Amphotericin B is a drug which can be used in the treatment of systemic fungal infections, including those attributed to Aspergillus species Isolates should be tested for susceptibility to amphotericin B to ensure the isolate is not resistant.
Standard microbiological technique is adequate for safety whilst carrying out this procedure.
Conidia producing isolates from a suitable medium, such as Sabouraud agar.
Materials (see Appendix for preparation of media)
- Phosphate buffered saline
- Tween® 80
- Amphotericin B is supplied by E. R. Squibb
- RPMI-1640 - Sigma (Cat No. R-8758)
- Morpholinopropanesulfonic acid (MOPS)
- Hydrochloric acid
- Vortex mixer
- Water bath at 60oC
- Sterile microtitre plate
- Sterile microtitre lid
- Glass bijou bottles (sterile)
- Digital pipettes and sterile pipette tips
- Sterile loops
Internal control procedure -
- A positive control (drug-free) is set up for each test isolate.
- A negative control (inoculum-free) is included with each batch of isolates to be tested, to ensure the sterility of the medium.
- 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.
- An organism with a known MIC to amphotericin B is tested with each batch. Aspergillus fumigatus isolate (12AF) is used in our laboratory. The MIC of 12AF is 0.5-0.1 mg/l.
Preparation of Drug Dilutions:
- Prepare stock solution of amphotericin B (1600 mg/l) by dissolving 0.035 g in 10 ml of sterile distilled water. (This allows for the content of sodium desoxycholate). This solution may be dispensed into 0.6 ml amounts and stored at -20° C protected from the light.
- Dilute the stock solution of amphotericin B in sterile water by 1/5 to give a solution of amphotericin B at 320mg/l. Prepare the start solution of amphotericin B at 32mg/l in RPMI medium by diluting the 320mg/l solution by 1/10. (200m l needed per isolate).
- To wells in columns 2-12, add 100m l of RPMI medium.
- To the first well in each row of the microtitre plate add 200m l of the amphotericin B start solution (32mg/l).
- Using the multichannel pipette double dilute 100m l volumes from columns 1-11, discarding the extra 100m l from wells in column 11. This will yield a final range of drug dilutions from 16-0.015 mg/l.
- Column 12 is the drug-free positive control.
- Inoculate a separate well with 200m l RPMI medium. This acts as the medium sterility control.
- 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.
- 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.
- 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. 100m l 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:
- For each isolate add 100m l of the inoculum into all wells in the appropriate row. (One row for each isolate). The final inoculum is now 5x105conidia/ml.
- Incubate the microtitre plate for 48 hours at 37oC in a moist chamber.
- The MIC is read visually. The concentration of drug in the first well in which there is no growth is the MIC value.
- 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.
- MFC = 100m l 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.
- 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.
Some organisms may not grow in RPMI medium. In such cases, report as "failed to grow in test medium - sensitivity not available".
Due to poorly defined endpoints, experience is required in reading the results.
- 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.
- Report the MIC in mg/l. Assign the following comments to the report form -
Therapeutic response may be a better guide to eventual outcome than MIC results for amphotericin B. No interpretations should be provided for this drug since breakpoints have not been established.
- Report the MFC in mg/l.
Preparation of drug dilutions:
(1) 10 min
(2-8) 15 min
(9-10) 10-20 min, for each isolate, depending upon experience
(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
(3) 10 min per isolate
(4) 48 h followed by 5 min per isolate
Denning DW, Radford SA, Oakley KL, Hall L, Johnson EM, Warnock DW. J Antimicrob Chemother 1997; 40:401-414.
Preparation of RPMI-1640 Medium with 2% Glucose (RPMI medium)
- Place 900ml of RPMI-1640 medium into a conical flask.
- Add 34.5g of morpholinopropanesulfonic acid powder.
- Stir to dissolve.
- Add 20g of glucose and continue stirring. Ensure that all powder has completely dissolved.
- Adjust the pH to 7.0 using 10M NaOH.
- Make up to 1 litre with RPMI-1640 medium and re-check the pH.
- Filter sterilise.
- Store in sterile medical flats at 4oC.
Preparation of PBS-Tween
- Dissolve tablets as per manufacturer's instructions in distilled water.
- Add 0.05% Tween 80 before sterilisation.
- Aliquot into 20ml amounts in universal bottles.
- Sterilise in an autoclave for 20 minutes at 121oC (15psi).
- Tighten the lids and store at room temperature.