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Antimicrobial resistance and susceptibility testing

Antimicrobial resistance. Antimicrobial resistance and susceptibility testing. May 2007. Learning objectives. At the end of the presentation, participants should: Identify antimicrobial susceptibility testing needs Understand standard antimicrobial susceptibility testing

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Antimicrobial resistance and susceptibility testing

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  1. Antimicrobial resistance Antimicrobial resistance and susceptibility testing May 2007

  2. Learning objectives At the end of the presentation, participants should: • Identify antimicrobial susceptibility testing needs • Understand standard antimicrobial susceptibility testing • Interpret antimicrobial susceptibility testing

  3. Leading global infectious diseases S. pneumonia: Up to 55% resistance to penicillin in some regions S. dyentariae: 90% resistance to cotrimoxazole S.Typhi: Outbreaks of multi-resistant strains in 11 countries HIV: Report of resistance to all marketed agents M. tuberculosis:Multi-drug resistant tuberculosis P. falciparum:Chloroquine resistance in 81/92 countries

  4. Antibiotic resistant infections

  5. Antimicrobial resistance Results from misuse, overuse, under/ inadequate use of antimicrobials • Costs money, lives and undermines effectiveness of health delivery programs • Threat to global stability and national security WHO Global Strategy for Containment of Antimicrobial Resistance: • Intervention framework to slow emergence and reduce the spread of antimicrobial resistant microorganisms

  6. Natural & acquired resistance Natural resistance • Chromosomic genetic support • Affect almost all species strains • Existed before antibiotic use (Enterobacter sp. - amoxicillin) Acquired resistance (mutation) • Chromosomic, plasmidic or transposon genetic support • Affects a fraction of strains • Increased with antibiotic use(extended spectrum beta-lactamase producing E. coli)

  7. Different acquired resistances Acquired to a population of strains in a given species • Extremely frequent in nosocomial infections Acquired under treatment; specific strain,specific patient • Relatively uncommon except for certain species (e.g., Enterobacter, Pseudomonas, Mycobacterium)

  8. Mechanisms of resistance Prevent antibiotic from reaching its target • Impaired cell membrane permeability • Efflux phenomenon Prevent the antibiotic from biding to its target • Supplementary targets • Decreased affinity by target modification Inactivation before reaching the target

  9. Genetic exchange of antimicrobial resistance genes Staphylococci Pseudomonas Enterococci Enterobacteriaceae Vibrio cholerae Pneumococci Campylobacter Streptococci

  10. 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

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

  12. 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)

  13. 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

  14. Reading E-tests Ciprofloxacin for Yersinia pestis Resistant > 4 ug/ml Intermediate 1-4 ug/ml Susceptible < 1 Upper reading

  15. Antimicrobial susceptibility tests Antimicrobial susceptibility testing is expensive (costs include all supplies) Kirby-Bauer • 12 discs panel = $1.35 E-test (Performed only in certain situations) • One strip = $2.50

  16. Different standards Use standardized reference National Committee for Clinical Laboratory Standards (USA) Other norms • Canadian • Chinese • National Do not confuse the different tables Choose one for everything

  17. 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

  18. 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

  19. 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

  20. 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

  21. The epidemiological concept for breakpoints Inherited resistance mechanism Wild type c C MIC

  22. 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

  23. 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

  24. 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

  25. 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

  26. 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

  27. 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/

  28. Common interpretation problems Problem with the size of the inoculumsSolution: • Use McFarland 0.5 photometer • Scale -> same tubes

  29. Common interpretation problems Contamination with another organism

  30. Common interpretation problems Bad manipulation Inoculation of the Muller Hinton • Swabbing • Not by flooding

  31. Common interpretation problems Problems with E-test reading

  32. 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

  33. 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

  34. 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

  35. 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/

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