Evaluation of fluorescence-based amplified fragment length polymorphism analysis for molecular typing in hospital epidemiology: comparison with pulsed-field gel electrophoresis for typing strains of vancomycin-resistant Enterococcus faecium.

Fluorescence-based amplified fragment length polymorphism (fbAFLP) is a novel assay based on the fluorescent analysis of an amplified subset of restriction fragments. The fbAFLP assay involves the selective PCR amplification of restriction fragments from a total digest of genomic DNA. The ligation of adapters with primer-specific sites coupled with primers containing selective nucleotides allowed the full potential of PCR to be realized while maintaining the advantages of restriction endonuclease analysis. Fluorescence-based fragment analysis with polyacrylamide gel electrophoresis provides the accurate band sizing required for homology assessment. The large number of phylogenetically informative characters obtained by fbAFLP is well suited for cluster analysis and database development. The method demonstrated excellent reproducibility and ease of performance and interpretation. We typed 30 epidemiologically well-characterized isolates of vancomycin-resistant enterococci from an outbreak in a university hospital by fbAFLP. Clustering of fbAFLP data matched epidemiological, microbiological, and pulsed-field gel electrophoresis data. This study demonstrates the unprecedented utility of fbAFLP for epidemiological investigation. Future developments in standardization and automation will set fbAFLP as the "gold standard" for molecular typing in epidemiology.

Outbreak of vancomycin-resistant enterococci in a burn unit.

OBJECTIVE: To investigate and control an outbreak of colonization and infection caused by vancomycin-resistant enterococci (VRE) in a burn intensive care unit (BICU). DESIGN: Epidemiological investigation, including multiple point-prevalence culture surveys of patients and environment, cultures from hands of healthcare workers (HCWs), pulsed-field gel electrophoresis (PFGE) typing of patient and environmental isolates, case-control study, and institution and monitoring of control measures. SETTING: BICU in an 800-bed university medical center in Galveston, Texas. RESULTS: Between June 6, 1996, and July 14, 1997, 21 patients were colonized by VRE, and 4 of these patients developed bacteremia. Of 2,844 environmental cultures, 338 (11.9%) were positive, but all hand cultures from HCWs were negative. PFGE typing indicated that the outbreak was clonal, with VRE isolates from patients differing by < or =4 bands from the index case. Thirteen of 14 environmental isolates varied by < or =4 bands from the pattern of the index case. A case-control study analyzed by exact logistic regression identified diarrhea (odds ratio [OR], 43.9; 95% confidence interval [CI95], 5.5-infinity; P=.0001) and administration of an antacid (OR, 24.2; CI95, 2.9-infinity; P=.002) as independent risk factors for acquisition of VRE. During a 5-week period in October and November 1996, all patient and 317 environmental cultures were negative for VRE. The outbreak recurred from a contaminated electrocardiogram lead that had not been identified during the prior 5 weeks. VRE were finally eradicated from the BICU in July 1997, using barrier isolation and a very aggressive environmental decontamination program. CONCLUSIONS: A VRE outbreak in a BICU over 13 months was caused by a single clone. After apparent eradication of VRE from a BICU, recrudescence of the outbreak occurred, evidently from a small inapparent source of environmental contamination. Changes in gastrointestinal (GI) tract function (motility) and administration of medications, other than antibiotics, that have an effect on the GI tract may increase the risk of GI tract colonization by VRE in burn patients. Application of barrier isolation and an aggressive environmental decontamination program can eradicate VRE from a burn population.

Comparison of three methods to recover vancomycin-resistant enterococci (VRE) from perianal and environmental samples collected during a hospital outbreak of VRE.

OBJECTIVE: To establish an efficient and sensitive technique for recovering vancomycin-resistant enterococci (VRE) from perianal and environmental samples collected during implementation of control measures for an outbreak of VRE. DESIGN: Perianal and environmental samples were collected in triplicate on sterile swabs. One swab was used to inoculate a selective broth medium containing 6 pg of vancomycin and 8 pg of ciprofloxacin per mL, one to inoculate Campylobacter agar containing 10 microg/mL of vancomycin, and one to inoculate Enterococcosel agar containing 8 microg/mL of vancomycin. SETTING: Samples were collected in the intensive care units of a 600-bed university hospital over a period of 2 months. SAMPLE SELECTION: Patients and their immediate environment were sampled if they resided in a ward with a patient known to be colonized or infected with VRE. RESULTS: Of the 88 perianal samples obtained from 63 patients, 37 were positive for VRE by broth culture, with 36 also recovered on both types of solid media (sensitivity, 97.3%; negative predictive value, 98.1%). Of the initial samples collected from each of the 63 patients, 20 were positive for VRE by all methods. Of the 500 environmental samples cultured, 139 were positive for VRE in broth, with only 33 recovered on Campylobacter agar (sensitivity, 23.7%; negative predictive value, 77.2%) and 22 on Enterococcosel agar (sensitivity, 15.8%; negative predictive value, 75.2%). CONCLUSIONS: Our data indicate that, when performing surveillance cultures during an outbreak of VRE, use of an enrichment broth medium is required to recover VRE contaminating environmental surfaces; however, direct inoculation to selective solid medium is adequate to recover VRE in patient perianal specimens.

Comparison of the Vitek GPS-TB card with disk diffusion testing for predicting the susceptibility of enterococci to vancomycin.

OBJECTIVE: To compare the ability of the Vitek GPS-TB card with disk diffusion testing for determining the susceptibility of enterococci to vancomycin. DESIGN: Vitek susceptibility testing was performed using the GPS-TB card and software version R05.03. Disk diffusion susceptibility testing was performed according to National Committee for Clinical Laboratory Standards guidelines. When discrepancies occurred between the interpretation of Vitek and disk diffusion, both tests were repeated and the epsilometer test (E test) and agar screen containing 6 microgram/mL vancomycin were performed. RESULTS: Of 415 isolates tested, 313 were susceptible to vancomycin and 97 were resistant to vancomycin by both test methods. Two isolates were intermediate by Vitek and resistant by disk diffusion, 2 were intermediate by Vitek and susceptible by disk diffusion, and 1 was susceptible by Vitek and intermediate by disk diffusion. All but 1 of these latter 5 isolates (intermediate by Vitek and susceptible by disk diffusion) were available for retesting. On repeat testing, the 2 isolates that were intermediate by Vitek and resistant by disk diffusion were resistant by both methods, the 1 isolate that was intermediate by Vitek and susceptible by disk diffusion was susceptible by both methods, and the isolate that was susceptible by Vitek and intermediate by disk diffusion was also susceptible by both methods. These results were confirmed by E test and agar screen. CONCLUSION: We found the results of the GPS-TB card compared well with disk diffusion. However, isolates with intermediate results by Vitek should be retested using another method, such as the E test.

Epidemiology of ventilator-acquired pneumonia based on protected bronchoscopic sampling.

We performed a prospective observational cohort study of the epidemiology and etiology of nosocomial pneumonia in 358 medical ICU patients in two university-affiliated hospitals. Protected bronchoscopic techniques (protected specimen brush and bronchoalveolar lavage) were used for diagnosis to minimize misclassification. Risk factors for ventilator-associated pneumonia were identified using multiple logistic regression analysis. Twenty-eight cases of pneumonia occurred in 358 patients for a cumulative incidence of 7.8% and incidence rates of 12.5 cases per 1, 000 patient days and 20.5 cases per 1,000 ventilator days. Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Hemophilus species made up 65% of isolates from the lower respiratory tract, whereas only 12.5% of isolates were enteric gram-negative bacilli. Daily surveillance cultures of the nares, oropharynx, trachea, and stomach demonstrated that tracheal colonization preceded ventilator-associated pneumonia in 93.5%, whereas gastric colonization preceded tracheal colonization for only four of 31 (13%) eventual pathogens. By multiple logistic regression, independent risk factors for ventilator- associated pneumonia were admission serum albumin <= 2.2 g/dl (odds ratio [OR] 5.9; 95% confidence interval [CI] 2.0-17.6; p = 0.0013), maximum positive end-expiratory pressure >= 7.5 cm H2O (OR, 4.6; 95% CI, 1.4 to 15.1; p = 0.012), absence of antibiotic therapy (OR, 6.7; 95% CI, 1.8 to 25.3; p = 0.0054), colonization of the upper respiratory tract by respiratory gram-negative bacilli (OR, 3.4; 95% CI, 1.1 to 10.1; p = 0.028), pack-years of smoking (OR, 2.3 for 50 pack-years; 95% CI, 1. 2 to 4.2; p = 0.012), and duration of mechanical ventilation (OR, 3. 4 for 14 d; 95% CI, 1.5 to 7.8; p = 0.0044). Several of these risk factors for ventilator-associated pneumonia appear amenable to intervention.

Nosocomial sinusitis in patients in the medical intensive care unit: a prospective epidemiological study.

A prospective observational cohort study of nosocomial sinusitis was carried out in two medical intensive care units. Sinusitis was diagnosed by computed tomographic scanning and the culture of sinus fluid obtained by puncture of a maxillary sinus. Clinical and epidemiological data were collected at the time of admission to the unit and daily thereafter. Specimens from the nares, oropharynx, trachea, and stomach were cultured on admission and daily thereafter. The cumulative incidence of nosocomial sinusitis was 7.7%, and the incidence rates were 12 cases per 1,000 patient-days and 19.8 cases per 1,000 nasoenteric tube-days. Risk factors for nosocomial sinusitis, as determined by multiple logistic regression analysis, included nasal colonization with enteric gram-negative bacilli (odds ratio [OR], 6.4; 95% confidence interval [95% CI], 2.2-18.8; P = .007), feeding via nasoenteric tube (OR, 14.1; 95% CI, 1.7-117.6; P = .015), sedation (OR, 15.9; 95% CI, 1.9-133.5; P = .011), and a Glasgow coma score of < or = 7 (OR, 9.1; 95% CI, 3.0-27.3; P = .0001).

Validation of surgical wound classification in the operating room.

OBJECTIVE: To determine the accuracy with which circulating nurses (CNs) classify surgical procedures by risk of contamination in the operating room. DESIGN: Classification of surgical procedures by CNs was compared with the classification of surgical procedures by a physician observer. SETTING: University-affiliated, tertiary care hospital. METHODS: Circulating nurses used the traditional wound classification system of clean, clean-contaminated, contaminated, and dirty-infected to classify surgical wounds in the operating room. A physician remained in the operating room throughout each of 100 surgical procedures and simultaneously classified surgical wounds without the knowledge of the CNs. RESULTS: Classification of surgical wounds by CNs was compared with classification by the physician observer for 50 cases in general surgery and 50 cases in trauma surgery. Compared with the physician observer, the overall accuracy of classification by CNs was 88% (95% confidence interval [CI] of 81.6% to 94.4%; Kappa statistic, 0.83). Classification of surgical wounds was more difficult in trauma surgery (accuracy of 82%) than in general surgery (accuracy of 94%). Accuracy increased for both services when surgical wounds were classified into just two categories (clean or clean-contaminated versus contaminated or dirty-infected). CONCLUSIONS: Surgical wounds can be classified in the operating room with a high degree of accuracy by CNs. Classification was more difficult in trauma than in general surgery, but classification in trauma surgery improved with feedback to and additional education of CNs. The accuracy of classification by CNs was even higher when classifications were divided into just two categories.

Validation of surgical wound surveillance.

OBJECTIVE: To determine the sensitivity and specificity of standard infection control surveillance techniques for the identification of surgical wound infections. DESIGN: Surveillance data collected by three infection control practitioners (ICPs) was compared to surveillance data collected simultaneously by a gold standard observer. SETTING: University-affiliated, tertiary care hospital. METHODS: Using standard infection control surveillance techniques (chart review and discussions with patients' nurses and physicians), ICPs collected surveillance data on patients on the General Surgery and Trauma Surgery Services on days 4 and 7 after surgery and then weekly for 30 days or until patients were discharged from the hospital. Simultaneously, a hospital epidemiologist collected surveillance data and examined each patient's wound daily. RESULTS: Nine hundred twenty-five surgical patients including 537 trauma cases and 388 elective general surgery cases were followed postoperatively. The ICPs identified 67 surgical wound infections, and the hospital epidemiologist identified 80 surgical wound infections for a sensitivity of 83.8% with a 95% confidence interval (CI95) of 75.7% to 91.9%. Specificity was 99.8% with a CI95 of 99% to 100%. The sensitivity was the same for trauma surgery and general surgery, but incisional wound infections were more difficult to identify than deep wound infections. During a second validation period, sensitivity was 92.3% with a CI95 of 62% to 100%. CONCLUSIONS: Standard infection control surveillance techniques have the same sensitivity for detection of surgical wound infections as they do for identification of other nosocomial infections. Accurate data on surgical wound infections can be collected without direct examination of surgical wounds.

Infected radial artery pseudoaneurysms occurring after percutaneous cannulation.

During a ten-month period in 1988 at our institution, we identified three infected radial artery pseudoaneurysms (RAPAs) associated with arterial lines. A retrospective chart review to 1983 identified three additional cases, all occurring in 1986. In the six-year period of 1983 through 1988, during which approximately 12,500 radial artery catheters were placed, the incidence of RAPA formation was 6/12,500 (0.048 percent). Five of the six cases were associated with Staphylococcus aureus infection. The duration of radial artery cannulation was significantly longer in patients who developed a pseudoaneurysm (12.5 days) than in those patients who did not suffer this complication (4.3 days). Patients in whom infected RAPAs occurred also tended to be older (mean, 71.6 years) than the average age (54 years) for all patients admitted to the intensive care unit (ICU). They also tended to have long stays in the ICU prior to development of RAPA, the shortest stay being 11 days and the average being 51 days. Risk factors for the development of this complication may include advanced age, longer duration of catheterization and hospitalization, and infection with S aureus.