EUCAST Technical Note on Voriconazole and Aspergillus spp.

The European Committee on Antimicrobial Susceptibility Testing Subcommittee on Antifungal Susceptibility Testing (EUCAST-AFST) has determined breakpoints for voriconazole against Aspergillus spp. This Technical Note is based on the EUCAST rationale document for voriconazole (available on the EUCAST website: http://www.eucast.org). Voriconazole breakpoints are based on epidemiological cut-off values, pharmacokinetic/pharmacodynamic data and clinical experience. Breakpoints will be reviewed regularly or when new data emerge.     

EUCAST Technical Note on Aspergillus and amphotericin B,itraconazole, and posaconazole

The European Committee on Antimicrobial Susceptibility Testing Subcommittee on Antifungal Susceptibility Testing (EUCAST-AFST) has determined breakpoints for amphotericin B, itraconazole and posaconazole for Aspergillus species. This Technical Note is based on the EUCAST amphotericin B, itraconazole and posaconazole rationale documents (available on the EUCAST website: http://www.eucast.org/antifungal_susceptibility_testing_afst/rationale_documents_for_antifungals/). The amphotericin B and itraconazole breakpoints are based on epidemiological cut-off values and clinical experience. The posaconazole breakpoints are also based on pharmacokinetic and pharmacodynamic data. Breakpoints will be reviewed regularly or when new data emerge.     

EUCAST technical note on anidulafungin

The European Committee on Antimicrobial Susceptibility Testing-Subcommittee on Antifungal Susceptibility Testing has determined breakpoints for anidulafungin for Candida spp. This Technical Note is based on the EUCAST anidulafungin rationale document (available at: http://www.eucast.org). Species-specific breakpoints for C. albicans are S ≤0.03 mg/L and R >0.03 mg/L and for C. glabrata, C. tropicalis and C. krusei S ≤0.06 mg/L and R >0.06 mg/L. C. parapsilosis was not regarded a good target for anidulafungin. There are insufficient data to set breakpoints for other species. The breakpoints are based upon pharmacokinetic data, epidemiological cut-off values and clinical experience. Breakpoints will be reviewed regularly.     

EUCAST technical note on posaconazole

The European Committee on Antimicrobial Susceptibility Testing-Subcommittee on Antifungal Susceptibility Testing (EUCAST-AFST) has determined breakpoints for posaconazole for Candida spp. This Technical Note is based on the EUCAST posaconazole rationale document (available on the EUCAST website: http://www.eucast.org). Species-specific breakpoints for C. albicans, C. parapsilosis and C. tropicalis are S: MIC ≤0.06 mg/L, R: MIC >0.06 mg/L. There are insufficient data to set breakpoints for C. glabrata and C. krusei as well as non-species-related breakpoints. The breakpoints are based upon pharmacokinetic data, epidemiological cut-off values and clinical experience. Breakpoints will be reviewed regularly.     

Antimicrobial susceptibility testing of Kingella kingae with broth microdilution and disk diffusion using EUCAST recommended media

ObjectivesIncreasing use of improved culture techniques and sensitive nucleic acid amplification assays have resulted in recognition of Kingella kingae as an important cause of invasive infections in young children, especially in septic arthritis, osteomyelitis, bacteraemia, and endocarditis. In 2016, EUCAST established clinical MIC breakpoints for K. kingae (published in EUCAST Clinical Breakpoint Tables v 7.0, 2017). The present study was carried out to produce MIC-zone diameter correlations for K. kingae on an international collection of isolates, with the aim of suggesting zone diameter breakpoints corresponding to the clinical MIC breakpoints.MethodsAntimicrobial susceptibility testing was performed for 18 clinically relevant agents on a collection of 159 clinical isolates of K. kingae. Broth microdilution MIC determination and disk diffusion were performed according to EUCAST recommendations for fastidious organisms.ResultsThe correlation between MICs and zone diameters was good for all agents with EUCAST breakpoints for K. kingae. β-lactamase was detected in 41 isolates (26%) and these isolates were resistant to aminopenicillins. These isolates were also resistant to trimethoprim-sulfamethoxazole. Resistance to tetracyclines was detected in 8% of all isolates. All resistant isolates were correctly categorized for these agents with the proposed zone diameter breakpoints. One isolate, resistant to erythromycin but susceptible to other macrolides, was categorized as susceptible with erythromycin disk diffusion. No resistance was detected for the cephalosporins, carbapenems, and fluoroquinolones tested.ConclusionBased on the results in this study, zone diameter breakpoints for K. kingae calibrated to EUCAST clinical MIC breakpoints were proposed and approved by EUCAST.     

Comparison of antimicrobial susceptibility interpretation among Enterobacteriaceae using CLSI and EUCAST breakpoints

PurposeTo determine the difference in antimicrobial susceptibility of various antibiotics using the CLSI & EUCAST breakpoints.MethodsIn this non interventional, retrospective observational study, we reviewed minimum inhibitory concentrations (MIC) of various antibiotics routinely reported for Enterobacteriaceae clinical isolates, from an automated microbiology identification system (VITEK-2). These MICs were then analysed using both CLSI 2019 and EUCAST 2019 guidelines and classified as per the breakpoints into various categories.ResultsThe concordance rates of the antimicrobial susceptibility for various drugs ranged from 78.2% to 100% among two breakpoints. Perfect agreement with κ = 1 (p < 0.001) was observed for only three antimicrobials ceftriaxone, levofloxacin and trimethoprim-sulfamethoxazole. The changes in antimicrobial susceptibility interpretation for cefepime, ciprofloxacin, amoxicillin clavulanic acid was majorly in Intermediate category.ConclusionThe change in interpretation of the susceptibility will lead to change in the usage of antibiotics especially due to recent change in definition of I by EUCAST. There is need of more studies in this aspect to ascertain clinical implication of change in antimicrobial susceptibility.     

EUCAST technical note on Amphotericin B

The European Committee on Antimicrobial Susceptibility Testing-Subcommittee on Antifungal Susceptibility Testing (EUCAST-AFST) has determined breakpoints for amphotericin B for Candida spp. This Technical Note is based on the EUCAST amphotericin B rationale document (available on the EUCAST website: http://www.eucast.org). Species-specific breakpoints for C. albicans, C. glabrata, C. krusei, C. parapsilosis and C. tropicalis are S: MIC ≤1 mg/L, R: MIC > 1 mg/L. There are insufficient data to set breakpoints for other species. The breakpoints are based upon pharmacokinetic data, epidemiological cut-ff values and clinical experience. Breakpoints will be reviewed regularly.     

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

The correlation between results obtained with the European Committee on Antibiotic Susceptibility Testing (EUCAST) antifungal susceptibility testing procedure (document 7.1) and four commercial systems was evaluated for a collection of 93 clinical isolates of Candida spp. Overall, agreement between the EUCAST procedure and the Sensititre YeastOne and Etest methods was 75% and 90.4%, respectively. The correlation indices (p < 0.01) between the EUCAST and commercial methods were 0.92 for Sensititre YeastOne, 0.89 for Etest, - 0.90 for Neo-Sensitabs, and 0.95 for Fungitest. Amphotericin B MICs obtained by Sensititre YeastOne were consistently higher than with the EUCAST method and, although very major errors were not observed, 91% of MICs were misclassified. Amphotericin B- and fluconazole-resistant isolates were identified correctly with Sensititre YeastOne, Etest and Fungitest. Neo-Sensitabs identified amphotericin B-resistant isolates, but misclassified > 5% of fluconazole-resistant isolates as susceptible. The commercial methods, particularly Etest and Fungitest, appeared to be suitable alternatives to the EUCAST procedure for antifungal susceptibility testing of clinical isolates of Candida.     

EUCAST expert rules in antimicrobial susceptibility testing

EUCAST expert rules have been developed to assist clinical microbiologists and describe actions to be taken in response to specific antimicrobial susceptibility test results. They include recommendations on reporting, such as inferring susceptibility to other agents from results with one, suppression of results that may be inappropriate, and editing of results from susceptible to intermediate or resistant or from intermediate to resistant on the basis of an inferred resistance mechanism. They are based on current clinical and/or microbiological evidence. EUCAST expert rules also include intrinsic resistance phenotypes and exceptional resistance phenotypes, which have not yet been reported or are very rare. The applicability of EUCAST expert rules depends on the MIC breakpoints used to define the rules. Setting appropriate clinical breakpoints, based on treating patients and not on the detection of resistance mechanisms, may lead to modification of some expert rules in the future.     

Validation of EUCAST zone diameter breakpoints against reference broth microdilution

The European Committee on Antimicrobial Susceptibility Testing (EUCAST) began harmonizing clinical breakpoints in Europe 2002. In 2009, work to develop a disc diffusion method began and the first disc diffusion breakpoints calibrated to EUCAST clinical MIC breakpoints were published in December 2009. In this study we validated EUCAST clinical zone diameter breakpoints against the International Standard Organization (ISO) reference broth microdilution. A collection of 544 isolates (238 Gram-negative and 306 Gram-positive) were tested against a panel of antimicrobial agents. Antimicrobial susceptibility testing was performed with broth microdilution as described by ISO and disc diffusion in accordance with EUCAST methodology. Inhibition zone diameters and MIC values were interpreted and categorized (S, I and R) according to EUCAST clinical breakpoint table version 2.0. Categorical agreement (CA) as well as minor (mD), major (MD) and very major (VMD) discrepancies were determined. There was in general good correlation between susceptibility test results obtained with disc diffusion and broth microdilution. Overall CA was 97.3% for all combinations of organisms and antimicrobial agents (<n = 5231) and the overall discrepancy rates were 110 (2.1%) mD, 24 (0.5%) MD and 7 (0.1%) VMD. The overall CA for Gram-positive and Gram-negative organisms were 98.7% (2346 tests) and 96.2% (2942 tests), respectively. Seven VMD were observed, five for Gram-positive organisms (coagulase negative staphylococci (<n = 2) and Staphylococcus aureus (<n = 3)) and two for Gram-negative organisms (<Pseudomonas aeruginosa). Minor discrepancies were mainly observed in Gram-negatives and were related to different antimicrobial agents and species.     

European Committee on Antimicrobial Susceptibility Testing (EUCAST) Technical Notes on antimicrobial susceptibility testing

The main objectives of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) are to harmonise breakpoints for antimicrobial agents in Europe, and to act as the breakpoint committee for the European Medicines Agency (EMEA) during the registration of new antimicrobial agents. Detailed EUCAST procedures for harmonising and setting breakpoints for antimicrobial agents are available on the EUCAST website. Beginning with the current issue, a series of EUCAST Technical Notes will be published in CMI, based on the rationale documents produced by EUCAST for each of the antimicrobial agents studied, with the aim of highlighting important background information underlying decisions on breakpoints made by EUCAST.     

EUCAST technical note on the EUCAST definitive document EDef 7.2: method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for yeasts EDef 7.2 (EUCAST-AFST)

The European Committee on Antimicrobial Susceptibility Testing-Subcommittee on Antifungal Susceptibility Testing (EUCAST-AFST) has revised the EDef 7.1 document on the method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for fermentative yeasts. Changes are: dimethylsulphoxide is now the recommended solvent for caspofungin, micafungin and fluconazole; the shelf-life of plates containing the echinocandins prepared from stock solutions in dimethylsulphoxide is extended to 6 months at ?80°C; testing of amphotericin and Cryptococcus has been incorporated; and minimum inhibitory concentration ranges for quality control strains and anidulafungin are included.     

Multicentre determination of rezafungin (CD101) susceptibility of Candida species by the EUCAST method

ObjectivesRezafungin (CD101) is a new long-acting echinocandin allowing weekly dosing, currently undergoing phase-II clinical trials for invasive candidiasis. The aim of this study was to assess rezafungin's in vitro activity against the most frequent Candida species following the EUCAST methodology.MethodsThe susceptibility of 2018 clinical Candida isolates was determined at four European laboratories. In parallel, six control strains were repeatedly tested. Wild-type upper limits (WT-ULs), defined as the MIC value where the wild-type distribution ends, were determined following the principles for EUCAST ECOFF-setting.ResultsThe lowest rezafungin MICs (geometric MIC (GM-MIC), MIC range (mg/L)) were observed for C. albicans (0.016, 0.002–0.125) and the highest for C. parapsilosis (1.657, 0.063->4). MICs for the remaining species were in between these values (GM-MICs 0.048–0.055). Visual and statistical WT-ULs were identical for C. glabrata (0.125), C. krusei (0.125), C. parapsilosis (4), and C. tropicalis (0.25). If adopting these WT-ULs for classification into WT and non-WT populations, 1/413 C. glabrata, 1/402 C. krusei, 1/398 C. parapsilosis, and 1/402 C. tropicalis isolates were categorized as non-WT, all of which derived from Laboratory 1. For C. albicans unexplained laboratory variation was observed (WT-UL: 0.063–0.125 in Laboratories 1 and 2 versus 0.016 in Laboratories 3 and 4). A similar systematic difference was observed comparing the MICs for the three C. albicans QC strains, specifically, obtained in Laboratories 1and 2 with those in Laboratories 3 and 4.DiscussionRezafungin displayed species-specific activity similar to other echinocandins. Interlaboratory variation was observed for the most susceptible species C. albicans clinical and QC strains, an observation that warrants further investigation.     

Factors influencing the trailing endpoint observed in Candida albicans susceptibility testing using the CLSI procedure.

The trailing endpoint phenotype observed during testing of Candida albicans susceptibility to azoles according to the CLSI procedure is defined as a difference in MIC depending on whether the result is obtained after 24 or 48 h. This study investigated whether intrinsic differences between the EUCAST and CLSI methods could explain trailing growth. The glucose concentration in the medium and the shape of the microtitre plate wells were both found to be involved. In order to reduce the incidence of trailing growth according to the CLSI procedure, the use of higher glucose concentrations and flat-bottomed microtitre plates could be valuable improvements.     

Development of a EUCAST disk diffusion method for the susceptibility testing of rapidly growing anaerobic bacteria using Fastidious Anaerobe Agar (FAA): a development study using Bacteroides species

ObjectivesAntimicrobial resistance among anaerobic bacteria is increasing, leading to a growing demand for inexpensive and reliable susceptibility testing methods. The aim of this study was to determine the suitability of Fastidious Anaerobe Agar (FAA) as a medium for disk diffusion for rapidly growing anaerobic bacteria.MethodsReproducibility of zone diameters and quality of growth were tested using six quality control (QC) strains. We compared four anaerobic incubation systems, two incubation temperatures (35°C and 37°C), and FAA from four manufacturers. The effect of incubation for 16–20 hours instead of 24 hours was tested on ten randomly selected isolates of the Bacteroides fragilis group. The final method was tested on 170 clinical B. fragilis-group isolates and compared to agar dilution MICs.ResultsAfter 24 hours' incubation, all QC strains demonstrated confluent growth. The different anaerobic incubation systems were equal regarding quality of growth and zone diameters. Incubation at 35°C resulted in slightly larger zones (1–2 mm) than at 37°C. Except for Acumedia FAA, the different manufacturers showed good agreement in zone diameters. All B. fragilis-group isolates displayed confluent growth after 16–20 hours. Metronidazole inhibition zones correlated well with the reference MICs. There was an area of poorer separation for meropenem and piperacillin–tazobactam between 19-27 and 14-23 mm respectively. Prolonged incubation (40–44 h) of clindamycin resulted in better separation and the area of overlap was reduced from 13 to 8 mm compared with 16–20 hours' incubation.ConclusionFAA is a suitable medium for disk diffusion of these rapidly growing anaerobic bacteria.