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

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.

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.

Evaluation of an enzyme immunoassay kit for the detection of Clostridium difficile enterotoxin.

The Premier Clostridium difficile toxin A enzyme immunoassay (EIA) kit was evaluated for the detection of C. difficile enterotoxin in fecal samples. A total of 314 samples was tested by culture, cytotoxin detection and EIA kit. Compared to a combined culture/cytotoxin result the Premier EIA kit had a sensitivity of 88.3%, a specificity of 100%, a predictive value positive of 100% and a predictive value negative of 87.4%. Test results were available within 3 hrs providing a rapid and reliable means of detecting C. difficile enterotoxin.

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 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.

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.

Ciprofloxacin and Clostridium difficile-associated diarrhoea.

Recent reports have implicated ciprofloxacin as a cause of Clostridium difficile-associated diarrhoea. This problem was examined in three ways. First, the MIC of ciprofloxacin for C. difficile was determined. The MIC range was 8-32 mg/L, with C. difficile were 'treated' with ciprofloxacin and clindamycin in a test-tube, and the growth of C. difficile monitored. The clindamycin-treated emulsions supported growth of C. difficile, while the ciprofloxacin-treated and control emulsions did not differ significantly and failed to support the growth of C. difficile. Finally, 213 patients on ciprofloxacin monotherapy were investigated. Twenty-nine patients were given ciprofloxacin as treatment for diarrhoea, while a further 15 patients developed diarrhoea while being treated. None of these 44 patients harboured C. difficile. Faecal samples from 73 of the remaining 169 patients who did not have or develop diarrhoea were investigated for C. difficile, but none was positive. It was concluded that ciprofloxacin is unlikely to promote C. difficile-associated diarrhoea.

Hepatitis C testing. Comparison of Ortho's EIA and RIBA II tests in 1,182 patients undergoing primary liver transplantation.

Plasma samples from 1,182 patients undergoing primary liver transplantation were tested for anti-hepatitis C (HCV) virus by two methods: Ortho HCV ELISA Test System (EIA) and Chiron RIBA HCV Test System (RIBA II). The EIA results, 0 or +, were recorded first, followed by RIBA results, N = negative, P = positive, or I = indeterminate. Concordant results--0N, + P, + I--were found in 1,076 (91%), and discordant results were found in 106 (9%). The EIA optical density did not relate to concordant or discordant results. Band patterns were described by stating the band position (1, 2, 3, or 4) and inserting a dash (-) if no band was visualized. Most + P samples fell into two patterns: 47% showed all four bands, pattern 1234, and 15% showed the two-band pattern, 34. When the EIA was negative, 0P, the opposite was seen: 8% showed the 1234 pattern and 81% showed the 34 pattern. There were 226 samples that formed bands (+ P, 149; 0P, 31; + I, 15; 0I, 31). The frequency of bands was as follows: 4, 32%; 3, 31%; 2, 19%; and 1, 18%. Band 2 and the EIA test detected antibodies to the same c100-3 fragment and showed 74% concordance. No explanation is apparent for the lower concordance rate here than that between the EIA test and bands 3 = 96% or 4 = 88%. The EIA and RIBA II tests, together with positive liver function tests and abnormal tissue pathologic findings, provide a basis for the diagnosis of HCV.

Limulus amoebocyte lysate assay in the diagnosis of peritonitis in patients receiving continuous ambulatory peritoneal dialysis.

AIMS: To evaluate the Limulus amoebocyte lysate (LAL) assay for differentiating Gram positive from Gram negative peritonitis in patients receiving continuous ambulatory peritoneal dialysis (CAPD). METHODS: One hundred and six patients with suspected peritonitis were studied. LAL assay was performed by adding 0.1 ml of CAPD fluid to 0.1 ml of LAL reagent and incubating in a heating block for 60 minutes at 37 degrees C. The sensitivity of the reaction was determined by: (i) diluting endotoxin in distilled water and used (filter sterilised) peritoneal dialysis fluid; and (ii) diluting a broth culture of E coli used in peritoneal dialysis fluid. A positive LAL assay was defined as the constant stability of the clot through an inversion of 180 degrees. RESULTS: Compared with bacterial culture, the LAL assay had a sensitivity of 65% and a specificity of 98%. The sensitivity of microscopy compared with culture of Gram negative organisms was 76%; overall sensitivity of microscopy in comparison was 80%. CONCLUSIONS: The Gram stain was more sensitive than has previously been reported; the LAL assay was specific but insensitive for the diagnosis of CAPD peritonitis. There was a correlation between reduced leucocyte count and culture; this was reduced in cases from which Gram negative organisms had been isolated. It is recommended that laboratories evaluate their Gram stain procedure to improve its sensitivity because the LAL assay is not a satisfactory substitute.

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.

Non-radioactive restriction fragment length polymorphism (RFLP) typing of Clostridium difficile.

A typing method for Clostridium difficile based on restriction fragment length polymorphisms (RFLP) is described. The technique utilizes commercially available Escherichia coli ribosomal ribonucleic acid (rRNA) as probe material. Probe labelling, hybridization and detection was performed using the Enhanced Chemiluminescence (ECL) gene detection system. The probe labelling procedure was easy to perform, taking only 20 min. The complete typing method was comparatively simple, reproducible and readily adaptable to most bacterial genera.

Evaluation of Clinitek 200 and Rapimat II/T for screening for urinary tract infection.

Two machines, the Clinitek 200 and the Rapimat II/T, were evaluated for their ability to screen urine samples for significant bacteriuria and other elements indicative of urinary tract pathology. The automated screening procedures were compared with a conventional approach of microscopy and quantitative culture for 1020 urine specimens obtained from patients in a 700 bed general hospital. When compared with the bacterial culture method both machines gave identical results with a negative predictive value of 0.99, while when compared with microscopy alone the Clinitek 200 and Rapimat II/T gave negative predictive values of 0.92 and 0.87, respectively. It is concluded that both machines would provide cost effective screening of urine specimens.

Laboratory diagnosis of Clostridium difficile-associated diarrhoea.

This paper reviews the various laboratory procedures available for the isolation and identification of Clostridium difficile and the detection of toxins produced by this organism. Laboratories should be selective in determining which patients require investigation for Clostridium difficile-associated diarrhoea. Transport and storage of stool specimens at 4 degrees 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 for Clostridium 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 of Clostridium difficile from patients with diarrhoeal disease should be considered paramount.

Vertebral osteomyelitis due to Staphylococcus warneri.

We report a case of vertebral osteomyelitis in a diabetic woman. This appears to be the first report of such an infection with the coagulase negative staphylococcus, S warneri.

Latex particle agglutination for detecting and identifying Clostridium difficile.

A total of 329 selective enrichment broth cultures were tested for detection of Clostridium difficile by latex particle agglutination (LPA), gas-liquid chromatography, and bacterial culture. Of 53 broths positive by LPA, 36 were positive by gas-liquid chromatography, and 42 were positive by bacterial culture. The sensitivity and specificity of LPA relative to bacterial culture was 95.6% and 96.3%, respectively, while the sensitivity and specificity of gas-liquid chromatography relative to bacterial culture was 84.6% and 100%, respectively. The high predictive value of a negative test (99%) should make LPA on broth cultures a good screening test for detecting C difficile. Of several other Clostridium spp tested in pure culture, strains of C sordellii and C bifermentans also gave a positive result by LPA. These results, together with the low cost and simple facilities required, suggest that the LPA test will be a useful procedure for the presumptive identification of C difficile in selective enrichment broths and for the identification of pure cultures.