Multiplex real-time PCR probe-based for identification of strains producing: OXA48, VIM, KPC and NDM

Abstract

The spread of multi-resistant enterobacteria, particularly carbapenem-resistant Enterobacteriaceae (CRE), in both community and hospital settings is a global problem. The phenotypic identification of CRE is complex, occasionally inconclusive and time consuming. However, commercially available molecular assays are very expensive, and many do not allow the simultaneous identification of all genetic markers of resistance that have been recognised in CRE (bla KPC, bla OXA-48, bla VIM and bla NDM). The aim of the present study is to describe a new test: a multiplex real time PCR probe-based assay designed for the simultaneous detection of KPC, OXA-48, VIM and NDM in a short time (no longer than 90 min from the extraction of DNA to detection). Our assay correctly identified 63 CRE isolates and all standard reference strains tested, in agreement with and extending the results of phenotypic identification tests; additionally, a KPC-VIM co-expressing Enterobacter aerogenes isolate was identified using the new assay, whereas traditional methods failed to detect it. The assay was also able to correctly detect 28 CRE-producers from 50 positive blood cultures, again detecting, in four specimens, the presence of CRE co-expressing KPC and VIM, which were only partially identified by traditional methods. Finally, when used directly on rectal swabs, the assay enabled the identification of CRE-carrier patients, for whom isolation is mandatory in a hospital setting.

Ceftazidime/Avibactam-Resistant Klebsiella pneumoniae subsp. pneumoniae Isolates in a Tertiary Italian Hospital: Identification of a New Mutation of the Carbapenemase Type 3 (KPC-3) Gene Conferring Ceftazidime/Avibactam Resistance

“…Identification of carbanemases (KPC, VIM, imipenemase [IMP], NDM, oxacillin-hydrolyzing [OXA] 48) was performed using the immunochromatographic (IC) assay NG CARBA (NG Biotech, Guipry, France) according to the manufacturer's instructions. Carbapenemases detected by IC assay were also confirmed by a multiplex real-time PCR probe-based assay (our patent ID MI2014A000327) designed for the simultaneous detection of KPC, OXA-48, VIM, and NDM in a short time (no longer than 90 min from the extraction of DNA to detection) [30].…”

Resistance to Ceftazidime/Avibactam in Klebsiella pneumoniae KPC-Producing Isolates: A Real-Life Observational Study

“…Identification of carbanemases (KPC, VIM, imipenase [IMP], NDM, oxacillin-hydrolysing [OXA-48]) was performed using the immunochromatographic (IC) assay NG CARBA (NG Biotech, Guipry, France) according to manufacturer's instructions. Carbapenemases detected by IC assay were also confirmed by molecular assay, using an in-house-developed multiplex real time PCR probe-based assay, able to simultaneously quickly detect KPC, OXA-48, VIM and NDM [31].…”

Microbiology and Clinical Outcome of Hospital-Acquired Respiratory Infections in an Italian Teaching Hospital: A Retrospective Study

“…Antimicrobial susceptibility testing was performed using Micronaut panels (Diagnostika Gmbh, Bornheim, Germany, now company of Bruker Daltonics, Billerica, MA, USA) run on MICRO MIB (Bruker Daltonics, Billerica, MA, USA) and interpreted following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoint (v 8.1_2018). The identification of carbapenemases (KPC, VIM, IMP, NDM, and OXA48) was performed using the immunochromatographic assay NG CARBA (NG Biotech, Guipry-Messac, France) according to the manufacturer’s instructions, and then confirmed by PCR assay [ 16 ]. The antibiotic resistance profiles were determined according to the definitions reported by Magiorakos et al [ 17 ]: multidrug-resistant (MDR) bacteria are non-susceptible to ≥1 agent in ≥3 antimicrobial categories; extensively drug-resistant (XDR) bacteria are non-susceptible to ≥1 agent in all but ≥2 categories; pandrug-resistant bacteria (PDR) are non-susceptible to all antimicrobial agents listed [ 17 ].…”