TITLE: Screening procedure to identify triazole-resistant Aspergillus spp. using agar plates
AUTHORS: Juliê  JA Alves

[DESCRIPTION]
The methodology of the screening procedure for detecting resistance to azoles in the genus Aspergillus spp. consists of an in house technique, with visual analysis of fungal growth on agar plates supplemented with azoles, Itraconazole at a concentration of 4 µg/ml, Voriconazole 2 µg/ml and Posaconazole 0.5 µg/ml. This protocol is based on the E.Def 10.1 from EUCAST

[BEFORE_START]
Separate all reagents and identify the plates

[STEPS]
SECTION: Preparation of antifungal solutions:
1. From the antifungal powder prepare stock solutions in DMSO, at concentrations at least 200 times higher than those tested on the agar plate 
SECTION: Preparation of antifungal solutions:
2. From the stock solution, prepare the working solutions at the following concentrations: 
Itraconazole (ITZ) - 400 µg/ml, 
Voriconazole (VRZ) - 200 µg/ml 
Posaconazole (PSZ) - 50 µg/ml

Storage Temperature: -25 to -18º C (Freezer)

For calculations, use the formula: 
Initial concentration x Initial volume = Final concentration x Final volume
  
       Concentração da Solução de Estoque      Volume da Solução de Estoque      Volume de DMSO      Concentração da Solução de Trabalho      Concentração Final no agar          µg/mL      µL      µL      µg/mL      µg/mL        ITC      1600      2500      7500      400      4        VRC      400      5000      5000      200      2        PSC      400      1250      8750      50      0,5   
 
 
SECTION: Preparation of the medium:
3. Weigh all reagents:
RPMI 1640 powder – 10.4 g 
D-Glucose 2% - 18 g 
Bacteriological agar – 20 g 
MOPS - 34.53 g (0.165 M)

SECTION: Preparation of the medium:
4. In a 1L becker, Add 900 mL of distilled water and RPMI powder 10.4 g, glucose 2% 18 g, Bacto Agar 20 g, MOPS 34.53 g 

SECTION: Preparation of the medium:
5. Microwave 5 min, little by little, stirring with a glass rod, until the solutes are completely dissolved
 
SECTION: Preparation of the medium:
6. Confirm the pH of 7.0 

  

SECTION: Preparation of the medium:
7. Add more 100 mL of distilled water
  

SECTION: Preparation of the medium:
8. 
Divide the medium into 4 Erlenmeyer flasks containing 250 mL each

SECTION: Preparation of the medium:
9. Autoclave the medium at 121º for 15 min.
SECTION: Preparation of the azole-containing plates:
10. Identify all the plates with the azole or "control"
 

SECTION: Preparation of the azole-containing plates:
12. Add 2.5 mL of each azole working solution (100% final concentration) to each of the vials and ensure proper mixing

SECTION: Preparation of the azole-containing plates:
13. The remaining antifungal free vial will be used for the growth control 
SECTION: Preparation of the azole-containing plates:
14. Pour the agar from each vial into the respective plates
 

SECTION: Preparation of the azole-containing plates:
11. Cool the agar down to 45°C
SECTION: Preparation of the azole-containing plates:
15. After solidification of the plates (approximately 20 min) Storage the plates at 4°C for up to 6 months from the date of preparation.
  

SECTION: Preparation of the inoculum:
16. For inoculum suspensions use fresh and mature cultures incubated for at least 2–7 days 
SECTION: Preparation of the inoculum:
18. Working in the biological safety cabinet, add 3 ml of the water/Tween 20 solution to the 3-7 day slope. Tween 20 will help to put the Aspergillus conidia into suspension.
SECTION: Preparation of the inoculum:
17. Make a diluent stock solution by adding 1 ml of Tween 20 to every 50 ml of distilled water
SECTION: Preparation of the inoculum:
16.1. The isolates should be cultured on  Sabouraud dextrose agar (with or without chloramphenicol) or other culture medium where the fungus is able to sporulate sufficiently and incubated at 34–37°C
  

SECTION: Preparation of the inoculum:
19. If necessary gently detach only the conidia from the culture with a handle until the solution becomes turbid
SECTION: Preparation of the inoculum:
20. Put 1.5 mL of this conidial suspension in identified eppendorfs
  

SECTION: Preparation of the inoculum:
20.1. This conidial suspension can be used in case of retest
SECTION: Preparation of the inoculum:
21. Use the residual solution from the tube from steps 17-20 to perform the next steps
SECTION: Preparation of the inoculum:
22. Add 2 mL of destilled water in a tube and transfer 1-2 drops of the residual solution with conidial 

SECTION: Preparation of the inoculum:
22.1. Or transfer 1-2 drops of the conidial suspension
SECTION: Preparation of the inoculum:
22.2. In some cases you will need more drops, for Aspergillus flavus for example
SECTION: Preparation of the inoculum:
23. Visually compare the suspension to the 0.5 McFarland Standard and make adjustments as described until the suspension visually matches the standard
  

SECTION: Preparation of the inoculum:
23.1. Adjustment is made by adding more destilled water if the suspension is very turbid and more conidia if the turbidity is very low.
SECTION: Inoculation and incubation of agar plates:
24. Dip a sterile cotton swab in the inoculum suspension and rub the entire surface of the control plate in a back-and-forth motion 
SECTION: Inoculation and incubation of agar plates:
25. Re-dip the cotton swab and rub the entire surface in the same way of each of the three remaining plates with the azoles
SECTION: Inoculation and incubation of agar plates:
26. Incubate the plates at 35-37°C for 2880 min and observe growth.

SECTION: Inoculation and incubation of agar plates:
26.1. Visual analysis score definition: 
0: no visible growth
1: weak/minimal growth (such as >5 tiny colonies or confluent weak growth where isolate was inoculated (covering ≤ half of the plate)
2: clearly visible growth with hyphal extension, but not covering entire plate 
3: prominent uninhibited growth covering most of the plate
SECTION: Analyzing the results
27. The presence or absence of fungal growth on the surface of the plates will yield the following preliminary classification:

Azole susceptible isolate: growth on the azole-free plate and absence of growth on the others plates

Potentially an azole non-susceptible isolate: growth on the azole-free plate and on any of the other supplemented plates.
 
    

SECTION: Preparation of the medium:
6.1. If necessary adjust with a solution of NaOH (10 M) to 7.0