ArticleLiterature Review

The 2014 Garrod Lecture: EUCAST - are we heading towards international agreement?

June 2015
Journal of Antimicrobial Chemotherapy 70(9)

Source
PubMed

Authors:

Karolinska Institutet

Antimicrobial susceptibility testing with phenotypic methods is based on the measurement of the MIC (mg/L) and breakpoints to categorize bacteria and fungi as susceptible, intermediate or resistant. Phenotypic antimicrobial susceptibility testing requires an agreement on breakpoints and a rigorous standardization of methods and materials. Requirements for defining breakpoints include a definition of doses and dose intervals, information on MIC distributions for the target organisms, definitions of the highest MIC for organisms devoid of phenotypically expressed resistance (the epidemiological cut-off) and information on resistance mechanisms, pharmacokinetics, pharmacodynamics and clinical outcome in trials. In 2001, the breakpoint committees of France, Germany, Norway, Sweden, the Netherlands and the UK were tasked with developing European breakpoints under the umbrella of EUCAST, organized by ESCMID and later also by ECDC. Breakpoints for previously established antibacterial and antifungal agents in Europe have now been harmonized. With the EMA, EUCAST has since 2006 determined breakpoints for new agents. All breakpoints are freely available on the EUCAST web site; these are used in semi-automated antimicrobial susceptibility testing devices and have been employed since 2010 in a EUCAST disc diffusion method. They have been or are now being implemented in most countries inside Europe and many countries outside it. Everything needed to perform and interpret antimicrobial susceptibility testing is freely available from the EUCAST web site, as are aggregated MIC distributions based on more than 26 000 distributions. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Updating the approaches to define susceptibility and resistance to anti-tuberculosis agents: implications for diagnosis and treatment

Article

Full-text available

Apr 2022

Inappropriately high breakpoints have resulted in systematic false-susceptible AST results to anti-TB drugs. MIC, PK/PD and clinical outcome data should be combined when setting breakpoints to minimise the emergence and spread of antimicrobial resistance. https://bit.ly/3i43wb6

On the Consequences of Poorly Defined Breakpoints for Rifampin Susceptibility Testing of Mycobacterium tuberculosis Complex

Article

Full-text available

Mar 2021
J CLIN MICROBIOL

In a recent report of a systematic review of critical concentrations (CCs), the World Health Organization (WHO) lowered the rifampin (RIF) CC for antimicrobial susceptibility testing (AST) of the Mycobacterium tuberculosis complex using Middlebrook 7H10 medium and the BACTEC Mycobacterial Growth Indicator Tube (MGIT) 960 system from 1 to 0.5 μg/mL. The previous RIF CC for 7H10 had been in use for over half a century. Because it had served as the de facto reference standard, it contributed to the endorsement of inappropriately high CCs for other AST methods, including the U.S. Food and Drug Administration (FDA)-approved MGIT system. Moreover, this resulted in confusion about the interpretation of seven borderline resistance mutations in rpoB (i.e., L430P, D435Y, H445L, H445N, H445S, L452P, and I491F). In this issue of the Journal of Clinical Microbiology , J. Shea, T. A. Halse, D. Kohlerschmidt, P. Lapierre, et al. (J Clin Microbiol 59:e01885-20, 2021, https://doi.org/10.1128/JCM.01885-20 ) provide evidence that the CC endorsed by the Clinical and Laboratory Standards Institute for the Sensititre MYCOTB system, which is not FDA-approved but is CE-IVD marked in the European Union, is likely also too high. These findings underscore the importance of calibrating AST methods against a rigorously defined reference standard, as recently proposed by the European Committee on Antimicrobial Susceptibility Testing, as well as the value of routine next-generation sequencing for investigating discordant AST results.

The Minimum Inhibitory Concentration of Antibiotics: Methods, Interpretation, Clinical Relevance

Article

Full-text available

Feb 2021

Inefficiency of medical therapies used in order to cure patients with bacterial infections requires not only to actively look for new therapeutic strategies but also to carefully select antibiotics based on variety of parameters, including microbiological. Minimal inhibitory concentration (MIC) defines in vitro levels of susceptibility or resistance of specific bacterial strains to applied antibiotic. Reliable assessment of MIC has a significant impact on the choice of a therapeutic strategy, which affects efficiency of an infection therapy. In order to obtain credible MIC, many elements must be considered, such as proper method choice, adherence to labeling rules, and competent interpretation of the results. In this paper, two methods have been discussed: dilution and gradient used for MIC estimation. Factors which affect MIC results along with the interpretation guidelines have been described. Furthermore, opportunities to utilize MIC in clinical practice, with pharmacokinetic /pharmacodynamic parameters taken into consideration, have been investigated. Due to problems related to PK determination in individual patients, statistical estimation of the possibility of achievement of the PK/PD index, based on the Monte Carlo, was discussed. In order to provide comprehensive insights, the possible limitations of MIC, which scientists are aware of, have been outlined.

Antimicrobial Susceptibility Testing of Antimicrobial Peptides to Better Predict Efficacy

Article

Full-text available

Jul 2020

During the development of antimicrobial peptides (AMP) as potential therapeutics, antimicrobial susceptibility testing (AST) stands as an essential part of the process in identification and optimisation of candidate AMP. Standard methods for AST, developed almost 60 years ago for testing conventional antibiotics, are not necessarily fit for purpose when it comes to determining the susceptibility of microorganisms to AMP. Without careful consideration of the parameters comprising AST there is a risk of failing to identify novel antimicrobials at a time when antimicrobial resistance (AMR) is leading the planet toward a post-antibiotic era. More physiologically/clinically relevant AST will allow better determination of the preclinical activity of drug candidates and allow the identification of lead compounds. An important consideration is the efficacy of AMP in biological matrices replicating sites of infection, e.g., blood/plasma/serum, lung bronchiolar lavage fluid/sputum, urine, biofilms, etc., as this will likely be more predictive of clinical efficacy. Additionally, specific AST for different target microorganisms may help to better predict efficacy of AMP in specific infections. In this manuscript, we describe what we believe are the key considerations for AST of AMP and hope that this information can better guide the preclinical development of AMP toward becoming a new generation of urgently needed antimicrobials.

Standards for MIC testing that apply to the majority of bacterial pathogens should also be enforced for Mycobacterium tuberculosis complex

Article

Feb 2019

What is resistance? Impact of phenotypic versus molecular drug resistance testing on multi- and extensively drug-resistant tuberculosis therapy

Article

Full-text available

Jan 2018
ANTIMICROB AGENTS CH

Rapid and accurate drug-susceptibility testing (DST) is essential for the treatment of multi- and extensively drug-resistant tuberculosis (M/XDR-TB). We compared the utility of genotypic DST assays with phenotypic DST (pDST) using BACTEC 960 MGIT or Löwenstein-Jensen to construct M/XDR-TB treatment regimens for a cohort of 25 consecutive M/XDR-TB patients and 15 possible anti-TB drugs. Genotypic DST results from Cepheid GeneXpert MTB/RIF (Xpert) and line probe assays (LPAs: Hain GenoType MTBDR plus 2.0 and MTBDR sl 2.0)] and whole genome sequencing (WGS) were translated into individual algorithm-derived treatment regimens for each patient. We further analysed if discrepancies between the various methods were due to flaws in the genotypic or phenotypic test using MIC results. Compared with pDST, the average agreement in the number of drugs prescribed in ‘genotypic' regimens ranged from just 49% (95% CI 39-59%) for Xpert and 63% (95% CI 56-70%) for LPAs to 93% (95% CI 88-98%) for WGS. Only the WGS regimens did not comprise any drugs to which pDST showed resistance. Importantly, MIC testing revealed that pDST likely underestimated the true rate of resistance for key drugs (rifampicin, levofloxacin, moxifloxacin, and kanamycin) because critical concentrations (CCs) were too high. WGS can be used to rule-in resistance even in M/XDR strains with complex resistance patterns, but pDST for some drugs is still needed to confirm susceptibility and construct the final regimens. Some CCs for pDST need to be re-examined to avoid systematic false-susceptible results in low-level resistant isolates.

Ancient and recent differences in the intrinsic susceptibility of Mycobacterium tuberculosis complex to pretomanid

Article

Full-text available

Mar 2022
J ANTIMICROB CHEMOTH

Objectives: To develop a robust phenotypic antimicrobial susceptibility testing (AST) method with a correctly set breakpoint for pretomanid (Pa), the most recently approved anti-tuberculosis drug. Methods: The Becton Dickinson Mycobacterial Growth Indicator Tube™ (MGIT) system was used at six laboratories to determine the MICs of a phylogenetically diverse collection of 356 Mycobacterium tuberculosis complex (MTBC) strains to establish the epidemiological cut-off value for pretomanid. MICs were correlated with WGS data to study the genetic basis of differences in the susceptibility to pretomanid. Results: We observed ancient differences in the susceptibility to pretomanid among various members of MTBC. Most notably, lineage 1 of M. tuberculosis, which is estimated to account for 28% of tuberculosis cases globally, was less susceptible than lineages 2, 3, 4 and 7 of M. tuberculosis, resulting in a 99th percentile of 2 mg/L for lineage 1 compared with 0.5 mg/L for the remaining M. tuberculosis lineages. Moreover, we observed that higher MICs (≥8 mg/L), which probably confer resistance, had recently evolved independently in six different M. tuberculosis strains. Unlike the aforementioned ancient differences in susceptibility, these recent differences were likely caused by mutations in the known pretomanid resistance genes. Conclusions: In light of these findings, the provisional critical concentration of 1 mg/L for MGIT set by EMA must be re-evaluated. More broadly, these findings underline the importance of considering the global diversity of MTBC during clinical development of drugs and when defining breakpoints for AST.

MDR M. tuberculosis outbreak clone in Eswatini missed by Xpert has elevated bedaquiline resistance dated to the pre-treatment era

Article

Full-text available

Nov 2020

Background Multidrug-resistant (MDR) Mycobacterium tuberculosis complex strains not detected by commercial molecular drug susceptibility testing (mDST) assays due to the RpoB I491F resistance mutation are threatening the control of MDR tuberculosis (MDR-TB) in Eswatini. Methods We investigate the evolution and spread of MDR strains in Eswatini with a focus on bedaquiline (BDQ) and clofazimine (CFZ) resistance using whole-genome sequencing in two collections ((1) national drug resistance survey, 2009–2010; (2) MDR strains from the Nhlangano region, 2014–2017). Results MDR strains in collection 1 had a high cluster rate (95%, 117/123 MDR strains) with 55% grouped into the two largest clusters (gCL3, n = 28; gCL10, n = 40). All gCL10 isolates, which likely emerged around 1993 (95% highest posterior density 1987–1998), carried the mutation RpoB I491F that is missed by commercial mDST assays. In addition, 21 (53%) gCL10 isolates shared a Rv0678 M146T mutation that correlated with elevated minimum inhibitory concentrations (MICs) to BDQ and CFZ compared to wild type isolates. gCL10 isolates with the Rv0678 M146T mutation were also detected in collection 2. Conclusion The high clustering rate suggests that transmission has been driving the MDR-TB epidemic in Eswatini for three decades. The presence of MDR strains in Eswatini that are not detected by commercial mDST assays and have elevated MICs to BDQ and CFZ potentially jeopardizes the successful implementation of new MDR-TB treatment guidelines. Measures to limit the spread of these outbreak isolates need to be implemented urgently.

Commentary: what is the role of the EUCAST reference method for MIC testing of the Mycobacterium tuberculosis complex?

Article

Aug 2020

Rapid antimicrobial susceptibility tests for sepsis; the road ahead

Article

Full-text available

May 2019

Current methods for antimicrobial susceptibility testing (AST) are too slow to affect initial treatment decisions in the early stages of sepsis, when the prescriber is most concerned to select effective therapy immediately, rather than finding out what will not work 1 or 2 days later. There is a clear need for much faster differentiation between viral and bacterial infection, and AST, linked to earlier aetiological diagnosis, without sacrificing either the accuracy of quantitative AST or the low cost of qualitative AST. Truly rapid AST methods are eagerly awaited, and there are several candidate technologies that aim to improve the targeting of our limited stock of effective antimicrobial agents. However, none of these technologies are approaching the point of care and nor can they be described as truly culture-independent diagnostic tests. Rapid chemical and genomic methods of resistance detection are not yet reliable predictors of antimicrobial susceptibility and often rely on prior bacterial isolation. In order to resolve the trade-off between diagnostic confidence and therapeutic efficacy in increasingly antimicrobial-resistant sepsis, we propose a series of three linked decision milestones: initial clinical assessment (e.g. qSOFA score) within 10 min, initial laboratory tests and presumptive antimicrobial therapy within 1 h, and definitive AST with corresponding antimicrobial amendment within an 8 h window (i.e. the same working day). Truly rapid AST methods therefore must be integrated into the clinical laboratory workflow to ensure maximum impact on clinical outcomes of sepsis, and diagnostic and antimicrobial stewardship. The requisite series of development stages come with a substantial regulatory burden that hinders the translation of innovation into practice. The regulatory hurdles for the adoption of rapid AST technology emphasize technical accuracy, but progress will also rely on the effect rapid AST has on prescribing behaviour by physicians managing the care of patients with sepsis. Early adopters in well-equipped teaching centres in close proximity to large clinical laboratories are likely to be early beneficiaries of rapid AST, while simplified and lower-cost technology is needed to support poorly resourced hospitals in developing countries, with their higher burden of AMR. If we really want the clinical laboratory to deliver a specific, same-day diagnosis underpinned by definitive AST results, we are going to have to advocate more effectively for the clinical benefits of bacterial detection and susceptibility testing at critical decision points in the sepsis management pathway.

‘Those who cannot remember the past are condemned to repeat it’. Drug-susceptibility testing for bedaquiline and delamanid

Article

Feb 2019
INT J INFECT DIS

Despite being fundamental to all treatment decisions, the breakpoints that define susceptibility and resistance to conventional anti-tuberculosis (TB) drugs were traditionally defined based on expert opinion as opposed to modern microbiological principles. As a result, the breakpoints for several key drugs (i.e. amikacin, levofloxacin, and moxifloxacin) were too high, resulting in the systematic misclassification of a proportion of resistant strains as susceptible. Moreover, a recent systematic review of clinical outcome data prompted the World Health Organization (WHO) to make significant changes to its treatment guidelines. For example, capreomycin and kanamycin are no longer recommended for TB treatment because their use correlates with worse clinical outcomes. This history notwithstanding, robust breakpoints still do not exist for bedaquiline and delamanid six years after their approval. This was compounded by the fact that access to both agents for drug-susceptibility testing had initially been restricted. It is incumbent upon the European Medicines Agency, the United States Food and Drug Administration, and WHO to ensure that drug developers generate the necessary data to set breakpoints as a prerequisite for the approval of new agents.

Isoniazid Resistance in Mycobacterium tuberculosis Is a Heterogeneous Phenotype Composed of Overlapping MIC Distributions with Different Underlying Resistance Mechanisms

Article

Full-text available

Jun 2019
ANTIMICROB AGENTS CH

MIC testing using the BACTEC 960 MGIT system of 70 phylogenetically diverse, isoniazid-resistant clinical strains of Mycobacterium tuberculosis revealed a complex pattern of overlapping MIC distributions. Whole-genome sequencing could explain most of the level of resistance observed. The MIC distribution of strains with only inhA promoter mutations was split by the current concentration that is endorsed by the Clinical Laboratory Standards Institute to detect low-level resistance to isoniazid and is therefore likely not optimally set.

Commercial Methods for Antifungal Susceptibility Testing of Saprophytic Molds: Can They Be Used to Detect Resistance?

Article

Full-text available

Mar 2024
J. Fungi

Commercial tests are often employed in clinical microbiology laboratories for antifungal susceptibility testing of filamentous fungi. Method-dependent epidemiological cutoff values (ECVs) have been defined in order to detect non-wild-type (NWT) isolates harboring resistance mechanisms. We reviewed the literature in order to find studies where commercial methods were used to evaluate for in vitro susceptibility of filamentous fungi and assess their ability to detect NWT isolates according to the available ECVs. Data were found for the gradient concentration strips Etest and MIC Test Strips (MTS), broth microdilution Sensititre YeastOne (SYO), Micronaut-AM and the agar dilution VIPcheck assays. Applying itraconazole, voriconazole and posaconazole Etest ECVs for A. fumigatus, Etest was able to detect 90.3% (84/93), 61.2% (90/147) and 86% (31/36) of isolates with known cyp51A mutations, respectively. Moreover, Etest also was able to detect 3/3 fks mutants using caspofungin ECVs and 2/3 micafungin mutant isolates. Applying the voriconazole and posaconazole SYO ECVs, 57.7% (67/116) and 100% (47/47) of mutants with known cyp51A substitutions were classified as NWT, respectively. VIPcheck detected 90.3% (159/176), 80.1% (141/176) and 66% (141/176)of mutants via itraconazole, voriconazole and posaconazole, respectively, whereas Micronaut-AM detected 88% (22/25). In conclusion, Etest posaconazole and itraconazole, as well as micafungin and caspofungin ECVs, detected A. fumigatus mutants. On the other hand, while the posaconazole SYO ECV was able to detect cyp51A mutants, similar data were not observed with the SYO voriconazole ECV.

A multicategory logit model detecting temporal changes in antimicrobial resistance

Article

Full-text available

Dec 2022
PLOS ONE

Monitoring and investigating temporal trends in antimicrobial data is a high priority for human and animal health authorities. Timely detection of temporal changes in antimicrobial resistance (AMR) can rely not only on monitoring and analyzing the proportion of resistant isolates based on the use of a clinical or epidemiological cut-off value, but also on more subtle changes and trends in the full distribution of minimum inhibitory concentration (MIC) values. The nature of the MIC distribution is categorical and ordinal (discrete). In this contribution, we developed a particular family of multicategory logit models for estimating and modelling MIC distributions over time. It allows the detection of a multitude of temporal trends in the full discrete distribution, without any assumption on the underlying continuous distribution for the MIC values. The experimental ranges of the serial dilution experiments may vary across laboratories and over time. The proposed categorical model allows to estimate the MIC distribution over the maximal range of the observed experiments, and allows the observed ranges to vary across labs and over time. The use and performance of the model is illustrated with two datasets on AMR in Salmonella .

Establishing Antimicrobial Susceptibility Testing Methods and Clinical Breakpoints for Inhaled Antibiotic Therapy

Article

Full-text available

Feb 2022

Inhaled antibiotics are a common and valuable therapy for patients suffering from chronic lung infection, with this particularly well demonstrated for patients with cystic fibrosis. However, in vitro tests to predict patient response to inhaled antibiotic therapy are currently lacking. There are indications that antimicrobial susceptibility testing (AST) may have a role in guidance of therapy, but which tests would correlate best still needs to be researched in clinical studies or animal models. Applying the principles of EUCAST methodology, the analysis of relevant and reliable data correlating different AST tests to patients’ outcomes may yield clinical breakpoints for susceptibility, but these data are currently unavailable. At present, we believe that it is unlikely that standard determination of minimal inhibitory concentration (MIC) will prove the best predictor.

Has resistance to chlorhexidine increased among clinically-relevant bacteria? A systematic review of time course and subpopulation data

Article

Full-text available

Aug 2021
PLOS ONE

Stephen Buxser

Chlorhexidine (CHX) was introduced for use as an antimicrobial more than 70 years ago. CHX has been and continues to be used broadly for disinfecting surfaces in medical and food service facilities as well as directly on skin of humans and animals. Considering its widespread use over many decades, questions of resistance to CHX have been raised. Additionally, questions of possible coincident resistance to the biocide and resistance to clinically relevant antibiotics have also been raised. A number of important questions remain, including is there consistent evidence of resistance, what is the degree of resistance, especially among clinically isolated microbial strains, and what is the degree of resistance compared to the typical concentrations of the biocide used? Data for microbial species isolated over the last 70+ years were compiled to construct as complete a picture as practical regarding possible resistance, especially among species in which resistance to commonly used antibiotics has been noted to be increasing. This is a compilation and analysis of individual MIC values for CHX reported in the literature, not a compilation of the conclusions individual authors reached. The data were analyzed using straight-forward and robust statistical procedures to detect changes in susceptibility to CHX over time, i.e. linear regression. Linear regression was supplemented with the use of nonlinear least squares regression analysis to detect the presence of population parameters associated with subpopulations of microbial strains which exhibit increased resistance to CHX. Pseudomonas aeruginosa , Klebsiella pneumoniae , and Acinetobacter baumannii were all found to have an increased resistance to CHX over time with the most profound change detected in A . baumannii . Additionally, subpopulations with log-normal distributions were found consistent with the presence of a baseline subpopulation of susceptible strains and a subpopulation with increased resistance to CHX. However, the CHX-resistant subpopulations did not correlate exactly with antibiotic resistance, so details of the relationship remain to be addressed. Increased resistance over time was not detected for Escherichia coli , Enterobacter faecalis , Staphylococcus aureus , or Candida albicans , although a subpopulation with greater than baseline resistance to CHX was detected among strains of E . faecalis and C . albicans . A difference in susceptibility to CHX was also detected between methicillin-resistant (MRSA) and methicillin-sensitive (MSSA) S . aureus strains. The levels of resistance to CHX detected were all markedly lower than concentrations routinely used in medical and food service applications. Reaching conclusions regarding the relationship between antibiotic and CHX resistance was complicated by the limited overlap between tests of CHX and antibiotic resistance for several species. The results compiled here may serve as a foundation for monitoring changes in resistance to CHX and possible relationships between the use of CHX and resistance to antibiotics commonly used in clinical medicine.

Update from the European Committee on Antimicrobial Susceptibility Testing (EUCAST)

Article

Full-text available

Mar 2022
J CLIN MICROBIOL

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 a software to display these distributions, the EUCAST concept of identifying epidemiological cut-off 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 – for AST in fungi (AFST), 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 cut off values, the relationship between phenotypic and genotypic resistance, 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 prevents 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.

Examination of Cefazolin Plasma Levels in Cardiac Surgery under a Revised Dutch Dosing Regimen

Article

Full-text available

Feb 2021

Introduction: Cefazolin is the first choice antibiotic prophylaxis during cardiac surgery and is widely used to prevent deep sternal wound infections (DSWI). Recently, the Dutch guideline has been changed where the cefazolin dose given at the onset of cardiopulmonary bypass (CPB), has been removed and the postoperative dosages are both halved. The aim of the study is to assess if adequate cefazolin plasma levels are obtained with the new Dutch guideline. Methods: Twenty-four adults undergoing cardiac surgery with cardiopulmonary bypass (CPB), receiving cefazolin were studied. The main goal is 100% fT >MIC during surgery and epidemiological cutoff is 2 mg/L. During the postoperative phase the goal is 40% fT >MIC. Per patient 9 to 11 blood samples were collected to measure plasma cefazolin concentrations. Results: During surgery 100% of the measured concentrations were above the ECOFF of 2mg/L. BMI was not significantly associated with unbound cefazolin concentrations. Sex, age, albumin levels preoperative and duration of CPB were also not significantly associated with cefazolin unbound concentration. Redose was significantly associated with higher total plasma concentration. In the postoperative phase 85% were above the ECOFF of 2mg/L. Conclusion: This study shows that the unbound cefazolin concentration during surgery is 100% above the ECOFF of 2mg/L, though sometimes close to the minimum. In the postoperative period more than 85% was above the ECOFF of 2mg/L.

Mutations in dnaA and a cryptic interaction site increase drug resistance in Mycobacterium tuberculosis

Article

Full-text available

Nov 2020
PLOS PATHOG

Genomic dissection of antibiotic resistance in bacterial pathogens has largely focused on genetic changes conferring growth above a single critical concentration of drug. However, reduced susceptibility to antibiotics—even below this breakpoint—is associated with poor treatment outcomes in the clinic, including in tuberculosis. Clinical strains of Mycobacterium tuberculosis exhibit extensive quantitative variation in antibiotic susceptibility but the genetic basis behind this spectrum of drug susceptibility remains ill-defined. Through a genome wide association study, we show that non-synonymous mutations in dnaA , which encodes an essential and highly conserved regulator of DNA replication, are associated with drug resistance in clinical M . tuberculosis strains. We demonstrate that these dnaA mutations specifically enhance M . tuberculosis survival during isoniazid treatment via reduced expression of katG , the activator of isoniazid. To identify DnaA interactors relevant to this phenotype, we perform the first genome-wide biochemical mapping of DnaA binding sites in mycobacteria which reveals a DnaA interaction site that is the target of recurrent mutation in clinical strains. Reconstructing clinically prevalent mutations in this DnaA interaction site reproduces the phenotypes of dnaA mutants, suggesting that clinical strains of M . tuberculosis have evolved mutations in a previously uncharacterized DnaA pathway that quantitatively increases resistance to the key first-line antibiotic isoniazid. Discovering genetic mechanisms that reduce drug susceptibility and support the evolution of high-level drug resistance will guide development of biomarkers capable of prospectively identifying patients at risk of treatment failure in the clinic.

Multicentre testing of the EUCAST broth microdilution reference method for MIC determination on Mycobacterium tuberculosis

Article

Full-text available

Oct 2020

Objectives The first objective of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) subcommittee for antimycobacterial susceptibility testing (AMST), launched in 2016, was to set a reference method for determining the MICs of antituberculous agents, since many protocols are used worldwide and a consensus one is needed for the determination of microbiological breakpoints. Methods During 2017 and 2018, MIC determination protocols were evaluated prospectively in a multicentre study within the four AMST laboratories. MIC results were obtained for isoniazid, levofloxacin and amikacin on the reference strain Mycobacterium tuberculosis H37Rv ATCC 27294. Broth microdilution (BMD) in Middlebrook 7H9 and solid medium dilution (SMD) in Middlebrook 7H10 were performed using two inoculum concentrations. MICs were interpreted with regard to visual and 99% inhibition after 7, 14 or 21 days of incubation for BMD and 21 days for SMD. Results Following the EUCAST reference protocol, intra- and inter-assay agreements were within ±1 MIC dilution for >95% of the observations for the three drugs in both methods. MIC values, presented as MIC mode (range) for BMD and SMD respectively, were: 0.03 (0.015–0.06) mg/L and 0.12 (0.06–0.25) mg/L for isoniazid, 0.25 mg/L (0.25–0.5) and 0.5 mg/L (0.12–0.5) for levofloxacin, and 0.5 mg/L (0.5–1.0) and 0.5 mg/L (0.5–1.0) for amikacin. Conclusions Both SMD and BMD were reproducible and eligible as a reference method for MIC determination of the Mycobacterium tuberculosis complex (MTBC). BMD was finally selected as the EUCAST reference method. From now on it will be used to set epidemiological cut-off values and clinical breakpoints of new and old antituberculous agents.

Towards individualised treatment of tuberculosis

Book

Full-text available

May 2019

Katarina Niward

EUCAST rapid antimicrobial susceptibility testing (RAST) in blood cultures: validation in 55 European laboratories

Article

Full-text available

Aug 2020
J ANTIMICROB CHEMOTH

Objectives: When bloodstream infections are caused by resistant bacteria, rapid antimicrobial susceptibility testing (RAST) is important for adjustment of therapy. The EUCAST RAST method, directly from positive blood cultures, was validated in a multi-laboratory study in Europe. Methods: RAST was performed in 40 laboratories in northern Europe (NE) and 15 in southern Europe (SE) from clinical blood cultures positive for Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus or Streptococcus pneumoniae. Categorical results at 4, 6 and 8 h of incubation were compared with results for EUCAST standard 16-20 h disc diffusion. The method, preliminary breakpoints and the performance of the laboratories were evaluated. Results: The total number of isolates was 833/318 in NE/SE. The number of zone diameters that could be read (88%, 96% and 99%) and interpreted (70%, 81% and 85%) increased with incubation time (4, 6 and 8 h). The categorical agreement was acceptable, with total error rates in NE/SE of 2.4%/4.9% at 4 h, 1.1%/3.5% at 6 h and 1.1%/3.3% at 8 h. False susceptibility at 4, 6 and 8 h of incubation was below 0.3% and 1.1% in NE and SE, respectively, and the corresponding percentages for false resistance were below 1.9% and 2.8%. After fine-tuning breakpoints, more zones could be interpreted (73%, 89% and 93%), with only marginally affected error rates. Conclusions: The EUCAST RAST method can be implemented in routine laboratories without major investments. It provides reliable antimicrobial susceptibility testing results for relevant bloodstream infection pathogens after 4-6 h of incubation.

Factors influencing susceptibility testing of antifungal drugs: a critical review of document M27-A4 from the Clinical and Laboratory Standards Institute (CLSI)

Article

Full-text available

Aug 2020

Due to the increasing numbers of fungal infections and the emergence of drug-resistant fungi, optimization and standardization of diagnostic methods for the measurement of antifungal susceptibility are ongoing. The M27-A4 document by the US Clinical and Laboratory Standards Institute (CLSI) is presently used for the interpretation of minimum inhibitory concentrations of major opportunistic yeast species as measured by broth microdilution testing in many countries. Although microdilution is considered a benchmark for reproducible and accurate results, increased testing capacity, and limited human bias, the method is often inaccessible to routine clinical laboratories and researchers, especially in low-income countries. Furthermore, several studies suggest that there are still a considerable number of factors that make the estimation of in vitro activity of antifungal agents challenging. This review article summarizes the limitations of the M27-A4 standard which, despite the advances and improvements obtained by the standardization of antimicrobial resistance testing methods by CLSI, still persist.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis complex isolates – The EUCAST broth microdilution reference method for MIC determination

Article

Full-text available

Aug 2020

Scope Several methods are used worldwide for antibiotic susceptibility testing (AST) for the Mycobacterium tuberculosis complex (MTBC). The variability in the results obtained with these methods hampers to set epidemiological cut-off (ECOFF) values and clinical breakpoints according to EUCAST guidelines. Methods for susceptibility testing and determination of the minimal inhibitory concentrations (MIC) need to be standardized for MTBC isolates for old and new agents. Our objective was to establish a standardized reference method for MIC determination of the MTBC. Methods The EUCAST antimycobacterial susceptibility testing subcommittee (AMST) compared protocols of MIC determination with regard to medium, inoculum preparation, antituberculous agent preparation, incubation, reading of the results and interpretation. Recommendations The EUCAST reference method of MIC determination for MTBC is the broth microdilution method in Middlebrook 7H9-10%OADC medium. The final inoculum is a 10⁵ CFU/ml suspension, from a 10-2 dilution of a 0.5 McFarland suspension prepared after vortexing bacterial colonies with glass beads before suspending them in water. The culture is maintained in a U-shaped 96-well polystyrene microtiter sterile plate with a lid incubated at 36°+1°C. Reading is done using an inverted mirror as soon as the 1:100 diluted control (i.e. 10³ CFU/ml) shows visual growth. The MIC, expressed in mg/L, is the lowest concentration which inhibits visual growth. M. tuberculosis H37Rv ATCC 27294 is used as the reference strain and its targeted MIC values are within the range 0.03-0.12 for isoniazid, 0.12-0.5 for levofloxacin and 0.25-1 mg/L for amikacin. Conclusions The EUCAST reference method for MTBC was endorsed by EUCAST after public consultation and will from now on be used to define EUCAST ECOFFs and clinical breakpoints. This reference method is not primarily intended to be used under routine conditions and the AST methods need to be calibrated against this reference method to be used with EUCAST breakpoints.

Interpretation of discordant rifampicin susceptibility test results obtained using GeneXpert versus phenotypic drug susceptibility testing

Article

Full-text available

Jul 2020

Background The three-month difference in turnaround time between Xpert and conventional phenotypic drug susceptibility testing (pDST) causes patient treatment challenges when pDST rifampin (RIF) susceptibility results and earlier Xpert results disagree, resulting in unnecessary tuberculosis (TB) patient exposure to toxic second-line drugs. Here, the prevalence of discordant RIF susceptibility test results, specifically Xpert (resistant) versus pDST (susceptible) results, was determined. Methods Tuberculosis patients enrolled between January 2015 and June 2018 at Beijing Chest Hospital who consecutively tested positive for RIF resistance using Xpert then negative using pDST were studied. DNA sequences and minimal inhibitory concentration (MIC) results provided insights for understanding discordant results. Results Of 26,826 patients with suggestive TB symptoms undergoing Xpert MTB/RIF testing, 728 diagnosed as RIF-resistant were evaluated. Of these, 118 (16.2%) exhibiting Xpert RIF resistance and phenotypic RIF susceptibility yielded 104 successfully subcultured isolates; of these, 86 (82.7%) harbored rpoB gene RRDR mutations and 18 (17.3%) did not. Leu511Pro (25.0%) and Leu533Pro (17.3%) mutants were most frequently associated with discordant RIF susceptibility test results. Of the 86 isolates with rpoB mutations, 42 (48.8%) with MICs ≤1.0 mg/L were assigned to the RIF-susceptible group, with Leu511Pro the most common mutation observed. Isolates with very low bacterial load were most frequently misdiagnosed as RIF-resistant by Xpert. Conclusion Approximately one-sixth of RIF-resistant TB isolates identified via Xpert yielded discordant pDST results due to questionable interpretation of specific “disputed” mutations. Thus, a diagnostic flow chart should be used to correctly interpret Xpert RIF-resistance results to best guide patient treatment.

How To Optimally Combine Genotypic and Phenotypic Drug Susceptibility Testing Methods for Pyrazinamide

Article

Full-text available

Aug 2020
ANTIMICROB AGENTS CH

False susceptible phenotypic drug-susceptibility testing (DST) results for pyrazinamide due to mutations with MICs close to the critical concentration (CC) confound the classification of pncA resistance mutations, leading to an underestimate of the specificity of genotypic DST. This could be minimised by basing treatment decisions on well-understood mutations and by adopting an area of technical uncertainty for phenotypic DST rather than only testing the CC, as is current practice for the Mycobacterium tuberculosis complex.

Comparative Assessment of DNA Targets and Amplification Methods for Leishmania (Viannia) Detection in Human Samples

Article

Full-text available

Mar 2020

Multiple PCR-based approaches have been developed for Leishmania detection in clinical and laboratory samples, and this diversity limits inter-study comparisons, meta-analyses, and generalization of findings. Toward the harmonization of a molecular tool for detection of Leishmania (Viannia) for research purposes, we evaluated the concordance of 18SrDNA qPCR and minicircle kinetoplastid DNA (mkDNA) PCR followed by Southern blot (PCR-SB) in in vitro infection systems and in lesion and mucosal swab samples from Colombian patients with cutaneous leishmaniasis caused by L. (Viannia). The lower limit of parasite detection of 18SrDNA qPCR and mkDNA PCR-SB was 10-1 promastigotes and one intracellular amastigote per reaction. From cutaneous lesions (n = 63), an almost perfect concordance was found between the methods (κ = 0.92, 95% CI: 0.82-1.00). Despite equal limits of detection, mkDNA PCR-SB was more efficient for parasite detection in mucosal samples than 18SrDNA qPCR or 18SrDNA digital droplet PCR. The high concordance, sensitivity, scaling potential, and feasibility of implementation of the 18SrDNA qPCR support its selection as the L. (Viannia) in research laboratories as a first step toward harmonization of research protocols in the region.

Phylogenetically informative mutations in genes implicated in antibiotic resistance in Mycobacterium tuberculosis complex

Article

Full-text available

Mar 2020

Background: A comprehensive understanding of the pre-existing genetic variation in genes associated with antibiotic resistance in the Mycobacterium tuberculosis complex (MTBC) is needed to accurately interpret whole-genome sequencing data for genotypic drug susceptibility testing (DST). Methods: We investigated mutations in 92 genes implicated in resistance to 21 anti-tuberculosis drugs using the genomes of 405 phylogenetically diverse MTBC strains. The role of phylogenetically informative mutations was assessed by routine phenotypic DST data for the first-line drugs isoniazid, rifampicin, ethambutol, and pyrazinamide from a separate collection of over 7000 clinical strains. Selected mutations/strains were further investigated by minimum inhibitory concentration (MIC) testing. Results: Out of 547 phylogenetically informative mutations identified, 138 were classified as not correlating with resistance to first-line drugs. MIC testing did not reveal a discernible impact of a Rv1979c deletion shared by M. africanum lineage 5 strains on resistance to clofazimine. Finally, we found molecular evidence that some MTBC subgroups may be hyper-susceptible to bedaquiline and clofazimine by different loss-of-function mutations affecting a drug efflux pump subunit (MmpL5). Conclusions: Our findings underline that the genetic diversity in MTBC has to be studied more systematically to inform the design of clinical trials and to define sound epidemiologic cut-off values (ECOFFs) for new and repurposed anti-tuberculosis drugs. In that regard, our comprehensive variant catalogue provides a solid basis for the interpretation of mutations in genotypic as well as in phenotypic DST assays.

CLSI – EUCAST: Comparison of antibiotic-susceptibility profile of Enterobacteriaceae of animal origin according to the standards

Article

Oct 2019

Antimicrobial resistance is a relevant "One Health" issue that shows the need of comparison of isolates of different origins. In this way, guidelines for antimicrobial-resistance evaluation in animals are relevant in relation to human sources. This work aims to compare antimicrobial-resistance results of animal isolates considering CLSI and EUCAST guidelines. The comparison shows considerable differences in the results, which include antibiotics used as primary options in hospital infections. EUCAST showed the higher number of samples with resistance profiles than CLSI that indicates a more efficient scenario to the EUCAST to screen antibiotic-resistant bacteria. EUCAST was more consonant to the expected phenotype for ESBL producers, with higher index of resistance to oxyimino-beta-lactam antibiotics. The study shows that there are differences in the interpretative results using different guidelines, where the susceptibility test results concerning Enterobacteriaceae of animal origin are not always coincident in CLSI and EUCAST. EUCAST has proved to be the most reliable alternative for profile screening of antibiotic resistance, when compared to CLSI. We might say the same with respect to the ESBL-producing Enterobacteriaceae, in which EUCAST has proved to be more efficient about the demonstration of expected resistance profiles for the ESBL producers. These differences show that guideline selection might influence the therapeutic option.

Genomic identification of cryptic susceptibility to penicillins and β-lactamase inhibitors in methicillin-resistant Staphylococcus aureus

Article

Full-text available

Oct 2019

Antibiotic resistance in bacterial pathogens threatens the future of modern medicine. One such resistant pathogen is methicillin-resistant Staphylococcus aureus (MRSA), which is resistant to nearly all β-lactam antibiotics, limiting treatment options. Here, we show that a significant proportion of MRSA isolates from different lineages, including the epidemic USA300 lineage, are susceptible to penicillins when used in combination with β-lactamase inhibitors such as clavulanic acid. Susceptibility is mediated by a combination of two different mutations in the mecA promoter region that lowers mecA-encoded penicillin-binding protein 2a (PBP2a) expression, and in the majority of isolates by either one of two substitutions in PBP2a (E246G or M122I) that increase the affinity of PBP2a for penicillin in the presence of clavulanic acid. Treatment of S. aureus infections in wax moth and mouse models shows that penicillin/β-lactamase inhibitor susceptibility can be exploited as an effective therapeutic choice for ‘susceptible’ MRSA infection. Finally, we show that isolates with the PBP2a E246G substitution have a growth advantage in the presence of penicillin but the absence of clavulanic acid, which suggests that penicillin/β-lactamase susceptibility is an example of collateral sensitivity (resistance to one antibiotic increases sensitivity to another). Our findings suggest that widely available and currently disregarded antibiotics could be effective in a significant proportion of MRSA infections.

Comprehensive antimicrobial susceptibility testing of a large collection of clinical strains of Aggregatibacter actinomycetemcomitans does not identify resistance to amoxicillin

Article

Full-text available

May 2019

Aim The present study aims to determine the susceptibility of Aggregatibacter actinomycetemcomitans to amoxicillin by investigating a large collection of oral strains of diverse geographical origin. Methods Two hundred and fifty‐seven A. actinomycetemcomitans strains were serotyped using a multiplex polymerase chain reaction, and minimal inhibitory concentration (MIC) values of amoxicillin were determined using the agar dilution method (range 0.25 to 8.0 mg/L). The plates were spot‐wise inoculated with approximately 10⁴ colony‐forming units, incubated in 5% CO2 at 37 C°, and visually inspected after 24 and 48 hours. A MIC ≤ 2.00 mg/L was categorised as susceptible using EUCAST interpretative criteria for Haemophilus species. Results Amoxicillin MIC‐values varied from 0.25 mg/L to 2.00 mg/L, and all tested strains, including strains previously reported as resistant, were susceptible to amoxicillin. The MIC50 was 1.00 mg/L and the MIC90 was 2.00 mg/L. Conclusion Meticulous investigation of strains including isolates previously reported as resistant could not confirm the emergence of resistance to β‐lactams in A. actinomycetemcomitans. Based on the present in vitro results, amoxicillin can be considered a key oral antimicrobial agent for treatment of A. actinomycetemcomitans. This article is protected by copyright. All rights reserved.

Time to switch from CLSI to EUCAST? A Southeast Asian perspective.

Article

Mar 2019

Time to switch from CLSI to EUCAST? A Southeast Asian perspective

Article

Full-text available

Apr 2019

Antimicrobial susceptibility testing (AST) of clinical isolates is essential for guiding therapy as well as for surveillance of antimicrobial resistance (AMR). The two most commonly used methodologies worldwide are those of the Clinical and Laboratory Standards Institute (CLSI) and the European Committee for Antimicrobial Susceptibility Testing (EUCAST). CLSI predominates in the United States and many regions outside Europe, where EUCAST is preferred. However, the global situation is evolving, with countries such as Australia recently switching to EUCAST [1].

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

Article

Full-text available

Mar 2019
BMC INFECT DIS

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.

Antimicrobial resistance due to the content of potentially toxic metals in soil and fertilizing products

Article

Full-text available

Dec 2018
Microb Ecol Health Dis

Potentially toxic metals (PTM), along with PTM-resistant bacteria and PTM-resistance genes, may be introduced into soil and water through sewage systems, direct excretion, land application of biosolids (organic matter recycled from sewage, especially for use in agriculture) or animal manures as fertilizers, and irrigation with wastewater or treated effluents. In this review article, we have evaluated whether the content of arsenic (As), cadmium (Cd), chromium (CrIII + CrVI), copper (Cu), lead (Pb), mercury (Hg), nickel (Ni), and zinc (Zn) in soil and fertilizing products play a role in the development, spreading, and persistence of bacterial resistance to these elements, as well as cross- or co-resistance to antimicrobial agents. Several of the articles included in this review reported the development of resistance against PTM in both sewage and manure. Although PTM like As, Hg, Co, Cd, Pb, and Ni may be present in the fertilizing products, the concentration may be low since they occur due to pollution. In contrast, trace metals like Cu and Zn are actively added to animal feed in many countries. In several studies, several different bacterial species were shown to have a reduced susceptibility towards several PTM, simultaneously. However, neither the source of resistant bacteria nor the minimum co-selective concentration (MCC) for resistance induction are known. Co- or cross-resistance against highly important antimicrobials and critically important antimicrobials were identified in some of the bacterial isolates. This suggest that there is a genetic linkage or direct genetic causality between genetic determinants to these widely divergent antimicrobials, and metal resistance. Data regarding the routes and frequencies of transmission of AMR from bacteria of environmental origin to bacteria of animal and human origin were sparse. Due to the lack of such data, it is difficult to estimate the probability of development, transmission, and persistence of PTM resistance. Abbreviations: PTM: potentially toxic metals; AMR: antimicrobial resistance; ARG: antimicrobial resistance gene; MCC: minimum co-selective concentration; MDR: multidrug resistance; ARB: antimicrobial resistant bacteria; HGT: horizontal gene transfer; MIC: minimum inhibitory concentration

Drug resistance mechanisms and drug susceptibility testing for tuberculosis

Article

Sep 2018

Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) is the deadliest infectious disease and the associated global threat has worsened with the emergence of drug resistance, in particular multidrug‐resistant TB (MDR‐TB) and extensively drug‐resistant TB (XDR‐TB). Although the World Health Organization (WHO) End‐TB Strategy advocates for universal access to antimicrobial susceptibility testing, this is not widely available and/or it is still underused. The majority of drug resistance in clinical MTB strains is attributed to chromosomal mutations. Resistance‐related mutations could also exert certain fitness cost to the drug‐resistant MTB strains and growth fitness could be restored by the presence of compensatory mutations. Understanding these underlying mechanisms could provide an important insight into TB pathogenesis and predict the future trend of MDR‐TB global pandemic. This review covers the mechanisms of resistance in MTB and provides a comprehensive overview of current phenotypic and molecular approaches for drug susceptibility testing, with particular attention to the methods endorsed and recommended by the WHO.

Ceftazidime-Avibactam Susceptibility Breakpoints Against Enterobacteriaceae and Pseudomonas aeruginosa

Article

Full-text available

Oct 2018
ANTIMICROB AGENTS CH

Clinical susceptibility breakpoints against Enterobacteriaceae and Pseudomonas aeruginosa for the ceftazidime-avibactam dosage regimen of 2000-500 mg every 8 hours (q8h) by 2-h intravenous infusion (adjusted for renal function) have been established by the FDA, CLSI and EUCAST as susceptible, MIC ≤8 mg/L, and resistant, MIC >8 mg/L. The key supportive data from PK/PD analyses, in vitro surveillance including molecular understanding of relevant resistance mechanisms, and efficacy in regulatory clinical trials, are collated and analyzed here.

Linking minimum inhibitory concentrations to whole genome sequence-predicted drug resistance in Mycobacterium tuberculosis strains from Romania

Article

Full-text available

Jun 2018

Mycobacterium tuberculosis drug resistance poses a major threat to tuberculosis control. Current phenotypic tests for drug susceptibility are time-consuming, technically complex, and expensive. Whole genome sequencing is a promising alternative, though the impact of different drug resistance mutations on the minimum inhibitory concentration (MIC) remains to be investigated. We examined the genomes of 72 phenotypically drug-resistant Mycobacterium tuberculosis isolates from 72 Romanian patients for drug resistance mutations. MICs for first- and second-line drugs were determined using the MycoTB microdilution method. These MICs were compared to macrodilution critical concentration testing by the Mycobacterium Growth Indicator Tube (MGIT) platform and correlated to drug resistance mutations. Sixty-three (87.5%) isolates harboured drug resistance mutations; 48 (66.7%) were genotypically multidrug-resistant. Different drug resistance mutations were associated with different MIC ranges; katG S315T for isoniazid, and rpoB S450L for rifampicin were associated with high MICs. However, several mutations such as in rpoB, rrs and rpsL, or embB were associated with MIC ranges including the critical concentration for rifampicin, aminoglycosides or ethambutol, respectively. Different resistance mutations lead to distinct MICs, some of which may still be overcome by increased dosing. Whole genome sequencing can aid in the timely diagnosis of Mycobacterium tuberculosis drug resistance and guide clinical decision-making.

Perspectives for personalized therapy for patients with multidrug-resistant tuberculosis

Article

May 2018

According to the World Health Organization (WHO), tuberculosis is the leading cause of death attributed to a single microbial pathogen worldwide. In addition to the large number of patients affected by tuberculosis, the emergence of Mycobacterium tuberculosis drug‐resistance is complicating tuberculosis control in many high‐burden countries. During the past five years, the global number of patients identified with multidrug‐resistant tuberculosis (MDR‐TB), defined as bacillary resistance at least against rifampicin and isoniazid, the two most active drugs in a treatment regimen, has increased by more than 20 percent annually. Today we experience a historical peak in the number of patients affected by MDR‐TB. The management of MDR‐TB is characterized by delayed diagnosis, uncertainty of the extent of bacillary drug‐resistance, imprecise standardized drug regimens and dosages, very long duration of therapy and high frequency of adverse events which all translate into a poor prognosis for many of the affected patients. Major scientific and technological advances in recent years provide new perspectives through treatment regimens tailor‐made to individual needs. Where availale, such personalized treatment has major implications on the treatment outcomes of patients with MDR‐TB. The challenge now is to bring these adances to those patients that need them most. This article is protected by copyright. All rights reserved.

MIC-based dose adjustment: Facts and fables

Article

Dec 2017
J ANTIMICROB CHEMOTH

Over recent decades, several publications have described optimization procedures for antibiotic therapy in the individual patient based on antimicrobial MIC values. Most methods include therapeutic drug monitoring and use a single MIC determination plus the relevant pharmacokinetics/pharmacodynamics to adjust the dose to optimize antimicrobial drug exposure and antibacterial effects. However, the use of an MIC obtained by a single MIC determination is inappropriate. First, routine clinical laboratories cannot determine MICs with sufficient accuracy to guide dosage owing to the inherent assay variation in the MIC test. Second, the variation in any MIC determination, whatever method is used, must be accounted for. If dose adjustments are made based on therapeutic drug monitoring and include MIC determinations, MIC variation must be considered to prevent potential underdosing of patients. We present the problems and some approaches that could be used in clinical practice.

Cryptococcus neoformans Epidemiological Cutoff Values

Article

Full-text available

Dec 2017

Purpose of Review The purpose of this review is to provide a current view of the importance of the determination and use of epidemiological cutoff values (ECVs) for Cryptococcus neoformans, since there are no clinical breakpoints (CBPs). Recent Findings ECVs have been proposed for some antifungal agents and C. neoformans, using standardized methodologies by the Clinical and Laboratory Standards Institute (CLSI) and by the European Committee for Antimicrobial Susceptibility Testing (EUCAST), based on the distribution of minimum inhibitory concentrations (MICs). There is no sufficient evidence for the determination of ECVs for C. neoformans using commercial methods; however, as these methods are routinely used in the microbiology laboratory, it is recommended for the establishment of local ECVs using these methods and following the criteria for their determination. Summary Due to the geographic and genetic variations inherent to C. neoformans, it is important to calculate ECVs, since they are useful in clinical practice to guide therapy in the absence of CBPs.

Treatment of onychomycosis in an era of antifungal resistance: Role for antifungal stewardship and topical antifungal agents

Article

Jan 2024

A growing body of literature has marked the emergence and spread of antifungal resistance among species of Trichophyton , the most prevalent cause of toenail and fingernail onychomycosis in the United States and Europe. We review published data on rates of oral antifungal resistance among Trichophyton species; causes of antifungal resistance and methods to counteract it; and in vitro data on the role of topical antifungals in the treatment of onychomycosis. Antifungal resistance among species of Trichophyton against terbinafine and itraconazole—the two most common oral treatments for onychomycosis and other superficial fungal infections caused by dermatophytes—has been detected around the globe. Fungal adaptations, patient characteristics (e.g., immunocompromised status; drug–drug interactions), and empirical diagnostic and treatment patterns may contribute to reduced antifungal efficacy and the development of antifungal resistance. Antifungal stewardship efforts aim to ensure proper antifungal use to limit antifungal resistance and improve clinical outcomes. In the treatment of onychomycosis, critical aspects of antifungal stewardship include proper identification of the fungal infection prior to initiation of treatment and improvements in physician and patient education. Topical ciclopirox, efinaconazole and tavaborole, delivered either alone or in combination with oral antifungals, have demonstrated efficacy in vitro against susceptible and/or resistant isolates of Trichophyton species, with low potential for development of antifungal resistance. Additional real‐world long‐term data are needed to monitor global rates of antifungal resistance and assess the efficacy of oral and topical antifungals, alone or in combination, in counteracting antifungal resistance in the treatment of onychomycosis.

Wild-type distributions of minimum inhibitory concentrations and epidemiological cut-off values-laboratory and clinical utility

Article

Dec 2023
CLIN MICROBIOL REV

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.

Interpretation of Antimicrobial Susceptibility Testing Using European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI) Breakpoints: Analysis of Agreement

Article

Full-text available

Mar 2023

Objectives: Breakpoints provided by European Committee on Antimicrobial Susceptibility Testing (EUCAST) are now being used in many countries. This study was planned to ascertain the agreement in antimicrobial susceptibility using the Clinical and Laboratory Standards Institute (CLSI) and EUCAST breakpoints during the Kirby-Bauer disk diffusion method. Methods: This was a prospective observational study. Clinical isolates belonging to the family Enterobacteriaceae recovered between January and December, 2022, were included in the analysis. The diameter of the zone of inhibition of the 14 antimicrobials (viz. amoxicillin/clavulanic acid, cefazolin, ceftriaxone, cefuroxime, cefixime, aztreonam, meropenem, gentamicin, amikacin, ciprofloxacin, levofloxacin, norfloxacin, trimethoprim/sulfamethoxazole and fosfomycin) was analysed. Antimicrobial susceptibility was interpreted using CLSI 2022 and EUCAST 2022 guidelines. Results: Susceptibility data from a total of 356 isolates showed a slight increase in the percentage of resistant isolates with most of the drugs using EUCAST guidelines. The level of agreement varied from almost perfect to slight. For two drugs, i.e., fosfomycin and cefazolin, the agreement was least among the drug analysed (kappa (κ) value < 0.5, p < 0.001). For Ceftriaxone and Aztreonam, with EUCAST, susceptible (S) isolates would have been categorised in the newly redefined "I" category. It would have indicated the use of higher dosages of drugs. Conclusion: Change in the breakpoints impacts the interpretation of the susceptibility. It can also lead to a change in the dosage of the drug used for treatment. Therefore, there is an urgent need to see the impact of recent modifications "I" category of EUCAST on the clinical outcome and usage of antimicrobials.

A new approach for the synthesis of tri-substituted pyrazole propionic acids derivatives:Anti-inflammatory, antimicrobial and molecular docking studies

Article

Mar 2023
J MOL STRUCT

Therapeutic drug monitoring of carbapenem antibiotics in critically ill patients: an overview of principles, recommended dosing regimens and clinical outcomes

Article

Mar 2023
Expet Rev Clin Pharmacol

Introduction: The importance of antibiotic treatment for sepsis in critically ill septic patients is well established. Consistently achieving the dose of antibiotic required to optimally kill bacteria, minimize the development of resistance, and avoid toxicity is challenging. The increasing understanding the pharmacokinetic and pharmacodynamic (PK/PD) characteristics of antibiotics, and the effects of critical illness on key PK/PD parameters, is gradually re-shaping how antibiotics are dosed in critically ill patients. Areas covered: The PK/PD characteristics of commonly used carbapenem antibiotics, the principles of the application of therapeutic drug monitoring (TDM), and current as well as future methods of utilizing TDM to optimally devise dosing regimens will be reviewed. The limitations, and evidence-base supporting the use of carbapenem TDM to improve outcomes in critically ill patients will be examined. Expert opinion: It is important to understand the principles of TDM in order to correctly inform dosing regimens. Although the concept of TDM is attractive, and the ability to utilize PK software to optimize dosing in the near future is expected to rapidly increase clinicians' ability to meet pre-defined PK/PD targets more accurately, current evidence provides only limited support for the use of TDM to guide carbapenem dosing in critically ill patients.

Synthesis, molecular docking, and biological evaluation of methyl-5-(hydroxyimino)-3-(aryl-substituted)hexanoate derivatives

Article

Full-text available

Jun 2022

Beta-aryl keto hexanoic acids (5a-l) were synthesized efficiently, followed by esterification that afforded beta-aryl keto methylhexanoates (6a-l). The chemo-selective ketoxime beta-aryl methyl hexanoates (7a-l) were isolated in good yields. Spectroscopic methods were used to characterize the obtained moieties. The antioxidant, anti-inflammatory, and antibacterial properties of the effectively synthesized compounds 7a-l were also investigated. The anti-inflammatory activity of the compounds 7c, 7f, 7i, and 7l was excellent, with a low IC50 value at micromolar concentration, which was much better than the reference diclofenac. All synthesized compounds 7a-l were assessed for their in vitro antibacterial activity against S. aureus, B. subtilis and E. coli. Most of the compounds exhibited promising activity against Gram-positive bacterial strain, compound 7i showed excellent activity compared to standard streptomycin and in the case of E. coli, compounds 7b, 7c, 7j, 7k and 7l have shown moderate activity. Further, the cytotoxic activities of the compounds were assessed against lung cancer cells (A549) by using MTT assay. The possible interaction mechanism of the molecules 7c and 7g with Gram-negative strain E. coli DNA gyrase B in complex with PDB ID: 4DUH was studied.

Need for standardization and harmonization of Helicobacter pylori antimicrobial susceptibility testing

Article

Feb 2022

Background and aims: As with other infectious diseases, Helicobacter pylori eradication regimens should be guided by susceptibility testing to achieve excellent success rate, especially in the era of high antibiotic resistance. However, susceptibility testing for H. pylori is rarely performed, which can be partly ascribed to the current lack of standardization of testing methods and the lack of unified consensus on the antibiotic resistance breakpoints. The aim of this review was to call for an international consensus on standardization and harmonization of H. pylori susceptibility testing. Methods: We summarize and compare the advantages and disadvantages of four different phenotypic antimicrobial susceptibility testing (AST) methods (agar dilution, E-test, disk diffusion, and broth microdilution) and the molecular susceptibility testing method for H. pylori. Results: The standard phenotypic testing methods and the molecular testing methods have their own advantages and disadvantages. Compared to the standard phenotypic methods, the molecular testing method does not require successful H. pylori culture, and therefore, is much more rapid and convenient for clinical use. However, the currently available molecular testing method is only suitable for detecting clarithromycin and quinolone susceptibility profiles in H. pylori. Although the standard AST is time-consuming, it is currently the only way to test the susceptibility of H. pylori to all the commonly used antibiotics. Conclusion: To make H. pylori susceptibility testing become a clinical routine, an international consensus on standardization and harmonization of H. pylori AST is needed. Future efforts are needed for optimizing broth culture of H. pylori, and developing commercial AST plates for achieving high throughput and automated susceptibility testing for H. pylori.

Evaluation expérimentale des concentrations critiques de la témocilline vis-à-vis de souches d'entérobactérales.

Thesis

Sep 2019

Kevin Alexandre

Dans le monde entier l’antibiorésistance des entérobactérales communautaires, notamment par production de ß-lactamase à spectre étendu (E-BLSE), conduit à une consommation préoccupante d’antibiotique de dernier recours tels les carbapénèmes. Dérivé de la ticarcilline la témocilline pourrait représentée une alternative y compris sur certaines entérobactérales productrices de carbapénèmase (EPC). Néanmoins, il existe une incertitude concernant les concentrations critiques distinguant les entérobactérales sensibles des résistantes avec trois valeurs selon les pays utilisateurs de témocilline (8 mg/L, 16 mg/L ou 32 mg/L) tandis qu’une harmonisation internationale reste en attente. En ce contexte trois travaux originaux ont été poursuivi ainsi qu’une revue de la littérature. Il fut d’abord étudié in vitro la sensibilité à la témocilline de 762 entérobactérales responsables d’infection urinaire communautaire. Dans un contexte de prévalence faible des E-BLSE (5%) et nulle des EPC, les trois méthodes de routine (disque, automate, Etest) se sont révélées très fiables, la borne épidémiologique pour la témocilline s’établissant à 8 mg/L. Ensuite, l’efficacité de la témocilline vis-à-vis d’entérobactérales productrices ou non de ßlactamases (E-BLSE ou EPC) a été évaluée dans deux modèles murins complémentaires. Il a été montré l’efficacité de la témocilline à un schéma reproduisant la posologie humaine de 2 g toutes 12 h vis-à-vis d’entérobactérales pour lesquels la CMI de la témocilline était de 8 mg/L. L’efficacité de ce schéma posologique, bien que significative, était moindre vis-à-vis des isolats pour lesquels la CMI de la témocilline était de 16 mg/L. Par contre, il ne fut pas observé d’efficacité significative de la témocilline vis-à-vis des isolats avec une CMI à 32 mg/L quelques soit le schéma posologique (2 g toutes les 8 ou 12 h). L’ensemble de ces résultats, ainsi qu’une revue exhaustive des données de la littérature, conduisent à proposer une concentration critique de 8 mg/L pour le schéma posologique de 2 g toutes les 12 h, et de 16 mg/L pour celui de 2 g toutes les 8h. Cette dernière proposition correspondrait au « sensible à forte exposition », nouvelle définition de la catégorisation « intermédiaire » selon les dernières recommandations de l’EUCAST.

The higher prevalence of extended spectrum beta-lactamases among Escherichia coli ST131 in Southeast Asia is driven by expansion of a single, locally prevalent subclone

Article

Full-text available

Sep 2019

The ST131 multilocus sequence type (MLST) of Escherichia coli is a globally successful pathogen whose dissemination is increasing rates of antibiotic resistance. Numerous global surveys have demonstrated the pervasiveness of this clone; in some regions ST131 accounts for up to 30% of all E. coli isolates. However, many regions are underrepresented in these published surveys, including Africa, South America, and Asia. We collected consecutive bloodstream E. coli isolates from three countries in Southeast Asia; ST131 was the most common MLST type. As in other studies, the C2/H30Rx clade accounted for the majority of ST131 strains. Clinical risk factors were similar to other reported studies. However, we found that nearly all of the C2 strains in this study were closely related, forming what we denote the SEA-C2 clone. The SEA-C2 clone is enriched for strains from Asia, particularly Southeast Asia and Singapore. The SEA-C2 clone accounts for all of the excess resistance and virulence of ST131 relative to non-ST131 E. coli. The SEA-C2 strains appear to be locally circulating and dominant in Southeast Asia, despite the intuition that high international connectivity and travel would enable frequent opportunities for other strains to establish themselves.

Widespread implementation of EUCAST breakpoints for antibacterial susceptibility testing in Europe

Article

Full-text available

Jan 2015
Euro Surveill

The European Committee on Antimicrobial Susceptibility Testing (EUCAST) was established to harmonise clinical antimicrobial breakpoints and to define breakpoints for new agents in Europe. Data from the European Antimicrobial Resistance Surveillance Network (EARS-Net) external quality assessment (EQA) exercises from 2009 to 2012, from the United Kingdom External Quality Assessment Scheme (UK NEQAS) from November 2009 to March 2013 and data collected by EUCAST through a questionnaire in the first quarter of 2013 were analysed to investigate implementation of EUCAST guidelines in Europe. A rapid change to use of EUCAST breakpoints was observed over time. Figures for implementation of EUCAST breakpoints at the end of the studied period were 61.2% from EARSNet data and 73.2% from UK NEQAS data. Responses to the EUCAST questionnaire indicated that EUCAST breakpoints were used by over 50% of laboratories in 18 countries, by 10 to 50% of laboratories in eight countries and by less than 10% in seven countries. The EUCAST disk diffusion method was used by more than 50% of laboratories in 12 countries, by 10 to 50% of laboratories in ten countries and by less than 10% in eleven countries. EUCAST guidelines implementation is essential to ensure consistent clinical reporting of antimicrobial susceptibility results and antimicrobial resistance surveillance.

Genotyping using whole-genome sequencing is a realistic alternative to surveillance based on phenotypic antimicrobial susceptibility testing

Article

Full-text available

Dec 2012
J ANTIMICROB CHEMOTH

Objectives: Antimicrobial susceptibility testing of bacterial isolates is essential for clinical diagnosis, to detect emerging problems and to guide empirical treatment. Current phenotypic procedures are sometimes associated with mistakes and may require further genetic testing. Whole-genome sequencing (WGS) may soon be within reach even for routine surveillance and clinical diagnostics. The aim of this study was to evaluate WGS as a routine tool for surveillance of antimicrobial resistance compared with current phenotypic procedures. Methods: Antimicrobial susceptibility tests were performed on 200 isolates originating from Danish pigs, covering four bacterial species. Genomic DNA was purified from all isolates and sequenced as paired-end reads on the Illumina platform. The web servers ResFinder and MLST (www.genomicepidemiology.org) were used to identify acquired antimicrobial resistance genes and MLST types (where MLST stands for multilocus sequence typing). ResFinder results were compared with phenotypic antimicrobial susceptibility testing results using EUCAST epidemiological cut-off values and MLST types. Results: A total of 3051 different phenotypic tests were performed; 482 led to the categorizing of isolates as resistant and 2569 as susceptible. Seven cases of disagreement between tested and predicted susceptibility were observed, six of which were related to spectinomycin resistance in Escherichia coli. Correlation between MLST type and resistance profiles was only observed in Salmonella Typhimurium, where isolates belonging to sequence type (ST) 34 were more resistant than ST19 isolates. Conclusions: High concordance (99.74%) between phenotypic and predicted antimicrobial susceptibility was observed. Thus, antimicrobial resistance testing based on WGS is an alternative to conventional phenotypic methods.

The role of pharmacokinetics/pharmacodynamics in setting clinical MIC breakpoints: The EUCAST approach

Article

Full-text available

Mar 2012
CLIN MICROBIOL INFEC

Clinical breakpoints are used in clinical microbiology laboratories to categorize microorganisms as clinically susceptible (S), intermediate (I) or resistant (R) dependent on the quantitative antimicrobial susceptibility as indicated by the MIC value determined in a well-defined standard test system. The laboratory report, with the designations of S, I or R for each antimicrobial agent, provides guidance to clinicians with respect to the potential use of agents in the treatment of patients, and clinical breakpoints should therefore distinguish between patients that are likely or unlikely to respond to antimicrobial treatment. In Europe, clinical breakpoints are set by the European Committee on Antimicrobial Susceptibility Testing (EUCAST), following a defined procedure. This includes evaluation of efficacy in experimental settings and clinical studies to derive pharmacodynamic targets such as the fAUC/MIC ratio or %fT > MIC required for efficacy, the pharmacokinetic properties of the agent, Monte Carlo simulations to estimate exposures of the antimicrobial agent in the target patient population and commonly used dosing regimens. The probability of target attainment is subsequently determined for a range of pharmacodynamic targets and the results from the Monte Carlo simulations. The breakpoints derived are subsequently evaluated with respect to the wild-type population of the target microorganisms, specific resistance mechanisms and other relevant data. In this paper, we provide an overview of the EUCAST process and considerations for setting pharmacokinetic/pharmacodynamic breakpoints. These are the breakpoints that in the EUCAST breakpoint tables are referred to as 'non-species-related breakpoints'.

EUCAST expert rules in antimicrobial susceptibility testing

Article

Full-text available

Oct 2011
CLIN MICROBIOL INFEC

Clin Microbiol Infect 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.

Normalized Resistance Interpretation as a Tool for Establishing Epidemiological MIC Susceptibility Breakpoints

Article

Full-text available

Jan 2010
J CLIN MICROBIOL

Göran Kronvall

Normalized resistance interpretation (NRI) utilizes the fact that the wild-type population on the sensitive side is not affected by resistance development, and therefore a normalized reconstruction of the peak can be performed. The method was modified for MIC distributions by the introduction of helper variables, in-between values assigned the mean of the neighboring numbers of isolates. This method was used on Staphylo- coccus aureus and Escherichia coli MIC distributions for 27 antimicrobials each and obtained from the EUCAST (European Committee on Antimicrobial Susceptibility Testing) website (http://www.eucast.org/mic_distributions/). The number of isolates in each of the 54 distributions ranged from 40 to 124,472. NRI produced normalized distributions in all cases. Cutoff values were calculated for +2.0 and +2.5 standard deviations (SD) above the means and then rounded up to nearest regular MIC dilution step. EUCAST also show cutoff values, ECOFF values, which were used as the reference. The NRI generated +2.0 SD values showed the best agreement with 26 of 27 within ±1 dilution step and 17 exactly on the ECOFF values for Staphylococcus aureus, and 25 of 27 within ±1 dilution step and 14 right on the ECOFF values for Escherichia coli. NRI offers an objective method for the reconstruction of the wild-type population in an MIC distribution for a given bacterial species and an antimicrobial agent. This method offers a new tool in comparative susceptibility studies such as global surveillance of resistance, as well as in quality control in individual laboratories.

Wild-Type MIC Distributions for Aminoglycoside and Cyclic Polypeptide Antibiotics Used for Treatment of Mycobacterium tuberculosis Infections

Article

Full-text available

Jan 2010
J CLIN MICROBIOL

The aminoglycosides and cyclic polypeptides are essential drugs in the treatment of multidrug-resistant tuberculosis, underscoring the need for accurate and reproducible drug susceptibility testing (DST). The epidemiological cutoff value (ECOFF) separating wild-type susceptible strains from non-wild-type strains is an important but rarely used tool for indicating susceptibility breakpoints against Mycobacterium tuberculosis. In this study, we established wild-type MIC distributions on Middlebrook 7H10 medium for amikacin, kanamycin, streptomycin, capreomycin, and viomycin using 90 consecutive clinical isolates and 21 resistant strains. Overall, the MIC variation between and within runs did not exceed +/-1 MIC dilution step, and validation of MIC values in Bactec 960 MGIT demonstrated good agreement. Tentative ECOFFs defining the wild type were established for all investigated drugs, including amikacin and viomycin, which currently lack susceptibility breakpoints for 7H10. Five out of seven amikacin- and kanamycin-resistant isolates were classified as susceptible to capreomycin according to the current critical concentration (10 mg/liter) but were non-wild type according to the ECOFF (4 mg/liter), suggesting that the critical concentration may be too high. All amikacin- and kanamycin-resistant isolates were clearly below the ECOFF for viomycin, and two of them were below the ECOFF for streptomycin, indicating that these two drugs may be considered for treatment of amikacin-resistant strains. Pharmacodynamic indices (peak serum concentration [Cmax]/MIC) were more favorable for amikacin and viomycin compared to kanamycin and capreomycin. In conclusion, our data emphasize the importance of establishing wild-type MIC distributions for improving the quality of drug susceptibility testing against Mycobacterium tuberculosis.

Breakpoints for Susceptibility Testing Should Not Divide Wild-Type Distributions of Important Target Species

Article

Full-text available

Jan 2009
ANTIMICROB AGENTS CH

The fluconazole MIC distributions for Candida glabrata from testing 34 different clinical isolates and performing 51 tests on a single isolate mirrored each other. Since what is perceived as biological variation in isolates without resistance mechanisms is mainly methodological variation, breakpoints which divide this distribution not only lack a sound biological basis but also result in poor reproducibility of susceptibility characterization. This makes 2, 4, 8, and possibly 16 μg/ml unsuitable breakpoints for C. glabrata and fluconazole.

European harmonization of MIC breakpoints for antimicrobial susceptibility testing of bacteria

Article

Full-text available

Sep 2003

Development of the EUCAST disk diffusion antimicrobial susceptibility testing method and its implementation in routine microbiology laboratories

Article

Aug 2013
CLIN MICROBIOL INFEC

With the support of ESCMID and European countries, EUCAST has developed a disk diffusion test with zone diameter breakpoints correlated with the EUCAST clinical MIC breakpoints. The development of the EUCAST disk diffusion method and quality control criteria are described, together with guidance on quality control and implementation of the method in clinical microbiology laboratories. The method includes the use of Mueller-Hinton agar without supplements for non-fastidious organisms and with 5% mechanically defibrinated horse blood and 20 mg/L β-NAD for fastidious organisms, a standardized inoculum resulting in confluent growth, an incubation time of 16-20 h, a reading guide on how to read zone diameters on individual species-agent combinations and zone diameter breakpoints calibrated to the EUCAST clinical MIC breakpoints. EUCAST recommendations are described in detail and updated regularly on the EUCAST website (http://www.eucast.org).

Validation of EUCAST zone diameter breakpoints against reference broth microdilution

Article

Oct 2013
CLIN MICROBIOL INFEC

The European Committee on Antimicrobial Susceptibility Testing (EUCAST) began harmonizing clinical breakpoints in Europe 2002. In 2009, work to develop a disk diffusion method began and the first disk diffusion breakpoints calibrated to EUCAST clinical minimum inhibitory concentration (MIC) breakpoints were published in December 2009. In this study we validated EUCAST clinical zone diameter breakpoints vs. 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 disk 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 disk 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 was observed, five for Gram-positive organisms (coagulase negative staphylcocci (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. This article is protected by copyright. All rights reserved.

Antimicrobial susceptibility in Escherichia coli of human and avian origin - A comparison of wild-type distributions

Article

Mar 2009
CLIN MICROBIOL INFEC

In the present study, the antimicrobial susceptibilities of 97 Escherichia coli isolates from birds, and 100 clinical isolates from blood cultures, were determined by disk diffusion. The wild-type distributions were defined by the normalized resistance interpretation method. It is shown that the avian and clinical inhibition zone diameter distributions of wild-type E. coli are indistinguishable. © 2009 The Authors Journal compilation © 2009 European Society of Clinical Microbiology and Infectious Diseases.

Antibiotic sensitivity testing - Report of an International Collaborative Study

Article

Feb 1971
Acta Pathol Microbiol Scand B Microbiol Immunol

Antibiotic Susceptibility Testing by a Standardized Single Disk Method

Article

Apr 1966

New method for normalized interpretation of antimicrobial resistance from disk test results for comparative purposes

Article

Mar 2003

To evaluate a calibration method for disk diffusion antibiotic susceptibility tests, using zone diameter values generated in the individual laboratory as the internal calibrator for combinations of antibiotic and bacterial species. The high-zone side of zone histogram distributions was first analyzed by moving averages to determine the peak position of the susceptible population. The accumulated percentages of isolates for the high zone diameter values were calculated and converted into probit values. The normal distribution of the ideal population of susceptible strains was then determined by using the least-squares method for probit values against zone diameters, and the ideal population was thereby defined, including mean and standard deviation. Zone diameter values were obtained from laboratories at the Karolinska Hospital (KS) and Växjö Hospital (VX), and from two laboratories (LabA, LabB) in Argentina. The method relies on well standardized disk tests, but is independent of differences in MIC limits and zone breakpoints, and does not require the use of reference strains. Resistance was tentatively set at below 3 SD from the calculated, ideal mean zone diameter of the susceptible population. The method, called normalized interpretation of antimicrobial resistance, was tested on results from the KS and VX clinical microbiology laboratories, using the disk diffusion method for antimicrobial susceptibility tests, and for two bacterial species, Staphylococcus aureus and Escherichia coli. In total, 114 217 test results were included for the clinical isolates, and 3582 test results for control strains. The methodology at KS and VX followed the standard of the Swedish Reference Group for Antibiotics (SRGA). Zone diameter histograms for control strains were first analyzed to validate the procedure, and a comparison of actual means with the calculated means showed a correlation coefficient of r = 0.998. Results for clinical isolates at the two laboratories showed an excellent agreement for 54 of 57 combinations of antibiotic and bacterial species between normalized interpretations and the interpretations given by the laboratories. There were difficulties with E. coli and mecillinam, and S. aureus and tetracycline and rifampicin. The method was also tested on results from two laboratories using the NCCLS standard, and preliminary results showed very good agreement with quality-controlled laboratory interpretations. The normalized resistance interpretation offers a new approach to comparative surveillance studies whereby the inhibition zone diameter results from disk tests in clinical laboratories can be used for calibration of the test.

Statistical characterisation of bacterial wild-type MIC value distributions and the determination of epidemiological cut-off values

Article

Jun 2006

MIC distribution data were obtained from a variety of international sources, and pooled after selection by a defined criterion. Sixty-seven of these datasets were subjected to a range of statistical goodness-of-fit tests. The log-normal distribution was selected for subsequent modelling. Cumulative counts of MIC distribution data were fitted to the cumulative log-normal distribution using non-linear least squares regression for a range of data subsets from each antibiotic-bacterium combination. Estimated parameters in the regression were the number of isolates in the subset, and (the log(2) values of) the mean and standard deviation. Optimum fits for the cumulative log-normal curve were then used to determine the wild-type MIC range, determined by calculating the MICs associated with the lower and upper 0.1% of the distribution, rounding to the nearest two-fold dilution, and calculating the probabilities of values higher and lower than these values. When plotted logarithmically, histograms of MIC frequencies appeared normal (Gaussian), but standard goodness-of-fit tests showed that the two-fold dilution grouping of MICs fits poorly to a log-normal distribution, whereas non-linear regression gave good fits to population (histogram) log-normal distributions of log(2) MIC frequencies, and even better fits to log-normal cumulative distributions. Optimum fits were found when the difference between the estimated and true number of isolates in the fitted subset was minimal. Sixteen antibiotic-bacterium datasets were fitted using this technique, and the log(2) values of the means and standard deviations were used to determine the 0.1% and 99.9% wild-type cut-off values. When rounded to the nearest two-fold dilution, > or = 98.5% of MIC values fall within the cut-off value range. Non-linear regression fitting to a cumulative log-normal distribution is a novel and effective method for modelling MIC distributions and quantifying wild-type MIC ranges.

Breakpoints for intravenously used cephalosporins in Enterobacteriaceae—EUCAST and CLSI breakpoints

Article

Feb 2008

Gunnar Kahlmeter

It has long been acknowledged that the cephalosporin breakpoints used in most European countries and the USA fail to detect many or most extended spectrum beta-lactamases (ESBLs) in Enterobacteriaceae and that all ESBLs are clinically significant. Therefore, microbiological laboratories have undertaken not only regular cephalosporin susceptibility tests based on breakpoints, but also special tests to detect all ESBLs. An increasing accumulation of clinical data implies that the clinical success of third generation cephalosporin therapy is related more to the minimum inhibitory concentration (MIC) than to the presence or absence of an ESBL. However, the breakpoints must be lower than those previously recommended by many breakpoint committees. In Europe, this adjustment has been achieved by EUCAST (European Committee on Antimicrobial Susceptibility Testing) through the ongoing process of harmonising European breakpoints. In the USA, the CLSI recently voted to adopt similar guidelines but are waiting to implement these while revising other beta-lactam breakpoints. As Enterobacteriaceae are becoming increasingly resistant, a less 'diehard' interpretation of the relationship among MICs, ESBLs and clinical outcome may provide therapeutic alternatives in difficult situations.

Subcommittee for Detection of Resistance Mechanisms and Specific Resistances of Clinical and/or Epidemiological Importance. EUCAST Guidelines for Detection of Resistance Mechanisms And Specific Resistances of Clinical and/or Epidemiological Importance, Version 1.0

C Giske

Giske C and the EUCAST Subcommittee for Detection of Resistance Mechanisms and Specific Resistances of Clinical and/or Epidemiological Importance. EUCAST Guidelines for Detection of Resistance Mechanisms And Specific Resistances of Clinical and/or Epidemiological Importance, Version 1.0, December 2013. http://www.eucast.org/fileadmin/src/media/ PDFs/EUCAST_files/Resistance_mechanisms/EUCAST_detection_of_ resistance_mechanisms_v1.0_20131211.pdf.

Escherichia coli vs Cefotaxime MICs (Accepted Distributions on Updates at

May 2015

Eucast
Database

18 EUCAST Database: Escherichia coli vs. Cefotaxime MICs (Accepted Distributions on 10 April 2015). Updates at: http://mic.eucast.org/ Eucast2/regShow.jsp?Id=3217.

The 2014 Garrod Lecture: EUCAST - are we heading towards international agreement?

Abstract

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