|Identification of Staphylococcus aureus with Peptide Nucleic Acid (PNA)-Fluorescence In Situ Hybridization (FISH)|
Staphylococcus aureus, particularly methicillin resistant strains (MRSA), have represented a likely target for molecular development, particularly in blood cultures. As more institutions implement patient screening protocols for MRSA, replacement of routine culture methods with molecular assays has gained increasing attention.PNA-FISH assays provide for the definitive identification of Staphylococcus aureus from positive blood culture vials. Peptide nucleic acid fluorescent in-situ hybridization is a relatively straight forward procedure that does not involve amplification and has limited equipment requirements. Procedurally it is easy to perform with minimal hands on time.PNA is a synthetic imitator of a nucleic acid sequence in which the backbone is a pseudopeptide rather than a sugar. PNA behaves similarly to DNA and will bind to complementary nucleic acid strands. A PNA probe is constructed, utilizing a complementary, hybridizing sequence for a known nucleic acid target sequence. The probe is typically bound to a fluorescent protein as a means of visualizing/detecting the target. In one commercially available method, once a blood culture vial demonstrates gram-positive cocci in clusters, a drop of the blood culture broth is added to fixation solution on a slide. Heat or methanol is used to fix the smear. After fixation, probe that targets species-specific ribosomal RNA is added to the smear, which is then cover-slipped.Slides are then incubated at 55oC. Post incubation, slides are immersed in a preheated wash solution and coverslips gently removed. After incubation in the wash solution, smears are air dried; a drop of mounting medium is added and the slide is cover-slipped again.The slides are examined with a fluorescent microscope, utilizing specific filters. Green fluorescing cocci in clusters are identified as Staphylococcus aureus. This identification would be available, depending on the routine identification system utilized, potentially 24 hours earlier than the norm.A significant number of blood cultures that demonstrate gram-positive cocci in clusters yield coagulase negative staphylococci (CNS), which represent potential contaminants, rather than significant infection. What is the significance of differentiating blood cultures that contain S. aureus from those that are growing CNS in a much earlier timeframe?Studies have shown that IF the differentiation of CNS from S. aureus is effectively communicated to clinicians and pharmacy/antimicrobial stewardship teams, active assessment can occur utilizing defined exclusion criteria for those patients whose cultures yielded CNS rather than S. aureus. In scenarios where contamination rather than infection is indicated, vancomycin can be discontinued earlier, and length of hospital stay is also shortened. Reduced antibiotic exposure, reduced risk of development of resistance, and reduced cost are all potential benefits.
|Development of Assays|
Cepheid was one of the first companies to market an assay for methicillin-resistant Staphylococcus aureus (MRSA), based on its SmartCycler® real-time PCR platform.Molecular detection of methicillin resistance in staphylococci is based on the detection of the mecA gene. However, since coagulase negative staphylococci (CNS) can also possess this gene, discrimination between CNS and MRSA must be achieved by the simultaneous detection of additional gene sequences specific for S. aureus. Cepheid's assay was a multiplex assay that did include targets for six variants of the mecA gene, as well as the S. aureus orfX gene. Despite this, independent investigators documented incidences of both false-positives and false-negatives. The BD GeneOhm™ MRSA assay is another real time assay designed for the SmartCycler® platform. This assay employs molecular beacons for detection. The probe has a hairpin shape, with a fluorophore at one end, and a quencher at the other. In the absence of the target, the hairpin is closed and fluorescence is quenched. In the presence of the target, the hairpin opens when the beacon hybridizes to the target, resulting in the emission of fluorescence, which is measured during each cycle of amplification. Result availability is similar to the Cepheid assay. As with the Cepheid assay, independent investigators documented some incidence of both false positives and false negatives, but noted the advantage of rapid availability of screening results for surveillance purposes.
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