Molecular epidemiology of endemic Clostridium difficile infection

This is the first study to provide a comprehensive insight into the molecular epidemiology of endemic Clostridium difficile and particularly that associated with a recently recognized epidemic strain. We DNA fingerprinted all C. difficile isolates from the stools of patients with symptomatic antibiotic-associated diarrhoea and from repeated samples of the inanimate ward environment on two elderly medicine hospital wards over a 22-month period. Notably, C. difficile was not recoverable from either ward immediately before opening, but was found on both wards within 1-3 weeks of opening, and the level of environmental contamination rose markedly during the first 6 months of the study period. C. difficile infection (CDI) incidence data correlated significantly with the prevalence of environmental C. difficile on ward B (r = 0.76, P < 0.05) but not on ward A (r = 0.26, P > 0.05). We found that RAPD and RS-PCR typing had similar discriminatory power, although, despite fingerprinting over 200 C. difficile isolates, we identified only six distinct types. Only two distinct C. difficile strains were identified as causing both patient infection and ward contamination. Attempts to determine whether infected patients or contaminated environments are the prime source for cross-infection by C. difficile had limited success, as over 90% of C. difficile isolates were the UK epidemic clone. However, a non-epidemic strain caused a cluster of six cases of CDI, but was only isolated from the environment after the sixth patient became symptomatic. The initial absence of this strain from the environment implies patient-to-patient and/or staff-to-patient spread. In general, routine cleaning with detergent was unsuccessful at removing C. difficile from the environment. Understanding the epidemiology and virulence of prevalent strains is important if CDI is to be successfully controlled.     

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.     

Emergence and control of fluoroquinolone-resistant, toxin A-negative, toxin B-positive Clostridium difficile.

BACKGROUND: Clostridium difficile is a major cause of infectious diarrhea in hospitalized patients. Between August 2003 and January 2004, we experienced an increase in the incidence of C. difficile-associated disease. We describe the investigation into and management of the outbreak in this article. METHODS: A total of 73 consecutive patients with nosocomial C. difficile-associated diarrhea were identified. C. difficile isolates were characterized using toxin-specific enzyme immunoassays, a tissue-culture fibroblast cytotoxicity assay, polymerase chain reaction (PCR), and antimicrobial susceptibility tests. Rates of recurrence and of C. difficile colitis were recorded. Changes in antibiotic use and infection control policies were documented. RESULTS: The incidence of C. difficile-associated diarrhea peaked at 21 cases per 1,000 patient admissions. Of the C. difficile isolates recovered, 85 (95%) were identical toxin A-negative and toxin B-positive strains, corresponding to toxinotype VIII and PCR ribotype 017. All clonal isolates were resistant to multiple antibiotics, including ofloxacin, ciprofloxacin, levofloxacin, moxifloxacin, and gatifloxacin (minimum inhibitory concentrations [MICs] of greater than 32 micro g/mL) and erythromycin, clarithromycin, and clindamycin (MICs of greater than 256 micro g/mL). Recurrent C. difficile-associated disease occurred in 26 (36%) of the patients. At least 10 (14%) of the patients developed C. difficile colitis. Additional infection control measures introduced included the use of ward memos, a hand-hygiene awareness campaign, increased environmental cleaning, attention to prescribing practices for antibiotics, increased awareness of diarrheal illness, and early isolation of affected patients. Total use of fluoroquinolones did not change throughout the study period. Despite persistence of this toxin-variant strain, the incidence of C. difficile-associated disease in our institution decreased to fewer than 5 cases per 1,000 admissions. CONCLUSIONS: We report on the emergence of a fluoroquinolone- and clindamycin-resistant, toxin A-negative, and toxin B-positive strain of C. difficile associated with an outbreak of C. difficile-associated disease in our institution during a 6-month period. We found that careful attention to improvement of infection control interventions was the most important means of controlling this nosocomial pathogen.     

Prospective evaluation of environmental contamination by Clostridium difficile in isolation side rooms.

We determined prospectively the frequency, persistence and molecular epidemiology of Clostridium difficile environmental contamination after detergent-based cleaning in side rooms used to isolate patients with C. difficile diarrhoea. Approximately one-quarter of all environmental sites in side rooms sampled over four-week periods were contaminated with C. difficile. The overall side room prevalence of environmental C. difficile declined from 35% initially, to 24% in week 2, 18% in week 3, and 16% in week 4. The bed frame was the most common site from which C. difficile was recovered, although the floor was the most contaminated site in terms of total numbers of colonies. C. difficile was recovered significantly more frequently from swabs plated directly on to C. difficile selective media containing lysozyme than from enrichment broth (P< 0.001), emphasizing the benefit of lysozyme supplementation. The great majority of C. difficile isolates (87% of all isolates, 84% of patient isolates) was indistinguishable from the UK epidemic strain (PCR ribotype 1). It thus could not be determined whether environmental contamination was a cause or a consequence of diarrhoea. Our findings highlight the need for improved approaches to hospital environmental hygiene, and call into question current UK guidelines that recommend detergent-based cleaning to remove environmental C. difficile. In particular, improved cleaning of frequently touched sites in the immediate bed space area is required.     

A molecular characterization of Clostridium difficile isolates from humans, animals and their environments.

It is generally accepted that most patients with Clostridium difficile-associated diarrhoea acquire the organism from the environment. Recently we demonstrated that household pets may constitute a significant reservoir of C. difficile through gastrointestinal carriage in up to 39% of cats and dogs. These findings suggested that direct transmission from household pets, or contamination of the environment by them, may be a factor in the pathogenesis of C. difficile-associated diarrhoea. To investigate this possibility, we examined isolates of C. difficile from humans, pets and the environment by restriction enzyme analysis (REA) and restriction fragment length polymorphism (RFLP) typing using enhanced chemiluminescence. Both REA and RFLP typing methods used Hind III digests of chromosomal DNA. A total of 116 isolates of C. difficile from pets (26), veterinary clinic environmental sites (33), humans (37) and hospital environmental sites (20) was examined. REA was far more discriminatory than RFLP typing and for all isolates there were 34 REA types versus 6 RFLP types. There was good correlation between the REA types found in isolates from pets and from the veterinary clinic environment, and between isolates from humans and from those found in the hospital environment. There was, however, no correlation between REA type of C. difficile found in pets and isolates of human origin. We conclude that there may still be a risk of humans acquiring C. difficile from domestic pets as these findings may be the result of geographical variation.     

The effect of Perasafe and sodium dichloroisocyanurate (NaDCC) against spores of Clostridium difficile and Bacillus atrophaeus on stainless steel and polyvinyl chloride surfaces

Clostridium difficile is an important cause of nosocomial diarrhoea. The aim of this study was to evaluate the potential for Perasafe, a recently introduced biocide, to contribute to control of C. difficile spores in the patient environment, in comparison with the chlorine-releasing agent sodium dichloroisocyanurate (NaDCC). These agents were evaluated against a water control, in a surface test on stainless steel and polyvinyl chloride (PVC) floor covering, materials commonly found in the hospital environment. The organisms studied were a toxigenic clinical isolate of C. difficile, and Bacillus atrophaeus (formerly B. subtilis var niger). The data indicate that in our in vitro system, Perasafe was significantly more active than NaDCC (1000 ppm available chlorine) against C. difficile spores dried on stainless steel surfaces, and against B. atrophaeus on PVC floor covering material, achieving mean log10 reduction factors in viable counts of 6 and 5.5, respectively, at 10 min exposures. Perasafe appeared to be less lethal in 10 min exposures to C. difficile spores fixed on PVC floor covering material. In general, 1000 ppm chlorine generated from NaDCC showed lower log10 reduction factors in viable counts at 10 min, ranging from 0.7 to 1.5, than Perasafe which ranged from 2.7 to 6.0. The potential efficacy of Perasafe in reducing the density of C. difficile spores in the patient environment in hospitals, nursing homes or other long-stay facilities should be evaluated in field studies.     

Lack of enhanced effect of a chlorine dioxide-based cleaning regimen on environmental contamination with Clostridium difficile spores

Spores of Clostridium difficile may play a significant role in transmission of disease within the healthcare environment and are resistant to a variety of detergents and cleaning fluids. A range of environmental cleaning agents has recently become available, many of which claim to be sporicidal. We investigated the effect of changing to a chlorine dioxide-based cleaning regimen on C.?difficile environmental contamination and patient infection rates. The prevalence of environmental contamination was unaffected with a rate of 8% (9/120) before and 8% (17/212) following the change. Rates of patient infection were also unchanged during these periods.     

Role of environmental cleaning in controlling an outbreak of Acinetobacter baumannii on a neurosurgical intensive care unit

An outbreak of Acinetobacter baumannii colonization and infection occurred in 19 patients over a 14-month period during 1998-1999 on a neurosurgical intensive care unit. During efforts to control the outbreak a significant correlation was observed between the number of environmental isolates of A. baumannii obtained during each monthly screening and the number of patients with A. baumannii colonization/infection in the same calendar month (P < 0.004). Use of 1000 ppm hypochlorite solution and the introduction of new cleaning protocols reduced the number of environmental isolates. Failure to maintain low levels of environmental contamination with A. baumannii resulted in increases in patient colonization. This study showed that high standards of cleaning play an integral role in controlling outbreaks of A. baumannii in the intensive care unit setting.     

Management of an outbreak of Clostridium difficile-associated disease among geriatric patients.

OBJECTIVE: To describe a nosocomial outbreak of Clostridium difficile-associated disease (CDAD). DESIGN: A traditional outbreak investigation. SETTING: Geriatric department of a tertiary care teaching hospital from March through April 2003. METHODS: The outbreak was detected by the C. difficile surveillance program of the infection control unit. CDAD was diagnosed by stool culture and fecal toxin A detection with a qualitative rapid immunoassay. Isolates of C. difficile were serotyped and genotyped using pulsed-field gel electrophoresis. RESULTS: The incidence of CDAD increased from 27 cases per 100,000 patient-days in the 6-month period before the outbreak to 99 cases per 100,000 patient-days during the outbreak. This outbreak involved 21 of 92 patients in 4 geriatric wards, which were located at 2 geographically distinct sites and staffed by the same medical team. The mean age of patients was 83 years (range, 71-100 years). Five (24%) of the 21 patients had community-acquired diarrhea, and secondary hospital transmission resulted in 3 clusters involving 16 patients. Serotyping and genotyping were performed on isolates in stool specimens from 19 different patients; 16 of these isolates were serotype A1, whereas 3 displayed profiles different from the outbreak strain. Management of this outbreak consisted in reinforcement of contact isolation precautions for patients with diarrhea, cohorting of infected patients in the same ward, and promotion of hand hygiene. Relapses occurred in 6 (29%) of 21 patients. CONCLUSION: Control of this rapidly developing outbreak of CDAD was obtained with early implementation of cohorting and ward closure and reinforcement of environmental disinfection, hand hygiene, and enteric isolation precautions.     

Hospital outbreak of Burkholderia stabilis bacteraemia related to contaminated chlorhexidine in haematological malignancy patients with indwelling catheters

Burkholderia cepacia complex (BCC) is an opportunistic pathogen that occasionally causes hospital outbreaks. This paper describes an outbreak of BCC bacteraemia in haematological malignancy patients related to a contaminated chlorhexidine gluconate solution. Eight BCC isolates were obtained from patients hospitalised in the same ward of a cancer centre in a Korean hospital. A further three BCC isolates were obtained from 0.5% chlorhexidine gluconate used in the same ward. The isolates were identified as B. stabilis and exhibited identical pulsed-field gel electrophoresis profiles. All patients with B. stabilis bacteraemia had indwelling intravenous catheters, which were treated with chlorhexidine to disinfect the catheters. Following identification of the source of contamination, strict controls regarding surveillance cultures for disinfectants have been enforced. No further B. stabilis infections have been found in the hospital.     

Prevalence of Clostridium difficile in the environment in a rural community in Zimbabwe

Clostridium difficile has been shown to be a nosocomial pathogen associated with diarrhoea and pseudomembranous colitis in hospitalised patients, but very little is known about its prevalence outside the hospital environment. The aim of this study was to determine the prevalence of C. difficile in faeces of domestic animals, soil and drinking water in a rural community. Water, animal faeces and soil were collected from homesteads in a rural community and the samples were cultured for C. difficile.Clostridium difficile isolates that produced toxins A or B were tested for their susceptibility to antimicrobial drugs. Clostridium difficile was isolated from 37.0% of 146 soil samples, 17.4% of 115 chicken faeces samples, 6.0% of 234 water samples and 4.3% of 161 faecal samples of other animals. Some of the C. difficile isolates from chickens (55.0%), soil (66.7%) and water (14.3%) were toxigenic. All toxigenic isolates were susceptible to metronidazole, vancomycin, doxycycline, chloramphenicol and tetracycline and all were resistant to cefotaxime, gentamicin, ciprofloxacin, norfloxacin and nalidixic acid. The results of the present study suggest that chickens kept by villagers are an important reservoir of C. difficile, which may act as a source of human infection.     

Difference in the incidence of Clostridium difficile among patients infected with human immunodeficiency virus admitted to a public hospital and a private hospital.

OBJECTIVE: To compare the occurrence of Clostridium difficile among inpatients infected with human immunodeficiency virus (HIV) in two different hospitals. DESIGN: Prospective, observational study. SETTING: Specialized HIV inpatient units. PATlENTS: HIV-infected inpatients at Cook County Hospital (CCH) and Rush Presbyterian St. Luke's Medical Center (RPSLMC). INTERVENTIONS: A clinical and epidemiologic assessment of patient risk factors for C. difficile was performed. C. difficile isolates found on stool, rectal, and environmental cultures were typed by pulsed-field gel electrophoresis. RESULTS: Twenty-seven percent of patients admitted to CCH versus 4% of patients admitted to RPSLMC had positive cultures for C. difficile (P = .001). At CCH, 14.7% of environmental cultures were positive versus 2.9% at RPSLMC (P = .002). Risk factors for C. difficile acquisition included hospitalization at CCH, more severe HIV, use of acyclovir and H2-blockers, and longer hospital stay. Patients admitted to CCH were taking more antibiotics, had longer hospital stays, and more frequently had a history of C. difficile infection. During the study, two strains (CD1A and CD4) extensively contaminated the CCH environment. However, only CD1A caused an outbreak. CONCLUSIONS: The C. difficile acquisition rate at CCH was sevenfold higher than that at RPSLMC, and CCH had a more contaminated environment. Differences in patient acquisition rates likely reflect a greater prevalence of traditional C. difficile risk factors and a concurrent outbreak at CCH. Although two strains heavily contaminated the environment at CCH, only one caused an outbreak, suggesting that factors other than the environment are important in initiating C. difficile outbreaks.     

Rapid detection of toxigenic Clostridium difficile from stool samples by a nested PCR of toxin B gene

Toxigenic Clostridium difficile is the aetiologic agent of most cases of antibiotic-associated diarrhoea and pseudomembranous colitis. The present standard method for C. difficile diagnosis is a cytotoxicity assay, performed on human fibroblast cultures. It is time consuming and requires special facilities. A nested-PCR assay detecting toxin B gene within a few hours was designed. One hundred and two stool samples were collected during four months. All samples were processed for toxin B-PCR, cultured for C. difficile and tested for cytotoxicity. This approach achieved 99% concordance with the cytotoxic assay. The sensitivity and specificity for the new PCR assay were 96.3% and 100% respectively. The procedure described is easy to perform, does not require special equipment and has produced excellent results. It deserves serious consideration for routine clinical microbiology laboratory use.     

A hospital outbreak of Clostridium difficile?

An increase in numbers of patients with Clostridium difficile and its toxin in their stools at a hospital in South-west London led to closure of a ward to admissions and to an investigation of a possible nosocomial outbreak. The findings suggested that the increase was not due to an outbreak of related cases but to increased investigation. The cost of the episode both in financial terms and in the effect on patient care, was considerable. This study highlights the need for caution in interpreting the significance of Cl. difficile in stool specimens. Laboratory data can only alert clinicians to the possibility of colitis; decisions about treatment and control of spread of infection should also be based on clinical criteria.     

Detection of Clostridium difficile toxins by enzyme immunoassay

An enzyme-linked immunosorbent assay (ELISA) for the rapid diagnosis of antibiotic-associated colitis (AAC) is presented. Commercially available antisera to Clostridium difficile toxins contain antibodies to other antigens found in non-toxigenic C. difficile and other bacteria. Removal of these unwanted antibodies by absorption increased the specificity of ELISA for detection of C. difficile toxins. Specimens tested included 40 faecal extracts positive for cytotoxicity from cases of AAC, 30 diarrhoeic and 30 well-formed stools negative for cytotoxicity and 50 culture filtrates of toxigenic and non-toxigenic C. difficile and other clostridial species. Use of absorbed sera reduced false-positive reactions observed with faecal specimens from 23 to 8%. About 90% of specimens that were positive by the tissue culture cytotoxicity test were positive by ELISA using the absorbed sera. The relative merits of ELISA and other methods for the rapid diagnosis of AAC are discussed.     

A large outbreak of Clostridium difficile-associated disease with an unexpected proportion of deaths and colectomies at a teaching hospital following increased fluoroquinolone use.

BACKGROUND AND OBJECTIVE: Fluoroquinolones have not been frequently implicated as a cause of Clostridium difficile outbreaks. Nosocomial C. difficile infections increased from 2.7 to 6.8 cases per 1000 discharges (P < .001). During the first 2 years of the outbreak, there were 253 nosocomial C. difficile infections; of these, 26 resulted in colectomy and 18 resulted in death. We conducted an investigation of a large C. difficile outbreak in our hospital to identify risk factors and characterize the outbreak. METHODS: A retrospective case-control study of case-patients with C. difficile infection from January 2000 through April 2001 and control-patients matched by date of hospital admission, type of medical service, and length of stay; an analysis of inpatient antibiotic use; and antibiotic susceptibility testing and molecular subtyping of isolates were performed. RESULTS: On logistic regression analysis, clindamycin (odds ratio [OR], 4.8; 95% confidence interval [CI95], 1.9-12.0), ceftriaxone (OR, 5.4; CI95, 1.8-15.8), and levofloxacin (OR, 2.0; CI95, 1.2-3.3) were independently associated with infection. The etiologic fractions for these three agents were 10.0%, 6.7%, and 30.8%, respectively. Fluoroquinolone use increased before the onset of the outbreak (P < .001); 59% of case-patients and 41% of control-patients had received this antibiotic class. The outbreak was polyclonal, although 52% of isolates belonged to two highly related molecular subtypes. CONCLUSIONS: Exposure to levofloxacin was an independent risk factor for C. difficile-associated diarrhea and appeared to contribute substantially to the outbreak. Restricted use of levofloxacin and the other implicated antibiotics may be required to control the outbreak     

Detection of Clostridium difficile toxins by enzyme immunoassay.

An enzyme-linked immunosorbent assay (ELISA) for the rapid diagnosis of antibiotic-associated colitis (AAC) is presented. Commercially available antisera to Clostridium difficile toxins contain antibodies to other antigens found in non-toxigenic C. difficile and other bacteria. Removal of these unwanted antibodies by absorption increased the specificity of ELISA for detection of C. difficile toxins. Specimens tested included 40 faecal extracts positive for cytotoxicity from cases of AAC, 30 diarrhoeic and 30 well-formed stools negative for cytotoxicity and 50 culture filtrates of toxigenic and non-toxigenic C. difficile and other clostridial species. Use of absorbed sera reduced false-positive reactions observed with faecal specimens from 23 to 8%. About 90% of specimens that were positive by the tissue culture cytotoxicity test were positive by ELISA using the absorbed sera. The relative merits of ELISA and other methods for the rapid diagnosis of AAC are discussed.     

Gaseous and air decontamination technologies for Clostridium difficile in the healthcare environment

The recent data for hospital-acquired infections suggest that infection rates for meticillin-resistant Staphylococcus aureus (MRSA) and Clostridium difficile are beginning to decrease. However, while there is still pressure to maintain this trend, the resistance of C. difficile spores to standard detergents continues to present a problem for many UK hospitals trying to prevent its spread or control outbreaks. Alternative disinfection technologies such as gaseous decontamination are currently being marketed to the healthcare sector as an alternative/supplement to manual disinfection, and have been shown to be effective in reducing environmental contamination. When used correctly, they offer a complementary technology to manual cleaning that increases the probability of an effective reduction in viability and provides a comparatively uniform distribution of disinfectant. Three gaseous decontamination technologies are examined for their suitability in reducing environmental contamination with C. difficile: gaseous hydrogen peroxide, chlorine dioxide and ozone. Air decontamination and UV-based technologies are also briefly described. We conclude that while there is a role to play for these new technologies in the decontamination of ward surfaces contaminated with C. difficile, the requirement for both a preclean before use and the limited 'in vivo' evidence means that extensive field trials are necessary to determine their cost-effectiveness in a healthcare setting.