Evaluation of the New NucliSENS EasyQ KPC Test for Rapid Detection of Klebsiella pneumoniae Carbapenemase Genes (bla_KPC)

Abstract

KPC-type carbapenemases are emerging in Klebsiella pneumoniae and other Gram-negative pathogens worldwide. Rapid and sensitive detection of these resistance determinants has become relevant to clinical management and infection control. We evaluated the bioMérieux EasyQ real-time PCR assay for bla_KPC detection with 300 members of the Enterobacteriaceae, including 29 control strains producing known carbapenemases and 271 nonreplicate clinical isolates. The EasyQ assay correctly detected all of the 111 isolates harboring bla_KPC genes, with no false positives, and results were available within 2 h.

TEXT

Klebsiella pneumoniae carbapenemases (KPCs) can confer resistance to virtually all β-lactam antimicrobials, not just carbapenems. For this reason, they are among the most challenging antibiotic resistance determinants that have emerged in the last decade (14, 17, 24). KPC-producing strains often exhibit multidrug resistance phenotypes and the ability to spread rapidly within hospital settings (13, 14, 25). These enzymes are most commonly produced by K. pneumoniae, although they have also been detected in other species of the Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter species (3, 7, 25, 27).

Rapid, reliable detection of KPC-producing members of the Enterobacteriaceae is essential both for clinical management of infected patients and for interventions aimed at limiting their spread (11, 19, 21). Phenotypic testing can putatively detect KPC-mediated resistance, but it requires at least an overnight incubation and cannot definitively confirm the nature of the carbapenemase gene (8, 10, 11, 22, 23, 26). PCR and sequencing are currently considered the gold standard to identify isolates harboring bla_KPC genes, but this approach is also time-consuming and requires considerable technical expertise. Many of these shortcomings can reportedly be overcome with new molecular methodologies. They offer considerably shorter turnaround times, and they may also prove to be accurate (2,4–6,15).

The EasyQ KPC test (bioMérieux, Marcy l'Etoile, France) is a novel real-time PCR assay that has recently been developed for bla_KPC detection. It is designed for use with the bioMérieux NucliSENS platform. Amplification, real-time detection, and automated interpretation of the test results are done on a NucliSens EasyQ analyzer. In this work we evaluated the performance of this new system in the identification of clinical isolates of bla_KPC-positive members of the Enterobacteriaceae.

Study design.

The study was conducted with a total of 300 bacterial isolates: K. pneumoniae (n = 157) and Escherichia coli (n = 143). These included 29 strains (28 K. pneumoniae strains and 1 E. coli strain) producing known carbapenemases belonging to β-lactamase class A (KPC-2 and -3 types), class B (VIM-1 and NDM-1), and class D (OXA-48), isolated from various clinical sources (Table 1). The remaining 271 isolates (129 K. pneumoniae and 142 E. coli isolates) were selected from a consecutive series of nonreplicate, multidrug-resistant K. pneumoniae and E. coli isolates recovered from blood cultures between January 2009 and December 2011 in the clinical microbiology laboratory of the Catholic University of the Sacred Heart Medical Center and had not been characterized for resistance determinants at the time of the study. Identification and antibiotic susceptibility profiling of these 271 isolates had initially been performed with ID-GN and NO89 cards and the Vitek 2 system (bioMérieux). Isolates were subsequently reidentified with the Bruker Daltonics MALDI BioTyper system (Bruker Daltonik GmbH, Leipzig, Germany). Vitek 2 antibiogram results for oxyiminocephalosporins and carbapenems were confirmed by Etest (bioMérieux) determination of MICs, and results were interpreted according to EUCAST breakpoints (document version 2.0, 2 January 2012; http://www.eucast.org/clinical_breakpoints). Multidrug resistance was defined as nonsusceptibility to at least one agent in three or more of the following antimicrobial categories: oxyiminocephalosporins, carbapenems, aminoglycosides (amikacin and/or gentamicin), and quinolones (ciprofloxacin and levofloxacin) (12).

Table 1.

Performance of EasyQ KPC for detection of bla_KPC genes in 111 KPC-producing and 189 non-KPC-producing Klebsiella pneumoniae and Escherichia coli isolates^a

Microorganism (nb)	No. of KPC-positive isolates identified by EasyQ as:		No. of KPC-negative isolates identified by EasyQ as:		% sensitivity	% specificity
	KPC positive	KPC negative	KPC positive	KPC negative
Reference isolates	18	0	0	11	100	100
Escherichia. colic (1)	0	0	0	1	NDe	ND
Klebsiella pneumoniaed (28)	18	0	0	10	100	100
Bloodstream isolates	93	0	0	178
Escherichia colif (142)	2	0	0	140	100	100
Klebsiella pneumoniaeg (129)	91	0	0	38	100	100
Total (300)	111	0	0	189	100	100

^aIsolates were classified as KPC positive when bla_KPC was identified by standard DNA sequence analysis.

^bn, no. of isolates.

^cE. coli with NDM-1 carbapenemase.

^dEighteen K. pneumoniae isolates were KPC producers: 14 (9 with KPC-2 and 5 with KPC-3) belonged to sequence type (ST) 258, 3 with KPC-3 belonged to ST 512, and one with KPC-2 belonged to ST 101, respectively. Ten KPC-negative isolates produced the VIM-1 (n = 9) or OXA-48 (n = 1) carbapenemase, respectively.

^eND, not determined. Sensitivity was not calculated when <5 isolates were found.

^fE. coli isolates with KPC-3 (n = 2), CTX-M-1 (n = 1), CTX-M-1/SHV-12 (n = 4), CTX-M-3 (n = 1), CTX-M-10 (n = 2), CTX-M-15 (n = 98), CTX-M-15/SHV-12 (n = 12), SHV-2 (n = 1), and SHV-12 (n = 21).

^gK. pneumoniae isolates with KPC-3 (n = 91), CTX-M-15 (n = 12), SHV-5 (n = 4), and SHV-12 (n = 22).

NucliSENS EasyQ KPC testing was performed according to the protocol suggested by the manufacturer. Briefly, colonies from plated cultures (on tryptic soy agar [TSA] agar plates; bioMérieux) were suspended in saline at a density of 0.5 McFarland standard. Suspensions were heated for 10 min at 95°C, and aliquots of 2.5 μl (each) were used for real-time PCR. Data were analyzed with the NucliSENS EasyQ Director software.

The comparison method for assessment of the NucliSENS EasyQ KPC test consisted of PCR amplification and sequencing, as described elsewhere, to identify genes for class A carbapenemases (KPC and GES enzymes), class B metallo-β-lactamases (VIM, IMP, and NDM enzymes), class D carbapenemases (OXA-23, -24/40, -48, -51, -55, -58, and -143), extended-spectrum β-lactamases (ESBLs) (TEM type, SHV type, CTX-M type, and OXA-2 and -10 ESBLs), and plasmid-mediated AmpC β-lactamases (CMY-2 group, DHA group, ACC-1, MOX-1, FOX-1, MIR-1, and ACT-1 group) (1, 9, 16, 18, 20, 28). This analysis was carried out with all 271 bloodstream isolates.

To define the analytical sensitivity of the NucliSENS EasyQ assay, we tested (in triplicate) 3 isolates harboring bla_KPC-2 or bla_KPC-3 genes. The detection limit assays were carried out by preparing saline suspension of each isolate to a density equivalent to a 0.5 McFarland turbidity standard. Suspensions were diluted in a 10-fold dilution series, and aliquots of 2.5 μl of each 10-fold dilution were then processed by the real-time bla_KPC assay. The numbers of CFU per suspension (CFU/ml) were evaluated by standard plating procedures.

Reproducibility was assessed by testing in triplicate 12 reference strains, including 6 with KPC-type, 4 with VIM-1, 1 with OXA-48, and 1 with NDM-1 carbapenemase genes. The turnaround time for the PCR assay was recorded.

All isolates were stored in Cryobank vials (Mast Group Ltd., Bootle, Merseyside, United Kingdom) at −70°C.

Performance of NucliSENS EasyQ assay with strains carrying known carbapenemase genes.

In preliminary testing, carried out with the 29 strains carrying known carbapenemase genes, the EasyQ KPC assay correctly detected the presence of the bla_KPC gene in all 18 strains producing KPC carbapenemases (Table 1). False-positive results were not observed. The detection limit of the real-time bla_KPC assay was 4 CFU per reaction (mean). The reproducibility was excellent, with no false negatives and no false positives in the replicate test with the 12 tested strains.

Performance of NucliSENS EasyQ assay with clinical isolates.

The results of the EasyQ KPC assay were concordant with those of the molecular comparison method for all the 271 bloodstream isolates tested (Table 1). The mean turnaround time for the PCR assay was 1.49 h. The cost per test for PCR assay was EUR 22.50, and the mean technician time was 8 min.

The 93 bla_KPC–positive isolates (91 K. pneumoniae and 2 E. coli) expressed high-level ertapenem resistance (MICs of >32 μg/ml), while imipenem and meropenem MICs ranged from 2 to >32 μg/ml and from 4 to >32 μg/ml, respectively. The remaining 178 isolates (38 K. pneumoniae and 140 E. coli) were susceptible to carbapenems while showing various levels of resistance to the oxyiminocephalosporins, cefepime, cefotaxime, and ceftazidime (MIC range, 0.5 to >64 μg/ml). These isolates were found to carry genes encoding various types of ESBLs (including SHV-2, SHV-5, SHV-12, and CTX-M-1, CTX-M-3, CTX-M-10, and CTX-M-15).

Concluding remarks.

To our knowledge, this is the first study evaluating the performance of the EasyQ KPC assay with a large series of reference and clinical isolates of KPC-producing bacteria. In our series of 300 well-characterized members of the Enterobacteriaceae, this test showed a sensitivity of 100% and a specificity of 100% compared with reference molecular tests.

The real-time PCR assay has several advantages. First, results can be obtained within 2 h (from the initiation of the procedure), which may help clinicians to provide more adequate therapy during the early period of clinical illness and facilitates rapid decisions regarding isolation of patients to prevent dissemination. Second, it is simple and easy to perform and requires minimal training. Third, its theoretical processing capacity (up to 48 samples in 24 h) makes it suitable for testing even large collections of isolates (as may be required with proactive surveillance programs in outbreak situations). On the whole, compared to standard DNA sequence analysis, the EasyQ KPC test should be of benefit since it appears to allow fast, accurate, and cost-effective detection of KPC-producing members of the Enterobacteriaceae with considerable savings in terms of time and work.

One limitation of this study is that bla_KPC allelic variants other than bla_KPC-2 and bla_KPC-3 were not investigated. However, this appears to be a minor limitation, since bla_KPC-2 and bla_KPC-3 are by far the most common allelic variants encountered in clinical settings (4, 7, 19, 24), while the sequence variability among known bla_KPC variants is very limited (GenBank database, accessed on 12 May 2012). Nonetheless, further investigation may be needed to confirm the reliability of the EasyQ KPC assay for detecting other bla_KPC variants.