Susceptibility testing of itraconazole

Introduction

Itraconazole is a drug which can be used in the treatment of various localised and systemic fungal infections, including those attributed to Aspergillus species. Isolates should be tested for susceptibility to itraconazole to ensure the isolate is not resistant.

Hazards

Standard microbiological technique is adequate for safety whilst carrying out this procedure.

Specimens

Conidia producing isolates from a suitable medium, such as Sabouraud agar.

Materials (see Appendix for preparation of media)

  • Tween® 80
  • Itraconazole - Janssen Pharmaceuticals, Beerse, Belgium.
  • RPMI-1640 - Sigma (Cat No. R-8758)
  • Morpholinopropanesulfonic acid (MOPS)
  • Glucose
  • Acetone
  • Hydrochloric acid

Equipment

  • Vortex mixer
  • Haemocytometer
  • Water bath at 60oC
  • Sterile microtitre plate
  • Sterile microtitre lid
  • Glass bijou bottles (sterile)
  • Digital pipettes and sterile pipette tips
  • Sterile loops

Quality Control

Internal control procedure -

  1. A positive control (drug-free) is set up for each test isolate.
  2. A negative control (inoculum-free) is included with each batch of isolates to be tested, to ensure the sterility of the medium.
  3. A solvent control is included. This contains no antifungal drug but a concentration of the solvent (used to dissolve the drug) equivalent to that in the well with the highest concentration of antifungal drug.
  4. An organism with a known MIC to itraconazole is tested with each batch. Aspergillus fumigatus isolate (12AF) is used in our laboratory. The MIC of 12AF is 0.125-0.25mg/l.

Procedure

Preparation of Drug Dilutions:

  1. Prepare stock solution of itraconazole (3200mg/l). Weigh out 0.032g of pure itraconazole powder into a glass universal and add 5ml of acetone. Then, and only after adding the acetone, add 5ml of 0.2M HCl. Vortex vigorously and place in a water bath at 60oC until dissolved, vortexing occasionally. Dispense into 1ml amounts and store at - 20oC.
  2. Dilute the stock solution of itraconazole in sterile water by 1/10 to give a solution of itraconazole at 320mg/l. Prepare the start solution of itraconazole at 32mg/l in RPMI medium by diluting the 320mg/l solution by 1/10. (200ml needed per isolate).
  3. To wells in columns 2-12 (excluding 11) add 100ml of RPMI medium.
  4. To the first well in each row of the microtitre plate add 200µl of the itraconazole start solution. (32mg/l)
  5. Using the multichannel pipette double dilute 100µl volumes from columns 1-10, discarding the extra 100µl from wells in column 10. This will yield a final range of drug dilutions from 16-0.03mg/l
  6. Column 11 is the solvent control - add 100µl of the solvent solution equivalent to the solvent contained in the 32mg/l drug dilution.
  7. Column 12 is the drug-free positive control.
  8. Inoculate a separate well with 200µl RPMI medium. This acts as the medium sterility control.

Inoculum Preparation:

  1. In a class I safety cabinet, moisten a sterile loop with PBS/Tween and gently move over the surface of the Aspergillus spores. Transfer the loopful of spores into 5ml of PBS/Tween. Vortex well.
  2. Count the number of spores using a haemocytometer. Calculate the number of spores present per ml of PBS/Tween. Adjust to 1x106 conidia/ml using RPMI medium.
  3. Viability counts are performed on each isolate. The spore suspension (1x106 conidia/ml) is diluted initially to 2x103 conidia/ml by diluting in PBS/Tween. The resulting suspension is diluted a further 1/10 and 1/100 in PBS-Tween. 100µl of the neat suspension (2x103 conidia/ml) and dilutions are plated and spread over blood agar plates. These plates are then incubated at 37oC for 48 hours.

Inoculation of assay plates:

  1. For each isolate add 100ml of the inoculum into all wells in the appropriate row. (One row for each isolate). The final inoculum is now 5x105 conidia/ml.
  2. Incubate the microtitre plate for 48 hours at 37oC in a moist chamber

Interpretations

  1. The MIC is read visually. The concentration of drug in the first well in which there is no growth is the MIC value.
  2. Check the viability counts to ensure that the initial inoculum was prepared correctly. The 10-1 dilution plate should have between 10-40 colonies, and the 10-2 dilution plate should have 1-4 colonies.
  3. MFC = 100ml from each well with no growth is placed on one half of a blood agar plate, after gentle pipetting of the contents of each well to mix the suspension. Use a fresh sterile tip for every well. The plates are left to dry and then streaked out, to avoid carry over of the drug.
  4. The plates are then incubated at 37oC for 48 hours. The MFC is the concentration of drug with no growth, or up to and including 5 colonies. This represents >99.99% kill.

Limitations

  1. Some organisms may not grow in RPMI medium. In such cases, report as "failed to grow in test medium - sensitivity not available".
  2. Due to poorly defined endpoints, experience is required in reading the results.

Trouble-shooting

Observation: growth is observed in the medium control. 
Cause: contaminated medium.
Action required: repeat the tests with a new batch of medium.

Observation: the MIC of the control organism does not fall within normal limits (0.125-0.25mg/l).
Cause: this may be due to faulty preparation of the drug or inoculum or contamination of the control organism.
Action required: repeat all tests, with new batches of drug stock and medium. A fresh culture of the control organism should also be used.

Observation: viability counts are not within the acceptable ranges.
Cause: inoculum and/or dilutions prepared incorrectly.
Action required: disregard results and repeat tests, including the control organism, after a careful review of all steps in the protocol.

Reporting

  1. Report the MIC in mg/l. Assign the following comments to the report form -

  • MIC = <2mg/l SENSITIVE
  • MIC = 4mg/l INTERMEDIATE
  • MIC = >8mg/l RESISTANT
  1. Report the MFC in mg/l. If the value is >16mg/l, type in "itraconazole is not fungicidal against this isolate".

Timetable

Preparation of drug dilutions:

  • (1) 1-2 h depending upon the length of time taken to dissolve in the water bath
  • (2-8) 15 min

Inoculum preparation:

  • (9-10) 10-20 min, for each isolate, depending upon experience

Viability counts:

  • (11) 15 min per isolate

Inoculation of assay plate:

  • (12) 2-3 min per isolate
  • (13) 48 h

Reading of assay plate:

  • (1-2) 5 min per isolate

MFCs:

  • (3) 10 min per isolate
  • (4) 48 h followed by 5 min per isolate

References

D. W. Denning, S. A. Radford, K. L. Oakley, L. Hall, E. M. Johnson and D. W. Warnock. (1997). J. Antimicrob. Chemother. 40, 401-414.

Appendix

Preparation of RPMI-1640 Medium with 2% Glucose (RPMI medium):

  1. Place 900ml of RPMI-1640 medium into a conical flask.
  2. Add 34.5g of morpholinopropanesulfonic acid powder.
  3. Stir to dissolve.
  4. Add 20g of glucose and continue stirring. Ensure that all powder has completely dissolved.
  5. Adjust the pH to 7.0 using 10M NaOH.
  6. Make up to 1 litre with RPMI-1640 medium and re-check the pH.
  7. Filter sterilise.
  8. Store in sterile medical flats at 4oC.

Preparation of PBS-Tween:

  1. Dissolve tablets as per manufacturer's instructions in distilled water.
  2. Add 0.05% Tween 80 before sterilisation.
  3. Aliquot into 20ml amounts in universal bottles.
  4. Sterilise in an autoclave for 20 minutes at 121oC (15psi).
  5. Tighten the lids and store at room temperature.

Year prepared: 

2003