Two talks from the Mycology References Laboratory at the National Aspergillosis Centre, Nichola Duddy on "What happens to my sample?" and Caroline Moore on "Antifungal drugs - are you getting enough?"

1. Led by Graham Atherton Supported by Georgina Powell, Marie Kirwan & Debbie Kennedy NAC Centre Manager Chris Harris Talks given by Nicola Duddy and Caroline Moore Mycology Diagnostics Lab UHSM, Manchester National aspergillosis centre UHSM Manchester Support Meeting for Aspergillosis Patients Fungal Research Trust

2. What happens to my sample?? Mrs Nichola Duddy Mycology Reference Centre University Hospital of South Manchester The University of Manchester

3. Sample types Sputum – culture, galactomannan, PCR BAL (bronchoalveolar lavage) – taken by bronchoscopy. Culture, galactomannan, PCR Blood – serology, antifungal levels

4. Culture Selective agar: nutrients, antibiotics and low pH Sputum digestion (1:2) Streak onto Sabouraud agar (10µL = 0.01mL) = 1:200 Incubate for up to 5 days at 30˚C and 45˚C

5. Identification Why? Which fungus is causing the disease – monitor which are most prevalent species Drug choice – A. terreus AMPHO How? Colony morphology – colour, size and texture

6. The Aspergilli – various colours and sizes Klich MA. Identification of common Aspergillus species (2002). CBS.

7. Aspergillus fumigatus Aspergillus flavus Aspergillus terreus Aspergillus niger

8. Identification Why? Which fungus is causing the disease – monitor which are most prevalent species Drug choice – A. terreus AMPHO How? Colony morphology – colour, size and texture Microscopy – slide preparation

9. Lactophenol Cotton Blue Slide Preparation Phenol – kills the fungus and the cells are not broken down Lactic acid – preserves the fungal structures Cotton blue – a dye that stains the chitin in the cell wall of the fungus Sellotape pressed against culture then lowered onto slide

10. Microscopic Structures

11. Microscopy Shape of the vesicle Presence of : phialides = uniseriate phialides+metulae= biseriate Position of phialides Size of conidia Roughened conidiophore

12. Vesicle shapes

13. Microscopy Shape of the vesicle Presence of : phialides = uniseriate phialides+metulae= biseriate Position of phialides Size and texture of conidia Roughened conidiophore

14. Uni- or biseriate?

15. Microscopy Shape of the vesicle Presence of : phialides = uniseriate phialides+metulae= biseriate Position of phialides Size and texture of conidia Roughened conidiophore

16. Cell arrangement

17. Microscopy Shape of the vesicle Presence of : phialides = uniseriate phialides+metulae= biseriate Position of phialides Size and texture of conidia Roughened conidiophore

18. Roughened conidiophore of A. flavus

19. They don`t always look like the pictures in the textbooks!

20. Susceptibility testing Aim: what concentration of a drug stops the aspergillus growing? - High concentration ‘Resistant’ - Low concentration ‘Susceptible’ Use a microbroth method to determine a MIC (Minimum Inhibitory Concentration)

21. Minimum Inhibitory Concentration (MIC) Low drug concentration High drug concentration Doubling dilution RPMI + Drug + Spore suspension

22. Azole resistance in A. fumigatus - Important for therapy choice - Due to different mechanisms; one mechanism is change in drug target Itraconazole recent literature 0 - 5% 7% RMLM 1992 – 2007 (n = 519) significant increase in resistance since 2004 – now 13% Itraconazole resistant isolates 58% cross-resistant to voriconazole 66% cross-resistant to posaconazole Data from Dr Susan Howard

23. Summary Cultured on SAB Identification by colony appearance and microscopy Susceptibility testing using CLSI method; detects resistance to aid therapy