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

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          Abstract

          Background

          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; bla OXA-48, bla VIM, bla IMP, bla NDM and bla KPC.

          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.

          Results

          Both sensitivity and specificity of the multiplex PCR assay was 100%, as established by results on the strain collection and the inter-laboratory comparisons.

          Conclusions

          In 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 bla OXA-48, bla VIM, bla IMP, bla NDM and bla KPC, and is suitable for screening of broth cultured rectal swabs and for identification of carbapenemase genes in cultures.

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          Molecular epidemiology of KPC-producing Klebsiella pneumoniae isolates in the United States: clonal expansion of multilocus sequence type 258.

          Christian G Giske, Ravi B. Patel, Arjun Srinivasan … (2009)
          Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae have become more common in the United States and throughout the world. We used pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) to examine the molecular epidemiology of KPC-producing K. pneumoniae isolates sent to the Centers for Disease Control and Prevention (CDC) for reference testing from 1996 to 2008. A dominant strain, sequence type 258 (ST 258), was found and likely accounts for 70% of the CDC's K. pneumoniae PFGE database. Isolates with PFGE patterns related to ST 258 were identified in 10 of the 19 U.S. states currently reporting KPC-producing K. pneumoniae, in addition to one isolate from Israel. KPC subtyping and analysis of the surrounding genetic environment were subsequently performed on 23 representative isolates. Thirteen isolates identified as ST 258 possessed either bla(KPC-2) or bla(KPC-3) and some variability in the Tn4401 element upstream of the bla(KPC) gene. Escherichia coli DH10B was successfully transformed by electroporation with KPC-encoding plasmid DNA from 20 of the 23 isolates. Restriction analysis of plasmid DNA prepared from transformants revealed a diversity of band patterns, suggesting the presence of different plasmids harboring the bla(KPC) gene, even among isolates of the same ST.
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            Value of the modified Hodge test for detection of emerging carbapenemases in Enterobacteriaceae.

            Delphine Girlich, Patrice Nordmann, Laurent Poirel (2012)
            The modified Hodge test has an excellent sensitivity for detecting enterobacterial isolates producing Ambler class A (KPC) and class D (OXA-48) carbapenemases. Its sensitivity is low for NDM-1 producers (50%) but is increased to 85.7% by adding ZnSO(4) (100 μg/ml) in the culture medium. However, this test has a low specificity and is time-consuming.
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              Outbreak of OXA-48-positive carbapenem-resistant Klebsiella pneumoniae isolates in France.

              Gaelle Cuzon, Jocelyne Ouanich, Remy Gondret … (2011)
              Seventeen Klebsiella pneumoniae isolates producing the OXA-48 carbapenemase, obtained from 10 patients hospitalized from April to June 2010, mostly in the medical intensive care unit of the Villeneuve-Saint-Georges Hospital in a suburb of Paris, France, were analyzed. Seven patients were infected, of whom five were treated at least with a carbapenem, and five patients died. Molecular analysis showed that the isolates belonged to a single clone that harbored a 70-kb plasmid carrying the blaOXA-48 gene and coproduced CTX-M-15 and TEM-1 β-lactamases. This is the first reported outbreak of OXA-48-producing K. pneumoniae isolates in France.
              Article 0 comments Cited 63 times – based on 0 reviews     Review now
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                Author and article information

                Journal
                Journal ID (nlm-ta): BMC Infect Dis
                Journal ID (iso-abbrev): BMC Infect. Dis
                Title: BMC Infectious Diseases
                Publisher: BioMed Central
                ISSN (Electronic): 1471-2334
                Publication date Collection: 2014
                Publication date (Electronic): 14 January 2014
                Volume: 14
                Page: 27
                Affiliations
                [1 ]Maasstad Laboratory, Molecular Diagnostics Unit, Maasstad Hospital, Rotterdam, The Netherlands
                [2 ]Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The Netherlands
                [3 ]Molecular Diagnostics, Jeroen Bosch Hospital, ‘s-Hertogenbosch, The Netherlands
                [4 ]Molecular Biology Laboratory, Slotervaart Hospital, Amsterdam, The Netherlands
                [5 ]Laboratory for Microbiology and Public Health, Enschede, The Netherlands
                [6 ]Department of Medical Microbiology and Infectious Diseases, Erasmus MC, Rotterdam, The Netherlands
                [7 ]Isala clinics, Laboratory for Medical Microbiology and Infectious Diseases, Zwolle, The Netherlands
                [8 ]Medical Centre Alkmaar, Alkmaar, The Netherlands
                Article
                Publisher ID: 1471-2334-14-27
                DOI: 10.1186/1471-2334-14-27
                PMC ID: 3897903
                PubMed ID: 24422880
                SO-VID: 2f7cf4b9-8ad4-4320-af9b-d4c84c9dd29b
                Copyright © Copyright © 2014 van der Zee et al.; licensee BioMed Central Ltd.
                License:

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                Date received : 3 September 2013
                Date accepted : 24 December 2013
                Categories
                Subject: Research Article

                ScienceOpen disciplines: Infectious disease & Microbiology
                Keywords: real-time multiplex pcr,kpc,imp,carbapenemases,ndm,oxa-48,vim
                Data availability:
                ScienceOpen disciplines: Infectious disease & Microbiology
                Keywords: real-time multiplex pcr, kpc, imp, carbapenemases, ndm, oxa-48, vim

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