Gradient Diffusion Tests

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.

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

<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 Tigecycline In Vitro Surveillance in Taiwan (TIST) study, initiated in 2006, is a nationwide surveillance program designed to longitudinally monitor the in vitro activity of tigecycline against commonly encountered drug-resistant bacteria. This study compared the in vitro activity of tigecycline against 3,014 isolates of clinically important drug-resistant bacteria using the standard broth microdilution and disk diffusion methods. Species studied included methicillin-resistant Staphylococcus aureus (MRSA; n = 759), vancomycin-resistant Enterococcus faecium (VRE; n = 191), extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (n = 602), ESBL-producing Klebsiella pneumoniae (n = 736), and Acinetobacter baumannii (n = 726) that had been collected from patients treated between 2008 and 2010 at 20 hospitals in Taiwan. MICs and inhibition zone diameters were interpreted according to the currently recommended U.S. Food and Drug Administration (FDA) criteria and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. The MIC(90) values of tigecycline against MRSA, VRE, ESBL-producing E. coli, ESBL-producing K. pneumoniae, and A. baumannii were 0.5, 0.125, 0.5, 2, and 8 μg/ml, respectively. The total error rates between the two methods using the FDA criteria were high: 38.4% for ESBL-producing K. pneumoniae and 33.8% for A. baumannii. Using the EUCAST criteria, the total error rate was also high (54.6%) for A. baumannii isolates. The total error rates between these two methods were <5% for MRSA, VRE, and ESBL-producing E. coli. For routine susceptibility testing of ESBL-producing K. pneumoniae and A. baumannii against tigecycline, the broth microdilution method should be used because of the poor correlation of results between these two methods.

Activity of telavancin and comparator antimicrobial agents tested against Staphylococcus spp. isolated from hospitalised patients in Europe (2007–2008)

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.

Antimicrobial susceptibility testing (AST) results are among the most important pieces of information a clinical microbiology lab can release and clinicians rely heavily on them to care for their patients. Due to the clinical importance of these results, it is imperative that testing is performed under optimal conditions with standardized approaches to quality control, interpretation and reporting. There are a variety of in vitro methods to determine bacterial antimicrobial susceptibilities as well as interpretive criteria and testing limitations that have been vetted through the Clinical and laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial susceptibility testing (EUCAST). The goal in Part I of this two part section is to review the general background, breakpoint development and Governance of AST testing and in Part II review the standardization of the various method of AST, their limitations and associated challenges and review the future of antimicrobial susceptibility testing using newer technologies.

Antimicrobial Resistance in the Intensive Care Unit: Mechanisms, Epidemiology, and Management of Specific Resistant Pathogens

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.

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.

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.

The European Committee on Antimicrobial Susceptibility Testing (EUCAST) is an international susceptibility testing committee, organized by the European Society for Clinical Microbiology and Infectious Diseases (ESCMID) and functioning as the breakpoint advisory committee of the European Medicines Agency (EMA). The original remit of EUCAST was to harmonize European clinical breakpoints, but very soon, the activities expanded beyond the borders of Europe and included newly licensed agents in Europe. Among the milestones were the aggregating of large numbers of MIC distributions, creating software to display these distributions, the EUCAST concept of identifying epidemiological cutoff values (ECOFF), and the development of a EUCAST disk diffusion method. The EUCAST Development Laboratory has played a critical role in the development of antimicrobial susceptibility testing (AST) methodology, including development work for novel antimicrobial agents and for rapid AST directly from blood culture bottles. EUCAST has several standing subcommittees, including for AST in fungi (AFST) and mycobacteria (AMST) and for microorganisms of veterinary interest (VetCAST), and ad hoc subcommittees on subjects such as anaerobic bacteria, MIC and zone diameter distributions and epidemiological cutoff values, the relationship between phenotypic and genotypic resistance, and expert rules and methods for the detection of resistance mechanisms. All EUCAST decisions are subjected to the EUCAST public consultation process, the only exception being breakpoints of novel antimicrobial agents where confidentiality agreements during the licensing process prevent public participation. EUCAST has recently revised the definitions of clinical susceptibility interpretive categories S, I, and R, acknowledging the intimate relationship between drug exposure and susceptibility reporting.

The development of new antimicrobial agents is essential to combat antimicrobial resistance. Reliable antimicrobial susceptibility testing (AST) methods should be established early and thoughtfully to ensure timely patient access to these agents. The standard reference method for AST is broth microdilution (BMD) in cation-adjusted Mueller-Hinton broth (CAMHB), as defined by CLSI M07 (CLSI. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. 12th ed. CLSI Standard M07. Clinical and Laboratory Standards Institute, 2024) and ISO 20776-1 (ISO. Susceptibility Testing of Infectious Agents and Evaluation of Performance of Antimicrobial Susceptibility Test Devices-Part 1: Broth Micro-dilution Reference Method for Testing the In Vitro Activity of Antimicrobial Agents Against Rapidly Growing Aerobic Bacteria Involved in Infectious Diseases. ISO 20776-1. International Organization for Standardization, 2019). While some agents may require modifications to this method to better reflect clinical activity, such changes must be scientifically justified. Modifications aimed solely at producing lower minimal inhibitory concentration (MIC) values-or to make one antimicrobial agent appear superior to others-are not scientifically valid and are strongly discouraged. Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) caution developers that unnecessary deviations from reference AST methods can lead to increased costs, regulatory hurdles, delays in test availability and reduced clinical adoption. Until joint guidance under development by CLSI and EUCAST is finalized, early and rigorous evaluation using the reference method is essential.

Development of a national EUCAST challenge panel for antimicrobial susceptibility testing

Tigecycline can be effective to treat infections of carbapenem-resistant Acinetobacter baumannii (CRAB) however, no interpretive criteria have been approved so far. The objectives of this study were to evaluate the proportion of CRAB isolates and to compare gradient test with a broth microdilution (BMD) method for tigecycline susceptibility testing of A. baumannii.This study included 349 multidrug-resistant (MDR) Acinetobacter spp. collected from Serbia, Montenegro, Bosnia and Herzegovina in 2016 and 2017. Antibiotic susceptibility testing was performed by disk diffusion, VITEK2, gradient, ComASP Colistin. Tigecycline susceptibilities were interpreted according to breakpoints of European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Food and Drug Administration (FDA).Majority of the tested isolates were CRAB (92.8%). Tigecycline MIC50/MIC90 values were 4/8 μg/mL by BMD and 0.5/4 μg/mL by gradient test. Essential agreement for BMD and gradient test amounted to 65.1%. With EUCAST breakpoints, categorical agreement (CA) was achieved in 38% isolates. Major discordance (MD-false susceptibility/resistance) and minor discordance (mD-false categorization involving intermediate results) were observed in 10% and 57% A. baumannii, respectively. With FDA breakpoints, CA, MD and mD were observed in 44%, 16% and 47% isolates, respectively. Colistin resistance was 2.1%.The study highlights a high proportion of CRAB and several discordances between BMD and gradient test which may lead to inappropriate therapy.