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Objectives The procedure for setting clinical breakpoints (CBPs) for antimicrobial susceptibility has been poorly standardized with respect to population data, pharmacokinetic parameters and clinical outcome. Tools to standardize CBP setting could result in improved antibiogram forecast probabilities. We propose a model to estimate probabilities for methodological categorization errors and defined zones of methodological uncertainty (ZMUs), i.e. ranges of zone diameters that cannot reliably be classified. The impact of ZMUs on methodological error rates was used for CBP optimization. Methods The model distinguishes theoretical true inhibition zone diameters from observed diameters, which suffer from methodological variation. True diameter distributions are described with a normal mixture model. The model was fitted to observed inhibition zone diameters of clinical Escherichia coli strains. Repeated measurements for a quality control strain were used to quantify methodological variation. Results For 9 of 13 antibiotics analysed, our model predicted error rates of < 0.1% applying current EUCAST CBPs. Error rates were > 0.1% for ampicillin, cefoxitin, cefuroxime and amoxicillin/clavulanic acid. Increasing the susceptible CBP (cefoxitin) and introducing ZMUs (ampicillin, cefuroxime, amoxicillin/clavulanic acid) decreased error rates to < 0.1%. ZMUs contained low numbers of isolates for ampicillin and cefuroxime (3% and 6%), whereas the ZMU for amoxicillin/clavulanic acid contained 41% of all isolates and was considered not practical. Conclusions We demonstrate that CBPs can be improved and standardized by minimizing methodological categorization error rates. ZMUs may be introduced if an intermediate zone is not appropriate for pharmacokinetic/pharmacodynamic or drug dosing reasons. Optimized CBPs will provide a standardized antibiotic susceptibility testing interpretation at a defined level of probability.

Background In principle, automated systems allow rapid reading of disc diffusion AST (rAST) within 6-8 h. Objectives This study analysed whether rAST can discriminate resistance phenotypes such as ESBL, carbapenemases and MRSA/methicillin-resistant Staphylococcus epidermidis from WT populations. We describe species-drug combinations that may require clinical breakpoint adaptions for early reading due to zone diameter changes during the incubation period. Methods In total, 1852 clinical strains [Escherichia coli (n = 475), Klebsiella pneumoniae (n = 375), Enterobacter cloacae (n = 301), Staphylococcus aureus (n = 407) and S. epidermidis (n = 294)] were included in this study comprising WT populations and important resistance phenotypes, e.g. ESBL, carbapenemases and MRSA. We assessed (i) separation of resistance phenotypes and WT populations after 6, 8 and 12 h as compared with the 18 h standard, and (ii) diameter changes of WT populations and associated putative epidemiological cut-offs during the incubation period. Disc diffusion plates were automatically streaked, incubated and imaged using the WASPLabTM system. Results and conclusions We demonstrated that important resistance phenotypes could reliably be separated from WT populations at early reading times for the most prevalent bacterial pathogens encountered in the clinical laboratory. Current AST expert rules and algorithms for identification of resistance mechanisms can readily be applied for rAST, e.g. EUCAST recommended rules for detection of ESBL, AmpC, carbapenemases and MRSA/methicillin-resistant S. epidermidis. However, several species-drug combinations may require clinical breakpoint adaptations when using rAST as the diameter, and hence the epidemiological cut-off, changes during the incubation period.

Background Antibiotic resistance poses a significant threat to patients suffering from infectious diseases. Early readings of antibiotic susceptibility test (AST) results could be of critical importance to ensure adequate treatment. Disc diffusion is a well-standardized, established and cost-efficient AST procedure; however, its use in the clinical laboratory is hampered by the many manual steps involved, and an incubation time of 16-18 h, which is required to achieve reliable test results. Methods We have evaluated a fully automated system for its potential for early reading of disc diffusion diameters after 6-12 h of incubation. We assessed availability of results, methodological precision, categorical agreement and interpretation errors as compared with an 18 h standard. In total, 1028 clinical strains (291 Escherichia coli , 272 Klebsiella pneumoniae , 176 Staphylococcus aureus and 289 Staphylococcus epidermidis ) were included in this study. Disc diffusion plates were streaked, incubated and imaged using the WASPLab TM automation system. Results and conclusions Our results demonstrate that: (i) early AST reading is possible for important pathogens; (ii) methodological precision is not hampered at early timepoints; and (iii) species-specific reading times must be selected. As inhibition zone diameters change over time and are phenotype/drug combination dependent, specific cut-offs and expert rules will be essential to ensure reliable interpretation and reporting of early susceptibility testing results.

Background We investigated the feasibility of rapid disc diffusion antibiotic susceptibility testing (rAST) with reading of inhibition zones after 6 and/or 8 h of incubation for Enterococcus faecalis, Enterococcus faecium, Pseudomonas aeruginosa and Acinetobacter baumannii. In addition, we evaluated discrimination of resistant populations from the WT populations at early timepoints and the requirement for clinical breakpoint adaptations for proper interpretation of rAST data. Methods In total, 815 clinical strains [E. faecalis (n = 135), E. faecium (n = 227), P. aeruginosa (n = 295) and A. baumannii (n = 158)] were included in this study. Disc diffusion plates were streaked, incubated and imaged using the WASPLabTM automation system. WT populations and non-WT populations were defined using epidemiological cut-offs. Results and conclusions rAST at 6 and 8 h was possible for A. baumannii and enterococci with readability of inhibition zones >90%. Overall categorical agreement of rAST at 6 h with AST at 18 h was 97.2%, 97.4% and 95.3% for E. faecalis, E. faecium and A. baumannii, respectively. With few exceptions, major categorization error rates were <1% for A. baumannii, and vancomycin-resistant E. faecium were clearly separated from the WT at 6 h. For P. aeruginosa the average readability of inhibition zones was 68.9% at 8 h and we found an overall categorical agreement of 94.8%. Adaptations of clinical breakpoints and/or introduction of technical buffer zones, preferably based on aggregated population data from various epidemiological settings, are required for proper interpretation of rAST.

Whole genome sequencing allows rapid detection of drug-resistant isolates. However, the availability of high-quality data linking quantitative phenotypic drug susceptibility testing (DST) and genomic data has thus far been limited.We determined drug resistance profiles of 176 genetically diverse clinical isolates from Democratic Republic of the Congo, Ivory Coast, Peru, Thailand and Switzerland by quantitative phenotypic DST for 11 antituberculous drugs using the BD BACTEC MGIT 960 system and 7H10 agar dilution to generate a cross-validated phenotypic DST readout. We compared DST results with predicted drug resistance profiles inferred by whole genome sequencing.Classification of strains by the two phenotypic DST methods into resistotype/wild type populations was concordant in 73-99 % of cases, depending on the drug. Our data suggests that the established critical concentration (5 mg/L) for ethambutol resistance (MGIT 960 system) is too high and may misclassify strains as susceptible, compared to 7H10 agar dilution. Increased minimal inhibitory concentrations were explained by mutations identified by whole genome sequencing. Using whole genome sequences, we were able to predict quantitative drug resistance levels for the majority of drug resistance mutations. Predicting quantitative levels of drug resistance by whole genome sequencing was partially limited due to incompletely understood drug resistance mechanisms. The overall sensitivity and specificity of whole genome-based DST were 86.8 % and 94.5 %, respectively.Despite some limitations, whole genome sequencing has the potential to infer resistance profiles without the need for time-consuming phenotypic methods.

Background We investigated the feasibility of rapid disc diffusion antibiotic susceptibility testing (rAST) with reading of inhibition zones after 6 and/or 8 h of incubation for Enterococcus faecalis, Enterococcus faecium, Pseudomonas aeruginosa and Acinetobacter baumannii. In addition, we evaluated discrimination of resistant populations from the WT populations at early timepoints and the requirement for clinical breakpoint adaptations for proper interpretation of rAST data. Methods In total, 815 clinical strains [E. faecalis (n = 135), E. faecium (n = 227), P. aeruginosa (n = 295) and A. baumannii (n = 158)] were included in this study. Disc diffusion plates were streaked, incubated and imaged using the WASPLabTM automation system. WT populations and non-WT populations were defined using epidemiological cut-offs. Results and conclusions rAST at 6 and 8 h was possible for A. baumannii and enterococci with readability of inhibition zones >90%. Overall categorical agreement of rAST at 6 h with AST at 18 h was 97.2%, 97.4% and 95.3% for E. faecalis, E. faecium and A. baumannii, respectively. With few exceptions, major categorization error rates were <1% for A. baumannii, and vancomycin-resistant E. faecium were clearly separated from the WT at 6 h. For P. aeruginosa the average readability of inhibition zones was 68.9% at 8 h and we found an overall categorical agreement of 94.8%. Adaptations of clinical breakpoints and/or introduction of technical buffer zones, preferably based on aggregated population data from various epidemiological settings, are required for proper interpretation of rAST.

Background Antibiotic resistance poses a significant threat to patients suffering from infectious diseases. Early readings of antibiotic susceptibility test (AST) results could be of critical importance to ensure adequate treatment. Disc diffusion is a well-standardized, established and cost-efficient AST procedure; however, its use in the clinical laboratory is hampered by the many manual steps involved, and an incubation time of 16-18 h, which is required to achieve reliable test results. Methods We have evaluated a fully automated system for its potential for early reading of disc diffusion diameters after 6-12 h of incubation. We assessed availability of results, methodological precision, categorical agreement and interpretation errors as compared with an 18 h standard. In total, 1028 clinical strains (291 Escherichia coli , 272 Klebsiella pneumoniae , 176 Staphylococcus aureus and 289 Staphylococcus epidermidis ) were included in this study. Disc diffusion plates were streaked, incubated and imaged using the WASPLab TM automation system. Results and conclusions Our results demonstrate that: (i) early AST reading is possible for important pathogens; (ii) methodological precision is not hampered at early timepoints; and (iii) species-specific reading times must be selected. As inhibition zone diameters change over time and are phenotype/drug combination dependent, specific cut-offs and expert rules will be essential to ensure reliable interpretation and reporting of early susceptibility testing results.

We have recently developed a scalable algorithm for ordering the instantaneous observations of a dynamical system evolving continuously in time. Here, we apply the method to long molecular dynamics trajectories. The procedure requires only a pairwise, geometrical distance as input. Suitable annotations of both structural and kinetic nature reveal the free energy basins visited by biomolecules. The profile is supplemented by a trace of the temporal evolution of the system highlighting the sequence of events. We demonstrate that the resultant SAPPHIRE (States And Pathways Projected with HIgh REsolution) plots provide a comprehensive picture of the thermodynamics and kinetics of complex, molecular systems exhibiting dynamics covering a range of time and length scales. Information on pathways connecting states and the level of recurrence are quickly inferred from the visualisation. The considerable advantages of our approach are speed and resolution: the SAPPHIRE plot is scalable to very large data sets and represents every single snapshot. This minimizes the risk of missing states because of overlap or prior coarse-graining of the data.

Background Antibiotic resistance poses a significant threat to patients suffering from infectious diseases. Early readings of antibiotic susceptibility test (AST) results could be of critical importance to ensure adequate treatment. Disc diffusion is a well-standardized, established and cost-efficient AST procedure; however, its use in the clinical laboratory is hampered by the many manual steps involved, and an incubation time of 16-18 h, which is required to achieve reliable test results. Methods We have evaluated a fully automated system for its potential for early reading of disc diffusion diameters after 6-12 h of incubation. We assessed availability of results, methodological precision, categorical agreement and interpretation errors as compared with an 18 h standard. In total, 1028 clinical strains (291 Escherichia coli , 272 Klebsiella pneumoniae , 176 Staphylococcus aureus and 289 Staphylococcus epidermidis ) were included in this study. Disc diffusion plates were streaked, incubated and imaged using the WASPLab TM automation system. Results and conclusions Our results demonstrate that: (i) early AST reading is possible for important pathogens; (ii) methodological precision is not hampered at early timepoints; and (iii) species-specific reading times must be selected. As inhibition zone diameters change over time and are phenotype/drug combination dependent, specific cut-offs and expert rules will be essential to ensure reliable interpretation and reporting of early susceptibility testing results.

Objectives The procedure for setting clinical breakpoints (CBPs) for antimicrobial susceptibility has been poorly standardized with respect to population data, pharmacokinetic parameters and clinical outcome. Tools to standardize CBP setting could result in improved antibiogram forecast probabilities. We propose a model to estimate probabilities for methodological categorization errors and defined zones of methodological uncertainty (ZMUs), i.e. ranges of zone diameters that cannot reliably be classified. The impact of ZMUs on methodological error rates was used for CBP optimization. Methods The model distinguishes theoretical true inhibition zone diameters from observed diameters, which suffer from methodological variation. True diameter distributions are described with a normal mixture model. The model was fitted to observed inhibition zone diameters of clinical Escherichia coli strains. Repeated measurements for a quality control strain were used to quantify methodological variation. Results For 9 of 13 antibiotics analysed, our model predicted error rates of < 0.1% applying current EUCAST CBPs. Error rates were > 0.1% for ampicillin, cefoxitin, cefuroxime and amoxicillin/clavulanic acid. Increasing the susceptible CBP (cefoxitin) and introducing ZMUs (ampicillin, cefuroxime, amoxicillin/clavulanic acid) decreased error rates to < 0.1%. ZMUs contained low numbers of isolates for ampicillin and cefuroxime (3% and 6%), whereas the ZMU for amoxicillin/clavulanic acid contained 41% of all isolates and was considered not practical. Conclusions We demonstrate that CBPs can be improved and standardized by minimizing methodological categorization error rates. ZMUs may be introduced if an intermediate zone is not appropriate for pharmacokinetic/pharmacodynamic or drug dosing reasons. Optimized CBPs will provide a standardized antibiotic susceptibility testing interpretation at a defined level of probability.

Background We investigated the feasibility of rapid disc diffusion antibiotic susceptibility testing (rAST) with reading of inhibition zones after 6 and/or 8 h of incubation for Enterococcus faecalis, Enterococcus faecium, Pseudomonas aeruginosa and Acinetobacter baumannii. In addition, we evaluated discrimination of resistant populations from the WT populations at early timepoints and the requirement for clinical breakpoint adaptations for proper interpretation of rAST data. Methods In total, 815 clinical strains [E. faecalis (n = 135), E. faecium (n = 227), P. aeruginosa (n = 295) and A. baumannii (n = 158)] were included in this study. Disc diffusion plates were streaked, incubated and imaged using the WASPLabTM automation system. WT populations and non-WT populations were defined using epidemiological cut-offs. Results and conclusions rAST at 6 and 8 h was possible for A. baumannii and enterococci with readability of inhibition zones >90%. Overall categorical agreement of rAST at 6 h with AST at 18 h was 97.2%, 97.4% and 95.3% for E. faecalis, E. faecium and A. baumannii, respectively. With few exceptions, major categorization error rates were <1% for A. baumannii, and vancomycin-resistant E. faecium were clearly separated from the WT at 6 h. For P. aeruginosa the average readability of inhibition zones was 68.9% at 8 h and we found an overall categorical agreement of 94.8%. Adaptations of clinical breakpoints and/or introduction of technical buffer zones, preferably based on aggregated population data from various epidemiological settings, are required for proper interpretation of rAST.

Background In principle, automated systems allow rapid reading of disc diffusion AST (rAST) within 6-8 h. Objectives This study analysed whether rAST can discriminate resistance phenotypes such as ESBL, carbapenemases and MRSA/methicillin-resistant Staphylococcus epidermidis from WT populations. We describe species-drug combinations that may require clinical breakpoint adaptions for early reading due to zone diameter changes during the incubation period. Methods In total, 1852 clinical strains [Escherichia coli (n = 475), Klebsiella pneumoniae (n = 375), Enterobacter cloacae (n = 301), Staphylococcus aureus (n = 407) and S. epidermidis (n = 294)] were included in this study comprising WT populations and important resistance phenotypes, e.g. ESBL, carbapenemases and MRSA. We assessed (i) separation of resistance phenotypes and WT populations after 6, 8 and 12 h as compared with the 18 h standard, and (ii) diameter changes of WT populations and associated putative epidemiological cut-offs during the incubation period. Disc diffusion plates were automatically streaked, incubated and imaged using the WASPLabTM system. Results and conclusions We demonstrated that important resistance phenotypes could reliably be separated from WT populations at early reading times for the most prevalent bacterial pathogens encountered in the clinical laboratory. Current AST expert rules and algorithms for identification of resistance mechanisms can readily be applied for rAST, e.g. EUCAST recommended rules for detection of ESBL, AmpC, carbapenemases and MRSA/methicillin-resistant S. epidermidis. However, several species-drug combinations may require clinical breakpoint adaptations when using rAST as the diameter, and hence the epidemiological cut-off, changes during the incubation period.