Development of a national EUCAST challenge panel for antimicrobial susceptibility testing

Meropenem-vaborbactam (MEV) is a novel carbapenem-beta-lactamase inhibitor combination antibiotic approved by the U.S. Food and Drug Administration (FDA) for treatment of complicated urinary tract infections, including pyelonephritis, in adults. In this study, we evaluated the performance of Etest MEV (bioMérieux, Marcy l'Etoile, France) compared to that of broth microdilution for 629 Enterobacterales and 163 Pseudomonas aeruginosa isolates. According to CLSI/FDA breakpoints, 13 Enterobacterales isolates (12 clinical and 1 challenge) were resistant to MEV. Overall, Etest MEV demonstrated 92.4% essential agreement (EA), 99.2% category agreement (CA), 0% very major errors (VME), 0% major errors (ME), and 0.8% minor errors (mE) with clinical and challenge isolates of Enterobacterales Individual species demonstrated EA rates of ≥80%, with the exception of Proteus mirabilis, for which clinical and challenge isolates demonstrated 34.3% EA, 97.1% CA, 0% ME, and 2.9% mE, precluding the use of Etest MEV with this species. Excluding P. mirabilis, MEV Etest MEV demonstrated 95.8% EA, 99.3% CA, 0% VME, 0% ME, and 0.7% mE with Enterobacterales isolates. When evaluated using European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints, Etest MEV performance with clinical (16 MEV resistant) and challenge (12 MEV resistant) isolates of Enterobacterales (excluding P. mirabilis) and P. aeruginosa demonstrated an unacceptably high VME rate of 7.1% despite 95.2% EA, 99.2% CA, and 0.5% ME compared to the reference method. In conclusion, we report that Etest MEV is accurate and reproducible for MEV susceptibility testing for P. aeruginosa and Enterobacterales, with the exception of P. mirabilis, using CLSI/FDA breakpoints. Etest MEV should not be used with P. mirabilis due to unacceptable analytical performance.

Early diagnosis and effective treatment of bloodstream infections significantly improve prognosis. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) has developed rapid antimicrobial susceptibility testing(RAST) based on the disk diffusion method. In the present study, we compared the data from the RAST study from positive blood cultures in the Autobio BC (Autobio-diagnostics, Zhengzhou, Chinese) blood culture system with those obtained using the standard disk diffusion method. A total of 234 spiked blood culture samples (Klebsiella penumoniae [n= 50], Escherichia coli [n= 50], Acinetobacter baumannii [n= 20], Pseudomonas aeruginosa [n= 13], Staphylococcus aureus [n= 51], and Enterococcus spp. [n= 50]) were collected. The RAST test was performed on positive bottles, and the results were measured at 4, 8, and 20 h. Standard disk diffusion (sDD) was performed, and zone diameters were measured from subculture samples obtained from blood culture bottles. The RAST zone diameters were compared with the sDD zone diameters. The categorical agreement based on the readable zone diameters for the 4th, 8th, and 20th h was K.pneumoniae (99.7 %,99.7 %,99.7 %), E.coli (99.4 %,99.6 %,99.7 %), A.baumannii (98.7 %,98.7 %,98.7 %), P.aeruginosa (100 %,99.3 %,100 %), S. aureus (100 %,100 %,100 %), and Enterococcus spp. (100 %,100 %,100 %). The minor error(mE), major error(ME), and very major error(VME) results obtained in the whole study were determined as 0.9, 0, and 1.3 for 4 h; 1.4, 0, and 1.3 for 8 h; 0.6, 0, and 1.3 for 20 h. The EUCAST RAST study can be performed from positive bottles in the Autobio BC blood culture system, resulting in reliable outcomes. To report earlier antibiotic susceptibility test results, laboratories using Autobio BC can combine RAST with quality control studies.

<i>Introduction</i>: The escalating prevalence of multidrug resistance is a global threat to human health particularly in critically ill patients with bloodstream infections (BSIs). Delay in the administration of the appropriate antimicrobial treatment is associated with higher mortality rates and adverse consequences. This study attempted to estimate the rapid antimicrobial susceptibility testing (RAST) breakpoints directly from flagged BacT/Alert blood culture bottles in clinical practice. <i>Material & Methods</i>: A descriptive, cross-sectional study conducted at a tertiary care hospital in Delhi over a period of two months. The RAST was performed directly from the clinical samples for blood cultures received in our laboratory in parallel with the routine antimicrobial testing as per standard CLSI guidelines. Blood cultures were routinely incubated in BacT/Alert 3D. The inhibition zones were read at 4, 6, 8 and 16-20 hour of incubation as per European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. The identification of the isolates was confirmed by Vitek-2 compact system. <i>Results</i>: In our study, the area of technical uncertainty (ATU) percentage was initially high at 4 hours but decreased significantly in later incubation periods. At 4 hours, none of the <i>S. aureus</i> isolates showed >90% categorical agreement (CA) for any antimicrobial tested. However, clindamycin achieved the highest CA (100%) at 6 hours and 90% thereafter, with no very major errors (VME) or major error (ME). Cefoxitin required 8 hours to reach >90% CA, with no VME observed at any time point, but up to 75% ME at 8 hours. At 4 hours, most antimicrobials had high (>1.5%) rates of VME among <i>Enterobacteriales</i>. By 6 hours, only Meropenem and Gentamicin had >90% CA, with no VME observed for other antibiotics. <i>Conclusion</i>: The RAST method is relatively easy to implement in clinical microbiology labs, offering cost-effectiveness, simplicity, and rapid results, especially in resource-limited settings. However, reporting RAST results can be complex due to potential challenges with CA, VME, and ME, particularly in the initial hours of incubation and within the ATU.

The accuracy and turnaround time (TAT) of the QuickMIC system (QMIC) for rapid antimicrobial susceptibility testing (AST) of Gram-negative pathogens from positive blood cultures were evaluated, in comparison with a standard-of-care workflow based on culture and AST with broth microdilution. Essential agreement (EA) and bias were evaluated according to the ISO 20776-2:2021, category agreement (CA), categorical overestimation (CO), and underestimation (CU) rates were evaluated according to the European Committee on Antimicrobial Susceptibility Testing(EUCAST) breakpoints, and major discrepancy (MD) and very major discrepancy (VMD) rates were evaluated according to the Food and Drug Administration (FDA) guidelines and breakpoints. QMIC yielded evaluable data for 103/118 blood cultures (87.3%), including Enterobacterales (N = 82), Pseudomonas aeruginosa (N = 16), and Acinetobacter baumannii complex (N = 5). Overall, 41 isolates (39.8%) were positive for extended-spectrum β-lactamases and/or carbapenemases. QMIC exhibited EA and CA values of 89.5% and 93.3%, respectively, with values <90% were reported for cefepime, meropenem, and piperacillin-tazobactam. Mean bias was -23.1%, with values >±30% observed with colistin and piperacillin-tazobactam. Overall, CO and CU were observed in 2.3% and 4.4% of cases. MD and VMD with FDA breakpoints were observed in 1.5% and 8.9% of cases, respectively. The lowest accuracy of QMIC in terms of CU was observed with cefepime, colistin, and gentamicin and in terms of VMD with ceftazidime-avibactam, meropenem, and piperacillin-tazobactam. The mean TAT for QMIC was 192 ± 18 min. QMIC demonstrated the potential for rapid AST, with a mean time to result of less than 4 hours, and an overall acceptable accuracy, except with some β-lactams. Further studies are warranted to assess the potential impact of QMIC implementation on clinical outcomes.IMPORTANCEThe introduction of rapid antimicrobial susceptibility testing (AST) systems for positive blood cultures (BCs) is one of the most promising technological advancements to improve bloodstream infections diagnosis, by providing faster results useful for antimicrobial stewardship. The value of such systems is expected to be higher in settings of high endemicity of antimicrobial resistance, where phenotype prediction is more difficult, and rates of inaccurate empirical therapies are expected to be higher. In this study, we investigated the accuracy and turnaround time (TAT) of a new rapid AST system for positive BCs, in comparison with a standard-of-care workflow based on broth microdilution, including several cases positive for extended-spectrum β-lactamase- and/or carbapenemase-producing Gram negatives. TAT was 192 ± 18 min, and a good accuracy was observed for testing some agents (amikacin, gentamicin, ciprofloxacin, colistin, and ceftazidime-avibactam) but a lower accuracy for testing other agents (cefepime, meropenem, and piperacillin-tazobactam). Further assessment of this technology, after improving accuracy, is warranted to evaluate the clinical impact.

Comparative evaluation of the QMAC-dRAST V2.0 system for rapid antibiotic susceptibility testing of Gram-negative blood culture isolates

Piperacillin-tazobactam (P/T) is a β-lactam-β-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.

To evaluate the performance of the Accelerate Pheno™ system for the identification and antimicrobial susceptibilitytesting(AST) of a panel of Gram-negative bacilli (GNB) with different resistance profiles (e.g. penicillinases, ESBLs, cephalosporinase overproduction, carbapenemases, impermeability) directly from positive blood cultures in <7 h. A panel of 105 clinical strains previously characterized for the presence of β-lactamase-encoding genes was tested. Approximately 100 cfu of each isolate was inoculated into sterile blood culture bottles and incubated in a BD BACTEC™ FX automated system (Becton Dickinson, USA). Positive blood cultures were subjected to parallel testing using the Accelerate Pheno™ system and conventional culture methods [identification of isolated colonies by MALDI-TOF and VITEK® 2 system (bioMérieux, France), and AST by disc diffusion and Etest following EUCAST recommendations]. The overall identification agreement between the Accelerate Pheno™ system and conventional culture methods was 100% (105/105). The overall categorical agreement between the system and culture-based AST was 94.9% (1169/1232), with rates for minor errors of 4.1% (51/1232), major errors 0.3% (4/1232) and very major errors 0.7% (8/1232). The Accelerate Pheno™ system produced AST results indicative of third-generation cephalosporinases (26/26) and carbapenem-resistant strains (52/55). The Accelerate Pheno™ system is an accurate, sensitive and easy-to-use test for the rapid identification and AST of MDR GNB in bloodstream infections. Given the burden of multidrug resistance, its implementation in the microbiology laboratory could be a useful tool for prompt management of sepsis.

The characterization of wild-type minimum inhibitory concentration (MIC) and zone diameter distributions with the setting of epidemiological cut-off values (ECOFFs or ECVs) provides a reference for the otherwise relative MIC values in the international system for antimicrobial susceptibility testing. Distributions of MIC values for a species and an agent follow a log-normal distribution, which in the absence of resistance mechanisms is monomodal and designated wild type (WT). The upper end of the WT distribution, the ECOFF, can be identified with statistical methods. In the presence of phenotypically detectable resistance, the distribution has at least one more mode (the non-WT), but despite this, the WT is most often identifiable using the same methods. The ECOFF provides the most sensitive measure of resistance development in a species against an agent. The WT and non-WT modes are independent of the organism´s response to treatment, but when the European Committee on Antimicrobial Susceptibility Testing (EUCAST) determines the clinical breakpoints, the committee avoids breakpoints that split WT distributions of target species. This is to avoid the poorer reproducibility of susceptibility categorization when breakpoints split major populations but also because the EUCAST has failed to identify different clinical outcomes for isolates with different MIC values inside the wild-type distribution. In laboratory practice, the ECOFF is used to screen for and exclude resistance and allows the comparison of resistance between systems with different breakpoints from different breakpoint organizations, breakpoints evolving over time, and different breakpoints between human and animal medicine. The EUCAST actively encourages colleagues to question MIC distributions as presented on the website (https://www.eucast.org/mic_and_zone_distributions_and_ecoffs) and to contribute MIC and inhibition zone diameter data.

Introduction: Acinetobacter species in clinical isolates cause severe infections including meningitis, bloodstream infection, ventilator-associated pneumonia, and surgical site infections. Objectives: In the present study, we evaluated Acinetobacter drug resistance using both European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical and Laboratory Standards Institute (CLSI) antimicrobial susceptibility test methods. Materials and Methods: Clinical specimens of 128 patients who were admitted in three referral tertiary care teaching hospitals were enrolled in 2014. Blood and other sterile fluid samples, endotracheal secretion, ulcer, urine and other clinical specimen cultures were included, and microbial resistance of Acinetobacter isolates was determined and compared with disk diffusion and E-test antimicrobial susceptibility methods, using both the EUCAST and CLSI standards. Cohen’s kappa coefficient was also reported. Results: The highest percentage of resistance (96.9%) was found for meropenem and imipenem antimicrobials, and the lowest resistance (82.8%) was found for amikacin. The highest kappa agreement coefficient was for ciprofloxacin (kappa coefficient = 0.783), and the lowest kappa was for amikacin (kappa coefficient = 0.21). Conclusion: According to the results, it is better to consider amikacin as a choice in combination with another effective antimicrobial for treatment of drug resistant Acinetobacter.

Oral second- and third-generation cephalosporins are increasingly used as transitional therapy for Escherichia coli bloodstream infection, particularly in patients with resistance and intolerance to first-line agents. However, many automated antimicrobial susceptibility testing (AST) systems only assess parenteral cephalosporins, potentially delaying transitions of care. Limited data exist on the correlation and surrogacy between parenteral and oral cephalosporin AST. We aimed to determine whether AST for parenteral cefazolin, ceftriaxone or cefotaxime can serve as surrogates to predict susceptibility for oral cephalosporins. E. coli blood isolates susceptible to parenteral third-generation cephalosporins were consecutively collected from unique patients from two health systems in Phoenix, AZ, USA from January 2021 to 2023. Broth microdilution was performed according to CLSI. Categorical agreement (CA) rates were calculated using cefazolin, ceftriaxone, or cefotaxime as the surrogate antibiotic for oral cephalosporins (cefuroxime, cefaclor, cefprozil, cefdinir, cefpodoxime, cefixime). Discrepancies were classified as minor errors (mE), major errors (ME), or very major errors (VME) per CLSI. Among 200 isolates, susceptibility to cefazolin at MIC ≤2 mg/L, cefazolin at MIC ≤16 mg/L, ceftriaxone and cefotaxime at MIC ≤1 mg/L were 43%, 91.5% and 100%, respectively. Cefazolin MIC ≤2 mg/L provided high CA across oral cephalosporins but excessive mE (25%-37%) and ME (20%-40%) rates. Ceftriaxone and cefotaxime achieved 100% CA for oral third-generation cephalosporins with mE rates within acceptable limits; however, CA was suboptimal for oral second-generation agents (83%-88%). No surrogate antibiotic met acceptable CLSI criteria due to lack of CA, however, ceftriaxone/cefotaxime achieved acceptable limits in CA, mE, ME and VME. Prediction of oral second-generation cephalosporins may warrant cefazolin MIC ≤2 mg/L, despite limitations regarding false resistance and susceptibility discordant. Ceftriaxone/cefotaxime demonstrates a more reliable surrogate for predicting oral third-generation cephalosporins susceptibility. Future investigations are necessary to clinically corroborate these findings.

The critical influence of the intermediate category on interpretation errors in revised EUCAST and CLSI antimicrobial susceptibility testing guidelines

Assessment of version 2.5 of QMAC-dRAST for rapid antimicrobial susceptibility testing with reduced sample-to-answer turnaround time and an integrated expert system

Predicting doripenem susceptibility based on meropenem and imipenem interpretation for Pseudomonas aeruginosa

Schizophyllum commune is a cosmopolitan, saprophytic basidiomycete known to cause respiratory tract infections following spore inhalation. Management of such infections remained poorly defined, and antifungal susceptibility testing may provide valuable guidance for therapeutic decisions. However, existing data are limited, and protocols are not optimized for non-sporulating molds such as S. commune, requiring methodological adaptations. This study aimed to (i) perform molecular characterization of isolates and (ii) determine antifungal susceptibility profiles of a collection of S. commune isolates using both Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) reference methods. A total of 113 fungal isolates were included, comprising environmental (n = 31), clinical French isolates (n = 74), and strains from fungal international collections (n = 8). Species identification was confirmed via the large subunit (LSU) region of the ribosomal DNA sequencing and by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Phylogenetic analysis was conducted on a subset of 20 isolates using partial sequences of three genes: LSU, EF-1α, and RPB2. Modifications to the CLSI and EUCAST broth microdilution methods included: inoculum preparation using standardized culture slices fragmented by bead-beating, incubation at 35°C for 96 h and endpoints reading at 100% of inhibition. Phylogenetic analysis confirmed that all tested isolates were S. commune and not S. radiatum. Amphotericin B (AMB) and voriconazole (VOR) demonstrated low geometrical mean minimal inhibitory concentrations (MICs) across both methods (EUCAST: AMB = 0.39 µg/mL, VOR = 0.24 µg/mL; CLSI: AMB = 0.1 µg/mL, VOR = 0.2 µg/mL). In contrast, terbinafine exhibited high MIC values (>8 µg/mL) in both protocols. Notable discrepancies were observed for posaconazole (POS), isavuconazole (ISA), and itraconazole (ITR), with MICs approximately two dilution steps higher in EUCAST compared to CLSI (POS: 4.22 µg/mL vs 0.9 µg/mL, ISA: 3.64 µg/mL vs 0.74 µg/mL, and ITR: 3.9 µg/mL vs 0.81 µg/mL, respectively). After standardization of both methods for this non-sporulating mold, VOR and AMB have the lowest MIC values.IMPORTANCESchizophyllum commune is a fungal pathogen increasingly associated with respiratory infections, yet therapeutic guidance remains unclear. This study provides the largest collection to date of clinical and environmental isolates (113 total) and applies standardized antifungal susceptibility testing using adapted EUCAST and CLSI protocols for this non-sporulating species. The results show that amphotericin B and voriconazole are the most active agents in vitro, while terbinafine is ineffective. These findings are critical for informing treatment decisions and interpreting susceptibility tests, especially in the absence of established guidelines. By introducing a reproducible methodology and delivering clinically relevant data, this work addresses a gap in medical mycology and supports improved management of rare fungal infections.

Correlation between the procedure for antifungal susceptibility testing for Candida spp. of the European Committee on Antibiotic Susceptibility Testing (EUCAST) and four commercial techniques