Characterization of a Vancomycin-resistant Enterococcus faecium Outbreak Caused by 2 Genetically Different Clones at a Neonatal Intensive Care Unit

In July 2010, we identified an outbreak of vancomycin-resistant enterococci (VRE) in our 26-bed neonatal intensive care unit. We performed an epidemiological investigation after clinical cultures of 2 neonates were positive for VRE. Identification, susceptibility testing, and molecular characterization were performed. Cultures of 3 surveillance stool samples of inpatients and 5 environmental samples were positive for VRE. All isolates were identified as Enterococcus faecium containing the vanA gene. Two distinct clones were identified by performing pulsed-field gel electrophoresis. The 2 clones exhibited different pulsotypes, but they represented identical Tn1546 types. Two sequence types, ST18 and ST192, were identified among all of the isolates with multilocus sequence typing. Our investigation determined that the outbreak in the neonatal intensive care unit was caused by 2 genetically different clones. The outbreak may have occurred through clonal spread and horizontal transfer of the van gene.

Evaluation of a Newly Developed Multiplex Real-time PCR Assay for the Detection of Vancomycin-Resistant Enterococci from Rectal Swabs / 대한임상미생물학회지

BACKGROUND: Asymptomatic vancomycin-resistant enterococci (VRE) colonization precedes infection. VRE-colonized patients serve as silent reservoirs of enterococci that go on to colonize other patients. Rapidly identifying colonized patients is crucial to prevent the spread of VRE. The culture-based method of VRE screening is time-consuming. We evaluated the diagnostic performance of a recently developed multiplex real-time PCR for the detection of VRE. METHODS: We obtained 105 rectal swabs from patients who were being monitored for carriage of VRE. After 24 hour incubation of swabs in enterococcosel broth (EB) supplemented with 6 microg/mL vancomycin, multiplex real-time PCR was performed using the Anyplex(TM) VanR Real-time Detection (VanR) kit (Seegene, Inc., Seoul, Korea). The results of multiplex real-time PCR were compared to those of culture. We evaluated the specificity and detection limits of multiplex real-time PCR using VanR for VRE. RESULTS: A total of 96/105 (91.4%) samples were VRE positive according to multiplex real-time PCR with EB while 85/105 (80.9%) samples were positive in culture. Eleven discordant results (10.4%) (multiplex real-time PCR positive, culture negative) were noted. All non-enterococcal bacteria and vancomycin-susceptible enterococci were negative. The DNA detection limits of VanR were 0.035 pg per reaction (3 microL) for Enterococcus faecium and 0.35 pg for Enterococcus faecalis. CONCLUSION: The application of multiplex real-time PCR after EB incubation allows rapid and sensitive detection in 26-28 hours for VRE screening from rectal swabs. This method could facilitate the timely implementation of contact isolation to prevent the spread of VRE.

Vancomycin-resistant Enterococci: Incidence, Antimicrobial Susceptibility, and Resistance Genotypes / 대한임상병리학회지

BACKGROUND: Infections caused by vancomycin-resistant enterococci (VRE) are becoming increasingly prevalent throughout the world. VRE can spread by direct patient-to-patient contact as well as on the hands of personnel and contaminated environmental surfaces. The purpose of this study was to examine the incidence of VRE among total enterococci from clinical specimen and investigate the antimicrobial characteristics and resistance genotypes of isolated VRE. METHODS: A total of 790 enterococcal isolates from patients over a period of 12 months were screened for vancomycin resistance using brain heart infusion agar plates supplemented with 6 g/mL of vancomycin. The incidence of VRE among enterococcal isolates was calculated from microbiology statistics program. Twenty three isolates of VRE were tested for minimal inhibitory concentrations (MIC) of vancomycin, penicillin, and gentamicin and resistance genotypes. RESULTS: In the first half period, the incidence of VRE was 1.9%, and in the second half, the incidence increased to 7.7%. Thirteen strains were found to be highly resistant to vancomycin, penicillin and gentamicin (MIC, >128 g/mL). According to the direct PCR analyses, the frequency of vanB, vanC1, and vanC2 types was 13, 7, and 3 strains, respectively. CONCLUSIONS: Continued vigilance, strict enforcement of infection control, and curtailment of vancomycin use seem to be our best approaches to controlling this increasingly important problem. For this purposes, accurate and timely detection of vancomycin-resistance and periodic investigation for incidence are essential.

Evaluation of Appropriateness of STAT Tests / 대한임상병리학회지

BACKGROUND: Apart from devaluing the basic skills of history taking and clinical examinations, the indiscriminate use of STAT tests is increasing in hospital practice. The purpose of this study is to evaluate the appropriateness of the STAT test requests in a tertiary care teaching university hospital. METHODS: We assessed the reasons for the STAT test requests on 644 patients (inpatients 338, emergency room patients 215, and outpatients 91), totaling 1,681 requests, during a 2 week period (between August 8 to 22, 1996) by discussing with the clinicians and nurses and/or reviewing the patient's records. RESULTS: Of 1,681 requests, 779 (46.3%) were considered inappropriate according to the criteria used to define categories. Inappropriate requests were detected in 45.1% (265/588) in wards, 49.0% (446/910) in the emergency room, and 37.2% (68/183) in the out patient department. The frequency of requests during the day showed two peaks: the first between 10 and 11 a.m., and the second between 3 and 4 p.m., which appears to indicate that the STAT tests are often requested for the convenience of the physician rather than true need of the patients. CONCLUSIONS: Many STAT tests are requested for reasons other than true emergencies. Inconsiderate, wasteful, and disruptive STAT test requests imposed an extra burden on the laboratory and resulted in a delay of reports on other true STAT or routine tests. Strategies to reduce the number of inappropriate STAT tests should be established in order to reserve the emergency service for situations of true need.

A Comparative Study of Biological and Analytical Variability of Automated Clinical Chemistry Tests / 대한임상병리학회지

BACKGROUND: Results of automated clinical chemistry tests are affected by many factors including analytical variability. In 1976, the College of American Pathologists (CAP) Conference on the analytical goals in clinical chemistry recommended that analytical variability should be less than 1/4 of the appropriate biological variability to improve distinction between normal and diseased populations. This study is conducted to evaluate whether automated clinical chemisty analyses performed in our laboratory is in conformance with the CAP's recommendation. METHODS: Routine chemistry and electrolyte tests were performed using Hitachi 747 automatic analyzer on 22 healthy volunteers. Blood samples were obtained from the volunteers' same vein twice in one week interval to study the total variability. Serum samples from 12 subjects were tested in duplicate immediately after blood collection for within-run analytical variability; and samples from another 10 subjects were repeated after 6 hours for within-day analytical variability. Within-run analytical variability plus within-day analytical variability make total analytical variability. Biological variability was defined as the difference between total variability and the analytical variability. Finally, ratios of analytical and biological variabilities were calculated. RESULTS: The ratios of analytical and biological variabilities of uric acid, glucose, and K were less than 0.25. But ratios of BUN, PO4, alkaline phosphatase, total bilirubin, AST, cholesterol, ALT, Cl, and protein exceeded 0.25. The ratios of Na, Ca, albumin, CO2, and creatinine could not be calculated. CONCLUSIONS: It is suggested that the analytical processes of the automated clinical chemistry tests be improved so as to be in conformity with the CAP's recommendation.

Dimethylnitrosamine-Induced Liver Cirrhosis and Expression of Hepatocyte Growth Factor (HGF), its Receptor c-Met, and the Transforming Growth Factor (TGF)-beta1 in Sprague-Dawley Rats

BACKGROUND: Liver fibrosis and cirrhosis are the ultimate histologic consequences of chronic liver damage. Efforts have been made to study the mechanisms of cirrhosis and to discover effective therapeutic strategies. However, to date, no animal model reproduces the disease in man. The purpose of this work is to establish a model of DMN-induced liver cirrhosis for treatment of liver cirrhosis, to understand the basic characteristics of DMN-induced liver cirrhosis, and to confirm the expression of HGF, its receptor c-Met, and TGF-beta1 in Sprague-Dawley rats. METHODS: Five-week-old male Sprague-Dawley rats (n=56) were used for this study. Liver cirrhosis was induced in the rats by using DMN (1 ml/kg body weight, i.p.) given 3 consecutive days a week for 6 weeks. Changes in the portal vein pressure were measured by a venous catheter during the duration of the DMN-treatment. The levels of serum albumin, bilirubin, and ammonia were determined in a clinical laboratory by routive methods. Pieces of the median lobe were cut and fixed in 10% buffered neutral formalin, embedded in paraffin, and stained by hematoxylin-eosin (H&E) & masson-trichrome (M&T). Changes in the extracellular matrix were measured by image analysis and hydroxyproline content. Immunohistochemical staining of alpa-smooth muscle actin was performed to confirm the activation ofhepatic stellate cells. Northern blot analyses were performed to confirm the expression of HGF and TGF-beta1 and western blotting was performed c-Met, HGF receptor. RESULTS: Pressures in the portal vein were significantly increased during the DMN-treatment time (p<0.05). Biochemical parameters were significantly correlated with the progression of liver cirrhosis. H&E staining of 4-week DMN-treated rats demonstrated fibrous tissue bridging between the periportal and the pericentral areas with gradual widening of fibrous bands. Both the extracellular matrix measured by image analysis of the M&T staining and the hydroxyproline content rose continuously throughout the 6 weeks of DMN treatment. alpa-smooth muscle actin was observed in the stellate cells of DMN-treated rats. The northern blot analyses showed that the expression of HGF mRNA decreased with the progression of DMN-induced liver cirrhosis but that of TGF-beta1 mRNA did not. The western blot analyses showed that the expression of the c-Met receptor protein increased continuously, but the expression of HGF mRNA a decreased. CONCLUSION:The model of cirrhosis induced by chronic, discontinuous treatment with a low dose of DMN in rats was simple and predictable and displayed many of the features of human cirrhosis. The decrease in the expression of HGF mRNA may be responsible for the reduced hepatocyte regeneration in liver cirrhosis. The expression of the c-Met protein was related with the decreased expression of HGF. The exact significance of TGF-beta1 was not determined in this study.