Direct detection of vanA and vanB genes in clinical specimens for rapid identification of vancomycin resistant enterococci (VRE) using multiplex PCR

Petrich, AK; Luinstra, KE; Groves, D. J.; Ma, Chaoyang; Jb, Mahony

doi:10.1006/mcpr.1999.0250

Cited by 30 publications

(22 citation statements)

References 28 publications

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“…Satake et al (39) first described the detection of vanA and vanB directly from fecal specimens using a multiplex PCR in a gel-based assay. Gel-based assays that detect vanA and vanB directly from fecal specimens or rectal swabs have reported sensitivities of 68 to 87% and specificities approaching 100% (34,39). However, gel-based assays, while they are capable of providing results within 6 to 8 h from the time of specimen collection, increase the risk of laboratory and sample contamination, require more technician time, and slow the time to detection, in comparison to real-time PCR-based assays.…”

Section: Detection Of Vre Directly From Patient Samplesmentioning

confidence: 99%

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

Diekema¹,

Dodgson²,

Sigurðardóttir³

et al. 2004

J Clin Microbiol

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Section: Detection Of Vre Directly From Patient Samplesmentioning

confidence: 99%

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

Diekema¹,

Dodgson²,

Sigurðardóttir³

et al. 2004

J Clin Microbiol

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“…For the simultaneous detection of the vancomycin resistance genes vanA and vanB by PCR, the annealing temperatures of the necessary primer pairs were chosen within the same temperature range to perform a so-called multiplex-PCR. In combination with microtitre plate hybridisation the multiplex PCR analysis of clinical isolates only took 8 hours instead of the required 24-48 hours when a culturing procedure is used [49]. By combining the results of three multiplex PCRs specific for genes coding for aminoglycoside acetyltransferases, nucleotidyltransferases and phosphotransferases, seven classes of aminoglycoside-resistant Acinetobacter baumannii strains responsible for nosocomial infections could be identified [44].…”

Section: Pcr-based Detection Of Antibiotic Resistance Genesmentioning

confidence: 99%

Molecular tools for the characterisation of antibiotic-resistant bacteria

Aarts¹,

Sidi-Boumedine

Nesme

et al. 2001

Vet. Res.

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-This review will discuss a number of molecular tools which are currently used as well as some innovative approaches for the characterisation of antibiotic-resistant bacterial strains. Various methods involved in the detection and characterisation of genes and mutations associated with antibiotic resistance and that are used for strain typing as part of epidemiological studies, are described. Furthermore, a few examples are discussed in which the results of both gene and strain characterisation are combined to investigate the underlying mechanism of the spread of antibiotic resistance. Some of the available molecular techniques are heavily supported by the existence of databases on the Internet. These databases either contain a fast growing amount of sequence information or a large number of allelic or fingerprint profiles. The current progress in applied DNA technology and the ongoing projects on the elucidation of the whole genomic sequence of bacterial species have lead and will further lead to the development and application of sophisticated new strategies for the analysis of antibiotic resistant bacterial strains.antibiotic resistance / molecular detection / molecular typing / horizontal gene transfer / new approaches Résumé -Outils moléculaires pour la caractérisation de bactéries résistantes aux antibiotiques. Cette revue présente les outils moléculaires utilisés actuellement ainsi que des nouvelles approches pour la caractérisation de souches bactériennes résistantes aux antibiotiques. Différentes méthodes de détection et de caractérisation des gènes et des mutations impliqués dans la résistance aux antibiotiques, utilisées dans des études épidémiologiques, sont décrites. Quelques exemples sont présentés où sont associées les caractérisations du gène et de la souche bactérienne dans l'étude des mécanismes de diffusion de résistances aux antibiotiques. Certaines techniques moléculaires font

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“…Culture-based screening methods for VRE are typically time-consuming and can take from 1 to 5 days to complete (9,11,15,16). The phenotypic methods used at present for the detection of glycopeptide resistance are also limited in their abilities to detect low-level glycopeptide resistance and to distinguish between the different Van types (3,6,17).…”

mentioning

confidence: 99%

“…Therefore, many microbiology laboratories have recently introduced PCR for confirmation of the presence of isolates of VRE to facilitate the rapid and accurate identification of these organisms. The application of PCR for the detection of VRE directly from clinical surveillance specimens or enrichment broths can further reduce the detection time (11,15,16,18). Those studies that have used conventional PCR have reported various degrees of sensitivity and high degrees of specificity, thus providing encouraging results for the direct detection of VRE in these specimens.…”

mentioning

confidence: 99%

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

et al. 2003

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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.

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Direct detection of vanA and vanB genes in clinical specimens for rapid identification of vancomycin resistant enterococci (VRE) using multiplex PCR

Cited by 30 publications

References 28 publications

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

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

Molecular tools for the characterisation of antibiotic-resistant bacteria

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

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