R.A. Bowman

Gastrointestinal carriage of Clostridium difficile in cats and dogs attending veterinary clinics

Cats and dogs being treated at two veterinary clinics were investigated for gastrointestinal carriage of Clostridium difficile using selective solid and enrichment media. Thirty-two (39.5%) of 81 stool samples yielded C. difficile. There were significant differences in isolation rates between clinics, 61.0% of animals being positive at one clinic compared to 17.5% at the other (Chi-square, P less than 0.005). Of 29 animals receiving antibiotics, 15 (52.0%) harboured C. difficile while 11 (23.9%) of 46 animals not receiving antibiotics were positive (Chi-square, P less than 0.01). There was no difference in carriage rate between cats (38.1%) and dogs (40.0%). The environment at both veterinary clinics was surveyed for the presence of C. difficile. Fifteen of 20 sites at one clinic were positive compared to 6 of 14 sites at the other clinic. Both cytotoxigenic and noncytotoxigenic isolates of C. difficile were recovered from animals and environmental sites. These findings suggest that household pets may be a potentially significant reservoir of infection with C. difficile.

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.

Laboratory diagnosis of Clostridium difficile-associated diarrhoea.

This paper reviews the various laboratory procedures available for the isolation and identification ofClostridium difficile and the detection of toxins produced by this organism. Laboratories should be selective in determining which patients require investigation forClostridium difficile-associated diarrhoea. Transport and storage of stool specimens at 4 °C is recommended when delays in processing may occur. Tissue culture techniques are still the best method for detection of cytotoxin and a variety of cell lines can be used. Other methods for detecting cytotoxin, and methods for detecting other toxins are not sufficiently developed yet to warrant introduction into diagnostic laboratories. Culture techniques remain the most sensitive for diagnosis, particularly since the development of a variety of enrichment techniques. Cycloserine cefoxitin fructose agar is still adequate, although reduced concentrations of antimicrobial agents are necessary, and improvements, such as the addition of sodium taurocholate, increase the recovery of spores. Enrichment cultures have markedly increased isolation rates forClostridium difficile but the significance of these isolates needs to be carefully evaluated. Until simpler and more reliable tests are available in clinical laboratories for the detection of toxins, the isolation ofClostridium difficile from patients with diarrhoeal disease should be considered paramount.

Outbreak of gentamicin-resistant Acinetobacter baumanii in an intensive care unit: clinical, epidemiological and microbiological features

&NA; The clinical, epidemiological and microbiological features of an outbreak of infection and colonisation caused by gentamicin‐resistant Acinetobacter baumanii (GRAB) in an 18‐bed intensive care unit (ICU) of a 680‐bed adult teaching hospital are described. A retrospective review of medical, laboratory and infection control records was followed by prospective surveillance. Typing of isolates was performed by restriction enzyme analysis (REA) of chromosomal DNA. The incidence of GRAB in the ICU increased from 1.26 cases per 1000 occupied bed days (OBDs) for January to June 1993, to 6.62 per 1000 OBDs for July to December 1993 (Chi square = 4.8, P < 0.05), confirming the existence of an outbreak. For the two year period, 1993 and 1994, a total of 45 cases of GRAB infection or colonisation was identified. Males and females were equally represented, with an age range of 16–79 years and a mean age of 51 years. Admitting diagnoses varied, with multiple trauma and head injury predominating (ten cases). For 35 of the 45 cases the initial site of GRAB isolation was sputum or other respiratory tract specimen. Specific treatment for GRAB was initiated in 23 patients, however no deaths were directly attributable to GRAB infection. The period of time between admission to the ICU and first isolation of GRAB ranged from three to 70 days with a median of nine days. Overall, ten (11%) of 91 staff hand samples and one of 37 (3%) environmental samples yielded GRAB. All GRAB isolates produced similar biochemical profiles and antibiotic resistance patterns, except for a group of five which were ciprofloxacin resistant. Thirty patient isolates, all ten staff hand isolates and the environmental isolate produced identical REA patterns. The remaining five patient isolates (all ciprofloxacin resistant) which were available for typing produced a different REA pattern. Our study has documented a moderate‐sized outbreak of GRAB in an ICU setting. Typing of isolates using REA was useful in delineating outbreak strains. Carriage of GRAB on staff hands was demonstrated as the most likely source of infection. Despite institution of infection control measures GRAB now appears endemic in the ICU.

Clostridium difficile-associated diarrhoea: Epidemiological data from Western Australia

The incidence of Clostridium difficile-associated diarrhoea (CDAD) was investigated retrospectively at a 690-bed teaching hospital for the period 1983-92. Our aims were to determine: (i) the distribution by age and sex of patients with CDAD, (ii) the possibility of a seasonal trend and, (iii) the influence of infection control procedures, contamination of the hospital environment and the use of third-generation cephalosporins. The laboratory diagnosis of CDAD was based on demonstration of the organism by stool culture and/or detection of specific cytotoxin in stool filtrates. C. difficile was detected in 917 patients who were being investigated for diarrhoeal illness. Yearly isolations varied from a low of 49 in 1983 to a high of 120 in 1990 (Chi square for linear trend 128.8; P < 0.005). Most patients were elderly, with 63% aged 60 years or more; the majority (59%) were female. The relationship between culture of C. difficile and detection of cytotoxin in faecal extracts was also examined. Sixty percent of a sample of 132 isolates from patients in whom faecal cytotoxin was not detected produced cytotoxin in vitro, suggesting that culture is a more sensitive indicator of infection with C. difficile than cytotoxin detection. When the total number of faecal specimens received in the laboratory was used as a denominator there was an increase in the number of incident cases of CDAD between 1983 and 1990, apart from 1986. When occupied bed days was used as the denominator a similar trend was observed with a peak in 1990.(ABSTRACT TRUNCATED AT 250 WORDS)

A selective broth for Clostridium difficile

Summary A selective broth for the isolation of Clostridium difficile from stool specimens is described. The broth contains gentamicin, cycloserine and cefoxitin (GCC broth) and gives rapid presumptive evidence of the presence of C. difficile using gas‐liquid chromatography. The broth may also be used for the detection of cytotoxin. Final recovery of C. difficile was significantly improved with an increase in isolation rate of 20% in patients in whom fecal cytotoxin could be detected and 125% in patients where fecal cytotoxin could not be detected. Until the pathogenesis of C. difficile‐associated diseases is more clearly defined we would stress the importance of isolating the organism, and we advocate the use of a selective broth such as GCC to improve the isolation rate.

In vitro bacterial adherence to hydrogel and poly(methyl methacrylate) intraocular lenses

Purpose: To compare the in vitro adherence of Staphylococcus epidermidis to poly(hydroxyethyl methacrylate) (polyHEMA) or hydrogel intraocular lenses (IOLs) and poly(methyl methacrylate) (PMMA) IOLs. Setting: Lions Eye Institute, Perth, Western Australia. Methods: One‐piece hydrogel lenses and one‐piece PMMA lenses were suspended for 60 minutes in standardized suspensions of a well‐characterized strain of S. epidermidis and then sonicated in a known quantity of balanced salt solution to remove the adherent bacteria. Quantitative cultures of the sonicates were performed and the results analyzed statistically. Results: The mean bacterial adherence of S. epidermidis to the PMMA IOLs (58,400 CFU) was more than 20 times greater than that to the hydrogel IOLs (1953 CFU). The difference was statistically significant (P < .001). Conclusions: Adherence of S. epidermidis to hydrogel IOLs is significantly lower than to PMMA IOLs. This suggests that the risk of postoperative endophthalmitis after cataract extraction and IOL implantation may be lower with the use of hydrogel IOLs.

Selective criteria for the microbiological examination of faecal specimens.

To assess the effectiveness of predetermined investigation criteria for the examination of faecal samples from inpatients, cultured stool specimens were prospectively examined for Salmonella spp, Shigella spp, Campylobacter spp and Clostridium difficile, and screened microscopically for intestinal parasites. Out of a total of 505 specimens, 421 (83%) fulfilled the criteria for examination for C difficile, 254 (50%) for Salmonella spp, Shigella spp, and Campylobacter spp, and 87 (17%) for intestinal parasites. Isolation rates for these organisms in those groups of patients where examination was indicated were 22.5% for C difficile and 9.1% for Salmonella spp, Shigella spp, and Campylobacter spp; the detection rate for parasites was 3.5%. In those patients where the criteria did not suggest investigation, the isolation or detection rates were 3.6% for C difficile, 0% for Salmonella spp, Shigella spp, and Campylobacter spp, and 1.7% for intestinal parasites, suggesting that the use of predetermined investigation criteria was effective.

Clostridium difficile in general practice and community health

The isolation rate for Clostridium difficile in diarrhoeal stools was investigated in patients from general practice and community health centres over a 14-month period. C. difficile or its cytotoxin was detected in specimens from 89 (4.7%) of 1882 patients studied and accounted for 30.3% of all enteropathogenic micro-organisms isolated. Overall C. difficile was second only to Giardia lamblia in frequency. Recovery rates in the different groups of patients surveyed varied from 3.6 to 27.5%. The relationship between stool culture results and stool cytotoxin assay also varied considerably between groups of patients studied. Coincident infections with a variety of enteropathogenic bacteria and intestinal parasites were diagnosed in 14 of the 89 patients. It was concluded that laboratories servicing this type of practice should be aware that C. difficile may be a cause of diarrhoea. An adequate clinical history should facilitate proper processing of the specimen.

Evaluation of three commercial enzyme immunoassay kits for detecting faecal Clostridium difficile toxins.

The detection of faecal cytotoxicity using tissue culture was compared with three commercial Clostridium difficile enzyme immunoassay (EIA) kits; Premier C difficile toxin A (Meridian Diagnostic, Inc.); CD-TOX C difficile toxin A (Porton Cambridge); and Cytoclone A+B EIA (Cambridge Biotech Corporation). Of 160 faecal samples examined by all four methods, 52 (32.5%) were cytotoxic, 44 (27.5%) were positive by Premier, 48 (30%) by CD-TOX EIA, and 50 (31.3%) with Cytoclone. When compared with detection of cytotoxicity by tissue culture assay, the following performance indices were obtained: Premier, sensitivity 84.1%, specificity 99.1%, positive predictive value (PPV) 97.8%, negative predictive value (NPV) 93%; CD-TOX, sensitivity 92.3%, specificity 88.0%, PPV 78.7%, NPV 95.9%; Cytoclone, sensitivity 96.2%, specificity 93.5%, PPV 87.7%, NPV 98.1%. EIA results were available within three hours, whereas the results of the cytotoxin assay were available after 24-48 hours. All three kits provided satisfactory results and, although relatively expensive, all could be used in the laboratory effectively to screen for diarrhoeal disease associated with C difficile.