The ability of clinical microbiology laboratories to reliably detect carbapenemase-producing carbapenem-resistant Enterobacteriaceae (CP-CRE) is an important element of the effort to prevent and contain the spread of these pathogens and an integral part of antimicrobial stewardship. All existing methods have limitations. A new, straightforward, inexpensive, and specific phenotypic method for the detection of carbapenemase production, the carbapenem inactivation method (CIM), was recently described. Here we describe a two-stage evaluation of a modified carbapenem inactivation method (mCIM), in which tryptic soy broth was substituted for water during the inactivation step and the length of this incubation was extended. A validation study was performed in a single clinical laboratory to determine the accuracy of the mCIM, followed by a nine-laboratory study to verify the reproducibility of these results and define the zone size cutoff that best discriminated between CP-CRE and members of the family Enterobacteriaceae that do not produce carbapenemases. Bacterial isolates previously characterized through whole-genome sequencing or targeted PCR as to the presence or absence of carbapenemase genes were tested for carbapenemase production using the mCIM; isolates with Ambler class A, B, and D carbapenemases, non-CP-CRE isolates, and carbapenem-susceptible isolates were included. The sensitivity of the mCIM observed in the validation study was 99% (95% confidence interval [95% CI], 93% to 100%), and the specificity was 100% (95% CI, 82% to 100%). In the second stage of the study, the range of sensitivities observed across nine laboratories was 93% to 100%, with a mean of 97%; the range of specificities was 97% to 100%, with a mean of 99%. The mCIM was easy to perform and interpret for Enterobacteriaceae, with results in less than 24 h and excellent reproducibility across laboratories.

The purpose of this study was to develop the modified carbapenem inactivation method (mCIM) for the detection of carbapenemase-producing (CP-PA) and carbapenemase-producing (CP-AB) and perform a multicenter evaluation of the mCIM and Carba NP tests for these nonfermenters. Thirty and 30 isolates previously characterized by whole-genome sequencing from the CDC-FDA Antibiotic Resistance Isolate Bank were evaluated, including CP isolates (Ambler class A, B, and D), non-carbapenemase-producing (non-CP) carbapenem-resistant isolates, and carbapenem-susceptible isolates. Initial comparison of a 1-μl versus 10-μl loop inoculum for the mCIM was performed by two testing sites and showed that 10 μl was required for reliable detection of carbapenemase production among and Ten testing sites then evaluated the mCIM using a 10-μl loop inoculum. Overall, the mean sensitivity and specificity of the mCIM for detection of CP-PA across all 10 sites were 98.0% (95% confidence interval [CI], 94.3 to 99.6; range, 86.7 to 100) and 95% (95% CI, 89.8 to 97.7; range, 93.3 to 100), whereas the mean sensitivity and specificity among CP-AB were 79.8% (95% CI, 74.0 to 84.9; range, 36.3 to 95.7) and 52.9% (95% CI, 40.6 to 64.9; range, 28.6 to 100), respectively. At three sites that evaluated the performance of the Carba NP test using the same set of isolates, the mean sensitivity and specificity of the Carba NP test were 97.8% (95% CI, 88.2 to 99.9; range, 93.3 to 100) and 97.8% (95% CI, 88.2 to 99.9; range, 93.3 to 100) for and 18.8% (95% CI, 10.4 to 30.1; range, 8.7 to 26.1) and 100% (95% CI, 83.9 to 100; range, 100) for Overall, we found both the mCIM and the Carba NP test to be accurate for detection of carbapenemase production among isolates and less reliable for use with isolates.

There is an urgent need for rapid, accurate detection and classification of carbapenemases. The current study evaluated the automated BD Phoenix CPO Detect and the manual bioMérieux Rapidec Carba NP tests for meeting these needs. Both tests were challenged with 294 isolates of Enterobacteriaceae spp., Pseudomonas aeruginosa, and Acinetobacter baumannii chosen to provide extreme diagnostic difficulty. Carbapenemases such as KPC, NMC-A, IMI, SME, NDM, SPM, IMP, VIM, and OXA-23, 40, 48, 58, 72, 181, and 232 were produced by 243 isolates and 51 carbapenemase-negative isolates included porin mutants and producers of extended-spectrum β-lactamases (ESBLs), AmpCs, K1, and broad-spectrum β-lactamases. Both tests exhibited high sensitivity of carbapenemase detection (>97%). Due to the highly challenging carbapenemase-negative isolates, specificities were lower than typical for evaluations involving mostly routine clinical isolates. BD Phoenix CPO Detect was 68.6% specific and Rapidec Carba NP was 60.8% to 78.4% specific, depending on how borderline results were interpreted. Only BD Phoenix CPO Detect classified carbapenemases. It correctly classified 85.0% of class A, 72.4% of class B, and 88.6% of class D carbapenemases. Importantly with respect to empirical therapy with new β-lactamase inhibitor combinations such as ceftazidime/avibactam, no class B carbapenemases were misclassified as class A carbapenemases. Both tests offer advantages. Used alone, without initial susceptibility tests, Rapidec Carba NP can provide positive results for some isolates after only 10 to 30 min incubation. BD Phoenix CPO Detect provides novel advantages such as automated carbapenemase detection, inclusion in susceptibility panels to eliminate delays and subjectivity in initiating carbapenemase tests, and classification of most carbapenemases.

This report describes the results of an 11-laboratory study to determine if a cefoxitin broth microdilution MIC test could predict the presence of mecA in staphylococci. Using breakpoints of <4 g/ml for mecA-negative and >6 or 8 g/ml for mecA-positive isolates, sensitivity and specificity based on mecA or presumed mecA for Staphylococcus aureus at 18 h of incubation were 99.7 to 100% in three cation-adjusted Mueller-Hinton broths tested. For coagulase-negative strains at 24 h of incubation, breakpoints of <2 g/ml for mecA-negative and >4 g/ml for mecA-positive isolates gave sensitivity and specificity of 94 to 99% and 69 to 80%, respectively.The use of a cefoxitin disk test to detect staphylococci that are likely to contain the mecA gene has been widely advocated since the test was first suggested (3, 6) and has been adopted by antimicrobial susceptibility testing organizations worldwide (http://www.bsac.org.uk/_db/_documents/version_6.1.pdf, http: //www.srga.org/, http://www.clsi.org/). For laboratories that do not use disk diffusion as their primary testing method, performing disk diffusion requires additional expense and reagents. Consequently, we performed studies to determine if a cefoxitin broth microdilution MIC breakpoint would correlate with the presence of the mecA gene in staphylococci.Cation-adjusted Mueller-Hinton broths (CAMHB) from three manufacturers (BBL, BD Diagnostic Systems, Sparks, MD; Difco, BD Diagnostic Systems, Sparks, MD; and Oxoid, Basingstoke, Hampshire, England) (cation content adjusted if necessary), were used to prepare frozen microdilution panels containing cefoxitin concentrations of 0.5 to 32 g/ml (including 6 g/ml) and oxacillin concentrations of 0.06 to 16 g/ml (in BBL CAMHB only). The frozen panels were shipped to all participants, along with 30-g cefoxitin disks (BBL). Each laboratory used its current lot of Mueller-Hinton agar for disk diffusion testing. ). Additional strains were tested at the CDC, totaling 167 S. aureus isolates and 22 CoNS. The total number of strains tested in all labs was 479 S. aureus isolates and 204 CoNS. All results were read after both 18-h and 24-h incubations. Endpoints were read as the lowest concentration at which no visible growth was observed. At 18 h of incubation, 97.3% of the MIC quality control results (for S. aureus ATCC 29213) and 97.9% of the disk diffusion quality control results (for S. aureus ATCC 25923) were within the range considered acceptable by the Clinical and Laboratory Standards Institute (CLSI).After testing, all strains were shipped to the CDC for further analysis. PCR testing for mecA was performed as previously described (8) on the following strains: (i) any S. aureus strain for which the cefoxitin MIC was Ͻ16 g/ml in any of the three media tested (n ϭ 180); (ii) any S. aureus strain for which the cefoxitin MIC was Ͼ8 g/ml and the oxacillin MIC was Ͻ4 g/ml in any of the three media tested (n ϭ 5); and (iii) a sample of the 304 isolates (n ϭ 16 [5%]) for which the cefoxitin MIC was Ͼ8 g/ml and the oxacillin MIC was Ͼ2 g...

A study conducted by 11 laboratories investigated the ability of four combinations of erythromycin (ERY) and clindamycin (CC) (ERY and CC at 4 and 0.5, 6 and 1, 8 and 1.5, and 0.5 and 2 g/ml) in a single well of a broth microdilution panel to predict the presence of inducible CC resistance. Each laboratory tested approximately 30 Staphylococcus aureus isolates and 20 coagulase-negative staphylococcus (CoNS) isolates in a panel using cation-adjusted Mueller-Hinton broth from three different manufacturers. Only the strains resistant to ERY and those susceptible or intermediate to CC were included in the analysis (S. aureus, n ‫؍‬ 333; CoNS, n ‫؍‬ 97). Results of the D-zone test were used as the gold standard. After an 18-h incubation, the combination of 4 g/ml ERY and 0.5 g/ml CC performed the best, with 98 to 100% sensitivity and 100% specificity for both organism groups. After a 24-h incubation, the ERY-CC combinations of 4 and 0.5, 6 and 1, and 8 and 1.5 g/ml correlated well with the D-zone test.The D-zone test, a disk approximation test using erythromycin (ERY) and clindamycin (CC) disks, which was proposed originally by Fiebelkorn et al. (5) and is currently described in the Clinical and Laboratory Standards Institute's (CLSI) antimicrobial susceptibility documents (1, 2, 4), has made the detection of inducible CC resistance in staphylococci an easy test for clinical microbiology laboratories to perform, especially if they are already performing disk diffusion tests. To further simplify the process for laboratories that routinely perform dilution susceptibility tests, a version using the broth microdilution test was investigated.In a previous study at the Centers for Disease Control and Prevention (CDC), a test combining ERY and CC in a single well of a broth microdilution panel using results of growth or no growth had produced promising results, but further work was suggested by the authors to define the most accurate concentrations for the two drugs (7). Results of a follow-up study at CDC, using a combination of 4 g/ml ERY and 0.5 g/ml CC in a single well, showed excellent correlation with the D-zone test results for 349 clinical isolates of staphylococci (335 Staphylococcus aureus and 13 coagulase-negative staphylococcus [CoNS] isolates), with a sensitivity of 97.4% and a specificity of 99.1% (unpublished data).In this report, we present the results of a multicenter laboratory evaluation undertaken to validate the preliminary findings at CDC. MATERIALS AND METHODSThe current study involved cation-adjusted Mueller-Hinton broth (CAMHB) from three manufacturers, the 4-g/ml ERY-0.5-g/ml CC combination described above, and three additional combinations of ERY and CC (6 and 1, 8 and 1.5, and 0.5 and 2 g/ml, respectively). Frozen broth microdilution panels containing ERY (8 to 0.12 g/ml) and CC (4 to 0.03 g/ml) (BBL CAMHB; BD, Sparks, MD) and the four combinations of ERY and CC in Mueller-Hinton broth from three different manufacturers (BBL, Difco [BD], and Oxoid [Basingstoke, Hampshire, England]; with cation c...