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Antimicrobial Agents and Chemotherapy, February 2004, p. 556-560, Vol. 48, No. 2
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.2.556-560.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Duplex Real-Time PCR Assay for Rapid Detection of Ampicillin-Resistant Enterococcus faecium

Stein Christian Mohn,^1,2* Arve Ulvik,³ Roland Jureen,¹ Rob J. L. Willems,⁴ Janetta Top,⁴ Helen Leavis,⁴ Stig Harthug,^1,5 and Nina Langeland^1,6

Institute of Medicine,¹ Center for Medical Genetics and Molecular Medicine,² Department of Pharmacology, University of Bergen,³ Departments of Infection Control,⁵ Medicine, Haukeland University Hospital, Bergen, Norway,⁶ Diagnostic Laboratory for Infectious Diseases and Perinatal Screening, National Institute for Public Health and the Environment, Bilthoven, The Netherlands⁴

Received 2 July 2003/ Returned for modification 2 October 2003/ Accepted 30 October 2003

Rapid and accurate identification of carriers of resistant microorganisms is an important aspect of efficient infection control in hospitals. Traditional identification methods of antibiotic-resistant bacteria usually take at least 3 to 4 days after sampling. A duplex real-time PCR assay was developed for rapid detection of ampicillin-resistant Enterococcus faecium (ARE). Primers and probes that are used in this assay specifically detected the D-Ala-D-Ala ligase gene of E. faecium and the modified penicillin-binding protein 5 gene (pbp5) carrying the Glu-to-Val substitution at position 629 (Val-629) in a set of 129 tested E. faecium strains with known pbp5 sequence. Presence of the Val-629 in the strain set from 11 different countries was highly correlated with ampicillin resistance. In a screening of hospitalized patients, the real-time PCR assay yielded a sensitivity and a specificity for the detection of ARE colonization of 95% and 100%, respectively. The results were obtained 4 h after samples were harvested from overnight broth of rectal swab samples, identifying both species and the resistance marker mutation in pbp5. This novel assay reliably identifies ARE 2 to 3 days more quickly than traditional culture methods, thereby increasing laboratory throughput, making it useful for rectal screening of ARE. The assay demonstrates the advantages of real-time PCR for detection of nosocomial pathogens.

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* Corresponding author. Mailing address: Institute of Medicine, University of Bergen, Haukeland University Hospital, N-5021 Bergen, Norway. Phone: (47) 95763424. Fax: (47) 55975890. E-mail: stein.christian.mohn{at}helse-bergen.no.

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Antimicrobial Agents and Chemotherapy, February 2004, p. 556-560, Vol. 48, No. 2
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.2.556-560.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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