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© 2008 Mayo Foundation for Medical Education and Research
Clinical Validation of a LightCycler™ Assay for the Rapid Detection of KPC Producing Enterobacteriaceae from Cultured IsolatesS.A. CUNNINGHAM1, P.C. KOHNER1, L.M. NYRE1, E.A. VETTER1, F.J. ROBBERTS1, G. WELDHAGEN2, R. PATEL1
1Division of Clinical Microbiology Mayo Clinic, Rochester, MN; 2Department of Internal Medicine Faculty of Health Sciences, University of Pretoria
Abstract
Method
Background: We evaluated the ability of an in-house developed blaKPC LightCycler™ 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 LightCycler™ (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 LightCycler™ 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%.
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.
34 isolates of Enterobacteriaceae with antimicrobial susceptibility results suggesting ESBL production with or without increased MIC’s to ertapenem were studied.
Modified Hodge Test: A 150mm Mueller Hinton agar plate was streaked for a lawn of growth with Escherichia coliATCC 21922. The plates were then inoculated with the test isolates in a single line from the center of the plate to the outer edge. A 10µg imipenem disk was placed on the center of the plate.
KPC production was based on the ability of the E.coli to grow along with the test isolate (figure 2).
LightCycler™: 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 (Eppendorf AG, Germany). The sample was centrifuged, and the resulting extract was tested using the KPC LightCycler™ (KPCLC) master mix.
Determination of blaKPC was based on the presence of a melt curve of 69.9°C ± 2°C (figure 1).
All samples included within this study were verified for patient permission for use under Minnesota Statue 144.335 before testing.
Background
Carbapenemase resistance, by means of KPC production, in Klebsiella pneumoniae and other members of the Enterobacteriaceae family is becoming more common. Detection of this resistance by the conventional phenotypic method (modified Hodge test) is known to be difficult and subjective. In addition, by only testing an isolate’s minimum inhibitory concentration (MIC) we are able to interpret the level of resistance of the isolate, but not the actual mechanism causing the resistance.
Here we evaluate a laboratory developed blaKPC LightCycler™ assay for rapid confirmation of KPC production in Enterobacteriaceae.
Results
Fifteen isolates tested positive for KPC by the LightCycler™assay.
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%.
Sequence alignment of the three KPC enzyme genotypes shows no mismatches in the target area eliminating the potential of false negatives.
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.
Conclusions
Acknowledgements
We would like to thank the staff of the Clinical Bacteriology Laboratory for their assistance in the collection of the samples.
We would also like to acknowledge Dr. Jean Patel and Karen Anderson at the Centers for Disease Control and Prevention’s Antimicrobial Resistance Laboratory for their assistance in providing strains for validation to our laboratory.
RESULTS
Figure 1: KPC Confirmation by LightCycler Hybridization Probe Melt
CurveFigure 2: KPC Confirmation by
Modified Hodge Testing
Modified HodgeMethod
POS NEG
POS 15 0
NEG 0 19KPCLC
Paper # D-291a
NEG≤4≤4≤24
NEG≤4≤4≤41
NEG≥8≥8≥41
NEG≤4≥8≥42
NEG≤4≤4≥410
POS≤4≤4≥41
POS≤4≥8≥41
POS≥8≥8≥414
Modified Hodge/
KPC PCR
MeropenemMIC
ImipenemMIC
ErtapenemMIC
Number of
Isolates
Positive/Positive≥8≥8≥41Citrobacter freundii
Negative/Negative≤4≤4≤21
Negative/Negative≤4≥8≥41
Positive/Positive≥8≥8≥41Enterobacter
cloacae
Negative/Negative≥8≥8≥41Serratia marcescens
Positive/Positive≤4≥8≥41Escherichia coli
Negative/Negative≤4≤4≤41
Negative/Negative≤4≥8≥41
Negative/Negative≤4≤4≤23
Negative/Negative≤4≤4≥410
Positive/Positive≤4≤4≥41
Positive/Positive≥8≥8≥412
Klebsiella pneumoniae
Modified Hodge/
KPC PCR
MeropenemMIC
ImipenemMIC
ErtapenemMIC
Number of
Isolates
Organism Identification
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