Very Major Errors Research Articles

Reporting antimicrobial susceptibilities and phenotypes of resistance to vancomycin in vancomycin-resistant Enterococcus spp. clinical isolates: A nationwide proficiency study

IntroductionThe ability of Spanish microbiology laboratories to (a) determine antimicrobial susceptibility (AS), and (b) correctly detect the vancomycin resistance (VR) phenotype in vancomycin-resistant Enterococcus spp. (VRE) was evaluated. MethodsThree VRE isolates representing the VanA (E. faecium), VanB (E. faecium) and VanC (E. gallinarum) VR phenotypes were sent to 52 laboratories, which were asked for: (a) AS method used; (b) MICs of ampicillin, imipenem, vancomycin, teicoplanin, linezolid, daptomycin, ciprofloxacin, levofloxacin and quinupristin–dalfopristin, and high-level resistance to gentamicin and streptomycin; (c) VR phenotype. Results(a) The most frequently used system was MicroScan; (b) according to the system, the highest percentage of discrepant MICs was found with gradient strips (21.3%). By antimicrobial, the highest rates of discrepant MICs ranged 16.7% (imipenem) to 0.7% (linezolid). No discrepant MICs were obtained with daptomycin or levofloxacin. Mayor errors (MEs) occurred with linezolid (1.1%/EUCAST) and ciprofloxacin (5.0%/CLSI), and very major errors (VMEs) with vancomycin (27.1%/EUCAST and 33.3%/CLSI) and teicoplanin (5.7%/EUCAST and 2.3%/CLSI). For linezolid, ciprofloxacin, and vancomycin, discrepant MICs were responsible for these errors, while for teicoplanin, errors were due to a misassignment of the clinical category. An unacceptable high percentage of VMEs was obtained using gradient strips (14.8%), especially with vancomycin, teicoplanin and daptomycin; (c) 86.4% of the centers identified VanA and VanB phenotypes correctly, and 95.0% the VanC phenotype. ConclusionMost Spanish microbiology laboratories can reliably determine AS in VRE, but there is a significant percentage of inadequate interpretations (warning of false susceptibility) for teicoplanin in isolates with the VanB phenotype.

Early Identification and Antimicrobial Susceptibility Testing of Short versus Standard Incubated Blood Cultures from a Tertiary Care Centre in Southern India

Introduction: Blood cultures play an important role in the early diagnosis of sepsis and its management. Early detection of pathogens in Blood Stream Infections (BSI) and their Antimicrobial Susceptibility Testing (AST) pattern, plays a vital role in the diagnosis of sepsis and is important for guidance of appropriate therapy. Aim: To evaluate the accuracy of shortly incubated blood cultures in comparison with standard method for an early Identification (ID) and AST. Materials and Methods: This was a prospective observational study undertaken from July 2015-June 2016 at Nizam’s Institute of Medical Sciences, Hyderabad, Telangana, India. The blood cultures were loaded in the BacT/Alert system. A total of 92 patients with two sets of blood cultures that flagged positive within 24 hours of collection were included in the study. Grams stain and subcultures of the broths were done. The culture plates were examined after four hours and then at hourly intervals for the presence of growth. Once the growth was sufficient it was processed immediately for ID and AST by Vitek 2C. Incubation of the plates was continued for the rest of the 24 hours at 37oC and was processed again. The mean time for detection were compared between short and standard cultures. Results: Gram negative pathogens were the predominant organisms isolated in 82/92 (89%) followed by Gram positive in 10/92 (10%). The short and standard cultures had comparable results with respect to ID of the isolates. But, the AST results were comparable only in 88/92 (95.6%) patients. Of the remaining four patients, the AST showed Very Major Error (VME) in 3 (3.3%) patients and Major Error (ME) in 1 (1.08%) patient. Conclusion: Short incubation of cultures enabled earliest ID and AST of the isolates from blood cultures than standard incubation

Multicenter Clinical Evaluation of ETEST Plazomicin (PLZ) for Susceptibility Testing of Enterobacterales.

ABSTRACTPlazomicin (PLZ), brand name ZEMDRI (Cipla Therapeutics), is a novel aminoglycoside antibiotic approved by the U.S. Food and Drug Administration (FDA) for treatment of complicated urinary tract infections including pyelonephritis. ETEST® is a gradient diffusion method that represents an alternative to the more laborious broth micro-dilution (BMD) method for performing antimicrobial susceptibility testing (AST). A multi-center evaluation of the performance of the new ETEST PLZ (bioMérieux) was conducted in comparison with BMD following FDA and International Standards Organization (ISO) recommendations using FDA-defined breakpoints. Clinical isolates of Enterobacterales (n = 598) were included. Fifty-three isolates were resistant to PLZ according to BMD. Overall, the ETEST PLZ demonstrated 99.0% essential agreement (EA), 92.8% category agreement (CA), 1.9% very major errors (VME), 0% major errors (ME), and 7.0% minor errors (mE) with both clinical and challenge isolates of Enterobacterales. The VME was found for a single Serratia marcescens strain. Individual species demonstrated EA rates ≥ 90%. In conclusion, we report that ETEST PLZ represents an accurate tool for performing PLZ AST of Enterobacterales.

Evaluation of MicroScan WalkAway for Determination of Ceftazidime-Avibactam and Ceftolozane-Tazobactam Susceptibility in Carbapenem-Resistant Gram-Negative Bacilli.

We evaluated the performance of ceftazidime-avibactam and ceftolozane-tazobactam MicroScan Neg multidrug-resistant MIC 1 (NMR1) panel for clinical carbapenem-nonsusceptible Gram-negative bacilli isolates. We evaluated 212 clinically significant carbapenem-nonsusceptible Gram-negative bacilli (139 Pseudomonas aeruginosa and 73 KPC-producing Enterobacterales) from 71 Brazilian hospitals (2013 to 2020). Ceftazidime-avibactam and ceftolozane-tazobactam MICs from the panel were compared with a broth microdilution (BMD) test as the reference method. Essential agreement (EA) and categorical agreement (CA) were assessed. For P. aeruginosa, antimicrobial susceptibility testing error rates were calculated using the error-rate bound method. Discrepancies were initially observed with 11 isolates; 4 resolved after retesting, 2 in favor of the NMR1 and 2 in favor of the BMD method. The ceftazidime-avibactam EA (overall and evaluable) was 100% for P. aeruginosa and Enterobacterales. The CA was 100% for Enterobacterales and 98.6% for P. aeruginosa. The ceftolozane-tazobactam EA was 98.6% and 100% (overall and evaluable, respectively), and the CA was 96.4% for P. aeruginosa. For ceftazidime/avibactam, no very major error (VME) was found, and the major error (ME) rate was 4.2% (2/48). For ceftolozane-tazobactam and P. aeruginosa, using the CLSI breakpoints, the minor error (mE) was 11.4%, and no VME or ME was found. While using EUCAST breakpoints, the VME was 11.4% with no ME. The mE becomes ME or VME in the absence of the intermediate category. All categorical errors were also within 1 log of MIC variation, and the adjusted error rate for CLSI/EUCAST was 0% (0/212). The NMR1 panel is an option to test ceftazidime-avibactam for KPC-producing Enterobacterales and carbapenem-nonsusceptible P. aeruginosa. When a MIC of 4 mg/liter for ceftolozane-tazobactam is obtained using this method, an alert could be created, and the results could be confirmed by an alternative method.

Performance Evaluation of the Gradient Diffusion Strip Method and Disk Diffusion Method for Ceftazidime–Avibactam Against Enterobacterales and Pseudomonas aeruginosa: A Dual-Center Study

Objectives: Ceftazidime–avibactam is a novel synthetic beta-lactam + beta-lactamase inhibitor combination. We evaluated the performance of the gradient diffusion strip method and the disk diffusion method for the determination of ceftazidime–avibactam against Enterobacterales and Pseudomonas aeruginosa.Methods: Antimicrobial susceptibility testing of 302 clinical Enterobacterales and Pseudomonas aeruginosa isolates from two centers were conducted by broth microdilution (BMD), gradient diffusion strip method, and disk diffusion method for ceftazidime–avibactam. Using BMD as a gold standard, essential agreement (EA), categorical agreement (CA), major error (ME), and very major error (VME) were determined according to CLSI guidelines. CA and EA rate > 90%, ME rate < 3%, and VME rate < 1.5% were considered as acceptable criteria. Polymerase chain reaction and Sanger sequencing were performed to determine the carbapenem resistance genes of all 302 isolates.Results: A total of 302 strains were enrolled, among which 182 strains were from center 1 and 120 strains were from center 2. A percentage of 18.21% (55/302) of the enrolled isolates were resistant to ceftazidime–avibactam. The CA rates of the gradient diffusion strip method for Enterobacterales and P. aeruginosa were 100% and 98.65% (73/74), respectively, and the EA rates were 97.37% (222/228) and 98.65% (73/74), respectively. The CA rates of the disk diffusion method for Enterobacterales and P. aeruginosa were 100% and 95.95% (71/74), respectively. No VMEs were found by using the gradient diffusion strip method, while the ME rate was 0.40% (1/247). No MEs were found by using the disk diffusion method, but the VME rate was 5.45% (3/55). Therefore, all the parameters of the gradient diffusion strip method were in line with acceptable criteria. For 31 blaKPC, 33 blaNDM, 7 blaIMP, and 2 blaVIM positive isolates, both CA and EA rates were 100%; no MEs or VMEs were detected by either method. For 15 carbapenemase-non-producing resistant isolates, the CA and EA rates of the gradient diffusion strips method were 100%. Whereas the CA rate of the disk diffusion method was 80.00% (12/15), the VME rate was 20.00% (3/15).Conclusion: The gradient diffusion strip method can meet the needs of clinical microbiological laboratories for testing the susceptibility of ceftazidime–avibactam drugs. However, the VME rate > 1.5% (5.45%) by the disk diffusion method. By comparison, the performance of the gradient diffusion strip method was better than that of the disk diffusion method.

Use of Fosfomycin Etest To Determine In Vitro Susceptibility of Clinical Isolates of Enterobacterales Other than Escherichia coli, Nonfermenting Gram-Negative Bacilli, and Gram-Positive Cocci.

Clinical isolates of Enterobacterales other than Escherichia coli (EOTEC), nonfermenting Gram-negative bacilli, and Gram-positive cocci were tested for susceptibility to fosfomycin using Etest and reference agar dilution. Applying EUCAST (v. 11.0, 2021) intravenous fosfomycin breakpoints, Etest MICs for EOTEC showed essential agreement (EA), categorical agreement (CA), major error (ME), and very major error (VME) rates of 70.4%, 88.4%, 4.1%, and 32.1%, respectively. No species of EOTEC tested with acceptable rates for all of EA (≥90%), CA (≥90%), ME (≤3%), and VME (≤3%). Etest MICs for Enterococcus faecalis, interpreted using CLSI oral/urine criteria (M100, 2021) showed EA, CA, minor error, ME, and VME rates of 98.5%, 81.2%, 18.8%, 0%, and 0%. Against Staphylococcus aureus, EA, CA, and ME rates were 84.1%, 98.7%, and 1.3% (EUCAST intravenous criteria). S. aureus isolates with fosfomycin MICs of >32 μg/ml (resistant) were not identified by agar dilution. We conclude that performing fosfomycin Etest on isolates of S. aureus will reliably identify fosfomycin-susceptible isolates with low, acceptable rates of MEs and VMEs. Testing of urinary isolates of E. faecalis by Etest is associated with an unacceptably high rate of minor errors (18.8%) but low, acceptable rates of MEs and VMEs when results are interpreted using CLSI criteria. Isolates of EOTEC tested by Etest with resulting MICs interpreted by EUCAST criteria were associated with an unacceptably high VME rate (32.1%). In vitro testing of clinical isolates beyond E. coli, E. faecalis, and S. aureus to determine susceptibility to fosfomycin is problematic with current methods and breakpoints.

Accuracy of gradient diffusion method for susceptibility testing of dalbavancin and comparators

ABSTRACT Objectives This multicenter study aimed to assess the performances of gradient diffusion (GD) method in comparison to broth microdilution (BMD) method for susceptibility testing of dalbavancin, daptomycin, vancomycin, and teicoplanin. Methods Minimum Inhibitory Concentrations (MICs) were retrospectively determined concomitantly by BMD and GD methods, for 93 staphylococci and enterococci isolated from clinical samples. BMD was considered as the gold standard. Essential (EA) and categorical agreements (CA) were calculated. Discordant categorical results were categorized as major (ME) and very major errors (VME). Results EA and CA were 95.7% and 96.8%, 82.8% and 100%, 97.8% and 96.8%, and 94.6% and 95.7% for dalbavancin, daptomycin, vancomycin, and teicoplanin respectively. Concerning dalbavancin, 3 ME without any VME were observed and discrepancies were low (≤ to 2 two-fold dilutions) between both methods. VME were noted in 1 and 3 cases for vancomycin and teicoplanin, respectively, and resulted from 1 two-fold dilution discrepancy in each case. EA was lower for daptomycin. When they were discrepant, BMD MICs were systematically higher than GD ones. Nevertheless, no categorical discrepancy was noted. Conclusions GD appears as an acceptable and convenient alternative for dalbavancin, vancomycin, and teicoplanin MICs determination. Our study also emphasizes how achieving accurate daptomycin MICs remains challenging.

Comparison of phenotypic antimicrobial susceptibility testing results and WGS-derived genotypic resistance profiles for a cohort of ESBL-producing Escherichia coli collected from Canadian hospitals: CANWARD 2007-18.

To determine whether the genotypic resistance profile inferred from WGS could accurately predict phenotypic resistance for ESBL-producing Escherichia coli isolated from patient samples in Canadian hospital laboratories. As part of the ongoing CANWARD study, 671 E. coli were collected and phenotypically confirmed as ESBL producers using CLSI M100 disc testing criteria. Isolates were sequenced using the Illumina MiSeq platform, resulting in 636 high-quality genomes for comparison. Using a rules-based approach, the genotypic resistance profile was compared with the phenotypic resistance interpretation generated using the CLSI broth microdilution method for ceftriaxone, ciprofloxacin, gentamicin and trimethoprim/sulfamethoxazole. The most common genes associated with non-susceptibility to ceftriaxone, gentamicin and trimethoprim/sulfamethoxazole were CTX-M-15 (n = 391), aac(3)-IIa + aac(6')-Ib-cr (n = 121) and dfrA17 + sul1 (n = 169), respectively. Ciprofloxacin non-susceptibility was most commonly attributed to alterations in both gyrA (S83L + D87N) and parC (S80I + E84V), with (n = 187) or without (n = 197) aac(6')-Ib-cr. Categorical agreement (susceptible or non-susceptible) between actual and predicted phenotype was 95.6%, 98.9%, 97.6% and 88.8% for ceftriaxone, ciprofloxacin, gentamicin and trimethoprim/sulfamethoxazole, respectively. Only ciprofloxacin results (susceptible or non-susceptible) were predicted with major error (ME) and very major error (VME) rates of <3%: ciprofloxacin (ME, 1.5%; VME, 1.1%); gentamicin (ME, 0.8%-31.7%; VME, 4.8%); ceftriaxone (ME, 81.8%; VME, 3.0%); and trimethoprim/sulfamethoxazole (ME, 0.9%-23.0%; VME, 5.2%-8.5%). Our rules-based approach for predicting a resistance phenotype from WGS performed well for ciprofloxacin, with categorical agreement of 98.9%, an ME rate of 1.5% and a VME rate of 1.1%. Although high categorical agreements were also obtained for gentamicin, ceftriaxone and trimethoprim/sulfamethoxazole, ME and/or VME rates were ≥3%.

Comparison of MICs in Escherichia coli isolates from human health surveillance with MICs obtained for the same isolates by broth microdilution.

ObjectivesHuman health surveillance and food safety monitoring systems use different antimicrobial susceptibility testing (AST) methods. In this study, we compared the MICs of Escherichia coli isolates provided by these methods.MethodsE. coli isolates (n = 120) from human urine samples and their MICs were collected from six medical laboratories that used automated AST methods based on bacterial growth kinetic analyses. These isolates were retested using broth microdilution, which is used by the food safety monitoring system. The essential and categorical agreements (EA and CA), very major errors (VME), major errors (ME) and minor errors (mE) for these two methods were calculated for 11 antibiotics using broth microdilution as a reference. For statistical analysis, clinical breakpoints provided by EUCAST were used.ResultsFive study laboratories used VITEK®2 and one MicroScan (Walkaway Combo Panel). Out of 120 isolates, 118 isolates (98.3%) were confirmed as E. coli. The 99 E. coli isolates from five study laboratories that used VITEK®2 showed high proportions of EA and CA with full agreements for gentamicin, meropenem, imipenem and ertapenem. Additionally, 100% CA was also observed in cefepime. Few VME (0.5%), ME (1.9%) and mE (1.5%) were observed across all antibiotics. One VME for ceftazidime (7.1%) and 12 MEs for ampicillin (29.4%), cefotaxime (2.4%), ciprofloxacin (3.2%), tigecycline (1.5%) and trimethoprim (22.2%) were detected.ConclusionsMICs from E. coli isolates produced by VITEK®2 were similar to those determined by broth microdilution. These results will be valuable for comparative analyses of resistance data from human health surveillance and food safety monitoring systems.

Evaluation of the performance of rapid antibiotic susceptibility test results using the disk diffusion directly from the positive blood culture bottles

PurposeIn this study, we aimed to evaluate the compliance of rapid antibiotic susceptibility test (RAST) and conventional laboratory procedures. MethodsThe RAST was performed directly from the blood cultures of 71 Gram negative bacilli (GNB) and 38 Gram positive cocci (GPC) isolates. The results were evaluated at fourth, sixth and eighth hour. Categorical agreement (CA), very major error (VME), major error (ME) and minor error (mE) were calculated and compared with the results of conventional Vitek-2 system. ResultsCategorical agreement was detected ≥90 in cefotaxime and meropenem at fourth hour in Escherichia coli isolates. An encourage positive CA results were obtained from meropenem, ceftazidime and ciprofloxacin at the fourth hour in Klebsiella pneumoniae isolates. CA was compatible in imipenem, ciprofloxacin, gentamicin, and tobramycin for Pseudomonas aeruginosa at sixth hour. CA was low (<90%) in piperacillin-tazobactam for E. coli and K. pneumoniae, and meropenem in P. aeruginosa isolates. A good CA (≥90) with all tested antibiotics were found at all hours for Acinetobacter baumannii and also very high CA (100%) was detected at sixth and eighth hour in Staphylococcus aureus isolates. CA remained below the standard criteria at fourth hour in vancomycin and high level gentamicin, in addition to imipenem at sixth hours in enterococci isolates. VME and ME were not detected and mE was 12.7% in GNB and 50% in GPC at eighth hour. ConclusionsEUCAST RAST at eighth hour will be beneficial in urgent patients due to their high CA rate, easy preparation, inexpensive, and could be performed with the available equipment and personnel.

Evaluation of the Vitek 2, Phoenix, and MicroScan for Antimicrobial Susceptibility Testing of Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia causes high-mortality infections in immunocompromised hosts with limited therapeutic options. Many U.S. laboratories rely on commercial automated antimicrobial susceptibility tests (cASTs) and use CLSI breakpoints (BPs) for S. maltophilia. However, contemporary data on these systems are lacking. We assessed performance of Vitek 2, MicroScan WalkAway, and Phoenix relative to that of reference broth microdilution for trimethoprim-sulfamethoxazole (SXT), levofloxacin (LEV), minocycline (MIN), and ceftazidime (CAZ) with 109 S. maltophilia bloodstream isolates. Using CLSI breakpoints, categorical agreement (CA) was below 90% on all systems and drugs, with the exception of SXT by MicroScan (98.1%) and Phoenix (98.1%) and MIN by MicroScan (100%) and Phoenix (99.1%). For SXT, Vitek 2 yielded a 77.1% CA. LEV and CAZ CA ranged from 67% to 85%. Very major errors (VME) were >3% for SXT (MicroScan, Phoenix), LEV (MicroScan), and CAZ (all systems). Major errors (ME) were >3% for SXT (Vitek 2), LEV (Phoenix), and CAZ (MicroScan, Phoenix). Minor errors were >10% for CAZ and LEV on all systems. Data were analyzed with EUCAST pharmacokinetic/pharmacodynamic CAZ, LEV, ciprofloxacin (CIP), and tigecycline (TGC) breakpoints when possible. CA was <90% for all. VME were >3% for CAZ (all systems), LEV (MicroScan), and TGC (Vitek 2), and ME were >3% for LEV (MicroScan), CAZ (all systems), ciprofloxacin (Vitek 2 and MicroScan), and TGC (Vitek 2, Phoenix). Minor errors (MI) were >10% for all agents and systems, by EUCAST breakpoints with an intermediate category (LEV, CAZ, CIP). Laboratories should use caution with cASTs for S. maltophilia, as a high rate of errors may be observed.

Surveillance of Antimicrobial Susceptibility of Anaerobe Clinical Isolates in Southeast Austria: Bacteroides fragilis Group Is on the Fast Track to Resistance.

Anaerobic bacteria play an important role in human infections. Bacteroides spp. are some of the 15 most common pathogens causing nosocomial infections. We present antimicrobial susceptibility testing (AST) results of 114 Gram-positive anaerobic isolates and 110 Bacteroides-fragilis-group-isolates (BFGI). Resistance profiles were determined by MIC gradient testing. Furthermore, we performed disk diffusion testing of BFGI and compared the results of the two methods. Within Gram-positive anaerobes, the highest resistance rates were found for clindamycin and moxifloxacin (21.9% and 16.7%, respectively), and resistance for beta-lactams and metronidazole was low (<1%). For BFGI, the highest resistance rates were also detected for clindamycin and moxifloxacin (50.9% and 36.4%, respectively). Resistance rates for piperacillin/tazobactam and amoxicillin/clavulanic acid were 10% and 7.3%, respectively. Two B. fragilis isolates were classified as multi-drug-resistant (MDR), with resistance against all tested beta-lactam antibiotics. The comparative study of 109 BFGI resulted in 130 discrepancies in 763 readings (17%) with a high number of Very Major Errors (VME) and Major Errors (ME). In summary, resistance rates, with the exception of clindamycin and moxifloxacin, are still low, but we are facing increasing resistance rates for BFGI. Surveillance studies on a regular basis are still recommended.

Performance of QMAC-dRASTTM (Direct Rapid Antimicrobial Susceptibility Testing) - a Newcomer in Phenotypic Automatic AST

Objective: QMAC-dRASTTM is a phenotypic automatized Antibiotic Susceptibility Testing (AST) system based on microfluidic chip technology enabling observation of changes in a single bacterial cell under antibiotic treatment conditions. The 96 wells plate with dried antibiotics comprises 19 and 17 antibiotics for the Gram-Negatives (GNs) and Gram-Positives (GPs), respectively. Categorical (Sensitive, Intermediate or Resistant) results were compared to results obtained by our laboratory standard susceptibility testing procedure and given as Categorical Agreement (CA). Methods: In a 3-month period (2019/2020), blood cultures detected positive were included. Excluded were known off-panel strains of QMAC-dRASTTM, such as Gram-positive bacilli, Streptococcus and Candida species. Percentages of CA (CA, %) between QMAC-dRASTTM and routine testing methods used in the laboratory (EUCAST disc diffusion and/or etest/Broth Micro Dilution MIC), were calculated. Results: 255 positive blood cultures from as many patients were examined. Of the positive blood culture strains, 144 were GNs, and 111 were GPs. An overall combined CA,% of 96.3 (2410 of 2502 determinations) was obtained, and discrepancies were noted in 92 of 2502 test results (3.7%). The percentage of very major errors (VMEs) was 0.7% for GNs and 2.2% for GPs. For 87% of blood culture specimens examined, susceptibility reports were available within 6-7 hours. Conclusion: The high CA,% for as well GNs as GPs are promising. The presented time to report data obtained by QMAC-dRASTTM in this study being of 3-8 hours for blood culture specimens examined strongly support a further possible improvement in the workflow for handling blood stream infections.

Performance Evaluation of BD Phoenix NMIC-413 Antimicrobial Susceptibility Testing Panel for Imipenem, Meropenem, and Ertapenem Against Clinical Carbapenem-Resistant and Carbapenem-Susceptible Enterobacterales.

Purpose: The infection of carbapenem-resistant Enterobacterales (CRE) has become a major clinical and healthcare problem worldwide. The screening methods of CRE have been extensively developed but still need improving [e.g., tests with accurate and simple minimum inhibitory (MICs)]. In this study, the performance of the BD Phoenix NMIC-413 AST panel was evaluated against clinical CRE and carbapenem-susceptible Enterobacterales (CSE) in China. The panel was first evaluated in the Chinese clinical lab.Methods: Antimicrobial susceptibility testing of 303 clinical Enterobacterales isolates were conducted by broth microdilution (BMD), Phoenix NMIC-413 AST panel, and disk diffusion method for imipenem, ertapenem, and meropenem. Considering BMD is a gold standard, essential agreement (EA), categorical agreement (CA), minor error (MIE), major error (ME), and very major error (VME) were determined according to CLSI guidelines. CA and EA > 90%, ME <3%, and VME <1.5% were considered as acceptable criteria. Polymerase chain reaction and sanger sequencing were performed to determine the β-lactamase genotypes of CRE isolates.Results: Three hundred and three isolates included 195 CREs and 108 CSEs were enrolled according to the BMD-MIC values of three carbapenems. Tested CREs showing 100 blaKPC−2-positive organisms, 31 blaIMP-positive organisms, 28 blaNDM-positive organisms, 5 blaVIM-positive organisms, 2 both blaIMP and blaVIM-positive organisms, 2 blaOXA−48-positive organisms, and 27 isolates without carbapenemase genes. For the Phoenix NMIC-413 method, CA and EA rates >93%, MIE rates <5%, ME rates <1.75%, and VME rates were 0%, across the three drugs. For the disk diffusion method, the CA rates for three drugs were all >93%, while the MIE and ME rates were all <5 and <3%, respectively. VME rate was 3.28% for imipenem, exceeded the cut-off value specified by CLSI M52, 0 and 0.56% for ertapenem and meropenem, separately.Conclusion: Based on the genomic data, the detection of CRE and CSE was more reliable using the BD Phoenix NMIC-413 panel compared to the BMD and disk approaches. Therefore, our study supports the use of BD Phoenix NMIC-413 panel as a suitable alternative to BMD for the detection of carbapenem resistant isolates in a clinical setting.

Comparative Evaluation of Seven Tigecycline Susceptibility Testing Methods for Carbapenem-Resistant Enterobacteriaceae.

PurposeCarbapenem-resistant Enterobacteriaceae (CRE) strains are extensively resistant to most antibiotics. Tigecycline is one of the few effective drugs that can be used to treat infections caused by CRE. The aim of this study was to evaluate the accuracy of different methods for detecting the susceptibility of CRE to tigecycline.MethodsSeven commonly used drug susceptibility testing methods were compared and evaluated for the ability to determine CRE tigecycline susceptibility: broth microdilution (BMD), agar dilution method (ADM), disk diffusion method, Etest, MicroScan, Vitek2 COMPACT, and BD Phoenix 100.ResultsThe minimum inhibitory concentration (MIC) of tigecycline to inhibit 50% and 90% of CRE growth (MIC50 and MIC90, respectively) assessed by ADM and BD Phoenix 100 was the same as that determined by the reference method, BMD. The MIC50 was 2 µg/mL, and the MIC90 was 4 µg/mL. The highest number of susceptible strains was detected by MicroScan, followed by BMD, Etest, ADM, BD Phoenix 100, Vitek2 COMPACT, and disk diffusion method, in descending order. No significant differences were observed among the tigecycline susceptibility results (P > 0.05) obtained from MicroScan, Etest, BD Phoenix 100, and BMD. BMD confirmed that 82.0% of strains were susceptible to tigecycline. ADM, MicroScan, and BD Phoenix 100 yielded the categorical agreement of 96%, 92%, and 93%, respectively. No method was found to present any very major errors (VMEs), and only the Vitek2 COMPACT yielded major errors (MEs) greater than 3%.ConclusionAmong the seven methods tested, the ADM, MicroScan, and BD Phoenix 100 methods were accurate for determining the tigecycline susceptibility of CRE. MicroScan was acceptable with better performance than other methods.

Evaluation of the Clinical Systems for Polymyxin Susceptibility Testing of Clinical Gram-Negative Bacteria in China.

ObjectivesThe performance of mainstream commercial antimicrobial susceptibility testing systems on polymyxins has not been well evaluated in China. In this study, three antimicrobial susceptibility testing systems were evaluated for polymyxin B and colistin.MethodsThe MICs of 257 Gram-negative strains collected from clinical cases and livestock were determined and analyzed. Using Broth Microdilution as the gold standard, the performance of VITEK 2® COMPACT, PhoenixTM M50, and Bio-kont AST System were evaluated. Essential agreement (EA), category agreement (CA), very major error (VME), and major error (ME) were calculated for comparison. The results of mcr-1 positive strains were separately discussed.ResultsThe EA, CA, VME, and ME to polymyxin B for Bio-kont were 83.5, 95.6, 13.1, and 0.6%, respectively. The EAs, CAs, VMEs, and MEs to colistin were as follows: Bio-kont, 86.7%/96.5%/7.2%/1.7%; Vitek 2, 64.2%/86.8%/41.0%/0%, and Phoenix M50, 92.9%/92.9%/21.7%/0%. The performance of Bio-kont to polymyxin B and colistin for Pseudomonas spp. (EA, CA < 90%, VME > 1.5%, ME = 5.6%/10%) and Enterobacter spp. (EA, CA < 90%, VME > 1.5% and ME = 0%), Vitek to colistin for most genera, and Phoenix to colistin for Enterobacter spp. (EA, CA < 90%, VME > 1.5%, ME = 0%) were unsatisfactory compared with other genera. The performance of Bio-kont to polymyxins for Escherichia spp. and Phoenix to colistin for Citrobacter spp., Escherichia spp., and Klebsiella spp., which all met the CLSI standard, were satisfactory. When the susceptibility of mcr-1 positive E. coli was tested, Bio-kont and Phoenix M50 presented excellent performance with no category errors, while Vitek 2 performed a high VME (25.5%).ConclusionWith relatively more accurate results for polymyxin B and colistin and lower VME, Bio-kont has an advantage in polymyxin antimicrobial susceptibility testing, especially for Escherichia spp., Klebsiella spp., Citrobacter spp. and Acinetobacter spp.

Evaluation of a Commercial Broth Microdilution Panel for Colistin Susceptibility Testing of Clinical Isolates of Escherichia coli and Klebsiella pneumoniae.

Colistin is among the last resort antibiotics for the treatment of infections caused by multidrug-resistant Gram-negative pathogens. Antimicrobial susceptibility testing of colistin is challenging due to its physicochemical properties. Broth microdilution (BMD) is the recommended method for colistin susceptibility testing. However BMD is not practical for clinical microbiology laboratories as manual preparation of BMD plates is time-consuming and labor intensive. Recently, some more user-friendly BMD products with commercial panels have become available. Our objective was to evaluate the performance of a commercial broth microdilution (BMD) product [Sensititre (Thermo Fisher Scientific)] for colistin MIC determination by comparison with reference BMD method using a collection of E. coli and K. pneumoniae isolates. A total of 323 unique patient isolates (102 E. coli, 221 K. pneumoniae) were included in the study. Isolates were stored at -70°C and subcultured twice on sheep blood agar before testing. Colistin MICs of the isolates were determined using Sensititre (a premade BMD product with dried antibiotics) and an 'in-house prepared BMD panel prepared in accordance with CLSI guidelines' (reference method). MIC determination with Sensititre was performed according to manufacturer's instructions. The reference method was performed using untreated 96-well sterile polystyrene plates. Colistin MIC results were interpreted according to EUCAST breakpoints (susceptible, ≤ 2 mg/L; resistant, > 2 mg/L). Overall susceptibility rate of isolates to colistin by reference BMD was 75.9%. Overall categorical agreement (CA), essential agreement (EA), very major error (VME), and major error (ME) rates for Sensititre were 98.5%, 72.5%, 3.8%, and 0.8%, respectively. The CA and EA between Sensititre and reference BMD for the isolates with reference colistin MICs close to the susceptibility breakpoint (2 - 8 mg/L) was 94.2% and 48.1%, respectively. Sensititre yielded a VME rate of 15% and ME rate of 0%, respectively, for this subset of isolates. In conclusion, Sensititre showed high CA but low EA with reference BMD for entire collection of isolates. The VME rate was just slightly above 3% and ME rate was acceptable. The rates of CA and EA were decreased and the rate of VME was increased when a subset consisting of more challenging isolates was used.

13. Evaluation of Etest for Antibiotic Susceptibility and Minimum Inhibitory Concentration (MIC) Agreement Against pseudomonas Aeruginosa (psa) from Patients with Cystic Fibrosis (CF)

BackgroundCF acute pulmonary exacerbations are often caused by PSA, including multi-drug resistant strains. Optimal antibiotic therapy is required to return lung function and should be guided by in vitro susceptibility results. There are sparse data on the performance of Etest relative to reference broth microdilution (BMD) for many newer drugs against CF PSA. Herein, we describe Etest performance with 10 anti-PSA antibiotics against CF isolates.MethodsContemporary, clinical PSA (n=105) isolated during pulmonary exacerbation from patients with CF were acquired from 3 US hospitals. MICs were assessed by BMD (reference) and Etest for aztreonam (ATM), cefepime (FEP), ceftazidime (CAZ), ceftazidime/avibactam (CZA), ceftolozane/tazobactam (C/T), ciprofloxacin (CIP), levofloxacin (LVX), meropenem (MEM), piperacillin/tazobactam (TZP), and tobramycin (TOB). Each respective MIC was completed in at least triplicate using the same inoculum between methods. Modal MICs for each method were compared by rates of essential agreement (EA), categorical agreement (CA), minor error (miE), major error (ME), and very major error (VME) rates. All miE, ME and VME were adjusted if within EA.ResultsOf the 105 PSA, 46% had a mucoid phenotype. Results are summarized in the Table. Median modal Etest MICs read 0–1 dilution higher (IQR: 0–1) than BMD. CA and EA ranged from 64–93% and 63–86%, respectively. Single VMEs occurred for ATM (2.9%) and CAZ (4.2%). For CZA, 2 VMEs were observed and both were within EA. Major errors were ≤3% except for ATM (3.3%), MEM (3.3%), CZA (5.3% with adjusted ME 2.1%*), and FEP (13%). Minor error rates were < 10% except for TZP, CIP, LEV, TOB, and FEP (13–29%), for which majority of miE were within EA (3/14, 11/16, 10/18, 13/19, 20/31, respectively). Performance was similar for non-mucoid and mucoid populations.Etest PerformanceConclusionEtest methods performed well for most antibiotics against this challenging collection of PSA from CF patients. Laboratories should be cautious of miE and ME that may occur with certain antibiotics. Furthermore, our observations suggest laboratories confirm CZA results for isolates with MICs near the breakpoint. Disclosures Joseph L. Kuti, PharmD, Allergan (Speaker’s Bureau)bioMérieux (Research Grant or Support, Other Financial or Material Support, Speaker Honorarium)Melinta (Research Grant or Support)Merck & Co., Inc. (Research Grant or Support)Paratek (Speaker’s Bureau)Summit (Other Financial or Material Support, Research funding (clinical trials)) David P. Nicolau, PharmD, Cepheid (Other Financial or Material Support, Consultant, speaker bureau member or has received research support.)Merck & Co., Inc. (Consultant, Grant/Research Support, Speaker’s Bureau)Wockhardt (Grant/Research Support)

9. Evaluation of Synergy Testing Methods for Clinical Labs to Determine Susceptibility of Extensively Drug-resistant Gram-negatives to Ceftazidime/ Avibactam and Aztreonam Combination Therapy

BackgroundCarbapenem-resistant Enterobacterales (CRE) and Pseudomonas aeruginosa (CR-PA) producing Metallo-β-lactamases (MBLs) cause severe nosocomial infections with no defined treatment. Combination therapy with ceftazidime/ avibactam (CZA) and aztreonam (ATM) is a potential option, but there is no approved, feasible, synergy testing method for clinical labs to guide clinical decision making. Here, we evaluate the performance of 4 synergy testing methods using gradient-strips or disks.MethodsWe used 10 representative Enterobacterales strains, namely, E. coli, K. pneumoniae, and E. cloacea, and 6 PA strains harboring MBL, GES or non-MBL enzymes (Fig 1). 4 strains were successfully treated with CZA-ATM in case reports, the rest were from the CDC AR Bank. Four synergy testing methods were evaluated, i) Disk stack (DS), ii) Disk elution (DE), iii) Gradient-strip Stack (SS), iv) Gradient-strip Cross (SX) (Fig 1). All methods were run side-by-side as per CLSI guidelines with broth microdilution (BMD) as the reference. Data is the mean of 3 replicates. Synergy is defined as a strain that is resistant (R) to ATM but drops to ≤ the susceptible (S) breakpoint (Table 1) in the presence of CZA (Fig 2). Categorical agreement (CA), very major error (VME), major error (ME), minor error (MI) were calculated across methods for CZA-ATM synergy relative to BMD.Summary of synergy testing methods evaluated CLSI Breakpoints used for this study ResultsAll CRE with NDM and PA with GES were ATM-R, CZA-R and S to the CZA-ATM combination. PA with NDM or VIM remained R to CZA-ATM likely due to other mechanisms of resistance. CA was high for DE (100%), SS (81%, MI 19%), and SX (88%, MI 13%) but low for DS (25%, ME 54%, MI 31%). Representative strains are shown (Fig 2, Table 2). Removing PA, CA for DE, SS, and SX was 100% and 20% for DS.Representative results of strains with each synergy testing method. Representative data of strains displaying synergy (green) or no synergy (red)ConclusionOverall, DE was the most reliable method for CZA-ATM synergy testing, and could be a valuable tool in low-resource labs. SS and SX were reliable but prone to technical error. DS had the worst performance. Disks and gradient-strips had identical performance across brands. We propose an algorithm for ATM-R, CZA-R, and MBL-positive CRE, where CZA-ATM synergy testing may be beneficial to guide therapy. These methods are reliable qualitative indicators of the presence or absence of synergy. Synergy testing is not recommended for CR-PA due to complex resistance profiles. Disclosures Cesar A. Arias, M.D., MSc, Ph.D., FIDSA, Entasis Therapeutics (Scientific Research Study Investigator)MeMed (Scientific Research Study Investigator)Merck (Grant/Research Support) William R. Miller, MD, Entasis Therapeutics (Scientific Research Study Investigator)Merck (Grant/Research Support)Shionogi (Advisor or Review Panel member)

Evaluation of Surrogate Tests for the Presence of mecA-Mediated Methicillin Resistance in Staphylococcus capitis, Staphylococcus haemolyticus, Staphylococcus hominis, and Staphylococcus warneri.

Testing of staphylococci other than Staphylococcus aureus (SOSA) for mecA-mediated resistance is challenging. Isolates of Staphylococcus capitis, Staphylococcus haemolyticus, Staphylococcus hominis, and Staphylococcus warneri were evaluated by cefoxitin and oxacillin broth microdilution (BMD), disk diffusion (DD), and PBP2a immunoassay, and the results were compared to mecA PCR results. No phenotypic susceptibility test correlated well with PCR results across all species, although the PBP2a immunoassay yielded 100% correlation. Oxacillin BMD testing by current Clinical and Laboratory Standards Institute (CLSI) SOSA breakpoints led to 2.1% very major errors (VMEs) and 7.1% major errors (ME). Adjusting this breakpoint up by a dilution (susceptible, ≤0.5 μg/ml; resistant, ≥1.0 μg/ml) led to 2.8% VMEs and 0.3% MEs. Among species evaluated, S. haemolyticus had unacceptable VMEs with this new breakpoint (6.4%), as did S. hominis (4.0%). MEs were acceptable by this new breakpoint, ranging from 0 to 1.2%. Oxacillin DD yielded high ME rates (20.7 to 21.7%) using CLSI or European Committee on Antimicrobial Susceptibility Testing breakpoints. VMEs ranged from 0 to 5.3%. Cefoxitin BMD led to 4.9% VMEs and 1.6% MEs. Cefoxitin DD performed best when interpreted with the CLSI SOSA breakpoint, with 1.0% VMEs and 2.9% MEs. This study led CLSI to adjust the oxacillin MIC breakpoints for SOSA. Laboratories should be aware that no individual phenotypic test correlates well across all species of SOSA with mecA PCR results. Molecular testing for mecA or evaluation for PBP2a is the preferred approach.