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Antimicrobial susceptibility testing
The document presents an extensive overview of antimicrobial susceptibility testing (AST), highlighting its significance in guiding clinical therapy, epidemiology, and monitoring resistance rates. It discusses various methods of AST, including phenotypic, genotypic, mechanistic, and expert rules, alongside the role of breakpoints in determining susceptibility and resistance. Eucast, as a key organization in establishing testing standards and guidelines, is detailed, including its history, recognition, and ongoing projects related to new agents and resistance detection.
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Antimicrobial susceptibility testing
- 1. Antimicrobial susceptibility testing GunnarKahlmeter Klinisk mikrobiologi, Kronoberg och Blekinge, Sverige Svenskt referenslaboratorium för fenotypisk resistensbestämning (SLIM) EUCAST Development Laboratory (EDL) EUCAST, ESCMID, BSAC, NEQAS Email: [email protected] Mobil: +46709844685 G Kahlmeter 2020
- 2. Susceptibility testing …. •To guide clinical therapy – we are often a bit late for that • To build a basis for empiric therapy • ”Penicillin resistance in S. pneumoniae in our area is 45%, so penicillin has ceased to be a reasonable first choice…” • For epidemiology • To follow and compare resistance rates • Over time • Geographical areas • Hospital vs. society (primary care) • To compare human, animal, agricultural, industrial etc settings • To detect and describe specific resistance mechanisms G Kahlmeter 2020
- 3. The correctness ofAST is the responsibility of the laboratory • Some test systems have problems with aminoglycosides, others with trimethoprimsulfa • Some types of tests do not cope well with some agents/bacteria (vancomycin, colistin, fosfomycin) • Some agents are just difficult (piperacillintazobactam)! • Some breakpoints are problematic (ciprofloxacin and Enterobacterales, colistin and Pseudomonas aeruginosa; MRSA and ceftaroline and ceftobiprole) • Some devices are generally problematic (gradient tests for several species/agent combinations; truncated concentrations series in semi-automated devices; difficulties QCing semiautomatic devices) • ……. EUCAST helps to identify problematic areas, but depending on the device and/or material used, the laboratory also needs to identify problematic areas. EUCAST 2020
- 4. Antimicrobial susceptibility testing(AST) • Phenotypic AST – based on MIC and breakpoints • Genotypic AST – detection of the gene (mecA, vanA/vanB, etc) • Mechanistic AST – detection of a resistance mechanism • Expert rules AST – susceptibility based on empiric knowledge. • Intrinsic resistance (or susceptibility) • IF – THEN rules G Kahlmeter 2020
- 5. Antimicrobial susceptibility testing(AST) Phenotypic AST – based on MIC and breakpoints • Predicts resistance and susceptibility • Quantitative • Reference method: broth microdilution (CLSI, EUCAST, ISO); Surrogate tests: disk diffusion, gradient tests, semiautomated devices, computeraided microscopy, microcalorimetri, flowcytometri • Genotypic AST – detection of the gene • Predicts resistance but cannot guarantee susceptibility • Not quantitative • Breakpoints not required, but validation does • No reference method • Mechanistic AST - detection of a R-mechanism • Predicts resistance but cannot guarantee susceptibility • Detection of a R-mechanism indicates resistance. • Not quantitative • No breakpoints • Expert rules AST • Predicts resistance OR susceptibility • Not quantitative • May change over time G Kahlmeter 2020
- 6. Antimicrobial susceptibility testing(AST) Phenotypic AST – based on MIC and breakpoints • Predicts resistance and susceptibility • Quantitative • Reference method: broth microdilution (CLSI, EUCAST, ISO); Surrogate tests: disk diffusion, gradient tests, semiautomated devices, computeraided microscopy, microcalorimetri, flowcytometri Genotypic AST – detection of the gene • Predicts resistance but cannot guarantee susceptibility • Not quantitative • Breakpoints not required, but validation does • No reference method • Mechanistic AST - detection of a R-mechanism • Predicts resistance but cannot guarantee susceptibility • Detection of a R-mechanism indicates resistance. • Not quantitative • No breakpoints • Expert rules AST • Predicts resistance OR susceptibility • Not quantitative • May change over time G Kahlmeter 2020
- 7. Antimicrobial susceptibility testing(AST) Phenotypic AST – based on MIC and breakpoints • Predicts resistance and susceptibility • Quantitative • Reference method: broth microdilution (CLSI, EUCAST, ISO); Surrogate tests: disk diffusion, gradient tests, semiautomated devices, computeraided microscopy, microcalorimetri, flowcytometri Genotypic AST – detection of the gene • Predicts resistance but cannot guarantee susceptibility • Not quantitative • Breakpoints not required, but validation does • No reference method Mechanistic AST - detection of a R-mechanism • Predicts resistance but cannot guarantee susceptibility • Detection of a R-mechanism indicates resistance. • Not quantitative • No breakpoints • Expert rules AST • Predicts resistance OR susceptibility • Not quantitative • May change over time G Kahlmeter 2020
- 8. Antimicrobial susceptibility testing(AST) Phenotypic AST – based on MIC and breakpoints • Predicts resistance and susceptibility • Quantitative • Reference method: broth microdilution (CLSI, EUCAST, ISO); Surrogate tests: disk diffusion, gradient tests, semiautomated devices, computeraided microscopy, microcalorimetri, flowcytometri Genotypic AST – detection of the gene • Predicts resistance but cannot guarantee susceptibility • Not quantitative • Breakpoints not required, but validation does • No reference method Mechanistic AST - detection of a R-mechanism • Predicts resistance but cannot guarantee susceptibility • Detection of a R-mechanism indicates resistance. • Not quantitative • No breakpoints Expert rules AST • Intrinsic resistance and IF/THEN rules • Not quantitative • Untrustworthy – prone to change G Kahlmeter 2020
- 9. Disk diffusion isoften straight forward, dependable and unproblematic G Kahlmeter 2020 Disk diffusion is • Cheap • Flexible • Rapidly adaptable • Continuous variable • Easy to QC
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- 11. The MIC Onur andErika in the next seminars G Kahlmeter 2020
- 12. The MIC isthe basis for determining clinical breakpoints. • Breakpoints are not scientifically calculated values • Breakpoints differ over time • Breakpoints differ depending on the availability of agents • If chlorampphenicol was the only available agent, we could not afford to be very stringent in our criteria • Breakpoints differ between agencies and committees • EMA, FDA, CLSI, EUCAST, Colleagues who know better • Breakpoints differ in relation to exposure (dose, admininistration, site) • Breakpoints differ between diseases • UTI, Systemic infections, CNS-infections, Endocarditis etc G Kahlmeter 2020
- 13. Breakpoints are determinedby.. • Medicines agencies (EMA, FDA, The Japanese Medicines agency, etc) • Committees (EUCAST, CLSI, ….) • Colleagues who know better G Kahlmeter 2020
- 14. EUCAST basic facts •EUCAST formed by ESCMID, 1996. • 2001 EUCAST reorganised and national breakpoint committees were given the main responsibility • 2001 – www.eucast.org (main web + MIC/Zone diameter distributions) • 2003 EMA accepts EUCAST breakpoint procedure for new agents • 2004 EU funding; later transformed into a series of ECDC contracts, funding from ESCMID • 2010 EUCAST Development Laboratory (bacteria, fungi) and network • 2010 EUCAST disk diffusion method launched • 2010 EUCAST first complete breakpoint table (v 1.0) published • 2010 – 2021 breakpoints and methods for new agents and new species EUCAST 2021 14
- 15. EUCAST is todayrecognized by…. • EMA • ECDC (only AST data generated with EUCAST criteria accepted since 2020) • EFSA – ECOFFs used for surveillance • WHO • National AST committees (NACs) • All European and many non-European countries • Pharmaceutical companies (all new agents since linezolid received breakpoints through EUCAST) EUCAST 2021 15
- 16. EUCAST Subcommittees • Antifungals •Antimycobacterials • Anaerobic bacteria • MIC distributions and ECOFFs • Subcommittee on the role of whole genome sequencing in antimicrobial susceptibility testing EUCAST 2021 16
- 17. EUCAST recent/current projects •New agents • Cefiderocol, tedizolid, delafloxacin • Betalactam betalactamase inhibitor agents and methods • Carbapenem carbapenemase inhibitor agents and methods • Species, methods and breakpoints • Achromobacter, Bacillus, B. pseudomallei, Vibrio spp, C. diphteriae • Stenotrophomonas and Burkholderia cepacia complex • Anaerobic bacteria - disk diffusion 16-20h for 5 fast-growing anaerobic bacteria (Bacteroides spp, Prevotella, F.necrophorum, Cl.perfringens, Cut.acnes). • Endocarditis pathogens (in collaboration with the French reference laboratory for streptococci) • Nocardia spp (in collaboration with France and Australia) – breakpoints and methods • Rapid (4, 6 and 8 h) disk diffusion AST directly from positive blood culture bottles - extensions • …. EUCAST 2021 17
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- 20. EUCAST 2021 20 TheEUCAST MIC and Zone distribution database Available at www.eucast.org ECOFF 2 mg/L ECOFF 17 mm
- 21. G Kahlmeter 2020 S.pneumoniae vs. benzylpenicillin
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- 23. The basis forall phenotypic susceptibility testing: EUCAST 2021 23 MIC The MIC is a relative measure influenced by variation in any parameter Medium Incubation time Incubation atmosphere Ph Ions etc Onur and Erika will speak to this!
- 24. MIC distributions andECOFFs on EUCAST • >0.5 million hits per year • >32 000 MIC distributions • Up to 100 000 MIC-values per distribution • Data from many investigators (1 – 100 per distrib.) • Data from many time periods (1950 - ) • Data from many geographical areas and projects (USA, Europe, Australia, Far East, South America, Sentry, Mystic, etc) • Data of multiple origin (Human clinical data, Surveillance programs, Veterinarian data, Wild life, Food safety programs) • Ownership: • Software and administration: ESCMID/EUCAST • Database: individual ownership of original data G Kahlmeter 2020
- 25. The use ofECOFFs • As a tool in the determination of clinical breakpoints • To avoid dividing wild type MIC distributions of important target organisms • As a surrogate clinical breakpoint when Pk/Pd data is incomplete and clinical data pertain only to wild type organisms • For sensitive detection of (screening for) resistance • oxacillin to detect all penicillin-R in S. pneumoniae • cefoxitin to detect methicillin resistance in S. aureus • benzylpenicillin to detect all betalactam resistance in H.influenzae • pefloxacin to detect quinolone resistance in Salmonella spp • meropenem to screen for KPC in Enterobacteriaceae • For surveillance of antimicrobial resistance when clinical breakpoints… • are not sensitive enough • have not been determined • change over time • differ between systems (CLSI, FDA, EUCAST etc) • differ between humans, cows, pigs, birds, fish and camels. • To exclude resistance G Kahlmeter 2020
- 26. G Kahlmeter 2020 Howclinical breakpoints are determined
- 27. General rationale behindEUCAST breakpoints • Microbiology • Defining target organisms (few or many) for the agent • Characterizing the MIC wild typ of target organisms. • to avoid that breakpoints split wild type distributions. • Infectious diseases • Defining target infections • Identifying clinical evidence for and against specific infections and organisms • Clinical outcome by MIC and organisms • PK/PD • Dosing • Standard and increased dosing (identify national and international differences) • Modes of administration and influence on PK/PD EUCAST 2021 28
- 28. G Kahlmeter 2020 Speciesspecific MIC distributions Target organisms for tobramycin • Enterobacterales (E.coli, K.pneumoniae…) • Pseudomonas aeruginosa • Staphylococcus aureus • …. Target infections for tobramycin • Several systemic infections • Infections originating from the urinary tract • ….
- 29. Clinical outcome byMIC – type response MIC No of cases Positive outcome 0.016 3 2 67 % 0.032 34 30 88 % 0.064 72 67 93 % 0.125 43 39 91 % 0.25 12 11 92 % 0.5 7 5 71 % 1 0 0 - 2 1 0 0 % 4 2 1 50 % 8 1 1 100 % EUCAST 2021 30
- 30. Clinical outcome byMIC – type response MIC No of cases Positive outcome (No) Positive outcome (%) 0.016 3 2 67 % 0.032 34 30 88 % 0.064 72 67 93 % 0.125 43 39 91 % 0.25 12 11 92 % 0.5 7 5 71 % 1 0 0 - 2 1 0 0 % 4 2 1 50 % 8 1 1 100 % EUCAST 2021 31
- 31. G Kahlmeter 2020 S,I, and R – definitions Susceptible Normal exposure Increased exposure Resistant
- 32. EUCAST 2021 Resistant Susceptible, increased exposure IR 33 (a) intrinsically lower sensitivity – exemplified by ciprofloxacin and Pseudomonas aeruginosa
- 33. Determining clinical breakpointsand ECOFFs. S – Susceptible, normal exposure I - Susceptible, increased exposure R - Resistant G Kahlmeter 2020
- 34. ATU An alert inthe laboratory! Take action! EUCAST 2020
- 35. MIC/zone correlates aremostly excellent…and unproblematic G Kahlmeter 2020
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- 38. • ATU isNOT a susceptibility category – it is to alert laboratory staff of a predictable issue with test or interpretation. • The ATU is NOT to compensate for poor methodological skills – on the contrary, AST today require more skills than ever before. • The ATU does NOT interfere with S, I and R interpretation. • The ATU is NOT a wide range of results – it is defined by a single MIC- value and/or a short range of zone diameter values. • How the ATU is dealt with depends on the situation (the sample and type of infection, the agent, the infecting organism). Area of Technical Uncertainty (ATU) EUCAST 2020
- 39. Results in ATU– alternative actions! • Repeat the test – if test failed technically, repeat test, preferably together with alternative test. • Confirm using an alternative test (MIC, PCR, PBP- agglutination…) – always repeat the original test, you will learn something. • Report the result with comment – “unreliable test result”. • Leave blank with a comment – “unreliable test result” • Discuss and explain – phone clinical colleague. • Report the problem to the breakpoint committee – it may well be a breakpoint problem. EUCAST 2020
- 40. Try hard tosolve IF..… •only few alternative antibiotics for therapy. •in a positive blood culture (or other serious infection). •frequently recurring problem •easy to solve. •requested to do so EUCAST 2020
- 41. Disk diffusion – theinhibition zone This will addressed by Onur and Erika G Kahlmeter 2020