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Premium member Presentation Transcript Quality assurance in Antibiotic sensitivity testing disc diffusion and E-tests : Dr.T.V.Rao MD Quality assurance in Antibiotic sensitivity testing disc diffusion and E-tests Dr.T.V.Rao MD 1What 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 What is the goal of Antibiotic Sensitivity testing ?Antimicrobial Susceptibility Tests: Dr.T.V.Rao MD 3 3 Antimicrobial Susceptibility Tests P rovide information for selection of an appropriate agent for antimicrobial therapyComponents of Antibiotic Sensitivity Testing: 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 4AST Methods Interpretation: A gar 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 concentration AST Methods Interpretation Dr.T.V.Rao MD 5Slide 6: Dr.T.V.Rao MD 6 AST MethodsAntimicrobial resistance: 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 Dr.T.V.Rao MD 7Antibiotic resistant infections : Antibiotic resistant infections Diseases Agent Resistances Pneumonia S pneumoniae Penicillin Dysentery S dysenteriae Multiple resistances Typhoid S typhi Multiple resistances Gonorrhea N gonorrhoeae Penicillin and tetracycline Tuberculosis M tuberculosis Rifampicine and INH Nosocomial infections S aureus Methicillin, vancomycin E species Vancomycin Klebsiella, Pseudomonas Multiple resistancesGenetic exchange of antimicrobial resistance genes: Genetic exchange of antimicrobial resistance genes Enterobacteriaceae Enterococci Staphylococci Pseudomonas Campylobacter Vibrio cholerae Pneumococci Streptococci Dr.T.V.Rao MD 9Antimicrobial susceptibility tests: 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 metho d Dr.T.V.Rao MD 10Critical points in quality assurance: 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 Dr.T.V.Rao MD 11The hands and heads - personnel: 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 resultsAgar disk diffusion method: Dr.T.V.Rao MD 13 13 Agar disk diffusion method Medium Mueller Hinton 4 mm thickness pH 7.2 to 7.4 Antibiotic storage -20 o C minimum disks temperature Inoculum McFarland 0.5 (10 8 bacteria/mL) Incubator temperature 35 o C atmosphere ambient airReference Strains: Dr.T.V.Rao MD 14 14 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” combinationsQuality Assurance in Antibiotic Susceptibility Testing with Control strains: Dr.T.V.Rao MD 15 Quality Assurance in Antibiotic Susceptibility Test ing with Control strains Susceptibility test with quality control strains for every new batch of Mueller-Hinton agar Staphylococcus aureus (ATCC 25923) Escherichia coli (ATCC 25922) Pseudomonas aeruginosa (ATCC 2785 3 )Susceptibility Testing Methods: Susceptibility Testing Methods Inoculate MH plate Place disks on agar plate Incubate plate 18-24 hr, 35 C Measure and record zone of inhibition around each diskSlide 17: Dr.T.V.Rao MD 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 resistantSelection of a Colony to Test: Dr.T.V.Rao MD 18 Selection of a Colony to Test 18Disk Diffusion Test: Disk Diffusion Test Select colonies Prepare inoculum suspension Prepare inoculum suspension Dr.T.V.Rao MD 19McFarland 0.5 and Adjusted Test Organism: Dr.T.V.Rao MD 20 McFarland 0.5 and Adjusted Test Organism 20Prepare the Material for Inoculation: Prepare the Material for Inoculation Standardize inoculum Suspension as per Mac farland standard Mix well Dr.T.V.Rao MD 21 Swab the plate with optimal sample: Swab the plate with optimal sample Remove sample Swab plate Dr.T.V.Rao MD 22Antibiotic discs: 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 swabbingSelect the Disks and Apply: Select the Disks and Apply Select disks Dr.T.V.Rao MD 24Incubate Overnight: Incubate Overnight Dr.T.V.Rao MD 25Disc Diffusion Method: Disc Diffusion Method P lace the appropriate drug-impregnated dis c on the surface of the inoculated agar plate Invert the plates and incubate them at 35 o C , o/n (18-24 h) Measure the diameters of inhibition zone in mm Dr.T.V.Rao MD 26Look at the Charts for establishing the zones of Sensitivity: Dr.T.V.Rao MD 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.Interpretation: 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 Susceptibl e Intermediate Resistant MIC < c ≤ MIC < C ≤ MIC Interpretation Dr.T.V.Rao MD 28Understanding breakpoints: 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 Dr.T.V.Rao MD 29The epidemiological concept for breakpoints : MIC c Wild type Inherited resistance mechanism C The epidemiological concept for breakpoints Dr.T.V.Rao MD 30The pharmacological concept for breakpoints: 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 Dr.T.V.Rao MD 31From breakpoints to interpretation: 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 MIC ≤ c Sensitive strain MIC > C Intermediate strain c < MIC ≤ C Resistant strain Dr.T.V.Rao MD 32Interpreting intermediate resistance : 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 Dr.T.V.Rao MD 33Disk Susceptibility Testing Problems: Dr.T.V.Rao MD 34 34 Disk Susceptibility Testing ProblemsDisk Susceptibility Testing Problems: Dr.T.V.Rao MD 35 Disk Susceptibility Testing ProblemsMeasuring Conditions: Dr.T.V.Rao MD 36 36 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 matterSlide 37: Dr.T.V.Rao MD 37 Factors Affecting Size of Zone of Inhibition Potency of antibiotic discs Composition of medium Acidic pH of medium Alkaline pH of medium Reading of zones Deterioration in contents leads to reduced size Affects rate of growth, diffusion of antibiotics and activity of antibiotics Tetracycline, novobiocin, methicillin zones are larger Aminoglycosides, erythromycin zones are larger Subjective errors in determining the clear edgeCommon interpretation problems: An agar gel that is too thick leads to smaller zones Common interpretation problems Source: http://www.who.int/csr/resources/publications/drugresist/WHO_CDS_CSR_RMD_2003_6/en/ Dr.T.V.Rao MD 38Common interpretation problems: Common interpretation problems Problem with the size of the inoculums Solution: Use McFarland 0.5 photometer Scale -> same tubesCommon interpretation problems: Contamination with another organism Common interpretation problems Dr.T.V.Rao MD 40Common interpretation problems: Common interpretation problems Bad manipulation Inoculation of the Muller Hinton Swabbing Not by floodingE-test: 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, expensi veSlide 43: MIC on a strip abbiodisk.com Be familiar with InstructionsEtest – antimicrobial gradient method: Dr.T.V.Rao MD 44 Etest – antimicrobial gradient method 44Reading E-tests: Reading E-tests Susceptible < 1 Resistant > 4 ug/ml Ciprofloxacin for Yersinia pestis Intermediate 1-4 ug/ml Upper reading Dr.T.V.Rao MD 45E test – MIC Reports are helpful in Critical management decisions: Dr.T.V.Rao MD 46 E test – MIC Reports are helpful in 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 .Antimicrobial Gradient Testing E-test®: Antimicrobial Gradient Testing E-test® Read plates after recommended Incubation Read MIC where elipse intersects scaleWhere errors can occur in susceptibility testing: media antimicrobials inoculum incubation equipment interpretation Where errors can occur in susceptibility testing Dr.T.V.Rao MD 48 48Common interpretation problems: 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 Quality control with standardized strains Condition and duration of incubation Dr.T.V.Rao MD 49Patient results may be incorrect if:: Dr.T.V.Rao MD 50 Patient results may be incorrect if: T he organism was misidentified A clerical error was made I nappropriate choice of antimicrobials were tested and reported T he wrong patient’s sample was examined T he wrong test was ordered T he sample was not preserved properlySlide 51: Dr.T.V.Rao MD 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 o C Use Mueller-Hinton medium for antibiotic sensitivity determination Use appropriate control cultures Use standard methodology for the testWhen things go wrong…: When things go wrong… Questions to ask? Is the procedure correct? Check test materials including test strains Check equipment Fridges Incubators Freezers Review technique of personnel Culture of general QC in lab essentialInvestigation on errors: Investigation on errors Quality of media should be investigated pH will affect macrolides, tetracycline's 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 batchSlide 54: Dr.T.V.Rao MD 54Need 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 test Dr.T.V.Rao MD 55 Need for Modified MethodsSelf 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 use Self correction of errors Dr.T.V.Rao MD 56What 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 What is the Role of Microbiology DepartmentsSlide 58: Created by Dr.T.V.Rao MD for ‘e’ learning resources for Microbiologists in Developing World Email email@example.com Dr.T.V.Rao MD 58 You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.