Transcript Slide 1

Diagnosis and Management of
Fungal Disease
Damian Downey & John E. Moore
Northern Ireland Regional Adult Cystic Fibrosis Centre
Belfast City Hospital
Diagnosis and Management of Fungal Disease
Cystic Fibrosis Trust Clinical Conference
 Background/Setting the Scene - John Moore
 Spectrum of fungal disease in CF
-
 Diagnostic Aspects  Discussion
-
Floor
 Identification of key issues:


Clinical?
Diagnostic?
 Next steps & Wrap-up -
Damian Downey
Damian Downey
John Moore
Infection in CF is very important
The Reason
No significant bugs
= 50 years
Pseudomonas aeruginosa
= 30 years
Burkholderia cenocepacia
=19 years
Aspergillus/Scedosporium = ??
UK CF Trust Newsletter
Fungal isolates from
hospital air
Sporidiobolus salmonicolor
Phaeococcomyces chersonesos
Emericella sp. Coniosporium sp.
Cladosporium sp.
Rhodotorula sp.
Fungi isolated
concurrently from
CF patients’
sputum
Aspergillus
versicolor
Phoma herbarum Blumeria sp.
Kondoa aeria
Trametes sp.
Rhexocercosporidium sp.
Sclerotinia sclerotiorum
Aspergillus
fumigatus
Sterem annosum Heterobasidion annosum
Penicillium sp.
Aureobasidium
pullulans
Paecilomyces sp. Aspergillus sydowii
Cryptococcus sp.
Cryptococcus magnus
Engyodontium album
Yarrowia lipolytica
Nagano Y, Elborn JS, Millar BC, Walker JM, Goldsmith CE, Rendall J, Moore JE.
Comparison of techniques to examine the diversity of fungi in adult patients with cystic fibrosis.
Med Mycol. 2010; 48:166-76.
Increased % positive sputum cultures for Aspergillus
( US CFF patient registry)
16
14
13.51
% positive for Aspergillus
12.87
13.45
13.02
12.16
12
11.44
10.88
10
9.42
8.8
8
6
7.31
6.18
4
2
0
1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005
Year
Bruce Montgomery; Personal communication
% Positive Aspergillus
in sputum
9
8
8
7.4
7
7
6
6.7
6
5.8
5.4
5
5
4
3
2
1
0
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
Year
CF Registry of Ireland (http://www.cfri.ie/)
ARTEFACTUAL
(i). Increased reporting
Efficacy/data capture
REAL EFFECT
(i). fungal virulence
(ii). change in epidemiology
(iii). increased risk factors
(ii). Improved laboratory
diagnosis
- culture
- NGS
- MALDI-TOF
- standardisation
(iii). Improvements in
radiological imaging
Spectrum of Fungal Disease in CF
Aspergillus fumigatus
 Aspergillus causes significant morbidity in CF
 Reported prevalence rates 6% to 58%
 Wide spectrum of disease
 Mean age of onset 12-14 years of age1
 Challenges over classification/diagnosis and
treatments
 Guidelines for ABPA2
1. Pihet et al. Med. Mycol. 2009
2. ABPA in CF – State of the Art: CFF Consensus Conference 2003
ABPA
 Manifests as a worsening of pulmonary disease with
wheezing, SOB, cough and chest pain
 Frequency varies due to differences in diagnosis
(1-15%)
ABPA
 Diagnostic criteria
 Clinical deterioration not attributable to another
aetiology
 Total IgE ˃1000 IU/ml
 Aspergillus SPT or specific IgE
 Precipitating antibody to Aspergillus or IgG
 New or recent abnormalities on CXR/CT not cleared
with antibiotics and PT
ABPA treatment
 Variation in therapeutic approaches
 Steroids are the mainstay of treatment1
 Triazole antifungals may have some steroid sparing
effect
 Combinations/length of treatment/monitoring
1. ABPA in CF – State of the Art: CFF Consensus Conference 2003
Triazole antifungals
 Decrease the fungal burden and antigenic
stimulation
 Itraconazole1
 Drug levels/interactions/SEs
 Azole resistance – 94 BAL samples2
 4.3% azole resistance
 Mixed population – sensitive/resistant
1. Wark et al. J Allergy Clin Immunol. 2003
2. Zhao et al. J Antimicrob Chemo. 2013
Voriconazole
 Voriconazole therapy in children with CF1
 Open label retrospective
 High oral bioavailability
 Absorption not affected by pH
 Plasma levels are variable2
 Drug interactions
 Cost implications
1. Hilliard et al. Journal of CF. 2005
2. Spriet et al. Eur J Clin Microbiol Dis. 2011
Cochrane Review 2012- Antifungal therapies for ABPA in
people with cystic fibrosis
 “At present, there are no randomised controlled
trials to evaluate the use of antifungal therapies
for the treatment of ABPA in people with cystic
fibrosis. Trials with clear outcome measures are
needed to properly evaluate this potentially
useful treatment for cystic fibrosis”
Cochrane Database Syst Rev. 2012 Jun 13;6:CD002204
Nebulised amphotericin
 Case series1,2
 Limited experience
 Tolerability
1. Proesmans et al. Adv in Resp Dis. 2010
2. Hayes et al. Paed Pulm 2010
Omalizumab therapy
 Recombinant monoclonal antibody targeting Fc receptor
of IgE and prevents binding of IgE to immune effector
cells
 Mediates inflammation and hypersensitive response
 Case series1,2
 Trial 16 ABPA patients (CF excluded)3
 Exacerbations reduced. No effect on lung function
 Pharma study in CF- RCT terminated early4
 Cochrane review 2013. Need for further studies 5
1.
5.
Wong et al. Paed respiratory reviews 2013
2. Brinkmann et al. Allergy 2009
3. Tille-Leblond et al. Allergy 2011
4. http://clinicaltrials.gov/show/NCT00787917
Cochrane Database Syst Rev. 2013 Sep 17;9:CD010288
Aspergillus spectrum
 Prevalence rates 6-58%
 Detection challenges
 Not all develop ABPA
 How do we define this group?
 Colonisation/infection/sensitisation
 Does it cause clinical deterioration?
 Does it affect lung function?
Aspergillus bronchitis
 Case series – 6 patients1
 Not ABPA by criteria
 But varying IgE
 None received steroids
 Clinical improvement with itraconazole
1.Shoseyov
et al. Chest 2006
Aspergillus colonisation
 Dutch study 20111
 61/259 children and adults
 Retrospective 2002-2007
 Defined as Aspergillus in >50% resp cultures that
year
 Categorized to the no. of years they met criteria
above
1. Vrankrijker et al. Clinical Microbiology and Infection 2010
Aspergillus fumigatus colonization in cystic fibrosis: implications
for lung function?
•Differences in FEV1 disappeared after adjustment for confounders
•Specific antibody data was not included
•Colonisation with Aspergillus was not independently associated with a decline in lung fn
•Aspergillus was independently associated with age and the use of inhaled antibiotics
Clinical Microbiology and Infection
Volume 17, Issue 9, pages 1381-1386, 16 DEC 2010 DOI: 10.1111/j.1469-0691.2010.03429.
The effect of chronic infection with Aspergillus
fumigatus on lung function and hospitalisation
 Retrospective paed study (1999-2006) Toronto
 Did not differentiate between infection/colonisation
 230 patients, 37 (16%) Aspergillus
 FEV1 (79% vs 86%)
 Increased ABPA, use of neb antibiotics, steroids, Pa
infection and inc trend of pulm exacerbations
1. Amin et al. Chest 2010
Aspergillus colonisation
 Separate colonisation and sensitisation?
 Toulouse Centre, 251 patients (complete data 206)1
 Retrospective 1995-2007
 ABPA (34), sensitisation group (63), persistent carriage
(37) and control (72)
 Groups were independently associated with FEV1 decline
 Some sensitised patients had episodes of ABPA
1. Fillaux et al. Scand J Infect Dis. 2012
Differences
 Definitions-colonisation/sensitisation/chronicity
 Transient vs persistent culture
 Comparing different groups
 Different analyses
 Retrospective
 Can we define different syndromes?
Classification of Aspergillus syndromes?
 Challenges in distinguishing colonisation,
sensitisation and Aspergillus bronchitis/infection
 146 patients in Manchester1
 Assessed serologic tests, RT-PCR, sputum GM
 39 (27%) culture positive
 108 (74%) RT-PCR positive (66 GM positive)
 68 (46%) GM positive
1. Baxter et al. Amer Acad of Allergy 2013
Class analysis
 Class 1
 ± asp in sputum, -ve GM and immunology
 Class 2
 serologic ABPA with +ve GM, serology and RT-PCR
 Class 3
 ± asp in sputum, IgE sensitised, -ve IgG and GM
 Class 4
 -ve IgE, +ve IgG, RT-PCR and GM
Treatment of Aspergillus in patients
with CF
 Double blind, placebo RCT, 24 weeks1
 35 Aspergillus +ve patients (non-ABPA)
 Difficult recruitment
 Exacerbations were the same in each group
 FEV1 declined in itraconazole group
 Therapeutic itraconazole levels not achieved in 43%
 No benefit
1. Aaron et al. PLoS one. 2012
Aspergillus
 Many challenges
 Diagnosis
 Definition of groups
 Effect of treatments
 Relationship to bacteria/viruses
Scedosporium apiospermum
 French 5 yr prospective study – 8.6% patients1
 14% of CF patients in a German Centre2
 Risk factors for acquisition are not clear
 Genotyping in a centre revealed no shared strains,
mainly single strain3
 Same strains can be present for years4
1.
2.
3.
4.
Cimon et al. Eur. J. Clin. Microbiol. Infect. Dis. 2000
Horre et al. Respiration 2009
Defontaine et al. J Clin Microbiol 2002
Bernhardt. J Cystic Fibrosis 2013 (in press)
Diagnostic Aspects
- increasing burden of organisms +
- increasing biofilm +
Phase I
Phase II
Phase III
Phase IV
Phase V
ANTIMICROBIAL
INTERVENTION
Acquistion:
environment
patients
Adherence early colonisation
established colonisation early infection chronic infection
Improved Detection Zone
Improvements: Radiological imaging (chest x—ray; HRCT; MRI;)
selective culture/selective enrichment (agars)
direct molecular detection (PCR, RT-PCR, qPCR)
serological/antibody detection (galactomannan)
Biomarkers via fluorescence correlation spectroscopy (FCS)
(Sahahzad et al. J Cell Mol Med. 2011; 15(12):2706-11 )
1st PA culture f
rom sputum
nth fungal culture
from sputum

- increasing burden of organisms +
- increasing biofilm +
Phase I
Phase II
Phase III
Phase IV
Phase V
ANTIMICROBIAL
INTERVENTION
Acquistion:
environment
patients
Adherence early colonisation
established colonisation early infection chronic infection
Improved Detection Zone
Improvements: Radiological imaging (chest x—ray; HRCT; MRI;)
selective culture/selective enrichment (agars)
direct molecular detection (PCR, RT-PCR, qPCR)
serological/antibody detection (galactomannan)
Biomarkers via fluorescence correlation spectroscopy (FCS)
(Sahahzad et al. J Cell Mol Med. 2011; 15(12):2706-11 )
1st PA culture f
rom sputum
nth fungal culture
from sputum

133,317 fungal pyrosequences
30 species or genera, including 24
micromycetes and 6 basidiomycetous
macroscopic fungi.
• variation in the initial processing with
lytic
• agent,
• volume of sputa used to inoculate
plates,
• type of media,
• length of incubation
• temperature,
Laboratory diagnosis of fungi from CF
patients
 Culture-based
 Non-culture-based
 PCR (specific & broad range),
 RT-PCR,
 qPCR, NGS, Sanger Sequencing
 Most CF labs performing fungal
cultures
 cheap
 Highly specialised
 Reference/Specialist laboratory
involvement
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. pan-resistant P. aeruginosa and B.
cepacia complex
2.
Develop medium that could be used for
quantitative of fungi
Media B +
Glucose
Agar
Yeast extract
Peptone6.8g
(per 1000ml)
16.7g
20g
30g
Cotrimoxazole 128mg/l
+
Chloramphenicol 50mg/l
Ceftazidime 32mg/l
Colistin 24mg/l
Ability to culture on Media B +
Fungi
Yeasts
Filamentous fungi
Growth
100%
100%
Bacteria
P. aeruginosa
B. cepacia complex
S. maltophilia
E. coli
H. influenzae
P. fluorescens
Morgarella morgarii
A. xylosoxidones
K. oxytoca
A. salmonicida
P. mirabilis
MRSA
S. aureus
0%
67% (poor growth)
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
SDA
Media B with
antibiotics
Media B
SDA
Media B
Media B with
antibiotics
SDA
Media B
Media B with
antibiotics
Selectivity and sensitivity: Medium B+ antibiotics > SDA > Medium B
Sensitivity
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.
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.
•
Selective isolation techniques were superior in detecting non-Aspergillus
hyphomycetes compared with conventional methods.
•
Although liquid media detected fewer strains of Exophiala, Pseudallescheria and
Scedosporium species, additional hyphomycete species not detected by other
methods were isolated.
•
Current conventional methods are insufficient to detect non-Aspergillus
hyphomycetes, especially Exophiala, Pseudallescheria and Scedosporium species,
in sputum samples of cystic fibrosis patients.
Conclusions:
•
These data suggest that standard microbiological media and procedures are not sufficient
to detect colonization of the respiratory tract by Pseudallescheria/Scedosporium in CF
patients.
•
By use of Sce-Sel+ agar, fungi belonging to this complex were isolated more frequently.
Therefore, this semiselective mycological isolation medium should be used for the detection
of these fungi in the respiratory tract of CF patients, especially in patients in whom a fungal
infection is assumed or who are scheduled for lung transplantation.
n=77 adult patients attend Regional Adult CF Centre, BCH
Median age:28.5 (18-59 years) Male: 48% Female: 52%
Fresh CF Sputum ( post physiotherapy)
1. Employing conventional
mycological culture
Plate onto
2. Mycological culture with CF-derived
fungal selective culture medium
Add 1:1 Sputalysin
Plate onto
+ Colistin
Ceftazidime
Cotrimoxazole
Chloramphenicol
SDA
3. Direct DNA extraction from sputum
(non-cultured) approach
SDA Media B
30 ˚C 1week
DNA extraction
PCR (ITS1-ITS4)
Nested PCR (ITS3-ITS4)
22 ˚C 2-3 weeks
Resend to mycology
specialist laboratory
DNA extraction
PCR (ITS1-ITS4)
Identification
One band
more than one band
Direct sequencing
Sequencing
Direct sequencing
Identification
Cloning
Identification
Nagano Y, Elborn JS, Millar BC, Walker JM, Goldsmith CE, Rendall J, Moore JE.
Comparison of techniques to examine the diversity of fungi in adult patients with cystic fibrosis.
Med Mycol. 2010; 48:166-76.
ITS1 region
18S rRNA gene
ITS2 region
5.8S rRNA gene
5’-TCC GTA GGT GAA CCT GCG G-3’
28S rRNA gene
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 rate of fungi in CF patients
100
97％
90
Yeast=6 genera
80
Filamentous fungi=9 genera
% patients positive
70
60
58％
50
40
39％
30
20
10
9.1％ 9.1％ 9.1％
6.5％
5.2％ 5.2％ 3.9％
3.9％ 2.6％ 2.6％
0
Yeasts and filamentous fungi
1.3％ 1.3％ 1.3％
1.3％ 1.3％ 1.3％ 1.3％
The number of fungi detected in CF patients
5 fungi
1%
3 fungi
16%
n=77
1 fungi=46
2 fungi=18
2 fungi
23%
1 fungi
60%
3 fungi=12
4 fungi=0
5 fungi=1
Method 1=14 (18%) [NHS standard]
Method 2=60 (78%) [Selective agar + molecular ID]
Method 3=77 (100%) [Full molecular workup]
80
70
60
% positive
50
(n=77)
40
30
20
10
0
Yeasts and filamentous fungi organisms
The classification of fungi related to CF
Decreasing clinical significance in CF patients
I
II
III
IV
very significant fungi
related to CF
relatively significant fungi
related to CF
potentially significant
fungi related to CF
not reported as human
pathogens
Candida albicans
Rhodotorula sp.
Aspergillus fumigatus
Candida dubliniensis
Saccharomyces
cerevisiae
Aspergillus versicolor
Candida parapsilosis
Aspergillus sydowii
Candida glabrata
Aspergillus spp.
Exophiala dermatitidis
Aureobasidium pullulans
Acremonium strictum
Cladosporium sp.
Scedosporium
apiospermum
Trichosporon sp.
Malassezia sp.
Penicillium 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
Fungal Workshop –
What are the diagnostic and
clinical challenges?
Fungal Workshop –
What are the diagnostic and
clinical challenges?
 How do we define Aspergillus groups with the tools
currently available?
 Should there be a nominated specialist laboratory to
support fungal workup in CF?
 What RCTs should be carried out?
Thank you
cysticfibrosis.org.uk