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

VIM and KPC are two major families of carbapenemases involved in nosocomial outbreaks of multidrug-resistant Gram-negative bacilli. To rapidly detect bla(VIM)- and bla(KPC)-encoding strains, three multiplex PCR-based methods were designed and validated: (i) a real-time PCR to detect all reported VIM alleles, namely bla(VIM-1-19, 23-37); (ii) a real-time PCR to identify bla(VIM)-type and bla(KPC) 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 ≥1 mg/L. The three methods displayed 100% sensitivity, specificity and concordance. Sequencing of the bla(VIM) amplicons revealed that 30 samples encoded bla(VIM-1) and 3 samples encoded bla(VIM-2). The real-time assay, optimised for the simultaneous detection of bla(VIM) and bla(KPC), identified 3 and 12 isolates positive for both bla(VIM)/bla(KPC) and for bla(KPC), 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.