Rapid detection of blaVIM-1–37 and blaKPC1/2–12 alleles from clinical samples by multiplex PCR-based assays

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Abstract
VIM and KPC are two major families of carbapenemases involved in nosocomial outbreaks of multidrug-resistant Gram-negative bacilli. To rapidly detect blaVIM- and blaKPC-encoding strains, three multiplex PCR-based methods were designed and validated: (i) a real-time PCR to detect all reported VIM alleles, namely blaVIM-1-19, 23-37; (ii) a real-time PCR to identify blaVIM-type and blaKPC carbapenemases in an ultrarapid single reaction; and (iii) a standard PCR to amplify and sequence all VIM alleles. All three methods detected 33 VIM-positive samples among 107 Gram-negative isolates with imipenem and meropenem minimum inhibitory concentrations ≥1mg/L. The three methods displayed 100% sensitivity, specificity and concordance. Sequencing of the blaVIM amplicons revealed that 30 samples encoded blaVIM-1 and 3 samples encoded blaVIM-2. The real-time assay, optimised for the simultaneous detection of blaVIM and blaKPC, identified 3 and 12 isolates positive for both blaVIM/blaKPC and for blaKPC, respectively. The analytical sensitivity of the real-time assays was linear over 6 log dilutions, with a reproducible detection limit of 1 CFU. No cross-reactivity was detected. The developed assays provide powerful tools for rapid identification of VIM and KPC carbapenemase producers, therefore contributing to the prevention and containment of resistance dissemination.