Correlation of disease severity with fecal toxin levels in patients with Clostridium difficile-associated diarrhea and distribution of PCR ribotypes and toxin yields in vitro of corresponding isolates

We investigated in vivo and in vitro yields of toxins A and B from and PCR ribotypes of Clostridium difficile isolates from 164 patients with differing severities of C. difficile-associated diarrhea (CDAD) (patients were grouped as follows: <3 loose stools per day, n = 45; 3 to 10 per day, n = 97; >10 per day, n = 22). The median fecal toxin levels in each group were 0.5, 6.8, and 149 U/g feces (P < 0.001), respectively. Patients with severe diarrhea also had more-frequent occurrence of blood in stool and vomiting, but there was no association with fecal toxin levels per se. There was no correlation between fecal toxin level and toxin yield in vitro for the corresponding C. difficile isolate or between its PCR ribotype and disease severity. A broad range of toxin yields among isolates belonging to major PCR ribotypes indicated a presence of many subtypes. We hypothesize that bacterial and host factors that affect C. difficile toxin levels in feces are important determinants of symptoms in CDAD patients. An inverse correlation between toxin yield and spore count (r = 0.66) in stationary-phase cultures supported the notion that toxin production and sporulation represent opposite alternative survival strategies for C. difficile cells facing nutrient shortage.     

Prevalence and clinical features of Clostridium difficile-associated diarrhea in a tertiary hospital in northern Taiwan.

BACKGROUND AND PURPOSE: Although the clinical manifestations of and risk factors for Clostridium difficile-associated diarrhea (CDAD) have been extensively investigated in western populations, data from Taiwanese patients are comparatively limited. This study investigated the incidence density of CDAD in Taiwanese patients and also the risk factors and clinical manifestations of CDAD. METHODS: From September 21, 2003 to December 21, 2003, patients hospitalized in 2 infection wards and 6 medical intensive care units at National Taiwan University Hospital who were older than 20 years, had a history of antibiotic usage within the prior 6 weeks, and developed diarrhea without another identified etiology were classified as having antibiotic-associated diarrhea (AAD), and were enrolled for further study. The diagnosis of CDAD was established when toxin A of C. difficile was detected in stool. RESULTS: The incidence density of AAD was 1/100 person-days of antibiotics usage. CDAD accounted for 12.5% of AAD. Fever and abdominal discomfort developed in only less than half of CDAD patients. Pus cell in the stool sample was found in 100 percent of patients with CDAD. Univariate analysis revealed that presence of malignancy and treatment with antifungal agents within the previous 6 weeks were risk factors for CDAD development. In multivariate analysis, use of antifungal agents was the only independent risk factor for CDAD. CONCLUSION: The incidence density of CDAD in this study of Taiwanese patients with AAD was 12.5%. Prior usage of antifungal agents was the only independent factor associated with subsequent CDAD development in patients with AAD.     

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.     

Risk factors for Clostridium difficile-associated diarrhea on an adult hematology-oncology ward

Nosocomial diarrhea caused by Clostridium difficile causes significant morbidity and mortality in an increasing proportion of hospitalized patients annually. This case-control study of patients admitted to the hematology-oncology ward of a tertiary academic medical center over a 2-year period demonstrates that patients with Clostridium difficile-associated diarrhea (CDAD) were 22 times more likely than ward-matched controls with diarrhea to have received any antibiotic either during hospitalization or in the month preceding admission (p < 0.005), and they were nearly three times as likely as controls to have received a cephalosporin during the same period (p < 0.005). Diarrhea among lung cancer patients was approximately three times more likely to be caused by this organism than to be due to other causes (p = 0.04). A trend towards CDAD patients receiving higher numbers of different antibiotics during hospitalization (3.3 vs. 2.6, 95%CI −1.42–0.02, p = 0.06) was noted. Administration of interleukin-2 either during hospitalization or in the 30 days preceding admission was seven times more likely to have occurred in CDAD cases (p = 0.04), raising the question of whether or not this agent increases risk.     

Clostridium difficile and its cytotoxin in the stools of young hospitalized children. Influence of antibiotic treatment

Clostridium difficile has been searched in 153 stool samples from 138 children aged 0 to 12 months. We divided the population in two groups depending on the antibiotic treatment. We have found C difficile in 39 samples (25%). The colonization rate increases with age ranging from 5% before 1 month, to 36% between 1 and 6 months and 54% between 6 and 13 months. An environmental sampling yielded once C difficile. Contamination may be related to the environment. 29% of the isolates produced a cytopathic toxin. Toxin titers in infants' stools range from 1/160 to 1/10240. One only of these children had diarrhea. C difficile and its toxin does not seem to infer any signs of enteric illness with infants. The results obtained with the group of non treated infants are not significantly different from the ones of the other group: the colonization rates are 21% in the non treated group and 29% in the other group. The rate of strains yielding a cytophatic toxin is similar in the 2 groups. It seems reasonable to agree that antibiotics do not influence the settlement of C difficile in infants' intestine.     

Isolation and molecular characterization of Clostridium difficile strains from patients and the hospital environment in Belarus

Toxigenic Clostridium difficile is the most common etiologic agent of hospital-acquired diarrhea in developed countries. The role of this pathogen in nosocomial diarrhea in Eastern Europe has not been clearly established. The goal of this study was to determine the prevalence of C. difficile in patients and the hospital environment in Belarus and to characterize these isolates as to the presence of toxin genes and their molecular type. C. difficile was isolated from 9 of 509 (1.8%) patients analyzed and recovered from 28 of 1,300 (2. 1%) environmental sites cultured. A multiplex PCR assay was used to analyze the pathogenicity locus (PaLoc) of all isolates, and strain identity was determined by an arbitrarily primed PCR (AP-PCR). The targeted sequences for all the genes in the PaLoc were amplified in all C. difficile strains examined. A predominantly homogeneous group of strains was found among these isolates, with five major AP-PCR groups being identified. Eighty-three percent of environmental isolates were classified into two groups, while patient isolates grouped into three AP-PCR types, two of which were also found in the hospital environment. Although no data on the role of C. difficile infection or epidemiology of C. difficile-associated diarrhea (CDAD) in this country exist, the isolation of toxigenic C. difficile from the hospital environment suggests that this pathogen may be responsible for cases of diarrhea of undiagnosed origin and validates our effort to further investigate the significance of CDAD in Eastern Europe.     

Epidemiology and molecular characterization of Clostridium difficile strains from patients with diarrhea: low disease incidence and evidence of limited cross-infection in a Swedish teaching hospital

We prospectively studied the epidemiology of Clostridium difficile-associated diarrhea (CDAD) in a 900-bed hospital over the course of 12 months by PCR-ribotyping of C. difficile isolates. A total of 304 cases were diagnosed, corresponding to an overall incidence of 7/1,000 admissions, with higher rates in nephrology, hematology, and organ transplantation wards (37, 30, and 21/1,000), and 72% were classified as hospital associated (onset in hospital or onset at home but after a hospital stay within 2 months). All 382 isolates from 227 of 304 (75%) patients available for PCR-ribotyping were typeable, yielding 70 PCR-ribotypes. The three most common types comprised 30% of hospital-associated and 34% of community-associated cases, indicating import via admitted patients as a major source of C. difficile strains occurring in the hospital. Of the 227 patients studied, 38% each contributed 2 to 13 fecal samples positive for C. difficile over the course of the study period. Repeat isolates of the same PCR-ribotype as the first isolate were found in 79% of these patients and in 95% of specimens delivered within 30 days, compared to 63% of those obtained at 31 to 204 days. Nosocomial acquisition of CDAD, defined as the proportion of cases sharing C. difficile type and admitted to the same ward within 2 or 12 months, was 20% and 32% of hospital-associated cases and 14% and 23% of all cases, respectively. Thus, most CDAD cases diagnosed over the course of the study period, including those associated with hospitalization, appeared to be caused by endogenous C. difficile strains rather than by strains truly being acquired in the hospital.     

Molecular epidemiology of hospital-associated and community-acquired Clostridium difficile infection in a Swedish county

All episodes of Clostridium difficile associated diarrhea (CDAD) diagnosed in a defined population of 274,000 including one tertiary and two primary hospitals and their catchment areas were studied during 12 months. The annual CDAD incidence in the county was 97 primary episodes per 100,000, and 78% of all episodes were classified as hospital associated with a mean incidence of 5.3 (range, 1.4 to 6.5) primary episodes per 1,000 admissions. The incidence among hospitalized individuals was 1,300-fold higher than that in the community (33,700 versus 25 primary episodes per 100,000 persons per year), reflecting a 37-fold difference in antibiotic consumption (477 versus 13 defined daily doses [DDD]/1,000 persons/day) and other risk factors. Three tertiary hospital wards with the highest incidence (13 to 36 per 1,000) had CDAD patients of high age (median age of 80 years versus 70 years for other wards, P < 0.001), long hospital stay (up to 25 days versus 4 days), or a high antibiotic consumption rate (up to 2,427 versus 421 DDD/1,000 bed days). PCR ribotyping of C. difficile isolates available from 330 of 372 CDAD episodes indicated nosocomial acquisition of the strain in 17 to 27% of hospital-associated cases, depending on the time interval between index and secondary cases allowed (2 months or up to 12 months), and only 10% of recurrences were due to a new strain of C. difficile (apparent reinfection). In other words, most primary and recurring episodes were apparently caused by the patient's endogenous strain rather than by one of hospital origin. Typing also indicated that a majority of C. difficile strains belonged to international serotypes, and the distribution of types was similar within and outside hospitals and in primary and relapsing CDAD. However, type SE17 was an exception, comprising 22% of hospital isolates compared to 6% of community isolates (P = 0.008) and causing many minor clusters and a silent nosocomial outbreak including 36 to 44% of the CDAD episodes in the three high-incidence wards.     

Multicenter evaluation of four methods for Clostridium difficile detection: ImmunoCard C. difficile, cytotoxin assay, culture, and latex agglutination.

A three-center study was undertaken to compare several test methods for the detection of Clostridium difficile, associated toxin, or related markers by using 927 stool specimens. Methods included direct assay of cytotoxin in stool by tissue culture, C. difficile bacterial culture followed by cytotoxin assay, bacterial culture alone, latex agglutination assay, and the ImmunoCard C. difficile test (Meridian Diagnostics, Inc.). The sensitivities, as determined against direct cytotoxin assay results, of the ImmunoCard C. difficile and latex agglutination assays were 84 and 67%, respectively (92 and 77%, respectively, when adjusted for bacterial culture outcomes). Evaluation for C. difficile-associated disease (CDAD) among 864 patients was based on clinical criteria for antibiotic-associated diarrhea combined with laboratory evidence of toxin or toxin-producing C. difficile in stool specimens. The sensitivity of each test method for screening of CDAD was as follows: bacterial culture, 95%; culture with cytotoxin assay of isolates, 90%; ImmunoCard C. difficile test, 83%; cytotoxin assay 82%; and latex agglutination assay, 67% (P < or = 0.05 versus all other methods). The standard deviations of the test sensitivity statistics between study sites were ranked as follows: cytotoxin assay (+/- 3.1%) < ImmunoCard C. difficile test (+/- 5.7%) < latex agglutination assay (+/- 12.3%) < culture (+/- 24.7%) < culture with cytotoxin assay (+/- 28.0%). The data support the use of the ImmunoCard C. difficile test as an adjunct for the diagnosis of CDAD.     

Relative frequency of Clostridium difficile in patients with diarrheal disease.

We have studied 161 patients with diarrheal disease to determine the frequency with which Clostridium difficile occurs in such patients. C. difficile or its toxin or both were detected in stools from 19 patients (11.9%), 17 of whom had previously received antimicrobial agents. Enteric pathogens other than C. difficile were recovered less frequently, with Salmonella sp., Giardia lamblia, and Campylobacter fetus being recovered from 4.1, 2.5, and 1.3%, respectively, of the patients studied. These data suggest that C. difficile may be frequently encountered in specimens obtained from patients with diarrhea who receive antibiotics and may play a role in diarrheal disease in such patients.     

Comparison of three enzyme immunoassays, a cytotoxicity assay, and toxigenic culture for diagnosis of Clostridium difficile-associated diarrhea.

Enzyme immunoassays (EIAs) based on monoclonal antibodies for the detection of Clostridium difficile toxins have recently been developed for clinical use. The aim of this study was to compare three commercially available EIAs, two for toxin A (Premier C. difficile Toxin A; Meridian, Osi, Elancourt, France; and Vidas C. difficile Toxin A; bioMérieux, Marcy l'Etoile, France) and one for toxins A and B (Cytoclone A + B EIA; Cambridge Biotech Corp., Codiapharm, Evian, France), with a cytotoxicity assay and toxigenic culture for the diagnosis of C. difficile-associated diarrhea (CDAD). The study was performed with 285 fresh stools from 285 patients with suspected CDAD. In case of disagreement, the tests were repeated on a frozen aliquot of the same stool sample, and the patient's chart was reviewed. CDAD diagnosis was established in 55 cases (incidence, 19.3%). The sensitivities and specificities of the methods were, respectively, 92.7 and 100% for the cytotoxicity assay, 96.4 and 99.1% for toxigenic culture, 75.5 and 97.8% for Cytoclone, 65.4 and 99.6% for Premier, and 65.4 and 100% for Vidas. The results were uninterpretable in 3.2% of cases with Cytoclone, 0.3% with Premier, and 2.5% with Vidas. We conclude that the cytotoxicity assay and toxigenic culture remain the best methods for the diagnosis of CDAD even though they lack standardization and require 48 to 96 h to obtain the result. Despite their rapidity and simplicity, EIAs are not sensitive enough to be relied on as the sole laboratory test.     

Etiology of childhood diarrhea in Korea.

To assess the role of recently recognized enteropathogens in childhood diarrhea in Korea, 231 children with diarrhea admitted to and 104 children without diarrhea seen at the well-baby clinic or the outpatient department of Hanyang University Hospital in Seoul, Korea, were evaluated during a 14-month period. Stools were cultured for bacterial pathogens, including enterotoxigenic (heat-labile and heat-stable enterotoxin-producing) and enteroadherent organisms. Only those stools obtained from patients with diarrhea were examined for rotavirus. All Escherichia coli isolates were screened for Shiga-like toxin (SLT) I, SLT-II, enterohemorrhagic E. coli fimbriae, and enteroinvasiveness by colony hybridization. One or more pathogens were identified in 75.8% of the children with diarrhea. Rotavirus was the most frequently identified pathogen, accounting for 47% of the cases. Other major enteropathogens were enterotoxigenic E. coli (22%), Clostridium difficile (16%), enteroadherent E. coli (15%), and enteropathogenic E. coli (6%). Shigella spp., Campylobacter jejuni, Salmonella spp., SLT-I-and enterohemorrhagic-E. coli-fimbria-probe-positive E. coli serotype O26:H11 and enteroinvasive E. coli were isolated from only a few patients. Aeromonas hydrophila and E. coli O157 were not isolated. Compared with those of the controls, the isolation rates of heat-stable-enterotoxin-producing E. coli (P less than 0.05), C. difficile (P less than 0.025), and enteroadherent E. coli (P less than 0.05) were significantly higher in the patients with diarrhea. The greatest number of rotavirus, enterotoxigenic E. coli, and C. difficile cases were identified during the cool, dry months of October and November.     

Pseudomonas aeruginosa as a potential cause of antibiotic-associated diarrhea.

Although Pseudomonas aeruginosa is not generally considered as a cause of antibiotic-associated diarrhea, several cases of diarrhea caused by P. aeruginosa have been reported. We experienced seven cases of nosocomial diarrhea presumably caused by P. aeruginosa, which was the predominant organism isolated from stool cultures. Clostridium difficile toxin was also positive in one patient. No other potential or recognized enteropathogens were identified from stools. All patients had underlying diseases and had been receiving antibiotics before the diarrheal onset. All of the seven P. aeruginosa isolates were resistant to previously given antibiotics. Diarrhea stopped three days after withdrawal of probable offending antibiotics without specific treatment in two patients. The other five patients having continuous diarrhea despite withdrawal of probable offending antibiotics, were successfully treated with antipseudomonal agents. The median duration of diarrhea after the initiation of treatment was 6.3 days. These data suggest that P. aeruginosa can be a potential cause of antibiotic-associated diarrhea. Further investigations are warranted to evaluate the possible etiologic role of P. aeruginosa in antibiotic-associated diarrhea.     

Human antibody response to Clostridium difficile toxin A in relation to clinical course of infection.

This study investigated whether differences in fecal and serum antitoxin A antibody levels may account for the duration of Clostridium difficile-associated diarrhea (CDAD) and the occurrence of relapses. By an enzyme linked-immunosorbent assay, we tested 40 patients with CDAD including 25 patients without immunodeficiency and 15 patients receiving antineoplastic drugs. Two hundred eighty serum samples and 80 normal stool samples were investigated as controls. In nonimmunocompromised patients, serum immunoglobulin (IgG) and fecal IgA antitoxin A antibody titers were significantly higher in patients who suffered a single episode (n = 21) than in those with relapsing CDAD (n = 4) whose titers were at control levels. Of these 25 patients, eight suffered from diarrhea which lasted for more than 2 weeks. These patients had significantly lower serum- and feces-specific antibody levels than the others who presented symptoms of shorter duration. In cytostatic-treated patients, antitoxin A antibody levels were similar to controls, but relapses occurred in a single case. These data suggest an association between a defective humoral response to toxin A and a more severe form of C. difficile infection. They also indicate that other host-related factors control the severity of CDAD and remain to be elucidated.     

Development and evaluation of a PCR method for detection of the Clostridium difficile toxin B gene in stool specimens

A PCR assay detecting Clostridium difficile toxin B gene in stool specimens was compared to the cytotoxicity assay as the reference standard for the diagnosis of C. difficile antibiotic-associated diarrhea (CDAD). Overall, 118 stool samples were tested. All of the specimens that were negative by the cytotoxicity assay (59 out of 118) were also negative by the PCR method (specificity of 100%). Of the 59 cytotoxin-positive samples, 54 were PCR positive (sensitivity of 91.5%). This PCR method is promising for rapid diagnosis of CDAD.     

Detection of toxigenic Clostridium difficile: comparison of the cell culture neutralization, Xpert C. difficile, Xpert C. difficile/Epi, and Illumigene C. difficile assays

Clostridium difficile is the most important cause of nosocomial diarrhea. Several laboratory techniques are available to detect C. difficile toxins or the genes that encode them in fecal samples. We evaluated the Xpert C. difficile and Xpert C. difficile/Epi (Cepheid, CA) that detect the toxin B gene (tcdB) and tcdB, cdt, and a deletion in tcdC associated with the 027/NAP1/BI strain, respectively, by real-time PCR, and the Illumigene C. difficile (Meridian Bioscience, Inc.) that detects the toxin A gene (tcdA) by loop-mediated isothermal amplification in stool specimens. Toxigenic culture was used as the reference method for discrepant stool specimens. Two hundred prospective and fifty retrospective diarrheal stool specimens were tested simultaneously by the cell cytotoxin neutralization assay (CCNA) and the Xpert C. difficile, Xpert C. difficile/Epi, and Illumigene C. difficile assays. Of the 200 prospective stools tested, 10.5% (n = 23) were determined to be positive by CCNA, 17.5% (n = 35) were determined to be positive by Illumigene C. difficile, and 21.5% (n = 43) were determined to be positive by Xpert C. difficile and Xpert C. difficile/Epi. Of the 50 retrospective stools, previously determined to be positive by CCNA, 94% (n = 47) were determined to be positive by Illumigene C. difficile and 100% (n = 50) were determined to be positive by Xpert C. difficile and Xpert C. difficile/Epi. Of the 11 discrepant results (i.e., negative by Illumigene C. difficile but positive by Xpert C. difficile and Xpert C. difficile/Epi), all were determined to be positive by the toxigenic culture. A total of 21% of the isolates were presumptively identified by the Xpert C. difficile/Epi as the 027/NAP1/BI strain. The Xpert C. difficile and Xpert C. difficile/Epi assays were the most sensitive, rapid, and easy-to use assays for the detection of toxigenic C. difficile in stool specimens.     

Risk factors of Clostridium difficile-associated diarrhea in hospitalized adults: Vary by hospitalized duration

BackgroundClostridium difficile is the leading cause of nosocomial infectious diarrhea. Hospitalized patients were at risk of C. difficile-associated diarrhea (CDAD). However the risk factors of CDAD in patients with different hospitalization period are not clear.Material and methodsA prospective investigation was conducted in medical wards of a district hospital in southern Taiwan, from January 2011 to January 2013. We arbitrary divided patients into two groups: hospitalized for at most 14 days and 15–30 days, and analyzed their risk factors for CDAD.ResultsOverall 451 patients were enrolled. The multivariable analysis of 19 (8.0%) patients developing CDAD within 14 days' hospital stay and 216 patients hospitalized for ≤ 14 days without CDAD showed malignancy (odds ratio [OR] 7.15, 95% confidence interval [CI] 1.82–28.09; P = 0.005), prior cephalosporin (OR 10.8, 95% CI 1.3–93.9; P = 0.03) and proton pump inhibitor (PPI; OR 7.1, 95% CI 2.1–24.7; P = 0.002) therapy were independently related to CDAD (Table 3), but hypertension (OR 0.2, 95% CI 0.1–0.7; P = 0.01) was reversely related to CDAD. However, of 9 (4.2%) patients developing CDAD later (15–30 days' hospital stay) and 207 patients with longer hospitalization (15–30 days) but free of CDAD, malignancy (OR 14.0, 95% CI 1.6–124.9; P = 0.02) and underlying diabetes mellitus (OR 20.5, 95% CI 2.9–144.9; P = 0.002) were independent risk factors of CDAD.ConclusionRisk factors for CDAD among hospitalized patients varied by the duration of hospital stay. Intervention strategies to prevent CDAD may be different in terms of hospital stay duration.     

Incidence and origin of Clostridium difficile in neonates

The stools of 65 of 92 (71%) infants in a special care nursery yielded Clostridium difficile on culture. Ninety percent of stools collected after 6 to 35 days in the unit were positive, and 36% of these also contained toxin. When tested in vitro, 94% of the isolates produced toxin. Of 110 swabs collected from the environment of the unit, 9% were positive for C. difficile, but the stools of 12 nurses working on the unit were negative. Thirty-five vaginal swabs collected from mothers just before delivery were negative for C. difficile on culture, but 16 of their infants had C. difficile in their stools. It was concluded that there is a high carriage rate in the stools of neonates of C. difficile acquired progressively during the course of their stay in the special care unit. Infection is mainly from environmental sources rather than maternal transmission.     

Asymptomatic carriage of Clostridium difficile and serum levels of IgG antibody against toxin A

BACKGROUND: Clostridium difficile infection can result in asymptomatic carriage, mild diarrhea, or fulminant pseudomembranous colitis. We studied whether antibody responses to C. difficile toxins affect the risks of colonization, diarrhea, and asymptomatic carriage. METHODS: We prospectively studied C. difficile infections in hospitalized patients who were receiving antibiotics. Serial stool samples were tested for C. difficile colonization by cytotoxin assay and culture. Serum antibody (IgA, IgG, and IgM) levels and fecal antibody (IgA and IgG) levels against C. difficile toxin A, toxin B, and nontoxin antigens were measured by an enzyme-linked immunosorbent assay (ELISA). RESULTS: Of 271 patients, 37 (14 percent) were colonized with C. difficile at the time of admission, 18 of whom were asymptomatic carriers. An additional 47 patients (17 percent) became infected in the hospital, 19 of whom remained asymptomatic. The baseline antibody levels were similar in the patients who later became colonized and those who did not. After colonization, those who became asymptomatic carriers had significantly greater increases in serum levels of IgG antibody against toxin A than did the patients in whom C. difficile diarrhea developed (P<0.001). The adjusted odds ratio for diarrhea was 48.0 (95 percent confidence interval, 3.4 to 678) among patients with colonization who had a serum level of IgG antibody against toxin A of 3.00 ELISA units or less, as compared with patients with colonization who had a level of more than 3.00 ELISA units. CONCLUSIONS: We find no evidence of immune protection against colonization by C. difficile. However, after colonization there is an association between a systemic anamnestic response to toxin A, as evidenced by increased serum levels of IgG antibody against toxin A, and asymptomatic carriage of C. difficile.