IVDT_In Vitro Diagnostics Technology

IVD Technology, November/December 2012

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of choice is to serially draw blood, typically two to four times. When cul- tures are positive from multiple blood draws, the positive predictive value (PPV) for bacteremia improves due to the diminishing probabilities that the same organism is present in each bottle by random chance.14 proposed that identity of true infection can be improved by determining spe- cies relatedness between blood culture bottles; if the organism in each bottle is identical, it is highly likely to be a true infection. However, phenotypic identifi cation methods require subcul- turing, adding signifi cant time to the process of identifying true infections. Additionally phenotypic methods are not highly accurate, and are, therefore, not likely to aid in identifying true infections for species that are diffi cult to reliably identify. Strain relatedness can be determined by RFLP; however, this requires subculturing and is a labor-intensive method that requires sophisticated analysis. It has been Staph ID/R Assay Design and Execution adapted the amplifi cation reaction to bpHDA, speeding amplifi cation ivdtechnology.com To address the above unmet need, we have developed Staph ID/R, which identifi es the major patho- genic staphylococci to the species level and detects the presence of the mecA gene, which imparts methicil- lin resistance. A multiplex bpHDA reaction is performed and the resultant amplicon is hybridized to an array of mecA gene and species-specifi c probes. Probes are printed that detect the seven most prevalent staphylo- cocci to the species level representing >98.5% of all positive staphylococcal blood cultures, with the remaining species detected by probes that cover groups of species. In a fi rst version of the application, we amplifi ed staphy- lococcal positive blood cultures using standard HDA. Th e data demon- strated excellent clinical sensitivity and specifi city, suggesting the approach is a potentially useful tool for patient management.15 We subsequently have and improving assay consistency, and have integrated the described test into the Portrait PA5000 analyzer. Th e bpHDA reaction for Staph ID/R requires the amplifi cation of both the tuf gene and mecA gene within staphylococci. As shown in fi gure 4 for the amplifi cation of MRSA blood cultures diluted into negative blood culture, within 10 minutes of bpHDA amplifi cation using real-time methods one can detect 100 copies of MRSA genomic DNA, whereas it takes 20 to 23 minutes to detect using stan- dard HDA. In model experiments we have determined a doubling time of 20 seconds for this assay allowing for the amplifi cation of a single copy of genomic DNA to detectable levels of amplicon within 15 minutes. An additional benefi t, as is seen in Figure 4, is improved consistency and quality of the amplifi cation. For HDA ampli- fi cation products, clean melt peaks are observed indicative of amplifi cation of the desired product; with standard HDA, signifi cant artifact formation is observed in the melt peaks. Th is eff ect is pronounced at lower input copies of target. To perform the Staph ID/R test, the operator simply pipettes positive blood culture media directly into the cartridge. After closing the sample port, the cartridge is inserted into the analyzer, sample information is entered, and the test is initiated using a graphical user interface. At the con- clusion of the test, results are displayed showing what staphylococcal species is present and whether or not the mecA gene was detected. Sample results from instrument runs are shown in Figure 5. Similar levels of sensitiv- ity and specifi city are observed in the instrument as on the bench. For example, as the test is designed there is a single base diff erence between S. aureus and S. warneri. As is seen in Figure 5, excellent discrimination is observed between these two species, with no cross-reactivity observed. Additionally, sensitivity is good for all species detected including two species (S. warneri and S. lugdunensis) that IVD TECHNOLOGY | NOVEMBER/DECEMBER 2012 23

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