Rapid Detection of vanA and vanB Genes Directly from Clinical Specimens and Enrichment Broths by Real-Time Multiplex PCR Assay

Abstract

A real-time PCR assay previously developed for use on the Roche LightCycler platform was investigated as an alternative to culture for the direct detection of vancomycin-resistant enterococci (VRE) in clinical specimens. PCR primers and fluorescence resonance energy transfer hybridization probes specific for the vanA and vanB genes were combined in a multiplex real-time PCR assay performed directly with fecal material obtained by rectal swabbing and with enrichment broth samples. DNA was prepared from the rectal swabs and enrichment broths with a commercially available DNA preparation column designed specifically for use with fecal specimens. One hundred eighty duplicate rectal swabs were obtained from 42 patients who were previously found to be positive for VRE and who were being monitored for carriage of VRE. Direct and enrichment broth cultures were performed with one swab, while PCR was performed with the other swab as well as any corresponding presumptive positive enrichment broth. In total, 100 specimens from 30 patients remained positive for VRE by at least one method. The multiplex real-time PCR was positive for 88 enrichment broths of rectal swabs from 27 patients but for only 45 rectal swabs from 15 patients. Direct culture was positive for VRE for only 43 specimens from 11 patients, while enrichment broth culture was positive for VRE for 75 specimens from 22 patients. Inhibition studies for the multiplex real-time PCR assay, performed by spiking the DNA extracts from 50 negative rectal swabs and the corresponding enrichment broths with between 1 and 10 CFU of a VanB Enterococcus faecium strain, detected inhibition rates of 55.1 and 10%, respectively. PCR performed directly with enrichment broths was found to be significantly more sensitive than enrichment broth culture (P < 0.025). Negative samples were identified significantly earlier by PCR than by culture alone.

Comparison of direct cultivation on a selective solid medium, polymerase chain reaction from an enrichment broth, and the BD GeneOhm™ VanR Assay for identification of vancomycin-resistant enterococci in screening specimens

“…This has led to increased interest in screening of patients for colonization as well as to increased interest in methods used for a fast, sensitive, and reliable VRE detection (Vonberg et al, 2007). Rapid screening methods for VRE using direct polymerase chain reaction (PCR) from rectal swabs or stool have been described ( [Palladino et al, 2003], [Petrich et al, 1999] and [Satake et al, 1997]); some of these assays are commercially available ( [Bourdon et al, 2010], [Stamper et al, 2007], [Sloan et al, 2004] and [Usacheva et al, 2010]). These tests have been evaluated in a few studies performed, and excellent sensitivity and specificity for detecting vanA enterococci were shown, and a low specificity due to comparably high rates of supposed false-positive vanB samples appeared as a problem ( [Stamper et al, 2007] and [Sloan et al, 2004]).…”

Rapid Detection of Antimicrobial-Resistant Organism Carriage: an Unmet Clinical Need

“…Of these, vanA and vanB predominate and are the only two that are of epidemiologic importance due to the transmissibility of the resistance genes (2). The vanA and vanB genes are found almost exclusively in Enterococcus faecium and E. faecalis, the enterococcal species that most commonly cause disease in humans, but have been detected in other species of Enterococcus (16,32); and vanB has been detected in resident anaerobic flora of the human bowel (41a). VanA-mediated resistance is associated with inducible high-level resistance to vancomycin and teicoplanin, while vanB-mediated resistance is associated with resistance to vancomycin but retained susceptibility to teicoplanin.…”

A 24-Hour Screening Protocol for Identification of Vancomycin-Resistant Enterococcus faecium

“…Our pediatric institution has a low prevalence of VRE, and we use conventional VRE screening methods, which require up to 5 days to identify VRE-positive patients (9) and 3 days to identify VREnegative patients. Other attempts to expedite VRE detection have included the use of 24-to 36-h selective broth cultures (9) and real-time PCR assays, using either swabs or broth for the identification of vanA and vanB genes encoding vancomycin resistance determinants (9,11). Preliminary work in our laboratory indicated that direct testing of rectal swabs with the Roche LightCycler VRE detection kit using DNA from our automated extraction protocol was inhibited in 50% of the samples tested.…”