commercially available latex agglutination test for Clostridium difficile was compared with a cell culture cytotoxin assay and bacteriological culture for the laboratory diagnosis of C. difficile-associated diarrhea and colitis (CAD). Stool specimens from 626 patients were tested by the three methods, and specimens from 118 patients (19%) were positive by at least one of the methods. The results of the three tests agreed in 88% of the specimens tested, overall, but they agreed in only 34% of the 118 positive specimens. Ninety-three patients were evaluated to assess the significance of positive and negative results for each assay. Of 40 patients found to have CAD, 70% were positive by the cytotoxin assay, 78% were positive by the latex agglutination test, and 90% were culture positive. Of 53 patients who did not have CAD, 2% were positive by the cytotoxin assay, 8% were positive by the latex test, and 4% were culture positive. The detection of CAD was improved by using the tests in combination, and 97% of specimens positive by two or three methods were from patients who had CAD. Testing of multiple specimens from individual patients also increased the sensitivity of detection of CAD. The results suggest that the latex agglutination test may be useful for rapid diagnosis of CAD, especially in laboratories that lack cell culture facilities. However, the accuracy of CAD detection is improved when the latex test is used in combination with culture or the cytotoxin assay.     

Isolation of Clostridium difficile from hospitalized patients without antibiotic-associated diarrhea or colitis.

Stool samples from 100 hospitalized patients and 21 healthy adults, obtained between March and June 1980, were cultured on a special selective medium containing cefoxitin and cycloserine to detect Clostridium difficile. This organism was isolated from 13 of the hospitalized patients and from 1 healthy subject. None of the patients with positive cultures had received antimicrobial therapy in the 3 preceding months. The observed rate of C. difficile isolation from adults not suffering from antibiotic-associated diarrhea or colitis is higher than previously reported. C. difficile culture is not recommended as a substitute for toxin assay in the evaluation of patients with intestinal disorders after antimicrobial chemotherapy.     

Clostridium difficile and its cytotoxin in feces of patients with antimicrobial agent-associated diarrhea and miscellaneous conditions.

Fecal specimens from 223 subjects were evaluated for the presence of Clostridium difficile by use of a selective medium developed in our laboratory and for the presence of C. difficile cytotoxin. C. difficile and cytotoxin were detected in 89 and 83%, respectively, of patients with antimicrobial agent-associated pseudomembranous colitis (PMC). In patients in whom PMC was not documented, C. difficile and cytotoxin were present in only 37 and 21%, respectively. C. difficile and cytotoxin were also recovered from the feces of 6 and 3, respectively, of 13 antimicrobial recipients who did not have diarrhea. Although C. difficile appears to be a major cause of PMC, it is not responsible for at least some two-thirds of cases of antimicrobial agent-associated diarrhea in which PMC is not documented. Neither the recovery of C. difficile nor the detection of its cytotoxin should be considered diagnostic for C. difficile-induced disease.     

Performance of the TechLab C. DIFF CHEK-60 enzyme immunoassay (EIA) in combination with the C. difficile Tox A/B II EIA kit, the Triage C. difficile panel immunoassay, and a cytotoxin assay for diagnosis of Clostridium difficile-associated diarrhea

We compared a recently marketed enzyme immunoassay for glutamate dehydrogenase (GDH), TechLab's C. DIFF CHEK-60 (TL-GDH), in combination with the C. difficile Tox A/B II enzyme immunoassay (Tox-A/B) with (i) the Triage C. difficile test, which detects both GDH (TR-GDH) and toxin A (TR-Tox-A); (ii) an in-house cytotoxin assay (C-Tox); and (iii) stool cultures for C. difficile. All C. difficile isolates were tested for the presence of the toxin genes by PCR. If a toxin gene-positive strain of Clostridium difficile was recovered and a toxin was detected by any method, the result was considered to be truly positive. Eighty-seven of 93 and 79 of 93 C. difficile culture-positive samples were also TL-GDH and TR-GDH positive, respectively. No test was able to detect toxin in all samples with true-positive results. Tox-A/B and TR-Tox-A in combination with the GDH detection tests and C-Tox were able to identify 52 and 50 samples with true-positive results. Tox-A/B and TR-Tox-A would have missed 15 and 31% of cases of C. difficile-associated diarrhea, respectively, if used alone.     

Molecular epidemiology of Clostridium difficile strains in children compared with that of strains circulating in adults with Clostridium difficile-associated infection

Molecular analysis of Clostridium difficile (28 isolates) from children (n = 128) in Oxfordshire, United Kingdom, identified eight toxigenic genotypes. Six of these were isolated from 27% of concurrent adult C. difficile-associated infections studied (n = 83). No children carried hypervirulent PCR ribotype 027. Children could participate in the transmission of some adult disease-causing genotypes.     

Laboratory diagnosis of Clostridium difficile-associated diarrhea and colitis: usefulness of Premier Cytoclone A+B enzyme immunoassay for combined detection of stool toxins and toxigenic C. difficile strains

Detection of Clostridium difficile toxins A and B in stools by Premier Cytoclone A+B enzyme immunoassay (EIA) was compared with detection by stool culture for C. difficile followed by detection of toxigenic isolates using the same EIA. Chart reviews were performed to evaluate the likelihood of C. difficile-associated diarrhea and colitis (CADC) for all patients with at least one positive toxin assay. While the toxins were detected in 58 of 85 consecutive CADC patients by both assays, CADC in 5 patients was detected only by stool toxin assay, and in 22 patients CADC was detected only by toxigenic culture. Our results suggest that for laboratories using a rapid toxin A+B EIA, direct toxin detection in stools should be combined with toxigenic culture in cases in which there is a negative stool toxin assay.     

Development of a rapid enzyme immunoassay for Clostridium difficile toxin A and its use in the diagnosis of C. difficile-associated disease.

A rapid (2.5 h) direct enzyme immunoassay (EIA) for Clostridium difficile toxin A was developed for clinical use. Specimen centrifugation and filtration were not required. The EIA detected toxin A levels in patient stool as low as 20 pg (2 ng/ml of stool). The test was 5,000 times more sensitive for toxin A than it was for toxin B and did not react with a panel of other bacterial species with the exception of one highly toxigenic strain of Clostridium sordellii. The EIA was compared with the cytotoxin assay, culture of toxigenic C. difficile (toxigenic culture), and latex agglutination by using 313 fresh stool specimens submitted from patients with suspected C. difficile-associated disease. Results read visually and with a plate reader were similar. Sixty-two specimens were positive by one or more tests, but only 22 (35%) were positive by all four laboratory methods. The EIA was 84.1% sensitive and 98.9% specific when it was compared with the cytotoxin assay. The use of toxigenic culture to referee discrepant results (EIA versus cytotoxin assay) showed the EIA sensitivity and specificity to be 95.1 and 99.3%, respectively, with respect to other laboratory methods. Patient charts were reviewed for antibiotic-associated diarrhea on 108 specimens, including all those that were positive by at least one test method. Of 34 patients determined to have C. difficile-associated disease, 29 (85.3%) were positive by EIA, 32 (94.1%) were positive by the cytotoxin assay, 27 (79.4%) were positive by toxigenic culture, and 20 (58.8%) were positive by latex agglutination. Seven patients with antibiotic-associated diarrhea had a positive latex result, but results were negative by EIA, the cytotoxin assay, and toxigenic culture. The EIA demonstrated high specificity and good sensitivity for C. difficile-associated disease cases. The test can be used alone or in combination with the cytotoxin assay or toxigenic culture to provide rapid and sensitive results.     

Immunoblot analysis of serum immunoglobulin G response to surface proteins of Clostridium difficile in patients with antibiotic-associated diarrhea.

We examined by immunoblot analysis the serum immunoglobulin G antibody response to EDTA-extracted surface proteins of Clostridium difficile in 16 patients with antibiotic-associated diarrhea. For each patient, paired serum samples were tested against proteins of the infecting strain and of a collection strain (C253) known to belong to the electrophoretic group 2 pattern. Eight patients, all harboring group 2 C. difficile strains, exhibited responses to the proteins of the infecting strain; six patients showed increases in the level of antibodies between acute-phase and convalescent-phase sera. A great variability in the antigens recognized was found; however, seven patients possessed antibodies directed against an antigen of about 35 kilodaltons, corresponding to the major protein of group 2 strains. The sera of these seven patients cross-reacted also with the 35-kilodalton and other proteins of strain C253. Our data show that C. difficile proteins other than toxins can elicit an immune response in patients with C. difficile-associated disease; in this group of patients, the major surface protein of the group 2 strains was the antigen most often recognized.     

Experimental reproduction of neonatal diarrhea in young gnotobiotic hares simultaneously associated with Clostridium difficile and other Clostridium strains.

Clostridium difficile, C. perfringens, and C. tertium are very often present simultaneously in the feces of conventional diarrheic young hares, whereas these three bacterial species are rarely encountered and never present simultaneously in the feces of healthy young hares. When a strain of each of the three bacterial species was monoassociated with axenic young hares, the appearance of pathological disorders was only observed in animals monoassociated with C. difficile, when the number of C. difficile exceeded 10(8) per g of fresh feces. When a strain of C. perfringens or a strain of C. tertium, or both, was associated with C. difficile, diarrhea and death occurred more rapidly than in hares monoassociated with C. difficile. C. difficile and C. perfringens became established more rapidly when disassociated than when monoassociated with axenic hares. The association of C. perfringens and C. tertium with axenic hares did not bring about any pathological disorders. It may be concluded that C. difficile is the causal agent of neonatal diarrhea in conventional and gnotobiotic young hares and that other strains of Clostridium enhance its pathogenic effect. C difficile alone or associated with C. perfringens or C. tertium does not play any pathogenic role in young rats, mice, or rabbits.