Ribotype 027 Clostridium difficile infections with measurable stool toxin have increased lactoferrin and are associated with a higher mortality.

We evaluated clinical and diagnostic indicators of severe C. difficile infection (CDI) and their association with poor clinical outcome. A total of 210 patients positive according to PCR (toxin B: tcdB) were included, with patients having a median age of 62 years and a Charlson co-morbidity index (CI) score of 5. Ninety-one percent (n = 191) were positive by toxigenic culture and 61% (n = 129) had stool toxin. Toxin-positive patients had significantly higher fecal lactoferrin (mean 316 µg/g versus 106 µg/g stool; p < 0.0001). Forty percent of patients (n = 85) were infected with ribotype 027 and significantly more of these patients had measurable stool toxin (79% vs. 50%; p < 0.0001). The mean fecal lactoferrin was significantly higher for toxin-positive 027 CDI compared with the 027 toxin-negative group (317 vs 60 µg/g; p = 0.0014). Ribotype 027 CDI with stool toxin showed a higher all-cause, 100-day mortality compared with non-027 with stool toxin (36 % vs 18%; p = 0.017). Logistic regression univariate analysis for odds ratio (OR) and p values revealed that age (OR = 1.1), intensive care unit treatment (OR = 2.7), CI (OR = 1.2), 027 CDI (OR = 2.1), white blood cell count (OR = 1.0), albumin level (OR = 0.1), and stool toxin-positive 027 CDI (OR = 2.5) were significantly associated with 100-day mortality (p < 0.05). In conclusion, CDI PCR-positive patients with 027 infection and stool toxin have increased lactoferrin and are at an increased risk of death.

Elevated lactoferrin is associated with moderate to severe Clostridium difficile disease, stool toxin, and 027 infection.

We evaluated blood and fecal biomarkers as indicators of severity in symptomatic patients with confirmed Clostridium difficile infection (CDI). Recruitment included patients with CDI based on clinical symptoms and supporting laboratory findings. Disease severity was defined by physician's assessment and blood and fecal biomarkers were measured. Toxigenic culture done using spore enrichment and toxin B detected by tissue culture were done as confirmatory tests. Polymerase chain reaction (PCR) ribotyping was performed on each isolate. There were 98 patients recruited, with 85 (87%) confirmed cases of toxigenic CDI (21 severe, 57 moderate, and seven mild), of which 68 (80%) were also stool toxin-positive. Elevated lactoferrin (p = 0.01), increased white blood cell (WBC) count (p = 0.08), and low serum albumin (p = 0.03) were all associated with the more severe cases of CDI. Ribotype 027 infection accounted for 71% of severe cases (p < 0.01) and patients with stool toxin had significantly higher lactoferrin levels and WBC counts (p < 0.05). Our findings show that elevated fecal lactoferrin, along with increased WBC count and low serum albumin, were associated with more severe CDI. In addition, patients infected with ribotype 027 and those with stool toxin had significantly higher fecal lactoferrin and WBC counts.

Glutamate dehydrogenase is highly conserved among Clostridium difficile ribotypes.

gluD was highly conserved and glutamate dehydrogenase (GDH) was readily expressed in vitro by all 77 Clostridium difficile ribotypes assayed. All ribotypes, including ARL 002, ARL 027, and ARL 106, were reactive in assays that detect C. difficile GDH.

Clostridium difficile prevalence rates in a large healthcare system stratified according to patient population, age, gender, and specimen consistency.

We evaluated Clostridium difficile prevalence rates in 2,807 clinically indicated stool specimens stratified by inpatient (IP), nursing home patient (NH), outpatient (OP), age, gender, and specimen consistency using bacterial culture, toxin detection, and polymerase chain reaction (PCR) ribotyping. Rates were determined based on the detection of toxigenic C. difficile isolates. We identified significant differences in the rates between patient populations and with age. Specimens from NH had a higher rate (46%) for toxigenic C. difficile than specimens from IP (18%) and OP (17%). There were no gender-related differences in the rates. Liquid specimens had a lower rate (15%) than partially formed and soft specimens (25%) and formed specimens (18%) for the isolation of toxigenic C. difficile. The nontoxigenic rate was lowest for NH (4%) and highest for patients<20 years of age (23%). We identified 31 different toxigenic ribotypes from a sampling of 190 isolates that showed the lowest diversity in NH. Fluoroquinolone resistance was observed in 93% of the 027 isolates, all of the 053 isolates, and in four other ribotypes. We observed different rates for toxigenic C. difficile in stratified patient populations, with the highest rate for NH, a low overall nontoxigenic rate, and fluoroquinolone resistance.

Cytotoxicity of Clostridium difficile toxin A for human colonic and pancreatic carcinoma cell lines.

The use of bacterial exotoxins may constitute novel adjuncts to treatment of gastrointestinal tract malignancies. Clostridium difficile toxin A was evaluated for its cytotoxic effect in vitro on 24 human cell lines and strains including carcinomas of the colon, pancreas, prostate, lung, breast, and lymphoid malignancies, as well as nonmalignant tissues. All nine colon and five pancreas cell lines were extraordinarily sensitive to the cytotoxic effect of Clostridium difficile toxin A at very low concentrations. This effect, which occurred rapidly and was dose dependent, was observed in all cells of seven colon and two pancreas cell lines at concentrations as low as 1-5 ng/ml (10(-12) to 10(-11) M), whereas cells derived from other sites required 60 to greater than 500 ng/ml to achieve an equivalent effect. The data suggest that Clostridium difficile toxin A may have potential therapeutic value in the treatment of some gastrointestinal tract cancers.

Immunization against experimental Pseudomonas aeruginosa and Serratia marcescens keratitis. Vaccination with lipopolysaccharide endotoxins and proteases.

Rabbits vaccinated with lipopolysaccharide endotoxins or with purified protease preparations from Pseudomonas aeruginosa and Serratia marcescens before corneal challenge with the viable bacteria exhibited significantly less corneal damage than rabbits not vaccinated with the bacterial products. However, the rabbits vaccinated with the lipopolysaccharide endotoxin preparations were significantly better protected than rabbits vaccinated with the bacterial proteases. Rabbits vaccinated with antisera raised against the proteases showed significantly less corneal damage than rabbits vaccinated with normal rabbit serum, and the passive protection was not significantly different than that elicited by active immunization against the bacterial proteases. The ability of the antiserum raised against the pseudomonas elastolytic protease to passively protect against severe corneal damage produced by experimentally induced pseudomonas keratitis was confirmed in mice. These findings support the idea that the bacterial endotoxins and proteases are virulence factors during the development of pseudomonas and serratia keratitis.

Comparison of culture, cytotoxicity assays, and enzyme-linked immunosorbent assay for toxin A and toxin B in the diagnosis of Clostridium difficile-related enteric disease.

Clostridium difficile culture, test tube, and microtiter cytotoxicity assays, and enzyme-linked immunosorbent assays (ELISAs) for toxin A and toxin B, were simultaneously performed on 113 fresh diarrheal stool specimens randomly selected from those submitted to our clinical laboratory for routine C. difficile testing. The performance of these tests in diagnosing C. difficile-related enteric disease (CDRED) was based on a clinical assessment of the likelihood of CDRED as determined by a systematic review of case histories blinded from the test results. Among 61 antibiotic recipients, both the microtiter cytotoxicity assay and the toxin A ELISA were highly specific for CDRED (95% and 100%, respectively). Specificities for the other procedures were much lower (tube cytotoxicity assay, 79%; culture, 74%; and toxin B ELISA, 56%). The high sensitivities of the culture (89%) and toxin B ELISA (83%) were somewhat negated by their low specificities. The only test that was both specific and had acceptable sensitivity (78%) was the microtiter cytotoxicity assay. This study indicates that ELISAs for detection of C. difficile toxins are not as reliable as the cytotoxicity assay in the laboratory diagnosis of CDRED, and that clinical correlation is essential in the evaluation of any new test for CDRED.

Deletions in the repeating sequences of the toxin A gene of toxin A-negative, toxin B-positive Clostridium difficile strains.

The repeating sequences of the toxin A gene from toxin A-negative, toxin B-positive (toxin A-, toxin B+) strains of Clostridium difficile which were isolated in geographically separated facilities in Japan and Indonesia were determined. All six strains tested had identical repeating sequences with two deletions (1548 and 273 nucleotides in size) in the toxin A gene. A PCR method was designed to detect the deletions and the deletions were confirmed in all 50 toxin A-, toxin B+ strains examined by this method. Western immunoblot analysis revealed that polyclonal antiserum against native toxin A did not react with the concentrated culture filtrates of the toxin A-, toxin B+ strains. These results may suggest that toxin A-, toxin B+ strains have deletions of the two thirds of the repeating regions of the toxin A gene, which encodes the epitopes fully responsible for the reaction with the polyclonal antiserum.

Production of monoclonal antibodies against Clostridium difficile cytotoxin using immunosorbent binding bioassay procedure.

In the following study, a novel screening approach was used to develop monoclonal antibodies specific for toxin B of Clostridium difficile. The approach, which consisted of an immunosorbent binding bioassay (ISBBA), is based on antigen immunocapture by monoclonal antibodies and detection of biological activity. Our results showed ISBBA, which uses unpurified antigen, to be more sensitive than the neutralization assay and ELISA for the detection of toxin B antibody.

Role of tumor necrosis factor and nitric oxide in the cytotoxic effects of Clostridium difficile toxin A and toxin B on macrophages.

Clostridium difficile, the bacterium involved in antibiotic-associated colitis, produces two exotoxins, toxin A (TxA) and toxin B (TxB). Although these toxins are well recognized as being cytotoxic to several mammalian cell types, the mechanisms involved are not fully understood. The aim of the present investigation was to examine the cytotoxicity of TxA and TxB to peritoneal macrophages in culture and to investigate whether tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) are involved in the process. As a control, the effect of E. coli LPS was also investigated. TxA, TxB and LPS were dose-dependently cytotoxic to macrophage monolayers, with TxB being the most potent. All of the toxins stimulated the release of TNF-alpha from macrophages. TxB was again the most active in inducing this response. The TNF-alpha released appears to be involved in the action of LPS and TxA, but not of TxB, since a mAb against TNF-alpha inhibited the cytotoxicity of the former two but had no effect on the latter. NO is not involved in the effects of TxA and TxB since these toxins did not induce the production of this mediator in macrophages, even in the presence of IFN-gamma. In addition, L-imino-ethyl-L-ornithine (L-NIO), a NO synthase inhibitor, did not modify the macrophage death caused by TxA or TxB. Although LPS was able to induce the production of high amounts of NO, NO did not mediate the LPS cytotoxicity since L-NIO did not influence the degree of macrophage death caused by LPS. TxA and TxB therefore appear to exert cytotoxic effects on cultured macrophages by different mechanisms. TNF-alpha is involved in TxA and LPS-mediated cytotoxicity but not in the toxicity caused by TxB. NO is not involved in the killing action of any of these toxins.

Stimulation of enzyme secretion from isolated pancreatic acini by Clostridium difficile toxin B.

Exposure of isolated rat pancreatic acini to increasing concentrations (10 ng - 800 ng/ml) of toxin B from Clostridium difficile produced a biphasic effect on the rate of secretion of amylase, trypsinogen, and chymotrypsinogen. Whereas doses of toxin B from 10-30 ng/ml increased enzyme secretion by 15-20%, doses between 30 ng and 60 ng/ml showed a regression of this effect, whereafter the rate of secretion of amylase, trypsinogen, and chymotrypsinogen increased with increasing concentrations of the toxin. Toxin B concentration of 800 ng/ml enhanced amylase, trypsinogen and chymotrypsinogen secretion by 119%, 185% and 195%, respectively, when compared with the basal level. Stimulation of enzyme secretion by toxin B was not affected by the presence of either actinomycin-D or cycloheximide, at a concentration which inhibited acinar RNA or protein synthesis by 80-90%. Although toxin B as well as CCK8, carbachol and secretin by themselves caused significant stimulation in amylase, trypsinogen and chymotrypsinogen secretion from isolated pancreatic acini, toxin B together with either CCK8, carbachol or secretin produced no further augmentation in enzyme secretion than what was observed with the secretagogues alone. It is concluded that toxin B of Cl. difficile exerts a direct effect on pancreatic acinar cells as evidenced by stimulation of enzyme secretion.

Vascular and glomerular effects of Clostridium difficile toxin A peptide on the isolated rat kidney.

Toxin A peptide from Clostridium difficile caused damage and secretion in the intestinal mucosa. These effects are mediated in part by pro-inflammatory substances. In order to evaluate and compare the biologic effect of toxin A on renal vascular, glomerular and tubular functions, we studied this toxin in isolated rat kidneys. Isolated kidneys from adult male Wistar rats (260-320 g) were perfused with Krebs-Henseleit solution containing 60 mg/ml dialyzed bovine serum albumin. We studied the effect of toxin A peptide (3.2 x 10(-6) M, injected into perfusate) on glomerular filtration rate (GFR), urinary flow rate (UF) and total sodium reabsorption (TNa+, %). All experiments were preceded by a 30-min basal period, and in another group of kidneys the time course of the variables was followed without toxin infusion for unpaired control. Toxin A (TxA) reduced the perfusion pressure (PP), from PPcontrol/30min = 124.89 +/- 1.91 to PPTxA/120min = 88.13 +/- 5.1 mmHg (N = 6, P < 0.01) with a maximal effect at 120 min after toxin infusion. TxA also caused a significant decrease in GFR with maximal effect at 90 min after toxin infusion (GFRcontrol/30min = 0.53 +/- 0.05 to GFRTxA/90min = 0.30 + 0.05 ml min-1g-1; N = 6, P < 0.01). TxA did not alter renal tubular sodium transport when compared with a control without toxin infusion. In addition, toxin-treated kidneys caused a time-dependent increase in urinary flow from UFcontrol/30min = 0.16 +/- 0.08 to UFTxA/120min = 0.35 +/- 0.1 ml min-1g-1 (N = 6, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Clostridium difficile ribotype 027 is most prevalent among inpatients admitted from long-term care facilities.

Intestinal inflammation was evaluated using faecal lactoferrin and ribotype in 196 hospitalized adults with Clostridium difficile infection to determine the impact of ribotype 027 in long-term care facilities (LTCFs). LTCF residents (n=28) had greater antibiotic use (P=0.049) and more ribotype 027 infection [odds ratio (OR): 4.87; 95% confidence interval (CI): 2.02-11.74; P<0.01], compared to those admitted from home. Patients infected with ribotype 027 strains had worse six-month mortality (OR: 1.90; 95% CI: 1.08-3.34; P=0.03) and more inflammation (95.26 vs 36.08 µg/mL; P=0.006), compared to those infected with non-027 strains. This study was not designed to determine acquisition site, but, in this population, suggests that the location from which the patient has been admitted is strongly associated with ribotype 027 and more severe C. difficile disease.

Importance of serratia protease in the pathogenesis of experimental Serratia marcescens pneumonia.

The results of studies to evaluate the possible importance of serratia proteases in the development of experimental Serratia marcescens pneumonia revealed the following. (i) Administration of a highly purified serratia protease to the lungs of guinea pigs and mice resulted in extensive pulmonary edema and hemorrhage similar to that observed in animals having an experimentally induced, acute serratia pneumonia. (ii) Guinea pigs subcutaneously vaccinated with the protease developed low levels of antiprotease antibodies and were partially protected against serratia pneumonia, as demonstrated by a significant increase in survival time. Mice intranasally vaccinated with the protease also developed antiprotease antibodies and were protected against serratia pneumonia, as demonstrated by a significant increase in survival time and an increase in the number of survivors. (iii) Serratia protease was detected in lung tissue extracts prepared from the lungs of guinea pigs dying of serratia pneumonia. Our findings support the idea that serratia protease(s) is involved in the pathogenesis of experimental serratia pneumonia.

Commercial latex test for Clostridium difficile toxin A does not detect toxin A.

The rapid latex test recently marketed by Marion Scientific (Div. Marion Laboratories, Inc., Kansas City, Mo.) for the detection of Clostridium difficile toxin A does not react with the toxin, based on the following findings: culture filtrates from nontoxigenic strains of C. difficile gave positive reactions in the test, culture filtrate in which toxin A had been removed gave positive reactions, purified toxin A did not react in the test, and the latex reagent bound an antigen which is distinct from toxin A and which is produced in various amounts by both toxigenic and nontoxigenic strains of C. difficile.

Enzyme-linked immunosorbent assay for Clostridium difficile toxin A.

Antibodies against Clostridium difficile toxin A were purified by affinity chromatography from antiserum prepared against crude C. difficile toxin preparations. The affinity-purified antibody preparation was free of detectable amounts of antibodies to other C. difficile antigens, as demonstrated by crossed immunoelectrophoresis, and specifically neutralized the cytotoxicity of toxin A. An indirect enzyme-linked immunosorbent assay (ELISA) was subsequently developed using the antibody preparation for the specific detection of toxin A. The ELISA, which could detect 1 ng (5 ng/ml) of toxin A, was used to quantitate the toxin in the culture supernatant fluids of strains of C. difficile. The ELISA values for toxin A closely correlated with the toxin A and B cytotoxic titers of the supernatant fluids. In addition, toxin A was detected by ELISA in human fecal specimens from persons with antibiotic-associated colitis, demonstrating that this toxin is produced during C. difficile colitis.

Identification of the latex test-reactive protein of Clostridium difficile as glutamate dehydrogenase.

Computer analysis showed that the gene encoding the latex test-reactive protein of Clostridium difficile exhibited high levels of homology with glutamate dehydrogenases from various sources. Further analysis demonstrated that the recombinant protein possessed glutamate dehydrogenase activity. Our results show that the protein that reacts in commercial latex tests for C. difficile is a glutamate dehydrogenase.

Monoclonal and specific polyclonal antibodies for immunoassay of Clostridium difficile toxin A.

Monoclonal antibody, affinity-purified antibody, and monospecific antiserum against toxin A were produced. The monoclonal antibody was an immunoglobulin G2a kappa chain isotype that immunoprecipitated toxin A, as shown by crossed immunoelectrophoresis. These antibodies were compared by counterimmunoelectrophoresis, latex agglutination, and indirect enzyme-linked immunosorbent assay for their sensitivity in detecting toxin A. Our findings indicate that these antibodies may be useful as immunodiagnostic reagents for Clostridium difficile disease.