Multi-centre evaluation of real-time multiplex PCR for detection of carbapenemase genes OXA-48, VIM, IMP, NDM and KPC

Anneke Van Der Zee,Adri Gm Van Der Zanden,René Te Witt,Lieuwe Roorda,Gerda Bosman,Paul Hm Smits,Mirjam Hermans,Ad C Fluit,James Cohen Stuart,Jacobus M Ossewaarde,Lesla Es Bruijnesteijn Van Coppenraet

doi:10.1186/1471-2334-14-27

Abstract

BackgroundResistance to carbapenem antibiotics is emerging worldwide among Enterobacteriaceae. To prevent hospital transmission due to unnoticed carriage of carbapenemase producing micro-organisms in newly admitted patients, or follow-up of patients in an outbreak setting, a molecular screening method was developed for detection of the most prevalent carbapenemase genes; blaOXA-48, blaVIM, blaIMP, blaNDM and blaKPC.MethodsA real-time multiplex PCR assay was evaluated using a collection of 86 Gram negative isolates, including 62 carbapenemase producers. Seven different laboratories carried out this method and used the assay for detection of the carbapenemase genes on a selection of 20 isolates.ResultsBoth sensitivity and specificity of the multiplex PCR assay was 100%, as established by results on the strain collection and the inter-laboratory comparisons.ConclusionsIn this study, we present a multiplex real-time PCR that is a robust, reliable and rapid method for the detection of the most prevalent carbapenemases blaOXA-48, blaVIM, blaIMP, blaNDM and blaKPC, and is suitable for screening of broth cultured rectal swabs and for identification of carbapenemase genes in cultures.

Highlights

Resistance to carbapenem antibiotics is emerging worldwide among Enterobacteriaceae
Rapid and accurate detection of Carbapenemase producing Enterobacteriaceae (CPE) is pivotal for adequate antibiotic therapy and infection control, especially in an outbreak setting
The most commonly used phenotypic CPE confirmation tests, the modified Hodge test and the carbapenemase inhibition tests with boronic acid or EDTA/DPA, have several disadvantages, because those tests require an overnight incubation step, do not provide information on the carbapenemase gene, and cannot differentiate OXA-48 producing isolates from ESBL and/or AmpC producing isolates with decreased permeability [4,5,6,7]

Summary

Introduction

Resistance to carbapenem antibiotics is emerging worldwide among Enterobacteriaceae. To prevent hospital transmission due to unnoticed carriage of carbapenemase producing micro-organisms in newly admitted patients, or follow-up of patients in an outbreak setting, a molecular screening method was developed for detection of the most prevalent carbapenemase genes; blaOXA-48, blaVIM, blaIMP, blaNDM and blaKPC. Methods: A real-time multiplex PCR assay was evaluated using a collection of 86 Gram negative isolates, including 62 carbapenemase producers. Seven different laboratories carried out this method and used the assay for detection of the carbapenemase genes on a selection of 20 isolates. Carbapenemase producing Enterobacteriaceae (CPE) are emerging worldwide, and have been implicated in numerous outbreaks [1,2,3]. Rapid and accurate detection of CPE is pivotal for adequate antibiotic therapy and infection control, especially in an outbreak setting. Phenotypic detection of CPE may be difficult because carbapenem MICs may be low (in the susceptible range), especially of OXA-48 producing Enterobacteriaceae. We describe a real-time PCR for detection of NDM, KPC, VIM, IMP and OXA-48 genes, which are currently the most prevalent carbapenemases [8]. An interlaboratory performance comparison of the PCR assay was initiated to investigate its robustness and reliability

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