Technical information

DIRECT OBSERVATION BY MICROSCOPY USING INDIA INK

India ink preparation

Dilute 1 part of India ink with 2 parts of distilled water in a vial with dropper and store at room temperature.

The solution is stable for at least 6 months.

Observation by microscopy

For diagnosis, direct observation is usually applied to CSF samples.

Mix a drop of concentrated CSF (centrifuged at 5000 rpm for 5 min) with a drop of India ink preparation on a

slide and cover it with a coverslip, then put another coverslip near to the first one and then a third in order to expand the solution.

Observe carefully all the preparation moving up and down and from left to right side.

The sample is positive for Cryptococcus if you see round cells surrounded by a halo (capsule), it is also important to confirm

the yeast nature of the observed cells finding budding cells.

ASSIMILATION OF MYO-INOSITOL

Cryptococcus is able to assimilate myo-inositol therefore when you have a CSF sample it is important also to culture part of the concentrated CSF on inositol agar with chloramphenicol to exclude or confirm the presence of the yeast.

Medium preparation

Yeast nitrogen base (YNB) 10X:

Dissolve 6.7 g of YNB in 100 ml of distilled water and filter sterilize the solution, then dispense 10 ml of the solution in 10 sterile vials.

Myo-inositol with chloramphenicol:

Dissolve 10 g of myo-inositol and 1 g of chloramphenicol (CAF) in 100 ml, boil the solution till complete dissolving and then filter sterilize and distribute 10 ml in 10 sterile vials.

2% agar:

Dissolve 2 g of agar in 100 ml of distilled water, boil for dissolving the solution and then distribute 18 ml in sterile vials. Sterilize by autoclaving.

Plate preparation

Mix 2 ml of YNB 10X, 1 ml of inositol plus CAF solution, and 18 ml of melted agar 2% in a 90 mm Petri dish and let it at room temperature until it is solidified.

Culture

Put a drop of the concentrated CSF on a plate containing YNB+ inositol+CAF and streak the sample on five sides to isolate the yeast. Incubate at 37°C for at least 5 days.

UREA HYDROLYSIS

Urease activity is tested culturing the yeast in urea broth medium.

Urea medium preparation (10X solution)

Dissolve 0.01 g yeast extract, 0.0091 g KH2PO4, 0.0095 g Na2HPO4, 2 g urea, 0.001 g phenol red, in 10 ml of distilled water and then filter sterilize the solution.

Paper disk preparation

Under a flow cabinet, put some sterile paper disks (about 6 mm diameter) onto a sterile Petri dish well separated each to the other, and then distribute a 50-µl drop

on each disk. Let the disks dry under the flow cabinet for some hours. When they are dried put the disks in a sterile vial and store them at 4-8°C.

The disks are stable for at least 6 months.

Test

Prepare a heavy concentrated suspension of the yeast in 0.5 ml of sterile distilled water. Put inside the suspension one disk embedded with urea broth and incubate at 37°C for at least 48h. If the suspension turns to red color then the test is positive and it means that the yeast have the urease enzyme, otherwise the solution remains yellow.

MELANIN PRODUCTION

Cryptococcus neoformans and C. gattii produce the enzyme phenoloxydase (laccase), which is able to convert phenolic compounds in melanin. In order to test if a yeast has this enzyme we need to culture it on a selective medium containing phenolic compounds. The most used media for this purpose are Niger seed agar or sunflower medium.

Niger seed agar or sunflower medium preparation

50 g Guizotia abyssinica (Niger seeds) or 50 g of Helianthus annuus (sunflower seeds)

1 g glucose

1 g KH2PO4

1 g creatinine

15 g agar

1000 ml distilled water

1. Grind the seeds as finely as possible with an electric mixer and add to 1000 ml distilled water in a stainless steel jug.

2. Boil for 30 minutes, pass through filter paper and adjust volume to 1000 ml.

3. Add remaining ingredients and dissolve.

4. Autoclave at 110 °C for 20 minutes.

5. For culture of environmental samples:

cool to 50°C and add 10 ml of CAF (0.1 g in 10 ml distilled water filter sterilized) and 0.3 ml benomyl (5 mg/ml DMSO)

6. Mix gently and pour into 90 mm plastic petri dishes.

Culture

Culture the yeast on the medium at 37°C for 5-7 days. If brown colonies grow, this means that the yeast is able to produce melanin.

DIFFERENTIAL MEDIUM TO DISTINGUISH C. NEOFORMANS FROM C. GATTII

Canavanine-glicine-bromothymol blue (CGB) agar medium

Solution A

Glycine 10 g

KH2PO4 1 g

MgSO4 1 g

Thiamine HCl 1 mg

L-canavanine sulphate 30 mg

Distilled water 100ml

1. Dissolve ingredients in small beaker and adjust pH to 5.6

2. Filter sterilize solution using 0.22 μm filter.

3. Store in refrigerator.

Solution B

Bromthymol blue 0.4 g

0.01N NaOH 64 ml

Distilled water 36 ml

1. Dissolve the bromthymol blue in the NaOH

2. Add to this the water.

Medium preparation

Distilled water 880 ml

Solution B 20 ml

Bacto agar 20 g

1. Autoclave to 121°C for 15 minutes, cool to 48°C.

2. For plates add 100 ml of the filtered solution A and mix. Dispense in plates.

Test

Streak a heavy inoculum of the yeast on the plate and incubate at 25°C for 2-7 days.

C. gattii turns the medium blue.

ISOLATE CONSERVATION

The isolates can be easily conserved dissolving 3 loopful of the fresh yeast in a sterile screw-cap vial containing 3 ml

of sterile distilled water and stored at room temperature. Prepare at least two vials for each isolate.

CRYPTOCOCCAL ANTIGEN DETECTION

The main commercial kits used for cryptococcal antigen detection in serum and CSF are produced by Meridian Biosciences Inc.:

- Cryptococcal Antigen Latex Agglutination System (CALAS®)

- Premier® Cryptococcal Antigen

DNA EXTRACTION FROM A CRYPTOCOCCUS CULTURE

Preparation of the reagents

SCS buffer

2 ml Sodio citrato (pH=5.8) 1 M

in 98 ml Sorbitolo 1M

Protoplasting solution

1 mg Chitinase

20 mg Beta-glucanase

in SCS buffer 1 ml

Lysing Buffer

100 mM Tris (pH=8.6) 1.576 g in 100 ml dH2O

1.5 M NaCl 8.766 g

8% DTAB 8 g

50 mM EDTA 1.861 g

CTAB solution

5% CTAB

in NaCl 0.4 M

NaCl solution

NaCl 1.2 M

TE solution

10 mM Tris (pH=7.4)

1 mM EDTA

Method

1. Culture 2 slants containing Sabouraud dextrose agar and incubate at 35°C for 48 h.

2. Suspend about 10^8 lieviti in 2 ml sterile distilled water gently washing the surface of the medium using a sterile Pasteur pipette.

3. Centrifuge the sample at 5000 rpm for 5 min, discard the supernatant and suspend the pellet in 1 ml SCS buffer. Mix using a vortex.

4. Repeat point 3.

5. Centrifuge the sample at 5000 rpm for 5 min.

6. Discard the supernatant and suspend the pellet in 1 ml Protoplasting Solution. Mix by vortex. Seal the vial with parafilm and incubate in a thermostatic bath at 37°C for 1 h.

7. Centrifuge the sample at 5000 rpm for 5 min.

8. Discard the supernatant and suspend the pellet in 600 µl Lysing Buffer. Mix by vortex and incubate in a thermostatic bath at 68°C for 30 min.

9. Add 900 µl cold chloroform and mix by inverting the vial.

10. Centrifuge the sample at 12000 rpm for 10 min.

11. Transfer 350 µl of the aqueous phase containing the DNA (upper phase) in a new 1.5 ml vial

12. Add 100 µl CTAB solution and then 700 µl of nuclease-free water

13. Gently mix by invertion and centrifuge the sample at 10000 rpm for 3 min.

14. Discard supernatant and add 150 µl NaCl 1.2 M, mix gently and add 500 µl cold ethanol 100%.

15. Mix gently inverting more times the vial and centrifuge the sample at 10000 rpm for 10 min.

16. Discard the supernatant and suspend the pellet in 500 µl cold ethanol 70% , and mix until the pellet detach from the bottom of the vial.

17. Centrifuge the sample at 10000 rpm for 10 min.

18. Discard the supernatant and air dry under the flow cabinet leaving the vial open (about 1 h and 30 min).

19. Suspend the DNA in 50 µl of nuclease-free water.

20. Store at 4°C if you have to use the DNA in a brief period.

21. If you have to store the DNA for long time after point 18 add 5 µl of TE 10X and store at -20°C.

DETERMINATION OF C. NEOFORMANS MATING TYPE ALLELIC PATTERN BY MULTIPLEX PCR

(Esposto et al. Clin Microbiol Infect, 2004;10:1089-1104)

MIX (50 µl)

Buffer 10X, 3mM MgCl2 (check if in your buffer there is MgCl2, in that case you have to adjust the

magnesium quantity to reach a final concentration of 3 mM), 400µM dNTPs, 20 pmol di each primer, 2.5 U di Taq polimerasi, 400 ng DNA sample.

Primers (usually I make a stock solution of 5 pmol/µl and then I put 4 µl of each primer in the mix):

STE20aAF TCCACTGGCAACCCTGCGAG

STE20aAR ATCAGAGACAGAGGAGCAAGAC

STE20alphaDF TGGGCGTATCCCAACCTACGA

STE20alphaDR TAACGACTCCGGTGCCGTGAA

NAD4F CATTTATCCGTATACGCATTCCC

NAD4R GCAAATTCAGCACCAGCAATAGT

NCP1F GGCAGTGATGGTTTCATCCGTA

NCP1R ATTGTCATCAGGGTCGACAAACA

Thermal cycling

Initial cycle: 5 min at 94°C

30 cycles:

30s at 94°C

10s at 60°C

1 min at 72°C

Final extension: 5 min at 72°C

Electrophoresis

Gel 1.4% at 70V for 1h 30 min

DETERMINATION OF C. NEOFORMANS MOLECULAR TYPE BY MULTIPLEX PCR

(Cogliati et al. , Med Mycol, 2000;38:97-103)

MIX (50 µl)

Buffer 10X, 3mM MgCl2 (check if in your buffer there is MgCl2, in that case you have to adjust the magnesium

quantity to reach a final concentration of 3 mM), 400µM dNTPs, 20 pmol di each primer except for 420F2 and B420R

that need only 10 pmol , 2.5 U di Taq polimerasi, 400 ng DNA sample.

Primers (usually I make a stock solution of 5 pmol/µl and then I put in the mix 4 µl of each primer except for 420F2 and B420R that need only 2 µl):

800F1 GTCTCTTAATTACTCAGC

800R1 GCTTTTTTTTCTGTCTCC

500F1 ACGGCAACCTTCTTATG

500R1 CGGTAGTGCGTTTTAAG

470F2 ACACAAAGGAAACTCGCT

470R3 ACAAACCCCCCTCTTC

420F2 ATTGTCGGTGCTTGCAT

B420R AACATCTCCCATTCCTC

Thermal cycling

Initial cycle: 5 min at 94°C

25 cycles:

30s at 94°C

10s at 52°C

1 min at 72°C

Final extension: 5 min at 72°C

Electrophoresis

Gel 1.4% at 70V for 1h 30 min

DETERMINATION OF C. GATTII MATING TYPE ALLELIC PATTERN BY MULTIPLEX PCR

(Cogliati et al. , Clin Microbiol Infect 2015; 21: 190.e1–190.e4)

MIX (50 µl)

Buffer 10X, 3mM MgCl2 (check if in your buffer there is MgCl2, in that case you have to adjust the magnesium quantity to reach a final

concentration of 3 mM), 400µM dNTPs, 20 pmol di each primer except for GEFCalphaF and GEFCalphaR that need only 10 pmol , 2.5 U di Taq polimerasi, 400 ng DNA sample.

Primers (usually I make a stock solution of 5 pmol/µl and then I put in the mix 4 µl of each primer except for GEFCalphaF and GEFCalphaR that need only 2 µl):

GEFCaF GAGATTGCGTGCTGCAGGTTACA

GEFCaR AGCTATTGGTGGTTGCGAATGAGA

STE12BaF GGTCCCACTGAATCTAAGTCATTG

STE12BaR GGAGCCGCGTGGCGCAAGC

VPSalphaBF GCTCGGTCTACTATGATGGCGAA

VPSalphaBR AGACAATTTTCAATTCCGACTTCCAT

GEFCalphaF GGGACAAGTCTCTGCACACGAG

GEFCalphaR TCAACGAAGCAATCTCCTCTTCGA

Thermal cycling

Initial cycle: 5 min at 94°C

28 cycles:

30s at 94°C

10s at 70°C

30s at 72°C

Final extension: 5 min at 72°C

Electrophoresis

Gel 1.6% at 70V for 2h

DETERMINATION OF C. GATTII MOLECULAR TYPE BY DUPLEX PCR

(Feng et al. , J Clin Microbiol, 2013;51:3110-12)

MIX (50 µl)

Buffer 10X, 3mM MgCl2 (check if in your buffer there is MgCl2, in that case you have to adjust the magnesium

quantity to reach a final concentration of 3 mM), 400µM dNTPs, 20 pmol di each primer, 2.5 U di Taq polimerasi, 400 ng DNA sample.

Primers (usually I make a stock solution of 5 pmol/µl and then I put 4 µl of each primer in the mix):

VACF AGCCCACGGCAAAATAGTG

VACR CACGGTCCAAAACTTGATTGTT

IGSF CCGAGGCAGGACACACATAC

IGSR GGCGGAATACAAATACTACTTACCT

Thermal cycling

Initial cycle: 5 min at 94°C

30 cycles:

30s at 94°C

30s at 56°C

20s at 72°C

Final extension: 6 min at 72°C

Electrophoresis

Gel 1.4% at 70V for 1h 30 min