Evaluation of phenotypic tests for detection of carbapenemases: New modifications with new interpretation.

INTRODUCTION: The emergence and spread of carbapenemase-producing Enterobacterales (CPE) is a worldwide public health threat. Rapid and accurate detection of CPE is essential to prevent their dissemination within health care settings. The aim of this study was to evaluate the performance of CIM, mCIM and mCIM with ammonium bicarbonate (mCIM-A) methods by using different interpretation criteria for detection of carbapenemases. METHODS: One hundred and fifty-three Klebsiella pneumoniae isolates previously characterized by molecular tests, including 133 carbapenemase producers and 20 non-carbapenemase producers, were collected in this study. CIM and mCIM tests were performed as described previously. mCIM-A by adding 50 mM ammonium bicarbonate to the bacterial suspension prepared in tryptic soy broth. The inhibition zone diameter of around meropenem disc was measured and interpreted as positive according to i) Pierce and colleagues (<19 mm), ii) EUCAST meropenem susceptibility breakpoint (<22). RESULTS: CIM, although seems to be good for carbapenemases other than OXA-48-like and NDM, is not satisfactory (42.3% and 83.4%, respectively) for those enzymes with any of the interpretation criteria. OXA-48-like and NDM were detected with a better performance (88.7% and 92.8, respectively) with mCIM when results were interpreted according to <22 mm zone diameter for OXA-48-like and NDM. The best results were obtained with mCIM-A using <22 mm criteria without any difference in the results of other enzymes and negative strains. CONCLUSIONS: mCIM-A method interpreted with <22 mm meropenem zone diameter seems to be preferable compared to CIM and mCIM. mCIM-A is simple and useful tool for identification of CPEs in clinical microbiology laboratories.

Comparison of the performance of three carbapenem inactivation methods for the detection of carbapenemase-producing gram-negative bacilli.

INTRODUCTION: The carbapenem inactivation method test (CIM) was developed as a method for detecting carbapenemase-producing Gram-negative bacilli, and the modified CIM (mCIM) was recommended by the CLSI for as an improved method in M100-S27. However, few studies have evaluated the influence of bacterial species and genotype on its sensitivity and specificity. In this study, we evaluate the performance of these improved modified CIM methods with mCIM. METHODS: As strains, clinical isolates from Naga Municipal Hospital and stored strains from the Study of Bacterial Resistance in the Kinki Region of Japan were used. The mCIM, CIM-Tris, and simple CIM (sCIM) test methods were applied to 120 Enterobacterales, 40 Pseudomonas aeruginosa, and 37 Acinetobacter spp. The procedure and criteria for each method were based on the original papers and the CLSI M - 100 S27 documents. RESULTS: The sensitivity of the test methods in the detection of carbapenemase in Enterobacterales, Pseudomonas spp., and Acinetobacter spp. was as follows: mCIM, 98.9%, 90.0%, and 76.5%, respectively; CIM-Tris, 94.4%, 100%, 100%; and sCIM 98.9%, 85.0%, 76.5%. All methods showed 100% specificity in Enterobacterales, Pseudomonas spp., and Acinetobacter spp. Each method performed well in the detection of metallo ß-lactamase-producing strains, however, the sensitivity tended to be low in the detection of the organisms producing serine-type carbapenemase, such as GES, OXA-23, and OXA-51. CONCLUSIONS: Care must be taken when selecting test methods because the sensitivity of the detection differs depending on the bacterial species and genotype.

Blood-Modified Carbapenem Inactivation Method: a Phenotypic Method for Detecting Carbapenemase-Producing Enterobacteriaceae Directly from Positive Blood Culture Broths.

A variant of the modified carbapenem inactivation method (mCIM) was developed to detect carbapenemase activity directly from positive blood culture broths. The method, termed "Blood-mCIM," was evaluated using Bactec blood culture bottles (Becton, Dickinson and Company, Franklin Lakes, NJ) inoculated with 27 different carbapenemase-producing Enterobacteriaceae (CPE) isolates and 34 different non-CPE isolates. The assay was positive for all blood culture broths inoculated with CPE isolates and negative for all blood culture broths inoculated with non-CPE isolates, corresponding to a diagnostic sensitivity and specificity of 100%, respectively. This assay is inexpensive using "off the shelf" reagents, does not require centrifugation or mechanical lysis, and can be readily implemented in any clinical microbiology laboratory. The Blood-mCIM should facilitate expedient administration of antimicrobial therapy targeted toward CPE bloodstream infections and assist infection control and public health surveillance.

Differences between meropenem and imipenem disk to detect carbapenemase in gram-negative bacilli using simplified carbapenem inactivation method.

The spread of carbapenemase-producing Enterobacteriaceae (CPE) is a major threat to public health. In the present study, we compared the difference between meropenem and imipenem disk for detecting carbapenemase-producing gram-negative bacilli using simplified carbapenem inactivation method (sCIM). 106 Enterobacteriaceae, including 74 CPE, 17 Pseudomonas aeruginosa including 10 carbapenemase-producing isolates and 36 Acinetobacter baumannii including 20 carbapenem-resistant isolates preserved in our laboratory were tested. Based on sCIM method, the test bacteria were tested with both meropenem and imipenem disk, respectively. In Enterobacteriaceae, the usage of both meropenem and imipenem disk showed high concordance (99.1%). Meropenem disk cannot identify positive isolates among the 10 P. aeruginosa and 20 A. baumannii isolates due to low carbapenem hydrolytic ability of the carbapenemase produced by these strains. Thus, meropenem disk was found to be similar to imipenem disk, presenting high specificity and sensitivity in the detection of carbapenemase in Enterobacteriaceae, but it cannot be used for the detection of carbapenemase in P. aeruginosa and A. baumannii.

EDTA-Modified Carbapenem Inactivation Method: a Phenotypic Method for Detecting Metallo-ß-Lactamase-Producing Enterobacteriaceae.

The increase in the prevalence and impact of infections caused by carbapenemase-producing Enterobacteriaceae is a global health concern. Therefore, rapid and accurate methods to detect these organisms in any clinical microbiology laboratory, including those in resource-limited settings, are essential to prevent and contain their spread. It is also important to differentiate between serine- and metal-dependent carbapenemases elaborated by carbapenemase-producing isolates for epidemiologic, infection control and prevention, and therapeutic purposes. Here, we describe the development and evaluation of the EDTA-modified carbapenem inactivation method (eCIM), an assay for discriminating between serine- and metal-dependent (i.e., metallo-ß-lactamases [MBLs]) carbapenemases when used in conjunction with the modified carbapenem inactivation method (mCIM). The eCIM had an overall sensitivity and specificity of 100% and was adopted by the Clinical and Laboratory Standards Institute as a method to use in combination with the mCIM to identify MBL-producing Enterobacteriaceae.

Comparative Evaluation of Four Phenotypic Methods for Detection of Class A and B Carbapenemase-Producing Enterobacteriaceae in China.

The objective of this study was to evaluate the performance of four phenotypic methods in the detection of carbapenemase-producing Enterobacteriaceae (CPE) in China. We evaluated the performance of four carbapenemase detection methods, the modified Hodge test (MHT), the Carba NP test, the meropenem hydrolysis assay (MHA) with 1- and 2-h incubation, and the modified carbapenem inactivation method (mCIM) with meropenem, imipenem, and ertapenem, on 342 carbapenem-resistant Enterobacteriaceae isolates (CRE) in China. PCR was used as the gold standard. The 2-h-incubation MHA performed the best in carbapenemase detection (overall sensitivity, specificity, positive predictive value, and negative predictive value all 100%). Second was the Carba NP test, with a sensitivity of 99.6%. The 1-h-incubation MHA performed poorly in Klebsiella pneumoniae carbapenemase (KPC) detection (sensitivity, 71.3%). For mCIM, the best performance was observed with the meropenem disk. The MHT exhibited the worst performance, with a specificity of 88.8%. All assays except 1-h-incubation MHA, which failed to identify 68 KPC-2s, had a sensitivity of >98% in the detection of 172 KPCs. Likewise, all assays had a sensitivity of >95% in the detection of 70 class B carbapenemases, except for MHT (82.9%). The 2-h-incubation MHA significantly improved the accuracy in CPE detection compared with that for 1-h incubation and performed the best in the detection of class A and B carbapenemases. Our findings suggest that the MHA is the most practical assay for carbapenemase detection. For those who cannot afford the associated equipment, both the Carba NP test and mCIM are good alternatives with regard to the practical requirements of time and cost.

Phenotypic Detection of Carbapenemase-Producing Organisms from Clinical Isolates.

The rapid spread of multidrug-resistant Gram-negative organisms constitutes one of the greatest challenges to global health. While Gram-negative organisms have developed several mechanisms to avert the bactericidal effects of commonly prescribed antibiotic agents, the increasing prevalence of carbapenemase-producing organisms (CPO) is particularly concerning given the rapid spread of mobile genetic elements containing carbapenemase genes, the limited treatment options for infections caused by these organisms, and the high mortality rates associated with CPO infections. Understanding if an organism is carbapenemase producing and, if so, the class of carbapenemase(s) produced has treatment implications, as some agents preferentially have activity against specific carbapenemases. Furthermore, CPO disseminate between patients with greater ease than non-CP-carbapenem-resistant organisms and warrant more intensive infection control measures than would be employed in the absence of carbapenemase production. Phenotypic assays currently used in clinical practice to detect CPO consist of the following: (i) growth-based assays which measure carbapenem resistance based on organism growth in the presence of a carbapenem antibiotic (e.g., modified Hodge test and modified carbapenem inactivation method), (ii) hydrolysis methods which detect carbapenem degradation products (e.g., Carba NP test and matrix-assisted laser desorption-ionization time of flight mass spectrometry), and (iii) lateral flow immunoassays which detect carbapenemase enzymes through the use of specific antibodies. Although there is no single phenotypic test that meets all specifications of the ideal test, as we describe in this review, there are a number of tests that are user-friendly, affordable, accurate, and feasible for implementation in clinical microbiology laboratories of all sizes.

Evaluation of the modified carbapenem inactivation method for the detection of carbapenemase-producing Enterobacteriaceae.

Carbapenemase-producing Enterobacteriaceae (CPE) are increasing worldwide. Rapid and accurate detection of CPE is necessary for appropriate antimicrobial treatment and hospital infection control. However, CPE contains some strains that are difficult to detect depending on genotype and MIC value of carbapenem, and a detection method has not been established. The recently reported modified carbapenem inactivation method (mCIM) has been developed in CLSI M100-S27 as a phenotypic technique for detecting carbapenemase activity. In the present study, we examined mCIM as a new CPE detection method using 207 Enterobacteriaceae isolates in comparison with the three existing screening methods of modified Hodge test, Carba NP test and carbapenem inactivation method and evaluated its performance. Consequently, both the sensitivity and specificity of mCIM were 100%, indicating better results than the conventional screening methods. The mCIM is a useful tool for microbiology laboratories due to its simplicity, clear criteria, cost-effectiveness and availability at any laboratory.

Molecular characteristics and evaluation of the phenotypic detection of carbapenemases among Enterobacterales and Pseudomonas via whole genome sequencing.

Background/purposes: The continuously increasing carbapenem resistance within Enterobacterales and Pseudomonas poses a threat to public health, nevertheless, the molecular characteristics of which in southern China still remain limited. And carbapenemase identification is a key factor in effective early therapy of carbapenem-resistant bacteria infections. We aimed to determine the molecular characteristics of these pathogens and compare commercial combined disc tests (CDTs) with the modified carbapenem inactivation method (mCIM) and EDTA-CIM (eCIM) in detecting and distinguishing carbapenemases using whole genome sequencing (WGS). Methods: A total of 78 Enterobacterales, 30 Pseudomonas were obtained from two tertiary hospitals in southern China. Susceptibility tests were conducted using an automated VITEK2 compact system with confirmation via the Kirby-Bauer method. The WGS was conducted on all clinical isolates and the molecular characteristics were analyzed by screening the whole genome sequences. CDTs with or without cloxacillin, mCIM, and eCIM, were performed and compared by taking WGS results as the benchmark. Results: A total of 103 carbapenem non-susceptible and 5 carbapenem susceptible bacteria were determined, with Klebsiella pneumoniae (42.7%), Pseudomonas aeruginosa (23.3%) and Escherichia coli (18.4%) being most prevalent. Carbapenemase genes were detected in 58 (56.3%) of the 103 carbapenem-non-susceptible clinical isolates, including 46 NDM, 6 KPC, 3 IMP, 1 IPM+VIM,1NDM+KPC, and 1 OXA-181. Carbapenemase-producing isolates were detected more frequently in Enterobacterales (76.3%). Among K. pneumoniae, the major sequence types were st307 and st11, while among E. coli and P. aeruginosa, the most prevalent ones were st410 and st242 respectively. For carbapenemase detection in Enterobacterales, the mCIM method achieved 100.00% (95% CI, 92.13-100.00%) sensitivity and 94.44% (70.63-99.71%) specificity (kappa, 0.96); for Pseudomonas, detection sensitivity was 100% (5.46-100.00%), and 100% (84.50-100.00%) specificity (kappa, 0.65). Commercial CDT carbapenemase detection sensitivity for Enterobacterales was 96.49% (86.84-99.39%), and 95.24% (74.13-99.75%) specificity (kappa, 0.90); for Pseudomonas, carbapenemase detection sensitivity was 100.00% (5.46-100.00%) and 37.93% (21.30-57.64%) specificity (kappa, 0.04). When cloxacillin testing was added, CDT specificity reached 84.61% (64.27-94.95%). Conclusion: The molecular epidemiology of carbapenem-non-susceptible isolates from pediatric patients in Southern China exhibited distinctive characteristics. Both the mCIM-eCIM combination and CDT methods effectively detected and differentiated carbapenemases among Enterobacterales isolates, and the former performed better than CDT among Pseudomonas.

Diagnostic Test Precision of Modified Carbapenem Inactivation Method and Carbapenemase Nordmann-Poirel Test for Phenotypic Detection of Carbapenemase Production in Enterobacterales: A Systematic Review.

Carbapenem-resistant Enterobacterales, particularly those that produce carbapenemases, pose a significant public health concern due to very limited treatment options. The timely identification of carbapenemase-producing Enterobacterales (CPE) is essential for putting in place efficient infection control measures and selecting appropriate antimicrobial therapies, thereby improving the clinical outcome of the patient. The purpose of this systematic review is to compare the diagnostic accuracy and practicality between two phenotypic tests, namely the modified carbapenem inactivation method (mCIM) and carbapenemase Nordmann-Poirel (Carba NP) test, in detecting carbapenemase production by Enterobacterales and thereby aiding the clinician in making a decision to choose an appropriate test for their phenotypic detection. This systematic review involved combining sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), accuracy, diagnostic odds ratio with 95% confidence interval (CIs), Forest plot for sensitivity and specificity, and plotting suitable summary receiver operating characteristic curve with the area under the curve. Of the 20 studies included in this review, the overall effect sizes of Carba NP and mCIM with 95% CIs were as follows: sensitivity, 91% (86-96%) and 97% (95-99%); specificity, 93% (88-97%) and 97% (93-100%); PPV, 97% and 98%; NPV, 79% and 90%; accuracy, 93% and 97%; diagnostic odds ratio, 1487.8879 and 8527.5541; and AUC, 0.85 and 1, respectively. In conclusion, the mCIM method showed superior sensitivity (97%), specificity (97%), and accuracy compared to the Carba NP test in detecting carbapenemase production, even though both these methods had a few technical limitations. The Carba NP test is rapid, affordable, and dependable, whereas mCIM is more accurate and cost-effective but time-consuming. We propose that both tests can be reliably used for screening of carbapenemase production in Enterobacterales, as endorsed by the Clinical and Laboratory Standards Institute even in resource-limited clinical laboratories, in the order of prioritizing the mCIM method first and then followed by the Carba NP test when situation demands expedited results.

The optimized carbapenem inactivation method for objective and accurate detection of carbapenemase-producing Acinetobacter baumannii.

The modified carbapenem inactivation method (mCIM) recommended by the Clinical and Laboratory Standards Institute is not applicable for detecting carbapenemases in Acinetobacter baumannii. Four currently reported phenotypic detection methods, namely, the modified Hodge test, the mCIM, the adjusted mCIM, and the simplified carbapenem inactivation method (sCIM), did not perform well in our 90 clinical A. baumannii isolates. Thus, the minimal inhibitory concentrations (MICs) of carbapenems and the existence and expression of carbapenemase-encoding genes were detected to explain the results. According to the E-test, which was more accurate than the VITEK 2 system, 80.0 and 41.1% were resistant to imipenem (IPM) and meropenem (MEM), respectively, and 14.4 and 53.3% exhibited intermediate resistance, respectively. Five ß-lactamase genes were found, of which blaOXA-51-like, blaTEM, and blaOXA-23-like were detected more frequently in 85 non-susceptible strains. The expression of blaOXA-23-like was positively correlated with the MIC values of IPM and MEM. Therefore, an improved approach based on the mCIM, designated the optimized CIM (oCIM), was developed in this study to detect carbapenemases more accurately and reproducibly. The condition was improved by evaluating the factors of A. baumannii inoculum, incubation broth volume, and MEM disk incubation time. Obvious high sensitivity (92.94%) and specificity (100.00%) were obtained using the oCIM, which was cost-effective and reproducible in routine laboratory work.

Comparison of the Performance of Phenotypic Methods for the Detection of Carbapenem-Resistant Enterobacteriaceae (CRE) in Clinical Practice.

In order to investigate the diagnostic performance characteristics of four phenotypic assays in detecting carbapenem-resistant Enterobacteriaceae (CRE), we collected the CRE strains from infected patients. The results of carbapenemase gene detection, blaKPC-2, blaOXA-23, blaNDM-1, blaNDM-4, blaNDM-5, blaIMP-4, and blaIMP-8, were used as a standard to evaluate the performances of combined disk test (CDT), modified carbapenem inactivation method(mCIM)/EDTA-modified carbapenem inactivation method(eCIM), NG-Test CARBA 5 (CARBA), and color developing immunoassay (CDI). The compliance of phenotype results based on CDT, mCIM/eCIM, CARBA, and CDI with genetic detection results was 94% (231/247), 95% (235/247), 98% (242/247), and 99% (246/247), respectively. CDT demonstrated a low specificity for carbapenemase detection, low negative predictive value (NPV), and low sensitivity for metallo-ß-lactamase (79%, 55%, and 88%, respectively); it also failed to accurately detect IMP. The mCIM/eCIM assay had serious problems in detecting OXA-23-like carbapenemases. The sensitivity and specificity of CARBA and CDI were higher than those of the first two methods. However, CARBA did not cover the detection of OXA-23, while CDI cannot detect IMP-8, resulting in low NPVs (70% and 88%, respectively). In conclusion, CARBA and CDI assays are highly accurate except individual rare genes and allow direct genotype detections. CDT and mCIM/eCIM assays are moderately accurate and can only distinguish serine-ß-lactamases from metallo-ß-lactamases. Laboratories should choose the appropriate method that meets their needs based on its characteristic.

The prediction values of carbapenemase detection methods and carbapenem susceptibility testing for clinical outcomes of patients with Acinetobacter bacteremia under carbapenem treatment.

BACKGROUND: Carbapenem-resistant Acinetobacter species have emerged as notorious pathogens causing nosocomial infections. Several phenotypic methods have been developed for detecting carbapenemase production in Enterobacteriaceae. The accuracy of these methods in the prediction of carbapenemase production in Acinetobacter species has not been studied well. METHODS: This retrospective study enrolled adult patients with Acinetobacter bacteremia from four medical centers in Taiwan between 2012 and 2016. Their demographics and clinical outcomes were recorded. The carbapenem susceptibility of the Acinetobacter species was determined using the agar diffusion method. The carbapenemase genes were detected by PCR. Four phenotypic methods, including the modified Hodge test (MHT), modified carbapenem inactivation method (mCIM), Carba NP test, and CarbAcineto NP test were carried out to determine the production of carbapenemase. RESULTS: We analyzed 257 adults who received initial carbapenem monotherapy for the treatment of Acinetobacter bacteremia. Shock within three days of bacteremia and acquisition of carbapenem non-susceptible isolates were independently associated with a higher 14-day and 30-day mortality in patients with Acinetobacter bacteremia. Among the four phenotypic tests for carbapenemase detection, MHT using the imipenem disc displayed the greatest sensitivity (94%; 95% confidence interval [CI], 89-97%) and specificity (81%; 95% CI, 73-88%) for predicting imipenem non-susceptibility. CONCLUSION: Carbapenem non-susceptibility and shock were independent risk factors for mortality in patients with Acinetobacter bacteremia. The MHT could predict the carbapenem susceptibility of Acinetobacter isolates. It is a cheap and quick assay, which could be applied in clinical practice.

mCIM test as a reliable assay for the detection of CRE in the Gulf region.

Introduction. Carbapenem resistant Enterobacterales (CRE) are one of the leading causes of systemic and nosocomial infections and are multidrug-resistant organisms producing different carbapenemases. There are many genotypic and phenotypic methods for detecting the carbapenemases; however, there is a limitation for each. Modified carbapenem inactivation method (mCIM) assay is a recent phenotypic method which has been published by the Clinical and Laboratory Standards Institute.Hypothesis / Gap Statement. mCIM assay could provide a reliable method for the detection of carbapenemases in CRE.Aim. Evaluation of the mCIM assay performance for the detection of carbapenemases in Enterobacterales and the identification of the common carbapenemase genes at Eastern Province of Saudi Arabia and Kingdom of Bahrain.Methodology. A collection of 197 non-duplicate carbapenem resistant Enterobacterales clinical isolates, were evaluated with the mCIM test comparing its performance to multiplex PCR. The minimum inhibitory concentration susceptibility testing was done by the Etest method for imipenem, meropenem, and ertapenem.Results. The sensitivity of the mCIM assay was 94 % (95 % CI, (89.3-97.1)). In Saudi Arabia and Bahrain, OXA-48 was the most prevalent carbapenemase gene followed by NDM. Coexistence of multiple carbapenemase genes is reported in eleven cases.Conclusion. These findings indicate that the mCIM test is a reliable and simple assay for detecting the activity of carbapenemase in Enterobacterales, especially in resource-limited laboratories.

Application of Modified Carbapenem Inactivation Method and Its Derivative Tests for the Detection of Carbapenemase-Producing Aeromonas.

PURPOSE: Infection and transmission of carbapenem-resistant Aeromonas is a serious threat to public health. Rapid and accurate detection carbapenem-resistant of these organisms is essential for reasonable treatment and infection control. This study aimed to find a simple and effective method to detect carbapenem-resistant phenotype in Aeromonas. METHODS: A total of 131 clinical preserved Aeromonas strains were used in this study. The carbapenemase genes were detected by PCR. Modified carbapenem inactivation method (mCIM) in conjunction with EDTA-modified carbapenem inactivation method (eCIM) and simplified carbapenem inactivation method (sCIM) were performed to detect carbapenemases. We also designed a simple method, carbapenem inactivation method using supernatant (CIM-s), to detect the carbapenemase activity in the medium. RESULTS: Of the 131 Aeromonas strains, 79 contained carbapenemase genes, including 68 blaCphA , 6 blaKPC-2 , 2 blaNDM-1 and 3 blaKPC-2+CphA . However, routine antibiotic susceptibility testing could not completely identify carbapenemase-producing Aeromonas. In phenotypic assays, the sensitivity and specificity of mCIM were 100%. The combined mCIM and eCIM could distinguish serine carbapenemase and metallo-ß-carbapenemases except co-producing organisms. The sensitivity and specificity of sCIM were 92.4% and 100%, respectively, which could not detect CphA totally. CIM-s results indicate that these carbapenemases could secrete into the medium to perform their hydrolytic activities and had a sensitivity and specificity of 97.5% and 100%, respectively. CONCLUSION: The combination of mCIM and eCIM can effectively detect and distinguish different types of carbapenemase in Aeromonas, and could be used as an important supplement approach to the antibiotic susceptibility testing.

Evaluation of the simplified carbapenem inactivation method as a phenotypic detection method for carbapenemase-producing Enterobacterales.

Carbapenemase-producing Enterobacterales (CPE) have become a global health concern. Current molecular detection methods require special equipment and reagents. Thus, there is an urgent need for a highly sensitive, specific, and simple method for phenotypic detection of CPE in clinical microbiology laboratories. A simplified carbapenem inactivation method (sCIM) was recently reported. However, its utility for CPE detection has not been sufficiently evaluated to date. We evaluated the sCIM and compared it with the modified CIM (mCIM), using 133 CPE strains (producing IMP, 92; NDM, 11; NDM and OXA-48-like, 1; KPC, 13; OXA-48-like, 12; GES-24, 3; Nmc-A, 1) and 82 non-CPE strains (extended spectrum ß-lactamase, 61; AmpC, 21). The sCIM was conducted by loading bacteria onto imipenem and meropenem disks. When imipenem disks with a 1+ bacterial load were used, the sensitivity and specificity of the sCIM were 97.0% and 100%, and those of the mCIM were 97.0% and 96.3%, respectively. The specificity of the sCIM decreased to 57.3% when the bacterial load on imipenem disks was increased to 2+. In contrast, when meropenem disks with a 1+ bacterial load were used, the sCIM had a lower sensitivity (78.2%) and an equivalent specificity (100%). When meropenem disks with a bacterial load of 2+ were used, the sensitivity and specificity of the sCIM increased to 96.2% and 93.9%, respectively. The diameter of the inhibition zone on meropenem disks was larger than that on imipenem disks, and the sCIM was less sensitive when meropenem disks were used. In addition, sCIM detection rates when using meropenem disks were particularly low for OXA-48-like producers (bacterial load 1+, 0/12; bacterial load 2+, 10/12). Our results indicate that the sensitivity and specificity of the sCIM was dependent on the bacterial load and that large bacterial loads led to false positives for AmpC and extended spectrum ß-lactamase producers. Thus, the sCIM has high sensitivity and specificity for appropriate bacterial loads when imipenem disks are used.

Carbapenemase detection by NG-Test CARBA 5-a rapid immunochromatographic assay in carbapenem-resistant Enterobacterales diagnosis.

BACKGROUND: The global spread of carbapenem-resistant Enterobacterales (CRE) represents a serious public health concern as these organisms are associated with limited treatment options, high mortality rate and rapid transmissibility. The identification of carbapenemase remains a challenge in microbiological laboratories as no single method is perfect when considering cost, carbapenemase coverage, accuracy, handling complexity and TATs together. METHODS: NG-Test CARBA 5 assay and modified carbapenem inactivation method in conjunction with EDTA carbapenem inactivation method (mCIM/eCIM) were challenged with a collection of 299 molecularly characterized CRE isolates in China in order to evaluate the performance in detecting five major carbapenemases (bla KPC, bla NDM, bla VIM, bla IMP, and bla OXA-48) among Enterobacterales. RESULTS: NG-Test CARBA 5 detected all KPC-, NDM-, VIM- and OXA-48-producing isolates perfectly with a weak false-positive signal for NDM in an IMP-4 producer, which makes the specificity for NDM decreases to 99.6%. The overall specificity/sensitivity were 99.9%/100% for NG-Test CARBA 5. mCIM/eCIM achieved high specificity of 100%/100% and sensitivity of 99.6%/97.4%, with one S. marcescens isolate harboring VIM-2 undetected. CONCLUSIONS: Both NG-Test CARBA 5 and mCIM/eCIM showed excellent results in the tested carbapenemase (bla KPC, bla NDM, bla VIM, bla IMP, and bla OXA-48) detection compared with molecular genotypic test. As every assay has its own limitations, suitable methods should be combined for the establishment of the CRE diagnostic pathways.

Diagnostic Accuracy and Agreement between Four Phenotypic Carbapenemase Detection Tests among Enterobacterales.

INTRODUCTION: Carbapenem-resistant Enterobacterales (CREs) are becoming increasingly popular as a cause of hospital-acquired infections that are difficult to treat and are frequently reported as causes of outbreaks in various hospitals. Conventional culturing techniques take at least 2 days to report a case as carbapenem resistant, and it is therefore important to detect such resistance mechanisms as early as possible. METHODS: This study aimed to compare the diagnostic performance of Carba NP, modified Hodge test (MHT), ethylenediaminetetraacetic acid (EDTA) disk synergy test (DST), and the modified carbapenem inactivation method (mCIM). This study was done at Microbiology Laboratory, Aga Khan University Hospital, Karachi. It was an observational study. Carba NP, MHT, EDTA DST, and the mCIM were performed on consecutive isolates of Enterobacterales. Sensitivity, specificity, and agreement between the four tests were calculated. RESULTS: Of 207 Enterobacterales isolated, 127 were resistant to carbapenems. One hundred and fourteen of these were tested by a polymerase chain reaction, and the sensitivities of the Carba NP, MHT, EDTA DST, and the mCIM were found to be 94.34%, 75.47%, 79.25%, and 98.11%, respectively. CONCLUSIONS: Due to increased rates of carbapenem resistance, there is a need to employ mechanisms in hospitals that can identify such organisms as early as possible, both from clinical and epidemiological standpoints. The Carba NP test is a rapid, cost-effective, and reliable method and mCIM is more accurate but time consuming and both can be safely used for the screening of CREs.

Adjustment of Modified Carbapenem Inactivation Method Conditions for Rapid Detection of Carbapenemase-Producing Acinetobacter baumannii.

BACKGROUND: The existing modified carbapenem inactivation methods (mCIMs) recommended by the CLSI for detecting carbapenemase production have not been applicable for Acinetobacter baumannii. We evaluated the influence of matrices used in mCIMs and CIMTris on the stability of the disks for detecting carbapenemase producers and suggested optimal mCIM conditions for detecting carbapenemase-producing A. baumannii. METHODS: Seventy-three A. baumannii isolates characterized for antimicrobial susceptibility and carbapenemase encoding genes were tested for carbapenemase production using mCIM and CIMTris. The influence of the matrices (Tryptic soy broth [TSB] and Tris-HCl) used in these methods on the stability of the meropenem (MEM) disk was also evaluated. The mCIM conditions were adjusted to enhance screening sensitivity and specificity for detecting carbapenemase-producing A. baumannii. RESULTS: The matrices had an impact on the stability of the MEM disk after the incubation period (two or four hrs). TSB nutrient broth is an appropriate matrix for mCIM compared with Tris-HCl pH 7.6, which leads to the loss of MEM activity in CIMTris. The sensitivity and the specificity of the optimal mCIM were both 100%. CONCLUSIONS: We established optimal mCIM conditions for simple, accurate, and reproducible detection of carbapenemase-producing A. baumannii.

Accuracy and applicability of different phenotypic methods for carbapenemase detection in Enterobacteriaceae: A systematic review and meta-analysis.

OBJECTIVES: Carbapenem-resistant Enterobacteriaceae (CRE) are a major public-health threat. The most important mechanism of carbapenem resistance in CRE is carbapenemase production. Early identification of carbapenemase-producing Enterobacteriaceae (CPE) leads to improved clinical outcomes. This systematic review aimed to assess the accuracy and applicability of the modified Hodge test (MHT), the carbapenemase Nordmann-Poirel (Carba NP) test, the modified carbapenem inactivation method (mCIM) and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) for CPE detection. METHODS: The meta-analysis included pooled sensitivity, specificity, diagnostic odds ratio, and summary receiver operating characteristic (SROC) curve and area under the curve (AUC). RESULTS: A total of 67 studies were included in the analysis. Pooled effect sizes (95% confidence interval) of the MHT, Carba NP, mCIM and MALDI-TOF/MS, respectively, were as follows: sensitivity, 92% (87-95%), 97% (94-98%), 99% (99-100%) and 99% (96-100%); specificity, 93% (86-97%), 100% (99-100%), 99% (96-100%) and 99% (96-100%); diagnostic odds ratio, 98.156 (48.175-199.995), 1277.710 (751.391-2172.692), 3597.352 (1287.575-10000) and 1781.360 (651.827-4868.228); and AUC, 0.97, 1, 1 and 1. CONCLUSION: Carba NP, mCIM and MALDI-TOF/MS all demonstrated high accuracy in CPE detection, whereas the MHT is not recommended owing to some clear drawbacks. We recommend the selection of carbapenemase detection tests in the order of mCIM, Carba NP and MALDI-TOF/MS according to their simplicity, cost, and equipment and skills involved.