34-Stuart Elborn - ISHAM

Comparison of Conventional and Molecular
Techniques to Examine the Diversity of Yeasts
and Filamentous Fungi in Adult Patients with
Cystic Fibrosis
J Stuart Elborn, Yuriko Nagano, Cherie Millar, John E Moore
Northern Ireland Public Health Laboratory,
Belfast City Hospital,
Centre of Infection and Immunity
Queen’s University, Belfast
Adult CF Centre City Hospital Belfast.

Prevalence of organisms in CF patients in U.K.
(UKCF Database, 2003) n=4,897 patients
100
90
80
70
%
60
50
55.7
*C: children A: adult
40
37
C: 20.2%
30
C: 10%
A: 15.6%
20
20.4
A: 11.6%
18.1
10
4.5
10.8
0
P. aeruginosa S. aureus H. influenzae B. cepacia
complex
Aspergillus
spp.
Candida
spp.
organisms

Increased % positive for Aspergillus in CF sputum
(CFF patient registry)
16
% positive for Aspergillus
14
12
10
8
6
4
6.18
7.31
8.8
9.42
10.88
11.44
12.16
12.87 13.02
13.51 13.45
2
0
1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005
Year

Overall objective
To improve clinical microbiology methods to aid in
the isolation, identification and characterization of
fungi from sputum of CF patients
Specific Aims
1. To develop an improved laboratory agar for the isolation of fungi
from CF sputum
2. To compare conventional and molecular techniques for the detection
of fungi in sputum samples from adult CF patients (n=77)
3. To investigate the prevalence and diversity of fungal species
Expected Outcomes
Appropriate monitoring / treatment
Reduced morbidity and mortality

Development of Improved Fungal
Agar
Problems associated with fungal isolation on agar
1. Previously, the inhibition of fungal growth by
P.aeruginosa and B. cepacia complex was reported
(J.R. Kerr, J infect. 1994 May; 28(3): 305-10; J Clin Micro. 1994 Feb; 525-527)
2. Overgrowth by rapidly growing bacterial organisms
Objectives of novel medium
1. Promote selectivity and sensitivity of yeasts and
filamentous fungi, whilst inhibiting co-flora (i.e.
resistant P. aeruginosa and B. cepacia complex
2. Develop medium that could be used for quantitative
assessment of fungi in sputum

Media B +
Glucose 16.7g
Agar
20g
Yeast extract 30g
Peptone 6.8g
(per 1000ml)
+
Cotrimoxazole 128mg/l
Chloramphenicol 50mg/l
Ceftazidime 32mg/l
Colistin 24mg/l
Nagano Y, Millar BC, Goldsmith CE, Walker JM, Elborn JS, Rendall J, Moore JE.
Development of selective media for the isolation of yeasts and filamentous fungi from the sputum of
adult patients with cystic fibrosis (CF).
J Cyst Fibros. 2008; 7(6):566-72

Ability to culture on Media B +
Fungi
Growth
Yeasts (n=7) 100%
Filamentous fungi (n=7) 100%
Bacteria
P. aeruginosa (n=16) 0%
B. cepacia complex (n=18) 67% (poor growth)
S. maltophilia (n=1) 0%
E. coli (n=1) 0%
H. influenza (n=1) 0%
P. fluorescens (n=2) 0%
Morgarella morgarii (n=2) 0%
A. xylosoxidones (n=2) 0%
K. oxytoca (n=1) 0%
A. salmonicida (n=1) 0%
P. mirobilis (n=1) 0%
MRSA (n=1) 0%
S. aureus (n=2) 0%

SDA
Media
B
Media B with
antibiotics

SDA
Media
B
Media B with
antibiotics
Selectivity and sensitivity: Medium B+ antibiotics > SDA > Medium B

Specificity of different fungal media
Fungal
Selective
Medium
Specificity Yeasts Filamentous
fungi
Combined
fungi
SDA 46.4% 76.0% 83.0% 84.6%
SDA + 89.2% 84.0% 33.0% 84.6%
Medium B + 85.7% 92.0% 83.0% 92.3%
Nagano Y, Millar BC, Goldsmith CE, Walker JM, Elborn JS, Rendall J, Moore JE.
Development of selective media for the isolation of yeasts and filamentous fungi from the sputum of adult patients with cystic fibrosis (CF).
J Cyst Fibros. 2008; 7(6):566-72.

n=77 adult patients attend Regional Adult CF Centre
Median age: 28.5 (18-59 years) Male: 48% Female: 52%
Fresh CF Sputum ( post physiotherapy)
1. Employing conventional
mycological culture
Plate onto
SDA
30 ˚C 1week
Resend to mycology
specialist laboratory
Identification
2. Mycological culture with CF-derived
fungal selective culture medium
Plate onto
SDA Media B
22 ˚C 2-3 weeks
DNA extraction
PCR (ITS1-ITS4)
Direct sequencing
Identification
+ Colistin
Ceftazidime
Cotrimoxazole
Chloramphenicol
3. Direct DNA extraction from sputum
(non-cultured) approach
Add 1:1 Sputalysin
DNA extraction
PCR (ITS1-ITS4)
Nested PCR (ITS3-ITS4)
One band
Direct sequencing
Identification
more than one band
Cloning
Sequencing
Nagano Y, Elborn JS, Millar BC, Walker JM, Goldsmith CE, Rendall J, Moore JE.
Comparison of conventional and molecular techniques to examine the diversity of yeasts and filamentous fungi in a population of adult patients with cystic
fibrosis. Med Mycology (in revision)

Primers used in this study
ITS1 region
ITS2 region
18S rRNA gene 5.8S rRNA gene 28S rRNA gene
5’-TCC GTA GGT GAA CCT GCG G-3’
5’-TCC TCC GCT TAT TGA TAT GC-3’
ITS 1 ITS 4
Primarily PCR
5’-GCA TCG ATG AAG AAC GCA GC-3’
ITS3 ITS 4
Nested PCR
Millar BC, Xu J, Earle JA, Evans J, Moore JE.
Comparison of four rDNA primer sets (18S, 28S, ITS1, ITS2) for the molecular identification of yeasts and
filamentous fungi of medical importance. Br J Biomed Sci. 2007;64(2):84-9.

Prevalence of fungi in CF patients
100
97
90
80
70
Yeast=6 genera
Filamentous fungi=9 genera
% patients positive
60
50
40
58
39
30
20
10
9.1 9.1 9.1 6.5
5.2 5.2 3.9 3.9 2.6 2.6 1.3 1.3 1.3 1.3 1.3 1.3 1.3
0
Candida spp.
Candia albicans
Candia dubliniensis
Candida parapsilosis
Aspergillus spp.
Penicilium sp.
Saccharomyces cerevisiae
Candida glabrata
Aspergillus fumigatus
Exophiala dermatitidis
Scedosporium apiospermum
Trichospororn sp.
Malassezia sp.
Rhodotorula sp.
Aureobasidium pullulans
Fuscoporia ferrea
Fusarium culmorum
Acremonium strictum
Thanatephorus cucumeris
Cladosporium sp.
Yeasts and filamentous fungi

The number of fungi detected in CF
patients
5 fungi
1%
2 fungi
23%
3 fungi
16%
1 fungi
60%
N=77
1 fungi=46
2 fungi=18
3 fungi=12
4 fungi=0
5 fungi=1

Comparison of three different
methods
80
70
60
Method 1=14 (18%)
Method 2=60 (78%)
Method 3=77 (100%)
% positive
(n=77)
50
40
30
20
10
0
Candida spp.
Candia albicans
Method 1 Method 2 Method 3
Candia dubliniensis
Candida parapsilosis
Candida glabrata
Exophiala dermatitidis
Rhodotorula sp.
Saccharomyces cerevisiae
Trichosporon sp.
Aspergillus spp.
Aspergillus fumigatus
Penicilium sp.
Scedosporium apiospermum
Aureobasidium pullulans
Fuscoporia ferrea
Fusarium culmorum
Acremonium strictum
Malassezia sp.
Thanatephorus cucumeris
Cladosporium sp.
Yeasts and filamentous fungi

Fungal air sampling from CF health
care environments
Collected 2min=1400L of air by an electrical slit air sampler (CF Casella Ltd., London, England)
Cultured on SDA and Media B at 22˚C for 2 weeks
In patient Out patient
1. Corridor 1. Corridor
2. Common room 2. Physiotherapy room
3. Physiotherapy room 3. Consulting room
4. Treatment room
5. Patient room
6. Shower & Toilet
7. Interview room

Comparison between fungi isolated in
the air and fungi isolated from CF
sputum
Fungal isolated from sputum
Fungal isolates from the air
Sporidiobolus salmonicolor
Thanatephorus cucumeris
Malassezia sp. Trichosporon sp.
Acremonium strictum
Fusarium culmorum
Fuscoporia ferrea
Cladosporium sp.
Rhodotorula sp.
Phaeococcomyces chersonesos
Emericella sp. Coniosporium sp.
Phoma herbarum Blumeria sp.
Kondoa aeria Trametes sp.
Rhexocercosporidium sp.
Scedosporium apiospermum
Saccharomyces cerevisiae
Exophiala dermatitidis
Candida glabrata
Candida parapsilosis
Candia dubliniensis
Aspergillus versicolor
Aspergillus fumigatus
Penicillium sp.
Aureobasidium pullulans
Sclerotinia sclerotiorum
Sterem annosum Heterobasidion annosum
Paecilomyces sp. Aspergillus sydowii
Cryptococcus sp.
Cryptococcus magnus
Engyodontium album
Candia albicans
Yarrowia lipolytica
*Green: isolated from only CF wards
*Blue: isolated from only clinic
*Black: isolated from both

Conclusions
1. Formulation of improved fungal isolation agar
-superior to SDA (78% vs. 18%)
-suppressed all G +ve and all G –ve, except B. cepacia complex organisms
-allowed proliferative growth of all yeasts and filamentous fungi tested
-recommendation: CF clinical microbiology laboratories employ medium B to improve
isolation of yeasts and filamentous fungi from CF sputum
2. Conventional vs. Molecular Results
% positive fungi
Genera isolated
Candida spp.
Conventional
SDA SDA+ Medium B
18% 78%
3 8
8/77 56/77
Molecular
100%
12
71/77
3. Candida spp. > Aspergillus spp. > Penicillium spp. > Saccharomyces
cerevisiae > Exophiala dermatitidis > Scedosporium apiospermum

The classification of fungi related to CF
Decreasing clinical significance in CF patients
I
very significant fungi
related to CF
II
relatively significant fungi
related to CF
III
potentially significant
fungi related to CF
IV
not reported as human pathogens
Aspergillus fumigatus
Aspergillus versicolor
Aspergillus sydowii
Aspergillus spp.
Candida albicans
Candida dubliniensis
Candida parapsilosis
Candida glabrata
Exophiala dermatitidis
Scedosporium apiospermum
Trichosporon sp.
Malassezia sp.
Penicillium sp.
Rhodotorula sp.
Saccharomyces cerevisiae
Aureobasidium pullulans
Acremonium strictum
Cladosporium sp.
Cryptococcus sp.
Fuscoporia ferrea
Fusarium culmorum
Thanatephorus cucumeris
Sporidiobolus salmonicolor
Phaeococcomyces chersonesos
Emericella sp.
Blumeria sp.
Phoma herbarum
trametes sp.
Coniosporium sp.
Kondoa aeria
Rhexocercosporidium sp.
Sclerotinia sclerotiorum
Sterem annosum
Heterobasidion annosum
Paecilomyces sp.
Engyodontium album
Yarrowia lipolytica

SDA
Media
B
Media B with
antibiotics

Reported infection caused by fungi in CF and non-CF
patients

Moore JE, Murphy A, Millar BC, Loughrey A, Rooney PJ, Elborn JS, Goldsmith CE.
Improved cultural selectivity of medically significant fungi by suppression of contaminating bacterial flora employing gallium (III) nitrate
J Microbiol Methods. 2009 ;76(2):201-203.

Prevalence of fungi in CF patients
100
97%
90
80
70
Candida albicans
Candida dubliniensis
% patients positive
60
50
40
58%
39%
Highest reported
rate is 11.1%
30
20
10
9.1% 9.1% 9.1% 6.5%
5.2% 5.2% 3.9% 3.9% 2.6% 2.6% 1.3% 1.3% 1.3% 1.3% 1.3% 1.3% 1.3%
0
Candida spp.
Candia albicans
Candia dubliniensis
Candida parapsilosis
Aspergillus spp.
Penicilium sp.
Saccharomyces cerevisiae
Candida glabrata
Aspergillus fumigatus
Exophiala dermatitidis
Scedosporium apiospermum
Trichospororn sp.
Malassezia sp.
Rhodotorula sp.
Aureobasidium pullulans
Fuscoporia ferrea
Fusarium culmorum
Acremonium strictum
Thanatephorus cucumeris
Cladosporium sp.
Yeasts and filamentous fungi organisms