Evaluation of the rapid carbapenem inactivation method (rCIM): a phenotypic screening test for carbapenemase-producing Enterobacteriaceae

Evaluation of the rapid carbapenem inactivation method (rCIM): a phenotypic screening test for... Abstract Objectives Fast and accurate diagnostic tests to identify carbapenemase-producing Enterobacteriaceae (CPE) are mandatory for proper antimicrobial therapy and implementing infection control measures. Here, we have developed a rapid Carbapenem Inactivation Method (rCIM) for CPE detection. Methods The rCIM consists of the incubation of a potential carbapenemase producer with meropenem discs and use of the resulting supernatant to challenge a susceptible indicator strain. Growth of the indicator strain is monitored using a nephelometer. The performances of the rCIM were compared with the CIM and Carba NP tests using a collection of 113 well-characterized carbapenem-resistant enterobacterial isolates, including 85 carbapenemase producers and 28 non-carbapenemase producers. In addition, rCIM was compared with the Carba NP test and PCR sequencing in a prospective analysis of 101 carbapenem-resistant enterobacterial isolates addressed to the French National Reference Center for Antimicrobial Resistance in July 2017. Results and discussion The rCIM correctly identified 84/85 carbapenemase producers and 28/28 non-carbapenemase producers, yielding a sensitivity of 99% and a specificity of 100%, slightly higher than the CIM and Carba NP test. In the prospective validation study, the rCIM showed a sensitivity and specificity of 97% and 95%, respectively. Two cephalosporinase-hyperproducing Enterobacter cloacae gave false-positive results, whereas an IMI-17-producing Enterobacter asburiae gave a false-negative result. The result was, however, positive when the isolate was grown on selective antibiotic-containing media. Conclusions The rCIM is a rapid (less than 3 h), cheap and accurate test for the detection of CPEs, which can be implemented in low-resource settings, making it a useful tool for microbiology laboratories. Introduction Infections caused by carbapenem-resistant Enterobacteriaceae (CRE) are increasingly reported worldwide,1–4 leading to significantly higher mortality rates and increased healthcare costs.5–7 Resistance to carbapenems can stem from production of carbapenemases or other mechanisms such as decreased permeability, overproduction of ESBLs or cephalosporinases, efflux pumps or combinations of these mechanisms.8 Carbapenemase-producing Enterobacteriaceae (CPE) are the most worrisome, as they leave clinicians with limited therapeutic options, owing to their hydrolytic spectrum as well as the associated resistance to other antibiotic classes and because of their propensity to spread rapidly in healthcare settings.9 In addition, as carbapenemase genes are frequently located on mobile genetic elements and plasmids, they can be transferred to other bacteria.10,11 CPEs were initially reported in hospitals but, in recent years, the problem has emerged also in the community.12 The existence of CPEs outside the hospital raises major concerns regarding the future of infection control, as it may speed up the spread to the general population. For antibiotic stewardship and infection control measures to be effective, rapid, reliable and cost-effective tests are required to detect CPE carriers or infections. Various phenotypic CPE detection tests have been developed, including inhibition tests of carbapenemase activity,13,14 detection of carbapenem hydrolysis using MALDI-TOF MS,15 biochemical tests (e.g. Carba NP test and derivatives)16,17 and the carbapenem inactivation method (CIM).18 These tests can detect the presence of a carbapenemase and sometimes discriminate between Ambler class A and Ambler class B carbapenemases (e.g inhibition tests and Carba NP test II and OXA-48 Disk Test).14,19 Immunochromatographic assays, aiming to detect OXA-48-like, IMP-like and OXA-48/KPC CPEs from solid cultures have shown their usefulness in rapid detection of these carbapenemases.20–22 Finally, molecular methods remain the gold standard for the detection of carbapenemase producers.13,23–25 Most commercially available molecular tools target only the five most prevalent carbapenemases and thus miss minor carbapenemases (GES, IMI, SME, etc.). Here, we describe the rapid Carbapenem Inactivation Method (rCIM), an improved variant of the CIM, which brings down the CPE detection time from more than 24 h, to less than 3 h. Materials and methods Bacterial isolates and antibiotic susceptibility testing A retrospective study of well-characterized enterobacterial strains from the collection of the French National Reference Center (NRC) for Antimicrobial Resistance was used to evaluate the rCIM and to determine the operating characteristics of the test. The strains were subcultured from the frozen stock (stored at –80 °C) on UriSelectTM 4 (BioRad, Marne-la-Coquette, France) for 24 h at 37 °C. The collection included 113 well-characterized carbapenem-resistant isolates, of which 85 were carbapenemase producers and 28 non-carbapenemase producers. The CPEs included 19 class A producers (10 KPCs, 3 IMIs, 2 GESs, 1 FRI-1, 2 SMEs and 1 NmcA) as well as 27 class B producers (12 NDMs, 8 VIMs, 6 IMPs and 1 GIM), and 39 class D producers (17 OXA-48s and 22 OXA-48 variants, including low-hydrolysing enzymes such as OXA-181 and OXA-244).26,27 MICs of carbapenems were determined using the Etest (bioMérieux, La Balmes les Grottes, France) and results were interpreted following EUCAST guidelines, as updated in 2016 (http://www.eucast.org).26 For the prospective study, all the isolates received by the NRC for CRE testing over a period of two weeks were included. Upon receipt by the NRC, enterobacterial isolates were cultured on UriSelectTM 4 (BioRad) for 24 h at 37 °C to assess their purity. Routine disc diffusion antibiograms were performed and interpreted according to EUCAST guidelines. Carbapenemase activity detection The CIM was performed as previously described.18,26 It consists of suspending a 10 μL loopful of bacteria in 400 μL of sterile water with one 10 μg meropenem disc and incubating for 2 h. The disc is then placed on a plate seeded with Escherichia coli ATCC 25922 and re-incubated for 18 h. If the strain is a carbapenemase producer, then the antibiotic in the disc is inactivated and leads to the appearance of a markedly diminished zone of inhibition (most frequently growth to the edge of the disc). The home-made Carba NP test was performed as previously described.19 The rCIM is derived from the CIM.18,26 Two 10 μL loopfuls of an overnight culture taken from solid media were homogenized into an Eppendorf tube containing 1 mL of sterile water. Two meropenem discs (Oxoid, UK) were added to the suspension and vortexed for 1 min. This allows for the diffusion of the meropenem from the discs into the suspension. The Eppendorf tube was incubated for 30 min at 37 °C, subsequently vortexed for an additional minute, and then centrifuged for 5 min at 10 000 rpm (for highly mucoid strains, a second centrifugation step may be needed). Five-hundred μL of the supernatant was added to 2500 μL of an E. coli ATCC 25922 resuspended in trypticase soy broth (bioMérieux, France) at a McFarland index of 1 (DEN-1B nephelometer, BioSan, Latvia). The tubes were incubated at 37 °C for 2 h with a nephelometer reading taken every 30 min to assess the growth of the indicator E. coli strain. Growth was expressed as baseline-subtracted growth in McFarland units. Positive and negative controls of Klebsiella pneumoniae ATCC BAA-1705 and BAA-1706, respectively, were used for each batch of tests. rCIM repeatability Repeatability of the rCIM was assessed through repeat testing of 10 carbapenemase-producing isolates from each Ambler class, as well as 10 isolates that were carbapenem resistant through other mechanisms. Technical replicates were done for each of the strains from overnight solid culture. Molecular identification of the carbapenemase genes An in-house PCR was performed as previously described.26,28 The targeted carbapenemase genes were blaOXA-48-like, blaKPC-like, blaNDM-like, blaIMP-like, blaIMI-like, blaVIM-like and blaGES-like. PCR products were sequenced using an automated Applied Biosystems sequencer (ABI PRISM 3100, Les Ullis, France). Statistical analysis The sensitivity, specificity and positive and negative predictive values were calculated, with their respective 95% CI, using the R v. 3.4.1 software suite and epiR v. 0.9–87. Test sensitivities and specificities were compared using McNemar’s χ2 test from the base R package. The gold standard was PCR followed by sequencing. Results and discussion Validation and definition of a positivity cut-off To establish the uninhibited growth of the E. coli indicator strain, mock challenge experiments were carried out with distilled water and growth was recorded every 30 min. To account for possible carry-over, we also performed experiments with centrifuged bacterial suspensions, without the addition of antibiotic discs. A similar strategy was undertaken for the negative controls, using incubated antibiotic discs and the E. coli indicator strain to account for carry-over. Carry-over was quantified by measuring the McFarland index before and after the supernatant challenge. This was found to be minimal, with a mean (±SD) value of –0.02 (±0.12) McFarland units. Furthermore, the use of chromogenic medium (colour of bacterial colonies) did not influence the reading of the results. In the presence of inactivated antibiotic, the indicator strain could grow unhindered, with median (min–max) baseline-subtracted growth at 1.5 and 2 h reaching 0.86 (0.84–0.89) and 1.34 (1.31–1.37), respectively. In the negative controls, growth at 1.5 and 2 h was 0.15 (0.12–0.17) and −0.02 (−0.05–0), respectively. Growth differences in positive and negative controls were not significantly different before 1.5 h, owing to residual indicator strain growth. We established a cut-off of 0.5 McFarland units as an a priori positive test and evaluated this hypothesis retrospectively on a collection of well-characterized enterobacterial isolates with reduced susceptibility to at least one carbapenem. Test repeatability To assess the reliability of the test we performed a repeatability analysis, including 10 strains from each of the carbapenemase Ambler classes as well as 10 non-carbapenemase-producing strains. The isolates comprised 1 Citrobacter koseri, 1 Enterobacter asburiae, 1 Morganella morganii, 4 Serratia marcescens, 7 Enterobacter cloacae, 8 E. coli and 18 K. pneumoniae, harbouring class A (FRI-1, GES-5, IMI-1, IMI-2, KPC-2, SME-1, SME-2), class B (GIM-1, IMP-1, IMP-4, IMP-11, NDM-1, NDM-4, VIM-1, VIM-2, VIM-19) or class D (OXA-48, OXA-162, OXA-181, OXA-244) and 10 non-carbapenemase producers (Table 1). Table 1. Results from the repeatability study, showing agreement between the rCIM, performed three times per isolate for a well-characterized set of 40 Enterobacteriaceae No.  Bacteria  Enzyme  ID  rCIM  Carbapenemase producers  1  E. cloacae  FRI-1  1I10  3  2  K. pneumoniae  GES-5  3B7  2+/1−  3  E. cloacae  IMI-1  1I3  2+/1−  4  E. asburiae  IMI-2  1I4  2+/1−  5  E. coli  KPC-2  1F3  3+  6  K. pneumoniae  1F5  3+  7  K. pneumoniae  1F9  3+  8  K. pneumoniae  1G1  3+  9  S. marcescens  SME-1  1I7  3+  10  S. marcescens  SME-2  1I8  3+  11  E. cloacae  GIM-1  1E10  3+  12  K. pneumoniae  IMP-1  1E2  3+  13  S. marcescens  IMP-11  1E9  3+  14  K. pneumoniae  IMP-4  3D4  3+  15  E. coli  NDM-1  1A1  3+  16  K. pneumoniae  1B1  3+  17  E. coli  NDM-4  1A6  3+  18  K. pneumoniae  VIM-1  1C7  3+  19  K. pneumoniae  VIM-19  1D4  3+  20  E. cloacae  VIM-2  3B8  3+  21  K. pneumoniae  OXA-162  2C2  3+  22  K. pneumoniae  OXA-181  2C5  3+  23  E. coli  OXA-244  2D10  3+  24  C. koseri  OXA-48  2B10  3+  25  E. coli  2A1  3+  26  E. coli  2A4  3+  27  E. coli  2A6  3+  28  K. pneumoniae  2A7  3+  29  K. pneumoniae  2A8  3+  30  K. pneumoniae  2A9  3+  Non-carbapenemase producers  31  E. cloacae  NA  2I10  3−  32  E. cloacae  2G3  3−  33  M. morganii  2H5  3−  34  E. cloacae  2F8  3−  35  E. coli  2F3  3−  36  K. pneumoniae  2F4  3−  37  K. pneumoniae  2I1  3−  38  K. pneumoniae  2I2  3−  39  K. pneumoniae  2I3  3−  40  S. marcescens  2J5  3−  No.  Bacteria  Enzyme  ID  rCIM  Carbapenemase producers  1  E. cloacae  FRI-1  1I10  3  2  K. pneumoniae  GES-5  3B7  2+/1−  3  E. cloacae  IMI-1  1I3  2+/1−  4  E. asburiae  IMI-2  1I4  2+/1−  5  E. coli  KPC-2  1F3  3+  6  K. pneumoniae  1F5  3+  7  K. pneumoniae  1F9  3+  8  K. pneumoniae  1G1  3+  9  S. marcescens  SME-1  1I7  3+  10  S. marcescens  SME-2  1I8  3+  11  E. cloacae  GIM-1  1E10  3+  12  K. pneumoniae  IMP-1  1E2  3+  13  S. marcescens  IMP-11  1E9  3+  14  K. pneumoniae  IMP-4  3D4  3+  15  E. coli  NDM-1  1A1  3+  16  K. pneumoniae  1B1  3+  17  E. coli  NDM-4  1A6  3+  18  K. pneumoniae  VIM-1  1C7  3+  19  K. pneumoniae  VIM-19  1D4  3+  20  E. cloacae  VIM-2  3B8  3+  21  K. pneumoniae  OXA-162  2C2  3+  22  K. pneumoniae  OXA-181  2C5  3+  23  E. coli  OXA-244  2D10  3+  24  C. koseri  OXA-48  2B10  3+  25  E. coli  2A1  3+  26  E. coli  2A4  3+  27  E. coli  2A6  3+  28  K. pneumoniae  2A7  3+  29  K. pneumoniae  2A8  3+  30  K. pneumoniae  2A9  3+  Non-carbapenemase producers  31  E. cloacae  NA  2I10  3−  32  E. cloacae  2G3  3−  33  M. morganii  2H5  3−  34  E. cloacae  2F8  3−  35  E. coli  2F3  3−  36  K. pneumoniae  2F4  3−  37  K. pneumoniae  2I1  3−  38  K. pneumoniae  2I2  3−  39  K. pneumoniae  2I3  3−  40  S. marcescens  2J5  3−  NA, not applicable, as these bacteria produce no carbapenem-hydrolysing enzymes. All of these strains had decreased permeability and either an ESBL or an overexpressed cephalosporinase. Overall, the repeatability was excellent, with the exception of low-hydrolysing strains and strains requiring induction of their β-lactamase. Two IMI-like-expressing strains, an E. asburiae and an E. cloacae, gave 2 out of 3 positive results with rCIM. A GES-5 K. pneumoniae strain tested positive 2 out of 3 times. Retrospective analysis At 1.5 h, the majority of growth indicators challenged with supernatant from carbapenemase-positive strains (82/85) had reached at least 0.5 McFarland units of growth. The strains which had a low growth index (around the diagnostic breakpoint of 0.5) at 1.5 h were further incubated for 30 mins. At the same time, one carbapenemase-negative isolate grew above the 0.5 threshold, but subsequently declined. After 2 h of incubation at 37 °C, the indicator strain, when challenged with inactivated antibiotic, grew to a median of about 2.5 McFarland units (range 0.38–3.13, IQR 1.85–2.69). The only strains that produced growth less than 1 McFarland unit were 3 OXA-244 E. coli isolates (0.38, 0.57 and 0.74, respectively) and one IMI-2 strain (0.86) (Figure 1, Table 2). In contrast, when challenged with active antibiotic, the median growth was of 0.13 McFarland units (range −0.13 to 0.47, IQR 0.05–0.22). One E. cloacae strain with cephalosporinase hyperproduction initially tested positive, and then tested negative twice. Table 2. Results from the retrospective study, showing agreement and differences between the rCIM, Carba NP and CIM for a well-characterized set of Enterobacteriaceae Enzyme  Species  No.  rCIM  CIM  Carba NP  Class A             KPC-2  E. cloacae  1  +  +  +  E. coli  3  +  +  +  K. oxytoca  1  +  +  +  K. pneumoniae  4  +  +  +   KPC-3  K. pneumoniae  1  +  +  +   IMI-2  E. aerogenes  1  +  +  −  E. asburiae  1  +  +  +   IMI-3  E. cloacae  1  +  +  +   GES-5  E. cloacae  1  +  +  +  K. pneumoniae  1  +  +  +   FRI-1  E. cloacae  1  +  +  +   NMCA  E. cloacae  1  +  +  +   SME-1  S. marcescens  1  +  +  +   SME-2  S. marcescens  1  +  +  +  Class B             NDM-1  K. pneumoniae  5  +  +  +  E. coli  2  +  +  +  P. rettgeri  1  +  −  +   NDM-4  E. coli  2  +  +  +   NDM-5  E. coli  1  +  +  +   NDM-6  E. coli  1  +  +  +   VIM-1  K. pneumoniae  3  +  +  +  E. coli  1  +  +  +   VIM-2  E. cloacae  1  +  −  −   VIM-4  E. cloacae  1  +  −  −  E. coli  1  +  +  +   VIM-19  K. pneumoniae  1  +  +  +   GIM-1  E. cloacae  1  +  +  +   IMP-1  E. coli  1  +  +  +  K. pneumoniae  1  +  +  +   IMP-4  K. pneumoniae  1  +  +  +   IMP-8  K. pneumoniae  1  +  +  +   IMP-10  S. marcescens  1  +  +  +   IMP-11  S. marcescens  1  +  +  +  Class D             OXA-48  C. koseri  2  +  +  +  E. cloacae  2  +  +  +  E. coli  4  +  +  +  K. oxytoca  4  +  +  +  K. pneumoniae  4  +  +  +  P. mirabilis  1  +  +  +   OXA-162  K. pneumoniae  1  +  +  +   OXA-181  E. coli  7  +  +  +  K. pneumoniae  2  +  +  +   OXA-204  E. coli  1  +  +  +  K. pneumoniae  1  +  +  +   OXA-232  E. coli  1  +  +  +   OXA-244  E. coli  8  7/8+  6/8+  5/8+   OXA-372  C. freundii  1  +  +  +  ESBL + decreased membrane permeability    K. pneumoniae  4  −  −  −  E. coli  2  −  −  −  Plasmid-mediated or chromosomal AmpC + decreased membrane permeability    E. aerogenes  1  −  −  −  E. cloacae  3  −  −  −  E. coli  1  −  −  −    K. pneumoniae  2  −  −  −    M. morganii  1  −  −  −  ESBL + plasmid-mediated or chromosomal AmpC + decreased membrane permeability    C. freundii  1  −  −  −  E. cloacae  3  −  −  −  Extended-spectrum oxacillinases    S. marcescens  1  −  −  −  Other mechanisms + decreased membrane impermeability    E. cloacae  1  −  −  −  E. coli  1  −  −  −  K. oxytoca  6  −  −  −  K. pneumoniae  1  −  −  −  Enzyme  Species  No.  rCIM  CIM  Carba NP  Class A             KPC-2  E. cloacae  1  +  +  +  E. coli  3  +  +  +  K. oxytoca  1  +  +  +  K. pneumoniae  4  +  +  +   KPC-3  K. pneumoniae  1  +  +  +   IMI-2  E. aerogenes  1  +  +  −  E. asburiae  1  +  +  +   IMI-3  E. cloacae  1  +  +  +   GES-5  E. cloacae  1  +  +  +  K. pneumoniae  1  +  +  +   FRI-1  E. cloacae  1  +  +  +   NMCA  E. cloacae  1  +  +  +   SME-1  S. marcescens  1  +  +  +   SME-2  S. marcescens  1  +  +  +  Class B             NDM-1  K. pneumoniae  5  +  +  +  E. coli  2  +  +  +  P. rettgeri  1  +  −  +   NDM-4  E. coli  2  +  +  +   NDM-5  E. coli  1  +  +  +   NDM-6  E. coli  1  +  +  +   VIM-1  K. pneumoniae  3  +  +  +  E. coli  1  +  +  +   VIM-2  E. cloacae  1  +  −  −   VIM-4  E. cloacae  1  +  −  −  E. coli  1  +  +  +   VIM-19  K. pneumoniae  1  +  +  +   GIM-1  E. cloacae  1  +  +  +   IMP-1  E. coli  1  +  +  +  K. pneumoniae  1  +  +  +   IMP-4  K. pneumoniae  1  +  +  +   IMP-8  K. pneumoniae  1  +  +  +   IMP-10  S. marcescens  1  +  +  +   IMP-11  S. marcescens  1  +  +  +  Class D             OXA-48  C. koseri  2  +  +  +  E. cloacae  2  +  +  +  E. coli  4  +  +  +  K. oxytoca  4  +  +  +  K. pneumoniae  4  +  +  +  P. mirabilis  1  +  +  +   OXA-162  K. pneumoniae  1  +  +  +   OXA-181  E. coli  7  +  +  +  K. pneumoniae  2  +  +  +   OXA-204  E. coli  1  +  +  +  K. pneumoniae  1  +  +  +   OXA-232  E. coli  1  +  +  +   OXA-244  E. coli  8  7/8+  6/8+  5/8+   OXA-372  C. freundii  1  +  +  +  ESBL + decreased membrane permeability    K. pneumoniae  4  −  −  −  E. coli  2  −  −  −  Plasmid-mediated or chromosomal AmpC + decreased membrane permeability    E. aerogenes  1  −  −  −  E. cloacae  3  −  −  −  E. coli  1  −  −  −    K. pneumoniae  2  −  −  −    M. morganii  1  −  −  −  ESBL + plasmid-mediated or chromosomal AmpC + decreased membrane permeability    C. freundii  1  −  −  −  E. cloacae  3  −  −  −  Extended-spectrum oxacillinases    S. marcescens  1  −  −  −  Other mechanisms + decreased membrane impermeability    E. cloacae  1  −  −  −  E. coli  1  −  −  −  K. oxytoca  6  −  −  −  K. pneumoniae  1  −  −  −  Grey shading indicates unexpected results (either false-positive or false-negative testing results). Figure 1. View largeDownload slide Dynamics of E. coli indicator strain growth in the retrospective study. Box and whisker plot showing the dynamics of growth of the E. coli ATCC 25922 indicator strain from the retrospective study (n = 113). Measurements were made every 30 min after supernatant challenge of the indicator strain. Carbapenemase producers and non-producers are presented side by side for each measurement time point. Results represent baseline-subtracted growth, and are expressed as McFarland units. Filled circles represent outliers. A horizontal line marks the 0.5 McFarland index cut-off chosen. Figure 1. View largeDownload slide Dynamics of E. coli indicator strain growth in the retrospective study. Box and whisker plot showing the dynamics of growth of the E. coli ATCC 25922 indicator strain from the retrospective study (n = 113). Measurements were made every 30 min after supernatant challenge of the indicator strain. Carbapenemase producers and non-producers are presented side by side for each measurement time point. Results represent baseline-subtracted growth, and are expressed as McFarland units. Filled circles represent outliers. A horizontal line marks the 0.5 McFarland index cut-off chosen. Thus, the sensitivity of the rCIM was 99% (95% CI 94%–100%) and the specificity was 100% (95% CI 88%–100%), using a simple indicator of baseline-subtracted growth of 0.5 McFarland units at 2 h. Using the same panel of isolates, the sensitivity and the specificity of the CIM test were 94% (95% CI 87%–98%) and 100% (95% CI 88%–100%), respectively. Among the undetected strains, there was one NDM-1-producing Providencia rettgeri, two OXA-244-producing E. coli, one VIM-4- and one VIM-2-producing E. cloacae. The Carba NP test had a sensitivity of 93% (95% CI 85%–97%) and a specificity of 100% (95% CI 88%–100%). The false-negative isolates were the three OXA-244-producing E. coli, one VIM-4- and one VIM-2-producing E. cloacae, and an IMI-2-producing E. asburiae (Table 3). Table 3. Performance characteristics for the rCIM in the retrospective study. Results for 2 h growth time of the indicator strain   Carbapenemase     Characteristic  positive  negative  Total  rCIM positive  84  0  84  rCIM negative  1 (OXA-244a)  28  29  Total  85  28  113    Carbapenemase     Characteristic  positive  negative  Total  rCIM positive  84  0  84  rCIM negative  1 (OXA-244a)  28  29  Total  85  28  113  a Also negative with CIM and Carba NP test. Prospective rCIM evaluation The 101 isolates of the prospective study included 34 K. pneumoniae, 23 E. coli, 21 E. cloacae, 12 Citrobacter freundii, 2 E. asburiae, 2 Proteus mirabilis, 2 P. rettgeri, 1 Providencia stuartii, 1 Citrobacter youngae, 1 Enterobacter hormaechei, 1 Hafnia alvei and 1 M. morganii. They expressed 45 OXA-48-like, 10 NDM-like, 5 VIM-like, 2 KPC-like and 2 IMI-like enzymes, 15 hyperproduced cephalosporinases, 12 ESBLs, 4 hyperproduced cephalosporinase and an ESBL and 6 others had non-enzymatic carbapenem resistance mechanisms. After WGS, the two IMI-like-producing E. asburiae were identified as being the same strain, producing a novel IMI variant (submitted to GenBank under the name IMI-17), and was counted only once. Thus, there were 100 isolates tested in total. PCR sequencing results are displayed in Table 4. Table 4. Results from the prospective study, showing agreement and differences between the rCIM and Carba NP, relative to molecular methods for 100 Enterobacteriaceae evaluated by the French NRC   Species  No.  rCIM  Carba NP  Class A           KPC-2  K. pneumoniae  1  +  +   KPC-3  K. pneumoniae  1  +  +   IMI-17  E. asburiae  1  −  −  Class B           NDM-1  P. rettgeri  1  +  +  P. rettgeri  1  +  −  P. stuartii  1  +  −  K. pneumoniae  1  +  +  E. cloacae  1  +  +   NDM-5  E. coli  4  +  +   NDM-6  K. pneumoniae  1  +  +   VIM-1  E. cloacae  2  +  +  K. pneumoniae  2  +  +   VIM-4  E. cloacae  1  +  +  Class D           OXA-23  P. mirabilis  1  +  +   OXA-48  C. youngae  1  +  +  C. freundii  6  +  +  E. coli  10  +  +  E. cloacae  4  +  +  K. pneumoniae  21  +  +   OXA-181  E. coli  1  +  +  E. coli  1  +  −  Non-carbapenemase producers         Case  C. freundii  4  −  −  E. cloacae  4  −  −  E. cloacae  2  +  −  E. hormaechei  1  −  −  H. alvei  1  −  −  K. pneumoniae  3  −  −   ESBL  C. freundii  2  −  −  E. coli  5  −  −  E. cloacae  1  −  −  K. pneumoniae  4  −  −   Case + ESBL  E. cloacae  4  −  −   Other  M. morganii  1  −  −  E. coli  2  −  −  E. cloacae  2  −  −  P. mirabilis  1  −  −    Species  No.  rCIM  Carba NP  Class A           KPC-2  K. pneumoniae  1  +  +   KPC-3  K. pneumoniae  1  +  +   IMI-17  E. asburiae  1  −  −  Class B           NDM-1  P. rettgeri  1  +  +  P. rettgeri  1  +  −  P. stuartii  1  +  −  K. pneumoniae  1  +  +  E. cloacae  1  +  +   NDM-5  E. coli  4  +  +   NDM-6  K. pneumoniae  1  +  +   VIM-1  E. cloacae  2  +  +  K. pneumoniae  2  +  +   VIM-4  E. cloacae  1  +  +  Class D           OXA-23  P. mirabilis  1  +  +   OXA-48  C. youngae  1  +  +  C. freundii  6  +  +  E. coli  10  +  +  E. cloacae  4  +  +  K. pneumoniae  21  +  +   OXA-181  E. coli  1  +  +  E. coli  1  +  −  Non-carbapenemase producers         Case  C. freundii  4  −  −  E. cloacae  4  −  −  E. cloacae  2  +  −  E. hormaechei  1  −  −  H. alvei  1  −  −  K. pneumoniae  3  −  −   ESBL  C. freundii  2  −  −  E. coli  5  −  −  E. cloacae  1  −  −  K. pneumoniae  4  −  −   Case + ESBL  E. cloacae  4  −  −   Other  M. morganii  1  −  −  E. coli  2  −  −  E. cloacae  2  −  −  P. mirabilis  1  −  −  Case, overexpressed cephalosporinase. Grey shading indicates unexpected results (either false-positive or false-negative testing results). Using the cut-off defined in the retrospective study, the sensitivity of the rCIM was 98% (95% CI 89%–99%) and the specificity was 95% (95% CI 82%–99%), with a positive predictive value of 97% (95% CI 89%–100%) and a negative predictive value of 97% (95% CI 85%–100%). Two cephalosporinase-hyperproducing E. cloacae isolates gave false-positive results, while a novel IMI variant-producing E. asburiae gave a false-negative result (Figure 2). Figure 2. View largeDownload slide Results of E. coli indicator strain growth in the prospective study. Box and whisker plot showing results of indicator growth at 1.5 and 2 h for the prospectively evaluated strains (n = 100). Carbapenemase producers and non-producers are presented side by side for each measurement time point. Results represent baseline-subtracted growth, and are expressed as McFarland units. Filled circles represent outliers. A horizontal line marks the 0.5 McFarland index cut-off chosen. Figure 2. View largeDownload slide Results of E. coli indicator strain growth in the prospective study. Box and whisker plot showing results of indicator growth at 1.5 and 2 h for the prospectively evaluated strains (n = 100). Carbapenemase producers and non-producers are presented side by side for each measurement time point. Results represent baseline-subtracted growth, and are expressed as McFarland units. Filled circles represent outliers. A horizontal line marks the 0.5 McFarland index cut-off chosen. In combination with the interpretative reading of the antibiogram, the Carba NP test is used as the reference method for the screening of carbapenemase activity at the French NRC. It had a sensitivity of 92% (95% CI 82%–97%), a specificity of 100% (95% CI 91%–100%), a positive predictive value of 100% (95% CI 89%–100%) and a negative predictive value of 88% (95% CI 74%–96%). A total of four strains tested Carba NP negative. These were an OXA-181-producing E. coli, the novel IMI variant-expressing E. asburiae, one NDM-1-producing P. rettgeri and one NDM-1-producing P. stuartii. The rCIM showed better sensitivity than the Carba NP test (98% versus 92%, P = 0.01), with the trade-off of a lower specificity (95% versus 100%, P < 0.001). rCIM false-negative results Even though a low positivity cut-off was chosen in order to ease the detection of low carbapenem-hydrolysing enzymes, such as OXA-244,27 three isolates gave false-negative rCIM results (1.6%): two OXA-244-producing E. coli strains and an E. asburiae producing a novel IMI variant (IMI-17). One of the OXA-244 isolates was positive on repeated testing (2+/1−) while the two others tested negative when taken directly off the UriSelectTM 4 medium. OXA-244 enzymes are difficult to detect, as recently shown by Hoyos-Mallecot et al.27 These enzymes are characterized by weak carbapenem hydrolysis that results in reduced susceptibility to carbapenems only. However, as OXA-244 may be plasmid encoded, horizontal gene transfer may occur into bacteria with impaired outer membrane permeability, and lead to high-level carbapenem resistance.29,30 Thus, it is of outmost importance to identify these enzymes using proper diagnostic algorithms (i.e. the use of temocillin resistance as a marker for OXA-48-like carbapenemases) and rapid diagnostic tests.31,32 The rCIM seems to be an excellent test in this respect, as six out of the seven OXA-244-producing E. coli isolates were correctly identified as carbapenemase producers. In the case of the E. asburiae isolate, the false-negative rCIM results could be explained by the low level and inducible nature of IMI production. These enzymes can be induced with imipenem or cefoxitin through the presence of the LysR-type regulator IMI-R.33 The IMI-producing E. asburiae isolate was negative with rCIM, Carba NP and CIM tests when grown on UriSelect™ 4 media. When the isolate was grown on Mueller–Hinton or on selective chromogenic media, such as CarbaSmart (bioMérieux), it gave a positive rCIM result. rCIM false-positive results Overall, the rCIM gave three false-positive isolates (1.6%); all corresponded to cephalosporinase-hyperproducing E. cloacae. One of them, in the retrospective study, proved negative on repeated testing (2−/1+). The two other isolates were from the prospective study and grew slightly above the baseline-subtracted McFarland threshold of 0.5 (0.89 and 1.29, respectively); on repeat testing, one gave consistent results over the 0.5 McFarland threshold (3+), while one gave mostly negative results (2−/1+). Laboratories have to keep in mind that cephalosporinase-hyperproducing E. cloacae may provide weak positive results and thus would require additional testing. Practical guidance In high incidence settings, the rCIM might be used to rapidly confirm the presence of a carbapenem-hydrolysing enzyme in various enterobacterial isolates. The rCIM can be interpreted after 1.5 h of indicator strain growth (roughly 2 h of total preparation). In our total experience of testing 213 isolates, a baseline-subtracted growth of more than 1 is highly indicative of carbapenemase production (131/148 CPEs and 1 false-positive isolate). For baseline-subtracted growth between 0.5 and 1, 30 min of additional incubation was beneficial, as nine other CPE isolates (140/148) were detected (Figure 3). Figure 3. View largeDownload slide Overall experience with the rCIM. Box and whisker plot showing overall experience with rCIM (n = 213). Results presented for 1.5 and 2 h of indicator strain growth. Carbapenemase producers and non-producers are presented side by side for each measurement time point. Grey zone marked between 0.5 and 1 McFarland represents a zone of uncertainty requiring further testing. Filled circles represent outliers. Rectangles highlight the results of the novel IMI variant-producing strain when tested directly from a chromogenic medium. Filled triangles represent the results obtained with the novel IMI variant-expressing strain giving a positive rCIM result when tested from the CarbaSmart medium. Figure 3. View largeDownload slide Overall experience with the rCIM. Box and whisker plot showing overall experience with rCIM (n = 213). Results presented for 1.5 and 2 h of indicator strain growth. Carbapenemase producers and non-producers are presented side by side for each measurement time point. Grey zone marked between 0.5 and 1 McFarland represents a zone of uncertainty requiring further testing. Filled circles represent outliers. Rectangles highlight the results of the novel IMI variant-producing strain when tested directly from a chromogenic medium. Filled triangles represent the results obtained with the novel IMI variant-expressing strain giving a positive rCIM result when tested from the CarbaSmart medium. At 2 h, isolates with baseline-subtracted growth between 0.5 and 1 should undergo further testing (using molecular or immune-enzymatic assays, such as the OKN K-SeT assay),34 or even retesting. Of all the carbapenemase producers tested, only OXA-244, KPC-2 and IMI-2 producers (n = 7) gave such results. Another isolate, an E. cloacae hyperproducing a cephalosporinase, also gave an indeterminate result. Once the antibiograms are obtained, phenotypic interpretation of the antibiogram and retesting from the Mueller–Hinton plate may clarify certain borderline cases, especially with IMI producers. In the absence of carbapenemase production, 63 out of 65 isolates were identified at 2 h. In 2016, the French NRC received 2839 carbapenem-resistant strains, of which 1551 harboured a carbapenemase. Class D carbapenemases were by far the most frequent, forming about three-quarters of the total number of isolates sent in for expert assessment (1187/1551; 76%). Of these, the majority were OXA-48 (n = 1078) and OXA-181 producers (n = 73). OXA-244 producers (n = 8) represented only 0.5% of the total carbapenemase producers. The epidemiology of strains tested in our prospective study mirror the national epidemiology. Of interest, one OXA-181 isolate tested in the prospective study was positive with rCIM but repeatedly negative with the Carba NP test. Class B carbapenemases follow at a distance (282/1551; 18%), with NDM-type enzymes predominating (n = 217), followed by VIM-type enzymes (n = 62), and very rarely IMP enzymes (n = 3). All these enzymes were accurately identified with all three phenotypic tests. Class A carbapenemases are relatively rare and account for less than 5% (53/1551). Of note, the IMI-type enzymes are very rarely isolated and represented less than 1% (14/1551) of the cases identified by the NRC. Lastly, some strains sent to the NRC expressed multiple carbapenemases. Thus, in daily clinical practice, the sensitivity of the rCIM would probably be even higher. One limit of the current study is the use of a set of conditions including incubation in sterile water and the use of trypticase soy broth media for the growth of the indicator. As a result, outcomes might differ when other buffers or broths might be used (e.g. Tris-buffered saline or Mueller–Hinton). Trypticase soy broth was chosen because it is a general-purpose nutritive medium, cheaper than Mueller–Hinton and should be more readily available to laboratories. Furthermore, the rCIM requires multiple hands-on steps (including multiple incubation steps and centrifugation), which should be taken into account when comparing with other commercial methods (Carba NP and β-Carba Test). We acknowledge that the extra hands-on time might increase the time-to-result, though we feel that this remains similar as compared with other methods. Lastly, the need for a big inoculum (two 10 μL loops) needs to be addressed as a limiting factor as large quantities of bacteria are not always recovered from screening media. Recently, another variation of the CIM, the mCIM, was published, showing good inter-laboratory agreement,35,36 but with the same drawback of the 24 h time frame necessary for detection. The rCIM resolves this major problem, requiring less than 3 h of total work time, which is compatible with the daily practice of a clinical microbiology laboratory. Compared with the Carba NP, the rCIM offers a more objective perspective of interpretation as it is a quantitative test. This may make it easier for laboratory personnel to quantify uncertainty and use additional tests, as appropriate. The cost of the rCIM is very low, comparable to the CIM and mCIM.18,26,35 Using the online prices from a major French distributor, we estimated the costs for the rCIM to be $0.13 for 3 mL of trypticase soy broth + $0.096 for two antibiotic discs (Table 5). Table 5. Comparative characteristics of phenotypic methods with price estimation per test and total work time Test  rCIM  CIM  mCIM  Carba NP  Initial incubation time (h)  0.5  2  4  —  Time-to-results (h)  2–2.5  8–24  24  2  Antibiotic used  meropenem  meropenem  meropenem  imipenem  Antibiotic amount/ concentration used  20 μg  10 μg  10 μg  3 mg/mL  Cost per test (US$)  0.25  0.45a  <1b  2–10a  Test  rCIM  CIM  mCIM  Carba NP  Initial incubation time (h)  0.5  2  4  —  Time-to-results (h)  2–2.5  8–24  24  2  Antibiotic used  meropenem  meropenem  meropenem  imipenem  Antibiotic amount/ concentration used  20 μg  10 μg  10 μg  3 mg/mL  Cost per test (US$)  0.25  0.45a  <1b  2–10a  a Gauthier et al., 2017.26 b Pierce et al., 2017.35 Unlike the CIM and mCIM, the rCIM does not require the manipulation of contaminated antibiotic discs with highly drug-resistant bacteria, eliminating the potential of cross-contamination. On the other hand, the rCIM requires the use of a nephelometer and a centrifuge, but we expect that these would be present in the vast majority of laboratories. Conclusions Overall, the high sensitivity and specificity of rCIM, as well as the fact that is a rapid test, make it a useful tool in clinical microbiology laboratories. The rCIM does not need trained personnel and the equipment needed is largely present in most microbiology laboratories worldwide. The rCIM resolves the main limitation of the CIM as it can provide same-day results, thus being able to guide the clinician regarding the treatment and making it compatible with efficient implementation of infection control measures. In conclusion, the rCIM is a quantitative test that has a price comparable with that of CIM and mCIM (<$1 per test) and a detection time comparable with that of Carba NP, making it accessible even in small laboratories. Funding This work was supported by the Assistance Publique – Hôpitaux de Paris, by a grant from the Université Paris-Sud (EA7361), and by the LabEx LERMIT supported by a grant from the French National Research Agency (ANR-10-LABX-33). This work was also funded in part by a grant from the Joint Programme Initiative on Antimicrobial Resistance (ANR-14-JAMR-0002). Transparency declarations None to declare. References 1 Nordmann P, Cuzon G, Naas T. The real threat of Klebsiella pneumoniae carbapenemase-producing bacteria. Lancet Infect Dis  2009; 9: 228– 36. Google Scholar CrossRef Search ADS PubMed  2 Munoz-Price LS, Poirel L, Bonomo RA et al.   Clinical epidemiology of the global expansion of Klebsiella pneumoniae carbapenemases. Lancet Infect Dis  2013; 13: 785– 96. Google Scholar CrossRef Search ADS PubMed  3 Logan LK, Weinstein RA. The epidemiology of carbapenem-resistant Enterobacteriaceae: the impact and evolution of a global menace. J Infect Dis  2017; 215: S28– 36. 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Antimicrob Agents Chemother  2014; 58: 2119– 25. Google Scholar CrossRef Search ADS PubMed  31 Robert J, Pantel A, Merens A et al.   Development of an algorithm for phenotypic screening of carbapenemase-producing Enterobacteriaceae in the routine laboratory. BMC Infect Dis  2017; 17: 78. Google Scholar CrossRef Search ADS PubMed  32 Bakthavatchalam YD, Anandan S, Veeraraghavan B. Laboratory detection and clinical implication of oxacillinase-48 like carbapenemase: the hidden threat. J Global Infect Dis  2016; 8: 41– 50. Google Scholar CrossRef Search ADS   33 Naas T, Dortet L, Iorga BI. Structural and functional aspects of class A carbapenemases. Curr Drug Targets  2016; 17: 1006– 28. Google Scholar CrossRef Search ADS PubMed  34 Glupczynski Y, Jousset A, Evrard S et al.   Prospective evaluation of the OKN K-SeT assay, a new multiplex immunochromatographic test for the rapid detection of OXA-48-like, KPC and NDM carbapenemases. J Antimicrob Chemother  2017; 72: 1955– 60. 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Evaluation of the rapid carbapenem inactivation method (rCIM): a phenotypic screening test for carbapenemase-producing Enterobacteriaceae

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Oxford University Press
ISSN
0305-7453
eISSN
1460-2091
D.O.I.
10.1093/jac/dkx519
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Abstract

Abstract Objectives Fast and accurate diagnostic tests to identify carbapenemase-producing Enterobacteriaceae (CPE) are mandatory for proper antimicrobial therapy and implementing infection control measures. Here, we have developed a rapid Carbapenem Inactivation Method (rCIM) for CPE detection. Methods The rCIM consists of the incubation of a potential carbapenemase producer with meropenem discs and use of the resulting supernatant to challenge a susceptible indicator strain. Growth of the indicator strain is monitored using a nephelometer. The performances of the rCIM were compared with the CIM and Carba NP tests using a collection of 113 well-characterized carbapenem-resistant enterobacterial isolates, including 85 carbapenemase producers and 28 non-carbapenemase producers. In addition, rCIM was compared with the Carba NP test and PCR sequencing in a prospective analysis of 101 carbapenem-resistant enterobacterial isolates addressed to the French National Reference Center for Antimicrobial Resistance in July 2017. Results and discussion The rCIM correctly identified 84/85 carbapenemase producers and 28/28 non-carbapenemase producers, yielding a sensitivity of 99% and a specificity of 100%, slightly higher than the CIM and Carba NP test. In the prospective validation study, the rCIM showed a sensitivity and specificity of 97% and 95%, respectively. Two cephalosporinase-hyperproducing Enterobacter cloacae gave false-positive results, whereas an IMI-17-producing Enterobacter asburiae gave a false-negative result. The result was, however, positive when the isolate was grown on selective antibiotic-containing media. Conclusions The rCIM is a rapid (less than 3 h), cheap and accurate test for the detection of CPEs, which can be implemented in low-resource settings, making it a useful tool for microbiology laboratories. Introduction Infections caused by carbapenem-resistant Enterobacteriaceae (CRE) are increasingly reported worldwide,1–4 leading to significantly higher mortality rates and increased healthcare costs.5–7 Resistance to carbapenems can stem from production of carbapenemases or other mechanisms such as decreased permeability, overproduction of ESBLs or cephalosporinases, efflux pumps or combinations of these mechanisms.8 Carbapenemase-producing Enterobacteriaceae (CPE) are the most worrisome, as they leave clinicians with limited therapeutic options, owing to their hydrolytic spectrum as well as the associated resistance to other antibiotic classes and because of their propensity to spread rapidly in healthcare settings.9 In addition, as carbapenemase genes are frequently located on mobile genetic elements and plasmids, they can be transferred to other bacteria.10,11 CPEs were initially reported in hospitals but, in recent years, the problem has emerged also in the community.12 The existence of CPEs outside the hospital raises major concerns regarding the future of infection control, as it may speed up the spread to the general population. For antibiotic stewardship and infection control measures to be effective, rapid, reliable and cost-effective tests are required to detect CPE carriers or infections. Various phenotypic CPE detection tests have been developed, including inhibition tests of carbapenemase activity,13,14 detection of carbapenem hydrolysis using MALDI-TOF MS,15 biochemical tests (e.g. Carba NP test and derivatives)16,17 and the carbapenem inactivation method (CIM).18 These tests can detect the presence of a carbapenemase and sometimes discriminate between Ambler class A and Ambler class B carbapenemases (e.g inhibition tests and Carba NP test II and OXA-48 Disk Test).14,19 Immunochromatographic assays, aiming to detect OXA-48-like, IMP-like and OXA-48/KPC CPEs from solid cultures have shown their usefulness in rapid detection of these carbapenemases.20–22 Finally, molecular methods remain the gold standard for the detection of carbapenemase producers.13,23–25 Most commercially available molecular tools target only the five most prevalent carbapenemases and thus miss minor carbapenemases (GES, IMI, SME, etc.). Here, we describe the rapid Carbapenem Inactivation Method (rCIM), an improved variant of the CIM, which brings down the CPE detection time from more than 24 h, to less than 3 h. Materials and methods Bacterial isolates and antibiotic susceptibility testing A retrospective study of well-characterized enterobacterial strains from the collection of the French National Reference Center (NRC) for Antimicrobial Resistance was used to evaluate the rCIM and to determine the operating characteristics of the test. The strains were subcultured from the frozen stock (stored at –80 °C) on UriSelectTM 4 (BioRad, Marne-la-Coquette, France) for 24 h at 37 °C. The collection included 113 well-characterized carbapenem-resistant isolates, of which 85 were carbapenemase producers and 28 non-carbapenemase producers. The CPEs included 19 class A producers (10 KPCs, 3 IMIs, 2 GESs, 1 FRI-1, 2 SMEs and 1 NmcA) as well as 27 class B producers (12 NDMs, 8 VIMs, 6 IMPs and 1 GIM), and 39 class D producers (17 OXA-48s and 22 OXA-48 variants, including low-hydrolysing enzymes such as OXA-181 and OXA-244).26,27 MICs of carbapenems were determined using the Etest (bioMérieux, La Balmes les Grottes, France) and results were interpreted following EUCAST guidelines, as updated in 2016 (http://www.eucast.org).26 For the prospective study, all the isolates received by the NRC for CRE testing over a period of two weeks were included. Upon receipt by the NRC, enterobacterial isolates were cultured on UriSelectTM 4 (BioRad) for 24 h at 37 °C to assess their purity. Routine disc diffusion antibiograms were performed and interpreted according to EUCAST guidelines. Carbapenemase activity detection The CIM was performed as previously described.18,26 It consists of suspending a 10 μL loopful of bacteria in 400 μL of sterile water with one 10 μg meropenem disc and incubating for 2 h. The disc is then placed on a plate seeded with Escherichia coli ATCC 25922 and re-incubated for 18 h. If the strain is a carbapenemase producer, then the antibiotic in the disc is inactivated and leads to the appearance of a markedly diminished zone of inhibition (most frequently growth to the edge of the disc). The home-made Carba NP test was performed as previously described.19 The rCIM is derived from the CIM.18,26 Two 10 μL loopfuls of an overnight culture taken from solid media were homogenized into an Eppendorf tube containing 1 mL of sterile water. Two meropenem discs (Oxoid, UK) were added to the suspension and vortexed for 1 min. This allows for the diffusion of the meropenem from the discs into the suspension. The Eppendorf tube was incubated for 30 min at 37 °C, subsequently vortexed for an additional minute, and then centrifuged for 5 min at 10 000 rpm (for highly mucoid strains, a second centrifugation step may be needed). Five-hundred μL of the supernatant was added to 2500 μL of an E. coli ATCC 25922 resuspended in trypticase soy broth (bioMérieux, France) at a McFarland index of 1 (DEN-1B nephelometer, BioSan, Latvia). The tubes were incubated at 37 °C for 2 h with a nephelometer reading taken every 30 min to assess the growth of the indicator E. coli strain. Growth was expressed as baseline-subtracted growth in McFarland units. Positive and negative controls of Klebsiella pneumoniae ATCC BAA-1705 and BAA-1706, respectively, were used for each batch of tests. rCIM repeatability Repeatability of the rCIM was assessed through repeat testing of 10 carbapenemase-producing isolates from each Ambler class, as well as 10 isolates that were carbapenem resistant through other mechanisms. Technical replicates were done for each of the strains from overnight solid culture. Molecular identification of the carbapenemase genes An in-house PCR was performed as previously described.26,28 The targeted carbapenemase genes were blaOXA-48-like, blaKPC-like, blaNDM-like, blaIMP-like, blaIMI-like, blaVIM-like and blaGES-like. PCR products were sequenced using an automated Applied Biosystems sequencer (ABI PRISM 3100, Les Ullis, France). Statistical analysis The sensitivity, specificity and positive and negative predictive values were calculated, with their respective 95% CI, using the R v. 3.4.1 software suite and epiR v. 0.9–87. Test sensitivities and specificities were compared using McNemar’s χ2 test from the base R package. The gold standard was PCR followed by sequencing. Results and discussion Validation and definition of a positivity cut-off To establish the uninhibited growth of the E. coli indicator strain, mock challenge experiments were carried out with distilled water and growth was recorded every 30 min. To account for possible carry-over, we also performed experiments with centrifuged bacterial suspensions, without the addition of antibiotic discs. A similar strategy was undertaken for the negative controls, using incubated antibiotic discs and the E. coli indicator strain to account for carry-over. Carry-over was quantified by measuring the McFarland index before and after the supernatant challenge. This was found to be minimal, with a mean (±SD) value of –0.02 (±0.12) McFarland units. Furthermore, the use of chromogenic medium (colour of bacterial colonies) did not influence the reading of the results. In the presence of inactivated antibiotic, the indicator strain could grow unhindered, with median (min–max) baseline-subtracted growth at 1.5 and 2 h reaching 0.86 (0.84–0.89) and 1.34 (1.31–1.37), respectively. In the negative controls, growth at 1.5 and 2 h was 0.15 (0.12–0.17) and −0.02 (−0.05–0), respectively. Growth differences in positive and negative controls were not significantly different before 1.5 h, owing to residual indicator strain growth. We established a cut-off of 0.5 McFarland units as an a priori positive test and evaluated this hypothesis retrospectively on a collection of well-characterized enterobacterial isolates with reduced susceptibility to at least one carbapenem. Test repeatability To assess the reliability of the test we performed a repeatability analysis, including 10 strains from each of the carbapenemase Ambler classes as well as 10 non-carbapenemase-producing strains. The isolates comprised 1 Citrobacter koseri, 1 Enterobacter asburiae, 1 Morganella morganii, 4 Serratia marcescens, 7 Enterobacter cloacae, 8 E. coli and 18 K. pneumoniae, harbouring class A (FRI-1, GES-5, IMI-1, IMI-2, KPC-2, SME-1, SME-2), class B (GIM-1, IMP-1, IMP-4, IMP-11, NDM-1, NDM-4, VIM-1, VIM-2, VIM-19) or class D (OXA-48, OXA-162, OXA-181, OXA-244) and 10 non-carbapenemase producers (Table 1). Table 1. Results from the repeatability study, showing agreement between the rCIM, performed three times per isolate for a well-characterized set of 40 Enterobacteriaceae No.  Bacteria  Enzyme  ID  rCIM  Carbapenemase producers  1  E. cloacae  FRI-1  1I10  3  2  K. pneumoniae  GES-5  3B7  2+/1−  3  E. cloacae  IMI-1  1I3  2+/1−  4  E. asburiae  IMI-2  1I4  2+/1−  5  E. coli  KPC-2  1F3  3+  6  K. pneumoniae  1F5  3+  7  K. pneumoniae  1F9  3+  8  K. pneumoniae  1G1  3+  9  S. marcescens  SME-1  1I7  3+  10  S. marcescens  SME-2  1I8  3+  11  E. cloacae  GIM-1  1E10  3+  12  K. pneumoniae  IMP-1  1E2  3+  13  S. marcescens  IMP-11  1E9  3+  14  K. pneumoniae  IMP-4  3D4  3+  15  E. coli  NDM-1  1A1  3+  16  K. pneumoniae  1B1  3+  17  E. coli  NDM-4  1A6  3+  18  K. pneumoniae  VIM-1  1C7  3+  19  K. pneumoniae  VIM-19  1D4  3+  20  E. cloacae  VIM-2  3B8  3+  21  K. pneumoniae  OXA-162  2C2  3+  22  K. pneumoniae  OXA-181  2C5  3+  23  E. coli  OXA-244  2D10  3+  24  C. koseri  OXA-48  2B10  3+  25  E. coli  2A1  3+  26  E. coli  2A4  3+  27  E. coli  2A6  3+  28  K. pneumoniae  2A7  3+  29  K. pneumoniae  2A8  3+  30  K. pneumoniae  2A9  3+  Non-carbapenemase producers  31  E. cloacae  NA  2I10  3−  32  E. cloacae  2G3  3−  33  M. morganii  2H5  3−  34  E. cloacae  2F8  3−  35  E. coli  2F3  3−  36  K. pneumoniae  2F4  3−  37  K. pneumoniae  2I1  3−  38  K. pneumoniae  2I2  3−  39  K. pneumoniae  2I3  3−  40  S. marcescens  2J5  3−  No.  Bacteria  Enzyme  ID  rCIM  Carbapenemase producers  1  E. cloacae  FRI-1  1I10  3  2  K. pneumoniae  GES-5  3B7  2+/1−  3  E. cloacae  IMI-1  1I3  2+/1−  4  E. asburiae  IMI-2  1I4  2+/1−  5  E. coli  KPC-2  1F3  3+  6  K. pneumoniae  1F5  3+  7  K. pneumoniae  1F9  3+  8  K. pneumoniae  1G1  3+  9  S. marcescens  SME-1  1I7  3+  10  S. marcescens  SME-2  1I8  3+  11  E. cloacae  GIM-1  1E10  3+  12  K. pneumoniae  IMP-1  1E2  3+  13  S. marcescens  IMP-11  1E9  3+  14  K. pneumoniae  IMP-4  3D4  3+  15  E. coli  NDM-1  1A1  3+  16  K. pneumoniae  1B1  3+  17  E. coli  NDM-4  1A6  3+  18  K. pneumoniae  VIM-1  1C7  3+  19  K. pneumoniae  VIM-19  1D4  3+  20  E. cloacae  VIM-2  3B8  3+  21  K. pneumoniae  OXA-162  2C2  3+  22  K. pneumoniae  OXA-181  2C5  3+  23  E. coli  OXA-244  2D10  3+  24  C. koseri  OXA-48  2B10  3+  25  E. coli  2A1  3+  26  E. coli  2A4  3+  27  E. coli  2A6  3+  28  K. pneumoniae  2A7  3+  29  K. pneumoniae  2A8  3+  30  K. pneumoniae  2A9  3+  Non-carbapenemase producers  31  E. cloacae  NA  2I10  3−  32  E. cloacae  2G3  3−  33  M. morganii  2H5  3−  34  E. cloacae  2F8  3−  35  E. coli  2F3  3−  36  K. pneumoniae  2F4  3−  37  K. pneumoniae  2I1  3−  38  K. pneumoniae  2I2  3−  39  K. pneumoniae  2I3  3−  40  S. marcescens  2J5  3−  NA, not applicable, as these bacteria produce no carbapenem-hydrolysing enzymes. All of these strains had decreased permeability and either an ESBL or an overexpressed cephalosporinase. Overall, the repeatability was excellent, with the exception of low-hydrolysing strains and strains requiring induction of their β-lactamase. Two IMI-like-expressing strains, an E. asburiae and an E. cloacae, gave 2 out of 3 positive results with rCIM. A GES-5 K. pneumoniae strain tested positive 2 out of 3 times. Retrospective analysis At 1.5 h, the majority of growth indicators challenged with supernatant from carbapenemase-positive strains (82/85) had reached at least 0.5 McFarland units of growth. The strains which had a low growth index (around the diagnostic breakpoint of 0.5) at 1.5 h were further incubated for 30 mins. At the same time, one carbapenemase-negative isolate grew above the 0.5 threshold, but subsequently declined. After 2 h of incubation at 37 °C, the indicator strain, when challenged with inactivated antibiotic, grew to a median of about 2.5 McFarland units (range 0.38–3.13, IQR 1.85–2.69). The only strains that produced growth less than 1 McFarland unit were 3 OXA-244 E. coli isolates (0.38, 0.57 and 0.74, respectively) and one IMI-2 strain (0.86) (Figure 1, Table 2). In contrast, when challenged with active antibiotic, the median growth was of 0.13 McFarland units (range −0.13 to 0.47, IQR 0.05–0.22). One E. cloacae strain with cephalosporinase hyperproduction initially tested positive, and then tested negative twice. Table 2. Results from the retrospective study, showing agreement and differences between the rCIM, Carba NP and CIM for a well-characterized set of Enterobacteriaceae Enzyme  Species  No.  rCIM  CIM  Carba NP  Class A             KPC-2  E. cloacae  1  +  +  +  E. coli  3  +  +  +  K. oxytoca  1  +  +  +  K. pneumoniae  4  +  +  +   KPC-3  K. pneumoniae  1  +  +  +   IMI-2  E. aerogenes  1  +  +  −  E. asburiae  1  +  +  +   IMI-3  E. cloacae  1  +  +  +   GES-5  E. cloacae  1  +  +  +  K. pneumoniae  1  +  +  +   FRI-1  E. cloacae  1  +  +  +   NMCA  E. cloacae  1  +  +  +   SME-1  S. marcescens  1  +  +  +   SME-2  S. marcescens  1  +  +  +  Class B             NDM-1  K. pneumoniae  5  +  +  +  E. coli  2  +  +  +  P. rettgeri  1  +  −  +   NDM-4  E. coli  2  +  +  +   NDM-5  E. coli  1  +  +  +   NDM-6  E. coli  1  +  +  +   VIM-1  K. pneumoniae  3  +  +  +  E. coli  1  +  +  +   VIM-2  E. cloacae  1  +  −  −   VIM-4  E. cloacae  1  +  −  −  E. coli  1  +  +  +   VIM-19  K. pneumoniae  1  +  +  +   GIM-1  E. cloacae  1  +  +  +   IMP-1  E. coli  1  +  +  +  K. pneumoniae  1  +  +  +   IMP-4  K. pneumoniae  1  +  +  +   IMP-8  K. pneumoniae  1  +  +  +   IMP-10  S. marcescens  1  +  +  +   IMP-11  S. marcescens  1  +  +  +  Class D             OXA-48  C. koseri  2  +  +  +  E. cloacae  2  +  +  +  E. coli  4  +  +  +  K. oxytoca  4  +  +  +  K. pneumoniae  4  +  +  +  P. mirabilis  1  +  +  +   OXA-162  K. pneumoniae  1  +  +  +   OXA-181  E. coli  7  +  +  +  K. pneumoniae  2  +  +  +   OXA-204  E. coli  1  +  +  +  K. pneumoniae  1  +  +  +   OXA-232  E. coli  1  +  +  +   OXA-244  E. coli  8  7/8+  6/8+  5/8+   OXA-372  C. freundii  1  +  +  +  ESBL + decreased membrane permeability    K. pneumoniae  4  −  −  −  E. coli  2  −  −  −  Plasmid-mediated or chromosomal AmpC + decreased membrane permeability    E. aerogenes  1  −  −  −  E. cloacae  3  −  −  −  E. coli  1  −  −  −    K. pneumoniae  2  −  −  −    M. morganii  1  −  −  −  ESBL + plasmid-mediated or chromosomal AmpC + decreased membrane permeability    C. freundii  1  −  −  −  E. cloacae  3  −  −  −  Extended-spectrum oxacillinases    S. marcescens  1  −  −  −  Other mechanisms + decreased membrane impermeability    E. cloacae  1  −  −  −  E. coli  1  −  −  −  K. oxytoca  6  −  −  −  K. pneumoniae  1  −  −  −  Enzyme  Species  No.  rCIM  CIM  Carba NP  Class A             KPC-2  E. cloacae  1  +  +  +  E. coli  3  +  +  +  K. oxytoca  1  +  +  +  K. pneumoniae  4  +  +  +   KPC-3  K. pneumoniae  1  +  +  +   IMI-2  E. aerogenes  1  +  +  −  E. asburiae  1  +  +  +   IMI-3  E. cloacae  1  +  +  +   GES-5  E. cloacae  1  +  +  +  K. pneumoniae  1  +  +  +   FRI-1  E. cloacae  1  +  +  +   NMCA  E. cloacae  1  +  +  +   SME-1  S. marcescens  1  +  +  +   SME-2  S. marcescens  1  +  +  +  Class B             NDM-1  K. pneumoniae  5  +  +  +  E. coli  2  +  +  +  P. rettgeri  1  +  −  +   NDM-4  E. coli  2  +  +  +   NDM-5  E. coli  1  +  +  +   NDM-6  E. coli  1  +  +  +   VIM-1  K. pneumoniae  3  +  +  +  E. coli  1  +  +  +   VIM-2  E. cloacae  1  +  −  −   VIM-4  E. cloacae  1  +  −  −  E. coli  1  +  +  +   VIM-19  K. pneumoniae  1  +  +  +   GIM-1  E. cloacae  1  +  +  +   IMP-1  E. coli  1  +  +  +  K. pneumoniae  1  +  +  +   IMP-4  K. pneumoniae  1  +  +  +   IMP-8  K. pneumoniae  1  +  +  +   IMP-10  S. marcescens  1  +  +  +   IMP-11  S. marcescens  1  +  +  +  Class D             OXA-48  C. koseri  2  +  +  +  E. cloacae  2  +  +  +  E. coli  4  +  +  +  K. oxytoca  4  +  +  +  K. pneumoniae  4  +  +  +  P. mirabilis  1  +  +  +   OXA-162  K. pneumoniae  1  +  +  +   OXA-181  E. coli  7  +  +  +  K. pneumoniae  2  +  +  +   OXA-204  E. coli  1  +  +  +  K. pneumoniae  1  +  +  +   OXA-232  E. coli  1  +  +  +   OXA-244  E. coli  8  7/8+  6/8+  5/8+   OXA-372  C. freundii  1  +  +  +  ESBL + decreased membrane permeability    K. pneumoniae  4  −  −  −  E. coli  2  −  −  −  Plasmid-mediated or chromosomal AmpC + decreased membrane permeability    E. aerogenes  1  −  −  −  E. cloacae  3  −  −  −  E. coli  1  −  −  −    K. pneumoniae  2  −  −  −    M. morganii  1  −  −  −  ESBL + plasmid-mediated or chromosomal AmpC + decreased membrane permeability    C. freundii  1  −  −  −  E. cloacae  3  −  −  −  Extended-spectrum oxacillinases    S. marcescens  1  −  −  −  Other mechanisms + decreased membrane impermeability    E. cloacae  1  −  −  −  E. coli  1  −  −  −  K. oxytoca  6  −  −  −  K. pneumoniae  1  −  −  −  Grey shading indicates unexpected results (either false-positive or false-negative testing results). Figure 1. View largeDownload slide Dynamics of E. coli indicator strain growth in the retrospective study. Box and whisker plot showing the dynamics of growth of the E. coli ATCC 25922 indicator strain from the retrospective study (n = 113). Measurements were made every 30 min after supernatant challenge of the indicator strain. Carbapenemase producers and non-producers are presented side by side for each measurement time point. Results represent baseline-subtracted growth, and are expressed as McFarland units. Filled circles represent outliers. A horizontal line marks the 0.5 McFarland index cut-off chosen. Figure 1. View largeDownload slide Dynamics of E. coli indicator strain growth in the retrospective study. Box and whisker plot showing the dynamics of growth of the E. coli ATCC 25922 indicator strain from the retrospective study (n = 113). Measurements were made every 30 min after supernatant challenge of the indicator strain. Carbapenemase producers and non-producers are presented side by side for each measurement time point. Results represent baseline-subtracted growth, and are expressed as McFarland units. Filled circles represent outliers. A horizontal line marks the 0.5 McFarland index cut-off chosen. Thus, the sensitivity of the rCIM was 99% (95% CI 94%–100%) and the specificity was 100% (95% CI 88%–100%), using a simple indicator of baseline-subtracted growth of 0.5 McFarland units at 2 h. Using the same panel of isolates, the sensitivity and the specificity of the CIM test were 94% (95% CI 87%–98%) and 100% (95% CI 88%–100%), respectively. Among the undetected strains, there was one NDM-1-producing Providencia rettgeri, two OXA-244-producing E. coli, one VIM-4- and one VIM-2-producing E. cloacae. The Carba NP test had a sensitivity of 93% (95% CI 85%–97%) and a specificity of 100% (95% CI 88%–100%). The false-negative isolates were the three OXA-244-producing E. coli, one VIM-4- and one VIM-2-producing E. cloacae, and an IMI-2-producing E. asburiae (Table 3). Table 3. Performance characteristics for the rCIM in the retrospective study. Results for 2 h growth time of the indicator strain   Carbapenemase     Characteristic  positive  negative  Total  rCIM positive  84  0  84  rCIM negative  1 (OXA-244a)  28  29  Total  85  28  113    Carbapenemase     Characteristic  positive  negative  Total  rCIM positive  84  0  84  rCIM negative  1 (OXA-244a)  28  29  Total  85  28  113  a Also negative with CIM and Carba NP test. Prospective rCIM evaluation The 101 isolates of the prospective study included 34 K. pneumoniae, 23 E. coli, 21 E. cloacae, 12 Citrobacter freundii, 2 E. asburiae, 2 Proteus mirabilis, 2 P. rettgeri, 1 Providencia stuartii, 1 Citrobacter youngae, 1 Enterobacter hormaechei, 1 Hafnia alvei and 1 M. morganii. They expressed 45 OXA-48-like, 10 NDM-like, 5 VIM-like, 2 KPC-like and 2 IMI-like enzymes, 15 hyperproduced cephalosporinases, 12 ESBLs, 4 hyperproduced cephalosporinase and an ESBL and 6 others had non-enzymatic carbapenem resistance mechanisms. After WGS, the two IMI-like-producing E. asburiae were identified as being the same strain, producing a novel IMI variant (submitted to GenBank under the name IMI-17), and was counted only once. Thus, there were 100 isolates tested in total. PCR sequencing results are displayed in Table 4. Table 4. Results from the prospective study, showing agreement and differences between the rCIM and Carba NP, relative to molecular methods for 100 Enterobacteriaceae evaluated by the French NRC   Species  No.  rCIM  Carba NP  Class A           KPC-2  K. pneumoniae  1  +  +   KPC-3  K. pneumoniae  1  +  +   IMI-17  E. asburiae  1  −  −  Class B           NDM-1  P. rettgeri  1  +  +  P. rettgeri  1  +  −  P. stuartii  1  +  −  K. pneumoniae  1  +  +  E. cloacae  1  +  +   NDM-5  E. coli  4  +  +   NDM-6  K. pneumoniae  1  +  +   VIM-1  E. cloacae  2  +  +  K. pneumoniae  2  +  +   VIM-4  E. cloacae  1  +  +  Class D           OXA-23  P. mirabilis  1  +  +   OXA-48  C. youngae  1  +  +  C. freundii  6  +  +  E. coli  10  +  +  E. cloacae  4  +  +  K. pneumoniae  21  +  +   OXA-181  E. coli  1  +  +  E. coli  1  +  −  Non-carbapenemase producers         Case  C. freundii  4  −  −  E. cloacae  4  −  −  E. cloacae  2  +  −  E. hormaechei  1  −  −  H. alvei  1  −  −  K. pneumoniae  3  −  −   ESBL  C. freundii  2  −  −  E. coli  5  −  −  E. cloacae  1  −  −  K. pneumoniae  4  −  −   Case + ESBL  E. cloacae  4  −  −   Other  M. morganii  1  −  −  E. coli  2  −  −  E. cloacae  2  −  −  P. mirabilis  1  −  −    Species  No.  rCIM  Carba NP  Class A           KPC-2  K. pneumoniae  1  +  +   KPC-3  K. pneumoniae  1  +  +   IMI-17  E. asburiae  1  −  −  Class B           NDM-1  P. rettgeri  1  +  +  P. rettgeri  1  +  −  P. stuartii  1  +  −  K. pneumoniae  1  +  +  E. cloacae  1  +  +   NDM-5  E. coli  4  +  +   NDM-6  K. pneumoniae  1  +  +   VIM-1  E. cloacae  2  +  +  K. pneumoniae  2  +  +   VIM-4  E. cloacae  1  +  +  Class D           OXA-23  P. mirabilis  1  +  +   OXA-48  C. youngae  1  +  +  C. freundii  6  +  +  E. coli  10  +  +  E. cloacae  4  +  +  K. pneumoniae  21  +  +   OXA-181  E. coli  1  +  +  E. coli  1  +  −  Non-carbapenemase producers         Case  C. freundii  4  −  −  E. cloacae  4  −  −  E. cloacae  2  +  −  E. hormaechei  1  −  −  H. alvei  1  −  −  K. pneumoniae  3  −  −   ESBL  C. freundii  2  −  −  E. coli  5  −  −  E. cloacae  1  −  −  K. pneumoniae  4  −  −   Case + ESBL  E. cloacae  4  −  −   Other  M. morganii  1  −  −  E. coli  2  −  −  E. cloacae  2  −  −  P. mirabilis  1  −  −  Case, overexpressed cephalosporinase. Grey shading indicates unexpected results (either false-positive or false-negative testing results). Using the cut-off defined in the retrospective study, the sensitivity of the rCIM was 98% (95% CI 89%–99%) and the specificity was 95% (95% CI 82%–99%), with a positive predictive value of 97% (95% CI 89%–100%) and a negative predictive value of 97% (95% CI 85%–100%). Two cephalosporinase-hyperproducing E. cloacae isolates gave false-positive results, while a novel IMI variant-producing E. asburiae gave a false-negative result (Figure 2). Figure 2. View largeDownload slide Results of E. coli indicator strain growth in the prospective study. Box and whisker plot showing results of indicator growth at 1.5 and 2 h for the prospectively evaluated strains (n = 100). Carbapenemase producers and non-producers are presented side by side for each measurement time point. Results represent baseline-subtracted growth, and are expressed as McFarland units. Filled circles represent outliers. A horizontal line marks the 0.5 McFarland index cut-off chosen. Figure 2. View largeDownload slide Results of E. coli indicator strain growth in the prospective study. Box and whisker plot showing results of indicator growth at 1.5 and 2 h for the prospectively evaluated strains (n = 100). Carbapenemase producers and non-producers are presented side by side for each measurement time point. Results represent baseline-subtracted growth, and are expressed as McFarland units. Filled circles represent outliers. A horizontal line marks the 0.5 McFarland index cut-off chosen. In combination with the interpretative reading of the antibiogram, the Carba NP test is used as the reference method for the screening of carbapenemase activity at the French NRC. It had a sensitivity of 92% (95% CI 82%–97%), a specificity of 100% (95% CI 91%–100%), a positive predictive value of 100% (95% CI 89%–100%) and a negative predictive value of 88% (95% CI 74%–96%). A total of four strains tested Carba NP negative. These were an OXA-181-producing E. coli, the novel IMI variant-expressing E. asburiae, one NDM-1-producing P. rettgeri and one NDM-1-producing P. stuartii. The rCIM showed better sensitivity than the Carba NP test (98% versus 92%, P = 0.01), with the trade-off of a lower specificity (95% versus 100%, P < 0.001). rCIM false-negative results Even though a low positivity cut-off was chosen in order to ease the detection of low carbapenem-hydrolysing enzymes, such as OXA-244,27 three isolates gave false-negative rCIM results (1.6%): two OXA-244-producing E. coli strains and an E. asburiae producing a novel IMI variant (IMI-17). One of the OXA-244 isolates was positive on repeated testing (2+/1−) while the two others tested negative when taken directly off the UriSelectTM 4 medium. OXA-244 enzymes are difficult to detect, as recently shown by Hoyos-Mallecot et al.27 These enzymes are characterized by weak carbapenem hydrolysis that results in reduced susceptibility to carbapenems only. However, as OXA-244 may be plasmid encoded, horizontal gene transfer may occur into bacteria with impaired outer membrane permeability, and lead to high-level carbapenem resistance.29,30 Thus, it is of outmost importance to identify these enzymes using proper diagnostic algorithms (i.e. the use of temocillin resistance as a marker for OXA-48-like carbapenemases) and rapid diagnostic tests.31,32 The rCIM seems to be an excellent test in this respect, as six out of the seven OXA-244-producing E. coli isolates were correctly identified as carbapenemase producers. In the case of the E. asburiae isolate, the false-negative rCIM results could be explained by the low level and inducible nature of IMI production. These enzymes can be induced with imipenem or cefoxitin through the presence of the LysR-type regulator IMI-R.33 The IMI-producing E. asburiae isolate was negative with rCIM, Carba NP and CIM tests when grown on UriSelect™ 4 media. When the isolate was grown on Mueller–Hinton or on selective chromogenic media, such as CarbaSmart (bioMérieux), it gave a positive rCIM result. rCIM false-positive results Overall, the rCIM gave three false-positive isolates (1.6%); all corresponded to cephalosporinase-hyperproducing E. cloacae. One of them, in the retrospective study, proved negative on repeated testing (2−/1+). The two other isolates were from the prospective study and grew slightly above the baseline-subtracted McFarland threshold of 0.5 (0.89 and 1.29, respectively); on repeat testing, one gave consistent results over the 0.5 McFarland threshold (3+), while one gave mostly negative results (2−/1+). Laboratories have to keep in mind that cephalosporinase-hyperproducing E. cloacae may provide weak positive results and thus would require additional testing. Practical guidance In high incidence settings, the rCIM might be used to rapidly confirm the presence of a carbapenem-hydrolysing enzyme in various enterobacterial isolates. The rCIM can be interpreted after 1.5 h of indicator strain growth (roughly 2 h of total preparation). In our total experience of testing 213 isolates, a baseline-subtracted growth of more than 1 is highly indicative of carbapenemase production (131/148 CPEs and 1 false-positive isolate). For baseline-subtracted growth between 0.5 and 1, 30 min of additional incubation was beneficial, as nine other CPE isolates (140/148) were detected (Figure 3). Figure 3. View largeDownload slide Overall experience with the rCIM. Box and whisker plot showing overall experience with rCIM (n = 213). Results presented for 1.5 and 2 h of indicator strain growth. Carbapenemase producers and non-producers are presented side by side for each measurement time point. Grey zone marked between 0.5 and 1 McFarland represents a zone of uncertainty requiring further testing. Filled circles represent outliers. Rectangles highlight the results of the novel IMI variant-producing strain when tested directly from a chromogenic medium. Filled triangles represent the results obtained with the novel IMI variant-expressing strain giving a positive rCIM result when tested from the CarbaSmart medium. Figure 3. View largeDownload slide Overall experience with the rCIM. Box and whisker plot showing overall experience with rCIM (n = 213). Results presented for 1.5 and 2 h of indicator strain growth. Carbapenemase producers and non-producers are presented side by side for each measurement time point. Grey zone marked between 0.5 and 1 McFarland represents a zone of uncertainty requiring further testing. Filled circles represent outliers. Rectangles highlight the results of the novel IMI variant-producing strain when tested directly from a chromogenic medium. Filled triangles represent the results obtained with the novel IMI variant-expressing strain giving a positive rCIM result when tested from the CarbaSmart medium. At 2 h, isolates with baseline-subtracted growth between 0.5 and 1 should undergo further testing (using molecular or immune-enzymatic assays, such as the OKN K-SeT assay),34 or even retesting. Of all the carbapenemase producers tested, only OXA-244, KPC-2 and IMI-2 producers (n = 7) gave such results. Another isolate, an E. cloacae hyperproducing a cephalosporinase, also gave an indeterminate result. Once the antibiograms are obtained, phenotypic interpretation of the antibiogram and retesting from the Mueller–Hinton plate may clarify certain borderline cases, especially with IMI producers. In the absence of carbapenemase production, 63 out of 65 isolates were identified at 2 h. In 2016, the French NRC received 2839 carbapenem-resistant strains, of which 1551 harboured a carbapenemase. Class D carbapenemases were by far the most frequent, forming about three-quarters of the total number of isolates sent in for expert assessment (1187/1551; 76%). Of these, the majority were OXA-48 (n = 1078) and OXA-181 producers (n = 73). OXA-244 producers (n = 8) represented only 0.5% of the total carbapenemase producers. The epidemiology of strains tested in our prospective study mirror the national epidemiology. Of interest, one OXA-181 isolate tested in the prospective study was positive with rCIM but repeatedly negative with the Carba NP test. Class B carbapenemases follow at a distance (282/1551; 18%), with NDM-type enzymes predominating (n = 217), followed by VIM-type enzymes (n = 62), and very rarely IMP enzymes (n = 3). All these enzymes were accurately identified with all three phenotypic tests. Class A carbapenemases are relatively rare and account for less than 5% (53/1551). Of note, the IMI-type enzymes are very rarely isolated and represented less than 1% (14/1551) of the cases identified by the NRC. Lastly, some strains sent to the NRC expressed multiple carbapenemases. Thus, in daily clinical practice, the sensitivity of the rCIM would probably be even higher. One limit of the current study is the use of a set of conditions including incubation in sterile water and the use of trypticase soy broth media for the growth of the indicator. As a result, outcomes might differ when other buffers or broths might be used (e.g. Tris-buffered saline or Mueller–Hinton). Trypticase soy broth was chosen because it is a general-purpose nutritive medium, cheaper than Mueller–Hinton and should be more readily available to laboratories. Furthermore, the rCIM requires multiple hands-on steps (including multiple incubation steps and centrifugation), which should be taken into account when comparing with other commercial methods (Carba NP and β-Carba Test). We acknowledge that the extra hands-on time might increase the time-to-result, though we feel that this remains similar as compared with other methods. Lastly, the need for a big inoculum (two 10 μL loops) needs to be addressed as a limiting factor as large quantities of bacteria are not always recovered from screening media. Recently, another variation of the CIM, the mCIM, was published, showing good inter-laboratory agreement,35,36 but with the same drawback of the 24 h time frame necessary for detection. The rCIM resolves this major problem, requiring less than 3 h of total work time, which is compatible with the daily practice of a clinical microbiology laboratory. Compared with the Carba NP, the rCIM offers a more objective perspective of interpretation as it is a quantitative test. This may make it easier for laboratory personnel to quantify uncertainty and use additional tests, as appropriate. The cost of the rCIM is very low, comparable to the CIM and mCIM.18,26,35 Using the online prices from a major French distributor, we estimated the costs for the rCIM to be $0.13 for 3 mL of trypticase soy broth + $0.096 for two antibiotic discs (Table 5). Table 5. Comparative characteristics of phenotypic methods with price estimation per test and total work time Test  rCIM  CIM  mCIM  Carba NP  Initial incubation time (h)  0.5  2  4  —  Time-to-results (h)  2–2.5  8–24  24  2  Antibiotic used  meropenem  meropenem  meropenem  imipenem  Antibiotic amount/ concentration used  20 μg  10 μg  10 μg  3 mg/mL  Cost per test (US$)  0.25  0.45a  <1b  2–10a  Test  rCIM  CIM  mCIM  Carba NP  Initial incubation time (h)  0.5  2  4  —  Time-to-results (h)  2–2.5  8–24  24  2  Antibiotic used  meropenem  meropenem  meropenem  imipenem  Antibiotic amount/ concentration used  20 μg  10 μg  10 μg  3 mg/mL  Cost per test (US$)  0.25  0.45a  <1b  2–10a  a Gauthier et al., 2017.26 b Pierce et al., 2017.35 Unlike the CIM and mCIM, the rCIM does not require the manipulation of contaminated antibiotic discs with highly drug-resistant bacteria, eliminating the potential of cross-contamination. On the other hand, the rCIM requires the use of a nephelometer and a centrifuge, but we expect that these would be present in the vast majority of laboratories. Conclusions Overall, the high sensitivity and specificity of rCIM, as well as the fact that is a rapid test, make it a useful tool in clinical microbiology laboratories. The rCIM does not need trained personnel and the equipment needed is largely present in most microbiology laboratories worldwide. The rCIM resolves the main limitation of the CIM as it can provide same-day results, thus being able to guide the clinician regarding the treatment and making it compatible with efficient implementation of infection control measures. In conclusion, the rCIM is a quantitative test that has a price comparable with that of CIM and mCIM (<$1 per test) and a detection time comparable with that of Carba NP, making it accessible even in small laboratories. Funding This work was supported by the Assistance Publique – Hôpitaux de Paris, by a grant from the Université Paris-Sud (EA7361), and by the LabEx LERMIT supported by a grant from the French National Research Agency (ANR-10-LABX-33). This work was also funded in part by a grant from the Joint Programme Initiative on Antimicrobial Resistance (ANR-14-JAMR-0002). Transparency declarations None to declare. References 1 Nordmann P, Cuzon G, Naas T. The real threat of Klebsiella pneumoniae carbapenemase-producing bacteria. Lancet Infect Dis  2009; 9: 228– 36. Google Scholar CrossRef Search ADS PubMed  2 Munoz-Price LS, Poirel L, Bonomo RA et al.   Clinical epidemiology of the global expansion of Klebsiella pneumoniae carbapenemases. Lancet Infect Dis  2013; 13: 785– 96. Google Scholar CrossRef Search ADS PubMed  3 Logan LK, Weinstein RA. The epidemiology of carbapenem-resistant Enterobacteriaceae: the impact and evolution of a global menace. J Infect Dis  2017; 215: S28– 36. 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Journal of Antimicrobial ChemotherapyOxford University Press

Published: Apr 1, 2018

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