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Prevalent PCR ribotypes of clinical and environmental strains of Clostridium difficile isolated from intensive-therapy unit patients in Kuwait.

Ninety-five isolates of Clostridium difficile from symptomatic and asymptomatic patients and 18 from their environment in the intensive-therapy units (ITUs) of four teaching hospitals in Kuwait were typed by PCR amplification of rRNA intergenic spacer regions (PCR ribotyping). A total of 32 different ribotypes was detected among the clinical isolates. The predominant ribotypes from the clinical isolates were types 097 and 078, which accounted for approximately 40 % of all isolates in the ITUs in Kuwait. Ribotypes 097 (toxigenic), 078 (toxigenic) and 039 (non-toxigenic) were three distinct clones that were circulating in all four hospitals. Ribotypes 097, 078 and 076 (i.e. 50 % of isolates from symptomatic patients) were the predominant isolates associated with C. difficile-associated disease (CDAD). The environmental isolates belonged to a diverse range of ribotypes, with no particular types common to all the hospitals. Ribotype 078 was found only in the patient environment in Mubarak hospital, while ribotype 097 was restricted to Amiri hospital. The hospital environment occupied by symptomatic as well as symptom-free patients was contaminated with C. difficile. Eight new strains that did not match any in the PCR ribotype library established at the PHLS Anaerobe Reference Unit, Cardiff, UK, were assigned ribotypes 105, 125, 128, 129, 131, 134, 140 and 141. These findings show that the isolates associated with CDAD in Kuwait are different from those found in the UK and some other European countries.

Clostridioides difficile↗

Comparison of the effect of detergent versus hypochlorite cleaning on environmental contamination and incidence of Clostridium difficile infection.

To determine how best to decontaminate the hospital environment of Clostridium difficile, we carried out a cross-over study on two elderly medicine wards to determine whether cleaning with a hypochlorite disinfectant was better than using neutral detergent in reducing the incidence of C. difficile infection (CDI). We examined 1128 environmental samples in two years, 35% of which grew C. difficile. There was a significant decrease of CDI incidence on ward X, from 8.9 to 5.3 cases per 100 admissions (P<0.05) using hypochlorite, but there was no significant effect on ward Y. On ward X the incidence of CDI was significantly associated with the proportion of culture-positive environmental sites (P<0.05). On ward Y the only significant correlation between CDI and C. difficile culture-positive environmental sites was in patient side-rooms (r=0.41, P<0.05). The total daily defined doses of cefotaxime, cephradine and aminopenicillins were similar throughout the trial. These results provide some evidence that use of hypochlorite for environmental cleaning may significantly reduce incidence of CDI, but emphasize the potential for confounding factors.

Aged↗

Saccharomyces boulardii for Clostridium difficile-associated enteropathies in infants.

Based on experimental evidence in animals showing that the oral administration of Saccharomyces boulardii is effective in reducing morbidity and mortality due to Clostridium difficile-induced pseudomembranous colitis, we conducted an open trial to examine the effects of the living yeast, given as primary therapy, in a selected group of infants and children with persistent intestinal symptoms related to toxinogenic C. difficile overgrowth. Over a period of 10 consecutive months, we studied 19 eligible patients (median age 8 months) who presented with enteral symptoms lasting for > 15 days and who had solely C. difficile in stools with positive cytotoxin B assay. Serotyping of the strains and determination in vitro of production of toxins A and B were performed subsequently. The patients presented with persistent or protracted diarrhea, malabsorption, and failure to grow (n = 8), or with repeated attacks of colics, emesis, and hypermeteorism without diarrhea (n = 4), or with both entities (n = 7). Patients with chronic protracted diarrhea (n = 3) had depressed jejunal disaccharidase activities and ultrastructural changes of enterocytes, including sparce and shortened microvilli. None had evidence of colitis. All the strains of C. difficile tested (n = 17) belonged to pathogenic serotypes (A1, A8, C, F, G, H, and K) and produced in vitro high levels of toxins A (n = 16) and B (n = 17). S. boulardii was given orally in a lyophilized form over 15 days (250 mg two to four times per day according to age).(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗

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.

Adolescent↗

[Evaluation of four rapid methods for the investigation of the toxigenic capacity of Clostridium difficile strains isolated in a selective medium].

BACKGROUND: Use of selective Clostridium difficile culture as a diagnostic method for C. difficile associated disease requires to prove the toxigenic ability of the isolates. Toxin B detection by cell culture assay after growing the microorganism in enriched broth is the standard method, but it delays the final diagnosis for 3-5 days. This study compares retrospectively four rapid techniques for detecting these toxigenic C. difficile strains. METHODS: 106 clinical isolates of C. difficile (72 toxigenic and 34 non-toxigenic), these and 16 clinical strains of other species of Clostridium were investigated. The four methods were performed directly from colonies growing on solid agar. They were: a) cytotoxin detection in cell culture; b) two PCR amplifications of toxin A and toxin B, respectively, and c) toxin A detection by an immunoenzymatic method (VIDAS CDA2). All these procedures were completed within a normal working day. RESULTS: Only the 72 toxigenic C. difficile strains gave positive results by cell culture and PCR techniques (sensitivity and specificity: 100%). A total of 14 out of 49 toxigenic C. difficile strains showed negative results by the VIDAS assay in the first run, but all them were positive in repeated tests. CONCLUSIONS: Although all methods performed well, the cytotoxicity assay done directly on colonies growing in CCFA is a simple and rapid technique, and appears to be well-suited for use in laboratories with access to cell cultures.

Agar↗

[Diarrhea associated with Clostridium difficile. One-year retrospective study at a tertiary hospital].

BACKGROUND: Diarrhea associated with Clostridium difficile is a health care problem of growing importance in the last few years specially in the hospital environment. The epidemiologic data and factors associated with this disease have not, to date, been sufficiently studied in Spain. METHODS: The cases of diarrhea associated with C. difficile reported in 1996 in a tertiary hospital of 1,500 beds were retrospectively reviewed, collecting clinical and epidemiologic data. The technique used for the detection of the C. difficile toxin was EIA Premier. RESULTS: One hundred thirty-two patients were included in the study, 83.3% of whom were over the age of 65 years, who had had 148 episodes of diarrhea associated with C. difficile. Most had been admitted into internal medicine (36%) or in the geriatric department (25%) and the remaining in the surgical departments (16.4%) or others (22.6%). The most frequently prescribed antibiotics were third generation cephalosporins (28.6%), clindamycin (17%), quinolones (11.1%) and macrolides (9.1%). Most of the patients received from 2 to 4 antibiotics prior to presenting diarrhea. Thirteen percent of the episodes of diarrhea associated with C. difficile were exclusively treated with withdrawal of the prescribed antibiotic, while the remaining cases were also given specific treatment which in 68.6% of the cases was with metronidazole and 31.4% with vancomycin. No significant difference was observed in the evolution of the patients according to the antibiotic prescribed. CONCLUSIONS: Diarrhea associated with C. difficile should be taken into account as a frequent complication of wide spectrum antibiotic treatment, specially in the elderly, immunosuppressed or in patients with pluripathology. With this study the authors wish to underline the need for the judicious use of antibiotics in the hospital environment and aid in the rapid diagnosis of this entity.

Adult↗

Nosocomial outbreak of Clostridium difficile diarrhea in a pediatric service.

An outbreak of nosocomial diarrhea that occurred in a pediatric orthopedic service between 1 December 1993 and 15 April 1994 is reported. A total of 37 patients (mean age, 9.6 years; range, 2 months-19.3 years) were involved in the outbreak, including six patients with bacteriologically documented Clostridium difficile infection. A multivariate analysis identified lincomycin treatment for at least three days as the only significant risk factor. Stool samples from four asymptomatic patients were also positive for Clostridium difficile and its cytotoxins. Isolates from all patients belonged to serogroup C, were highly resistant to lincomycin, and exhibited the same restriction pattern by pulsed-field gel electrophoresis. The outbreak ended after treatment with lincomycin was discontinued and hygiene control measures were implemented.

Adolescent↗

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.

Animals↗

Infectious gastro-enteritis: an uncommon cause of diarrhoea in adult allogeneic and autologous stem cell transplant recipients.

The incidence and aetiology of acute diarrhoea in 60 adult allogeneic or autologous stem cell transplant (SCT) recipients was determined in a prospective study. Stool specimens were obtained prior to SCT and on days +20, +40, +60 and +100 post transplant. Microbiological evaluation was performed for pathogenic bacteria, fungi, parasites and viruses. Forty-seven patients were evaluable of whom 31 had a total of 48 acute diarrhoeal episodes. Diarrhoea occurred in 79% of allogeneic and 47% of autologous SCT recipients (P < 0.05). Intestinal infections were found in three of 48 (6%) diarrhoeal episodes. Clostridium difficile with positive toxin was cultured twice and one stool specimen was positive for cryptosporidium. Intestinal pathogens were identified in 13 out of 172 stool specimens from asymptomatic patients and included: rotavirus (4), adenovirus (3), C. difficile, toxin positive (2), and others (4). Graft-versus-host disease was confirmed by biopsy in two of 36 episodes of diarrhoea in allogeneic patients, and in three patients a relationship between reactivation of cytomegalovirus and diarrhoea was suspected. In 40 of 48 (83%) episodes of diarrhoea no clear aetiology could be found.

Adult↗

Regulated transcription of Clostridium difficile toxin genes.

The Clostridium difficile toxA and toxB genes, encoding cytotoxic and enterotoxic proteins responsible for antibiotic-associated colitis and pseudomembranous colitis, were shown to be transcribed both from gene-specific promoters and from promoters of upstream genes. However, the gene-specific transcripts represented the majority of tox gene mRNAs. The 5' ends of these mRNAs were shown to correspond to DNA sequences that had promoter activity when fused to the Escherichia coli beta-glucuronidase (gusA) gene and introduced into C. perfringens. The appearance of tox mRNA in C. difficile was repressed during exponential growth phase but increased substantially as cells entered stationary phase. When glucose or other rapidly metabolizable sugars were present in the medium, the stationary phase-associated induction was inhibited, indicating that the toxin genes are subject to a form of catabolite repression. This glucose effect was general to many toxinogenic strains having varying levels of toxin production.

Bacterial Toxins↗

An investigation of a nosocomial outbreak of Clostridium difficile by pyrolysis mass spectrometry.

Isolates from a presumptive nosocomial outbreak of Clostridium difficile infection at a large teaching hospital were typed by pyrolysis mass spectrometry (PMS) and antibiograms. One isolate, from the putative index case, was dissimilar from the outbreak strain, but 24 isolates from 16 patients were indistinguishable by both methods. The outbreak centred on two wards for the acute care of the elderly, with a few cases elsewhere. Transfer of patients appeared to be the route of transmission between wards. There was a significant fall in the incidence of cases following intervention by the Infection Control Unit. This included ward inspection, advice on antibiotic usage and advice on prevention of faecal-oral transfer, particularly by proper handwashing. Subsequent monitoring of C. difficile infection showed a background of sporadic, dissimilar isolates with occasional apparent cross-infection incidents limited to a few patients. In suspected outbreaks, patterns of antibiotic susceptibility may be useful in initial screening, before referral for more sophisticated typing. There was excellent correlation between PMS results, antibiograms and epidemiological information.

Aged↗

Subunit composition of the glycyl radical enzyme p-hydroxyphenylacetate decarboxylase. A small subunit, HpdC, is essential for catalytic activity.

p-Hydroxyphenylacetate decarboxylase from Clostridium difficile catalyses the decarboxylation of p-hydroxyphenylacetate to yield the cytotoxic compound p-cresol. The three genes encoding two subunits of the glycyl-radical enzyme and the activating enzyme have been cloned and expressed in Escherichia coli. The recombinant enzymes were used to reconstitute a catalytically functional system in vitro. In contrast with the decarboxylase purified from C. difficile, which was an almost inactive homo-dimeric protein (beta(2)), the recombinant enzyme was a hetero-octameric (beta(4)gamma(4)), catalytically competent complex, which was activated using endogenous activating enzyme from C. difficile or recombinant activating enzyme to a specific activity of 7 U.mg(-1). Preliminary results suggest that phosphorylation of the small subunit is responsible for the change of the oligomeric state. These data point to an essential function of the small subunit of the decarboxylase and may indicate unique regulatory properties of the system.

Amino Acid Sequence↗

Use of arbitrary primer PCR to type Clostridium difficile and comparison of results with those by immunoblot typing.

An arbitrarily primed PCR (AP-PCR) assay was used to type Clostridium difficile isolates from a hospital outbreak of antibiotic-associated diarrhea. Forty-one isolates were separated into nine groups, with 66% falling into one group; no other group contained more than 10%. Comparison of AP-PCR grouping with that when the immunoblot technique was used showed agreement for 33 of 34 isolates typed by both techniques, and AP-PCR grouped seven isolates that were not typeable by immunoblotting.

Base Sequence↗

[Clostridium difficile: a typing method for epidemiological studies].

One hundred and twenty two strains of Clostridium difficile were studied: 33 strains were isolated from stools of neonates or children, 89 cases of antibiotic associated diarrhea. The strains were identified by biochemical tests and gaz liquid chromatography of the fermentation products. A typing scheme for Clostridium difficile were proposed by using a polyacrylamide gel electrophoresis of whole cell proteins. We studied the correlation between this typing and the serogrouping scheme based on slide agglutination with a rabbit antisera and previously described by Delmée. The serogrouping defines 10 groups, PAGE allows the differentiation of 12 profiles, with a good correlation between the two methods for some serogroups except for the heterogenous serogroup A.

Bacterial Proteins↗

Laboratory diagnosis of Clostridium difficile disease.

The laboratory diagnosis of Clostridium difficile-associated disease (CDAD) is based on culture and toxin detection in fecal specimens. Culture is performed on a commercially available selective media. C. difficile colony morphology is typical when viewed under a dissecting microscope. Definitive identification is best obtained by gas liquid chromatography. Culture is very sensitive but, when used alone without toxin testing, it leads to low specificity and misdiagnosis of CDAD when high rates of asymptomatic carriage exist. Toxin detection by a tissue culture cytotoxin assay followed by neutralisation with specific antiserum is often considered the standard. However, this approach lacks sensitivity and has not detected up to 30% of patients with confirmed CDAD. Multiple enzyme immunoassays (EIAs) have been introduced by various manufacturers for the detection of toxin A alone or for both toxins A and B. Some of these are designed to give results in less than 1 h. Comparative studies of EIA kits reported that the sensitivity and specificity are slightly lower than cytotoxin assays. Toxigenic culture tests C. difficile isolates for toxin production: colonies isolated on selective media are tested for in-vitro toxin production either by a cytotoxicity assay or by direct EIA. It has higher sensitivity than the cytotoxicity assay and equivalent specificity. In the routine laboratory, culture and toxin detection should be performed on every specimen and, in culture-positive and fecal toxin-negative cases, toxigenic cultures should be performed on isolated colonies.

Antibodies, Bacterial↗

Toxigenic status of Clostridium difficile in a large Spanish teaching hospital.

The aim of this study was to evaluate the toxigenic status of circulating strains of Clostridium difficile in a large teaching hospital. Overall 220 isolates were studied of which 199 (90.5 %) produced both large clostridial toxins detected by conventional methods. Ten more strains (4.5 %) had toxin A and B genes detectable by PCR. Eleven (5.0 %) variant strains (A- B+) were detected among the isolates studied and 10 strains (4.5 %) had the binary toxin genes (cdtA and cdtB).

Bacterial Proteins↗

HIV and diarrhea in the era of HAART: 1998 New York State hospitalizations.

BACKGROUND: This study reflects an attempt to identify the causes of diarrheal illness in hospitalized HIV patients in light of therapeutic advancements in HIV management. METHODS: The study identifies the various etiologies associated with diarrhea among HIV patients hospitalized in New York State in 1998. Data for this study were extracted from the New York State Department of Health Statewide Planning and Research Cooperative System. Pathogens recognized to cause diarrhea in persons with HIV and general codes identifying diarrhea were examined by using the principal and all secondary diagnoses based on the International Classification of Diseases 9th Revision Clinical Modification codes. RESULTS: Based on the Statewide Planning and Research Cooperative System data set, more than 15,000 patients with HIV were hospitalized in 1998. Among the HIV patients hospitalized, 2.8% were admitted with a diarrheal diagnosis. The following diagnoses occurred the most frequently among HIV patients hospitalized with a diarrheal illness: Clostridium difficile (51.3%), other protozoal diseases (18.1%), and other organisms, not elsewhere specified (11.7%). CONCLUSIONS: In the era of highly active antiretroviral therapy, diarrhea is still an occurring symptom in HIV patients. Despite the relatively small percentage of hospitalizations attributed to diarrhea, clinicians must remember that even "mild" to "moderate" diarrhea can have a debilitating impact among persons with the symptom.

AIDS-Related Opportunistic Infections↗

The role of Clostridium difficile and viruses as causes of nosocomial diarrhea in children.

OBJECTIVE: We report surveillance of nosocomial diarrhea in children at our institution during the past decade and note different epidemiology of diarrhea due to viruses and Clostridium difficile. DESIGN: A prospective cohort study. SETTING: A university-affiliated pediatric hospital with 180 beds serving an urban area and providing referral care for the Maritime Provinces of Canada. PARTICIPANTS: Children younger than 18 years. METHODS: Surveillance was conducted from 1991 to 1999 using personal contact with personnel and review of microbiology and medical records. Nosocomial diarrhea was defined as loose stools occurring more than 48 hours after admission, with at least two loose stools in 12 hours and no likely non-infectious cause. RESULTS: Nosocomial diarrhea was the third most common nosocomial infection (217 of 1,466; 15%), after bloodstream and respiratory infections, with from 0.5 to 1 episode per 1,000 patient-days. Of 217 nosocomial diarrhea episodes, 122 (56%) had identified pathogens: C. difficile (39 of 122; 32%), rotavirus (38 of 122; 31%), adenovirus (36 of 122; 30%), and other viral (9 of 122; 7%). The median age was 1.3 years (range, 11 days to 17.9 years), 0.80 year for children with viral diarrhea, 3.9 years for children with C. difficile, and 1.5 years for children with diarrhea without a causative organism identified (P< .0001). Most children with nosocomial diarrhea were incontinent (diapered) at the time of their first episode (138 of 185; 75%), but preexisting incontinence was more common in those with viral diarrhea (93%) compared with those with no organism identified (71%) or those with C. difficile-associated diarrhea (CDAD) (49%) (P <.0001). CONCLUSIONS: C. difficile is the single most common cause of nosocomial diarrhea in our tertiary-care center, although all viral pathogens account for 69% of cases. Diapered status appears to be a risk factor for CDAD in children, and CDAD occurs more often in older children than viral nosocomial diarrhea. Further characterization of risk factors for, and morbidity associated with, nosocomial CDAD in children is warranted.

Child↗