Antibiotic sensitivity testing Quality Control

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Antibiotic sensitivity testing Quality Control

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Antibiotic sensitivity testing Quality Control

  1. 1. QUALITY ASSURANCE IN ANTIBIOTIC SENSITIVITY TESTING DISC DIFFUSION AND E-TESTS Dr.T.V.Rao MDDR.T.V.RAO MD 1
  2. 2. WHAT IS THE GOAL OF ANTIBIOTIC SENSITIVITY TESTING? • The goal of antimicrobial susceptibility testing is to predict the in vivo success or failure of antibiotic therapy. Tests are performed in vitro, and measure the growth response of an isolated organism to a particular drug or drugs. The tests are performed under standardized conditions so that the results are reproducible. The test results should be used to guide antibiotic choice. The results of antimicrobial susceptibility testing should be combined with clinical information and experience when selecting the most appropriate antibiotic for our patients.DR.T.V.RAO MD 2
  3. 3. ANTIMICROBIAL SUSCEPTIBILITY TESTSProvide information for selection ofan appropriate agent forantimicrobial therapy3 Dr.T.V.Rao MD 3
  4. 4. COMPONENTS OF ANTIBIOTIC SENSITIVITY TESTING • 1.The identification of relevant pathogens in exudates and body fluids collected from patients • 2. Sensitivity tests done to determine the degree of sensitivity or resistance of pathogens isolated from patient to an appropriate range of antimicrobial drugs • 3. Assay of the concentration of an administered drug in the blood or body fluid of patient required to control the schedule of dosage.DR.T.V.RAO MD 4
  5. 5. AST METHODS INTERPRETATION• Agar disk diffusion method provides qualitative interpretive category results of susceptible, intermediate, and resistant• Micro dilution and agar gradient diffusion methods provide a quantitative result, a minimum inhibitory concentrationDR.T.V.RAO MD 5
  6. 6. AST Methods6 Dr.T.V.Rao MD
  7. 7. ANTIMICROBIAL RESISTANCEResults from misuse, overuse, under/ inadequate use ofantimicrobials• Costs money, lives and undermines effectiveness of health delivery programs• Threat to global stability and national securityWHO Global Strategy for Containment of AntimicrobialResistance:• Intervention framework to slow emergence and reduce the spread of antimicrobial resistant microorganismsDR.T.V.RAO MD 7
  8. 8. ANTIBIOTIC RESISTANT INFECTIONSDiseases Agent ResistancesPneumonia S pneumoniae PenicillinDysentery S dysenteriae Multiple resistancesTyphoid S typhi Multiple resistancesGonorrhea N gonorrhoeae Penicillin and tetracyclineTuberculosis M tuberculosis Rifampicine and INHNosocomial infections S aureus Methicillin, vancomycin E species Vancomycin Klebsiella, Multiple resistances Pseudomonas
  9. 9. GENETIC EXCHANGE OF ANTIMICROBIAL RESISTANCE GENES Pseudomonas Staphylococci Enterobacteriaceae Enterococci Vibrio cholerae Pneumococci Campylobacter StreptococciDR.T.V.RAO MD 9
  10. 10. ANTIMICROBIAL SUSCEPTIBILITY TESTSMinimum inhibitory concentration [MIC] • The smallest concentration of antibiotic that inhibits the growth of organismLiquid media (dilution) allows MIC estimationSolid media (diffusion) • Disk diffusion (Kirby-Bauer) • E-tests • Allows MIC estimationBeta lactamase production: quick screening metho d DR.T.V.RAO MD 10
  11. 11. CRITICAL POINTS IN QUALITY ASSURANCE1. Culture media: Muller-Hinton2. Reagents: disks3. Size of the inoculums4. Incubation condition5. Control with reference strains6. Reading inhibition diameters (accurate measurement)7. Knowledge of staff DR.T.V.RAO MD 11
  12. 12. THE HANDS AND HEADS - PERSONNEL• Essential that everyone is aware of the importance of QC• Training of personnel in correct technique • Storage of discs • Preparation of a standard inoculum • Swabbing of plates • Choice and storage of media • Timing and methods of incubation of plates • Measurement of zone sizes • Recording of results
  13. 13. 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 13 Dr.T.V.Rao MD 13
  14. 14. REFERENCE STRAINS  E. coli ATCC 25922  S. aureus ATCC 25923  P. aeruginosa ATCC 27853QC organisms must be obtained fromreputable sourceUse specific QC organisms to test differentgroups of “drug-bug” combinations14 Dr.T.V.Rao MD 14
  15. 15. QUALITY ASSURANCE IN ANTIBIOTIC SUSCEPTIBILITY TESTING WITH CONTROL STRAINS• Susceptibility test with quality control strainsfor every new batch of Mueller- Hinton agar • Staphylococcus aureus (ATCC 25923) • Escherichia coli (ATCC 25922) • Pseudomonas aeruginosa (ATCC 27853 ) DR.T.V.RAO MD 15
  16. 16. SUSCEPTIBILITY TESTING METHODS Incubate plate Inoculate Place disks 18-24 hr, 35 C MH plate on agar plate Measure and record zone of inhibition around each disk
  17. 17. Disc Diffusion Method Measurement of the diameters of inhibition zone  Measure from the edge where the growth stats, BUT there are three exceptions  With sulfonamides and co-trimoxazole, ignore slight growth within the zone  Certain Proteus spp. may swarm into the area of inhibition  When beta-lactamase producing Streptococci are tested, zone of inhibition are produced with a heaped-up, clearly defined edge, regardless of the size of the inhibition zone, they should be reported as resistant DR.T.V.RAO MD 17
  18. 18. SELECTION OF A COLONY TO TEST18 Dr.T.V.Rao MD 18
  19. 19. DISK DIFFUSION TEST Prepare inoculum suspension Prepare inoculum Select colonies suspensionDR.T.V.RAO MD 19
  20. 20. MCFARLAND 0.5 AND ADJUSTED TEST ORGANISM20 Dr.T.V.Rao MD 20
  21. 21. PREPARE THE MATERIAL FOR INOCULATIONStandardize inoculumSuspension as per Mac farland standard Mix well DR.T.V.RAO MD 21
  22. 22. SWAB THE PLATE WITH OPTIMAL SAMPLERemove sample Swab plateDR.T.V.RAO MD 22
  23. 23. ANTIBIOTIC DISCS• Stored and handled correctly • Refrigeration – taken out 1 hour before use• Expiry dates noted• Discs at room temperature before use • Avoid condensation• Placing of discs within 15 minutes of swabbing
  24. 24. SELECT THE DISKS AND APPLY Select disksDR.T.V.RAO MD 24
  25. 25. INCUBATE OVERNIGHTDR.T.V.RAO MD 25
  26. 26. DISC DIFFUSION METHOD Place the appropriate drug-impregnated disc on the surface of the inoculated agar plate Invert the plates and incubate them at 35 oC, o/n (18-24 h) Measure the diameters of inhibition zone in mmDR.T.V.RAO MD 26
  27. 27. LOOK AT THE CHARTS FOR ESTABLISHING THE ZONES OF SENSITIVITY• The zone sizes are looked up on a standardized chart to give a result of sensitive, resistant, or intermediate. Many charts have a corresponding column that also gives the MIC (minimal inhibitory concentration) for that drug.DR.T.V.RAO MD 27
  28. 28. INTERPRETATIONThe 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 Susceptible Intermediate Resistant MIC < c ≤ MIC C ≤ MIC < DR.T.V.RAO MD 28
  29. 29. UNDERSTANDING BREAKPOINTSWords 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 countryBreakpoints are determined using two approaches • Pharmacological concept • Epidemiological concept DR.T.V.RAO MD 29
  30. 30. THE EPIDEMIOLOGICAL CONCEPT FOR BREAKPOINTS Wild type Inherited resistance mechanism c C MICDR.T.V.RAO MD 30
  31. 31. THE PHARMACOLOGICAL CONCEPT FOR BREAKPOINTSThe concentration range tested for a drug and theinterpretative criteria for various categories are based onextensive studies that correlate with • Serum achievable levels for each antimicrobial agent • Particular resistance mechanisms • Successful therapeutic outcomeIn practice situations the entire range may not be used fordecision making and therefore the concept of breakpointconcentration DR.T.V.RAO MD 31
  32. 32. FROM BREAKPOINTS TO INTERPRETATION MIC ≤ c Sensitive strain MIC > C Intermediate strain c < MIC ≤ C Resistant strain 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 monitoringDR.T.V.RAO MD 32
  33. 33. INTERPRETING INTERMEDIATE RESISTANCESometime 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 DR.T.V.RAO MD 33
  34. 34. DISK SUSCEPTIBILITY TESTING PROBLEMS34 Dr.T.V.Rao MD 34
  35. 35. DISK SUSCEPTIBILITY TESTING PROBLEMS35 Dr.T.V.Rao MD
  36. 36. MEASURING CONDITIONS Calipers Rulerread with good light, and from the back of the platezone size reading is drug specificmagnification may helpmillimeters matter 36 Dr.T.V.Rao MD 36
  37. 37. Factors Affecting Size of Zone of Inhibition Potency of antibiotic discs  Deterioration in contents leads to reduced size Composition of medium  Affects rate of growth, diffusion of antibiotics and activity of antibiotics Acidic pH of medium  Tetracycline, novobiocin, methicillin zones are larger Alkaline pH of medium  Aminoglycosides, erythromycin Reading of zones zones are larger  Subjective errors in determining the clear edge DR.T.V.RAO MD 37
  38. 38. 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/DR.T.V.RAO MD 38
  39. 39. COMMON INTERPRETATION PROBLEMSProblem with the size of the inoculumsSolution:• Use McFarland 0.5 photometer• Scale -> same tubes
  40. 40. COMMON INTERPRETATION PROBLEMSContamination withanotherorganismDR.T.V.RAO MD 40
  41. 41. COMMON INTERPRETATION PROBLEMSBad manipulationInoculation of theMuller Hinton • Swabbing • Not by flooding
  42. 42. E-TESTPlastic strips with apredefined gradient of • One antibiotic • One antifungalOnly one manufacturerOne strip per antibioticWide range of antibioticsEasy to useStorage at -20°CShort shelf life, expensive
  43. 43. MIC on a stripabbiodisk.comBe familiar withInstructions
  44. 44. ETEST – ANTIMICROBIAL GRADIENT METHOD44 Dr.T.V.Rao MD 44
  45. 45. READING E-TESTS Ciprofloxacin for Yersinia pestis Resistant > 4 ug/ml Intermediate 1-4 ug/ml Susceptible < 1 Upper readingDR.T.V.RAO MD 45
  46. 46. E TEST – MIC REPORTS ARE HELPFULIN CRITICAL MANAGEMENT DECISIONS • Quantitative MIC data is a prerequisite for the management of critical infections, including sepsis, especially among critical care patients. Etest is particularly valuable in such situations, when on- scale MICs are needed for treatment decisions.DR.T.V.RAO MD 46
  47. 47. ANTIMICROBIAL GRADIENT TESTING E-TEST® Read plates afterrecommended Incubation Read MIC where elipse intersects scale
  48. 48. WHERE ERRORS CAN OCCUR IN SUSCEPTIBILITY TESTING• media• antimicrobials• inoculum• incubation• equipment• interpretationDR.T.V.RAO MD 48 48
  49. 49. COMMON INTERPRETATION PROBLEMSResults depends on the technique usedMany factors influence results • Lack of standardization of the inoculums • Thickness and quality of the culture media • Quality and conservation of the disks • Quality control with standardized strains • Condition and duration of incubation DR.T.V.RAO MD 49
  50. 50. 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 50 Dr.T.V.Rao MD
  51. 51. Quality Assurance in Antibiotic Susceptibility Test Salient features of quality control  Use antibiotic discs of 6 mm diameter  Use correct content of antimicrobial agent per disc  Store supply of antimicrobial discs at -20 oC  Use Mueller-Hinton medium for antibiotic sensitivity determination  Use appropriate control cultures  Use standard methodology for the test DR.T.V.RAO MD 51
  52. 52. WHEN THINGS GO WRONG…• Questions to ask? • Is the procedure correct? • Check test materials including test strains • Check equipment • Fridges Culture of general QC in lab essential • Incubators • Freezers • Review technique of personnel
  53. 53. INVESTIGATION ON ERRORS• Quality of media should be investigated • pH will affect macrolides, tetracyclines and aminoglycoside • In this case the pH was too low – acidic • Ideal pH 7.2-7.4• TMP-SMX is affected by the amount of thymidine in media • This QC may indicate the presence of excess thymidine in the agar which will allow the bacteria to bypass the inhibitory effects of TMP-SMX • Corrective action should be taken in house or with the manufacturer and QC repeated with a new batch
  54. 54. DR.T.V.RAO MD 54
  55. 55. NEED FOR MODIFIED METHODS • Modified Methods in Disc diffusion for Antibiotic sensitivity testing to be used for detections of following bacterial isolates • 1 MRSA • 2 ESBL • 3 Enterobacteriaceae and Gram negative bacteria and Carbapenems resistant using Modified Hodge testDR.T.V.RAO MD 55
  56. 56. SELF CORRECTION OF ERRORS • AST QC needs a culture of general QC in the laboratory • Systems for performing, recording and troubleshooting should be documented in writing • Any errors should be investigated timeously and systematically • Results can then be reported with confidence and permit appropriate and safe antimicrobial useDR.T.V.RAO MD 56
  57. 57. WHAT IS THE ROLE OF MICROBIOLOGY DEPARTMENTS• Each laboratory should have a staff member with the time, interest, and expertise to provide leadership in antibiotic testing and resistance. This person would read relevant publications, network with other laboratories, and evaluate potentially useful tests to detect new forms of resistance before new CLSI- recommended tests become available ”• - Ken Thomson, Emerging Infect. Dis., 2001 DR.T.V.RAO MD 57
  58. 58. • Created by Dr.T.V.Rao MD for „e‟ learning resources for Microbiologists in Developing World • Email • doctortvrao@gmail.comDR.T.V.RAO MD 58

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