An increase in vancomycin-resistant enterococcal (VRE) bacteremia in hemato-oncological patients (n=19) in our institution from 2000 through 2001 led us to analyze the molecular epidemiologic patterns and clinical features unique to our cases. The pulsed field gel electrophoresis of the isolates revealed that the bacteremia was not originated from a single clone but rather showed endemic pattern of diverse clones with small clusters. A different DNA pattern of blood and stool isolates from one patient suggested exogenous rather than endogenous route of infection. Enterococcus faecium carrying vanA gene was the causative pathogen in all cases. Patients with VRE bacteremia showed similar clinical courses compared with those with vancomycin-susceptible enterococcal (VSE) bacteremia. Vancomycin resistance did not seem to be a poor prognostic factor because of similar mortality (5/8, 62.5%) noted in VSE bacteremia. Initial disease severity and neutropenic status may be major determinants of prognosis in patients with VRE bacteraemia.
Adolescent ; Adult ; Bacteremia/*drug therapy/microbiology ; Bacterial Proteins/genetics ; Carbon-Oxygen Ligases/genetics ; Electrophoresis, Gel, Pulsed-Field ; Enterococcus/*drug effects/genetics ; Female ; Humans ; Male ; Middle Aged ; *Vancomycin Resistance

BACKGROUND: We developed and evaluated the utility of a multiplex real-time PCR assay that uses melting curve analysis and allows simultaneous identification of vancomycin-resistant genotypes and clinically relevant enterococci. METHODS: The specificity of the assay was tested using 4 reference strains of vancomycin-resistant enterococci (VRE) and 2 reference strains of vancomycin-susceptible enterococci. Ninety-three clinical isolates of enterococci with different glycopeptide-resistant phenotypes were genotyped and identified using a multiplex real-time PCR assay and melting curve analysis. RESULTS: Representative melting curves were obtained for Enterococcus faecium, Enterococcus faecalis, vanA-containing E. faecium, vanB-containing E. faecalis, Enterococcus gallinarum, and Enterococcus casseliflavus. Phenotypic and genotypic analysis of the isolates revealed same results for 82 enterococcal isolates, while in 4 isolates, the glycopeptide-resistant phenotypes were inconsistent with the glycopeptide-resistant genotypes and in the 4 other isolates, species could not be accurately identified. Three isolates with mixed strains, which were detected by the PCR assay, could not be correctly identified using phenotypic methods. CONCLUSIONS: VRE genotyping and identification of clinically relevant enterococci were rapidly and correctly performed using multiplex real-time PCR assay and melting curve analysis.
Bacterial Proteins/genetics ; Carbon-Oxygen Ligases/genetics ; DNA, Bacterial/genetics ; Enterococcus/genetics/*isolation &purification ; Enterococcus faecalis/genetics/isolation &purification ; Enterococcus faecium/genetics/isolation &purification ; Genotype ; Nucleic Acid Denaturation ; Peptide Synthases/genetics ; Phenotype ; *Polymerase Chain Reaction ; Vancomycin Resistance/*genetics

BACKGROUND: Active screening for vancomycin-resistant enterococci (VRE) using rectal specimens is recommended to limit the spread of antimicrobial resistance within certain high-risk populations. We evaluated the diagnostic performance of Vancomycin Resistance 3 Multiplexed Tandem PCR assay (AusDiagnostics, Australia), a rapid multiplex real-time PCR assay that detects vanA and/or vanB. METHODS: Two-hundred-and-eleven rectal swabs from Hematology and Oncology unit were submitted for VRE surveillance via direct detection of vanA and/or vanB by culture and by using Vancomycin Resistance 3 Multiplexed Tandem PCR assay. Enterococci were identified to the species level by using standard biochemical tests and BD Phoenix Automated Microbiology System (BD Diagnostic Systems, USA). Vancomycin susceptibility of enterococci was determined using Etest (BioMerieux, France). RESULTS: Compared to the culture method, Vancomycin Resistance 3 Multiplexed Tandem PCR assay had a sensitivity of 84.0%, specificity of 98.8%, positive predictive value (PPV) of 91.3%, and negative predictive value (NPV) of 97.6%. The assay failed to detect 18 (8.5%) specimens because of the presence of PCR inhibitors; of the remaining 193 specimens, 25 (12.9%) were positive, 23 for vanA, and 2 for vanB. Although both sensitivity and specificity for vanA VRE was 100% compared to the culture method, all vanB-positive specimens tested negative by VRE culture. CONCLUSIONS: Vancomycin Resistance 3 Multiplexed Tandem PCR assay is a rapid and laborsaving option for VRE surveillance for direct use on rectal swabs. However, the high rate of PCR failure owing to the inhibitors in the specimens and the low specificity for vanB should be considered when interpreting the results.
Bacterial Proteins/genetics ; Carbon-Oxygen Ligases/genetics ; DNA, Bacterial/*analysis ; Enterococcus/*drug effects/*genetics/growth & development/metabolism ; Humans ; *Multiplex Polymerase Chain Reaction ; Reagent Kits, Diagnostic ; Rectum/*microbiology ; Sensitivity and Specificity ; Vancomycin/*pharmacology ; Vancomycin Resistance/*genetics

BACKGROUND: Recently, the iNtRON VRE vanA/vanB real-time PCR (iNtRON; iNtRON Biotechnology, Korea) assay, a multiplex real-time PCR method, was introduced. In this prospective study, we compared the iNtRON assay with the Seeplex VRE ACE detection kit (Seeplex; Seegene, Korea), a conventional multiplex PCR assay. METHODS: A chromogenic agar-based culture, in which pre-selected vancomycin-resistant enterococci (VRE) was grown and subsequently plated on blood agar with vancomycin disks, was regarded as the reference method. A total of 304 consecutive rectal swab specimens were tested for VRE by culture and by iNtRON and Seeplex PCR assays. For the PCR assays, specimens were enriched for 16-24 hr before PCR. RESULTS: VRE were isolated from 44 (14.5%) specimens by chromogenic agar-based culture. The clinical sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the iNtRON assay were 100% (95% confidence interval: 89.8%-100%), 99.2% (96.9%-99.9%), 95.6% (83.6%-99.2%), and 100% (98.2%-100%), respectively, while those of the Seeplex assay were 97.7% (86.2%-99.9%), 99.6% (97.5%-99.9%), 97.7% (86.2%-99.9%), and 99.6% (97.5%-99.9%), respectively. The iNtRON assay had a detection limit of 3,159 copies/microL and 13,702 copies/microL for the vanA and vanB genes, respectively. No cross-reactivity was observed in 11 non-VRE bacterial culture isolates. CONCLUSIONS: The overall performance of the iNtRON assay was comparable to that of a chromogenic agar-based culture method for prompt identification of VRE-colonized patients in hospitals. This assay could be an alternative or supportive method for the effective control of nosocomial VRE infection.
Bacterial Proteins/*genetics ; Bacterial Typing Techniques/*methods/standards ; Carbon-Oxygen Ligases/*genetics ; DNA, Bacterial/*metabolism ; Gram-Positive Bacterial Infections/microbiology ; Humans ; Reagent Kits, Diagnostic ; *Real-Time Polymerase Chain Reaction ; Vancomycin Resistance/genetics ; Vancomycin-Resistant Enterococci/*genetics/isolation & purification

BACKGROUND: The aim of this study was to evaluate a newly developed PCR-based reverse blot hybridization assay (PCR-REBA), REBA Sepsis-ID (M&D, Wonju, Korea), to rapidly detect the presence of bacteremia and antimicrobial resistance gene in blood culture samples. METHODS: One thousand four hundred consecutive blood culture samples from patients with a delta neutrophil index greater than 2.7% were selected from March to July in 2013. Three hundred positive and 1,100 negative for bacterial growth in blood culture bottles samples were tested by conventional and real-time PCR-REBA, respectively. RESULTS: The overall agreement between the conventional identification test and the REBA Sepsis-ID test was 95.3% (286/300). Agreement for gram-positive bacteria, gram-negative bacteria, fungi, and polymicrobials was 94.5% (190/201), 97.3% (71/73), 100% (14/14), and 91.7% (11/12), respectively. The detection rate of the mecA gene from methicillin-resistant Staphylococcus isolates was 97.8% (90/92). The vanA gene was detected in one blood culture sample from which vancomycin-resistant Enterococcus was isolated. When the cycle threshold for real-time PCR was defined as 30.0, 2.4% (26/1,100) of negative blood culture samples tested positive by real-time PCR. CONCLUSIONS: The REBA Sepsis-ID test is capable of simultaneously and quickly detecting both causative agents and antimicrobial resistance genes, such as mecA and van, in blood culture positive samples.
Bacteremia/microbiology ; Bacterial Proteins/*genetics ; Bacteriological Techniques/*methods ; Carbon-Oxygen Ligases/*genetics ; Drug Resistance, Bacterial/genetics ; Enterococcus/*genetics/isolation & purification ; Humans ; Methicillin-Resistant Staphylococcus aureus/*genetics/isolation & purification ; *Nucleic Acid Hybridization ; RNA, Ribosomal, 16S/analysis ; Reagent Kits, Diagnostic ; *Real-Time Polymerase Chain Reaction

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.
Anti-Bacterial Agents/pharmacology ; Bacterial Proteins/genetics ; Bacterial Typing Techniques ; Carbon-Oxygen Ligases/genetics ; DNA, Bacterial/analysis ; *Disease Outbreaks ; Electrophoresis, Gel, Pulsed-Field ; Enterococcus faecium/drug effects/*genetics/isolation & purification ; Feces/microbiology ; Genotype ; Gram-Positive Bacterial Infections/diagnosis/epidemiology/*microbiology ; Humans ; Infant, Newborn ; Intensive Care Units, Neonatal ; Male ; Multilocus Sequence Typing ; Vancomycin/pharmacology ; *Vancomycin Resistance