Use of Molecular and Reference Susceptibility Testing Methods in a Multicenter Evaluation of MicroScan Dried Overnight Gram-Positive MIC Panels for Detection of Vancomycin and High-Level Aminoglycoside Resistances in Enterococci

Abstract

Modified MicroScan gram-positive MIC no. 8 panels (PM-8) were analyzed for their improved ability to detect vancomycin resistance (VR) and high-level aminoglycoside resistance (HLAR) in enterococci. A validation study design that utilized selected challenge strains, recent clinical isolates, and reproducibility experiments in a multicenter format was selected. Three independent medical centers compared the commercial panels to reference broth microdilution panels (RBM) and Synergy Quad Agar (QA). Resistance was verified by demonstration of VR and HLAR genes by PCR tests. The study was conducted in three phases. (i) In the challenge phase (CP), two well-characterized sets of enterococci were obtained from the Centers for Disease Control and Prevention; one set contained 50 isolates for VR testing and one contained 48 isolates for HLAR testing. In addition, a set of 47 well-characterized isolates representing diverse geographic areas, obtained from earlier national surveillance studies, was tested at the University of Iowa College of Medicine (UICM). (ii) In the efficacy phase (EP), each laboratory tested 50 recent, unique clinical isolates by all methods. (iii) In the reproducibility Phase (RP), each laboratory tested the same 10 strains by all methods in triplicate on three separate days. All isolates from the EP were sent to the UICM for molecular characterization of vanA, -B, -C 1 , -C 2-3 , and HLAR genes. In the CP, the ranking of test methods by error rates (in parentheses; very major and major errors combined, versus PCR results) were as follows: for high-level streptomycin resistance (HLSR), QA (12.0%) > PM-8 (5.2%) > RBM (1.6%); for high-level gentamicin resistance (HLGR), RBM (3.7%) > PM-8 (3.1%) > QA (2.6%); and for VR, RBM ‫؍‬ QA (3.0%) > PM-8 (1.2%). In the EP, agreement between all methods and the reference PCR result was 98.0% for HLSR, 99.3% for HLGR, and 98.6% for VR. In the RP, the percentages of results ؎ 1 log 2 dilution of the all-participant mode were as follows: for VR, 100% (PM-8), 98.9% (QA), and 90.0% (RBM); for HLSR, 99.6% (RBM), 98.5% (PM-8), and 82.2% (QA); and for HLGR, 99.6% (RBM), 99.3% (PM-8), and 98.1% (QA). The ability of the PM-8 to detect VR and HLAR in enterococci was comparable to those for reference susceptibility and molecular PCR methods and was considered acceptable for routine clinical laboratory use.

Real Time PCR for the Rapid Detection of vanA Gene in Surface Waters and Aquatic Macrophyte by Molecular Beacon Probe

“…Many reports are available in the literature regarding the identification of VRE using conventional microbiological methods, which require time, resources, and space − . The application of conventional polymerase chain reaction technique for detection of VRE, although an improvement over conventional microbiological or biochemical tests, lacks absolute quantitation and requires time-consuming post PCR analysis , .…”

Evaluation of the Revised MicroScan Dried Overnight Gram-Positive Identification Panel To Identify Enterococcus Species

“…Enterococcus faecalis and E. faecium account for 80 to 90% and 10 to 15% of enterococcal infections, respectively, and other species including E. gallinarum, E. casseliflavus, E. raffinosus, E. durans, E. hirae, and E. avium, account for a majority of the remainder (14)(15)(16)(17)19). Identification systems, such as Vitek and MicroScan, have been utilized to identify only a few of the Enterococcus species, including E. faecalis, E. faecium, E. durans, and E. avium (1,4,24). Recently, MicroScan revised the gram-positive identification panel by adding Enterococcus species (E. casseliflavus, E. gallinarum, and E. raffinosus and combining E. hirae with E. durans as E. durans/hirae) to the database, reformulating biochemical tests, and including new tests.…”