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.

Control of methicillin-resistant Staphylococcus aureus in a neonatal intensive-care unit: use of intensive microbiologic surveillance and mupirocin.

OBJECTIVE: To describe the epidemiology and the interventions used to control two methicillin-resistant Staphylococcus aureus (MRSA) epidemics involving 46 infants with two fatalities in a neonatal intensive care unit (NICU). SETTING: A 50-bed, level III NICU in a university hospital. INTERVENTIONS: After traditional interventions failed to stop the first epidemic, an intensive microbiologic surveillance (IMS) program was developed. Cultures were obtained on all infants each week, and those colonized with MRSA were isolated. When an infant was found to be colonized with MRSA, cultures immediately were obtained on all surrounding infants. This was continued until no MRSA-colonized infants were found in the area. During the first epidemic, mupirocin was used in an attempt to eradicate the organism from the unit. RESULTS: All infants, colonized and noncolonized, and parents of and personnel working with colonized infants were treated simultaneously with 5 days of mupirocin. This failed to eradicate MRSA in colonized infants. The spread of MRSA ceased in the unit, but a second epidemic occurred 4 months later. This time, IMS alone was successful in quickly containing the epidemic, and MRSA disappeared from the unit after all colonized infants were discharged. Plasmid analysis demonstrated that the same strain was responsible for both outbreaks. CONCLUSIONS: IMS and isolation are effective in containing the spread of MRSA in an NICU. The use of mupirocin failed to eradicate the organism.

Impact of technological developments and organizational strategies on clinical laboratory cost reduction.

Health care reform efforts, largely under the aegis of managed health care initiatives, have prompted clinical laboratories to increase efficiency and reduce both expenditures and test turnaround times. The adoption of newer technologies is viewed as a mechanism of achieving the latter objectives, but direct and indirect costs and outcomes are often difficult to project. Issues explored in this article include the impact on a large university hospital-based clinical microbiology laboratory following the application of various technological approaches to organism recognition and susceptibility testing and the consolidation of certain testing services. Included are applications of an automated blood culture system; radiometric detection, identification, and susceptibility testing of mycobacteria; and the use of molecular probes to identify various microorganisms. Assessment was made through retrospective review of direct costs, estimates of average test report turnaround times, work flow changes, and real or perceived outcomes. Both the application of technology per se and consolidation of an independent virology service into the general microbiology laboratory enabled improvement in test report times and led to direct or indirect cost reduction. Managerial strategies to bring about organization changes throughout all clinical laboratories in response to a major hospital-wide cost reduction program are also presented together with financial outcomes achieved.

The pull-through procedure: technical factors in influencing outcome, with emphasis on pouchitis.

BACKGROUND: The purpose of the study was to review those features that we believed to be critical to the successful performance of the ileal pouch-anal anastomosis, or pull-through, procedure, and specifically the complication of pouchitis. METHODS: The charts of 205 patients who successfully underwent ileal pouch-anal anastomosis procedure were reviewed. No follow-up was available in five patients; therefore, the basis of this report and its analysis was based on 200 consecutive procedures in which at least two of the three surgeons participated. Particular emphasis was placed on continence, particularly nighttime continence. The incidence of pouchitis, either a single episode or intermittent episodes, was surveyed. Particular attention was paid to the level of rectal mucosectomy and anastomosis at the top of the columns of Morgagni, thus retaining the transitional zone. RESULTS: Only 5% of patients were incontinent in the absence of pouchitis. Twenty-five patients (13%) wore a pad at night, but only nine (5%) wore a pad during the day. Of those patients with pouchitis, 6% (12) have had a single episode and 12% (23) were intermittently on medication. Therapy of pouchitis was usually carried out with ciprofloxacin 500 mg by mouth everyday or twice a day. CONCLUSIONS: Ileal pouch-anal anastomosis is an excellent procedure, provided technical details are adhered to. Satisfactory outcome with respect to nighttime continence can be achieved with rectal mucosectomy with minimal manipulation and retaining the transitional epithelium, performing the pouch anastomosis at the top of the columns of Morgagni. The incidence of pouchitis is disappointing but need not be inhibiting of either patients or carrying out this life-saving procedure in patients with ulcerative colitis and familial polyposis.

Recurrent epidemics caused by a single strain of erythromycin-resistant Staphylococcus aureus. The importance of molecular epidemiology.

OBJECTIVE--To compare the accuracy of clinical epidemiologic findings with results of molecular epidemiologic analysis in identifying the source of recurrent epidemics of erythromycin-resistant Staphylococcus aureus (ERSA) infections in a well-baby nursery. DESIGN--Epidemic investigations with retrospective and prospective microbiological surveillance. Staphylococcus aureus isolates were evaluated by plasmid analysis and genotyping. SETTING--A well-baby nursery in a 700-bed university teaching hospital with approximately 250 deliveries per month. PATIENTS--Newborn infants who developed ERSA infections during 1990 and 1991. INTERVENTION--Traditional infection control measures, including cohorting of infected infants and hand washing, were implemented. Personnel were cultured for nasal carriage of S aureus during both epidemics. Employees carrying ERSA were removed from the nursery and treated with mupirocin. RESULTS--In the first epidemic, 15 infants were infected with ERSA. A nursing assistant who cared for most of the infants was found to be a carrier of ERSA. She was removed from the nursery and the epidemic resolved. Fifteen months after the first epidemic ended, an epidemic that involved 11 infants began. The attending physician had a facial furuncle and was found to be a carrier of ERSA. The physician was treated and the epidemic resolved. Plasmid and genotyping showed the ERSA organisms from both epidemics were the same. The employee implicated in the first epidemic did not have the epidemic strain, but the physician who attended during both epidemics did. CONCLUSIONS--Traditional epidemic investigations may lead to false conclusions that can only be recognized with molecular epidemiologic techniques. For these techniques to be useful in the control of outbreaks, plasmid analysis and/or genotyping must be readily available.

Collaborative evaluation of the Radiometer Sensititre AP80 for identification of gram-negative bacilli.

A multicenter trial of the Sensititre AP80 panel read on the Sensititre AutoReader (Radiometer America, Westlake, Ohio) for the automated identification of gram-negative bacilli was conducted with 1,023 clinical isolates (879 members of the family Enterobacteriaceae plus 144 nonenteric organisms). Assignment of taxa was based on the computer-assisted interpretation of the results of a series of reactions with fluorogenic enzyme substrates after 5 h of incubation, with an incubation interval of approximately 18 h used when indicated. Accuracy was determined initially by comparison with the results obtained with the API 20E or Rapid NFT system (Analytab Products, Plainview, N.Y.). Isolates showing discrepancies were identified by using conventional biochemical profiles. Identifications were available after 5 h of incubation for 918 isolates (90%). Agreements with reference results for members of the family Enterobacteriaceae were 95.3 and 92.5% at the genus and species levels, respectively, and for the nonmembers of the family Enterobacteriaceae, the agreements with reference results were 95.1 and 84.7%, respectively. The Sensititre AP80 panel was found to be simple and convenient to use, allowed for the testing of three isolates per panel, required minimal supplementary testing for completion of identification, performed in a reproducible fashion, and demonstrated an accuracy of same-day identification comparable to that reported for other automated systems. The AP80 panel appears well suited for routine use in the clinical microbiology laboratory as an automated means of identifying both members of the family Enterobacteriaceae and nonenteric gram-negative bacilli.

Colonization by enteroaggregative Escherichia coli in travelers with and without diarrhea.

Enteroaggregative Escherichia coli (EAggEC) has been found to be associated with pediatric diarrhea in developing countries. In order to determine the role of EAggEC as an agent of traveler's diarrhea, we used a sensitive and specific DNA probe for EAggEC to screen bacterial colony blots from 278 volunteers before and after travel. Colonization with EAggEC was infrequent (2.5%) prior to travel but rose to 27 to 33% after travel in volunteers who took either placebo or trimethoprim-sulfamethoxazole. Travelers who took trimethoprimsulfamethoxazole were colonized with organisms that were uniformly resistant to that antimicrobial agent; when volunteers received ciprofloxacin, colonization with EAggEC was prevented (2.0%). Although colonization rates were high in the placebo and trimethoprim-sulfamethoxazole groups, only a minority of travelers who were colonized with EAggEC experienced diarrhea. On the basis of our data, we suggest that colonization with EAggEC alone is not sufficient to cause traveler's diarrhea.

Niacin-positive Mycobacterium kansasii isolated from immunocompromised patients.

Niacin-positive Mycobacterium kansasii was isolated from three patients, two with respiratory infections and one with a perirectal abscess. The isolates were phenotypically similar to other strains of M. kansasii, differing only in their ability to produce niacin. This phenotype has been reported only twice in the literature, during the 1960s.

Survival of Streptococcus pneumoniae in corneal storage media.

The Cincinnati Eye Bank had six corneoscleral rims in which Streptococcus pneumoniae was cultured after preservation in corneal storage media. To determine the survival of this organism under conditions common for corneal storage, gentamicin-supplemented McCarey-Kaufman (M-K) medium and chondroitin sulfate/Dextran medium (Dexsol, Ciron Ophthalmics, Irvine, CA, U.S.A.) were inoculated with S. pneumoniae and kept at 4 degrees C. Thioglycollate broth plus 10% rabbit serum (Thio-S) and tryptic soy broth (TSB) served as growth controls. At day 14 after inoculation of 10(5) colony-forming units (CFU)/ml, Dexsol showed a 1-log decrease in bacterial concentration, the M-K medium a 2-log decrease and Thio-S a 4-log decrease, whereas TSB showed no detectable organisms. By day 21 Dexsol had only a 2-log decrease in bacteria. These data suggest that corneal storage medium supplemented with gentamicin does not exert bactericidal activity against S. pneumoniae and may actually support its survival at 4 degrees C.

Dose effects of LPS on neutrophils in a whole blood flow cytometric assay of phagocytosis and oxidative burst.

Whole blood phagocytosis (P) and oxidative burst (OB), a rapid and sensitive flow cytometric method for quantifying neutrophil activation, was modified for single laser systems by using propidium iodide labeled Staphylococcus aureus and 2',7' dichlorofluorescein diacetate. The purpose of the present study was to characterize this assay with respect to the stimulatory activity of bacterial lipopolysaccharide (LPS) on phagocytosis. Blood from healthy donors was preincubated with log doses of bacterial LPS B (0.1-1,000 ng/ml) or sterile pyrogen-free saline at 37 degrees C from 0-120 minutes. LPS increased both P and OB in a dose-dependent manner (up to 62 and 121%, respectively) at all time points tested, and this effect on P and OB could be detected even with no preincubation. This LPS-induced phagocytic activity could be blocked by the addition of polymyxin B (10 micrograms/ml) during preincubation. The priming effect of LPS was maximal at 45 min. P and OB were inhibited by preincubation with EDTA at doses greater than 2 mM (60 and 80% inhibition, respectively). These observations are consistent with the exquisite sensitivity of the neutrophil to endotoxin. This method can evaluate neutrophil response to immunomodulatory and chemotherapeutic agents in a physiological milieu. These findings re-emphasize the necessity of using pyrogen-free reagents in any study of neutrophil function.

Reemergence of epidemic methicillin-resistant Staphylococcus aureus in a general hospital associated with changing staphylococcal strains.

An epidemic of methicillin-resistant Staphylococcus aureus (MRSA) infections involving 323 patients occurred at the University of Cincinnati Hospital from 1977 to 1981. Subsequently, endemic MRSA persisted in the hospital for 6 years, until 1987, when a new epidemic began with 223 patients becoming infected over 3 years. Between the two epidemics, there was a major change in the MRSA recovered from infected patients, as demonstrated by three epidemiologic markers. Antibiograms showed that the tetracycline-resistant MRSA involved in the first epidemic was replaced by tetracycline-susceptible MRSA in the second epidemic; bacteriophage typing indicated that the original epidemic strain, D11/83A/85, had been replaced by new strains, many of which were susceptible to phage 54; and restriction endonuclease analysis of plasmid DNA confirmed that a single strain was involved in the first epidemic and that multiple strains were present in the second epidemic. The epidemiology of MRSA infections in the hospital changed with the change in staphylococcal strains. The first epidemic was hospital based with most infections occurring in surgical patients, and the burn unit was the major reservoir. In contrast, 28% of the patients in the second epidemic had community-acquired infections, and nursing home patients were an important source of these infections. Also, 29% of the hospital-acquired infections in this epidemic occurred in nonsurgical patients. This time the burn unit was not a reservoir of infection, but other intensive care units were. The increased diversity of strains of MRSA in the second epidemic might be related to increased transmission in the community and more widespread transmission in the hospital.

Wide variability in Pseudomonas aeruginosa aminoglycoside results among seven susceptibility testing procedures.

Seven commonly used antimicrobial susceptibility testing methods were used to test the susceptibility of 150 isolates of Pseudomonas aeruginosa against gentamicin, tobramycin, amikacin, carbenicillin, and piperacillin. Results were compared with respect to the susceptibility characteristics of the population of isolates as defined by each method. Conventional methods included agar disk diffusion and agar dilution, carried out in accordance with current recommendations of the National Committee for Clinical Laboratory Standards, as well as broth microdilution testing with cation-supplemented Mueller-Hinton broth (CSMHB). Methods in which instrumentation was used for result determination included the Autobac I, Avantage, Sensititre Autoreader (using a breakpoint panel at 18 h of incubation), and Vitek (AMS-240, using the GNS susceptibility card). When necessary for comparison, MIC data were converted to categorical interpretations (susceptible, intermediate, and resistant). With respect to gentamicin, no significant differences were noted among the results of disk diffusion, broth microdilution, Sensititre Auto breakpoint, or Vitek methods which characterized 60 to 67% of isolates as susceptible, 16 to 22% as intermediate, and 13 to 17% as resistant. In contrast, agar dilution, Autobac, and Avantage, although yielding gentamicin results similar to those of one another, were each significantly different in result reporting from the other four methods above for gentamicin results, and they characterized the Pseudomonas population largely as susceptible (88 to 97%), with 0 to 6% intermediate and only 3% to 6% resistant. More isolates were characterized as being resistant to gentamicin in the Avantage test if an assay broth supplemented with increased amounts of calcium was used. Cation impregnation of Autobac disks did not appreciably change Autobac results. The geometric mean MIC of gentamicin was 4 micrograms/ml lower in the agar dilution method than in the CSMHB microdilution method, despite monitoring of the agar for cation content through performance disk diffusion testing with P. aeruginosa ATCC 27853. Tobramycin activity was greater than gentamicin activity, and susceptibility to tobramycin ranged from 89 to 97%, with few statistically significant differences noted among the seven methods studied. Differences in MIC distribution and geometric mean MIC between agar dilution and CSMHB microdilution testing were minimal and suggested less of a cation influence on tobramycin than gentamicin results. Although amikacin was also more active than gentamicin (83 to 99% of isolates were susceptible), differences in the amikacin results among methods tended to reflect the same trends in reporting as seen with gentamicin testing, with the exception that results of Avantage testing were similar to those of disk diffusion, CSMHB microdilution, Sensititre, and Vitek. A difference in geometric mean MIC of 5 micrograms/ml between CSMHB testing and agar dilution testing suggested the influence of divalent cations on amikacin results. Few highly significant differences were noted among methods when isolates were tested against carbenicillin and piperacillin, except that Avantage piperacillin results (66% susceptible) and Autobac piperacillin results (98% susceptible) were noticeably different from the percent piperacillin susceptibility (range, 85 to 92%) measured by the other methods. Method-dependent variability among aminoglycoside susceptibility results, particularly when testing gentamicin, prevents meaningful comparison of Pseudomonas susceptibility trends among hospitals when different methods are used and promotes confusion and frustration among clinical microbiologists and clinicians owing to the uncertainties of clinical meaning of these data.

Bioassay of teicoplanin in serum containing rifampin or a beta-lactam antibiotic.

The purpose of this study was to develop bioassays for the measurement of teicoplanin in serum containing rifampin or a beta-lactam antibiotic. Use of rifampin-resistant Bacillus subtilis as indicator organism or pretreatment of the serum sample with Bacillus cereus penicillinase Type I (nafcillin, ticarcillin, mezlocillin) or Type II (cefazolin, cefuroxime, ceftazidime, ceftriaxone) effectively eliminated assay interference. Validation bioassays performed on two separate days utilizing triplicate coded serum samples containing 0 to 200 micrograms teicoplanin in combination with 40 micrograms/ml rifampin or 200 to 500 micrograms/ml beta-lactam showed no significant differences (p greater than 0.05, two-way analysis of variance) in analyte recovery between assay days. Regression analysis of each teicoplanin/rifampin or teicoplanin/beta-lactam data set yielded slope values of 0.92 to 1.01, intercept values of -0.45 to 0.84 and correlation coefficients of 0.9925 to 0.9990. Thus, serum teicoplanin can be quantitated accurately, precisely, and reproducibly in patients receiving concomitant rifampin or beta-lactam chemotherapy.

Legionella cincinnatiensis sp. nov. isolated from a patient with pneumonia.

A Legionella-like organism (strain 72-OH-H [= ATCC 43753]) was isolated from an open-lung biopsy specimen from a hemodialysis patient with end-stage renal disease and bronchopneumonia. Growth characteristics and gas-liquid chromatographic profiles of the isolate were consistent with those for Legionella spp. The isolate was presumptively identified as a Legionella longbeachae serogroup 1 strain by direct immunofluorescence staining. However, the organism was serologically distinct in the slide agglutination test with absorbed antisera. DNA hybridization studies showed that strain 72-OH-H constitutes a new Legionella species, which is named Legionella cincinnatiensis (ATCC 43753).

Rapid MIC testing with the sensititre autoreader.

Automated microdilution MIC results, obtained with the Autoreader (Sensititre, Inc., Salem, N.H.) following 5 h of incubation, were compared with manually read, concurrent control MICs following 18 h of incubation in a three-laboratory comparative study. A total of 704 members of the family Enterobacteriaceae or similar gram-negative organisms were tested against 17 antimicrobial agents. Autoreader MICs were within 1 doubling dilution of control values in 92.9% of instances. Discrepancies of +/- 2 doubling dilutions and +/- 3 or greater doubling dilutions were noted in 4.5 and 2.6%, respectively, of the 7,687 drug-organism combinations analyzed. The majority of errors occurred when beta-lactam antimicrobial agents were tested with a variety of different species. MICs at 5 h, when Pseudomonas aeruginosa was used, were possible in only half the isolates tested and yielded data on only a limited number of drugs in the remaining instances. Excluding results obtained with penicillin and ampicillin, which were uniformly poor, Staphylococcus aureus Autoreader values were within +/- 1 doubling dilution of control values in 93.6% of instances, 5.4% varied by +/- 2 dilutions, and only 1% of test values by +/- 3 or more dilutions from control values for 82 isolates tested against nine antimicrobial agents. Of eight additional S. aureus isolates tested that were resistant to methicillin, only one was read correctly by the Autoreader, with results on the remaining seven appearing as either insufficient growth or as total resistance to all drugs tested. Interlaboratory reproducibility was excellent for selected isolates of S. aureus and gram-negative bacilli. The accuracy of the Sensititre Autoreader MIC results was comparable to that of other same-day quantitative systems for members of the family Enterobacteriaceae and S. aureus, while the economic and procedural advantages of the broth microdilution method was maintained.

Microbiology of adult cellulitis.

Needle aspiration of cellulitis sites is commonly advocated to assist in the identification of causative organisms. Twenty-five nondiabetic, adult patients with a clinical diagnosis of cellulitis had site aspirations and blood cultures obtained before antibiotic therapy was initiated. Site cultures were positive in 6 of 25 patients. Blood cultures were positive in 4 of 25 patients. All organisms except one (Enterobacter agglomerans) were staphylococci or streptococci. The gram-negative bacilli were not believed to be a pathogen based on the patient's prompt response to nafcillin. In adult patients who do not have complications, the use of needle aspiration was not supported. Empiric treatment of cellulitis aimed at gram-positive cocci appears to be sufficient.

Sensititre autoreader for same-day breakpoint broth microdilution susceptibility testing of members of the family Enterobacteriaceae.

The Sensititre Autoreader system is an instrument-assisted broth microdilution susceptibility test procedure based on the detection of fluorogenic growth substrate metabolism by test bacteria with different concentrations of antimicrobial agents. In the current investigation, this system was assessed as a means for predicting the in vitro activity of 17 antimicrobial agents versus numerous species of the family Enterobacteriaceae and Pseudomonas aeruginosa by using a breakpoint broth microdilution test format. Same-day and overnight determinations of susceptibility were made with the Sensititre Autoreader system, and in both cases, the results were compared with those obtained with a manual overnight breakpoint broth microdilution susceptibility test. Among a total of 6,086 organism-antimicrobial agent comparisons with Enterobacteriaceae, concordance was noted between the results of the same-day Autoreader system and the manual overnight test in 94.4% of cases. The same-day Autoreader results with members of the Enterobacteriaceae other than Proteus spp. were determined after 4 h of incubation; with Proteus spp. the same-day Autoreader results were determined after 5 h of incubation. When the Enterobacteriaceae Autoreader results were determined after 18 h of incubation, concordance was noted in 97.2% of comparisons. Among a total of 1,377 organism-antimicrobial agent comparisons with P. aeruginosa after 18 h of incubation, agreement of results from the manual overnight test and the Autoreader system was achieved in 92.2% of cases.