D-291a. Clinical Validation of a LightCycler Assay for the Rapid Detection of KPC Producing Enterobacteriaceae from Cultured Isolates
Session: Poster Session: β-Lactamase Detection in the Clinical Laboratory
Saturday, October 25, 2008: 12:00 AM
Room: Hall C
Background: We evaluated the ability of an in-house developed blaKPC LightCyclerTM assay to rapidly detect KPC producing isolates of Enterobacteriaceae in comparison to the conventional modified Hodge testing method. Method: Thirty-four isolates of Enterobacteriaceae with antimicrobial susceptibility results suggesting ESBL production with or without increased MICs to ertapenem were studied. An inoculating needle was used to touch several colonies and then swirled into a neutralization buffer/bead processing (NB) tube. This tube was boiled and disrupted on an Eppendorf Thermomixer R thermomixing device. The sample was centrifuged, and the resulting extract was tested using the KPC LightCyclerTM (KPCLC) master mix. The KPCLC master mix uses FRET hybridization probes and was optimized for use on the LightCycler™ 2.0 instrument. All samples included within this study were verified for patient permission for use under Minnesota Statue 144.335 before testing. Results: Fifteen isolates tested positive for KPC by the LightCyclerTM assay (table). Of these 15, all were also modified Hodge positive. All 19 isolates negative for KPC by the PCR assay were also modified Hodge negative. The sensitivity, specificity, and positive and negative predictive values of the assay were 100%.
Number of IsolatesErtapenem
MIC
Imipenem
MIC
Meropenem
MIC
Modified Hodge/
KPC PCR
14≥4≥8≥8POS
1≥4≥8≤4POS
1≥4≤4≤4POS
10≥4≤4≤4NEG
2≥4≥8≤4NEG
1≥4≥8≥8NEG
1≤4≤4≤4NEG
4≤2≤4≤4NEG
Conclusion: The KPC PCR assay offers a method for rapid detection of Enterobacteriaceae producing KPC, eliminating the additional 16-24 hours needed to detect these by the modified Hodge test and the associated laborious and subjective interpretation.
Emily Vetter, BS1, Frans Robberts, PhD2, Gerhard Weldhagen3, Lisa Nyre4, Peggy Kohner, BS1, Robin Patel, MD, FIDSA1, Scott Cunningham4 and  S. A. Cunningham, None., (1)Mayo Clinic, Rochester, MN, (2)Mayo Foundation, (3)AMPATH National Laboratory Services, (4)Mayo Clinic