Detection Of Carbapenemase-producing Enterobacterales Research Articles

Carba PBP: a novel penicillin-binding protein-based lateral flow assay for rapid phenotypic detection of carbapenemase-producing Enterobacterales.

Rapid phenotypic detection assays, including Carba NP and its variants, are widely applied for clinical diagnosis of carbapenemase-producing Enterobacterales (CPE). However, these tests are based on the acidification of the pH indicator during carbapenem hydrolysis, which limits test sensitivity and speed, especially for the detection of CPE producing low-activity carbapenem (e.g., OXA-48 variants). Herein, we developed a novel rapid and sensitive CPE detection method (Carba PBP) that could measure substrate (meropenem) consumption based on penicillin-binding protein (PBP). Meropenem-specific PBP was used to develop a competitive lateral flow assay (LFA) for meropenem identification. For the detection of carbapenemase activity, meropenem concentration was optimized using a checkerboard assay. The performance of Carba PBP was evaluated and compared with that of Carba NP using a panel of 94 clinical strains characterized by whole-genome sequencing and carbapenem susceptibility test. The limit of detection of PBP-based LFA for meropenem identification was 7 ng mL-1. Using 10 ng mL-1 meropenem as the substrate, Carba PBP and Carba NP could detect 10 ng mL-1 carbapenemase within 25 min and 1,280 ng mL-1 CPE in 2 h, respectively. The sensitivity and specificity were 100% (75/75) and 100% (19/19) for Carba PBP and 85.3% (64/75) and 100% (19/19) for Carba NP, respectively. When compared with Carba NP, Carba PBP showed superior performance in detecting all the tested CPE strains (including OXA-48-like variants) within 25 min and presented two orders of magnitude higher analytical sensitivity, demonstrating potential for clinical diagnosis of CPE. IMPORTANCE This study successfully achieved the goal of carbapenemase activity detection with both high sensitivity and convenience, offering a convenient lateral flow assay for clinical diagnosis of carbapenemase-producing Enterobacterales.

Model-based evaluation of admission screening strategies for the detection and control of carbapenemase-producing Enterobacterales in the English hospital setting

BackgroundGlobally, detections of carbapenemase-producing Enterobacterales (CPE) colonisations and infections are increasing. The spread of these highly resistant bacteria poses a serious threat to public health. However, understanding of CPE transmission and evidence on effectiveness of control measures is severely lacking. This paper provides evidence to inform effective admission screening protocols, which could be important in controlling nosocomial CPE transmission.MethodsCPE transmission within an English hospital setting was simulated with a data-driven individual-based mathematical model. This model was used to evaluate the ability of the 2016 England CPE screening recommendations, and of potential alternative protocols, to identify patients with CPE-colonisation on admission (including those colonised during previous stays or from elsewhere). The model included nosocomial transmission from colonised and infected patients, as well as environmental contamination. Model parameters were estimated using primary data where possible, including estimation of transmission using detailed epidemiological data within a Bayesian framework. Separate models were parameterised to represent hospitals in English areas with low and high CPE risk (based on prevalence).ResultsThe proportion of truly colonised admissions which met the 2016 screening criteria was 43% in low-prevalence and 54% in high-prevalence areas respectively. Selection of CPE carriers for screening was improved in low-prevalence areas by adding readmission as a screening criterion, which doubled how many colonised admissions were selected. A minority of CPE carriers were confirmed as CPE positive during their hospital stay (10 and 14% in low- and high-prevalence areas); switching to a faster screening test pathway with a single-swab test (rather than three swab regimen) increased the overall positive predictive value with negligible reduction in negative predictive value.ConclusionsUsing a novel within-hospital CPE transmission model, this study assesses CPE admission screening protocols, across the range of CPE prevalence observed in England. It identifies protocol changes—adding readmissions to screening criteria and a single-swab test pathway—which could detect similar numbers of CPE carriers (or twice as many in low CPE prevalence areas), but faster, and hence with lower demand on pre-emptive infection-control resources. Study findings can inform interventions to control this emerging threat, although further work is required to understand within-hospital transmission sources.

Use of a chromogenic medium with and without selective enrichment to screen for carbapenemase-producing Enterobacterales (CPE) from canine and feline fecal specimens during an outbreak of NDM-5-producing Escherichia coli.

Carbapenemase-producing Enterobacterales (CPE) are one of the most urgent threats to human healthcare globally. Descriptions of CPE outbreaks in veterinary hospitals suggest the need for screening strategies for CPE from companion animals. Our aim was to optimize a chromogenic agar method with and without selective enrichment to isolate CPE from companion animal feces in an ongoing outbreak of New Delhi metallo-β-lactamse-5 Escherichia coli. A limit of detection (LOD) assay for spiked canine and feline feces was performed for both methods using a carbapenamase-producing E. coli (24213-18); the LOD (1.5 × 103 cfu/g of feces) was equivalent to that reported for human fecal specimens. We screened 1,247 companion animal fecal specimens for carriage of CPE by 1) direct plating to chromogenic agar and 2) plating to chromogenic agar following selective enrichment. Twenty-one specimens were positive for CPE by both direct culture and enrichment culture. No specimens were positive with selective enrichment and negative by direct culture. A selective enrichment step did not result in any increased recovery of CPE from companion animals, which suggests that enrichment broth may not be necessary for outbreak surveillance testing. It is important to continue to validate methods for the detection of CPE in companion animals as outbreaks become more common in veterinary facilities.

Detection of carbapenemase-producing Enterobacterales by means of matrix-assisted laser desorption ionization time-of-flight mass spectrometry with ertapenem susceptibility-testing disks as source of carbapenem substrate.

MALDI-TOF mass spectrometry has become widely used in clinical microbiology and has proved highly accurate for detection of carbapenemases in Gram-negative bacteria. However, the use of carbapenem-hydrolysis assays in routine diagnostics is hampered by the need for antibiotic substances and for making their fresh solutions each time an assay is conducted. Here, we evaluated the use of commercial antibiotic susceptibility-testing disks as source of ertapenem substrate in MALDI-TOF MS-based assay for detection of carbapenemase-producing Enterobacterales (CPE). The assay was validated on 48 CPE isolates of 8 different species expressing NDM-, VIM-, KPC- and OXA-48-type carbapenemases and exhibiting various levels of resistance to carbapenems (MIC range: 0.25- > 32 mg/l), as well as on 48 carbapenemase-non-producing isolates. The assay conditions were optimized as follows: 10-μl loopful of bacterial colonies was suspended in 150 μl 0.01 M Na-PBS buffer, pH 7.4, a 10 μg ertapenem susceptibility-testing disk was immersed in the suspension and incubated 3 h at 35°C, after which supernatant was obtained by centrifugation and applied on a target plate with alpha-cyano-4-hydroxycinnamic acid matrix. Mass spectra were analyzed between 440 and 560 m/z. Carbapenemase activity was detected in all tested CPE isolates by the appearance of m/z peaks corresponding to ertapenem hydrolysis products: [Mh + H]+:494.2, [Mh + Na]+:516.2, [Mh + 2Na]+:538.2, [Mh/d + H]+:450.2, [Mh/d + Na]+:472.2, and simultaneous decrease or loss of peaks of intact antibiotic: [M + H]+:476.2, [M + Na]+:498.1, [M + 2Na]+:520.1. No hydrolysis peaks or loss of intact ertapenem peaks were observed for carbapenemase-negative strains. We therefore report the development of a sensitive, specific and cost-effective MALDI-TOF MS-based assay for detection of CPE, which makes use of antibiotic disks readily available in most laboratories.

Carbapenem-Resistant Gram-Negative Bacilli Causing Ventilator Associated Pneumonia: Study of MASTDISCS Combi Carba Plus for Detection of Carbapenemase Producing Enterobacterales.

Ventilator-associated pneumonia (VAP) caused by carbapenem-resistant gram-negative bacteria has been proven to be an escalating public health challenge in Egypt owing to its high mortality rate and raised health care costs. Detection of carbapenem-resistant gram-negative bacilli among VAP patients, genotypic identification of carbapenemase genes in the isolated strains with evaluation of their impact on patient outcome and detection of carbapenemase-producing enterobacterales by MASTDISCS combi Carba plus disc system. Broncho-alveolar lavage fluid (BALF) and endotracheal aspirate were collected aseptically from clinically suspected VAP patients. Pathogen identification and antibiotic sensitivity testing were done. Carbapenemase-encoding genes (bla KPC, bla NDM, and bla OXA-48) were tested by PCR in all carbapenem-resistant gram-negative isolates. Performance of MASTDISCS combi Carba plus in isolated Enterobacterales was assessed in relation to the PCR results. Eighty-three carbapenem-resistant gram-negative isolates were detected. The most frequent pathogens were Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa representing 34.9%, 20.5% and 18.1%, respectively. bla KPC was the predominant gene. Patients with persistent mechanical ventilation less than 15 days and Pseudomonas aeruginosa infection were significantly associated with a higher death rate. MAST-Carba plus had the highest sensitivity, specificity, positive and negative predictive values for detecting OXA-48 carbapenemases representing 81.8%, 92.5%, 75% and 94.9%, respectively. Worse outcome in VAP patients was associated with carbapenem-resistant gram-negative bacilli. MASTDISCS combi Carba plus is an efficient simple method for identification of different carbapenemases among enterobacterales.

Synergistic effects of length of stay and prior MDRO carriage on the colonization and co-colonization of methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, and carbapenemase-producing Enterobacterales across healthcare settings.

To characterize the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and carbapenemase-producing Enterobacterales (CPE) co-colonization and to compare risk factors between healthcare facility types. We conducted a 3-year cross-sectional study among patients admitted to an acute-care hospital (ACH) and its 6 closely affiliated intermediate- and long-term care facilities (ILTCFs) in Singapore in June and July of 2014-2016. Specimens were concurrently collected from nares, axillae, and groins for MRSA detection, and from rectum or stool for VRE and CPE detection. Co-colonization was defined as having >1 positive culture of MRSA/VRE/CPE. Multinomial logistic regression was performed to determine predictors of co-colonization. Of 5,456 patients recruited, 176 (3.2%) were co-colonized, with higher prevalence among patients in ITCFs (53 of 1,255, 4.2%) and the ACH (120 of 3,044, 3.9%) than LTCFs (3 of 1,157, 0.3%). MRSA/VRE was the most common type of co-colonization (162 of 5,456, 3.0%). Independent risk factors for co-colonization included male sex (odds ratio [OR], 1.96; 95% confidence interval [CI], 1.37-2.80), prior antibiotic therapy of 1-3 days (OR, 10.39; 95% CI, 2.08-51.96), 4-7 days (OR, 4.89; 95% CI, 1.01-23.68), >7 days (OR, 11.72; 95% CI, 2.81-48.85), and having an open wound (OR, 2.34; 95% CI, 1.66-3.29). Additionally, we detected the synergistic interaction of length of stay >14 days and prior multidrug-resistant organism (MDRO) carriage on co-colonization. Having an emergency surgery was a significant predictor of co-colonization in ACH patients, and we detected a dose-response association between duration of antibiotic therapy and co-colonization in ILTCF patients. We observed common and differential risk factors for MDRO co-colonization across healthcare settings. This study has identified at-risk groups that merit intensive interventions, particularly patients with prior MDRO carriage and longer length of stay.

Evaluation of the VITEK2 AST-XN17 card for the detection of carbapenemase-producing Enterobacterales in isolates primarily producing metallo \u03b2-lactamase

Carbapenemase-producing Enterobacterales (CPE) are not always resistant to carbapenem antimicrobial susceptibility testing (AST) and can be difficult to detect. With the newly created VITEK2 AST-XN17 card, the types of antibiotics measured in AST can be increased. In this study, we evaluated the detectability of CPE using the results of AST with multiple antimicrobial agents with additional measurements of the AST-XN17 card. In addition, we evaluated the CPE detectability of comments on CPE using the VITEK2 Advance Expert System (AES). In total, 169 Enterobacterales samples, including 76 non-CPE and 93 CPE, collected from multiple medical institutions in the Kinki region of Japan, were used in this investigation. AST with VITEK2 was performed by adding the AST-XN17 card in addition to the AST-N268 or AST-N404 card. Measurement results were identified using cutoff values, primarily Clinical and Laboratory Standards Institute breakpoints, and the CPE detection capability of each antibiotic was evaluated in several terms, including sensitivity and specificity. The drugs highly sensitive to CPE detection were faropenem (FRPM) > 2 µg/mL at 100% and meropenem > 0.25 µg/mL at 98.9%; the highest specificity to CPE detection was for avibactam/ceftazidime (AVI/CAZ) > 8 µg/mL at 100%. The sensitivity and specificity of each card in the AES output were 86.2% and 94.7% for AST-N404 and AST-XN17 and 91.5% and 90.8% for AST-N268 and AST-XN17, respectively. AST using the VITEK2 AST-XN17 card is a useful test method of screening for CPE.

Comparison of sCIM and Other Phenotypic Detection Methods for Carbapenemase-Producing Enterobacterales.

ABSTRACTRapid detection and reporting of carbapenemase-producing Enterobacterales (CPE) is one of the top priorities of clinical microbiology laboratories. The Clinical and Laboratory Standards Institute recommends the modified carbapenem inactivation method (mCIM) as the preferred method for this purpose, but it requires a broth incubation process which can be cumbersome. Here, we compared the performance of mCIM with three alternative rapid CPE detection methods against a collection of genetically defined CPE, with most carrying blaIMP, and non-CPE clinical isolates. The sensitivities of mCIM, simplified carbapenem inactivation method (sCIM), Rapidec Carba NP, and NG-Test Carba 5 were 98.0%, 54.9%, 90.2%, and 72.5%, whereas the specificities were 89.5%, 84.2%, 89.5%, and 100%, respectively. Modification of the interpretive criteria of sCIM increased its sensitivity to 88.2% and specificity to 89.5%. The results suggest that mCIM is currently the optimal method for CPE detection in an epidemiological setting where CPE-producing IMP group carbapenemase is predominant. While sCIM is easier to perform, it requires further validation before it can be widely adopted as an alternative to mCIM in the clinical laboratory.IMPORTANCE Simple identification methods for carbapenemase-producing Enterobacterales are required for the clinical laboratory. The simplified carbapenem inactivation method (sCIM) is a carbapenemase detection method that can be performed with less hands-on time than mCIM, but its sensitivity and specificity were suboptimal compared with other phenotypic detection methods when tested against a collection of IMP-producing CPE. Insufficient inactivation of imipenem from inadequate inoculation was suspected as the cause. While sCIM is easier to perform, it requires optimization before it can be widely adopted as an alternative to mCIM in the clinical laboratory.

Evaluation of the Performances of the Rapid Test RESIST-5 O.O.K.N.V Used for the Detection of Carbapenemases-Producing Enterobacterales.

Background: The emergence of carbapenemase-producing Enterobacterales (CPE) is a public health problem, requiring rapid and reliable diagnostic methods. The aim is to compare the new rapid immunochromatographic (IC) test: RESIST-5 O.O.K.N.V with PCR and the predictive model of EUCAST algorithm for the detection of CPE. Methods: A longitudinal cross-sectional study was carried out in the bacteriology-virology laboratory of the Ibn Rochd-Casablanca University Hospital, from 1 February 2019 to 28 February 2020, concerning strains with reduced sensitivity to Ertapenem. The identification of bacterial species was carried out according to the standard criteria of microbiology and antibiogram according to CASFM-EUCAST 2019 recommendations. The sensitivity and specificity of the rapid IC test were calculated. Results: The results of the new IC test showed a sensitivity and specificity of 100% for the detection of OXA-48 and NDM. These carbapenemases were detected simultaneously with a sensitivity and specificity of 100%. OXA-48 was the most common carbapenemas found (36%), followed by NDM (24%) and (13.4%) cases of OXA-48 and NDM coexistence. Conclusion: The rapid IC test could be a rapid and effective diagnostic tool for detecting the most common carbapenemases in our context, and to accelerate the implementation of adequate antibiotic therapy and infection control measures in patients with CPE infections

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 β-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 β-lactamase producers. Thus, the sCIM has high sensitivity and specificity for appropriate bacterial loads when imipenem disks are used.

Optimization of the rapid carbapenem inactivation method for use with AmpC hyperproducers.

Detection of carbapenemase-producing Enterobacterales (CPEs) is sometimes difficult with AmpC-hyperproducing Enterobacterales (AHEs), as they may falsely be classified as CPEs. Here, we present a rapid Carbapenem Inactivation Method (rCIM) optimized for AmpC producers (rCIM-A) that allows rapid and easy discrimination between AHEs and CPEs. Enterobacterales (n = 249), including natural AmpC producers, AHEs, CPEs and non-carbapenemase-producing carbapenem-resistant control strains were evaluated, using Carba NP, rCIM and rCIM-A. The rCIM-A differs from the rCIM by the addition of cloxacillin (400 μg/mL) to the initial antibiotic incubation step. The rCIM-A yielded a sensitivity and specificity of 84.26% (95% CI: 76.00%-90.55%) and 99.29% (95% CI: 96.11%-99.98%), respectively, while those of the rCIM were 86.11% (95% CI: 78.13%-92.01%) and 80.85% (95% CI: 73.38%-86.99%), respectively; those of Carba NP were lower at 84.04% (95% CI: 75.05%-90.78%) and 91.37% (95% CI: 85.41%-95.46%), respectively, due to indeterminate results. The rCIM-A was capable of discriminating between AHEs and true CPEs, but still failed to identify OXA-23-producing Proteus mirabilis isolates and remained only partially reliable for identifying IMI-like producers and a few MBL (2 NDM-1, 1 LMB-1, 1 TMB-1 and 1 IMP-13) producers. One chromosomally encoded AmpC variant, MIR-10, gave repeatedly positive results using all three tests and was thus considered a false positive. Specificity for AHEs greatly improved with the rCIM-A without altering the test performance for the other resistance mechanisms. It may replace the rCIM as a cheap, easy, rapid and accurate CPE detection test.

Detection of carbapenemase producing enterobacteria using an ion sensitive field effect transistor sensor

The timely and accurate detection of carbapenemase-producing Enterobacterales (CPE) is imperative to manage this worldwide problem in an effective fashion. Herein we addressed the question of whether the protons produced during imipenem hydrolysis could be detected using an ion sensitive field effect transistor (ISFET). Application of the methodology on enzyme preparations showed that the sensor is able to detect carbapenemases of the NDM, IMP, KPC and NMC-A types at low nanomolar concentrations while VIM and OXA-48 responded at levels above 100 nM. Similar results were obtained when CPE cell suspensions were tested; NDM, IMP, NMC-A and KPC producers caused fast reductions of the output potential. Reduction rates with VIM-type and especially OXA-48 producing strains were significantly lower. Based on results with selected CPEs and carbapenemase-negative enterobacteria, a threshold of 10 mV drop at 30 min was set. Applying this threshold, the method exhibited 100% sensitivity for NDM, IMP and KPC and 77.3% for VIM producers. The OXA-48-positive strains failed to pass the detection threshold. A wide variety of carbapenemase-negative control strains were all classified as negative (100% specificity). In conclusion, an ISFET-based approach may have the potential to be routinely used for non OXA-48-like CPE detection in the clinical laboratory.

Genomic Epidemiology of Carbapenemase-Producing Enterobacterales at a Hospital System in Toronto, Ontario, Canada, 2007 to 2018.

At a hospital system (H1) in Ontario, Canada, we investigated whether whole-genome sequencing (WGS) altered initial epidemiological interpretation of carbapenemase-producing Enterobacterales (CPE) transmission. We included patients with CPE colonization/infection identified by population-based surveillance from October 2007 to August 2018 who received health care at H1 in the year before/after CPE detection. H1 reported epidemiological transmission clusters. We combined single nucleotide variant (SNV) analysis, plasmid characterization, and epidemiological data. Eighty-five patients were included. H1 identified 7 epidemiological transmission clusters, namely, A to G, involving 24/85 (28%) patients. SNV analysis confirmed transmission clusters C, D, and G and identified two additional cases belonging to cluster A. One was a travel-related case that was the likely index case (0 to 6 SNVs from other isolates); this case stayed on the same unit as the initially presumed index case 4 months prior to detection of the initially presumed index case on another unit. The second additional case occupied a room previously occupied by 5 cluster A cases. Plasmid sequence analysis excluded a case from cluster A and identified clusters E and F as possibly two parts of a single cluster. SNV analysis also identified a case without direct epidemiologic links that was 18 to 21 SNVs away from cluster B, suggesting possible undetected interhospital transmission. SNV and plasmid sequence analysis identified cases belonging to transmission clusters that conventional epidemiology missed and excluded other cases. Implementation of routine WGS to complement epidemiological transmission investigations has the potential to improve prevention and control of CPE in hospitals.

The effect of intestinal microbiota dysbiosis on growth and detection of carbapenemase-producing Enterobacterales within an in vitro gut model

Carbapenemase-producing Enterobacterales (CPE) can colonize the gut and are of major clinical concern. Identification of CPE colonization is problematic; there is no gold-standard detection method, and the effects of antibiotic exposure and microbiota dysbiosis on detection are unknown. Based on a national survey we selected four CPE screening assays in common use. We used a clinically reflective invitro model of human gut microbiota to investigate the performance of each test to detect three different CPE strains under different, clinically relevant antibiotic exposures. Twelve gut models were seeded with a pooled faecal slurry and exposed to CPE either before, after, concomitant with, or in the absence of piperacillin-tazobactam (358 mg/L, 3× daily, seven days). Total Enterobacterales and CPE populations were enumerated daily. Regular screening for CPE was performed using Cepheid Xpert® Carba-R molecular test, and with Brilliance™ CRE, Colorex™ mSuperCARBA and CHROMID® CARBA SMART agars. Detection of CPE when the microbiota are intact is problematic. Antibiotic exposure disrupts microbiota populations and allows CPE proliferation, increasing detection. The performances of assays varied, particularly with respect to different CPE strains. The Cepheid assay performed better than the three agar methods for detecting a low level of CPE within an intact microbiota, although performance of all screening methods was comparable when CPE populations increased in a disrupted microbiota. CPE strains differed in their dynamics of colonization in an invitro gut model and in their subsequent response to antibiotic exposure. This affected detection by molecular and screening methods, which has implications for the sensitivity of CPE screening in healthcare settings.

Evaluation of Disk carbapenemase test using improved disks for rapid detection and differentiation of clinical isolates of carbapenemase-producing Enterobacterales

ObjectivesRapid detection of carbapenemase-producing Enterobacterales (CPE) is important to control spread of the resistance. We previously reported that imipenem disks prepared from injectable imipenem-cilastatin could rapidly detect KPC- and NDM-type carbapenemases. In the present study, we evaluated performance of disks of IPM and combined disks of imipenem-tazobactam and imipenem-EDTA, which were prepared from powders of imipenem and inhibitors. MethodsIsolates of Enterobacterales were recovered from specimens of patients at a tertiary care hospital in Korea during January 2017 and March 2018. Routine CPE detection was performed by the CPE surveillance personnel whereas evaluation of the Disk carbapenemase test (DCT) was performed by the other personnel without knowing the results of surveillance. The DCT was carried out by pressing disks on to colonies and rehydrating in Petri plates and observing color change. ResultsThe DCT differentiated 688 of 694 (sensitivity 99.1%) carbapenemase-producing isolates in 2.5–20 min: 630 with KPC, 51 with NDM, three with IMP, one with VIM, two with KPC and IMP, and one with NDM and OXA-181. The DCT failed to detect six OXA- 48-like enzyme-producing isolates, but the modified method using 96-well flat-bottom microplates with mineral oil cover detected all 29 OXA-48-like enzyme-producing isolates in 20–120 min. The DCT was negative for all 440 ertapenem-nonsusceptible, carbapenemase gene-negative isolates (specificity 100%). ConclusionThe procedure of DCT is simple and can differentiate isolates of Enterobacterales with KPC-, NDM-, IMP- and VIM-type carbapenemases rapidly, and the modified DCT can detect isolates with OXA-48-like enzymes rapidly.

Comparison of Four Phenotypic Assays and Check-Direct CPE for Detection of Carbapenemases-Producing Enterobacterales.

Early and accurate detection of carbapenemase-producing Enterobacterales (CPE) is fundamental to prevent their spread in hospital environment. Our objective was to compare between four commonly used phenotypic assays and Check-Direct CPE (CDCPE) multiplex PCR in CPE detection. We examined stool samples or rectal swabs for CPE, samples collected from 23 Jan 2017 to 23 Jul 2017 from patients in intensive-care units (ICUs) of our hospital. A panel of 98 non-repetitive Enterobacterales isolates with reduced susceptibility to carbapenems were analyzed by means of (i) Modified Hodge Test (MHT), (ii) Blue Carba test (BCT), (iii) Combined Disc Test (CDT), and (iv) The Carbapenem Inactivation Method (CIM). All these phenotypic tests compared with CDCPE. Confirmation and validation of results was achieved by classical PCR and sequencing. Of the 98 non-repetitive Enterobacterales isolates, ninety-one were K. pneumoniae (93%), three K. oxytoca (3%), three E. cloacae (3%) and one E. coli (1%). By classic PCR the carbapenem resistance genes in K. pneumoniae isolates distributed as the followings; 49 blaOXA-48, 34 both blaOXA-48 and blaNDM-1, seven blaNDM-1 and one blaKPC. K. oxytoca; two blaOXA-48, one blaNDM-1. E. cloacae; two blaOXA-48, one blaNDM-1. E. coli; one isolate with both blaOXA-48 and blaNDM-1. The most common carbapenemase gene detected was blaOXA-48 rate of 54% (n = 53) followed by a combination of both blaOXA-48 and blaNDM-1 with rate of 36% (n = 35), only blaNDM-1 9% (n = 9) and blaKPC 1% (n = 1). Among phenotypic tests, we found CIM, MHT, and BCT correctly identified carbapenemase producers with sensitivity of 100%, 98%, and 90.8%, respectively. Rapid and accurate detection of CRE can be achieved by combination of both phenotypic and molecular tests. Surveillance studies are important both in terms of epidemiology and regulation of the treatment of patients.

Evaluation of CARBA PAcE, a novel rapid test for detection of carbapenemase-producing Enterobacterales.

Introduction Carbapenemase-producing Enterobacterales (CPE) are an increasing threat to global health. Fast detection is crucial for patient management and outbreak control.Hypothesis/Gap statementRecently, a new commercial colorimetric test, CARBA PAcE, was released that has not yet been scientifically evaluated.Aim Our goals were to evaluate the performance of CARBA PAcE using a large variety of different CPE.Methodology CARBA PAcE was challenged with 107 molecularly characterized CPE and 53 non-CPE controls. Isolates were grown on Mueller-Hinton agar (MHA); in the case of a false-negative result, isolates were additionally inoculated on Columbia blood agar (CBA) and CARBA PAcE was repeated. The test was performed according to the manufacturer’s protocol.Results CARBA PAcE showed an overall sensitivity and specificity of 72 % [confidence interval (CI) 62–80 %] and 91 % (CI 79–97 %), respectively, when isolates were grown on MHA. With growth on CBA, detection improved (especially of metallo-β-lactamases), resulting in an extrapolated sensitivity of 89 % (CI 81–94 %) for all carbapenemases and 96 % (CI 89–99 %) for the four major carbapenemases (NDM, OXA-48-like, KPC, VIM).Conclusion CARBA PAcE is a simple and very rapid test for the detection of CPE which performs well for the major carbapenemases when isolates are grown on CBA. Laboratories should be aware of the limitations of this assay, such as moderate sensitivity when isolates are grown on more challenging agars such as MHA and the poor detection of some rare carbapenemases (e.g. IMI, OXA-58).

Genomic Epidemiology of Carbapenemase-Producing Enterobacterales (CPE) in Toronto, Canada

Objective: We investigated whether whole-genome sequencing (WGS) data altered interpretations of clonal transmission as determined by conventional epidemiology and pulsed-field gel electrophoresis (PFGE) at a tertiary-care hospital (hospital Z, HZ). Methods: We included all carbapenemase-producing Enterobacterales (CPE)–colonized or –infected patients identified via population-based surveillance from 2007 through 2018, who were admitted to HZ during and/or in the year prior to or following CPE detection. HZ reported clonal transmission clusters using epidemiology and PFGE for CPE identified at HZ or reported to HZ by other hospitals as potentially acquired at HZ. We assessed single-nucleotide polymorphism (SNP) phylogenies and case epidemiology. Results: Overall, 85 CPE-colonized or -infected patients were included: 50 were detected at HZ and 35 were detected at another local hospital but were admitted to HZ in the previous or following year. HZ reported 6 transmission clusters (Table 1). SNP analyses confirmed clusters B, C, E, and F. In cluster A, SNP analyses cast doubt on 2 of 9 cases (possibly representing plasmid transmission) but also identified 2 additional cases with isolates highly related (0–3 SNP differences) to other isolates. One case may be the index case: a travel-related case who stayed on the same unit as case 1, 4 months before case 1 detection. The second case stayed in a room previously occupied by 5 cluster A cases. In cluster D, SNP analyses found 1 additional case whose isolate was highly related (ie, 17–19 SNP differences) to other isolates. This case was identified a year before cluster D at another hospital that shares patients with HZ; however, the case’s admission to HZ was after all cluster D cases were detected and no direct epidemiologic link was identified. Conclusions: WGS data can identify cases belonging to transmission clusters that conventional epidemiologic methods missed.Funding: NoneDisclosures: Allison McGeer reports funds to her institution from Pfizer and Merck for projects for which she is the principal investigator. She also reports consulting fees from Sanofi-Pasteur, Sunovion, GSK, Pfizer, and Cidara

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

IntroductionThe 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. MethodsOne 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). ResultsCIM, 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.

Detecting carbapenemase-producing Enterobacterales (CPE): an evaluation of an enhanced CPE infection control and screening programme in acute care.

The transmission of carbapenemase-producing Enterobacterales (CPE) poses an increasing healthcare challenge. A range of infection prevention activities, including screening and contact precautions, are recommended by international and national guidelines. We evaluated the introduction of an enhanced screening programme in a multisite London hospital group. In June 2015, an enhanced CPE policy was launched in response to a local rise in CPE detection. This increased infection prevention measures beyond the national recommendations, with enhanced admission screening, contact tracing and environmental disinfection, improved laboratory protocols and staff/patient education. We report the CPE incidence and trends of CPE in screening and clinical cultures and the adoption of enhanced CPE screening. All non-duplicate CPE isolates identified between April 2014 and March 2018 were included. The number of CPE screens increased progressively, from 4530 in July 2015 to 10 589 in March 2018. CPE detection increased from 18 patients in July 2015 (1.0 per 1000 admissions) to 50 patients in March 2018 (2.7 per 1000 admissions). The proportion of CPE-positive screening cultures remained at approximately 0.4% throughout, suggesting that whilst the CPE carriage rate was unchanged, carrier identification increased. Also, 123 patients were identified through positive CPE clinical cultures over the study period; there was no significant change in the rate of CPE from clinical cultures per 1000 admissions (P = 0.07). Our findings suggest that whilst the enhanced screening programme identified a previously undetected reservoir of CPE colonization in our patient population, the rate of detection of CPE in clinical cultures did not increase.