Evaluation of the KPC K-SeT® immunochromatographic assay for the rapid detection of KPC carbapenemase producers from positive blood cultures

Sir, The global dissemination of carbapenemase-producing Gram-negative pathogens is a matter of major concern, given their very complex MDR phenotypes and the high morbidity and mortality associated with invasive infections caused by these strains.1,2 When dealing with these pathogens, rapid detection and identification of the carbapenemase type is a crucial step for antimicrobial stewardship and also for the implementation of adequate infection control practices, thus improving patient outcomes and contrasting the diffusion of carbapenem-resistant strains.3 The KPC K-SeT® assay (Coris BioConcept, Gembloux, Belgium) is a CE-marked in vitro diagnostic test, based on fast immunochromatography, able to rapidly detect KPC-type carbapenemases from bacterial cultures.4 The system exploits monoclonal antibodies specific for KPC-type enzymes and is able to provide a result in <15 min, requiring only a minimal hands-on time and no additional instrumentation.5 In this work, we evaluated the performance of the KPC K-SeT® assay for the detection of KPC-type carbapenemases directly from positive blood cultures. The evaluation was performed at the Clinical Microbiology and Virology Unit of the Careggi University Hospital of Florence (central Italy), a setting of relatively high endemicity for KPC-producing Klebsiella pneumoniae.6 Analysis was carried out on 110 positive blood cultures from different patients, in which the presence of Gram-negative bacilli was detected by Gram staining. To enrich for the presence of positive specimens, 10 of these cases were selected after rapid detection of KPC-positive K. pneumoniae by molecular testing with the Verigene Gram-Negative Blood Culture Test (BC-GN) (Nanosphere, Northbrook, IL, USA). All specimens were also processed by the standard protocol employed in the laboratory, including isolation on Chocolate Agar PolyViteX plates (bioMérieux, Marcy-l’Étoile, France) and CHROMagar Orientation Medium plates (BD, Franklin Lakes, NJ, USA), identification by MALDI-TOF MS (Vitek®MS, bioMérieux) and antimicrobial susceptibility testing by commercial dehydrated 96-well Sensititre ITGNEGF panels (Thermo Fisher Scientific, Cleveland, OH, USA) or Vitek®2 cards (AST-N202) (bioMérieux). In all carbapenem-non-susceptible strains, the presence of carbapenemase genes, including blaKPC, blaVIM, blaNDM and blaOXA-48-like, was also investigated by means of a real-time PCR protocol as described previously.7 Testing with the K-SeT® system was carried out with positive blood cultures within a mean time of 20.0 h (median 16.3 h; range 0.2–71.3 h) from culture positivity reported by the blood culture system (BD BACTEC™ FX blood culture system), following the protocol proposed by the manufacturer with some modifications. Briefly, 40 μL of the positive blood culture was gently mixed with 10 drops of LY-A buffer, provided by the manufacturer, and then 3 drops of this mixture were dispensed into the well of the immunochromatographic cassette and incubated at room temperature for a maximum of 15 min. The results obtained with the 110 positive blood cultures investigated in this work are detailed in Table 1. Overall, 24 cultures were positive for a carbapenem-non-susceptible strain and 18 of them were positive for a KPC-producing strain, including K. pneumoniae (n = 17) and Proteus mirabilis (n = 1). The KPC K-SeT® assay detected the presence of a KPC-producing microorganism in all the confirmed KPC-positive blood cultures (sensitivity, 100%). Only in one case, positive for Escherichia coli, did the KPC K-SeT® assay repeatedly yield a positive result which, because this was not confirmed by routine method, was considered to be a false positive (specificity, 98.9%). In this case, the KPC K-SeT® assay was also repeated for the isolated strain following the manufacturer’s instructions, and yielded a negative result, which was also confirmed by the real-time PCR assay performed from total DNA extracted directly from positive blood culture with Nuclisens easyMag® (bioMérieux). Results of real-time PCR for blaKPC, of testing with KPC K-SeT® and of carbapenem susceptibility for the 110 non-replicate blood cultures positive for Gram-negative bacilli investigated in this study Test performed on blood culture. Based on MIC of meropenem according to EUCAST breakpoint (i.e. MIC ≤2 mg/L). Results of real-time PCR for blaKPC, of testing with KPC K-SeT® and of carbapenem susceptibility for the 110 non-replicate blood cultures positive for Gram-negative bacilli investigated in this study Test performed on blood culture. Based on MIC of meropenem according to EUCAST breakpoint (i.e. MIC ≤2 mg/L). All tested samples were scored as valid due to the positivity of the internal control within 15 min. The positivity of the KPC K-SeT® assay was always apparent in 2 min, at most. Altogether, results from this study suggested that the KPC K-SeT® assay could directly detect the presence of KPC carbapenemase producers in positive blood cultures, with an excellent sensitivity (100%) and good specificity (98.9%). The high sensitivity is consistent with the limit of detection of KPC K-SeT® assay with cultures of KPC-producing strains (∼106 cfu/mL; P. Bogaerts, S. Evrard, T. D. Huang and Y. Glupczynski, unpublished results), which is a value substantially lower than the bacterial load normally found in positive blood cultures (2 × 107–7 × 109 cfu/mL).8 The use of the KPC K-SeT® assay in this setting could allow the rapid detection of KPC-producers in positive blood cultures, providing valuable information for antimicrobial stewardship. The reliability of the test, its rapid and simple setting-up requiring minimal hands-on time and no expensive equipment, along with the low cost (retail price of ∼€6/test)5 make this product particularly useful in settings with high endemicity of KPC-producing Enterobacteriaceae. Further investigations aimed at evaluating the performance of the K-SeT® assay for the rapid detection of other carbapenemases (i.e. OXA-48 and NDM) from positive blood cultures would be of interest, especially for settings with a different carbapenemase epidemiology. In this perspective, the KPC K-SeT® assay could easily be implemented in the routine workflow of the clinical microbiology laboratory, and unlike other rapid methods imposes only a slight extra burden in terms of personnel and reagent costs. This study was supported by internal funding. None to declare.