Aspergillosis Patient Support Meeting April 2011 - Nichola Duddy

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

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

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

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

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

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

Aspergillus fumigatus
Aspergillus flavus
Aspergillus terreus
Aspergillus niger

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

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

Microscopic Structures

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

Vesicle shapes

Microscopy
Size and texture of conidia

Uni- or biseriate?

Microscopy

Cell arrangement

Microscopy

Roughened conidiophore of A. flavus

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

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)

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

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

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

Aspergillosis Patient Support Meeting April 2011 - Nichola Duddy

by Graham Atherton on Jul 28, 2011

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Aspergillosis Patient Support Meeting April 2011 - Nichola Duddy — Webinar Transcript