363. Evaluation of a Real-Time blaZ PCR Compared to the Penicillin Disk Zone-Edge Test for Detection of Beta-Lactamase Producing Staphylococcus aureus
Session: Poster Abstract Session: MRSA, MSSA, Enterococci
Thursday, October 3, 2013
Room: The Moscone Center: Poster Hall C
  • blaZ PCR poster_363.pdf (866.2 kB)
  • Background: The 2012 CLSI document M100-S22 describes a new penicillin disk zone-edge test for detecting β-lactamase producing S. aureus. Our hospital has a relatively high rate of penicillin susceptible S. aureus (18%) as determined by an in-house developed real-time PCR method to detect the blaZ gene in penicillin-susceptible isolates.  The aim of this study was to compare the accuracy of in-house real-time PCR test to the new penicillin disk zone-edge test.

    Methods: 100 clinical isolates of S. aureus from different patients with penicillin MICs of <=0.12 ug/ml and a ten sample challenge set, provided by the Centers for Disease Control (CDC), were tested by both  methods to detect β-lactamase positive isolates.  The penicillin disk zone-edge test was performed as described by CLSI (M100-S22). The penicillin zone edge was interpreted as positive for β-lactamase production only if full sized colonies were forming a sharp edge. Any amount of tapered/fuzzy edge was interpreted as negative. An in-house real-time PCR with a single primer set, using SYBR green chemistry for melting curve assessment, was performed on colonies.  A melting curve showing peaks with intensity 0.5 or higher was considered positive. 

    Results: Of 110 isolates, 103 (93.6%) gave congruent results with both methods.  The sensitivity of blaZ PCR was 100% (9/9).  All nine positive isolates had narrow melting peaks.  The specificity of blaZ PCR was 93.1% (94/101). Of seven isolates with discrepant results, six showed broad melting peaks.  If a narrow melt peak was included in the positivity criteria, the specificity of blaZ PCR would be 99% (100/101).

    Conclusion: Using the penicillin disk zone-edge as the gold standard, our in-house real-time blaZ PCR detected all the β-lactamase producing S. aureus.  However, further testing is required to resolve PCR-positive, penicillin disk zone-edge negative results.  Inclusion of the melting peak narrowness in the positivity criteria may increase the specificity of blaZ PCR.

    Nancy Watz, CLS (ASCP), M.S.1, Shirley De La Rama, CLS (ASCP)1, Niaz Banaei, MD2, Bertha Ballesteros Silva1 and Martha Moreno Mendez1, (1)Clinical Microbiology, Stanford Hospital and Clinics, Palo Alto, CA, (2)Departments of Pathology and Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University, Palo Alto, CA


    N. Watz, None

    S. De La Rama, None

    N. Banaei, Specific Technologies: Collaborator, Research support

    B. Ballesteros Silva, None

    M. Moreno Mendez, None

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