Automated Nucleic Acid Detection System Rapidly Resolves Gram-Positive Blood Cultures

Results obtained during a multicenter study that compared an automated diagnostic system and standard culture methods confirmed that the automated system was capable of accurately detecting most leading causes of Gram-positive bacterial blood stream infections as well as genetic markers of methicillin and vancomycin resistance directly from positive blood cultures in significantly less time than the standard procedures.

Investigators at the Medical College of Wisconsin (Milwaukee, USA) used the Nanosphere (Northbrook, IL, USA) Verigene Gram-Positive Blood Culture (BC-GP) Test to analyze 1,252 blood cultures containing Gram-positive bacteria that had been collected and tested previously at five clinical centers between April 2011 and January 2012. An additional 387 contrived blood cultures containing uncommon targets (e.g., Listeria spp., S. lugdunensis, vanB-positive Enterococci) were included to fully evaluate the performance of the BC-GP test.

The Verigene BC-GP test is a multiplexed, automated nucleic acid assay for the identification of genus, species, and genetic resistance determinants for a broad panel of the most common Gram-positive blood culture isolates. The Verigene System’s unique instrumentation allows for true random access test processing, enabling on-demand testing directly from positive blood culture bottles with less than five minutes of user hands-on time per test.

The specific targets identified by the BC-GP test include Staphylococcus spp., S. aureus, S. epidermidis, S. lugdudensis, Streptococcus spp., S. pyogenes, S. agalactiae, S. anginosus group, S. pneumoniae, E. faecalis, E. faecium, and Listeria spp. as well as the mecA, vanA, and vanB genes.

BC-GP analysis was performed by laboratory technicians who had been trained by the manufacturer. A single use extraction tray was inserted into the instrument's sample processor (SP), and an aliquot of positive blood culture broth containing Gram-positive organisms was transferred to the sample well within the extraction tray. Nucleic acid was extracted from blood culture samples using magnetic bead-based extraction and reagents contained in the extraction tray. No amplification of nucleic acid was performed. Purified nucleic acid was automatically hybridized to complementary nucleic acid capture probes immobilized on a glass microarray slide within the SP. Capture probes for each BC-GP test target were present in triplicate on the array. Detection of target sequence relied on hybridization of a second, nanoparticle-conjugated, detection probe. This method allowed up to 1,000-fold greater sensitivity than fluorescent probes and required comparatively simple excitation and detection optics. Automated sample processing (nucleic acid extraction and array hybridization) in the SP required 2.5 hours. Reading of the array was conducted in the Verigene Reader following processing and took 30 to 60 seconds.

Compared to the reference culture method, the sensitivity of the Verigene BC-GP test for the bacterial species included in the test ranged from 92.6% to 100%; its specificity was 94.5%–100%. Identification of the mecA gene (a genetic marker for methicillin resistance) in cultures that contained Staphylococcus aureus or S. epidermis (Gram-positive bacteria that are often methicillin resistant) was 98.6% sensitive and 94.3% specific. Identification of the vanA gene (a genetic marker for vancomycin resistance) was 100% sensitive and specific in cultures containing Enterococcus species.

In a retrospective analysis of turn-around-time, identification of methicillin-resistant S. aureus and vancomycin-resistant Enterococcus species with the Verigene BC-GP test was about 42 hours faster than with reference methods.

The investigators concluded that, "The high sensitivity and specificity characteristics of this test, coupled with on-demand testing capability and a [two-hour turnaround time] enable reporting of both the identification and antimicrobial resistance genes of bacteria obtained from blood culture significantly faster than using routine culture methods."

The study evaluating the Verigene Gram-Positive Blood Culture Test was published in the July 2, 2013, online edition of the journal PLOS Medicine.

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Medical College of Wisconsin
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