Methods: TaqMan® Assays were designed for use on a high-throughput real-time platform (QuantStudio® 12K Flex). DNA was isolated from samples using a KingFisher™ Flex instrument, followed by twelve-step cycling with a panel-specific PreAmp mix. Pre-amplification products were then processed on custom OpenArray® Real-Time PCR Plates.
Results: The target specificity of TaqMan® Assays were validated using clinical isolates (n=109) with known, sequence-validated resistance profiles and patient samples (n=517) for which phenotypic data was available. Clinical isolates yielded 100% correlation when compared to resistance profiles. One isolate, characterized as blaNDM-1 positive, was not detected by this panel, but repeated sequencing indicated loss of the NDM-1 plasmid. Screening of patient samples revealed a complex pattern of up to fourteen genes encoding resistance for four major classes of antibiotics, with TEM and ermA-C being the most prevalent (48% and 91% in respiratory and gastrointestinal samples, respectively). CTX-M Groups 1, 2, and 9; SHV; ACT/MIR; and qnrA and qnrS were also detected.
Conclusion: Molecular testing is a practical approach for the rapid detection of drug resistance. While phenotypic susceptibility methods are widely accepted in clinical practice, these tests are laborious, time-consuming, and results may be subjective. The direct testing for multidrug resistant organisms with a high-throughput TaqMan® Assay panel provides a powerful screening tool that can be utilized in clinical settings to improve antibiotic stewardship programs and improve patient care.
S. Brzezinski, Diatherix lab: Employee , Salary
D. Stalons, Diatherix Lab: Investigator , Salary
A. Hassoun, None
P. Brozka, Thermo Fisher: Employee , Salary
N. Fantin, Thermo fisher: Employee , Salary
K. Varma, Thermo fisher: Employee , Salary
J. Trotta, Thermo fisher: Employee , Salary
E. Grigorenko, Diatherix lab: Investigator , Salary