title: Rapid detection of blaKPC carbapenemase genes by internally controlled real-time PCR assay using bactec blood culture bottles.
abstract: Rapid detection of drug-resistant bacteria in clinical samples plays an instrumental role in patients' infection management and in implementing effective infection control policies. In the study described in this report, we validated a multiplex TaqMan real-time quantitative PCR (qPCR) assay for the detection of bla(KPC) genes and the human RNase P gene in Bactec blood culture bottles. The MagNA Pure LC (version 2.0) instrument was utilized to extract nucleic acids from the inoculated broth, while bovine serum albumin (BSA) was utilized as the PCR inhibitor reliever. The multiplex assay, which was specific for the detection of bla(KPC) genes, had a limit of detection of 19 CFU per reaction mixture with human blood-spiked Bactec bottles. Of the 323 Bactec blood culture sets evaluated, the same 55 (17%) blood cultures positive for carbapenem-resistant bacteria by culture were also positive by the validated qPCR assay. Thus, the sensitivity, specificity, positive predictive value, and negative predictive value of the qPCR assay compared to the results of culture were all 100%. bla(KPC) genes were also detected from the same Bactec bottle broth after manual extraction with a QIAamp DNA minikit; however, there was an average 3-threshold-cycle delay in the qPCR readings. With the limited therapeutic options available, the accurate and rapid detection of bla(KPC)-possessing bacteria by the described bla(KPC)/RNase P assay will be a crucial first step in ensuring optimal clinical outcomes and infection control.
authors.affiliation: Central Virology Laboratory, Public Health Services, Ministry of Health, Chaim Sheba Medical Center, Tel-Hashomer, Israel. hindiyeh@yahoo.com
authors.forename: Musa
authors.initials: M
authors.lastname: Hindiyeh
authors.forename: Gill
authors.initials: G
authors.lastname: Smollan
authors.forename: Zehava
authors.initials: Z
authors.lastname: Grossman
authors.forename: Daniela
authors.initials: D
authors.lastname: Ram
authors.forename: Jana
authors.initials: J
authors.lastname: Robinov
authors.forename: Natasha
authors.initials: N
authors.lastname: Belausov
authors.forename: Debbie
authors.initials: D
authors.lastname: Ben-David
authors.forename: Ilana
authors.initials: I
authors.lastname: Tal
authors.forename: Yehudit
authors.initials: Y
authors.lastname: Davidson
authors.forename: Ari
authors.initials: A
authors.lastname: Shamiss
authors.forename: Ella
authors.initials: E
authors.lastname: Mendelson
authors.forename: Nathan
authors.initials: N
authors.lastname: Keller
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D001431:Bacteriological Techniques / Q000379:methods*; D001769:Blood / Q000382:microbiology; D004755:Enterobacteriaceae / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011237:Predictive Value of Tests; D012680:Sensitivity and Specificity; D013997:Time Factors; D001618:beta-Lactamases / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D001618:beta-Lactamases; C063912:carbapenemase