Evaluation of the New NucliSENS EasyQ KPC Test for Rapid Detection of Klebsiella pneumoniae Carbapenemase Genes (blaKPC) (original) (raw)
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American Journal of Clinical Pathology, 2012
Klebsiella pneumoniae carbapenemase (KPC)producing Enterobacteriaceae are endemic in New York City hospitals and have been associated with serious infections globally. A real-time polymerase chain reaction (RT-PCR) assay was developed to detect carbapenem resistance attributable to KPC from blood culture bottles positive for gram-negative bacilli. Culture confirmation of carbapenemase production included automated imipenem and meropenem susceptibility testing and ertapenem susceptibility testing by disk-diffusion. A total of 323 Enterobacteriaceae isolates were tested, of which 8.7% (n = 28) demonstrated carbapenem-resistance by automated and manual susceptibility testing methods or by RT-PCR. The sensitivity, specificity, and positive and negative predictive values of the RT-PCR assay when compared with the automated method were 92.9%, 99.3%, 92.9%, and 99.3%, respectively, and 96.4%, 99.7%, 96.4%, and 99.7%, respectively, when compared with the ertapenem disk-diffusion method. RT-PCR is a rapid and reliable means of detecting carbapenem resistance due to KPC-plasmids in Enterobacteriaceae directly from blood culture bottles.
American Journal of Clinical Pathology, 2011
Transfer of the bla KPC genes encoding the Klebsiella pneumoniae carbapenemase (KPC) are increasingly responsible for emerging carbapenem resistance. The modified Hodge test (MHT) is recommended for the detection of KPC. We compared MHT with a real-time polymerase chain reaction (PCR) assay targeting common subtypes of bla KPC , using previously described forward and reverse primer sequences. The PCR product was detected using SYBR Green (Applied Biosystems, Foster City, CA) and confirmed by melt curve analysis. PCR was positive in 96% (52/54) of isolates that were MHT+, 90% (28/31) of MHT-isolates were PCR-, and the results were strongly correlated (P = .0001; Fisher exact test). The PCR assay is a sensitive, specific, and rapid test for detecting bla KPC genes. It could help optimize patient care by reducing the time taken to institute appropriate antimicrobial therapy and so help improve patient outcomes. Carbapenems are highly efficacious drugs for treating infections with extended-spectrum β-lactamase-producing gram-negative bacteria. 1 Previously, resistance to carbapenems has been rare; however, the emergence of transmissible carbapenem resistance is now a growing concern. 1-9 An increasingly common mechanism of carbapenem resistance is the class-A, Klebsiella pneumoniae carbapenemase (KPC). 2,8,10-13 KPCs have been reported in K pneumoniae and in Klebsiella oxytoca, Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis, Citrobacter freundii, Enterobacter spp, Serratia spp, and Salmonella spp. 2,8,14-21 The bla KPC genes that encode KPCs are present on transferable plasmids and are flanked by transposable elements, thus allowing for the gene to move from plasmid to the bacterial chromosome and back. 8,17,22,23 This potential to disseminate resistance to several classes of β-lactam antibiotics has been demonstrated in several reported outbreaks with high mortality rates. 9,13,21,24,25 Laboratory detection of KPC in clinical isolates is, therefore, crucial to limit this spread. Automated susceptibility testing methods do not reliably detect KPC-mediated resistance. 7,15 In 2009, the Clinical and Laboratory Standards Institute (CLSI) guidelines (M100) recommended use of the modified Hodge test (MHT) to definitively identify KPC producers. 26 Alternatively, the bla KPC genes have been detected by conventional and real-time polymerase chain reaction (PCR) methods. 15,20,27-29 However, there are limitations to using PCR: (1) It is more technically challenging and prone to inhibition. 30 (2) It is target-specific and may miss new variants of KPC owing to genetic mutation. Carbapenemase-like patterns of nonsusceptibility leading to false-positive MHT results have also been described in association with endemic CTX-M production and AmpC hyperproduction. 31,32 Upon completion of this activity you will be able to: • recognize emerging mechanisms for resistance to β-lactam antibiotics. • cite the current Clinical and Laboratory Standards Institute standard for laboratory detection of Klebsiella pneumoniae carbapenemasemediated carbapenem resistance. • discuss the advantages and disadvantages of the currently recommended method for detection of carbapenem resistance.
International journal of advanced biological and biomedical research, 2020
Background: The prevalence of carbapenem-resistant Enterobacteriaceae, especially Klebsiella pneumoniae carbapenemase (KPC), has been recently reported worldwide. Therefore, there is an indispensable need for precise and rapid detection of these carbapenemases. Objectives: This study was aimed to propose an accurate and rapid method for detecting K. pneumoniae carbapenemase genes from clinical samples, using reverse transcriptionpolymerase chain reaction (RT-PCR) and to evaluate the expression of these genes in the presence of β-lactam antibiotic by real-time PCR assay. Methods: One hundred and eighty-one K. pneumoniae strains were collected from patients presenting to Firoozgar Hospital of Tehran, Iran. The strains were tested using the disk diffusion method, the modified Hodge test (MHT), and E-test minimum inhibitory concentration (MIC). Next, reverse transcription-PCR method was applied for the identification of bla OXA-23 and bla OXA-48 genes. Finally, expression of genes was measured by real-time PCR assay in the presence and absence of β-lactam antibiotic. Results:Phenotypic testing showed a high level of antibiotic resistance, while the genotypic methods indicated the presence and expression of carbapenemase genes. Conclusions: The findings suggest revisions in the current antibiotic therapy protocols, considering the high expression level of resistant carbapenemases to K. pneumoniae strains.
Journal of Clinical Microbiology, 2011
Carbapenem resistance mediated by plasmid-borne Klebsiella pneumoniae carbapenemases (KPC) is an emerging problem of significant clinical importance in Gram-negative bacteria. Multiple KPC gene variants ( bla KPC ) have been reported, with KPC-2 ( bla KPC-2 ) and KPC-3 ( bla KPC-3 ) associated with epidemic outbreaks in New York City and various international settings. Here, we describe the development of a multiplex real-time PCR assay using molecular beacons (MB-PCR) for rapid and accurate identification of bla KPC variants. The assay consists of six molecular beacons and two oligonucleotide primer pairs, allowing for detection and classification of all currently described bla KPC variants ( bla KPC-2 to bla KPC-11 ). The MB-PCR detection limit was 5 to 40 DNA copies per reaction and 4 CFU per reaction using laboratory-prepared samples. The MB-PCR probes were highly specific for each bla KPC variant, and cross-reactivity was not observed using DNA isolated from several bacterial s...
Journal of infection in developing countries, 2017
INTRODUCTION The emergence of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-Kpn) isolates is attracting significant attention in nosocomial infection settings. K. pneumoniae is the main pathogen that harbours blaKPC genes. METHODOLOGY This study evaluated 54 K. pneumoniae carbapenem-resistant isolates from patients hospitalized at the University Hospital of Londrina, between July 2009 and July 2010. The isolates were phenotypically screened for carbapenemase production and submitted for genotypic confirmation by polymerase chain reaction (PCR) for KPC, metallo-β-lactamases, OXA-48, and extended-spectrum beta-lactamase genes. The absence of outer membrane proteins (OMP) was investigated by SDS-PAGE. The susceptibility profile was determined by broth microdilution, according to Clinical and Laboratory Standards Institute protocol. RESULTS All isolates were phenotypically positive for class A carbapenemase production, but negative for metallo-β-lactamase activi...
2019
Introduction: Carbapenem-resistant Enterobacteriaceae (CRE) bacteria are difficult to treat because of their high antibiotic resistance levels that can be mediated by carbapenemase enzymes such as Klebsiella Pneumoniae Carbapenemase (KPC). The purposes of this study were to determine the genetic and resistance patterns and to detect of KPC enzyme in carbapenem-resistant strains of Enterobacteriaceae isolates. Materials and methods: In this study, antibiotic resistant pattern and genotyping of carbapenem-resistant Enterobacteriaceae isolates and frequency of KPC enzyme were investigated. During 16 months of conducting the study (December 2016 until April 2018), strains of Escherichia coli, Enterobacter spp. and Citrobacter spp. were isolated and identified from different clinical specimens and antibiotic susceptibility test was determined. In addition, the prevalence of the KPC enzyme was determined by PCR and two phenotypic methods including Modified Hodge Test (MHT) and the combina...