Real-Time PCR Detection of MRSA and Other Staphylococci in Positive Blood Culture Bottles
S Mortuza, OV Martinez, TJ Cleary. University of Miami/Jackson Memorial Hospital, Miami, FL; University of Miami, Miami, FL
Background: Coagulase-negative Staphylococci (CNS) and Staphylococcus aureus (SA) account for over 50% of all positive blood culture isolates at our institution. Staphylococcus bacteremia can cause significant mortality and morbidity in hospitalized patients; therefore, rapid identification of the Staphylococci and methicillin-susceptibility (MS) or resistance (MR) has been shown to improve patient care. We evaluated a multiplex PCR SybrGreen assay for the identification of SA, CNS, and MS/MR in 159 single patient blood cultures positive for Gram-positive cocci-in-clusters (GPC-Cl).
Design: One drop of blood culture fluid with GPC-Cl was added to a lysis tube containing 0.15 garnet beads (MO BIO, Carlsbad, CA) and 500ul of TRIS/EDTA buffer. The PCR master mix contained previously described oligonucleotide primer sets for the amplification of genes specific for the Staphylococcus genus (TStaG, 370bp, Tm 85C), SA (SA442, 179bp, Tm 82C), and MS (mecA, 98bp, Tm 79C). The concentration of the various primer sets were weighted for the detection of SA and mecA. Amplification was performed in the SmartCycler instrument (Cepheid, Inc, Sunnyvale, CA). Three distinct peaks may be found by melt curve analysis of the amplified product. If the sample contained 3+ hemolysis after lysis, it was diluted 1:20 if the original PCR was negative.
Results: Of 18 patient samples positive for MRSA, PCR was positive in 17. The negative sample was identified as MRSA upon repeat testing of the original lysate. Fourteen patient samples were positive for MSSA. The two negative samples in this subset were identified as MS-CNS. These isolates may represent deletion mutations that are not detected by the SA442 PCR assay (JCM 41:493, 2003).
Culture and PCR results from blood culture bottles
|MRSA||MSSA||CNS||>1 staphylococcal spp|
CNS, alone, was found in 108 patient samples. Of these, 104 were detected by PCR. Two samples in this subset were positive for CNS when the original lysates were retested. Most species were MR. Ten patient samples were positive for two different Staphylococcal species. All of these samples were PCR positive. Nine patient samples grew organisms other than Staphylococci; none of these samples were PCR positive.
Conclusions: The real-time PCR assay was rapid, sensitive, and specific for the detection of MRSA in blood cultures with GPC-Cl. In addition, the assay also readily distinguished MSSA, CNS-MR, and CNS-MS.
Tuesday, March 10, 2009 11:45 AM
Platform Session: Section G2, Tuesday Morning