Antimicrobial Susceptibility Tests

1. Antimicrobial Susceptibility Tests

2. Antimicrobial Susceptibility Tests provide information for selection ofan appropriate agent for antimicrobial therapy QC Antimicrobial Susceptibility Testing - Module 8 2

3. Antimicrobial susceptibility tests Minimum inhibitory concentration [MIC] • The smallest concentration of antibiotic that inhibits the growth of organism Liquid media (dilution) allows MIC estimation Solid media (diffusion) • Disk diffusion (Kirby-Bauer) • E-tests • Allows MIC estimation Beta lactamase production: quick screening method QC Antimicrobial Susceptibility Testing - Module 8

4. AST Methods Interpretation • agar disk diffusion method provides qualitative interpretive category results of susceptible, intermediate, and resistant • microdilution and agar gradient diffusion methods provide a quantitative result, a minimum inhibitory concentration QC Antimicrobial Susceptibility Testing - Module 8

5. AST Methods QC Antimicrobial Susceptibility Testing - Module 8

6. MIC Dilution in liquid broth • Tubes containing increasing antibiotic concentrations • Incubation during 18 hr at 37°C Bacterial growth Inhibition 0 (Control) 0,25 0,50 1 2 4 8 mg/l QC Antimicrobial Susceptibility Testing - Module 8

7. Kirby-Bauer disc testing Antibiotic-impregnated discs placed on an agar plate at the interface between test organism and susceptible control organism Resulting zones of inhibition compared, use of controls Susceptibility is inferred (standard tables) QC Antimicrobial Susceptibility Testing - Module 8

8. E-test Plastic strips with a predefined gradient of • One antibiotic • One antifungal Only one manufacturer One strip per antibiotic Wide range of antibiotics Easy to use Storage at -20°C Short shelf life, expensive QC Antimicrobial Susceptibility Testing - Module 8

9. Reading E-tests Ciprofloxacin for Yersinia pestis Resistant > 4 ug/ml Intermediate 1-4 ug/ml Susceptible < 1 Upper reading QC Antimicrobial Susceptibility Testing - Module 8

10. Where errors can occur in susceptibility testing media antimicrobials inoculum incubation equipment interpretation QC Antimicrobial Susceptibility Testing - Module 8 10

11. Agar disk diffusion method Medium Mueller Hinton 4 mm thickness pH 7.2 to 7.4 Antibiotic storage -20oC minimum disks temperature Inoculum McFarland 0.5 (108 bacteria/mL) Incubator temperature 35oC atmosphere ambient air QC Antimicrobial Susceptibility Testing - Module 8 11

12. Reference Strains E. coli ATCC 25922 S. aureus ATCC 25923 P. aeruginosa ATCC 27853 QC organisms must be obtained from reputable source Use specific QC organisms to test different groups of “drug-bug” combinations QC Antimicrobial Susceptibility Testing - Module 8 12

13. QC Antimicrobial Susceptibility Testing - Module 8

14. QC Antimicrobial Susceptibility Testing - Module 8

15. BAP are enriched, differential media used to isolate fastidiousorganisms and detect hemolytic activity. β-hemolytic activity will show lysis and complete digestion of red blood cell contents surrounding colony QC Antimicrobial Susceptibility Testing - Module 8

16. Hemolyses of Streptococcus spp. (left) α-hemolysis (S. mitis); (middle) β-hemolysis (S. pyogenes); (right) γ-hemolysis (= non-hemolytic, S. salivarius)

17. Macconkey agar with lactose (left) and non-lactose (right) fermenters

18. Selection of a Colony to Test QC Antimicrobial Susceptibility Testing - Module 8 18

19. MacFarland 0.5 and Adjusted Test Organism QC Antimicrobial Susceptibility Testing - Module 8 19

20. Disk Susceptibility Testing Problems QC Antimicrobial Susceptibility Testing - Module 8 20

21. Disk Susceptibility Testing Problems QC Antimicrobial Susceptibility Testing - Module 8

22. Measuring Conditions Ruler Calipers • read with good light, and from the back of the plate • zone size reading is drug specific • magnification may help • millimeters matter QC Antimicrobial Susceptibility Testing - Module 8 22

23. Etest – antimicrobial gradient method QC Antimicrobial Susceptibility Testing - Module 8 23

24. Patient results may be incorrect if: • the organism was misidentified • a clerical error was made • inappropriate choice of antimicrobials were tested and reported • the wrong patient’s sample was examined • the wrong test was ordered • the sample was not preserved properly QC Antimicrobial Susceptibility Testing - Module 8

25. Critical points in quality assurance • Culture media: Muller-Hinton • Reagents: disks • Size of the inoculums • Incubation condition • Control with reference strains • Reading inhibition diameters (accurate measurement) • Knowledge of staff QC Antimicrobial Susceptibility Testing - Module 8

26. Standard strains for quality assurance Precision and accuracy ensured through control strains • Known susceptibility to antimicrobial agents Standard strains include • Staplylococcus aureus ATCC 25923 • Escherichia coli ATCC 25922 • Pseudomonas aeruginosa ATCC 27853 QC Antimicrobial Susceptibility Testing - Module 8

27. The main concept is the “clinical categorisation" Strains are sorted according to level of Minimal Inhibitory Concentration (MIC) versus reference breakpoints c and C are the minor and major breakpoints Interpretation QC Antimicrobial Susceptibility Testing - Module 8

28. Understanding breakpoints Words of laboratory specialists • It is not possible to work alone • Breakpoints are the expression of a consensus among the scientific community at a given time in a country Breakpoints are determined using two approaches • Pharmacological concept • Epidemiological concept QC Antimicrobial Susceptibility Testing - Module 8

29. The epidemiological concept for breakpoints Inherited resistance mechanism Wild type c C MIC QC Antimicrobial Susceptibility Testing - Module 8

30. The pharmacological concept for breakpoints The concentration range tested for a drug and the interpretative criteria for various categories are based on extensive studies that correlate with • Serum achievable levels for each antimicrobial agent • Particular resistance mechanisms • Successful therapeutic outcome In practice situations the entire range may not be used for decision making and therefore the concept of breakpoint concentration QC Antimicrobial Susceptibility Testing - Module 8

31. From breakpoints to interpretation Measuring antimicrobial sensitivity of a strain isolated from a patient, to determine its status as S, I or R is an individual problem Defining the status of a bacterial species or genus is an epidemiological problem distributed across time and space that requires monitoring QC Antimicrobial Susceptibility Testing - Module 8

32. Host factors affecting treatment Diffusion in tissues Serum protein binding Drug interactions Immune system Multiple simultaneous infections Virulence of organism Site and severity of infection QC Antimicrobial Susceptibility Testing - Module 8

33. Interpreting intermediate resistance Sometime the agent can still be used • Higher doses required to ensure efficacy • Agent may be efficacious if concentrated in vivo in an infected body fluid (e.g., urine) Sometimes there is uncertainty • Intermediate resistance may represent a “buffer” zone that prevents strains with borderline susceptibility from being incorrectly categorized as resistant QC Antimicrobial Susceptibility Testing - Module 8

34. Common interpretation problems Results depends on the technique used Many factors influence results • Lack of standardization of the inoculums • Thickness and quality of the culture media • Quality and conservation of the disks • Wuality control with standardized strains • Condition and duration of incubation QC Antimicrobial Susceptibility Testing - Module 8

35. Common interpretation problems An agar gel that is too thick leads to smaller zones Source: http://www.who.int/csr/resources/publications/drugresist/WHO_CDS_CSR_RMD_2003_6/en/ QC Antimicrobial Susceptibility Testing - Module 8

36. Common interpretation problems Problem with the size of the inoculumsSolution: • Use McFarland 0.5 photometer • Scale -> same tubes QC Antimicrobial Susceptibility Testing - Module 8

37. Common interpretation problems Contamination with another organism QC Antimicrobial Susceptibility Testing - Module 8

38. Common interpretation problems Bad manipulation Inoculation of the Muller Hinton • Swabbing • Not by flooding QC Antimicrobial Susceptibility Testing - Module 8

39. Common interpretation problems Problems with E-test reading QC Antimicrobial Susceptibility Testing - Module 8

40. Cost of anti-microbial resistance Cheap antimicrobials become ineffective Individual treatment failure Prolonged illness, hospitalization Need to switch to more expensive, complex drugs that are often not even available in resource-poor settings Need to develop new antimicrobials Good antimicrobial susceptibility testing saves lives and money QC Antimicrobial Susceptibility Testing - Module 8

41. WHO/CDS/CSR/APH/2000.4 Distr. : General English only WHO Global Principles For The Containment of Antimicrobial Resistance In Animals Intended for Food Report of a WHO Consultation with the participation of the Food and Agriulture Organization and the Office International des Epizooties Geneva, Switzerland 5 – 9 June 2000 Department for Communicable Diseases Surveillance and Response World Health Organization QC Antimicrobial Susceptibility Testing - Module 8

42. Antimicrobial resistance Developed by the Department of Epidemic and Pandemic Alert and Response of the World Health Organization with assistance from: European Program for Intervention Epidemiology Training Canadian Field Epidemiology Program Thailand Ministry of Health Institut Pasteur

43. References • Manual for the laboratory Identification and Antimicrobial Susceptibility Testing of Bacterial Pathogens of Public Health Importance in the Developing World WHO/CDS/CSR/RMD/2003.6 http://www.who.int/csr/resources/publications/drugresist/WHO_CDS_CSR_RMD_2003_6/en/ QC Antimicrobial Susceptibility Testing - Module 8