Laboratory Aspects of Antimicrobial Chemotherapy

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
Kirby-Bauer Method

• Developed in the USA in 1966.
• Based on NCCLS ( National Committee for Clinical
  Laboratory Standards ) data.
• Use Mueller-Hinton agar.
• Use standard 0.5 McFarland (BaSO4) inoculum.
• Streak inoculum in 3 directions or rotary plate.

11
Kirby-Bauer Method

• Use standard discs incubation conditions.
• Use standard NCCLS tables to interpret zone sizes
  as S, I or R.
• Interpretation based on regression line analysis
  of zone diameter size to MIC.
• Interpretation based on confluent growth of the
  organism.

12
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.

13
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.

14
Stokes method
Control organism
Test organism
Antibiotic discs
15
Stokes Comparative Method
16
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.

17
Disadvantages of Stokes Method

• Interpretation not valid for ß 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.

18
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
  (ESBLs).
• No consistent method between labs, therefore no
  consistent epidemiology data.

19
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 www.bsac.org.uk.
• Use ISA and/or ISA 5 horse blood 20mg/l NAD.
• Use standard 0.5 McFarland inoculum

20
Preparation of 0.5 McFarland
21
BSAC Method

• Use dilution protocol of 0.5 McFarland standard
  as specified by BSAC.
• Neat 0.5 Neisseria gonorrhoeae.
• 110 dilution of 0.5 Staphylococci.
• 110 dilution of 0.5 for Streptococci.
• 1100 dilution of 0.5 for Coliforms.
• 1100 dilution of 0.5 for Haemophilii

22
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.
• http//www.bsac.org.uk/_db/_documents/version_7_1_
  february_2008.pdf

23
Examples of BSAC Method
24
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.

25
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)

26
Template for interpreting zone diameters
The test plate is placed over the template and
the zones of inhibition are examined in
relationship to the template zones. If the zone
of inhibition of the test strain is within the
area marked with an R, the organism is
resistant. If the zone of inhibition is equal to
or larger than the marked area, the organism is
susceptible
27
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

28
Agar Breakpoint Method

• Uses published breakpoint concentrations.
• Antibiotic concentrations higher lower than the
  breakpoint are incorporated into agar.
• The test organism is inoculated incubated.
• Growth at the higher concentration indicates
  resistance.
• Growth at the lower but not the higher indicates
  intermediate resistance.
• No growth at the lower concentration indicates
  susceptibility.

29
Agar Breakpoint Method

• Can use agar in 96 well microtitre trays.
• Can use multipoint inoculation.
• Method can be automated for susceptibility
  identification e.g Mastascan Elite (Mast).
• The disadvantages are that it cannot detect
  emerging resistance problems it cannot be
  adequately controlled.

30
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

31
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.

32
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.

33
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.

34
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.

35
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.

36
Examples of E-tests
37
Examples of E-tests
38
Laboratory E-tests

• In the lab we use E-tests routinely to check-
• Monitoring development of resistance in
  endocarditis isolates
• Mupirocin susceptibility (MRSA).
• Confirmation of teicoplanin and/or vancomycin
  resistance (GISA, VRE).
• Isolates of Salmonella typhi / paratyphi
  ciprofloxacin susceptibility.
• Confirmation of penicillin resistant
  Streptococcus pneumoniae.

39
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.

40
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.

41
Vitek cards
42
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

43
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.

44
Vitek modules
45
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.

46
Molecular Methods

• Application of genotypic methods can allow rapid
  detection of resistance genes direct from the
  sample.
• Examples include-
• mecA gene detection by PCR denotes resistance to
  methicillin in Staph aureus (MRSA).
• Rifampicin isoniazid resistance in MDR
  Mycobacterium tuberculosis can be detected using
  a DNA probe (rpoB).
• Antiviral drug resistance due to genetic point
  mutations can be examined by PCR for HIV, CMV
  HCV.

47
How do we detect resistance mechanisms?

• ß-lactamases the are many types
• Selectively destroy the ß-lactam ring component
  of the antibiotic.
• The lab uses the nitrocefin test for detection of
  TEM ß-lactamase.
• It is a chromogenic cephalosporin that changes
  colour from yellow to red when the ß-lactam ring
  is hydrolysed.
• It is used on isolates of Haemophilus, Moraxella
  Neisseria.

48
Extended Spectrum ß-lactamases (ESBLs)

• ß-lactamases that inactivate the action of
  third-generation cephalosporins.
• A variety of detection methods are available,
  most based on measuring zone sizes of
  ceftazidime, cefotaxime cefpodoxime /-
  clavulanate.
• Interpretation is made by zone diameter
  difference /- clavulanate ESBLs are inhibited
  by clavulanate a ß-lactamase inhibitor

49
Combination disc method cefpodoxime 10 ?g
clavulanate vs. cefpodoxime 10 ?g.
50
ESBL detection

• ESBLs are important in cross-infection, may
  become multi-resistant cause life-threatening
  infection.

51
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.

52
Dosing interval and steady state plasma
concentration
Therapeutic failure
Drug concentration in plasma
Therapeutic range
Therapeutic failure
Time
53
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

54
Bioassay

• Use a specific strain of bacteria susceptible to
  the drug being assayed.
• Seed a large square bioassay agar plate.
• Cut wells from the agar using a cork-borer.
• Inoculate known control standards of the
  antibiotic to be assayed.
• Inoculate the patient pre post drug serum
  samples in triplicate.

55
Bioassay

• Incubate the plate overnight.
• Measure the zone diameter of the controls
  tests.
• Plot the zone of inhibition versus the log of the
  antibiotic control concentration.
• Read the test results by extrapolation of the
  graph.
• The bioassay is an inexpensive technique, however
  it has low specificity is very time-consuming.

56
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.

57
TDX Analyser
58
TDX Principles

• The TDX combines fluorescence polarisation
  technology competitive binding immunoassay.
• Serum antibiotic competes with fluorescein-labelle
  d antibiotic (tracer) in test kit for antibody
  binding
• The technology reads the proportion of bound
  unbound fluorescein.
• Excess larger bound molecules results in a high
  emission of polarised light i.e low test .
• Excess small unbound molecules results in a low
  emission of polarised light i.e high test.

59
TDX Principles
60
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.

61
TDX Dose Response Curve
62
Summary
63
References

• British Society for Antimicrobial Chemotherapy
  www.bsac.org.uk
• 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