Rapid detection of blaKPC carbapenemase genes by internally controlled real-time PCR assay using bactec blood culture bottles

Musa Hindiyeh, Gill Smollan, Zehava Grossman, Daniela Ram, Jana Robinov, Natasha Belausov, Debbie Ben-David, Ilana Tal, Yehudit Davidson, Ari Shamiss, Ella Mendelson, Nathan Keller

Research output: Contribution to journalArticlepeer-review

28 Scopus citations

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 blaKPC 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 blaKPC 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%. blaKPC 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 blaKPC-possessing bacteria by the described blaKPC/RNase P assay will be a crucial first step in ensuring optimal clinical outcomes and infection control.

Original languageEnglish
Pages (from-to)2480-2484
Number of pages5
JournalJournal of Clinical Microbiology
Volume49
Issue number7
DOIs
StatePublished - Jul 2011
Externally publishedYes

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