Evaluation of Automated Disk Diffusion Antimicrobial Susceptibility Testing Using Radian® In-Line Carousel.

Antimicrobial susceptibility testing (AST) by disk diffusion provides an accurate image of bacterial growth, enabling the detection of culture purity, heterogeneous growth, and antibiotic interactions. However, this manual method is time-consuming and visual interpretation is prone to errors. To overcome these disadvantages, the Radian® In-Line Carousel (Copan, Brescia, Italy) was launched, which is a WASPLab® module dedicated to full automation of (pre)-analytical steps as well as interpretation of disk diffusion AST. However, until now, no evaluation of Radian® against manual disk diffusion has been performed. We assessed the categorical agreement (CA) between standardized disk diffusion (reference method) and Radian® using EUCAST 2021 breakpoints. We tested 135 non-duplicate strains, selected from the National EUCAST challenge panel, clinical strains, and external quality controls. The strains included Enterobacterales (n = 63), Enterococcus faecalis (n = 3), Enterococcus faecium (n = 10), Pseudomonas aeruginosa (n = 16), Staphylococcus aureus (n = 19), coagulase-negative staphylococci (n = 4), and Streptococcus spp. (n = 20). Furthermore, we explored antibiotic disk thermolability in the WASP Radian® carousel by testing 10 ATCC® strains up to 7 days. The observed CA was 95.3%, 96.3%, 93.8%, 97.3% and 98.0% for Enterobacterales, Enterococcus spp., P. aeruginosa, Staphylococcus spp. and Streptococcus spp., respectively, resulting in an acceptable overall CA for all groups. (Very) major error rates were ≤ 5% for all antibiotics. Antibiotic disk thermostability was confirmed up to 4 days in the WASP Radian® In-Line Carousel. The Radian® In-Line Carousel provides a fully automated solution for accurate disk diffusion AST, reducing workload and improving standardization and traceability. In addition, our study demonstrated the thermostability of antibiotic disks up to 4 days in the WASP Radian® In-Line Carousel.

Performance of disk diffusion method for aztreonam in combination with avibactam against Enterobacteriales.

OBJECTIVES: To evaluate the performance of an in-house developed disk diffusion method for aztreonam in combination with avibactam against Enterobacteriales. METHODS: The in vitro antibacterial activity of aztreonam with avibactam against 204 carbapenemase-producing Enterobacteriales was determined by a disk diffusion method, with a broth microdilution method as a reference. RESULTS: The optimal S/R breakpoints for disk diffusion tests of 30/20 and 10/4 µg disks, calculated by the dBETs software using the model-based approaches, were ≥22/≤21 and ≥12/≤11 mm, respectively. On the basis of the estimated breakpoints, the CAs for disk diffusion tests of 30/20 and 10/4 µg aztreonam/avibactam disks were both 98.0%, with 0.5% major error and 37.5% very major error. CONCLUSIONS: The home-made disk diffusion method is an economical and practical method for clinical microbiology laboratories to determine the antibacterial susceptibility of aztreonam with avibactam against Enterobacteriales.

Re-assessment of the disk diffusion technique for routine antimicrobial susceptibility testing for Helicobacter pylori.

BACKGROUND: The aim of this study was to assess the disk diffusion technique against E-test as a routine antibiotic susceptibility testing method for Helicobacter pylori. MATERIALS AND METHODS: Susceptibilities of 301 H pylori clinical isolates were simultaneously profiled by E-test and disk diffusion method for levofloxacin (5-µg disk), clarithromycin (15-µg disk), metronidazole (5-µg disk), amoxicillin (10-µg disk), and tetracycline (30-µg disk). Furazolidone susceptibility was evaluated using a 100-µg disk only. The correlation between MICs by E-test and inhibition zone diameters by disk diffusion was assessed by linear regression analysis. RESULTS: Correlation between inhibition zone diameters and MICs was found for levofloxacin (r = -.932), clarithromycin (r = -.894), and to a minor extent metronidazole (r = -.820). Using the linear regression analysis, the inhibition zone diameter breakpoints were calculated to be 29 mm for levofloxacin, 41 mm for clarithromycin, and 15 mm for metronidazole corresponding to the EUCAST-recommended MIC breakpoints. The susceptibility agreement between E-test and disk diffusion for levofloxacin, clarithromycin, and metronidazole was 98.6%, 96.0%, and 96.7%, respectively. The inhibition zone diameters recorded for the amoxicillin, tetracycline, and furazolidone were large (approximately 60 mm in mean), and a poor correlation was found between inhibition zone diameters and MICs for amoxicillin (r = -.594) and tetracycline (r = -.490). CONCLUSIONS: The disk diffusion can be used as a routine H pylori susceptibility testing method for levofloxacin, clarithromycin, and metronidazole in clinical practice under the described technical conditions. The use of disk diffusion for amoxicillin, tetracycline, and furazolidone susceptibility testing needs to be further studied.

Antifungal susceptibility testing practices in mycology laboratories in France, 2018.

A survey of mycology laboratories for antifungal susceptibility testing (AFST) was undertaken in France in 2018, to better understand the difference in practices between the participating centers and to identify the difficulties they may encounter as well as eventual gaps with published standards and guidelines. The survey captured information from 45 mycology laboratories in France on how they perform AFST (number of strains tested, preferred method, technical and quality aspects, interpretation of the MIC values, reading and interpretation difficulties). Results indicated that 86% of respondents used Etest as AFST method, with a combination of one to seven antifungal agents tested. Most of the participating laboratories used similar technical parameters to perform their AFST method and a large majority used, as recommended, internal and external quality assessments. Almost all the participating mycology laboratories (98%) reported difficulties to interpret the MIC values, especially when no clinical breakpoints are available. The survey highlighted that the current AFST practices in France need homogenization, particularly for MIC reading and interpretation.

Area of technical uncertainty for susceptibility testing of amoxicillin/clavulanate against Escherichia coli: analysis of automated system, Etest and disk diffusion methods compared to the broth microdilution reference.

OBJECTIVES: The European Committee on Antimicrobial Susceptibility Testing (EUCAST) recently warned about an area of technical uncertainty (ATU) of amoxicillin/clavulanate (AMX/C) disk susceptibility testing against members of the Enterobacterales. Thus, we aimed to compare the reliability of three routine methods and to evaluate the impact of the ATU. METHODS: 286 Escherichia coli strains (including 159 AMX-resistant strains) were categorized for the two EUCAST AMX/C breakpoints by disk diffusion (Bio-Rad), the Phoenix automated system (Becton Dickinson) and the Etest (AES) compared to the broth microdilution reference method. RESULTS: By microdilution, 84.2% of strains were AMX/C-susceptible using the urinary breakpoint (MIC ≤32 mg/L) and 62.2% using the systemic breakpoint (MIC ≤8 mg/L), with 63.6% of MICs between 4 and 16 mg/L. For the systemic breakpoint, category agreement (CA) and very major error (VME) were unacceptable for the Etest (71.7% and 27.3%), disk (73.1% and 23.4% at 19-mm cut-off) and to a lesser extent for the Phoenix system (83.6% and 10.5%). For disks, an unacceptable VME rate was observed for diameters up to 22 mm, probably due to overcharged disks. For the Etest, VMEs were high at 6 mg/L (46/63) and 8 mg/L (22/29). For the urinary breakpoint, CA was more acceptable for disk (88.9%) and Etest (84.3%) but was unevaluable for Phoenix. CONCLUSION: AMX/C susceptibility testing of E. coli for systemic breakpoint was unreliable with the three routine methods, explained mainly by the high prevalence (~60%) of strains with microdilution MICs around the breakpoint (8 mg/L). Our data confirmed the EUCAST 19-20-mm ATU for disk and suggest introducing ATU for Etest MIC values of 6 and 8 mg/L.

Quality and suitability of antimicrobial discs: theoretical and practical sources of error and variability.

Introduction: The use of antimicrobial discs remains one of the main methods for assessing antibiotic activity. Most discs are made to one of three main standards (FDA, WHO, DIN); these all describe an assay method for assessing the quality of discs using a linear method. Theory predicts a curved relationship, and this is backed up in many cases in practice. In such cases, the assays are potentially invalid. Other sources of uncertainty arising from the manufacturing processes employed are also discussed.Areas covered: This includes error arising from applying FDA, WHO, and DIN standards, the manufacturing techniques employed, and variation in the materials used in production. The need for a specification that relates directly to the use of the discs is also discussed.Expert opinion: Manufactured discs, some of which may be out of specification due to curvature and other sources of error, have been used to establish quality zone sizes. Quality zone sizes have then been used to measure the quality of discs. This circular quality system where there is no quantitative check is potentially unsafe. In the many decades of their use, there has been no comprehensive check on the quality of manufactured discs using quantitative, validated assays.

EUCAST disc diffusion criteria for the detection of mecA-Mediated ß-lactam resistance in Staphylococcus pseudintermedius: oxacillin versus cefoxitin.

OBJECTIVES: Until recently, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommended the cefoxitin disc to screen for mecA-mediated ß-lactam resistance in Staphylococcus pseudintermedius. A recent study indicated that cefoxitin was inferior to oxacillin in this respect. We have re-evaluated cefoxitin and oxacillin discs for screening for methicillin resistance in S. pseudintermedius. METHODS: We included 224 animal and human S. pseudintermedius isolates from Europe (n = 108) and North America (n = 116), of which 109 were mecA-positive. Disc diffusion was performed per EUCAST recommendations using 30-µg cefoxitin and 1-µg oxacillin discs from three manufacturers and Mueller-Hinton agar from two manufacturers. RESULTS: Cefoxitin inhibition zones ranged from 6 to 33 mm for mecA-positive S. pseudintermedius (MRSP) and from 29 to 41 mm for mecA-negative S. pseudintermedius (MSSP). The corresponding oxacillin zone intervals were 6-20 mm and 19-30 mm. For cefoxitin 16% (95% CI 14.8-18.0%) of the isolates were in the area where positive and negative results overlapped. For oxacillin the corresponding number was 2% (1.6-2.9%). For oxacillin a breakpoint of susceptible (S) ≥ 20 mm and resistant (R) <20 mm resulted in only 0.4% and 1.1% very major error and major error rates respectively. CONCLUSIONS: This investigation confirms that the 1-µg oxacillin disc predicts mecA-mediated methicillin resistance in S. pseudintermedius better than the 30-µg cefoxitin disc. For a 1-µg oxacillin disc we propose that 20 mm should be used as cut off for resistance, i.e. isolates with a zone diameter <20 mm are resistant to all ß-lactam antibiotics except those with activity against methicillin-resistant staphylococci.

Performance and potential clinical impact of Alfred60AST (Alifax®) for direct antimicrobial susceptibility testing on positive blood culture bottles.

Rapid pathogen identification (ID) and antimicrobial susceptibility testing (AST) of bacteria-causing bloodstream infections can improve patients' outcome. In this study, we evaluated the performance of Alfred60AST (Alifax) which provides AST directly on positive blood culture (BC) bottles by light scattering. In a selected group of patients with a clinical suspicion of severe sepsis or at risk for infections with multiresistant organisms, we compared Alfred60AST AST results with traditional AST results (Vitek2 (bioMérieux) or disk diffusion). Discrepancy analysis was performed by Etest (bioMérieux) or broth microdilution. In total, 222 samples were evaluated. On 595 susceptibility determinations, 93.4% showed categorical agreement (CA) with the standard method. Eighty-one percent of isolates showed a 100% categorical agreement (CA) which increased to 84.3% after discrepancy analysis. There were 8 very major discrepancies (VMD), 18 major discrepancies (MD), and 13 minor discrepancies (MiD). Most discrepant results were observed for piperacillin-tazobactam (15.6%) and clindamycin (18.9%). Analysis time was 6-6.5 h for a complete Alfred60AST AST result. In addition, we evaluated the behavior of clinicians in adjusting antibiotic therapy according to the routine AST results. In 37% of all patients, antibiotic therapy was altered after reporting of AST result and adjustment was more frequent for Gram-negative than for Gram-positive isolates. With some improvements, Alfred60AST provides accurate and rapid preliminary AST results for organisms causing bloodstream infections and may have at least a potential clinical benefit in about one-third of patients with severe sepsis, by delivering faster results compared with conventional methods.

Evaluation of Bio-Rad® discs for antimicrobial susceptibility testing by disc diffusion and the ADAGIO™ system for the automatic reading and interpretation of results.

The disc diffusion test is used for antimicrobial susceptibility testing worldwide. In this study, the performance of both Bio-Rad® antibiotic discs (as compared with Oxoid® discs) and the ADAGIO™ automated system for the reading of disc diffusion test results was evaluated with American Type Culture Collection (ATCC) quality control (QC) and wild strains of bacteria. Inhibition zones of both disc brands were read manually and through use of the ADAGIO™ system. Categorized interpretation of the results for each strain and antibiotic combination was summarized according to the Clinical Laboratory Standards Institute MS-100 (2017 update) manual and ADAGIO™ readings. Eight ATCC QC strains and 120 different wild strains were evaluated, to give a total of 1226 antibiotic/bacteria combinations and 2486 manual readings. One major error and four minor errors (0.08% and 0.34%, respectively) were detected via manual readings of the Bio-Rad® discs as compared with the Oxoid® discs. For the same number of antibiotic/bacteria combinations, five minor errors and one major error (0.42% and 0.08%, respectively) were detected with the Bio-Rad® discs read by the ADAGIO™ system. In addition, the number of times the automatic reading needed manual edition with Bio-Rad® discs was statistically lower than it did with Oxoid® discs (3.7% vs. 5.7%, p < 0.05). These findings support the hypothesis that Bio-Rad discs are not inferior to Oxoid® discs, and the performance of the ADAGIO™ system is comparable to that of manual readings with both disc brands.

Multicenter Evaluation of the New Etest Gradient Diffusion Method for Piperacillin-Tazobactam Susceptibility Testing of Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii Complex.

Piperacillin-tazobactam (P/T) is a ß-lactam-ß-lactamase inhibitor combination frequently used in the hospital setting. Etest is a gradient diffusion method that represents an alternative to broth microdilution (BMD) for performing antimicrobial susceptibility testing. We conducted a multicenter evaluation of the performance of the new P/T Etest compared to that of BMD following U.S. Food and Drug Administration (FDA) and International Standards Organization (ISO) standard ISO 20776-2 criteria using Clinical and Laboratory Standards Institute (CLSI)-FDA and European Committee on Antimicrobial Susceptibility Testing (EUCAST) interpretive breakpoints, respectively. A total of 977 isolates (775 Enterobacterales isolates, 119 Pseudomonas aeruginosa isolates, and 83 Acinetobacter baumannii complex isolates) were tested. Overall essential agreement (EA) was 96.4% and 96.6% for Enterobacterales when FDA and ISO 20776-2 criteria, respectively, were followed. EA was 98.3% for P. aeruginosa and 91.6% for the A. baumannii complex when both the FDA and ISO criteria were followed. Applying CLSI-FDA breakpoints, categorical agreement (CA) reached 93.0%, 93.3%, and 89.2% for the Enterobacterales, P. aeruginosa, and the A. baumannii complex, respectively. Two very major errors (VMEs; 1.1%) were found among the Enterobacterales (for 2 Klebsiella pneumoniae isolates). No additional major errors (MEs) or VMEs were found. Applying EUCAST breakpoints, CA was 94.8% and 95.8% for Enterobacterales and P. aeruginosa, respectively (no breakpoints are currently available for the A. baumannii complex). No VMEs were observed among the Enterobacterales, but 2 (0.4%) MEs were found. Among the P. aeruginosa isolates, 2 (6.9%) VMEs and 3 (3.3%) MEs were observed. These errors resulted when P/T Etest MICs were 1 doubling dilution apart from the BMD MICs. In conclusion, the new P/T Etest represents an accurate tool for performing antimicrobial susceptibility testing of Enterobacterales, P. aeruginosa, and A. baumannii complex isolates with limited category errors.

Multicenter Evaluation of Colistin Broth Disk Elution and Colistin Agar Test: a Report from the Clinical and Laboratory Standards Institute.

Susceptibility testing of the polymyxins (colistin and polymyxin B) is challenging for clinical laboratories. The Clinical and Laboratory Standards Institute (CLSI) Antimicrobial Susceptibility Testing Subcommittee evaluated two methods to enable accurate testing of these agents. These methods were a colistin broth disk elution (CBDE) and a colistin agar test (CAT), the latter of which was evaluated using two inoculum volumes, 1 µl (CAT-1) and 10 µl (CAT-10). The methods were evaluated using a collection of 270 isolates of Enterobacterales, 122 Pseudomonas aeruginosa isolates, and 106 Acinetobacter spp. isolates. Overall, 94.4% of CBDE results were in essential agreement and 97.9% in categorical agreement (CA) with reference broth microdilution MICs. Nine very major errors (VME; 3.2%) and 3 major errors (ME; 0.9%) were observed. With the CBDE, 98.6% CA was observed for Enterobacterales (2.5% VME, 0% ME), 99.3% CA was observed for P. aeruginosa (0% VME, 0.7% ME), and 93.1% CA was observed for Acinetobacter spp. (5.6% VME, 3.3% ME). Overall, CA was 94.9% with 6.8% VME using CAT-1 and improved to 98.3% with 3.9% VME using CAT-10. No ME were observed using either CAT-1 or CAT-10. Using the CAT-1/CAT-10, the CA observed was 99.4%/99.7% for Enterobacterales (1%/0.5% VME), 98.7%/100% for P. aeruginosa (8.3%/0% VME), and 88.5%/92.3% for Acinetobacter spp. (21.4%/14.3% VME). Based on these data, the CLSI antimicrobial susceptibility testing (AST) subcommittee endorsed the CBDE and CAT-10 methods for colistin testing of Enterobacterales and P. aeruginosa.

Diagnostic accuracy of phenotypic assays for determining antimicrobial resistance status in Staphylococcus pseudintermedius isolates from canine clinical cases.

This study evaluated the diagnostic test accuracy of disc diffusion relative to broth-microdilution for clinical Staphylococcus pseudintermedius isolated from dogs in Australia (n = 614). Accuracy of disc diffusion and broth-microdilution for oxacillin relative to mecA real-time PCR was also assessed. Each isolate had paired minimum inhibitory concentration and zone diameter values for ten antimicrobial agents. Data was dichotomised using Clinical and Laboratory Standards Institute susceptible and resistant clinical breakpoints. Test accuracy was reported using relative diagnostic sensitivity (RSe), specificity (RSp), likelihood ratio pairs, diagnostic odds ratio, and area-under-the receiver-operating characteristic (ROC AUC) analysis. Disc diffusion was found to have high test accuracy for most antimicrobials (ROC AUC range: 0.96 - 0.99) except rifampicin (ROC AUC = 0.80). The RSp of disc diffusion was high for all antimicrobials (range, 97.1%-100%). However, RSe was considerably variable (range, 35.7%-98.8%), particularly for amoxicillin-clavulanic acid (51.5%, 95% CI, 38.9%, 64.0%), cefoxitin (35.7%, 95% CI, 12.8%, 64.9%), and cephalothin (43.6%, 95% CI, 27.8%, 60.4%). When disc diffusion and broth-microdilution were compared to mecA real-time PCR, the overall accuracy of both assays was similar (ROC AUC, 0.99 respectively). However, the RSe for broth-microdilution (96.1%, 95% CI, 88.9%, 99.2%) was significantly higher than for disc diffusion (86.8%, 95% CI, 77.1%, 93.5%) (McNemars mid-p value 0.01). Overall, these findings demonstrate that for most antimicrobials, disc diffusion performed according to CLSI guidelines can be used to differentiate clinical S. pseudintermedius isolates that might otherwise be assessed by broth-microdilution, provided consideration is given to the performance estimates reported here.

Development of Broth Microdilution MIC and Disk Diffusion Antimicrobial Susceptibility Test Quality Control Ranges for the Combination of Cefepime and the Novel ß-Lactamase Inhibitor Enmetazobactam.

Third-generation cephalosporin resistance among Enterobacteriaceae, mediated by the spread of extended-spectrum ß-lactamases (ESBLs), is a very serious medical concern with limited therapeutic options. Enmetazobactam (formerly AAI101) is a novel penicillanic sulfone ß-lactamase inhibitor active against a wide range of ESBLs. The combination of enmetazobactam and cefepime has entered phase 3 development in patients with complicated urinary tract infections. Using the Clinical and Laboratory Standards Institute (CLSI) M23 tier 2 study design, broth microdilution MIC and disk diffusion quality control (QC) ranges were determined for cefepime-enmetazobactam. Enmetazobactam was tested at a fixed concentration of 8 µg/ml in the MIC assay, and a cefepime-enmetazobactam disk mass of 30/20 µg was used in the disk diffusion assay. Escherichia coli ATCC 25922, E. coli ATCC 35218, E. coli NCTC 13353, Klebsiella pneumoniae ATCC 700603, and Pseudomonas aeruginosa ATCC 27853 were chosen as reference strains. The CTX-M-15-producing E. coli NCTC 13353 isolate is recommended for routine testing to control for inhibition of ESBL activity by enmetazobactam. Broth microdilution MIC QC ranges spanned 3 to 4 doubling dilutions and contained 99.6% to 100.0% of obtained MIC values for the five reference strains. Disk diffusion yielded inhibition zone diameter QC ranges that spanned 7 mm and encompassed 97.1% to 100.0% of the obtained values. Quality control ranges were approved by the CLSI in 2017 (broth microdilution MIC) and 2019 (disk diffusion). The established QC ranges will ensure that appropriate assay performance criteria are attained using CLSI reference methodology when determining the susceptibility of clinical isolates to cefepime-enmetazobactam.

Ten years of external quality assessment (EQA) of Neisseria gonorrhoeae antimicrobial susceptibility testing in Europe elucidate high reliability of data.

BACKGROUND: Confidence in any diagnostic and antimicrobial susceptibility testing data is provided by appropriate and regular quality assurance (QA) procedures. In Europe, the European Gonococcal Antimicrobial Susceptibility Programme (Euro-GASP) has been monitoring the antimicrobial susceptibility in Neisseria gonorrhoeae since 2004. Euro-GASP includes an external quality assessment (EQA) scheme as an essential component for a quality-assured laboratory-based surveillance programme. Participation in the EQA scheme enables any problems with the performed antimicrobial susceptibility testing to be identified and addressed, feeds into the curricula of laboratory training organised by the Euro-GASP network, and assesses the capacity of individual laboratories to detect emerging new, rare and increasing antimicrobial resistance phenotypes. Participant performance in the Euro-GASP EQA scheme over a 10 year period (2007 to 2016, no EQA in 2013) was evaluated. METHODS: Antimicrobial susceptibility category and MIC results from the first 5 years (2007-2011) of the Euro-GASP EQA were compared with the latter 5 years (2012-2016). These time periods were selected to assess the impact of the 2012 European Union case definitions for the reporting of antimicrobial susceptibility. RESULTS: Antimicrobial susceptibility category agreement in each year was ≥91%. Discrepancies in susceptibility categories were generally because the MICs for EQA panel isolates were on or very close to the susceptibility or resistance breakpoints. A high proportion of isolates tested over the 10 years were within one (≥90%) or two (≥97%) MIC log2 dilutions of the modal MIC, respectively. The most common method used was Etest on GC agar base. There was a shift to using breakpoints published by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) in the latter 5 years, however overall impact on the validity of results was limited, as the percentage categorical agreement and MIC concordance changed very little between the two five-year periods. CONCLUSIONS: The high level of comparability of results in this EQA scheme indicates that high quality data are produced by the Euro-GASP participants and gives confidence in susceptibility and resistance data generated by laboratories performing decentralised testing.

Polymyxin B Etest® compared with gold-standard broth microdilution in carbapenem-resistant Enterobacteriaceae exhibiting a wide range of polymyxin B MICs.

OBJECTIVES: Polymyxins have been revitalized to combat carbapenem-resistant Enterobacteriaceae (CRE). However, evaluating the activity of these agents by traditional broth dilution methods is not practical for busy clinical laboratories. We compared polymyxin B activity using two quantitative susceptibility testing methods, Etest® and broth microdilution (BMD), against CRE isolates from patients at an academic medical centre. METHODS: Polymyxin B activity against 70 CRE clinical isolates was determined by Etest® according to the manufacturer and by BMD according to CLSI guidelines. Pseudomonas aeruginosa ATCC® 27853 and Escherichia coli NCTC 13846 served as quality control strains. The EUCAST colistin susceptibility breakpoint of Enterobacteriaceae (≤2 mg/L) was used. Essential agreement was isolates with an MIC within 1 log2 dilution over total isolates. Categorical agreement was number of isolates in the same susceptibility category (susceptible or resistant) over total isolates. Major and very major error rates were calculated using number of susceptible and number of resistant isolates, respectively, as the denominator. McNemar's test was used for determining a difference in susceptibility between methods. RESULTS: The CRE isolates were primarily Klebsiella spp. (49%) and Enterobacter spp. (36%). Polymyxin B susceptibility was significantly higher by Etest® compared with BMD (97% versus 77%; p 0.0001). Categorical agreement was 80%, but essential agreement was low (10%). False non-susceptibility was never observed by Etest® (BMD reference), but the very major errors were high (88%). CONCLUSIONS: Etest® reporting of false susceptibility may result in inappropriate antibiotic use and treatment failure clinically. We do not recommend using Etest® for polymyxin B susceptibility testing for routine patient care.

The quality of antimicrobial discs from nine manufacturers-EUCAST evaluations in 2014 and 2017.

OBJECTIVES: Antimicrobial discs for susceptibility testing can be obtained from many manufacturers. We evaluated the quality of discs from nine manufacturers in 2014 and 2017. METHODS: Antimicrobial discs of 16 agents from nine manufacturers were evaluated using EUCAST criteria. Discs were tested in triplicate on Müller-Hinton medium against EUCAST quality control (QC) strains. Mean values were compared with targets and ranges in the EUCAST QC tables. RESULTS: Three manufacturers (Becton Dickinson, Mast and Oxoid) demonstrated excellent and consistent disc quality both in 2014 and 2017. Manufacturers with discs of inadequate quality improved their results between the two periods. Overall, 92% (795/861) versus 97% (1038/1071) of zone diameter readings were within QC ranges and 58% (497/861) versus 75% (806/1071) were within the QC target ± 1 mm, for the first and second studies, respectively. One manufacturer (HiMedia) had major quality problems with 33% (26/78) of readings out of range in the first study and 17% (20/120) in the second study. Discs from some manufacturers showed unexpected variation in inhibition zone diameters (4-9 mm) for discs within the same vial. CONCLUSIONS: Antimicrobial discs from three of nine manufacturers exhibited excellent and reproducible quality. The discs of the other six manufacturers demonstrated various quality issues, some of which were severe. After presenting the results to manufacturers and users, all managed to improve the quality. Our study points to the need for more stringent criteria for disc manufacturing. Criteria should not only address the nominal potency of discs but also define the end result.

Prospective evaluation of rapid antimicrobial susceptibility testing by disk diffusion on Mueller-Hinton rapid-SIR directly on blood cultures.

BACKGROUND: With the worldwide spread of antibiotic resistance, delivering antibiotic susceptibility test (AST) results in a timely manner represents a major challenge. In cases of sepsis, rapid AST may facilitate early optimization of empiric antibiotic therapy. Disc diffusion is a well-standardized AST method, however 16 to 24 h are required to achieve an overall AST profile according to antimicrobial societies. METHODS: In this prospective pilot study, we evaluated the performance of Mueller-Hinton-Rapid-SIR (MHR-SIR) agar after 6-8 h of incubation in comparison with standard MH agar after 16 h of incubation directly on positive blood cultures caused by Enterobacteriaceae and Staphylococcus aureus from routine clinical microbiology. A total of 133 positive blood samples including 110 Enterobacteriaceae (83%) and 23 Staphylococcus aureus (17%) were tested in parallel by two direct AST methods, each using EUCAST breakpoints. For each combination bacterium and antibiotic, we compared the categorical agreement and the correlation between the diameters obtained by MHR-SIR and by standard MH. RESULTS: Our results showed 97.7% categorical agreement for Enterobacteriaceae, with 1.4% minor errors, 0.4% major errors and 0.5% very major errors. For S. aureus, we observed 97.8% categorical agreement, 1.9% minor errors, 0.3% major errors and no very major errors. CONCLUSION: Our results showed excellent categorical agreement and correlations between diameters for MHR-SIR and standard MH methods. MHRSIR can predict the result of overall AST profile within 6-8 h with reliable results. AST is obtained on the same day the blood culture becomes positive, with a very moderate cost.

Colistin susceptibility test evaluation of multiple-resistance-level Pseudomonas aeruginosa isolates generated in a morbidostat device.

Objectives: Colistin is a last-resort antibiotic against the critical-status pathogen Pseudomonas aeruginosa. There is still uncertainty regarding how to accurately measure colistin susceptibility in P. aeruginosa. Evaluation of antimicrobial susceptibility testing (AST) methods is largely hampered by the lack of resistant isolates and those around the susceptibility breakpoint. The aim of this study was to generate such strains in a morbidostat device for use in AST method evaluation. Methods: A morbidostat device was used to cultivate susceptible clinical strains into isolates with a wide range of colistin MICs. Subsequently, five commercial AST methods were compared against the gold standard broth microdilution (BMD) method: MICRONAUT-S, SensiTest, Sensititre, Rapid Polymyxin Pseudomonas and Etest. Results: A total of 131 P. aeruginosa isolates were used for colistin susceptibility test evaluation (100 colistin susceptible and 31 colistin resistant). The 31 colistin-resistant isolates evolved resistance in the morbidostat to different MIC ranges (4-512 mg/L, 100% resistance generation efficacy). The categorical agreement (CA) rates for MICRONAUT-S, SensiTest and Rapid Polymyxin Pseudomonas were 94.7%, 93.9% and 92.4%, respectively. The Sensititre achieved the highest CA score (96.9%), whereas the Etests had the lowest CA score (84%). The very major discrepancy (VMD) rates for all tests were between 3.2% and 67.7%. Conclusions: The morbidostat device can efficiently provide laboratories with colistin-resistant strains for test evaluation. Although CA rates were high for commercial AST methods except for Etests, none met the ≤1.5% CLSI limit for VMD rates. Performance was generally inferior when using isolates with low-level resistance.