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Journal of Clinical Microbiology, April 2004, p. 1751-1752, Vol. 42, No. 4
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.4.1751-1752.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Evaluation of the Velogene Genomic Assay for Detection of vanA and vanB Genes in Vancomycin-Resistant Enterococcus Species

Department of Pathology, Keck School of Medicine, University of Southern California,¹ Los Angeles County and University of Southern California Medical Center, Los Angeles, California 90033²

Received 19 September 2003/ Returned for modification 15 November 2003/ Accepted 2 January 2004

	ABSTRACT

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The Velogene (VEL) genomic assay is a qualitative DNA probe for vanA and vanB in enterococci. 150 clinical isolates were tested with the VEL assay and characterized by pigment production, catalase levels, motility, growth in 6 µg of vancomycin/ml, vancomycin and teicoplanin susceptibility, API 20S assay, and genotyping by multiplex PCR. The VEL assay identified all enterococcal strains with vanA and vanB genes.

	TEXT

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During the last three decades, the prevalence of enterococci as nosocomial pathogens has been increasing. Vancomycin-resistant enterococci (VRE) were first recognized in 1986 (4) and are now a worldwide clinical problem, especially in acute care hospitals and intensive care units (3).

There are three predominant genotypes of VRE (2). The VanA gene confers high-level vancomycin resistance (vancomycin MIC 64 µg/ml), with accompanying teicoplanin resistance (>16 µg/ml). The vanB gene confers low- to high-level vancomycin resistance (vancomycin MICs between 16 and 1,000 µg/ml) and no teicoplanin resistance. Both vanA and vanB are acquired and transferable and are most commonly seen in Enterococcus faecalis and E. faecium. VanC1 (associated with E. casseliflavus), vanC2 (associated with E. gallinarum), and vanC3 (associated with E. flavescens) confer intrinsically low-level vancomycin resistance (3, 6).

Conventional testing with the vancomycin screen agar containing 6 µg of vancomycin/ml and conventional vancomycin susceptibility tests do not reliably differentiate between vanC and low-level vanB resistance. Identification of the isolated species of VRE requires a minimum of 18 to 24 h. Hospitals that would implement infection control measures to control VRE with vanA and vanB genes, but not those with vanC genes, need the differentiation. Homebrew PCR assays may detect resistance genes, but they are available only to those laboratories with the expertise to develop their own tests. An efficient, simple, and reliable test to differentiate genotypes would help rapid treatment and initiation of appropriate infection control procedures (3).

The Velogene (VEL) assay (ID Biomed, Bothell, Wash.) is based on a cycling probe technology that can be used to detect small amounts of DNA (1). Bacterial DNA is released from cells by suspending 1 loopful of growth from an 18- to 24-h culture in a lysing reagent that contains 20 mM TES [N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid] (pH 6.8), 0.05% Triton X-100, 150 U of achromopeptidase/ml, and 50 U of mutanolysin/ml. The DNA is denatured at 95°C for 5 min. When the target DNA (vanA and vanB genes) is hybridized with a fluorescein-labeled, biotinylated DNA-RNA-DNA chimeric probe, the RNase H cleaves the RNA portion of the probe. The mixture is transferred into streptavidin-coated microwells, and the uncleaved probe (negative reaction) is detected with the addition of fluorescein antibody conjugated with horseradish peroxidase, which converts the substrate to a blue end product. A positive-reaction product is colorless. The results can be detected visually or by spectrophotometer reading of the optical density at 650 nm. The probe test takes 90 min.

The VEL manufacturer provided a panel of 50 VRE isolates (representing major phenotypes and genotypes encountered in the United States) and a proficiency panel of 10 reference strains. After demonstrating proficiency with the VEL assay, we assayed 150 clinical isolates from individual patients hospitalized at the Los Angeles County and University of Southern California Medical Center. All the isolates were coded so that their vancomycin susceptibility characteristics were unknown to the technologist performing the VEL assay. The isolates were also tested for pigment and catalase production, motility, and growth on brain heart infusion agar containing 6 µg of vancomycin/ml. MICs of vancomycin and teicoplanin were determined by broth microdilution. The strains were identified with API 20S Streptococcus identification kits (bioMerieux, Marcy l'Etoile, France), with additional tests needed for a final identification of some strains. Genotypes were identified by multiplex PCR as previously described (5).

The VEL assay correctly detected all vanA and vanB VRE stains in the reference panels. For 10 strains (9 E. gallinarum strains and 1 E. casseliflavus strain) of the 150 clinical strains tested which grew on the vancomycin screening plate, teicoplanin had MICs of 0.25 to 1 µg/ml and vancomycin had MICs of 4 to 8 µg/ml; these 10 strains were VEL negative (Table 1). Vancomycin had MICs 2 µg/ml for another 67 strains which did not grow on the vancomycin screening plate; these strains were VEL negative also (Table 2). The remaining 73 strains were resistant to vancomycin (vancomycin MIC 16 µg/ml) and were VEL positive (Table 3). The multiplex PCR tests confirmed all VEL results. Susceptibility and sensitivity levels were 100%.

	ACKNOWLEDGMENTS

 
This work was supported by ID Biomedical, Bothell, Wash.

	FOOTNOTES

 
* Corresponding author. Mailing address: USC Pathology Reference Laboratory, 2250 Alcazar St., Room 109, Los Angeles, CA 90033. Phone: (949) 212-7559. Fax: (323) 442-2982. E-mail: mapplema{at}usc.edu.

	REFERENCES

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1. Bekkaoui, F., I. Poisson, W. Crosby, L. Cloney, and P. Duck. 1996. Cycling probe technology with RNnase H attached to an oligonucleotide. BioTechniques 20:240-248.[Medline]
2. Cetinkaya, Y., P. Falk, and C. G. Mayhall. 2000. Vancomycin-resistant enterococci. Clin. Microbiol. Rev. 13:686-707.[Abstract/Free Full Text]
3. Hayden, M. K. 2000. Insights into the epidemiology and control of infection with vancomycin-resistant enterococci. Clin. Infect. Dis. 31:1058-1065.[CrossRef][Medline]
4. Leclercq, R., E. Derlot, J. Duval, and P. Courvalin. 1988. Plasmid-mediated resistance to vancomycin and teicoplanin in Enterococcus faecium. N. Engl. J. Med. 319:157-160.[Medline]
5. Modrusan, Z., C. Marlowe, D. Wheeler, M. Pirseyedi, and R. N. Bryan. 1999. Detection of vancomycin resistant genes vanA and vanB by cycling probe technology. Mol. Cell. Probes 13:223-231.[CrossRef][Medline]
6. Rice, L. B. 2001. Emergence of vancomycin-resistant enterococci. Emerg. Infect. Dis. 7:183-187.[Medline]

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