Evaluation of two commercial assays for the rapid confirmation of OXA-48 like carbapenemases produced by Klebsiella pneumoniae
Evaluation of two commercial assays for the rapid confirmation of OXA-48 like carbapenemases produced by Klebsiella pneumoniae
Over the past decade, carbapenemase-producingEnterobacteriaceae (CPE) have emerged worldwideas significant pathogens (1). Rapid and accurateconfirmation of carbapenemase production is essentialto guide antimicrobial therapy and to ensure promptimplementation of infection control measures to preventspread of CPE (2).The aim of this study was to assess the performance of2 commercially available assays for the rapid detection ofCPE with OXA-48 like carbapenemases. These included acommercially-available PCR, i.e. the BDMAX CRE assay(Becton Dickinson, Canada), and a commercially availableimmunochromatographic assay, i.e. Resist-3 O.K.N K-SeT(Coris BioConcept, Belgium).In a previous CPE surveillance program at the HacettepeUniversity Adult and Oncology Hospitals, Turkey, 279isolates of Klebsiella pneumoniae were recovered fromrectal screening swabs from unique patients. Two hundredand seventy consecutive isolates produced OXA-48 likecarbapenemases and one produced IMP carbapenemase,as confirmed by in-house PCR methods (3). Speciesidentification was confirmed by MALDI-TOF MS(Bruker, Coventry, UK). One hundred and ninety-nineof these isolates were selected for inclusion in this studyincluding 192 isolates with OXA-48 like carbapenemasesand 7 isolates without any carbapenemase. The MIC ofcarbapenems for these isolates ranged widely from 32 mg/L (3).Prior to testing for carbapenemase activity, each isolatewas retrieved from storage at –80 °C and subculturedon Mueller–Hinton agar (Oxoid) with incubation for18 h at 37 °C in air. Each isolate was tested with the2 commercial assays in exact accordance with themanufacturer’s instructions. The BD MAX CRE assay is aresearch use only automated PCR assay allowing detectionand distinction of the 3 most commonly encounteredcarbapenemase genes: KPC, OXA-48, and NDM. Theassay automates sample extraction, amplification, anddetection by using real-time PCR with a total running timeof 90–120 min (depending on batch size). There is sampleprocessing control in the extraction tube that undergoesthe extraction, concentration, and amplification steps tomonitor the assay inhibition as well as process inefficiencydue to instrument or reagent failure.The RESIST-3 O.K.N. K-SeT is animmunochromatographic cartridge assay that employs amembrane technology with colloidal gold nanoparticles.This test also allows detection and distinction of KPC,NDM, and OXA-48 like carbapenemases from a singlecolony of Enterobacteriaceae. The result is visible within15 min in the form of red lines on the strip. For bothassays, the preanalytical handling time was 5–7 min.In blind testing, both the BD MAX CRE and theRESIST-3 O.K.N. K-SeT successfully detected OXA-48 likecarbapenemases in 192 isolates and gave negative resultsfor the 7 negative controls. No other carbapenemaseswere detected and no repeat tests were necessary. Thus thesensitivity and specificity of the 2 assays were 100%.No instrumentation is required for the RESIST-3O.K.N. K-SeT and a result can be generated in around 20min, compared with at least 95 min for the BD MAX CREassay (including handling time). The cost of the BD MAXassay is approximately 28.7 Euros per sample comparedwith 10.5 Euros for the RESIST-3 O.K.N. K-SeT.A range of laboratory assays has been developedfor detection of carbapenemases that can be applied tobacterial colonies. These include matrix-assisted laserdesorption/ionisation time-of-flight mass spectrometry(MALDI-TOF MS), immunochromatographic assays,susceptibility tests that employ β-lactamase inhibitors and chromogenic tests such as the CARBA-NP test (4–6). Alternatively, the presence of carbapenemases maybe inferred by detection of target genes using molecularassays, e.g., using PCR or microarrays (7). OXA-48carbapenemase was first reported in Klebsiella pneumoniaeisolated in İstanbul in 2001 (8). Since then, OXA-48 hasbecome the predominant carbapenemase type in Turkeyand many European countries. In some phenotypic assays,detection of some OXA-48 producers has proven to beproblematic due to a low level of carbapenemase activity(resulting in low levels of resistance to carbapenems)and the lack of a specific inhibitor to assist detection (9).Recent epidemiological studies have shown that OXA-48is the most prevalent carbapenemase in many Europeancountries such as France, Spain, and Belgium and alsoin the Middle East, North Africa, and Turkey (10). Inthe present study, we showed that both commercialassays are highly effective for detection of OXA-48 likecarbapenemase producers and that either assay couldconstitute a suitable first line test for investigation ofsuspected CPE. A limitation of both tests is their inabilityto detect VIM carbapenemase and, to a lesser extent,IMP carbapenemase, and a negative test might thereforenecessitate a second test to rule out the presence of thesemetalloenzymes. In a setting such as Turkey, where OXA-48 is by far the dominant carbapenemase enzyme, this isan acceptable limitation. High levels of performance werealso reported by Glupczynski et al. (11), who challengedthe assay with a well-defined collection of 112 carbapenemresistantEnterobactericeae including a range of specieswith OXA-48-like, NDM, and KPC enzymes.When comparing the 2 commercial assays there areseveral advantages of the RESIST-3 O.K.N. K-SeT. Noinstrumentation is required for the RESIST-3 O.K.N.K-SeT and a result can be generated in around 20 min,compared with at least 95 min for the BD MAX CREassay. The cost of the BD MAX assay is approximately28.7 Euros per sample compared with 10.5 Euros for theRESIST-3 O.K.N. K-SeT. Compared with phenotypicassays that detect general carbapenemase activity (e.g., themodified Hodge test, the CARBA NP test, or MALDI-TOFMS), both assays have the inherent limitation that somecarbapenemases will not be detected, but they have theadvantage that they allow for differentiation of the 3 majortypes of carbapenemases. We conclude that both assaysare convenient, accurate, an
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