Teresa Spanu Istituto di Microbiologia

1. Teresa Spanu Istituto di Microbiologia Rilevazione delle resistenze antimicrobiche

2. Defintion of antimicrobial resistance << Biological resistance is defined by the presence of acquired or mutational resistance mechanisms to the drug • Genotypic methods • Phenotypic methods Clinical resistance is mainly based on the bacterial response to treatment. • Phenotypic methods MacGowan et al. J Antimicrob Chemother 2008 www.eucast.org

3. Simply put, resistance is the continued growth of microorganisms in the presence of cytotoxic concentrations of antibiotics. Resistance is therefore relative, and as a result is defined operationally. Antimicrobial resistance can be detected by using • Phenotypic methods -Growth inhibition assays - Mechanism-specific tests such as beta-lactamase detection test and chromogenic cephalosporin test • Genotypic methods - Detection of resistance genes

4. Antibiogram • The profile of an organism’s susceptibility/resistance to a panel of antibiotics 

5. Antibiotic Sensitivity Testing Methods • Bacteriostatic assay • Minimal inhibitory concentration or MIC: the lowest concentration at which the isolate is completely inhibited (as evidenced by the absence of visible bacterial growth). Anne E Clatworthy, Emily Pierson & Deborah T Hung Nature Chemical Biology 3, 541 - 548 (2007)

6. DISK DIFFUSION METHOD Inoculum: 0.5 Mc Farland E-test

7. What Does the laboratory Need to Know about Antimicrobial susceptibility testing • Which organism to test • What methods to use • What antibiotics to test? • How to report results?

8. To achieve harmonization of antimicrobial breakpoints, the six National Committees have now organised themselves in EUCAST (European Committee on Antimicrobial Susceptibility Testing). Kahlmeter et al – JAC 2003

9. Detectingantimicrobialresistancemaybeproblematic EUCAST defines test systemsand determines breakpoints Crucialparameters Interpretative criteria Selectionofdrug and organimstobetested Methods and procedures

10. HOT TOPIC/ Microbiological diagnostic stewardship, Management, and Prevention and Control of Antimicrobial Resistant Healthcare-Associated Infections Detection of AMR infections Culture –basedmethod Screening of carriers Detection of infecting strains Species identification Detection of AMR Phenotypic methods Growth inhibition assays Mechanism-specific tests Immunochromatographic test Genotypic methods AMR, antimicrobial resistance

11. Biological resistance can be defined 1. With reference to the normal population of bacteria that exist before exposure to the antimicrobial agent MacGowan et al J Antimicrob Chemother2008) 62, Suppl. 2, ii105–ii114

12. HOT TOPIC/ Microbiological diagnostic stewardship, Management, and Prevention and Control of Antimicrobial Resistant Healthcare-Associated Infections Detection of AMR infections Culture –basedmethod Screening of carriers Detection of infecting strains Species identification Detection of AMR Phenotypic methods Growth inhibition assays Mechanism-specific tests Immunochromatographic test Genotypic methods AMR, antimicrobial resistance

13. EUCAST determinesepidemiologicalcut-off values • An MIC value that separates bacterial population in those with or without acquired and/or mutational resistance mechanisms http://www.eucast.org.

14. Used to signal the emergence of resistant strains Wild type No ‘R’ mechanism Non Wild type YES ‘R’ mechanism

15. Derek Brown (Cambridge, UK and Gunnar Kahhlmeter (Växjö, Sweden)

16. Clinical resistance can be defined on the bacterial response to treatment (in terms of adverse clinical outcomes related to uncontrolled infection if a patient receives that antimicrobial). MacGowan et al J Antimicrob Chemother2008) 62, Suppl. 2, ii105–ii114

17. The success or failure of antimicrobial therapy in bacterial and fungal infections is predicted ideally by antimicrobial susceptibility testing (AST), in which microorganisms are divided into treatable and nontreatable categories on the basis of MIC breakpoints EUCAST determinesepidemiologicalclinicalbreakpoints Breakpoints provide the basis for categorizing the results of in vitro susceptibility tests into predictions of outcome.

18. EUCAST definitions of clinical resistance/susceptibility Generally, three MIC categories are defined: susceptible (S), intermediate (I), and resistant (R). These terms, when provided in susceptibility test reports to clinicians charged with caring for patients with infection, clearly drive therapeutic decision-making. X >Y MIC MIC MIC susceptible intermediate resistant A microorganism is categorized as susceptible (S), intermediate (I) or resistant (R) by applying the appropriate breakpoint in a defined phenotypic test system CMI: Concentrazione Minima Inibente

19. 2011

20. The efficacy of antibiotics will evidently be affected by the concentration that reaches their target area. Long-term persistence of bacteria in the presence of antibiotics might not only result from expression or acquisition of genetic mechanisms of resistance but also be affected by the microenvironment that exists and develops in the site of infection 2011

21. http://www.eucast.org.

22. Definitions 2002 – 2018 (”the old definition”) Redefining S, I and R 2019 - www.eucast.org

23. In the old definition it is unclear which part is valid in the individual AST report. “A microorganism is defined as intermediate by a level of antimicrobial agent activity associated with uncertain therapeutic effect. It implies that an infection due to the isolate may be appropriately treated in body sites where the drugs are physiologically concentrated or when a high dosage of drug can be used; it also indicates a buffer zone that should prevent small, uncontrolled, technical factors from causing major discrepancies in interpretations.” Redefining S, I and R 2019 - www.eucast.org

24. The old definition of intermediate has four definitions rolled into one. • uncertain therapeutic effect (pharmacology/microbiology) • where the drugs are physiologically concentrated (pharmacokinetics) • when a high dosage of drug can be used (pharmacology/toxicology) • a buffer zone to prevent technical errors … (methodology) Redefining S, I and R 2019 - www.eucast.org

25. Intermediate results thus encompass both… • Uncertainty • uncertain therapeutic effect • uncertain laboratory result • Exposure • agent physiologically concentrated • Dosing strategy (dose, frequency, mode of administration) Redefining S, I and R 2019 - www.eucast.org

26. Uncertainty and Exposure • Uncertainty • responsibility of breakpoint committees • Breakpoints should avoid dividing wild type MIC distributions of important species; otherwise reproducibility in AST cannot be achieved • responsibility of the laboratory • Laboratories are responsible for using appropriate methods and interpretative criteria and for the quality control (QC) of test results. Redefining S, I and R 2019 - www.eucast.org

27. Uncertainty and Exposure • Exposure • responsibility of breakpoint committees • breakpoint committees should inform users of dosing strategies relevant to the breakpoints and under what other conditions breakpoints are valid. • responsibility of the clinician • It is possible to adjust the level of exposure by changing the dosing strategy; individual dose, frequency of dosing, from oral to intravenous, from intermittent to continuous infusion. Redefining S, I and R 2019 - www.eucast.org

28. The achievablelevelof exposure* depends on manyfactors. Individualdifferences in pharmacokineticsareallowed for in the calculations leading up to pharmacodynamicindicesfollowing population simulation. Othersfactors as followsaredetermined by the the site ofinfection or can be variedduringtherapy:1. Site ofinfection– concentration in certaintissues and body fluids may be high (urine, bile, lymphatictissues).2. Dose and dosingfrequency3. Mode of administration (Oral, Intravenous, IV infusion etc)*Exposure is a function of how the mode of administration, dose, dosing interval, infusion time, as well as distribution, metabolism and excretion of the antimicrobial agent will influence the infecting organism at the site of infection. All clinicalbreakpointsarerelated to the achievablelevelof exposure* of the microorganism. Redefining S, I and R 2019 - www.eucast.org

29. Dosing and mode of administration are in the EUCAST breakpoint table. EUCAST breakpoints are related to the doses and modes of administration listed by EUCAST in rationale documents and in the breakpoint table, ”Dosing” tab. With regimens other than those listed in the EUCAST tables, breakpoints may be invalid. For this reason EUCAST has made every effort to consult with all countries to ascertain that the doses and modes of administration listed in EUCAST documents are representative of international practices. Redefining S, I and R 2019 - www.eucast.org

30. The efficacy of antibiotics will evidently be affected by the concentration that reaches their target area. • Long-term persistence of bacteria in the presence of antibiotics might not only result from expression or acquisition of genetic mechanisms of resistance but also be affected by the microenvironment that exists and develops in the site of infection << GD Wright. NATURE REVIEWS MICROBIOLOGY 2007, 5:175

31. How is it that a quantitative in vitro measure of antibiotic effect, i.e., an MIC, can be converted into a predictor of therapeutic effect? Concentrazioni di antibiotico raggiungibili nei tessuti

32. EUCAST decision 2018 • To change the definition ofS, I and R. • To retain the abbreviations S, I and R. • To emphasise the relationship between the exposure of the microorganism at the site ofinfections and the breakpoint and to task National AST Committees (NAC) withinformingcolleaguesabout the relationship betweendosingpractices and breakpoints. • To task laboratorieswithtaking the responsibility for and deal with ”technical variation and errors”. Redefining S, I and R 2019 - www.eucast.org

33. With the modified definition of the ”I-category”…. ….the onlydifferencebetween ”S” and ”I” is the amountofdrug at the site of the infectionnecessary to achieve an adequateclinicalresponse. The term ”intermediate” is replaced by the term ”Susceptible, increased exposure” but the abreviation in reports is still ”I”. Redefining S, I and R 2019 - www.eucast.org

34. Redefining S, I and R 2019 - www.eucast.org Susceptible, standard dosing regimen ( S ) S - Susceptible, standard dosing regimen: A microorganism is categorised as Susceptible, standard dosing regimen*, when there is a high likelihood of therapeutic success using a standard dosing regimen of the agent. * Exposure is a function of how the mode of administration, dose, dosing interval, infusion time, as well as distribution, metabolism and excretion of the antimicrobial agent will influence the infecting organism at the site of infection.

35. Redefining S, I and R 2019 - www.eucast.org Susceptible, increased exposure ( I ) I – Susceptible, increased exposure: A microorganism is categorised as Susceptible, Increased exposure* when there is a high likelihood of therapeutic success because exposure to the agent is increased by adjusting the dosing regimen or by its concentration at the site of infection. * Exposure is a function of how the mode of administration, dose, dosing interval, infusion time, as well as distribution, metabolism and excretion of the antimicrobial agent will influence the infecting organism at the site of infection.

36. Redefining S, I and R 2019 - www.eucast.org Resistant ( R ) R - Resistant: A microorganism is categorised as Resistant when there is a high likelihood of therapeutic failure even when there is increased exposure*. * Exposure is a function of how the mode of administration, dose, dosing interval, infusion time, as well as distribution, metabolism and excretion of the antimicrobial agent will influence the infecting organism at the site of infection.

37. SIR – the old definitions Intermediate Uncertaineffect. Bufferzone for technical variation. For a highdose. Whereconcentrated for pharmacokineticreasons. Susceptible Resistant Redefining S, I and R 2019 - www.eucast.org

38. SIR - new definitions 2019 Susceptible Resistant Normal exposure Increased exposure Redefining S, I and R 2019 - www.eucast.org

39. Redefining S, I and R 2019 - www.eucast.org With the modified definition of the ”I-category”…. ….the onlydifferencebetween ”S” and ”I” is the amountofdrug at the site of the infectionnecessary to achieve an adequateclinicalresponse. The term ”intermediate” is replaced by the term ”Susceptible, increased exposure” but the abreviation in reports is still ”I”.

41. Redefining S, I and R 2019 - www.eucast.org Surveillance of antimicrobial resistance It has been common practice to combine susceptibility categories ´Resistant´ and ´Intermediate´, as non-susceptible, when reporting antimicrobial resistance rates. From 2019, this is no longer appropriate. • For surveillance purposes, avoid combining categories – present S, I and R separately. • If there is a need to combine, then combine S and I and present R separately.

42. Redefining S, I and R 2019 - www.eucast.org Laboratorytechnical variation and uncertainresults • The old definition ofIencompasses a degreeofuncertainty and/or uncontrolledtechnical variation. Where and to whatdegreewas not defined. • This part of the definition has beenremoved and EUCAST has identifiedobvious situations wherelaboratories must takespecific action to avoidreportinghighlyuncertainresults. • Thereare situations wherepoorreproducibilityofresults is predictable.

43. Redefining S, I and R 2019 - www.eucast.org Breakpoint committees and laboratories are tasked with minimising technical problems in AST. Technical problems typically appear when • a breakpoint bisects the wild type. • a breakpoint bisects a resistant population. • there is uncontrolled testing variation. • Poor quality of AST material (broth, agar, disks, devices etc). • Poor calibration/validation of AST procedures. • Poor QC practices in the laboratory.

44. PreliminaryATUs in Enterobacterales, Pseudomonas and Staphylococcus Preliminary ATUs

45. Redefining S, I and R 2019 - www.eucast.org ATU – alternative actions for the laboratory • repeat the test – this is only if there is reason to suspect a technical error. • perform an alternative test (perform an MIC, a PCR, a test to determine the resistance mechanism) – this is relevant when the alternative test is conclusive (PCR to detect a vanA or vanB gene in enterococci, a Bla test in H. influenzae). • report results in the ATU as “uncertain” – this can be achieved by leaving the interpretation blank + comment. Or by developing the LIS to deliver an asterix (instead of an S, I or R) which refers to a comment explaining the uncertainty. • report results in the ATU as “R”. If there are several good alternatives in the AST report this may be the easiest and safest option. • take the opportunity to discuss the results with the clinician.

46. Redefining S, I and R 2019 - www.eucast.org ATU – the appropriate action mayvarywithcircumstances • IF few antibiotics available to the clinician, THEN try to achieve trustworthy categorisation. • IF in a blood culture, THEN try to achieve trustworthy categorisation. • IF can be solved with an alternative method without delay, THEN try to achievetrustworthycategorisation. • IF many alternative antibiotics available, THEN report R (with or without a comment). • IF the result must be reported, THEN include a comment to discuss uncertainty.