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Laboratory Aspects of Antimicrobial Chemotherapy. Patrick Kimmitt. Aims of Presentation. Answers to the following questions:- (1) Why do we test antimicrobial susceptibility? (2) How do we perform antimicrobial susceptibility tests? (3) How can we detect resistance mechanisms?

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    1. Laboratory Aspects of Antimicrobial Chemotherapy Patrick Kimmitt

    2. Aims of Presentation • Answers to the following questions:- • (1) Why do we test antimicrobial susceptibility? • (2) How do we perform antimicrobial susceptibility tests? • (3) How can we detect resistance mechanisms? • (4) Why & how do we assay antimicrobial serum levels?

    3. Deciding on whether to use an antibiotic: • Day 1 Clinical assessment • Day 2 Positive microbiology • Day 3 Antibiotic sensitivity tests • Day 1: guess the disease, guess the bug, guess the sensitivity pattern • Day 2: guess the sensitivity pattern • Day 3: know the full picture: BUT TOO LATE?

    4. Antibiotics and the Laboratory • If there was no antibiotic resistance, there would be no need for a microbiology lab! • Infections could be treated “syndromically”

    5. Why do we test antimicrobial susceptibility? • To direct & predict antimicrobial chemotherapy. • To review & monitor epidemiological trends. • To set national & local antibiotic policies. • To test the activity of a new antimicrobial agent. • To presumptively identify isolates.

    6. But remember… • Other factors are very important when we choose an antibiotic • Will it get to where the infection is? • Bioavailability • Cost • Toxicity • Likelihood of development of resistance • Etc…

    7. How do we perform antimicrobial susceptibility tests? • We can use a number of methods including:- • Disc susceptibility tests - Kirby-Bauer - Stokes’ - BSAC. • Agar Breakpoint method. • Minimum Inhibitory Concentration (MIC) – Tube MIC or E-tests. • Automated methods – Vitek. • Molecular methods – PCR.

    8. Disc Susceptibility Tests • Agar surface evenly inoculated with the test organism. • Antibiotic filter paper discs applied to the plate. • Plates are incubated & antibiotics diffuse into the agar. • Antibiotic concentration decreases at increasing distance from disc. • Circular zone of growth appears. • Size of zone of inhibition indicates susceptibility of organism.

    9. Disc dispenser & inoculated plate

    10. Stokes’ Comparative Method • Developed in the U.K (1972). • A variety of media can be used including Iso-sensitest agar (ISA), ISA & 5% horse blood & Chocolate ISA. • Based on dense not confluent growth. • Use suspension of organism in broth equivalent in density to an overnight broth culture. • Inoculate fastidious organisms direct.

    11. Stokes’ Comparative Method • Use NCTC (National Collection of Type Cultures) controls e.g. NCTC 6571 Staph aureus, NCTC 10602 Ps. aeruginosa, NCTC 10418 E.coli. • Using a rotary plater apply the control suspension on the outer edge & the test suspension in the centre, leaving a gap for the discs.

    12. Stokes’ method Control organism Test organism Antibiotic discs

    13. Stokes’ Comparative Method

    14. Stokes’ Comparative Method • Interpretation based on comparison between zones seen with the test organism & those of the known sensitive control. • Sensitive = zone radius of test, larger, equal, or not more than 3mm smaller than the control. • Intermediate = zone radius more than 3mm, but smaller than the control by more than 3mm. • Resistant = zone radius of 3mm or less.

    15. Disadvantages of Stokes’ Method • Interpretation not valid for β lactamase-producing staphylococci (research for practical) or for tests with polymyxin, augmentin, teicoplanin or ciprofloxacin. • No correlation of zone diameter with the MIC of the organism. • No standard method for inoculum preparation- a heavy inoculum decreases zone of inhibition.

    16. Disadvantages of Stokes’ Method • No standard method for media or incubation conditions- pre-incubation decreases the zone of inhibition, pre-diffusion increases the zone of inhibition. • Unreliable for detection of resistance to new antibiotics or newer resistance mechanisms (ESBL’s). • No consistent method between labs, therefore no consistent epidemiology data.

    17. BSAC Method • Developed in the U.K in 1998 by BSAC working party, through statistical regression analysis of zone diameter & MIC data, on hundreds of strains. • A full up-to-date version of the method is available at • Use ISA and/or ISA & 5% horse blood & 20mg/l NAD. • Use standard 0.5 McFarland inoculum

    18. BSAC Method • Use this diluted inoculum to seed the media (using a rotary plater or by streaking in 3 directions). • Use standard inoculation & incubation criteria. • Use standard antibiotic quality controlled discs. • Use published BSAC tables to interpret zone sizes. •

    19. Examples of BSAC Method

    20. BSAC Method • Interpretation is based on semi-confluent growth of the organism. • Zones sizes can be measured using a template / ruler / electronic callipers / automated zone reader with a scanner & camera (Aura Image, Oxoid). • The method is subject to weekly NCTC control checks for each panel of antibiotics tested. • These controls are checked against published values.

    21. Rationale of BSAC method • Based on the relationship between the zone diameter of the disc diffusion test and the MIC. • The clinical breakpoint can be set as equivalent to a stated zone diameter (mm)

    22. Advantages of the BSAC Method • BSAC working party continually review & update the data. • Standardised method of reporting. • Increases the accuracy of epidemiological data. • Attempting to standardise protocols and breakpoints throughout Europe (EUCAST) – April 2008

    23. Minimum Inhibitory Concentration (MIC) • The MIC is the lowest concentration of the antimicrobial required to inhibit growth of the organism. • It is used to determine the quantitative activity of an antimicrobial. • It is used to confirm resistance or equivocal results. • It is used in cases of prolonged treatment or endocarditis to adjust the dose of therapy. • It is used to determine the susceptibility of slow-growing organisms e.g anaerobes

    24. Tube MIC • Set up a series of antibiotic doubling dilutions in tubes containing liquid media (ISA or ISA with lysed blood). • For example 128mg/l, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0. • Set up tubes for test organism & NCTC control organism.

    25. Tube MIC • Add standard organism inoculum to each tube. • Include an antibiotic free tube i.e organism only. • Include an organism free tube i.e antibiotic only. • Incubate tubes overnight. • Examine for presence of growth by shaking each tube & observing turbidity.

    26. Tube MIC • Check antibiotic free tube has growth. • Check organism free tube has no growth. • Check the NCTC control gives the recommended MIC. • The MIC is the first tube dilution without visible growth. • The tube MIC is very labour intensive, difficult to get right & prone to error.

    27. E-tests (AB Biodisk(Sweden)) • A commercial alternative to tube MIC. • Consists of a plastic strip 6cm by 0.5cm in size. • Exponential gradient of antimicrobial dried on one side. • MIC scale printed on the other side. • The range corresponds to fifteen 2-fold dilutions.

    28. E-tests • Follow manufacturers’ instructions for inoculum preparation, media recommendations & incubation conditions. • MIC interpretation made where growth of inhibition ellipses the strip. • Most E-test require examination with a hand-lens to look for minute colonies intersecting the strip.

    29. Examples of E-tests

    30. Examples of E-tests

    31. Automated Methods • Three main methods are in use in the U.K. • These include the Vitek (Biomerieux), the Phoenix (Becton-Dickinson) & the Mastascan Elite (Mast). • This presentation will focus on the Vitek, please research the other two methods.

    32. Vitek II • The Vitek I was originally designed by NASA for use as an on-board space exploration test system. • It is based on the use of small thin plastic cards each containing many wells linked by capillaries. • These cards are available as susceptibility & identification cards.

    33. Vitek cards

    34. Vitek modules • The Vitek II consists of:- • a robotic filling module whereby a standard suspension of the organism in saline is drawn up via a vacuum into the card. • an incubator / reader module containing a carousel to hold the cards & a photometer to measure optical density of the sensitivity cards & the biochemical colour changes of the identification cards

    35. Vitek modules • A computer module analyses the growth curve & generates an algorithm devised MIC value. It also analyses the biochemical id & compares it to a database. • An “expert” software analysis module recognises new / unusual / inconsistent results, highlights “alert” organisms (e.g. MRSA, VRE, Gentamicin resistance). • The “expert” system has built-in antibiotic interpretation rules.

    36. Vitek modules

    37. Vitek advantages / disadvantages • Can give rapid “expert” results for identification & susceptibility. • It can be directly linked to the LIMS. • It decreases the incidence of operator error & can be operated by an MLA. • It is very expensive, approx £7 for an id & sens. • It needs regular software updates & expert analysis by a BMS.

    38. Why & how do we assay antimicrobial serum levels? • We assay to ensure adequate therapeutic levels & to avoid the accumulation of toxic levels. • Most antimicrobial agents have a large therapeutic index & are given in large doses without causing harm. • The aminoglycosides, the glycopeptides & some antifungals have a narrow therapeutic range which can be close to the toxic range.

    39. Dosing interval and steady state plasma concentration Therapeutic failure Drug concentration in plasma Therapeutic range Therapeutic failure Time

    40. Antibiotic Assays • These agents can damage the 8th cranial nerve = ototoxicity = deafness. • They can also damage the kidneys = nephrotoxicity = renal failure. • Serum samples are often taken pre-dose to determine toxicity & post-dose to determine therapeutic activity. • We can assay antibiotics in 3 main ways – bioassay, immunoassay and HPLC

    41. Immunoassays • There are several commercial immunoassay methods available. • Most are based on competitive binding of antibody for antigen (serum) & labelled antigen (kit). • The most common commercial method is the TDX.

    42. TDX Analyser

    43. Calibration curve • A calibration curve of polarisation versus concentration is set up within the TDX using known concentration calibrators. • Internal controls are run with each assay to ensure the curve is correct. • The assay level is calculated by the TDX via extrapolation from the calibration curve.

    44. Summary

    45. References • British Society for Antimicrobial Chemotherapy • Medical Bacteriology: A Practical Approach Ed P.Hawkey & D.Lewis, Oxford University Press, 2003. • Antibiotics & Chemotherapy: Anti-Infective Agents & their use in therapy R.Finch et al Churchill Livingstone, 2002. • Textbook of Diagnostic Microbiology C.R Mahon & G.Manuselis Saunders & Co Ltd 2000. • Antimicrobial Chemotherapy D. Greenwood Oxford Medical Publications 1999. • Clinical Microbiology; E. J. Stokes, G.L. Ridgway & M.W.D Wren: Arnold; 1993