Assessing pure isolates of gram-negative bacilli for mechanism of carbapenem resistance
Real-Time Polymerase Chain Reaction (PCR) Using LightCycler with Amplified Product Detection Using Fluorescent Resonance Energy Transfer (FRET) Hybridization Probes
Varies
1. See Infectious Specimen Shipping Guidelines in Special Instructions for shipping information.
Organism identification and specimen source are required.
Question ID | Description | Answers |
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SRC53 | Specimen source |
The high sensitivity of amplification by PCR requires the specimen to be processed in an environment in which contamination of the specimen by Klebsiella pneumoniae (KPC) or New Dehli metallo-beta-lactamase (NDM) DNA is unlikely.
Supplies: Infectious Container, Large (T146)
Specimen Volume: Isolate
Collection Instructions:
1. Isolate the bacteria.
2. Bacterial organism must be in pure culture, actively growing. Do not submit mixed cultures.
If not ordering electronically, complete, print, and send a Microbiology Test Request (T244) with the specimen.
Other | Agar plate, mixed cultures |
Specimen Type | Temperature | Time | Special Container |
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Varies | Ambient (preferred) | ||
Refrigerated |
Assessing pure isolates of gram-negative bacilli for mechanism of carbapenem resistance
Nonsusceptibility to carbapenems in gram-negative bacilli by means of the enzyme KPC (Klebsiella pneumoniae carbapenemase) or NDM (New Dehli metallo-beta-lactamase) is becoming more common. The genes blaKPC and blaNDM encode KPC and NDM enzyme production, respectively. In addition to KPC and NDM production, there are other mechanisms of resistance to carbapenems in gram-negative bacilli, including production of other carbapenemases, or plasmid-encoded AmpC, or extended beta-lactamase production combined with decreased membrane permeability. Detection of carbapenemases by the modified Hodge test may be subjective and is not rapid. Testing for the minimum inhibitory concentration (MIC) determines the level but not the mechanism of resistance. PCR is a sensitive, specific, and rapid means of detecting of a specific portion of the genes encoding KPC and NDM production.
Not applicable
This PCR detects and differentiates both blaKPC and blaNDM. A positive KPC (Klebsiella pneumoniae carbapenemase) PCR indicates that the isolate carries blaKPC. A positive NDM (New Dehli metallo-beta-lactamase) PCR indicates the isolate carries blaNDM.
A negative result indicates the absence of detectable blaKPC or blaNDM DNA; however, false-negative results may occur due to inhibition of PCR, sequence variability underlying primers and, or loss of a plasmid carrying blaKPC and blaNDM.
This assay should be used for testing of isolates of gram-negative bacilli. Request KNSRP / KPC (blaKPC) and NDM (blaNDM) Surveillance PCR, if testing directly from rectal or perirectal swabs is desired.
The assay was validated using 159 gram-negative bacillus isolates, including 135 carbapenemase-producers (105 blaNDM positive and 30 blaKPC positive). The assay had 100% sensitivity and specificity for isolate testing compared with reference methods, including the modified Hodge test, testing for blaKPC using KPC (Klebsiella pneumoniae carbapenemase) PCR and testing for blaNDM by NDM (New Dehli metallo-beta-lactamase) PCR at the Health Protection Agency (HPA), London, UK.
1. Cunningham SA, Noorie T, Meunier D, et al: Rapid and simultaneous detection of genes encoding Klebsiella pneumoniae carbapenemase (blaKPC) and New Delhi metallo-beta-lactamase (blaNDM) in Gram-Negative Bacilli. J Clin Microbiol 2013;51:66-69
2. Multiplex Real-Time PCR Detection of Klebsiella pneumoniae Carbapenemase (KPC) and New Delhi Metallo-beta-lactamase (NDM-1) genes. Centers for Disease Control and Prevention 2011 (unpublished)
3. CLSI Document M100-S23, Vol.33 No.1, 2013. CLSI, Wayne, PA
4. New Carbapenem-Resistant Enterobacteriaceae Warrant Additional Action by Healthcare Providers. Centers for Disease Control and Prevention Health Alert Network, February 14, 2013
Isolates are lysed in buffer to release their DNA. This assay amplifies and detects a specific portion of the genes encoding the KPC (Klebsiella pneumoniae carbapenemase) and NDM (New Dehli metallo-beta-lactamase) enzymes. The LightCycler instrument amplifies and monitors target nucleic acid sequences by fluorescence during PCR cycling. This is an automated PCR system that can rapidly detect amplified product development through stringent air-controlled temperature cycling and capillary cuvettes. The detection of amplified products is based on the fluorescent-resonance energy transfer (FRET) principle. For FRET product detection, a hybridization probe with a donor fluorophore, fluorescein, on the 3' end is excited by an external light source, which emits light that is absorbed by a second hybridization probed with an acceptor fluorophore LC-Led 610 (blaKPC specific) and LC-red 670 (blaNDM specific), on the 5' end. The acceptor fluorophore then emits a light of a different wavelength that can be measured with a signal that is proportional to the amount of specific PCR product. The detection process is completed in less than 1 hour using a closed tube system.(Cunningham SA, Noorie T, Meunier D, et al: Rapid and simultaneous detection of genes encoding Klebsiella pneumoniae carbapenemase (blaKPC) and New Delhi metallo-beta-lactamase (blaNDM) in gram-negative bacilli. J Clin Microbiol 2013;51:66-69)
Monday through Friday
This test was developed, and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. This test has not been cleared or approved by the US Food and Drug Administration.
87150 x2
Test Id | Test Order Name | Order LOINC Value |
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KPNRP | KPC and NDM PCR | 85502-3 |
Result Id | Test Result Name |
Result LOINC Value
Applies only to results expressed in units of measure originally reported by the performing laboratory. These values do not apply to results that are converted to other units of measure.
|
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SRC53 | Specimen source | 31208-2 |
35168 | KPC PCR | 49617-4 |
35169 | NDM PCR | 73982-1 |