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The Xpert® vanA test performed on the GeneXpert® Instrument Systems is a qualitative in vitro diagnostic test designed for detection of the vanA gene sequence associated with vancomycin resistance in bacteria obtained from rectal swab specimens from patients at risk for intestinal colonization with vancomycin-resistant bacteria. The test utilizes automated real-time polymerase chain reaction (PCR) to detect the vanA gene that is frequently associated with vancomycin-resistant enterococci (VRE). The Xpert vanA test is intended to aid in the recognition, prevention, and control of vancomycin-resistant organisms that colonize patients in healthcare settings. The Xpert vanA test is not intended to diagnose infections caused by vancomycin-resistant bacteria nor to guide or monitor treatment for vancomycin-resistant bacterial infections. Concomitant cultures are necessary to recover organisms for identification of vancomycin-resistant bacteria, antimicrobial susceptibility testing, and for epidemiological typing.

The Xpert van4 test is an automated in vitro diagnostic test for the qualitative detection of the vanA gene sequence associated with vancomycin resistance in bacteria obtained directly from rectal swab specimens. The specimen is collected on a double swab, one of which is placed in a tube containing sample reagent. Following brief vortexing, the content of the sample reagent is transferred to the uniquely labeled Sample Chamber of a disposable fluidic cartridge (the Xpert vanA cartridge). The user initiates a test from the user interface of the GeneXpert® instrument system platform and places the cartridge with sample into the GeneXpert® instrument system which performs hands-off real-time, multiplex polymerase chain reaction (PCR) for the detection of vanA DNA. In the GeneXpert® Instrument Systems (comprised of the GeneXpert® Dx System, GeneXpert® System with Touchscreen, and GeneXpert® Infinity System), sample preparation, amplification, and real-time detection are all fully automated and completely integrated. The platform requires the use of single-use disposable cartridges that hold the PCR reagents and host the PCR process. Because the cartridges are self-contained, cross-contamination between samples is minimized. Depending on the specific instrument, a GeneXpert® instrument system may contain 1-80 modules, each of which are randomly accessible and capable of performing separate sample preparation and real-time PCR tests. Each module contains a syringe drive for dispensing fluids (i.e., the syringe drive activates the plunger that works in concert with the rotary valve in the cartridge to move fluids between chambers), an ultrasonic horn for lysing cells or spores, and a proprietary I-CORE thermocycler for performing real-time PCR and detection. The Xpert van4 test includes reagents for the detection of the gene for vancomycin resistance (van4) as well as an internal sample processing control (SPC) to control for adequate processing of the target bacteria and to monitor the presence of inhibitor(s) in the PCR assay. The SPC also ensures that the PCR conditions (temperature and time) are appropriate for the amplification reaction and that the PCR reagents are functional. The Probe Check Control (PCC) verifies reagent rehydration, PCR tube filling in the cartridge, probe integrity, and dye stability. The Xpert van 4 test performed on the GeneXpert® Instrument Systems provides results in less than 45 minutes. Each instrument in the GeneXpert® instrument family is equipped with a Windows OS-based personal computer that is preloaded with software applications for running the tests and viewing the results, as described in Table 1.

The IMDx VanR for Abbott m2000 assay is an in vitro diagnostic assay that uses polymerase chain reaction (PCR) amplification for the qualitative detection of nucleic acids encoding the vancomycin resistance genes vanA and/or vanB. The assay is performed directly on human perirectal swabs, rectal swabs, or stool specimens from patients at risk for Vancomycin Resistant Enterococcus (VRE) colonization. The IMDx VanR for Abbott m2000 assay detects the presence of vanA and vanB genes that can be associated with vancomycin-resistant enterococci. The IMDx VanR for Abbott m2000 assay can be used as an aid to identify. prevent and control vancomycin-resistant colonization in healthcare settings. The IMDx VanR for Abbott m2000 assay is not intended to diagnose VRE infection nor to guide or monitor treatment of infection. Culture methods are necessary to recover organisms for epidemiology typing and confirmation testing.

The IMDx VanR for Abbott m2000 assay is a PCR-based assay that targets regions unique to the vanA and vanB vancomycin resistance genes that may be associated with vancomycin resistant Enterococcus (VRE). Detection of the vanA and vanB genes is measured by the presence of fluorescently-labeled oligonucleotide probes that generate a fluorescent signal when specifically bound to amplified vanA and/or vanB PCR products. Differentiation of vanA from vanB is attained by labeling the oligonucleotide probes with different colored fluorescent dyes. The amplification cycle at which fluorescent signal is detected by the Abbott m2000rt is inversely proportional to the vanA and vanB DNA target level present in the sample. The IMDx VanR for Abbott m2000 assay includes reagents for the detection of the assay process control, which contains inactivated bacteria, unrelated to enterococci, and is introduced into each specimen during sample preparation. The process control (also acting as an internal control (IC)) is co-extracted with the specimen and co-amplified in the same PCR reaction as the vanA and vanB targets. The IC demonstrates that the entire assay process has proceeded within specification.

The BD GeneOhm™ VanR Assay is a qualitative in vitro test for the rapid detection of vancomycin-resistance (vanA and vanB) genes directly from perianal or rectal swabs. The BD GeneOhm™ VanR Assay detects the presence of the vanA and vanB genes that can be associated with vancomycin-resistant enterococci (VRE). The assay is performed on an automated real-time PCR instrument with perianal or rectal swabs from individuals at risk for VRE colonization. The BD GeneOhm™ VanR Assay can be used as an aid to identify, prevent and control vancomycin-resistant colonization in healthcare settings. The BD GeneOhm™ VanR Assay is not intended to diagnose VRE infections nor to guide or monitor treatment for VRE infections. Concomitant cultures are necessary to recover organisms for epidemiological typing, susceptibility testing and for further confirmatory identification.

Following specimen lysis, the vanA and vanB genetic targets, if present, are amplified. Amplification of the Internal Control (IC), a DNA fragment of 294-bp including a 254-bp sequence not found in VRE, will also take place unless PCR inhibitory substances are present. The amplified DNA targets are detected with molecular beacon probes, hairpin-forming single-stranded oligonucleotides labelled at one end with a quencher and at the other end with a fluorescent reporter dye (fluorophore). In the absence of target, the fluorescence is quenched. In the presence of target, the hairpin structure opens upon beacon/target hybridization, resulting in emission of fluorescence. For the detection of the vanA amplicon, the molecular beacon probe contains the fluorophore FAM at the 5' end and the nonfluorescent quencher moiety DABCYL at the opposite 3' end of the oligonucleotide. For the detection of the vanB amplicon, the molecular beacon probe contains the fluorophore Texas Red at the 5' end and the quencher DABCYL at the 3' end. For the detection of the IC amplicon, the molecular beacon probe contains the fluorophore TET at the 5' end and the quencher DABCYL at the 3' end. Each beacon-target hybrid fluoresces at a wavelength characteristic of the fluorophore used in the particular molecular beacon probe. The amount of fluorescence at any given cycle, or following cycling, depends on the amount of specific amplicon present at that time. The SmartCycler® software simultaneously monitors the fluorescence emitted by each molecular beacon probe, interprets all data, and provides a final result at the end of the cycling program.

The Cepheid Xpert® vanA Assay performed in the GeneXpert® Dx System is a qualitative in vitro diagnostic test designed for rapid detection of the vanA gene sequence associated with vancomycin resistance in bacteria obtained from rectal swab specimens from patients at risk for intestinal colonization with vancomycin-resistant bacteria. The test utilizes automated real-time polymerase chain reaction (PCR) to detect the vanA gene that is frequently associated with vancomycin-resistant enterococci (VRE). The Xpert vanA Assay is intended to aid in the recognition, prevention, and control of vancomycin-resistant organisms that colonize patients in healthcare settings. The Xpert vanA Assay is not intended to diagnose infections caused by vancomycin-resistant bacteria nor to guide or monitor treatment for vancomycin-resistant bacterial infections. Concomitant cultures are necessary to recover organisms for identification of vancomycin-resistant bacteria, antimicrobial susceptibility testing and for epidemiological typing.

The Cepheid Xpert vanA Assay is a rapid, automated in vitro diagnostic test for qualitative detection of the vanA gene sequence associated with vancomycin resistance in bacteria obtained directly from rectal swab specimens. The Xpert vanA Assay system performs real-time multiplex polymerase chain reaction (PCR) for detection of DNA after an initial sample processing step. The assay is performed on the Cepheid GeneXpert® Dx System. The specimen is collected on a double swab, one of which is placed in a tube containing elution reagent. Following brief vortexing, the eluted material and two single-use reagents (Reagent 1 and Reagent 2) that are provided with the assay are transferred to different, uniquely-labeled chambers of the disposable fluidic cartridge (the Xpert vanA cartridge). The user initiates a test from the system user interface and places the cartridge into the GeneXpert® Dx System instrument platform, which performs hands-off realtime, multiplex polymerase chain reaction (PCR) for detection of DNA. In this platform, additional sample preparation, amplification, and real-time detection are all fullyautomated and completely integrated. The GeneXpert® System consists of a GeneXpert instrument, personal computer, and the multi-chambered fluidic cartridges that are designed to complete sample preparation and real-time PCR for detection of the vanA gene that is associated with vancomycin-resistant enterococci (VRE) in less than 45 minutes. Each instrument system has 1 to 16 randomly accessible modules that are each capable of performing separate sample preparation and real-time PCR tests. Each module contains a syringe drive for dispensing fluids, an ultrasonic horn for lysing cells or spores, and a proprietary I-CORE® thermocycler for performing real-time PCR and detection. The Xpert vanA Assay includes reagents for the detection of the vanA resistant gene as well as an internal sample processing control (SPC) to control for adequate processing of the target bacteria and to monitor the presence of inhibitor(s) in the PCR assay. The SPC also ensures the PCR conditions (temperature and time) are appropriate for the amplification reaction and that the PCR reagents are functional. The Probe Check Control (PCC) verifies reagent rehydration, PCR tube filling in the cartridge, probe integrity, and dye stability.

The IDI-VanR® Assay is a qualitative in vitro test for the rapid detection of vancomycinresistance (vanA and vanB) genes directly from rectal swabs. The IDI-vanR® Assay detects the presence of the vanA and vanB genes that can be associated with vancomycin-resistant enterococci (VRE). The assay is performed on an automated realtime PCR instrument with rectal swabs from patients at risk for VRE colonization. The IDI-VanR® Assay can be used as an aid to identify, prevent and control vancomycinresistant colonization in healthcare settings. Concomitant cultures are necessary to recover organisms for epidemiological typing, susceptibility testing and for further confirmatory identification. The IDI-VanR® Assay is not intended to diagnose VRE infections nor to guide or monitor treatment for VRE infections.

Following specimen lysis, amplification of the vanA and vanB targets occurs. Amplification of the IC, a DNA fragment of 294-bp including a 254-bp sequence not found in VRE, will also take place unless there are PCR inhibitory substances. The amplified DNA targets are detected with molecular beacons, a hairpin-forming single-stranded oligonucleotides labelled at one end with a quencher and at the other end with a fluorescent reporter dye (fluorophore). In the absence of target, the fluorescence is quenched. In the presence of target, the hairpin structure opens upon beacon/target hybridization, resulting in emission of fluorescence. For the detection of vanA amplicons, the molecular beacon contains the fluorophore FAM at the 5' end and the non-fluorescent quencher moiety DABCYL at the opposite end of the oligonucleotide. For the detection of the vanB amplicons, the molecular beacon contains the fluorophore Texas Red at the 5' end and the quencher DABCYL at the 3' end. For the detection of the Internal Control (IC) amplicons, the molecular beacon contains the fluorophore TET at the 5' end and the quencher DABCYL at the 3' end. Each beacon-target hybrid fluoresces at a wavelength characteristic of the fluorophore used in the particular molecular beacon. The amount of fluorescence at any given cycle, or following cycling, depends on the amount of specific amplicons present at that time. The SmartCycler® software simultaneously monitors the fluorescence emitted by each beacon, interprets all data, and provides a final result at the end of the cycling program.

For the visual or spectrophotometric detection of the vanA and vanB genes in determining vancomycin resistance in enterococci isolated from culture.

The Velogene™ Genomic Identification Assay for VRE (Vancomycin Resistant Enterococci) is an in vitro, DNA probe based, diagnostic device that utilizes Cycling Probe™ Technology (CPT) to generate a spectrophotometric or visual result. Results can be generated 90 minutes after primary isolation. The Velogene™ Genomic Identification Assay for VRE (hereafter may be referred to as the Velogene™ assay) utilizes a fluorescein labeled, biotinylated DNA-RNA-DNA chimeric probe providing an RNase H cleavable linkage when bound to the complementary sequence of the van A or vanB gene. RNase H cleaves the RNA portion of the chimeric probe when it is hybridized to the target DNA. The uncleaved probe (vanA and vanB negative) is detected by binding of the fluoresceinated probe to a solid surface and attachment of an antifluorescein antibody conjugated with horseradish peroxidase, which converts a substrate to a colored end product. Cleavage of the probe (vanA or vanB positive) prevents binding of the probe-anti-fluorescein antibody enzyme complex, thus preventing formation of the colored end product. A vancomycin-resistant isolate (i.e. vanA or vanB gene is present) will produce a colorless result (or OD650 of ≤0.14). A vancomycin-sensitive isolate (i.e. vanA and vanB gene are absent) will produce a distinctly blue color (or OD650 of >0.14). Each Velogene™ assay kit contains supplies sufficient to process 48 samples and consists of two separate reagent kits: a VRE Lysis/Cycle Kit and a VRE Microwell Detection Kit.